Interim Guidelines for Smallpox Response and Management in Scotland in the Post-Eradication era

This Appendix provides guidelines for the development of appropriate specimen handling and diagnostic procedures to enable safe, accurate and timely diagnosis. It should be used in conjunction with clinical and public health plans.
Two centres in the UK, the Central Public Health Laboratory (CPHL) and the Centre for Applied Microbiological Research (CAMR) have established a full range of poxvirus diagnostic services based on EM, PCR, culture and serology. Clinical specimens for confirmation of a diagnosis should be referred to these Reference laboratories. Procedures for packaging and shipment of specimens can be found in Appendix 1 and any clarification can be obtained directly. Contact details are available in the Annex.
Rapid diagnostic tests based on EM will be available in the designated Category 3 laboratory in Scotland, and the time saved in making the diagnosis in this way may be critical in triggering an appropriate public health response. In Scotland the designated Category 3 laboratory is the West of Scotland Regional Virus Laboratory, Gartnavel General Hospital, Glasgow.
In the future, validated real time PCR assays will be available for these laboratories.

Patient categorisation and sample assessment

Smallpox is a Hazard Group 4 organism and must be handled accordingly. Clinical samples from suspected cases must be handled with due regard to the likelihood that smallpox is present, and the appropriate procedures observed. Should it be necessary to conduct work other than in a Category 4 laboratory, a full risk assessment must be conducted.
Specimens from suspected cases in Scotland will be sent to the Scottish designated Category 3 laboratory for diagnosis. Investigation will require:

Handling of samples in a Class I or III cabinet based within a Level 3 laboratory, preferably by vaccinated staff.
Inactivation of the sample by 5% formalin for EM or inactivation of the sample by guanadinium for PCR, to be undertaken within a Class I or III cabinet in a Level 3 laboratory (see next section).

One poxvirus infection, molluscum contagiosum, with a similar morphology to smallpox, is endemic in the UK. If orthopox particles are visualised by EM, the result should be discussed urgently with the referring clinician.

Standard Operating Procedures for EM on vesicular fluids

The PHLS has produced a draft SOP for conducting EM on vesicular fluids (V.SOP13; available on PHLS website). [ DN Is it on SCIEH website?]
Paragraphs 17 to 20 below are supplementary to V.SOP13, to enable safe working with higher risk specimens. A detailed description of specimen collection and transport can be found in Appendix 1.
Unpublished studies at CPHL have shown that reconstitution of dried samples using 5% formalin does not disrupt virus particles, but does make the sample safe.
To facilitate improved poxvirus diagnostics a quality assurance (QA) scheme for EM and VZV/HSV PCR on vesicular fluid samples will be required.
EM equipment and training and competencies of staff at the designated laboratory in Scotland will be reviewed to ensure that EM results from suspected smallpox cases are reliable.
EM is a reasonably sensitive technique in the right hands but it is critically dependent on good vesicle fluid collection. All specimens (both positive and negative) should be referred on to CPHL or CAMR for confirmation. It will be important to conduct differential tests for VZV and HSV at the same time to enable a rapid risk assessment.

Occupational health and safety issues

Staff at the designated Scottish laboratory will be vaccinated at Alert Level 0 (see Section VIII).
Wider vaccination of laboratory staff is not justified at Alert Level 0, since the risks of adverse effect from vaccination outweigh those from smallpox.
However, staff liable to be involved in diagnostic work at Alert Levels 2 to 4 should be identified. They will then be vaccinated at Alert Level 1 or 2 (see Section VIII).

Guidance notes on EM of suspected smallpox specimens

These guidance notes are to complement PHLS SOP: Investigation of clinical specimens using the floatation (direct) method (V.SOP13).
Suspected smallpox specimens must be inactivated before processing. All work must be carried out in a cabinet (Class I or III).
Vesicle fluid specimens.

Rehydrate the specimen in 10ul to 50 ul solution of 5% formalin in distilled water.
Continue with staining and grid preparation according to local protocol.
Reconstitution of the specimen in formalin does not disrupt the virus particles but does destroy infectivity.
Some detail of surface structure may be lost, but herpes group and pox group viruses should be clearly recognisable.
It may not be possible to distinguish between orthopox virus and molluscum contagiosum (although infection with these viruses should be clinically distinct).

Place crust in a plastic Griffith's tube with a few drops of 5% formalin in distilled water and grind to disrupt.
Alternatively, disrupt crusts on a clean microscope slide with a few drops of 5% formalin solution using forceps.
The resulting homogenate may be used for grid preparation.

Other liquid material; eg. TCF. Mix equal volumes of sample and 10% formalin solution to inactivate virus. Process as usual.
Examination of grids. Grids should be examined for 20 minutes before being considered negative. At least two virus particles must be seen and photographed before reporting as positive.
Formalin. 5% x 10% (v/v) solutions of formalin should be freshly prepared from 40% (v/v) stock solution just prior to use.

The Scottish Government