Monkeypox Virus

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Mpox is a viral illness endemic to parts of Central and West Africa. 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 (formerly Monkeypox virus (Poxviridae family, Orthopoxvirus genus) is not endemic, including Canada, have documented clusters of cases of MPXV infection. All infections characterized so far among the recent clusters have been due to Clade II (formerly the West African clade).

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

Testing Indications

Who to test:
Individuals with a compatible clinical illness where MPXV is suspected should be tested. Currently, 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 aerology) is not available at PHO.

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit


Lesion fluid, crust material or scab2

Not Applicable

Sterile tube/ container or Virus Culture Collection kit order#: 390081


Swab of lesion2,4,5,6

Not Applicable

Place swab in a sterile tube/ container6 or Virus Culture Collection kit order#: 390081


Nasopharyngeal and/or throat swab3,5,6

Particularly useful specimens if testing during the prodrome (e.g. pre-rash in febrile patients who are contacts of cases, patients with macular or papular rash) – and may be collected on all patients

Sterile tube/container or Virus Respiratory Collection kit order#: 390082



≥0.5 ml

Red top or serum separator tubes


Cerebrospinal fluid (CSF)

≥0.5 ml may be submitted on patients with meningitis/encephalitis

Sterile tube /container



50 ml 

Sterile container


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 MPX infection.


Nasopharyngeal/throat swabs and blood specimens are generally not recommended in patients who have skin lesions that can be swabbed, or have skin lesion material that can be submitted for testing (e.g., scab). Skin lesions usually have higher viral loads, therefore skin swabs or lesion material are more sensitive (approximately 90%) for MPXV detection. However, blood should always be submitted along with a nasopharyngeal (NP) swab or throat swab on patients suspected of MPXV infection who do not have a skin rash (e.g., a close contact of a case with a febrile illness but no rash), 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 MPX, 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.


Extracted nucleic acid (minimum volume 50 µl) may be submitted for a Mpox test provided that the specimen was heat inactivated for 60 minutes at 60°C prior to off-board lysis. Please indicate (i) the heat treatment duration and temperature and (ii) the extraction method/kit used on the requisition when extracted nucleic acid is submitted.


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 MPX.


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 a Mpox test 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, including:

  • Requesting health care provider (CPSO number)
  • Complete mailing address
  • Telephone and fax number
  • Patient’s full name, date of birth, and health card number where applicable (must match specimen label)
  • Test(s) requested and indication for testing
  • Clinical information including symptom onset date
  • Specimen type and collection

If available, please also provide

  • Travel history (if applicable)
  • Exposure history and details (include smallpox vaccination history if applicable)

Failure to complete all requisition fields appropriately may result in rejection or testing delay.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Testing is performed daily Monday to Friday during regular lab hours.

After hours and weekend testing can be arranged after approval from a PHO Microbiologist. Contact PHO Customer Service (416-235-6556; 1-877-604-4567) or the on-call Duty Officer (416-605-3113).

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

Test Methods

Mpox PCR testing at PHO is a nucleic acid amplification test (NAAT) that uses a real-time multiplex PCR assay to detect 2 targets - a generic MPXV (panmonkeypox) target that detects both MPXV Clades I and II [formerly the Congo Basin/Central African clade and the West African clade, respectively] 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.

Orthopoxvirus testing at PHO is based on molecular nucleic acid amplification testing (NAAT) using 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, and final reporting of all results.

Note: Mpox serology and antigen testing are not being offered, nor are they currently available or recommended in Canada


As of June 23, 2022, PHO routinely tests all specimens requesting a Mpox test using the Mpox PCR assay. Orthopoxvirus PCR will only be used in circumstances when the Mpox PCR assay results require further clarification. All Mpox PCR results from PHO will be considered final.

^ 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 will also usually 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.

Invalid Mpox PCR results from testing conducted at PHO are also reported as final, in which case, submission of additional specimen(s) is recommended if clinically indicated.

Note: MPVX is the only virus belonging to the Orthopoxvirus genus recently detected in Canada. Other Orthopoxvirus known to infect humans butnot currently circulating in Canada include Variola (eradicated worldwide), Vaccinia (component of the smallpox vaccine, rare post-vaccine transmission), Cowpox (rare zoonotic infection), and Camelpox (rare zoonotic infection).


Monkeypox virus PCR Test Result Interpretation

Monkeypox virus (panmonkeypox)@ Clade II Monkeypox virus Interpretation on report Comments
Not detected Not detected Monkeypox virus NOT detected by real-time PCR Monkeypox virus is not detected.
Detected Detected Low Detected Detected Low Monkeypox virus DETECTED or DETECTED LOW by real-time PCR. Monkeypox virus is detected. See footnote.*
Detected or Low Level Detected Not detected Monkeypox virus DETECTED or LOW LEVEL DETECTED; by real-time PCR Monkeypox virus is detected. See footnote.*
Not detected Detected or Low Level Detected Monkeypox virus DETECTED or LOW LEVEL DETECTED by real-time PCR Monkeypox virus is detected. See footnote.*
Detected or Low Level Detected Indeterminate# Monkeypox virus  DETECTED or LOW LEVEL DETECTED; by real-time PCR Monkeypox virus is detected. See footnote.*
Indeterminate# Detected or Low Level Detected Monkeypox virus  DETECTED or LOW LEVEL DETECTED by real-time PCR Monkeypox virus is detected. See footnote.*
Indeterminate# Not detected Monkeypox virus INDETERMINATE; by real-time PCR See footnote.*
Not detected Indeterminate # Monkeypox virus INDETERMINATE by real-time PCR See footnote.*
Indeterminate# Indeterminate # Monkeypox virus INDETERMINATE; by real-time PCR See footnote.*
Invalid& Invalid& Monkeypox virus PCR test INVALID Invalid

@ Detection of either Monkeypox virus PCR target is sufficient for laboratory confirmation of Monkeypox virus infection.

*Specimen(s) with low level detected (cycle threshold 35.01-38.00) or indeterminate (cycle threshold 38.01-39.99) result(s) in one or both targets (pan Monkeypox virus, clade II) will have this information noted in the report comments. These results 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. Further investigation may be required. If clinically indicated, please submit additional specimen(s) for testing.

# The indeterminate range for this assay includes cycle threshold values from 38.01 to 39.99. 

& The Mpox PCR test is reported as invalid when there is failed amplification of the extraction control. Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition.


In cases where Orthopoxvirus PCR test is performed after Mpox PCR testing is complete, result interpretation is outlined in the table below.

Orthopoxvirus Target Gene Cycle Threshold (Ct)# Result Interpretation Additional Notes
Ct 40 Detected Detection of Orthopoxvirus by real-time PCR. Monkeypox virus is the only virus belonging to the Orthopoxvirus genus recently detected in Canada.
Ct between 40.01 and 44.99 Indeterminate Orthopoxvirus DNA indeterminate by real-time PCR. 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.
Ct 45 with valid internal control gene amplification Not Detected Orthopoxvirus DNA NOT DETECTED by real-time PCR Negative Orthopoxvirus PCR results are Monkeypox virus negative and final.
Ct 45 with no detection of internal control gene amplification Invalid Orthopoxvirus real-time PCR result is invalid. Test is invalid due to failed amplification of the extraction control.  Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition. Submit another specimen for testing if clinically indicated.


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 monkeypox virus are reported to the Medical Officer of Health as per Health Protection and Promotion Act.

Similar on the Health Protection and Promotion Act, R.S.O. 1990, c. H.7 (

Data and Analysis

Mis à jour le 17 juin 2024