Mpox: diagnostic testing

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This guidance is aimed at healthcare workers and clinical diagnostic laboratories both in the public and private sectors. It is to be used for all suspected cases, irrespective of whether the case is being managed as a high consequence infectious disease (HCID) case or not; however, cases meeting the definition for suspected HCID mpox should be discussed with the Imported Fever Service (IFS) prior to testing.

Note that as of August 2024, the operational case definition has been updated to reflect the emergence of clade I (HCID) cases beyond countries in Central Africa. Users are advised to check the up-to-date [case definitions for suspected HCID mpox] (https://www.gov.uk/guidance/monkeypox-case-definitions#hcid-definition), and in particular the countries considered at risk of HCID cases, prior to sending samples.

Mpox diagnostic testing is available in UKHSA laboratories and some NHS and private laboratories. For testing in non-UKHSA laboratories please also refer to the guidance provided by that laboratory.

Information is available on the mpox case definitions, and will continue to be updated. There is also information available on the classification of contacts of clade II mpox cases and follow-up advice.

Suspected clade I (HCID) cases

These should be discussed initially with local infection services, and if considered high risk of clade I, the IFS should be contacted to ensure testing is expedited.

Personal protective equipment (PPE)

The minimum recommended PPE for healthcare workers who need to be within 1 metre of a suspected case of mpox outlined in the NHS guidance on infection prevention and control measures for clinically suspected and confirmed cases of mpox in healthcare settings. For suspected clade I (HCID) mpox cases this is airborne PPE precautions; for suspected clade II (non-HCID) mpox cases this is droplet and contact precautions, with eye protection where there is a possibility of splash (for example taking diagnostic samples such as throat swab or deroofing lesions). See the NHS guidance for further details.

Information on the minimum recommended level of PPE for inpatient care is available in the NHS guidance on infection prevention and control measures for clinically suspected and confirmed cases of mpox in healthcare settings, which complements the national manual.

The national infection prevention and control manual also outlines the minimum recommended level of PPE for inpatient care.

Sampling for diagnostic testing

Mpox is diagnosed by PCR test for MPXV on a viral swab taken from a vesicle or ulcer. Swabs should be sent in viral transport media. We recommend that 2 samples are taken for each patient to ensure sufficient material for confirmatory testing.

Follow the instructions below:

1. Ensure that you are wearing the minimum appropriate PPE as above.

2. Take a viral swab in viral culture medium or viral transport medium (for example Virocult®) from an open sore or from the surface of a vesicle. If other wounds are present, ensure that the sample is definitely taken from a vesicle, an ulcer or a crusted vesicle. Rub the swab over the lesion and place the swab in the collection tube. If there are pharyngeal lesions, a throat swab should also be taken. Label the tube with the patient’s name and date of birth, the date and site of the sample. Note that unlabelled tubes cannot be processed.

3. A viral throat swab can be taken for high-risk contacts of a confirmed or highly probable case who have developed systemic symptoms but do not have a rash or lesions that can be sampled. Please note that even if the throat swab is negative, the individual must continue with monitoring and isolation as instructed by their local health protection team, and should be reassessed and sampled if further symptoms develop.

4. Other samples (blood, semen, scabs) should only be sent if directed by the IFS.

5. For suspected HCID cases, urgent local testing for MPXV can be done in Containment Level 3 per local risk assessment; however, in order to ensure timely clade testing, samples should be sent concurrently to the Rare and Imported Pathogens Laboratory (RIPL). If local testing is positive, this ensures that samples are already in RIPL for urgent clade testing. If local testing is negative, testing can be cancelled, or other testing (for example for rickettsial infection) can be done more quickly. If MPXV testing is not provided locally, the laboratory should also forward the samples urgently to RIPL for MPXV PCR.

6. Samples for investigation of other infections, including sexually transmitted infections, should be packaged separately, with separate request forms for local testing.

7. Samples should be sent to your normal local laboratory. If HCID mpox is suspected, please alert the laboratory to arrange for urgent processing, and to organise for samples to be sent to RIPL via a Category B courier. Category A is not required either for suspected or confirmed mpox samples, unless transporting viral cultures. This is per the Department for Transport Multilateral Agreement M347 under section 1.5.1 of ADR on the carriage of MPXV.

Clinicians should remember that infections such as chickenpox, herpes simplex virus (HSV), molluscum contagiosum and syphilis are all still circulating and may present with similar lesions. Diagnostic testing for these infections should be undertaken as usual, with testing following normal local procedures.

Clade identification for locally confirmed PCR positive cases

Following the global increase in incidence of HCID clade I mpox in 2024, including by sexual transmission, there is an increased risk of mpox HCID infection circulating unrecognised on the background of clade II infections. Additionally, the emerging clade Ib strain may give false negative results with commercial clade assays.

All positive samples where HCID mpox is not suspected on clinical or epidemiological grounds should still be forwarded to RIPL for clade testing as part of ongoing surveillance. This can be done through routine sample referral systems such as DX.

Note that this is an update on previous guidance, which specified that clade testing could be done locally.

Sampling for monitoring confirmed or highly probable cases

If follow-up testing is required from a confirmed or highly probable case (a highly probable case is a case where only orthopox testing has been done), either because of clinical deterioration or to inform discharge from isolation to an inpatient setting, additional samples should be taken and should include the following:

a lesion swab and throat swab in viral transport medium
a blood sample in an EDTA tube
a urine sample in a universal sterile container

Samples should be sent to a laboratory capable of processing these samples for MPXV.

Submitting diagnostic samples to RIPL for testing

1. Download and complete the MPXV testing request form. If you require urgent reporting of results (usually only for suspected HCID cases), please ensure that you provide a direct contact number for the physician designated to receive results, which may be out of hours.

2. Package the samples carefully following the guidance in the RIPL user manual. You must ensure that stoppers and lids are firmly attached, the tubes are labelled with the sample type (for example swab left palm, penile lesion), patient name and date of birth. Do not use an internal study number. Samples that have leaked in transit cannot be processed. Samples that are not adequately labelled cannot be processed.

3. Send the samples to your normal local laboratory.

4. Inform your local laboratory that the samples for MPXV testing should be forwarded to RIPL for testing. This should be through a licensed Category B courier to:

Rare and Imported Pathogens Laboratory

UKHSA

Manor Farm Road

Porton Down

Salisbury SP4 0JG

DX 6930400, Salisbury 92 SP

The working hours contact for RIPL is 01980 612 348 from 9am to 5pm on weekdays. You can also email ripl@ukhsa.gov.uk but do not include patient identifiable information.

5. If urgent testing is required, including out of hours, this must be discussed and agreed with the on-call Imported Fever Service consultant (0844 778 8990). Urgent testing will normally be reserved only for suspected HCID cases (see definition for suspected HCID mpox).

Test results

Urgent results for suspected HCID cases will be phoned to the referring clinician.

All results will be sent to the referring laboratory through the standard electronic reporting system.

The RIPL office can answer enquiries about results during opening hours. If chasing the whereabouts of a sample, please first contact your courier to ensure that the samples have been delivered. If the sample has been sent correctly via a Category B courier, the courier should be able to confirm delivery time.

Semen testing for viral DNA

There is a body of evidence documenting the detection of RNA and DNA viruses in semen after acute infection. MPXV DNA has been detected in semen up to day 11 after acute infection in men in Italy, although longitudinal sampling after this point was not carried out.

It remains unclear whether this is from seminal carriage as the testes are immunologically-protected sites (as with other viral infections such as Ebola virus disease), or from resolving meatal or urethral lesions that disappear as the skin lesions resolve.

However, in view of this, current UKHSA advice takes a cautious approach and recommends that men who have had a confirmed or highly probable diagnosis of mpox according to the UKHSA case definition should use condoms during sex for 12 weeks after complete recovery from mpox infection. This is in line with advice from the World Health Organization (WHO).

It should be recognised that there is currently no evidence to support this or any alternative advice.

Following the initial 12 weeks and up to 6 months after recovery from infection, UKHSA recommends performing MPXV PCR on semen samples (and where necessary, oropharyngeal and/or rectal swabs) if the patient:

is undergoing fertility treatment or planning pregnancy
is undergoing planned semen storage (for example prior to chemotherapy)
has an immunocompromised sexual partner (including a pregnant partner)
is concerned about transmission to sexual partner or partners for any other reason and requests a test from their clinician

Testing can also be offered on patient request following discussion with the local infection or genitourinary team and then with RIPL.

A single negative PCR after 12 weeks post recovery is considered adequate, with recognition that this is based on expert opinion in the absence of a clear evidence base.

It should also be noted that a positive PCR does not necessarily indicate that the patient is potentially infectious, only that fragments of DNA have been detected. However, since viral culture is not feasible for clearance testing, if the PCR is positive it is prudent to consider the patient potentially infectious.

Repeat samples should be submitted every 2 weeks thereafter, and the patient should continue to use condoms until a negative test result is obtained.

Any samples sent without a minimum serial interval of 2 weeks will not be processed without prior discussion with RIPL.

Any testing that is requested prior to 12 weeks post recovery must first be discussed and agreed with RIPL prior to sending. Samples received without discussion will not be processed. For semen tested prior to 12 weeks post-recovery, 2 samples collected at least 48 hours apart should be tested, and both need to be negative to be deemed clear.

Sample collection must take place in a facility capable of arranging transport of a Category B sample. These are usually hospitals where cases of mpox are cared for and not genitourinary medicine (GUM) clinics.

Samples should be submitted in a universal sterile container and sent to the RIPL address in the Submitting diagnostic samples to RIPL for testing section.

Samples must be packaged and sent as a Category B infectious substance. Please ensure that samples are properly packaged and sealed. Leaking samples will not be processed. Samples will only be tested during working hours Monday to Friday. Please include a completed MPXV testing request form stating interval post infection as outlined above.

Appropriate counselling from clinicians should include that this is an unvalidated diagnostic test.

It is expected that evidence will emerge for duration of MPXV DNA detection in semen which will allow this guide to be updated, and earlier testing may be offered if the evidence supports this.

Information for laboratories handling samples potentially containing MPXV

MPXV is a Hazard Group (HG) 3 pathogen. Under normal circumstances, any procedure with HG3 pathogens involving potentially infectious material, where there is a risk of generating aerosols, droplets or splashes, must be performed within a microbiological safety cabinet (MSC) at Containment Level (CL) 3. However, some diagnostic samples may be handled at CL2 subject to local risk assessment. The following table provides the minimum containment level for handling samples suspected or confirmed to contain MPXV.

Specimen type/test	Suspect/probable/possible case	Confirmed/highly probable case
Nucleic acid extraction for the molecular detection of MPXV	MSC I, II or III at CL3 until after virus inactivation.	MSC I, II or III at CL3 until after virus inactivation.
Sample transport	Transported via Category B route	Transported via Category B route
Nucleic acid extraction of skin lesion swabs for molecular assays of infections other than MPXV (for example herpes simplex virus or syphilis)	MSC I, II or III at CL2 until after virus inactivation	MSC I, II or III at CL3 until after virus inactivation
Respiratory samples	MSC I, II or III at CL2	MSC I, II or III at CL2
Other microbiological samples where manual manipulation is required, for example plating of culture swabs, urine antigen testing, manipulation of blood cultures or urine	MSC I, II or III at CL2	MSC I, II or III at CL3
Routine laboratory blood tests	Autoanalysers at CL2	Autoanalysers at CL2
Waste disposal	Standard protocols for disinfection and waste disposal	Standard protocols for disinfection and waste disposal
Routine blood tests outside of an autoanalyzer	Open bench at CL2 with PPE (gloves, laboratory coat +/- eye protection subject to risk assessment) Samples must be centrifuged using sealed centrifuge rotors or sample cups which are loaded and unloaded in an MSC	Open bench at CL2 with PPE (gloves, laboratory coat +/- eye protection subject to risk assessment) Samples must be centrifuged using sealed centrifuge rotors or sample cups which are loaded and unloaded in an MSC
Point of care / near patient testing of lesion swabs	Generally not to be performed but is possible with a local risk assessment which identifies correct PPE to protect the healthcare worker	Generally not to be performed but is possible with a local risk assessment which identifies correct PPE to protect the healthcare worker
Point of care / near patient testing of respiratory samples	Should not be analysed unless a local risk assessment has been completed and shows that it can be undertaken safely. Near-patient tests for viral nucleic acid amplification vary widely in their general safety and where aerosols or droplets may be generated. If a local risk assessment can show that any aerosol or droplet generation occurs within a closed analyser, and external surfaces can be cleaned with detergent-based disinfectant, then these tests may be used	Not to be performed
Point of care tests / near patient testing of other samples, for example blood gases	May be used subject to local risk assessment regarding the potential for the generation of splashes and droplets, and the appropriate use of PPE and disinfection	May be used subject to local risk assessment regarding the potential for the generation of splashes and droplets, and the appropriate use of PPE and disinfection

Laboratory exposure may occur via needlestick injury, direct specimen contact, or inhalation of aerosols. These exposures can be avoided if standard biosafety precautions are taken.

It is important that other diagnostic testing is not delayed while waiting for the results of MPXV testing so as not to delay diagnosis of other illness that may require urgent treatment.

Other procedures, such as extraction or procedures which may generate aerosols, should be performed in a CL3 facility with staff wearing cuffed, back-fastening gowns, gloves and goggles.

For PCR testing of specimens from suspected mpox cases (for example testing for syphilis or HSV), the specimens should be opened in an appropriate microbiological safety cabinet in a CL2 facility.

Samples can be inactivated before extraction by heating for 1 hour at 60°C in a validated water-bath or block designed to ensure even heat distribution throughout standard specimen tubes.

Guanidine/guanidinium buffers such as AVL can also be used but work best with additional non-denaturing surfactants (see Inactivation of orthopoxvirus for diagnostic PCR analysis by Vinner and Fomsgaard, and PAHO/WHO guidance).

Information on inactivation studies performed by UKHSA on some commonly used sampling buffers for mpox is available.

Inactivated samples can be handled safely with standard laboratory precautions.

Samples from confirmed and highly probable mpox cases should be labelled appropriately so that laboratories can ensure that they are handled correctly with the necessary additional precautions as outlined above.

For routine testing of blood samples from suspected or confirmed mpox cases (for example for biochemistry or haematology), standard clinical laboratory precautions can be followed, but aerosol generating procedures should be avoided. The risk of infection from these samples is less than that for hepatitis B or any other similar organism.

Surfaces should be decontaminated with standard disinfectants. Waste should be autoclaved and disposed of as per standard laboratory procedures.

Guidance for laboratories seeking to establish mpox diagnostic testing capability

UKHSA provides a primary diagnostic testing service and a clade confirmation service.

The primary diagnostic service is chargeable, except for suspected HCID cases referred through the IFS. Clade confirmation is not charged for; however, you will be charged if you do not state clearly on the form that the sample has already tested positive and that only clade confirmation is required.

UKHSA does not restrict NHS or private laboratories from establishing their own MPXV testing service, but any such service would be provided at their own expense. Please note that all laboratories, including private laboratories, must forward samples from positive cases to RIPL for clade confirmation.

Laboratories planning on establishing local diagnostic testing should ensure they remain compliant with relevant legislation, perform appropriate risk assessments, and assure ongoing quality. Laboratories should be aware that MPXV is schedule V restricted by the Anti-terrorism, Crime and Security Act 2001.

Laboratories should also ensure that their testing provision is able to maintain compliance with the different operational definitions of HCID for mpox within their testing pathways, including the ability to distinguish or escalate for confirmation cases that may be classified as an HCID.

UKHSA does not approve, endorse or recommend a specific commercial MPXV diagnostic test, although an MPXV-specific nucleic acid amplification test is recommended, and use of an orthopox-only test is discouraged.

The use of multi-target assays is recommended. Reports of genomic deletions in assay target regions that may result in false negatives have been reported. Similarly, the recently reported clade Ib strain lacks the PCR target for the standard clade I assay and may be misidentified by the usual clade tests.

Please discuss the case with RIPL if you suspect you have a case with an assay target dropout (for example orthopox positive/MPXV negative).

At present, only nucleic acid amplification tests (typically PCR-based) are recommended for provision of testing services. Other test types are available on the market, however their performance characteristics in this outbreak are uncertain and are not recommended by UKHSA.

A list of tests on the commercial market is maintained by FIND.

Recommended process prior to testing

To ensure confidence and compliance with the current notifiable diseases regulations, it is recommended that UK laboratories follow the process outlined below prior to commencing with an mpox testing service.

1. Submit a copy of their nucleic acid assay verification file and reporting criteria for peer review to the UKHSA mpox Diagnostics team (contact details below), along with the intended go live date and predicted capacity.

2. Technical specialists at UKHSA Porton Down will provide a peer review that assay selection is reasonable for the service offered and remove the need for confirmatory testing of positive samples by RIPL. This peer review is not an assay approval and laboratories should ensure they follow their standard local assay approval processes including through relevant accredited bodies.

3. Laboratories should work with their UKHSA regional laboratory surveillance Second Generation Surveillance System (SGSS) leads to ensure data feeds from their laboratory systems are reporting correctly to SGSS data system.

4. For monitoring purposes, laboratories should provide both MPXV negative and positive results into SGSS.

5. It is critical that data collection and reporting systems are in place and tested with mpox data analytics teams before the service goes live to ensure this feeds into the national surveillance programme.

6. A turnaround time within 24 hours from sample receipt, and preferably same day, is desirable.

If a laboratory intends to provide testing using an alternative testing methodology, please contact the UKHSA mpox Diagnostics team (contact details below) to discuss prior to offering the service, as this may have significant implications for public health monitoring purposes.

UKHSA Porton Down may be able to supply control material to assist in assay verification activities (contact through the UKHSA mpox Diagnostics team, see below).

External Quality Assurance (EQA) providers are providing mpox proficiency schemes for laboratories to assure their testing services.

UKHSA mpox Diagnostics team contact details: ripl@ukhsa.gov.uk

Mpox: diagnostic testing

Applies to England

Suspected clade I (HCID) cases

Personal protective equipment (PPE)

Sampling for diagnostic testing

Clade identification for locally confirmed PCR positive cases

Sampling for monitoring confirmed or highly probable cases

Submitting diagnostic samples to RIPL for testing

Test results

Semen testing for viral DNA

Information for laboratories handling samples potentially containing MPXV

Guidance for laboratories seeking to establish mpox diagnostic testing capability

Recommended process prior to testing

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Mpox: diagnostic testing

Applies to England

Suspected clade I (HCID) cases

Personal protective equipment (PPE)

Sampling for diagnostic testing

Clade identification for locally confirmed PCR positive cases

Sampling for monitoring confirmed or highly probable cases

Submitting diagnostic samples to RIPL for testing

Test results

Semen testing for viral DNA

Information for laboratories handling samples potentially containing MPXV

Guidance for laboratories seeking to establish mpox diagnostic testing capability

Recommended process prior to testing

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