Chart 2: Laboratory Testing for Acute, Generalized Vesicular or Pustular Rash Illness

1. A patient presents with an acute, generalized vesicular or pustular rash illness and is evaluated by a healthcare practitioner, infectious disease, or dermatology specialist.
   1. Take digital photos of the rash presentation for clinical consultation.
   2. Obtain consultation with local and/or state public health department.
2. If patient evaluation determines the patient has a low or moderate risk of smallpox, the specimens for testing will also be low or moderate risk and will be tested by a sentinel and/or an LRN Reference Laboratory.
   1. Use BSL-2 facilities to perform diagnostic assay(s) appropriate for etiologic agent detection. Options are:
      1. DFA
      2. PCR (assays with the same cycling conditions can run in parallel)
      3. Electron microscopy (if available)
      4. Viral culture (if appropriate)
   2. If varicella is suspected begin testing; consider using Tzanck smear.
   3. If etiologic agent detection assay(s) other than Orthopoxvirus are positive:
      1. Orthopoxvirus is ruled out. No further testing is needed unless clinically indicated. The patient has a low risk of smallpox.
   4. If etiologic agent detection assay(s) other than Orthopoxvirus are negative:
      1. Orthopoxvirus is possible.
      2. Perform the following tests:
         1. Non-variola Orthopoxvirus PCR
         2. Orthopoxvirus PCR
      3. If the Non-variola Orthopoxvirus PCR and Orthopoxvirus PCR are both negative, Orthopoxvirus is ruled out.
         1. Re‑evaluate the patient’s condition and assess the need for dermatologic consultation and/or other diagnostic testing.
         2. Consider histologic testing for erythema multiforme, immune or drug reactions.
      4. If the Non-variola Orthopoxvirus PCR and the Orthopoxvirus PCR are both positive:
         1. Orthopoxvirus infection other than variola virus (e.g., vaccinia, monkeypox, or cowpox virus) is possible.
         2. If needed, contact CDC to submit specimen(s) for confirmatory testing and species typing. The CDC Emergency Operations Center is available 24/7 at 770-488-7100.
3. If patient evaluation determines the patient has a HIGH risk of smallpox:
   1. Contact CDC Emergency Operations Center at 770-488-7100 before testing to discuss patient clinical history.
   2. If specimens are at a non-variola testing laboratory:
      1. Initiate chain-of-custody at FBI direction. Refer to variola testing laboratory.
   3. If specimens are at a variola testing laboratory:
      1. Use enhanced BSL-3 facilities. It is required that testing personnel be up‑to‑date with their smallpox vaccination.
      2. Simultaneously split specimens for testing at CDC.
   4. Rule out variola prior to other testing. Perform the following:
      1. Variola virus PCR
      2. Orthopoxvirus PCR
      3. Non-variola Orthopoxvirus PCR
      4. Electron microscopy (if available). Perform electron microscopy at local facility AFTER BSL-3 preparation of grids.
   5. If the Non-orthopoxvirus PCR is negative, the Orthopoxvirus PCR is negative, and Variola virus PCR is negative:
      1. Orthopoxvirus is ruled out.
      2. Re‑evaluate the patient’s condition and assess the need for dermatologic consultation and/or other diagnostic testing.
      3. Consider histologic testing for erythema multiforme, immune or drug reactions.
   6. If the Non-orthopoxvirus PCR is positive, the Orthopoxvirus PCR is positive, and Variola virus PCR is negative:
      1. Orthopoxvirus infection other than variola virus (e.g., vaccinia, monkeypox, or cowpox virus) is possible.
      2. If needed, contact CDC to submit specimen(s) for confirmatory testing and species typing. The CDC Emergency Operations Center is available 24/7 at 770-488-7100.
   7. If the Non-orthopoxvirus PCR is negative, the Orthopoxvirus PCR is positive, and Variola virus PCR is negative:
      1. Smallpox infection is possible (though this could also represent different sensitivities of the assays)
      2. Contact CDC immediately PRIOR to release of results. Call CDC Emergency Operations Center at 770-488-7100 (available 24/7)

NOTE: If patient symptoms progress to more closely resemble smallpox, refer all specimens to a Variola Testing Laboratory and CDC. Sequester all viral cultures and specimens. Contact PHL for transport of specimens.