Specimen Collection and Transport Guidelines for Suspect Smallpox Cases

The information contained on this webpage regarding collection of specimens for variola virus (smallpox) diagnostic testing and their transportation replaces the previous document known as “Guide D – Specimen Collection and Transport Guidelines.”

Evaluating Patients and Reporting Suspected Cases of Smallpox

The poster “Evaluating Patients for Smallpox: Acute, Generalized Vesicular or Pustular Rash Illness Protocol”  is a tool designed to address testing needs when no smallpox outbreak has been detected or declared. Physicians should use this algorithm to categorize a patient’s risk of smallpox –low, moderate, or high– when they present with an acute, generalized vesicular or pustular rash illness.

Report a suspected case of smallpox immediately to the appropriate local, state or territorial health department.If, after their review, the patient meets the criteria for high-risk of smallpox, the case should be immediately reported to the CDC Emergency Operations Center (770-488-7100).

Because the likelihood of reintroduction of smallpox is extremely low, and acknowledging that there are many other causes of vesicular and pustular rash illnesses, healthcare providers evaluating such cases should also familiarize themselves with diseases that can be confused with smallpox (e.g., varicella, herpes simplex, drug reactions, erythema multiforme), as well as the clinical manifestations of smallpox disease. In this way, in the unlikely event of a smallpox case, the disease will be clearly and quickly recognized.

Diagnostic Testing

Real-time polymerase chain reaction (PCR) is the preferred method for detecting variola virus.

• PCR testing on specimens from low- to moderate-risk patients is available across the United States through reference level Laboratory Response Network (LRN) member laboratories.
• Variola virus-specific PCR testing on specimens from high-risk patients is limited to select reference level LRN member laboratories meeting additional facility and vaccination requirements and the CDC.
• CDC should be consulted prior to initiation of any testing on high-risk specimens.

The type of specimen collected will vary depending upon disease progression.

• See table below for a list of acceptable specimens.
• Whole blood alone is not a suitable specimen for smallpox diagnosis by PCR as viremia generally concludes with rash onset.

Although electron microscopy and antibody detection can aid in diagnosis, these assays alone are not definitive for variola virus. Review the Negative Staining Electron Microscope Protocol for Rash Illness.

Preparation for Specimen Collection

Collection of specimens can begin after appropriate consultations have been made.

• Effective communication and precautionary measures between specimen collection teams and laboratory staff is essential to maximize safety during the manipulation of specimens from a suspected case of smallpox; this is especially relevant in hospital settings, where laboratories routinely process specimens from patients with a variety of infectious and/or noninfectious conditions.
• A labeling system should clearly distinguish all specimens which require special handling.
• Laboratory exposures to poxviruses occur primarily through needle-stick injuries and/or direct contact with the virus or aerosols that may be generated by laboratory procedures. Do not include sharps with specimens, and dispose of them in an appropriate puncture-resistant containers for autoclave of infectious waste.

Review smallpox vaccination history and potential contraindications to vaccination for personnel on specimen collection teams. All personnel caring for suspected patients should wear a fit-tested N95 respirator.

• Specimen collection from suspected cases of smallpox should be conducted by personnel who have been successfully vaccinated against smallpox within the past 3 years.
• If vaccinated personnel are not available, then only those without contraindications to vaccination should collect specimens.
• If smallpox is confirmed, personnel collecting specimens should be vaccinated immediately. The state health department will coordinate with CDC to obtain smallpox vaccine.

Ante-mortem Collection of Specimens for Smallpox Diagnosis

Collect all specimens listed by disease stage. When possible, use plastic rather than glass materials for specimen collection. It is extremely important not to cross-contaminate specimens and to collect an amount of material sufficient to permit effective testing.

The following types of specimens should be collected according to the stage of disease:

Disease Stage	Specimens to Collect
Prodrome	Tonsillar tissue swab Nasopharyngeal swab Acute serum and whole blood
Rash*
Macules or Papules	Tonsillar tissue swab Lesion biopsy Acute serum and whole blood
Vesicles or Pustules	Lesion fluid, roof, or biopsy Electron microscopy grid (if supplies available) Acute serum and whole blood
Scabs or Crusts	Lesion scab or crust Acute serum and whole blood
Post-Rash	Convalescent serum

* More than one lesion should be sampled, preferably from different locations on the body and/or from different looking lesions.

Post-mortem Collection of Specimens for Smallpox Diagnosis

All post-mortem procedures require adherence to standard precautions with the use of personal protective equipment (PPE) and facilities with appropriate safety features. Contact CDC’s Emergency Operations Center (770-488-7100) prior to autopsy to review the containment features of individual autopsy suites as well as autopsy and disinfection procedures.

When possible, personnel with an up-to-date smallpox vaccination (within 3 years) should participate in autopsy or mortuary care for patients with confirmed or suspected smallpox. If unvaccinated personnel must be used, persons without contraindications to vaccination are preferred. If smallpox is confirmed, personnel collecting specimens should be vaccinated immediately. The state health department will coordinate with CDC to obtain smallpox vaccine.

Transfer of Human Remains

Personnel who perform post-mortem care of remains should wear PPE as recommended for standard and contact precautions. Prepare the body following routine healthcare facility procedures for cleaning and containing body fluids. Prior to transferring the body to the autopsy suite, wrap the body in a primary impervious plastic bag or disaster pouch that is sealed airtight with tape and a secondary impervious plastic bag. Wrapping should be done in a manner that prevents contamination of the outside of the bag; a change of gown and gloves may be necessary. Persons transporting prepared and covered human remains should wear gloves, but other PPE is not required.

Personal Protective Equipment

Personnel who perform or assist with the autopsy should wear the following PPE as required for standard and contact precautions in an autopsy setting:

• Protective garments:
  • Scrub suit, surgical cap, impervious gown with full sleeve coverage (no personal clothing should be worn)
  • Shoe covers or footwear restricted to contaminated areas
  • Double set of gloves with an interposed layer of cut-proof synthetic mesh gloves
  • Eye and face protection (goggles or face shield)
• Respiratory protection is required for all personnel, including those with recent, successful vaccination:
  • N95 or N100 respirators, or
  • Powered air-purifying respirators (PAPR) equipped with a high efficiency particulate air (HEPA) filter (recommended for any procedure that may result in aerosol generation
  • Autopsy personnel who cannot wear N95 respirators because of facial hair or other fit-limitations should wear PAPRs.

Facility Design for Autopsy

Conduct autopsies in facilities with the following engineering and facility designs. If your facility does not meet these engineering designs, refer the body to a facility that does.

• Air handling systems: autopsy suites must have adequate air-exchanges per hour, and correct directionality and exhaust of airflow.
  • Autopsy suites should have a minimum of 12 air-exchanges per hour and should be at a negative pressure relative to adjacent passageways and office spaces.
  • Air should not be returned to the building interior, but should be exhausted outdoors, away from areas of human traffic or gathering spaces (e.g., off the roof) and away from other air intake systems. For autopsies, local airflow control (i.e., laminar flow systems) can be used to direct aerosols away from personnel; however, this safety feature does not remove the need for appropriate PPE.
• Doors and windows: during autopsy, keep doors and windows to autopsy suite closed.
• Containment devices: biosafety cabinets should be available for handling and examination of smaller specimens. Oscillating saws are available with vacuum shrouds to reduce the amount of particulate and droplet aerosols generated. These devices should be used whenever possible to decrease the risk of occupational exposure.

Autopsy Procedures and Specimen Collection

Use the following safety procedures for autopsies and post-mortem assessment of confirmed or suspect smallpox cases:

• Prevention of percutaneous injury:
  • Handle all sharps carefully.
  • Never recap, bend, or cut needles.
  • Ensure that appropriate sharps containers are available.
• Specimen collection procedures:
  • Minimize the number and extent of procedures, both to decrease opportunities for worker risk and the potential for environmental contamination.
  • Perform autopsies only to the extent required to obtain needed information.
  • Omit examinations that generate aerosols and increase the risk of environmental contamination (e.g., the use of oscillating saws to open the skull).
  • All major organs should be sampled. Particular attention and extensive sampling should include tissues demonstrating gross pathology or involvement as suggested by clinical presentation. Autopsy specimens should include:
    • Skin
    • Spleen
    • Lymph nodes and tonsils
    • Liver
    • Lung
    • Kidney
    • Heart
  • Place representative tissues in 10% buffered formalin and store at room temperature. Formalin-fixed tissue for histopathology and immunohistochemistry should never be frozen.
  • Representative fragments of tissues listed above should be collected using sterile techniques and placed in sterile 1.5 to 2 mL screw-capped plastic vials with O-ring. DO NOT ADD ANY VIRAL TRANSPORT MEDIA. Specimens should be refrigerated at 4⁰C if shipment will occur within 24 hours, otherwise specimens may be frozen.
• Post-collection procedures:
  • Remove protective outer garments when leaving the immediate autopsy area and discard them in appropriate laundry or waste receptacles, either in an antechamber to the autopsy suite or immediately inside the entrance if an antechamber is not available.
  • Place all non-reusable specimen collection and barrier protection materials in biohazard bags for handling as medical waste.
  • Clean and disinfect all reusable equipment according to standard laboratory procedures.
  • Clean all surfaces thoroughly with 0.5% sodium hypochlorite or other EPA-approved high-level disinfectant.
  • After autopsy, the body should be double-bagged as described above, in a new set of large, impervious plastic bags.
  • After removing gloves, wash hands.

Packaging and Transporting High-Risk Specimens

Package, label, and ship high-risk specimens as a Category A infectious substance affecting humans (UN 2814) in accordance with the U.S. Department of Transportation’s Hazardous Materials Regulations and the International Air Transport Association Dangerous Goods Regulations.

Packaging and Transporting Low or Moderate-Risk Specimens

Package, label, and ship low- or moderate-risk specimens as a Category B Infectious substance (UN 3373) in accordance with the U.S. Department of Transportation’s Hazardous Materials Regulations and the International Air Transport Association Dangerous Goods Regulations.