Non-Variola Orthopox (including Monkeypox) (Sendout)

Real-Time Polymerase Chain Reaction (RT-PCR)

Serology testing, if performed: ELISA

Cotton swabs and swabs in media designed for bacterial preservation and/or transport may cause PCR inhibition and should not be used. Use of viral transport media dilutes DNA quantities. Specimens with insufficient human DNA will be resulted as inconclusive.

Blood collection in tubes with either heparin and/or ethylenediaminetetraacetic acid (EDTA) may interfere with results. Detection of immunoglobulin M and G antibodies is dependent upon the number of days the specimen was collected post-symptom onset. A previous history of smallpox vaccination or orthopoxvirus exposure may affect result interpretation.

Important: This assay does not distinguish between Orthopoxvirus (e.g. monkeypox, vaccinia, orf, pseudocowpox, and bovine papular stomatitis virus) except variola species. If positive, additional testing at the CDC may be performed.

Prior to Collection: Requires pre-approval from the Laboratory Medicine Resident (LMR) or Microbiology fellow on-call. Contact the LMR via the paging operator at 206-598-6190.

Collection Guidelines: Samples that are positive for Orthopoxvirus spp. via WA PHL testing will be forwarded to the CDC for Monkeypox virus specific testing. Deviations from the below guidance may result in sample rejection or recollection.

• A minimum of 2 swabs must be submitted for testing.
• Each swab should be used to sample multiple lesions, if present.
• Each swab must be packaged in its own sterile container (e.g. 15mL falcon tube, VTM tube, urine specimen cup) with its cap sealed by paraffin film or by similar means (e.g. tape).
• Each container must be labeled with a minimum of 2 patient identifiers and unique description for the topography of the swabbed lesions (e.g. right hand, left arm).

Acceptable Sample Types: Lesion material is required for persons with an active lesion or rash. Lesion material includes lesion fluid, lesion roof, lesion tissue, or crust. Use appropriate sterile technique and skin sanitation.

• Wet Swabs of Lesion:
  • Use sterile nylon, polyester, or Dacron swabs with a plastic or thin aluminum shaft for collection.
  • Swab the the lesion vigorously to collect adequate DNA. It is not necessary to de-roof the lesion before swabbing.
  • Wet swabs must be stored in Viral Transport Media (VTM) only. Universal Transport Media (UTM) is not accepted.
  • There should be no more than 3mL of VTM present.
• Dry Swabs of Lesion:
  • Use sterile nylon, polyester, or Dacron swabs with a plastic or thin aluminum shaft for collection.
  • Swab the the lesion vigorously to collect adequate DNA. It is not necessary to de-roof the lesion before swabbing.
  • Break off the end of each swab’s applicator into a dry, sterile screw-capped plastic vial, ideally with O-ring.
  • “Dry” swabs refer to swabs that are not placed in liquid (e.g. UTM, saline, gel).
• Crusts of Lesion:
  
  • Open and remove the top of the lesion using a sterile scalpel or 26-gauge needle.
  • Place the vesicle or pustule skin "roof" in a dry, sterile screw-capped plastic vial, ideally with O-ring.
  • Sample the crust or scab from at least 2 body locations.

• Blood (if requested): Collect 3 mL blood in RED TOP or GOLD SST tube.

Reject Due To:

• Unaccepted sample type
• Specimen not refrigerated or frozen properly
• Insufficient specimen volume
• Leaky specimen
• Swab on wooden shaft
• Approval from LHJ (Local Health Jurisdiction) or DOH CDE (Communicable Diseases Epidemiologist) not obtained
• Failure to follow specific Shipping & Handling requirements

Blood: Centrifuge sample in sealed rotors, and then transfer the sealed rotor to a biological safety cabinet. Under the BSC, open the centrifuge rotor and aliquot 1 mL serum (min. 0.5 mL) to a separate plastic vial (preferably with O-ring). Freeze at -20°C.

Storage: Freeze samples (serum, swabs, tissue) at -20°C, preferably within an hour of collection.

SPS: Accession a separate order for each sample type received. At UW-ML or HMC, notify UW-ML or HMC Sendouts immediately. Samples collected at UW-NW should be collected and shipped to WAPHL by SPS.

Login: SPMSND-;FREEZE

• MSNDTO: WPHL
• MSPTYP: [specimen type]
• MTSTRQ: ;Non Variola Orthopox (includes Monkeypox)

Sendouts:

• Place the order in the WA DOH ETOR (Electronic Test Ordering & Results Portal).
  • Manual requisitions are no longer accepted by WAPHL.
• Select test: Rule Out Rule out Non-variola Orthopox Virus under Emergency Response/Bioterrorism.
• Print the resulting requisition and package it with the sample.
• Package samples on dry ice according to Category B procedures.
• Shipping questions may be directed to Hannah Groeneveld, Sentinel Laboratory Coordinator, at (206) 418-5670.
• Contact WA PHL prior to sending samples outside of normal business hours.

Transportation Note: Specimens from patients with suspected or confirmed monkeypox are classified as “UN 3373 Biological Substance, Category B” per CDC.

Biosafety: Follow Departmental guidance in MediaLab; consult LMR/Micro Fellow if questions. For patients considered high risk for monkeypox, measures should be taken to minimize the risk of laboratory transmission when testing lesional specimens: Label all lesional specimens with a Bio-Alert sticker.

Stability (Frozen):

• Wet Swabs/Lesion Crusts: 7 days.
• Dry Swabs: 60 days.

Reject Due To:

• Unaccepted sample type
• Specimen not refrigerated or frozen properly
• Insufficient specimen volume
• Leaky specimen
• Swab on wooden shaft
• Failure to follow specific Shipping & Handling requirements

Samples should be sent to WAPHL for testing. WAPHL will forward to CDC for confirmatory testing, as appropriate.

• WAPHL Bioterrorism Division: 206-418-5562