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1 PHLN Summary Laboratory Definition
1.1 Condition:Acute smallpox infection.
1.1.1 Definitive Criteria
- Isolation of variola virus from clinical material; or
- Detection of variola virus DNA in clinical material.
1.1.2 Suggestive Criteria
- Detection of a poxvirus resembling variola virus by electron microscopy from clinical material.
1.1.3 CommentsSmallpox was eradicated globally in 1979. Official laboratory stocks are held in secure reference collections at the Centers for Disease Control and Prevention (CDC), Atlanta USA and the State Research Centre of Virology and Biotechnology in Novosibirsk, Russia. It is possible that other clandestine stocks may exist.
Laboratory diagnosis requires specimen processing in a Physical Containment level 4 (PC4) laboratory. Access to laboratory testing should be through the Chief Medical Officer. Requests for testing should come via the Chief Health Officer of the state or territory once satisfied that grounds for testing exist i.e.:
(i) Credible clinical smallpox as judged by expert physician, and Credible smallpox exposure; within 3 weeks (associated with a specific threat, a known or suspected bioweapon release, or close exposure allowing respiratory/mucous membrane transmission to material that is credible as viral cell culture derived and in which smallpox can’t be excluded) OR
(ii) Credible clinical smallpox judged by expert physician;
Relevant differential diagnoses including herpes simplex virus, varicella-zoster virus and enterovirus, excluded by laboratory testing;
Smallpox testing recommended in the absence of overt smallpox exposure after expert case review.
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A single suspected smallpox case meeting these criteria is an international emergency requiring immediate notification to Commonwealth and jurisdictional public health authorities. Because variola virus is a potential bioterrorism agent, appropriate ‘chain of custody’ should be observed for the handling of any specimens from suspect smallpox cases.
Isolation of variola virus in vitro is not available in Australia.
Validation and experience with nucleic acid tests for detection of variola virus is limited.
Variola, vaccinia, cowpox, monkeypox virions cannot be definitively distinguished by electron microscopy.
2 IntroductionVariola or smallpox virus is a double stranded DNA virus. It is a member of the family Poxviridae, genus orthopoxvirus. Variola virus has a host range strictly limited to humans. Three of the many other animal orthopoxviruses (vaccinia, monkeypox, and cowpox) may also infect humans. All have brick shaped virions between 220 to 450 nm in length which cannot be definitively visually distinguished from one another 1-3.
Variola virus was the cause of smallpox, a severe disease eradicated globally by vaccination in 1979. Smallpox manifested as two diseases associated with different variola variants. Variola major circulated in Asia and had a case fatality rate of 15–45% 1, 3, including 3% among the vaccinated 4. Variola minor circulated in South Africa, the Americas and Europe and had a case fatality rate of approximately 1%. Smallpox had winter and early spring seasonality similar to varicella and measles outbreaks.
The virus is shed from the oropharynx and skin, and transmitted mainly via droplets to the respiratory mucosa of contacts. Approximately 30% to 80% of close contacts of both variola major and minor were likely to be infected 5. Infectivity was typically from rash onset for 7 to 10 days until scab formation. Indirect spread by fomites or fine particle aerosol was less common. Variola aerosols might survive for between 6 and 24 hours 1. Variola is much more durable in scabs, surviving weeks to even years, but scabs are thought to be minimally infectious for humans 1.
After 12–14 days incubation period (range 7–17 days) 1, 6 the variola major patient typically experienced a 2–3 day prodrome of high fever, malaise and prostration with headache and backache. A maculopapular rash appeared initially on the oral and pharyngeal mucosa, face and forearms and spread to trunk and legs. This rash became vesicular within 1–2 days and finally pustular. The rash was centrifugal in distribution, and lesions at any site were in the same developmental stage. With recovery, scabs separated and scarring developed. Haemorrhagic and malignant forms of smallpox also existed. Variola minor manifested a less severe illness with few constitutional symptoms and a relatively sparse rash.
Presumptive laboratory diagnosis could rapidly be achieved by expert electron microscopic examination of vesicle fluid, or cells from a lesion base 1-3. Monkeypox, cowpox and vaccinia, while visually similar, could usually be excluded on clinical grounds or from the history. Definitive identification required growth of virus on chorioallantoic egg membrane 2, 3, or detection of viral DNA by Polymerase Chain Reaction (PCR), which has become possible since the smallpox era 2, 7, 8. Smallpox laboratory diagnosis now requires specimen processing in a PC4 laboratory1. Access to such facilities, availability of smallpox diagnostic tests, and experience with their use are all extremely limited. A variety of serological tests exist for detection of poxvirus antibodies but their potential use in diagnosis of variola is limited 2.
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3.1 Access to diagnostic testingWhen a patient with suspected smallpox is identified during a Response Code 1 or Response Code 2 alert (see Appendix A), the National High Security Quarantine Laboratory (NHSQL) at the Victorian Infectious Diseases Reference Laboratory – (VIDRL) will carry out testing of specimens.
NHSQL should be notified through the relevant State or Territory Chief Quarantine Officer. However, direct contact with the medical microbiologist on call at VIDRL is essential to arrange receipt of specimens and obtain advice on specimen collection, safe packaging and transport. In the event of ongoing cases, VIDRL will advise on which Physical Containment level 3 (PC3) or level 4 (PC4) laboratories can receive specimens in the relevant jurisdiction.
The VIDRL on-call microbiologist can be contacted on (03) 9342 2600, or on mobile 0438 599 437. If attempts to reach the VIDRL on call medical microbiologist via these numbers fail the designated back up is the VIDRL on call laboratory manager 0438 599 439, or failing this the Royal Melbourne Hospital switchboard (03) 9342 7000 who will attempt to reach VIDRL staff on the caller’s behalf.
PCR is the primary diagnostic modality employed for detection of smallpox by the NHSQL, and collection of appropriate vesicle swabs for this purpose is a priority. Electron microscopy is used as an adjunct to PCR by the NHSQL.
3.2 Specimen collectionSkin lesions are considered the best source of specimens for laboratory diagnosis of poxvirus infections 1-3. Specimens should be collected by smallpox immune staff (vaccination that day will suffice) wearing gown, gloves and a mask. There are vaccinated staff in many Australian laboratories due to the use of vaccinia constructs in molecular biological research.
In the smallpox era, laboratory diagnosis was applied only to material derived from clinical cases, so the negative predictive value of these laboratory tests when applied to environmental material has not been established. Notably the infectious dose of variola virus may be as low as 10–100 virions, while the analytical sensitivity of electron microscopy is approximately 105 virions. Culture is generally considered less sensitive than electron microscopy 2.
3.2.1 Equipment for collection of specimen
(i) PCRAppropriate equipment includes:
- Personal protective equipment
- A small scalpel blade or tuberculin needle for removing the roofs and upper tissue from lesions
- Eppendorf tubes or bijou bottles containing viral transport medium
- Dry swabs
- A fine tip permanent marker pen
- A waterproof sharps container for needles, syringes, scalpels
- Waterproof plasters
- A sealable plastic specimen bag. Absorbent packaging material and a strong metal outer container plus biohazard tape to seal it and 0.1% hypochlorite solution to clean the outside before transport to the laboratory
- ‘High Risk’ labels
- A clinical waste bag for disposal of discarded dressings and personal protective equipment
(ii) Electron microscopyAppropriate equipment for obtaining specimens includes:
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- Personal protective equipment
- A ‘tuberculin’ syringe and needle for aspirating fluid from vesicles
- A small scalpel blade for removing the roofs and upper tissue from lesions, and for lifting scabs
- Clean plastic microscope slides
- Eppendorf tube or bijou container for transporting crusts
- A permanent marker pen
- A slide container for the safe transfer of slides
- A waterproof sharps container for needles, syringes, scalpels and unused slides
- Waterproof plasters
- A sealable plastic specimen bag, absorbent packaging material and a strong metal outer container, plus biohazard tape to seal the bag
- Fresh 0.1% hypochlorite solution to clean the outside of the container before transport to the laboratory
- ‘High-Risk’ labels
- A clinical waste bag for the disposal of discarded dressings and personal protective equipment.
3.2.2 Procedure for collection of specimens
(i) PCRThe procedure for collecting specimens of vesicle fluid is as follows:
- Put on personal protective equipment
- Gently de-roof a vesicle using a scalpel blade tuberculin needle
- Firmly rub a dry swab on the base of the lesion using a rotary motion. The objective is to absorb fluid from the vesicle onto the swab, and to dislodge cellular material from the lesion base which will also adhere to the swab.
- Place the swab into an Eppendorf tube or bijou bottle of viral transport medium, carefully break or snip the swab shaft to allow closure of the tube, and replace the lid.
- At least 3 vesicles containing clear (i.e. non-postular) fluid should be sampled in the manner and the swabs pooled in a single Eppendorf or bijou.
- Label the tube with patient identifying data, place it in zip-lock plastic specimen bag and seal.
- Repeat to generate a duplicate set of 3 swabs in a second tube, labeled and bagged as above.
(ii) Electron microscopyThe procedure for collecting specimens of vesicle fluid is as follows:
- Put on personal protective equipment
- Puncture a vesicle with the tuberculin syringe, draw up fluid and express it onto a clean plastic microscope slide
- Cover the punctured vesicle with a waterproof plaster
- Allow the slide to air dry—do not wave it in the air
- Mark the slide clearly to indicate the surface used and the position of the sample
- Use a plastic slide carrier to transfer the slide to the laboratory
- Do not submit vesicle fluids to the laboratory in hypodermic syringes or in capillary tubes, as this could be hazardous to laboratory staff extracting the specimen.
- Swabs or specimens in viral transport medium are not suitable for electron microscopy.
- Vesicle crusts may be removed and sent for examination in a small sealed container (Eppendorf tube or bijou).
3.2.3 Transport of specimens to the laboratoryThe outside of each specimen container should be swabbed with disinfectant (0.1% hypochlorite solution) and a label should be attached bearing the patient’s name, hospital identification, the date of collection and the nature of the suspected infection. The specimens should be double bagged in secure, airtight and watertight bags, which have been similarly labeled. Bags containing specimens should be sponged with disinfectant before removed from the patient’s room.
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Samples should be identified as: “Infectious Substances Affecting Humans (smallpox sample)” and packaged and handled as required by the International Air Transport Association (IATA) packing instruction 602.
The specimens should be packaged as follows:
- Place the specimens for transport in a tightly sealed, watertight container, such as a screw-cap plastic tube or vial, and seal the cap with tape. Ensure plastic containers are resistant to temperatures as low as -80 0C.
- Wrap the primary container in sufficient absorbent material (e.g. tissue) to absorb the entire contents in case the container leaks or breaks.
- Place the wrapped, sealed primary container into a durable, watertight, screw-cap mailing tube or metal can. This secondary container should be sealed with tape.
- Several primary containers may be placed in one secondary container to a maximum of 50 mL of specimen material.
- On the outside of the secondary container, attach the specimen labels and other relevant information.
- Place the second container in a secure box or mailing tube addressed to:
- National High Security Quarantine Laboratory
Victorian Infectious Diseases Reference Laboratory
10 Wreckyn Street
North Melbourne Vic 3051
Notify the on-call VIDRL medical microbiologist of the dispatch of the specimen and flight time and number, courier or airway bill number as appropriate. If transport is by air, a dangerous goods declaration must be made (refer to the IATA Dangerous Goods regulations)
3.3 Laboratory procedures
3.3.1 Specimen handlingClinical samples from suspected cases must be handled with due regard to the likelihood that variola virus is present, and the appropriate procedures observed. Appendix A provides guidance on the Smallpox Response Codes and the roles of designated laboratories at the different phases. Should it be necessary to conduct work other than in a PC4 laboratory, a full risk assessment must be conducted.
During Smallpox Response Code 0, specimens from suspected cases will be sent to one of the designated PHLN regional laboratories for differential diagnosis of varicella-zoster virus (VZV), herpes simplex virus (HSV) and enterovirus, with testing for smallpox by Electron Microscopy (EM) if negative for these. Investigation will require:
- Handling of samples in a Class I or II cabinet based within PC3 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 PC3 laboratory (see next section).
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3.3.2 Occupational health and safety issuesStaff at the PHLN regional laboratories will be vaccinated at smallpox Response Code 0. Wider vaccination of laboratory staff is not justified at Response Code 0, since the risks of adverse effects from vaccination outweigh those from smallpox. However, staff liable to be involved in diagnostic work at Response Code 3 should be identified and screened in advance for suitability for vaccination.
3.3.3 Nucleic acid testsSpecimens should be referred to the NHSQL for analysis by PCR. Preparation of variola nucleic acid for PCR testing should be undertaken only in a PC4 laboratory. Once non-infectious nucleic acid has been prepared the specimen can be removed from the PC4 laboratory for further processing.
Protocols for extraction of orthopoxvirus nucleic acid from lesion material, and specific PCR amplification for variola, vaccinia, cowpox and monkeypox among others, have recently been published 7, 8. These have the potential to provide rapid and sensitive laboratory diagnosis of these viruses. Validation and experience with variola diagnosis is limited however. The availability of positive control material for variola is problematic for most laboratories.
PCR protocols have been developed targeting the acidophilic inclusion protein (ATI) or haemagglutinin (HA) of orthopoxviruses, with subsequent specific identification by restriction digest of PCR amplicons 7, 8.
The ATI-PCR targets a region prone to extreme hypervariability which may limit the effectiveness of the assay 8, and only identification of 5 orthopoxviruses (cowpox, vaccinia, ectromelia, camelpox and monkeypox) has been described 7.
The HA-PCR employing primers EACP1&2 and restriction digest with Taq 1 distinguishes 7 “old world” orthopoxviruses including vaccinia, variola and monkeypox 7, 8. Variola major and minor viruses are not distinguished by this method 8. Other “new world” orthopoxviruses not affecting humans may be detected with a second primer set and restricted digest protocol.
A second PCR approach also targets the HA gene of orthopoxviruses using specific primer pairs to selectively amplify variola, vaccinia, monkeypox, cowpox and others 7, 8. Some retrospective validation of primer pair VAR1/VAR2 has been carried out, successfully amplifying 15 stored smallpox scab specimens from the CDC Smallpox Virus Specimen Repository 8. These PCR primers rely on high stringency of PCR amplification conditions for their specificity, with the potential for loss of sensitivity or specifity if the reaction conditions are varied. Nucleic acid sequencing of PCR amplification products is recommended to provide definitive virus identification 8.
3.3.4 Electron microscopyElectron microscopic examination should be undertaken by an operator expert in application of this technique to viral diagnosis. Grids should be examined for 20 minutes before being considered negative. At least two convincing virus particles must be seen and photographed before reporting as positive. All possible viruses and virus-like particles should be photographed.
Viewed by electron microscope the virions of variola, vaccinia, cowpox and monkeypox viruses are brick-shaped and approximately 220 to 450mm in length nm in size. In the smallpox era, clinical presentation and history allowed cowpox and vaccinia to the distinguished from smallpox. Monkeypox only occurs naturally in tropical rainforest areas of Africa and could also be excluded on the basis of history.
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Large numbers of poxvirus particles are present in vesicular lesions, provided an adequate volume of specimen is referred, thus electron microscopic examination is considered highly reliable in smallpox diagnosis 1, 2.
For additional technical detail regarding electron microscopy see appendix C.
3.3.5 Virus growth assaysPoxvirus virus growth assays are laborious and slow, and rely on expertise that is not generally available in Australia. They also tend to be relatively insensitive 2. Culture work can only be undertaken in a PC4 laboratory. Generally culture of poxviruses is being supplanted by nucleic acid testing for diagnostic purposes 2, 5 in the few laboratories undertaking this type of work.
Culture may be attempted from vesicle fluid which contains large numbers of viral particles, or from lesion scrapings.
(i) Chorioallontoic membrane culture
Definitive detection of orthopoxviruses traditionally relied on growth on the choriallontoic membrane of 12 day old chicken embryos. Morphologically characteristic pocks typical of each virus were produced after incubation for 72 hours at 35 oC.
(ii) Cell culture
Variola will grow in a variety of cell lines, including human embryonic lung, primary rhesus monkey kidney, Vero, LLC-MK2. After several days a cytopathic effect appears consisting of ballooning, cell fusion and destruction. Subculture to choriallontoic membrane allowed definitive identification of other poxviruses by the characteristic pock morphology each produced.
3.3.6 SerologyA variety of serological assays have been described for detecting exposure to orthopoxviruses. Generally serological tests do not distinguish between antibodies to the various orthopoxviruses 2, and depend on measuring antibody rises between acute and convalescent sera. Their usefulness in acute diagnosis of variola infection is therefore limited. At present only neutralisation tests, Western blot and enzyme immunoassay are regularly used, and then generally for research purposes only by laboratories such as CDC, Atlanta.
4. Quality assuranceNo quality assurance programs are currently generally available.
- Henderson DA, Inglesby TV, Bartlett JG. (1999). Smallpox as a biological weapon, medical and public health management. JAMA. 281:p 2127-37.
- Buller M. (2000). Poxviruses. Chapter 34. In: Specker S, Hodinka R, Young S, (Eds). Clinical Virology Manual, 3rd Edition, ASM, Washington.
- Behbehani A. (1999). Human Poxviruses. Chapter 35. In: Lennette E, Smith T, (Eds). Laboratory diagnosis of viral infections, 3rd Edition, Marcel Detter, New York.
- Eitzen E, Paulin J, Cieslak T, Christopher G, Culpepper R. (1998). Medical Management of Biological Casualties Handbook. Fort Derick, Frederick, Maryland.
- Ropp S, Esposito J, Loparev V. (1999). Poxviruses infecting humans. Chapter 91. In: Murray P, Baron E, Pfuller M, Tenover F, Yolken R, (Eds). Manual of Clinical Microbiology, 7th Edition, ASM, Washington.
- Center for Disease Control and Prevention. (2001). Vaccinia (smallpox) vaccine: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. 50 (No RR-10):
- Meyer M, Ropp S, Esposito J. (1998). Poxviruses. Chapter 15. In: Stephenson J, Warnes A, (Eds). Methods in Molecular Medicine, Vol 12: Diagnostic Virology Protocols, Humana Press, New Jersey.
- Ropp S, Jin Q, Knight J. (1995). PCR strategy for identification and differentiation of smallpox and other orthopoxviruses. J. Clin. Microbiol. 33:p 2069-76.
Appendix A - Laboratory test procedures for smallpox by response code
National Alert Level
Testing characterized by
Response Code 0
Smallpox remains eradicated
Decentralised exclusion of smallpox:
Response Code 1
Centralised screening for the first smallpox case:
Response Code 2
Release case or cluster of cases in Australia
Centralised screening for the first smallpox case:
Response Code 3
Community transmission of smallpox in Australia
Decentralised confirmation of widespread smallpox:
Case epidemiologically linked to laboratory proven smallpox
No edpidemiologic link
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APPENDIX B. GUIDANCE NOTES ON EM OF SUSPECTED SMALLPOX SPECIMENSSuspected smallpox specimens must be inactivated before processing. All work must be carried out in a cabinet (class I or II)
FormalinSolutions of formalin at 5% and 10% (v/v) should be freshly prepared from 40% (v/v) stock solution just before use.
Vesicle fluid specimens
- Rehydrate the specimen in 10 1 to 50_ 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 orthopoxvirus and molluscum contagiosum (although infection with these viruses should be clinically distinct).
Vesicle crust specimens
- 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 (e.g. TCF)Mix equal volumes of sample and 10% formalin solution to inactivate virus. Process as usual.
MicroscopeAs a guide viruses measure roughly 200 by 250mm, so an electron microscope with good resolution is required. An electron microscopist will normally scan a grid (on the large fluorescent screen) at a screen magnification of x 40 000 and check any possible virus using the binoculars and small screen. Viruses would normally be photographed at between x 40 000 and x 60 000. The microscope must be able to resolve well beyond x 40 000. The tested resolution of the EM should be better than 1 nm.
CalibrationThe microscope should be properly calibrated, using a cross grating and if possible catalase crystals or similar. Do not trust the magnification given by the instrument: old microscopes, in particular, can give incorrect readings unless calibrated. Size is important in virus identification. Smallpox measures about 250 nm x 200 nm.
Stains and gridsUsually a negative stain such as phosphotungstic acid is used. Good results are achieved with pH7. Homemade Formvar carbon-coated copper 400-mesh girds are best.
LiteratureThere are a number of good atlases of virus morphology. Recommended tests are Virus Morphology (Madeley and Field 1988) and Electron Microscopy in Diagnostic Virology (Doane and Anderson 1987)
1. PC4 facilities to guarantee containment, and central diagnostic responsibility, are important during screening for initial cases in the elevated-risk Response Code 1 and 2 phases.
2. PC3 facilities and immune staff provide acceptable staff safety, maximal laboratory capacity and simplified logistics, both in the low-risk Response Code 0 phase and later in Response Code 3 when widespread community circulation of smallpox makes high-security laboratory containment irrelevant. Top of page