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. 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:

Poxviruses (Excluding MPXV and Variola Virus) Testing Information

*Uncontrolled print copy. Valid only on day of print: 01 May 2024.

Testing Indications

Who to test:
Individuals with a compatible clinical illness where MPXV is suspected should be tested. Consult with Public Health Ontario (PHO) if you have questions regarding testing indications, specimen collection or transportation.

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 scab²	Not Applicable	Sterile tube/ container or Virus Culture Collection kit order#: 390081
Mpox	General Test Requisition	Swab of lesion^2,4,5,6	Not Applicable	Place swab in a sterile tube/ container⁶ or Virus Culture Collection kit order#: 390081
Mpox	General Test Requisition	Nasopharyngeal and/or throat swab^3,5,6	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
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	Urine¹¹	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

Approval for testing and notification of specimen submission is not required. Contact PHO Customer Service (416-235-6556/1-877-604-4567) or the on-call Duty Officer (416-605-3113) after hours if you wish to consult prior to sample collection and shipment.

Submit a maximum of three skin lesion specimens per patient – based on PHO’s laboratory 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.

Deroofing 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, Varicella) and Enterovirus may be ordered on the same specimens being tested for MPXV – these will be performed once the Mpox test is completed. If any other tests are required, submit additional specimens, as specimens submitted for a Mpox test will not be routinely processed for additional tests other than Herpesviruses and Enterovirus.

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’s laboratory 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 Sunday.

Mpox PCR test results will be issued up to 2 days from receipt at PHO’s Laboratory testing site in Toronto.

Test Methods

Mpox PCR testing at PHO’s laboratory 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’s laboratory for clinical testing.

Orthopoxvirus testing at PHO’s laboratory 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’s laboratory 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.

Algorithm

As of June 23, 2022, PHO’s laboratory 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’s laboratory will be considered final.

Specimens from female patients and pediatric patients (< 18 years old) for which either or both of the two MPXV targets were detected and/or indeterminate will undergo review and further investigation.

Invalid Mpox PCR results from testing conducted at PHO’s laboratory 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).

Interpretation

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, West African clade) 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.

^{^}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.

^& 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.

For an explanation of cycle threshold values, see An Overview of Cycle Threshold Values and their Role in SARS-CoV-2 Real-Time PCR Test Interpretation.

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. At this time all Mpox-related testing results are reported to the Medical Officer of Health.