WEBCAST TRANSCRIPT

Transcript of "Smallpox Vaccination Laboratory Support"\
Presented by Dr. Russell Regnery, 6 December 2002, on the satellite
broadcast of "CDC Bioterrorism Update: Smallpox Preparedness"

(Associated graphics can be found at\
[www.bt.cdc.gov/agent/smallpox/training/webcast/dec2002/files/laboratory-module.ppt](http://www.bt.cdc.gov/agent/smallpox/training/webcast/dec2002/files/laboratory-module.ppt)
and
[www.bt.cdc.gov/agent/smallpox/training/webcast/dec2002/files/laboratory-module.pdf](http://www.bt.cdc.gov/agent/smallpox/training/webcast/dec2002/files/laboratory-module.pdf).)

Russell Regnery, PhD, Virologist, Poxvirus Section, Viral and
Rickettsial Diseases, National Centers for Infectious Diseases, CDC.

(Slides 1 and 2 are title and objectives, respectively)

# REGNERY:

The intent of this Laboratory Support presentation is to provide
background information for people who might be involved with smallpox
preparedness and response. Of course, thorough clinical evaluation and
patient history may be all that is required to conclude that a person
has had or is having an adverse reaction to smallpox vaccination.
However, persons involved with the vaccination program may be expected
to have some degree of familiarity with the basis for laboratory
testing, in the event of an adverse reaction, even though they
themselves may not be responsible for making sure the testing is
facilitated. This talk will try to help: provide some background on the
specific tests currently in place within the state\'s Laboratory
Response Network; what sort of specimens would be needed for testing;
and how to prepare those specimens for transport to the LRN sites.

## Slide 3

You\'ve already heard about the febrile vesicular rash algorithm and its
application for enhancing rapid diagnosis of smallpox look-alike
diseases. Many of the same principles apply to diagnosis of vaccine
adverse reactions caused by vaccinia.

## Slide 4

The algorithm provides significant diagnostic benefits even in the
absence of smallpox and encourages careful diagnosis of other rash
illnesses such as vaccinia.

## Slide 5

The use of such an algorithm minimizes the number of cases that require
intensive investigation and hopefully focuses attention where it is most
needed and justified. This is an important consideration for
laboratories, just as it is in the clinical setting. It allows for a
more rapid, thorough response to highly suspect cases. And in the case
of smallpox, of course, the diagnosis of smallpox would result in
initiation of vaccination.

## Slide 6

As I mentioned the essential features of the febrile vesicular rash
disease algorithm can be applied to the clinical diagnosis of possible
vaccinia-associated adverse events especially since such events would be
associated with histories of vaccination or contact with vaccinees. Many
of the laboratory testing requirements for vaccinia infection are also
very similar to those expected for variola or smallpox testing. Much of
what I say today applies to both vaccinia and variola testing. Rapid
implementation of methods for laboratory confirmation of
vaccinia-associated adverse events, in such cases where the clinical
picture may not be sufficient to provide a positive diagnosis, has
become a high priority for the anticipated vaccination program. Such
testing methods have been developed and validated and include real-time
PCR analysis of poxvirus DNA. It is anticipated that as vaccination
proceeds, additional methods of point-of-care diagnosis will become
available as they are developed and approved for clinical use.

## Slide 7

As additional background, there are a large number of poxviruses
currently recognized to exist in nature including poxviruses in both
vertebrates and arthropods. Today we\'re only concerned with those that
belong to the closely related orthopoxviruses, especially variola (the
agent of smallpox) and vaccinia (the agent that\'s used for smallpox
vaccination).

## Slide 8

Poxviruses are large viruses that replicate within the cytoplasm of
cells and their genome is a molecule of double-stranded DNA. The
presence of virus-specific DNA sequences provides an important target
for diagnostic testing.

## Slide 9

There are several closely related forms of infectious orthopoxvirus
particles, which are distinguished by how many membranes surround each
virion. It is interesting that some orthopoxviruses have limited host
ranges, such as variola, which only infects humans, whereas other
closely related viruses may have wider ranges, such as vaccinia. The
reasons for these host-range differences are not completely understood.
Importantly these viruses are genetically and antigenically very similar
and provide cross protection immunity from infection ;hence the basis
for vaccination with vaccinia to prevent variola infections (i.e.,
smallpox).

## Slide 10

Among orthopoxviruses vaccinia and cowpox cause typically cause
localized infections in human hosts with normal immune responses.
Variola and monkeypox typically cause systemic diseases. Variola is the
only orthopoxvirus for which man is recognized as the only naturally
occurring host.

## Slide 11

There are a variety of laboratory methods that can be used to confirm an
orthopoxvirus diagnosis. Many of these are currently limited to a few
reference laboratories including several of the serologic assays that
are listed at the bottom of this slide. However, highly sensitive
methods for vaccinia DNA identification in the form of real-time PCR
assays have been deployed to every state through the Laboratory Response
Network. In addition, a number of states have the capacity to do
electron microscopic analysis of orthopox specimens as well as
histopathologic evaluation.

## Slide 12

The specific real-time PCR assay that is currently deployed for analysis
of vaccinia adverse reactions detects the presence of the DNA polymerase
gene of vaccinia virus, the so-called E9L gene. This assay will also
detect cowpox virus and monkeypox viruses, two viruses that do not occur
naturally in North America. From a North American diagnostic perspective
then, this assay can be considered diagnostic for vaccinia infections.

## Slide 13

An almost identical assay will also detect the DNA of variola virus and
therefore can be used for the diagnosis of smallpox. This is
accomplished by simply changing specific fluorescent labeled probes.
Both assays incorporate internal controls. A wide variety of similar
assays, targeting a variety of poxvirus genes, are currently being
evaluated and readied for deployment. This is very much an ongoing
process.

## Slide 14

This is an example of experimental data from a real-time PCR test that
incorporated calibrated amounts of DNA at increasing dilutions together
with an unknown test sample containing orthopox DNA. Depending on the
amount of original starting viral DNA, fluorescent signal develops at
varying times over the duration of the experiment. Detection of virus
DNA from a patient sample is indicated by the arrow.

## Slide 15

Once again, just to emphasize, if smallpox were to re-emerge in the
human population, the E9L real-time PCR assay would be modified for
variola use simply by changing to an alternate probe.

## Slide 16

This is an example of a negatively stained electron micrographic
preparation of vaccinia virus. You can see the characteristic
brick-shaped particle of the orthopoxvirus and the characteristic
surface morphology (example seen on the left). The EM process for
orthopoxvirus identification may be accomplished relatively quickly by a
skilled observer and can be used to differentiate generic
orthopoxviruses from other groups of viral agents. However, it may not
be as sensitive as real-time PCR and cannot differentiate between
variola and vaccinia.

## Slide 17

Cell culture isolation can be an important and very sensitive method for
detection of vaccinia since it also amplifies the virus for further
characterization. It should be noted that specimens with high suspicion
for variola should not be subjected to culture in a pre-event setting
due to concerns for biocontainment and laboratory safety.

## Slide 18

The best specimens for many of the orthopox laboratory tests are the
"roofs" or crusts from the lesions, which contain large amounts of
orthopoxvirus material. Vesicular fluids from the lesions are also
convenient sources of diagnostic material. Vesicular fluids are another
good starting materials for electron microscopy. Whichever tests are
considered for diagnosis, multiple lesions should be sampled for both
roof of lesions and vesicular fluids from the lesions since not all
lesion specimens are equally suitable for virus detection. Collection of
biopsies can be done with local anesthetic if histopathologic exam is
considered. Histopathologic evaluation is especially important for
successful diagnosis of several of the orthopox look-alike syndromes.

## Slide 19

This is a good example of one of several vaccinia lesions associated
with the laboratory-acquired case of disseminated vaccinia in a
previously unvaccinated laboratory worker.

## Slide 20

Collection procedures for vaccinia in the event of an adverse reaction
to vaccination, or variola in the event of a terrorist release of
smallpox virus, are essentially identical. Specifics for collection
techniques can be found at the CDC website. Updates are expected in the
future so check the website occasionally.

## Slide 21

Typical practices associated with collection of patient specimens are
appropriate for collection of orthopoxvirus lesions as well. These
include wearing personal protective equipment, including gloves and
sanitizing the site prior to collection. If alcohol is used to prepare
the lesion for collection it is important to allow the lesion to dry
before it is collected.

## Slide 22

These are examples of lab materials useful for collection of orthopox
specimens for laboratory testing including the plastic vials in which
lesion crusts would be shipped to the LRN site for analysis.

## Slide 23

For collection of vesicles, it\'s suggested to use a scalpel or needle
to unroof the vesicle. The skin or scab that constitutes the roof goes
to the collection tube and is sent otherwise dry. One procedure suggests
gently scraping the base of the vesicle with a blunt end of a scalpel or
wooden applicator and trying to smear some of this on a microscope
slide. An electron microscope grid, with ultra-thin plastic covering,
can be gently touched down (shiny side or plastic film-side) against the
lesion. This can be repeated perhaps three times per lesion (resulting
in three EM grids).

## Slide 24

Touch preparations are made by repetitively touching a glass microscope
slide to a lesion. The slide and/or EM grid are allowed to air dry for
ten minutes. Store in slide in a slide holder and an EM grid in the
appropriate box.

## Slide 25

This is an example of lifting a crust -- in this case an mature scab
from a vaccination site.

## Slide 26

This is the same scab being prepared to be put into a vial as sterilely
as possible.

## Slide 27

This is a simulated orthopox lesion and the making of a touch-prep with
a glass or plastic microscope slide. The same lesion is touched 3 times
with the same slide.

Slide 28

These are electron microscope grids, forceps for handling the grids, and
the box for the grids.

## Slide 29

Here\'s the electron microscopic grid is being used to touch down on the
lesion.

## Slide 30

Close-up of the EM grids and the box.

## Slide 31

Biopsy specimens should either be split in two or taken in duplicate so
that one specimen can be fixed in formaldehyde for histopathology, while
the other is used for DNA detection or virus isolation. Serum, if
necessary, can be collected as well.

## Slide 32

For vaccinia testing, standard shipping guidelines are appropriate.
Standard diagnostic specimen shipping guidelines are available at the
website listed on the slide here
(www.bt.cdc.gov/labissues/packaginginfo.pdf). If serum is collected, it
is highly desirable to separate the serum from the blood on site,
however, if this is not possible, one can send refrigerated whole blood
to the LRN laboratory.

## Slide 33

Formalin-fixed tissue must be shipped at room temperature, not frozen.
Electron microscopic grids must be shipped at room temperature.

## Slide 34

All other virus-containing material must be stored and shipped frozen.
However, if overnight transportation to the LRN lab can be arranged,
freezing of fresh viral specimens is not necessarily required. Keep all
virus-containing material out of direct sunlight.

## Slide 35

Suspect vaccinia samples should be sent to the closest state or regional
LRN laboratory. Contact your state\'s public health laboratory
director\'s office for specific address information. It might be prudent
to obtain this local contact information prior to onset of large-scale
vaccination programs.

## Slide 36

Specimens with high suspicion of smallpox diagnosis need to come
directly to CDC and to selected LRN labs with smallpox surge potential.
As part of the febrile vesicular rash disease algorithm, it is
anticipated that state health departments would be in contact with CDC
regarding such a specimen and its transport.

## Slide 37

What about the past? It's encouraging to note that smallpox was
eradicated as a naturally occurring disease in the absence of high-tech
diagnostic tools. During the time when smallpox was epidemic, clinical
diagnosis drove the immediate medical response, and presumably this
would be expected to reoccur if smallpox were to re-emerge in the
future. Diagnostic electron microscopy capability was more common in the
past, and relatively low-tech gel diffusion serological assays were
available.

## Slide 38

What about the future? Additional sensitive diagnostic tests are
currently being developed and will be deployed. It is anticipated that
in addition to a wide variety of PCR-based tests for detection of DNA,
relatively simple tests will be developed that can be used at point of
care. It is also worth considering that in the event of a validated
outbreak of smallpox, expectations for smallpox diagnosis would change
considerably. As previously mentioned, it is likely that there would be
an increased emphasis on clinical patient evaluation and subsequent
initiation of vaccination even in the absence of laboratory
confirmation.

I hope this brief introduction to vaccinia and variola diagnostic
testing has been useful to you. Of course there\'s much more information
available at the CDC website.

END

For more information, visit
[www.cdc.gov/smallpox](http://www.cdc.gov/smallpox), or call the CDC
public response hotline\
at (888) 246-2675 (English), (888) 246-2857 (Español), or (866) 874-2646
(TTY)