Monkeypox: diagnostic testing

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

Infections such as chickenpox, HSV, molluscum contagiosum and syphilis are all still circulating. Diagnostic testing for these infections should be undertaken as usual, with testing for monkeypox proceeding in parallel.

Diagnostic tests

Personal protective equipment (PPE)

The minimum recommended PPE for healthcare workers who need to be within 1 metre of a suspected case of monkeypox is droplet and contact precautions, with eye protection where there is a possibility of splash (for example taking diagnostic tests such as throat swab or deroofing lesions) as outlined in the NHSE national infection prevention and control manual.

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

Sampling for diagnostic testing

Monkeypox is diagnosed by PCR test on a viral swab taken from one or more vesicles or ulcers, or from a dry scraping of the scab. Swabs should be sent in viral transport media. Scab scrapings should only be taken if there are no other lesions and should be sent in a standard universal container. Testing of scrapings may take longer than a lesion swab.

Follow the instructions below:

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

2. The most important sample to take is 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 an ulcer, vesicle, or crusted vesicle. Rub the swab over the lesion and place the swab in the viral culture tube or viral transport medium. Label the tube with the patient’s name and date of birth, the date and site of the sample. Please note that unlabelled tubes cannot be processed.

3. If all the lesions are crusted, scrape scab material into a dry plain universal container and label as above.

4. If the patient has fever or widespread rash, sore throat or other systemic symptoms, you should also take an EDTA blood sample and a throat swab. Urine samples may also be sent in a universal container and will be tested if deemed necessary for clinical management.

5. For high risk contacts of a confirmed case who have developed systemic symptoms but do not have a rash or lesions for sampling, you should take a throat swab in viral transport media. Note that even if the throat swab is negative, the individual must continue with monitoring and isolation as instructed, and should be reassessed and sampled if further symptoms develop.

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

7. Samples should be sent to your normal local laboratory for forwarding to the Rare and Imported Pathogens Laboratory (RIPL) (for monkeypox testing) and for local processing (all other tests).

8. Samples from suspected cases of monkeypox should be sent by Category B transport.

Sampling for monitoring confirmed cases

If follow up testing is required, either because of clinical deterioration or for discharge planning, additional samples should be taken and should include all of 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

These samples are needed to assess clinical status and monitor progress.

Samples from confirmed cases of monkeypox must be sent by Category A transport to the High Containment Microbiology unit at Colindale. Separate details have been provided to HCID units to arrange this testing.

Submitting diagnostic samples for testing

1. Download and complete the RIPL request form. Please provide a direct line contact number for the physician designated to receive results.

2. Package the samples carefully following the guidance on page 17 of 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, urine), patient name and date of birth. Do not use an internal study number. Samples that have leaked in transit cannot be processed if there is insufficient material left.

3. Send the samples to your normal local laboratory. Any additional samples for sexually transmitted infections should be packaged separately. Inform your local laboratory that the samples for monkeypox testing should be forwarded for testing. This should be through a licensed Category B courier to:

DX 6930400, Salisbury 92 SP

The working hours contact for RIPL is 01980 612 348 from 9am to 5pm on weekdays. Outside these times, medical staff only should contact the monkeypox clinical support line.

4. Samples arriving in the laboratory by 9am will processed and reported that day. Samples arriving before 12pm will be usually reported first thing the following morning.

5. For patients who are seriously unwell the consultant or registrar should contact the monkeypox clinical support line on 0344 225 0602 (24 hours a day, medical practitioners only).

Test results

Positive results will be phoned to the designated clinicians: please provide the relevant contact number when submitting the specimen. All results will be sent to the referring laboratory through the standard electronic reporting system.

The RIPL office can answer results enquiries during opening hours.

The monkeypox helpline operates 24 hours for advice regarding results.

Information for laboratories handling samples potentially containing monkeypox virus

Monkeypox 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 an MSC at CL3. 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 monkeypox.

Specimen type / test	Suspect/probable/possible case	Confirmed case
Nucleic acid extraction for the molecular detection of monkeypox virus. Propagation of monkeypox virus	MSC I or III at CL3 until after virus inactivation. (Likely referred to UKHSA RIPL for now but other regional centres in the future) Transported via Category B route	MSC I or III at CL3 until after virus inactivation. (Likely referred to UKHSA RIPL for now but other regional centres in the future) Transported via Category A route
Nucleic acid extraction of skin lesion swabs for molecular assays of infections other than monkeypox virus (for example herpes simplex virus or syphilis)	MSC I or III at CL2 until after virus inactivation	MSC I or III at CL3 until after virus inactivation
Respiratory samples	MSC I or III at CL2	MSC I 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 or III at CL2	MSC I or III at CL3
Routine laboratory blood tests	Autoanalysers at CL2. Standard protocols for disinfection and waste disposal	Autoanalysers at CL2. 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 a 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 a 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 monkeypox 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 monkeypox 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).

Inactivated samples can be handled safely with standard laboratory precautions.

Samples from confirmed monkeypox 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 monkeypox cases (for example for biochemistry, 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 disease.

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