Smallpox: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, treatment, and prophylaxis

Last updated May 29, 2008

Agent
Pathogenesis
Epidemiology
—Occurrence of Smallpox in the Pre-eradication Era
—Global Eradication of Smallpox
—Reservoir/Modes of Transmission/Communicability
—Use of Smallpox as a Biological Weapon
—Antiterrorism Legislation
Clinical Features of Variola Major
—Ordinary Smallpox
—Flat-Type (Malignant) Smallpox
—Hemorrhagic Smallpox
—Smallpox in Children
Clinical Features of Variola Minor
Differential Diagnosis
— Differential Diagnosis of the Rash Illness
—Monkeypox
—Distinguishing Features Between Smallpox, Monkeypox, and Chickenpox
Diagnostic Issues
—Criteria for Determining the Likelihood of Smallpox
Laboratory Diagnosis
—Specimen Collection and Handling
—Laboratory Response Network (LRN)
—Tests for Detection and Identification of Variola Virus
—Rapid Tests for Diagnosis of VZV and HSV
—Testing in Areas With Confirmed Smallpox
—Inadvertent Discovery of Variola Virus in a Laboratory Specimen
Treatment
Smallpox Vaccination
— Historical Perspective
—Dryvax Vaccinia Vaccine
—ACAM2000 Vaccine
—New Vaccines
—Recommendations for Use of Vaccinia Vaccines
—2002 Recommendations for Vaccination of Healthcare Workers
—Current Status of the US Smallpox Vaccination Program
—Vaccination Schedule
—Dosage and Route of Administration
—Local Reaction to Vaccination
—Contraindications and Precautions
—Vaccine Distribution and Storage
—Smallpox Vaccination Clinic Implementation
—Liability Issues Following Smallpox Vaccine Administration
—Documented Adverse Reactions to Smallpox Vaccine
—Treatment of Vaccine Adverse Reactions
—Risk of Contact Vaccinia
—Use of Vaccine for Postexposure Prophylaxis
—Use of Vaccine During a Smallpox Emergency
Infection Control
Issues Related to Autopsies and Burial
Public Health Reporting and Case Definitions
References

Agent

Variola virus classification:

DNA virus
Family Poxviridae, subfamily Chordopoxviridae, genus Orthopoxvirus

Virion morphology:

Brick-shaped virion approximately 200 nm in diameter, 250 to 300 nm long, and 250 nm high (see References: International Committee on Taxonomy of Viruses), about the size of a bacterial spore
Enveloped
Dumbbell-shaped core containing nucleic acid and surrounded by a series of membranes
Replicates in the cytoplasm of host cells, forming B-type inclusion bodies (Guarnieri bodies), unlike varicella or herpes viruses, which replicate in the nucleus

Genetic composition:

The genome is composed of a single, linear, double-stranded DNA covalently closed at each end.
Average genome has 200,000 base pairs (200 kbp) and is among the largest animal viruses.
The genome includes genes that encode for viral DNA-dependent RNA polymerase and thymidine kinase
Genome has low G+C content (36% to 37%).
The genome of several strains has been completely sequenced, and efforts are under way to assess the genetic diversity of existing variola viruses and differentiate them (see References: National Center for Biotechnology Information, LeDuc 2001, Gubser 2004). A Web-based poxvirus genomic resource database has been established (see References: Lefkowitz 2005).
Comparative genomic analysis of 45 epidemiologically varied variola virus isolates from the past 30 years indicates low sequence diversity, suggesting little difference in functional genes. Analysis of viral linear DNA suggests that variola evolution involved direct descent and DNA end-region recombination events (see References: Esposito 2006).
Extensive cross-neutralization between orthopoxviruses exists; therefore, neutralization tests are not useful in distinguishing variola virus from other orthopoxviruses (this feature also accounts for the protection against smallpox afforded by vaccination with cowpox and vaccinia viruses).

Variola viruses traditionally have been classified as variola major and variola minor on the basis of the severity of clinical illness caused by infection. Recognized variola minor strains include:

Alastrim
Amass
Kaffir

There are many viruses in the family Poxviridae with vertebrate host ranges that do not include humans; related viruses that can cause natural infections in humans include:

Other Orthopoxvirus species

Monkeypox virus
Vaccinia virus
Cowpox virus

Other Chordopoxviridae genera

Yatapoxviruses: tanapox virus, Yaba monkey tumor virus, and Yaba-like disease virus of monkeys
Parapoxviruses: Orf virus
Molluscipoxvirus: agent of molluscum contagiosum

Pathogenesis

The pathogenesis of smallpox involves the following steps (see References: Fenner 1988: Chapter 3; Henderson: Smallpox as a biological weapon):

The portal of entry for variola virus is usually through the oropharyngeal or respiratory mucosa; variola virus also can enter through the skin, and rarely, through the conjunctiva or placenta (see References: Fenner 1988: Chapters 1 and 3).
The virus migrates rapidly to regional lymph nodes.
Asymptomatic viremia occurs on the 3rd or 4th day after infection, with further dissemination of the virus to spleen, bone marrow, and other lymph nodes.
Secondary viremia occurs by the 8th to 12th day after initial infection; this is followed by onset of fever and toxemia.
The virus localizes in small blood vessels of the dermis and oropharyngeal mucosa, leading to initial onset of the enanthem and exanthem, at which point (about day 14) the patient becomes infectious. The spleen, lymph nodes, kidneys, liver, bone marrow, and other viscera also may contain large amounts of virus (see References: Breman 2002).
The development and evolution of skin lesions involves the following steps:

Dilatation of the capillaries in the papillary layer of the dermis occurs initially, followed by swelling of the endothelial cells in the vessel walls. Perivascular cuffing with lymphocytes, plasma cells, and macrophages can be seen.
Lesions then develop in the epidermis, where the cells become swollen and vacuolated; characteristic B-type inclusion bodies can be found in the cytoplasm.
The cells increase in size and the cell membranes rupture, leading to vesicular lesions.
Pustulation results from the migration of polymorphonuclear cells into the vesicle.
The contents of the pustule gradually become desiccated, leading to crusting or scabbing of the lesions.
Re-epithilialization and scarring occur as the lesions heal.

Death most commonly results from overwhelming toxemia, probably associated with circulating immune complexes.

Epidemiology

Occurrence of Smallpox in the Pre-eradication Era

Smallpox likely originated in Egypt or India over 3,000 years ago (see References: WHO: Fact sheet on smallpox). Egyptian mummies dating from as early as 1500 BC showed characteristic pox-like skin lesions suggestive of smallpox.
Smallpox initially was introduced to the native populations of the Western Hemisphere by explorers from Europe and later by African slaves. The first recorded epidemic of smallpox in the New World occurred in 1507 on the island of Hispaniola (see References: Fenner 1988: Chapter 5). Eventually the disease spread throughout the hemisphere with devastating consequences for many native tribes.
By the mid-1700s, smallpox was a major endemic disease throughout the world, except in Australia, where it was first introduced in 1789 and again in 1829.
Following the famous observations of Edward Jenner at the end of the 18th century, vaccination against smallpox using cowpox virus became a widespread practice in Europe and the United States. During the 19th century, cowpox virus was gradually replaced by vaccinia virus as the agent used in vaccination (see Smallpox Vaccination: Historical Perspective). During the first half of the 20th century, smallpox vaccination using vaccinia virus was widespread, particularly in Europe and the United States.
By the early 1950s, endemic smallpox had been eradicated from Europe, the USSR, and North and Central America (see References: Fenner 1988: Chapter 5). Most of the outbreaks that occurred in Europe and North America after World War II were small and involved fewer than 50 cases (see References: Bhatnagar 2006). However, the disease remained endemic throughout most of the developing world, with an estimated 50 million cases occurring each year (see References: WHO: Fact sheet on smallpox).

Global Eradication of Smallpox

In 1959, the 12th World Health Assembly of the World Health Organization (WHO) passed the first resolution for global eradication of smallpox; however, it was not until 1967 that substantial resources were dedicated to the project.
The basic strategy of smallpox eradication included: (1) mass smallpox vaccination campaigns and (2) surveillance and containment of outbreaks.
After an extensive, sustained, international collaboration over a 12-year period, the International Commission for the Global Certification of Smallpox Eradication declared in December 1979 that smallpox had been globally eradicated (see References: Fenner 1988: Chapter 27).
The last reported case of endemic smallpox occurred in Somalia in 1977, and the last case human case, which involved accidental laboratory exposure, occurred in Birmingham, England, in 1978 (see References: CDC: Laboratory associated smallpox—England; CDC: Smallpox surveillance—worldwide).
The following epidemiologic features of smallpox facilitated global eradication (see References: Fenner 1988: Chapter 4):

Humans are the only natural reservoir for variola virus.
Vectorborne transmission of the virus does not occur.
The virus does not survive in nature for prolonged periods of time.
The full-blown clinical illness is easily recognizable, allowing for accurate clinical surveillance of the disease.
The infectivity of variola virus is relatively low (ie, transmission generally requires relatively close face-to-face contact except in uncommon circumstances), making it possible to effectively interrupt chains of transmission.
Generally, only persons who develop the characteristic rash illness transmit the virus; subclinical illness is rare and transmission from subclinical cases is not of epidemiologic importance.
No chronic carrier state of the virus occurs.
An effective vaccine exists.
The incubation period (ie, 10 to 12 days) is long enough for a vaccination/containment strategy to be effective.

Reservoir/Environmental Survival/Modes of Transmission/Communicability

Reservoir

Before global eradication, the only reservoir for variola virus was humans. No natural reservoir for the virus currently exists.
Stocks of variola virus have been retained in two WHO-approved collaborating centers: the Centers for Disease Control and Prevention (CDC) in Atlanta and the Russian State Centre for Research on Virology and Biotechnology, Koltsovo, Novosibirsk Region, Russian Federation) (see References: WHO 2001).
There are concerns that not all the smallpox preparations developed in the Russian bioweapons program can be accounted for and that unknown caches of variola virus may exist (see References: Henderson 1998).

Environmental Survival

Survival in the environment appears to be inversely proportional to temperature and humidity. In the pre-eradication era, smallpox had a higher incidence in the winter and spring in those climates where these seasons had low temperature and humidity.
Variola virus has been shown to remain stable for 2 to 4 months in scab material from smallpox patients (see References: MacCallum 1957).
Variola virus apparently can persist on fomites (such as linen and clothing) for extended periods of time (months to possibly years) (see References: Henderson 1999).
Vaccinia virus released as an aerosol is almost completely destroyed in an atmosphere of high temperature (31°C to 33°C) and humidity (80%). In cooler temperatures and lower humidity, vaccinia virus aerosol survives as long as 24 hours (see References: Henderson 1999). A recent study has shown that vaccinia virus is susceptible to germicidal ultraviolet light and that susceptibility increased with decreasing relative humidity (see References: McDevitt 2007). Variola virus is presumed to behave in a similar fashion.
Studies with vaccinia virus suggest that the virus can survive on selected food and environmental surfaces as long as 2 weeks and in water at 4.5ºC as long as 166 days (see References: Essbauer 2007). Although comparable data are not available for variola virus, the authors postulate that other orthopoxviruses may behave similarly to vaccinia virus.

Modes of Transmission

Variola virus is predominantly transmitted person-to-person via inhalation of droplet nuclei (see References: Fenner 1988: Chapter 4). Transmission occurs most commonly among those with close face-to-face contact with an infected patient (particularly household contacts, since patients are usually ill enough to be confined to bed during the period of infectiousness).
Airborne transmission has been documented in two outbreaks that occurred in hospitals in the Federal Republic of Germany (one in 1961 and one in 1970) (see References: Wehrle 1970).

In the first outbreak, the index patient transmitted the virus to 19 persons, 10 of whom had no direct contact with the patient. The index patient had severe confluent skin involvement, ulcerative pharyngitis, and a barking cough.
In the second outbreak, the index patient transmitted the virus to 17 persons, none of whom had direct contact with the patient. The index patient had severe confluent skin lesions, severe bronchitis, and cough. Investigators noted that the relative humidity in the hospital was low (which may have facilitated survival of the virus) and that the design of the hospital set up strong air currents throughout the building (which may have facilitated dissemination of viral particles).

Fomite transmission (eg, from laundry and bedding) has been reported (see References: Dixon 1962, Kiang 2003). Contaminated fomites (ie, blankets) were used for intentional transmission of smallpox during the French-Indian wars in the United States in the 1700s (see References: Stearn 1945).
Transmission via direct contact with skin lesions and infected body fluids also has been recognized (see References: Kiang 2003).

Communicability

The infectious dose is presumed to be low (10 to 100 organisms) (see References: Franz 1997).
Most epidemiologic data suggested that infectiousness in smallpox correlated with rash onset, with patients in the prodromal phase generally not considered infectious (see References: Henderson: Smallpox as a biological weapon). This is distinct from varicella infection (ie, chickenpox), in which patients are infectious before rash onset. However, patients with smallpox should be considered infectious from the time of onset of fever, because virus is present in, and shed from, the oral lesions as they ulcerate during the 1 to 2 days of fever preceding rash onset (see References: CDC: Smallpox response plan and guidelines: Guide A; Breman 2002).
Infectiousness is considered to be highest during the first week after rash onset when lesions in the mouth ulcerate and release large amounts of virus into the saliva. Frequency of secondary transmission has been estimated (using a likelihood-based estimation procedure) as being highest between 3 and 6 days after onset of fever (see References: Nishiura 2007).
The observed secondary attack rates among unvaccinated close contacts have varied from 37% to more than 88% (see References: Arnt 1972, Heiner 1971, Kiang 2003, Rao 1968). The quantity of virus excreted in oropharyngeal secretions, the number of face-to-face contacts, and the extent of face-to-face exposure are considered key factors in determining infectiousness (see References: Kiang 2003).
The average number of cases infected by a primary case is estimated at 3.5 to 6 (see References: Gani 2001). This observation was consistent across analyses of outbreaks in isolated pre–20th century populations and in 30 outbreaks in 20th-century Europe. In these settings, herd immunity was low. This estimate suggests that in populations with little herd immunity, the transmission potential of smallpox would produce a rapid rise in outbreak cases before control measures could be applied.
The period of communicability lasts until all the lesions have scabbed over and the scabs have fallen off. Viable viral particles can be detected in scabs (see References: Wolff 1968; Fenner 1988: Chapter 2); however, scabs are considered relatively noninfectious, since the viral particles are bound in the fibrin matrix of the scab.

Use of Smallpox as a Biological Weapon

Historical Perspective

Smallpox was used as a biological weapon during the French-Indian wars in the United States (1754-1767), when British soldiers gave the Indians blankets that had been used by smallpox patients (see References: Stearn 1945).
In 1972, more than 140 countries signed the Biological and Toxin Weapons Convention, which called for termination of all offensive biological weapons research and development and destruction of existing biological weapons stocks.
Despite participating in the 1972 convention, the former Soviet Union continued to expand its biological-weapons program throughout the 1980s and early 1990s. During that time, the Soviet Union reportedly developed weaponized variola virus that could be mounted in intercontinental ballistic missiles and bombs for strategic use (see References: Alibek 1999). A recent report from the Center for Nonproliferation Studies suggests that a 1971 outbreak of smallpox in Kazakhstan involving 10 people (three of whom died) may have resulted from an open-air test of a Soviet smallpox biological weapon on Vozrozhdeniye Island in the Aral Sea (a top-secret Soviet bioweapons testing site) (see References: Tucker 2002).
Currently, variola virus is known to be stored in two facilities (at the CDC in Atlanta and at the Russian State Centre for Research on Virology and Biotechnology, Koltsovo, Novosibirsk Region, Russian Federation).
In the early 1980s, WHO recommended that all existing stocks of variola virus held in other countries be either destroyed or shipped to one of the two WHO-approved collaborating centers. All countries reported compliance; however, there has been no systematic way to assure that all countries actually did comply with the WHO recommendations (see References: Henderson 2001). Also, there is no way to be certain that the virus has not fallen into the hands of rogue nations or potential terrorists (see References: Henderson 1998).
On several occasions, WHO has recommended that the remaining stores of variola virus be destroyed. However, in December 2001, the WHO Advisory Committee on Variola Virus Research recommended that existing stocks of the virus be retained for the time being so that various research goals can be achieved. The World Health Assembly has continued to authorize specific research projects that utilize the stocks, while acknowledging destruction of the stocks as its eventual goal (see References: WHO 2001, WHO 2005).

Impact of a Smallpox Release

Smallpox is of concern as a biological weapon for several reasons: much of the population is susceptible to infection, the virus carries a high rate of morbidity and mortality, vaccine is not yet available for general use, and past experience has demonstrated that introduction of the virus creates a great deal of havoc and panic (see References: Henderson 1998, O'Toole 2002).
Aerosol release of virus (such as into an airport or subway system) would be the most efficient form of release and would likely result in the highest number of cases. Other possibilities include use of "human vectors" (ie, persons who have been deliberately infected with smallpox) and use of fomites (eg, contamination of letters sent through the mail) (see References: Kiang 2003).
Several studies have used modeling to examine the impact of a deliberate release of smallpox virus. In a recent stochastic model, investigators estimated that 100 index smallpox cases in a city of 2 million would result in 730 additional cases, assuming that control measures begin at 25 days after release and that the outbreak is controlled with ring vaccination and case isolation (see References: Viboud 2003). Given the recent attention to smallpox, ongoing global vigilance to rapidly detect any recurrence through accidental or intentional release is necessary (see References: Breman 2003). Furthermore, even if all stocks of naturally occurring smallpox virus are destroyed, it is now possible to genetically engineer a similar viral agent in the laboratory setting. This capability requires that the medical and public health communities maintain smallpox preparedness into the foreseeable future (see References: Bray 2004).
A recent study suggested that donated blood could be screened using real-time polymerase chain reaction (PCR) methods to prevent the spread of variola virus through blood supplies, should it be introduced by an act of bioterrorism. In an experimental setting, the procedure (RealArt Orthopox LC PCR kit) demonstrated 100% specificity and sensitivity of 1,590 copies/mL for vaccinia in positive controls (see References: Schmidt 2005).

Antiterrorism Legislation

The Intelligence Reform and Terrorism Prevention Act, designed to improve efforts to fight terrorism, was signed into law on Dec 17, 2004 (see References: Enserink 2005; Congressional Reports 2004; HoR 2004: Report 108-796). The legislation contained an amendment ("variola amendment") inserted at the last minute that imposes severe penalties for attempts to engineer or synthesize the smallpox virus. The amendment defines smallpox virus as any virus that contains more than 85% of the gene sequence of variola major or variola minor (see References: HoR 2004: Report 108-796, Title VI: Terrorism prevention). Penalties include fines of up to $2 million and prison terms ranging from 25 years to life) (see References: Enserink 2005; HoR 2004: Report 108-796). The National Science Advisory Board for Biosecurity (NSABB) decided that the variola amendment is too vague to be useful and stated that it hurts research, because as written the amendment covers several pox viruses, including a strain used for making vaccines (see References: Enserink 2005).
The NSABB has urged the government to repeal the law that bans synthesis of smallpox virus and to revise its select agents list. The NSABB has further suggested that potentially dangerous gene sequences, rather than specific pathogens, be regulated. They have urged the government to require companies to screen orders for synthetic DNA against the genomes of select agents and maintain a record of purchase orders (see References: Bhattacharjee 2006). The NSABB is developing guidelines for safeguards against wrongful application of life sciences research and has issued drafts of guidance documents that may help clarify issues (see References: NSABB 2006).

Clinical Features of Variola Major

Variola major can be further classified into five clinical types on the basis of differences in rash characteristics and density; the prognosis differs among the types (see References: Fenner: Chapter 1). These are:

Ordinary smallpox
Flat-type (or malignant) smallpox
Hemorrhagic smallpox
Modified smallpox
Variola sine eruptione

In the pre-eradication era, diagnosing smallpox and distinguishing its type took into account clinical illness pattern, epidemiologic considerations, and laboratory findings. Although there is some overlap between ordinary, flat-type (or malignant) and hemorrhagic smallpox, their clinical and epidemiologic features are sufficiently distinct to warrant separate consideration (see below), particularly to enhance clinicians' awareness of the various clinical manifestations of what should be an extinct disease.

Modified smallpox was like ordinary smallpox but had an accelerated course and was a milder illness with fewer skin lesions and a low case-fatality rate; it was more likely to occur in persons with some immunity from past vaccination. Variola sine eruptione occurred in vaccinated contacts of cases and was characterized by sudden onset of fever, headache, and backache. Illness resolved in 1 to 2 days without development of a rash.

Case-fatality rates in the pre-eradication era for the various types of smallpox were high; however, such rates may be lower with modern medical management and intensive care.

Images of smallpox rashes are available from CDC (see References: CDC: Smallpox: images).

Ordinary Smallpox

Ordinary smallpox was the most common form of variola major infection and accounted for at least 90% of cases in the pre-eradication era.
The case-fatality rate was usually about 30% in unvaccinated persons (range, 15% to 45%) (see References: Fenner 1988: Chapter 1). Death resulted from hypotension and toxemia (associated with circulating immune complexes).
The rash illness of ordinary smallpox is somewhat similar to varicella, although disease severity is greater (see References: Henderson 1999: Smallpox: clinical and epidemiologic features).
The rash consists of firm, raised pustules and can be confluent, semiconfluent, or discrete.

Clinical features of ordinary smallpox are shown in the table below. In July 2003, CDC posted a risk-evaluation algorithm on their Smallpox Web site to help clinicians determine if a patient with rash illness is at low or high risk of having smallpox on the basis of the clinical features of the illness (see References: CDC: Evaluate a rash illness suspicious for smallpox).

Clinical Features of Ordinary Smallpox
Feature	Characteristics
Incubation period*	—10-13 days (usually about 12 days) —May be as short as 7 days and as long as 19 days
Prodrome	—Lasts 2-4 days —Frequency of prodromal symptoms in one large case series†: ~Fever, 100% ~Chills, 60% ~Headache, 90% ~Backache, 90% ~Vomiting, 50% ~Pharyngitis, 15% ~Abdominal pain, 13% ~Diarrhea, 10%
Rash*	—Enanthem on mucosa of mouth and pharynx usually begins about 24 hr before skin lesions appear (initially papular, then vesicular, then ulcerative over several days) —First few skin lesions often appear on face ("herald spots") —Lesions spread to trunk and proximal extremities and then to distal extremities —Lesions prominent on face and distal extremities, including palms and soles, in centrifugal pattern —Lesions initially maculopapular (days 1-2), then vesicular (days 3-5), then pustular (days 7-14); pustules gradually scab over by end of second week or during third week —Vesicular lesions often have central umbilication which may persist into pustular stage, but as lesions progress they gradually flatten —Pustules often described as "shotty" (ie, like hard, round foreign bodies embedded in skin) —Lesions extend deep into skin, often are painful, and pitted scarring occurs as they heal —Lesions may be discrete (relatively few in number), semiconfluent, or confluent —Lesions generally progress at same rate with relatively synchronous onset —In partially immune persons, clinical course may be much less severe and rash may be atypical with fewer lesions and more rapid healing (ie, "modified smallpox")
Laboratory features*	—Relative or absolute increase in lymphocytes may be noted —Granulocytopenia may occur
Complications†‡	—Massive amounts of subcutaneous fluid may accumulate during vesicular and pustular stages of rash, leading to severe fluid and electrolyte disturbances, including renal failure —Massive skin desquamation can occur in cases of confluent disease; patients may clinically and metabolically resemble severe burn victims —Viral bronchitis/pneumonitis occurs relatively commonly —Other less common complications: ~Corneal ulceration (about 1% of cases) and/or keratitis (about 0.25% of cases) (may cause corneal scarring and blindness) ~Secondary bacterial infections (particularly skin and pulmonary infections) ~Encephalitis (0.2% of cases) ~Osteomyelitis or arthritis (about 1.7% of cases; usually in children) ~Orchitis (rare, 0.1%)
Case-fatality rates§	—Overall case-fatality rate for ordinary smallpox, 15%-45%* —Likelihood of death varies by type of disease (ie, confluent, semiconfluent, or discrete).‡ —Observed case-fatality rates by type of disease among unvaccinated patients in one large series†: ~Overall rate, 30% ~Confluent disease, 62% ~Semiconfluent disease, 37% ~Discrete disease, 9%
*See References: Fenner 1988: Chapter 1. †See References: Rao 1972. ‡See References: Koplan 1979. §Case-fatality rates are based on historical data from pre-eradication era; such rates may be lower with modern medical management and intensive care.

Flat-Type (Malignant) Smallpox

Flat-type smallpox accounted for about 6% of cases in the pre-eradication era and occurred most commonly in children; illness was usually fatal.
The rash seen in flat-type smallpox involves flattened, confluent lesions rather than the characteristic firm pustules seen with ordinary smallpox.
Flat-type smallpox is thought to be associated with a deficient cellular immune response to the virus, although immunologic data are generally lacking (see References: Henderson 1999: Smallpox as a biological weapon).

Clinical features are shown in the table below.

Clinical Features of Flat-Type Smallpox
Feature	Characteristics
Incubation period	—Similar to ordinary smallpox (mean, 12 days; usual range, 10-14 days)
Prodrome	—Similar to ordinary smallpox (ie, fever, headache, backache, abdominal pain) —Lasts 2-4 days —Severe toxemia may occur
Rash illness*†	—Lesions develop slowly —Lesions rarely progress to pustular stage but remain soft and flattened —Lesions may be "velvety" to touch by 4th or 5th day —Lesions often confluent —Lesions and surrounding skin warm to the touch and tender to slight pressure —If patient survives, lesions gradually disappear without forming scabs and without scarring —Skin peeling or desquamation may occur as lesions heal
Laboratory features	—Similar to ordinary smallpox —Relative or absolute increase in lymphocytes may be noted —Granulocytopenia may occur
Case-fatality rate‡	—Case-fatality rate 97% in one series involving 236 patients§
*See References: Fenner 1988: Chapter 1. †See References: Dixon 1948. ‡Case-fatality rates are based on historical data from the pre-eradication era; such rates may be lower with modern medical management and intensive care. §See References: Rao 1972.

Hemorrhagic Smallpox

Hemorrhagic smallpox was rare and accounted for between 2% and 3% of cases in the pre-eradication era. In one series, 200 cases occurred out of 6,942 hospitalized patients (see References: Rao 1972).
Illness was more common in adults, and pregnant women appeared to be at greater risk.
Hemorrhagic smallpox involved hemorrhages into the skin and/or mucous membranes. Early-onset and late-onset forms were described (see References: Fenner 1988: Chapter 1).
The pathologic features of hemorrhagic smallpox are consistent with disseminated intravascular coagulation (see References: Mitra 1976, Mehta 1967).
As with malignant smallpox, a defective immune response is suspected as the cause; however, immunologic data generally are lacking (see References: Henderson 1999: Smallpox as a biological weapon). Several studies have found lower antibody responses among patients with hemorrhagic disease compared with those with ordinary disease (see References: Sarkar 1967, Downie 1969).

Clinical features are outlined in the table below.

Clinical Features of Hemorrhagic Smallpox
Feature	Characteristics
Incubation period	—Similar to ordinary smallpox (mean, 12 days; usual range, 10-14 days).
Prodrome*	—Early-onset form: illness onset sudden, with high fever, severe headache and backache, and toxemia; hemorrhages often noted by day 2 —Late-onset form: illness begins with a typical prodrome, lasting 3-4 days
Rash illness*†	—Early-onset form: generalized dusky erythema, petechiae, and ecchymoses occur soon after illness onset —Late-onset form: lesions begin as macules and develop into pustules; bleeding at base of skin lesions occurs
Hemorrhagic manifestations	—In both forms, bleeding may occur from mucosal surfaces —Features in one series of nine patients with early-onset form‡: ~Subconjunctival hemorrhage, 67% ~Hematuria, 56% ~Epistaxis, 33% ~Hematemesis and/or melena, 33% ~Hemoptysis, 33% ~Bleeding from gums, 33%
Laboratory features‡§	—Relative or absolute increase in lymphocytes may be noted —Granulocytopenia may occur —Features consistent with disseminated intravascular coagulation are common: ~Thrombocytopenia ~Hypofibrinogenemia ~Clotting-factor deficiency ~Prolonged prothrombin time
Case-fatality rate**	—In one series of 85 patients, case-fatality rate was 96%.† —Death usually occurs during the first week of illness
See References: Fenner 1988: Chapter 1. †See References: Rao 1972. ‡See References: Mitra 1976. §See References: Mehta 1967. *Case-fatality rates are based on historical data from pre-eradication era; such rates may be lower with modern medical management and intensive care.

Smallpox in Children

The clinical picture of smallpox in children generally is similar to that seen in adults. However, in one series of 100 cases among children in India, the frequency of various signs and symptoms varied somewhat from those classically described (see References: Sheth 1971). For example, headache and backache were less common, whereas vomiting, conjunctivitis, and cough were somewhat more common. Signs, symptoms, and complications identified in that series are shown in the table below. Of the 100 patients, 66 had confluent disease, 25 had discrete disease, six had flat-type smallpox, and three had hemorrhagic smallpox. Overall, 34 children died (including all of those with flat-type or hemorrhagic smallpox).

The case-fatality rate in infants may be somewhat higher than in older children or adults (ie, >40%) (see References: Fenner 1988: Chapter 1). In one case series, the case-fatality rate for infants was 85% (see References: Guha Mazumder 1975).

Infection in pregnant women often leads to premature labor and death of the fetus (see References: Fenner 1988: Chapter 1). The overall case-fatality rate for pregnant women estimated from analysis of mid-20th century outbreaks was calculated to be about 34.3%. The proportion of miscarriage or premature birth was found to be 39.9%, but no clear pattern was discernable. Premature birth was highest during the last trimester of pregnancy (see References: Nishiura 2006: Smallpox during pregnancy) No clear congenital syndrome has been associated with smallpox infection in utero.

Signs, Symptoms, and Complications of Smallpox in Children

Symptoms

Headache (15%)
Backache (15%)
Retro-orbital pain (15%)
Prostration (75%)

Signs

Fever (100%)
Vomiting (83%)
Conjunctivitis (77%)
Hepatosplenomegaly (75%)
Hypotonia (75%)
Cough (71%)
Hoarseness (71%)
Edema (71%)
Delirium (64%)
Convulsions (7%)

Complications

Constipation (66%)
Bronchopneumonia (37%)
Alopecia (19%)
Osteomyelitis (4%)
Subcutaneous abscess (3%)
Diarrhea (2%)

Adapted from Sheth 1971 (see References).

Clinical Features of Variola Minor

Variola minor is a milder form of smallpox that is caused by distinct strains of variola virus. Variola minor was first recognized in the late 1800s; during the early 20th century, it was the most prevalent form of smallpox in the United States and Great Britain. The illness may be difficult to distinguish from variola major infection in partially immune persons.

Distinguishing Features of Variola Major and Variola Minor
Feature	Variola Major	Variola Minor*†
Prodrome	—Constitutional symptoms severe	—Constitutional symptoms tend to be mild
Rash illness	—Lesions often confluent or semiconfluent —Rash evolves over 2-3 wk	—Lesions usually discrete —Rash evolves over 1-2 wk
Complications	—Flat-type or hemorrhagic disease occurs more commonly (6% and 2%, respectively, in one large series‡)	—Hemorrhagic disease rare (<0.5%)
Case-fatality rate	—May be high (15%-45%)	—Fatal outcomes rare (<1%)
*See References: Fenner 1988: Chapter 1. †See References: Ker 1967, Marsden 1948. ‡See References: Rao 1972.

Differential Diagnosis

Differential Diagnosis of the Rash illness

Other rash illnesses, outlined in the table below, are included in the differential diagnosis of smallpox.

Differential Diagnosis for Smallpox
Condition	Agent	Distinguishing features
Ordinary Smallpox*
Chickenpox	VZV	See Distinguishing Features of Smallpox and Chickenpox below
Human monkeypox	Monkeypox virus	See Monkeypox below
Disseminated herpes zoster	VZV	—Usually occurs in immunocompromised hosts —Past history of chickenpox
Impetigo	Staphylococcus aureus Streptococcus pyogenes	—Lesions often pruritic and not painful —Lesions focal and not usually disseminated —Lesions not "shotty" —Gold-colored crusted plaques are classic —Lesions superficial and not embedded into dermis —Constitutional symptoms generally absent or minimal —Usually occurs in children
Hand, foot, and mouth disease	Coxsackievirus	—Usually occurs in children <10 yr of age —Has autumn seasonal pattern —Lesions may be confined to hands and feet (although dissemination may occur)
Disseminated herpes simplex	Herpes simplex virus	—Usually occurs in immunocompromised hosts —Lesions are vesicular and do not progress to pustules
Secondary syphilis	Treponema pallidum	—Rash generally does not include vesicular phase —Lesions not "shotty" —Constitutional symptoms relatively mild —Lesions generally evolve slowly from macules to papules to pustules (often over several weeks)
Molluscum contagiosum	Molluscipoxvirus	—Usually occurs in healthy children or HIV-positive adults —In healthy adults, lesions generally occur in genital area —Lesions are painless —Constitutional symptoms generally are absent —Lesions may persist for several months (or longer in immunocompromised patients)
Erythema multiforme major (including Stevens-Johnson syndrome)	Associated with various infectious and noninfectious processes	—Constitutional symptoms and rash usually appear at same time —Rash evolves rapidly —Bullae or "bull's-eye" lesions common —Extensive mucous membrane involvement, including conjunctivitis, common
Drug eruptions	Noninfectious	—Lesions generally not pustular —History of drug exposure —Fever may be present, but severe toxemia usually absent
Bullous pemphigoid	Noninfectious	—Tense bullae characteristic —Occurs most commonly in elderly —Intense pruritis may be present —Constitutional symptoms usually absent —Peripheral eosinophilia may be noted
Other skin conditions	Noninfectious	—Acne —Insect bites —Contact dermatitis
Hemorrhagic Smallpox
Meningococcemia	Neisseria meningitidis	Rapid progression to shock and often death
Hemorrhagic varicella	VZV	Usually occurs in immunocompromised children
Rocky mountain spotted fever	Rickettsia rickettsii	—Tick exposure history may be obtained —Occurs April through May —Most US cases occur in southeastern and south-central states
Ehrlichiosis	Ehrlichia chaffeensis Erhlichia phagocytophilia	—Tick exposure history may be obtained —Petechial rash uncommon —Peripheral blood smear may show morulae in neutrophils of patients with human granulocytic ehrlichiosis
Septicemia caused by gram-negative bacteria	Various bacterial agents	—Underlying illness usually present
Abbreviation: VZV, varicella-zoster virus. Other rash illnesses (eg, measles, rubella, scabies, scarlet fever) also may be considered in the differential diagnosis, although the rashes caused by these conditions generally are not characteristic of smallpox. Adapted from Fenner 1988: Chapter 1; CDC: Acute, generalized vesicular or pustular rash illness protocol; Moore 2006 (see* References).

Monkeypox

Historical perspective

Human monkeypox is caused by monkeypox virus, which, like variola virus, is in the Orthopoxvirus genus. Monkeypox is similar to smallpox, but illness is generally milder. Recognized cases have occurred predominantly in Western and Central Africa. Pertinent historic information about monkeypox in Africa is as follows:

The illness in humans is similar to discrete or semiconfluent ordinary smallpox (see References: Jezek 1987). A prodrome (fever, headache, backache) lasting 1 to 3 days occurs, followed by eruption of a smallpox-like rash that lasts 2 to 4 weeks.
Monkeypox cases tend to have prominent lymphadenopathy, which generally is not a feature of either chickenpox or smallpox (see References: Arita 1985, Breman 1980, Jezek 1987). This can be an important distinguishing characteristic between the three conditions.
The first human case was recognized in 1970; since then, sporadic cases and outbreaks have been recognized in Africa, although the illness appears to be relatively uncommon.
Natural animal reservoirs in Africa include several squirrel species and forest-dwelling primates (see References: Khodakevich 1988). Lagomorphs (rabbits) and other rodents including prairie dogs, also appear to be susceptible to infection. The ecological requirements and geographic distributions have been identified, and these may support further field studies and guide public health intervention strategies (see References: Levine 2007).
The case-fatality rate was 11% in one series of 282 patients (see References: Jezek 1987) and was 3% in one outbreak involving 71 cases (see References: CDC: Human monkeypox), suggesting that the illness is less severe than smallpox. In both investigations, all deaths occurred in children less than 10 years of age (who had not received earlier smallpox vaccination).
Person-to-person transmission has been demonstrated (see References: Arita 1985; Breman 1980; CDC: Human monkeypox; Jezek 1986; Jezek 1988). Secondary attack rates of 7.2%, 7.5%, and 15% have been reported among household contacts who had not received prior smallpox vaccination (see References: Arita 1985, Jezek 1986, Jezek 1988). These secondary attack rates are lower than those observed for smallpox and reflect the lower propensity for spread of monkeypox compared with smallpox.

US 2003 outbreak

In June 2003, an outbreak of monkeypox was recognized in the Midwestern United States (see References: CDC: Multistate outbreak of monkeypox—Illinois, Indiana, Kansas, Missouri, Ohio, and Wisconsin, 2003; Reed 2004). Key findings from the outbreak are as follows:

Seventy-one cases were reported to CDC; 18 (26%) patients were hospitalized but no fatalities occurred.
Most of the human cases involved contact with prairie dogs; while some patients were exposed to others with monkeypox, person-to-person transmission was not documented.
No exposed healthcare workers developed monkeypox symptoms, although one worker had serologic evidence of recent orthopoxvirus infection; that person had received smallpox vaccine during the previous year (see References: Fleischauer 2005).
Thirty exposed persons received smallpox vaccine to prevent monkeypox; three reported rash within 2 weeks after vaccination and one of these persons was confirmed as having monkeypox.
In one report, three exposed persons were identified who had no symptoms but had serologic evidence of recent monkeypox infection. All three had received smallpox vaccine in the past (13, 29, and 48 years earlier). These findings suggest long-term persistence of cross-protective immunity to orthopoxvirus infection following smallpox vaccination in these individuals (see References: Hammarlund 2005).
The outbreak was traced to contact with infected prairie dogs; the prairie dogs became infected through contact with six species of African rodents (including Gambian giant rats, rope squirrels, tree squirrels, brushtail porcupines, striped mice, and dormice).
In November 2003, the Food and Drug Administration (FDA) and CDC issued an interim final rule prohibiting importation of rodents from Africa, including species responsible for the monkeypox outbreak. In addition, the rule established or modified restrictions on the import, capture, transport, sale, barter, exchange, distribution, and release of prairie dogs and implicated species of African rodents.
A recently published risk assessment from the FDA has concluded that the potential for new domestically acquired human cases is low; however, if the disease were to become established domestically via escaped or illegally bred animals, the disorder could have substantial public health impact (see References: Bernard 2006).
CDC has published guidance for autopsy and safe handling of human remains of monkeypox patients (see References: CDC 2003: Interim guidance for autopsy and safe handling).

Distinguishing Features Between Smallpox, Monkeypox, and Chickenpox

Smallpox and monkeypox are generally quite similar, although smallpox often is more severe and the case-fatality rate is higher. Early in the clinical course, smallpox or monkeypox may be mistaken for chickenpox if the clinical suspicion for orthopoxvirus infection is low. Also, smallpox in partially immune patients may be mild and may resemble chickenpox. An assessment of suspected smallpox cases referred to CDC between 2002 and 2004 found that chickenpox accounted for more than half of the cases (see References: Seward 2004). Distinguishing features of the three illnesses are outlined in the table below.

Distinguishing Features Between Smallpox, Chickenpox, and Monkeypox
Feature	Smallpox (Variola Major)*	Chickenpox	Monkeypox
Prodrome†	Lasts 2-4 days, with high fever, headache, backache, severe prostration; vomiting and severe abdominal pain may occur	Prodrome often absent; if present, it is mild and brief (ie, about 1 day)	Lasts about 2 days and is similar to that seen with smallpox
Distribution of rash	Begins on oral mucosa, spreads to face, then expands in centrifugal pattern (ie, most dense on face and distal extremities)	Begins on trunk and expands in centripetal pattern (ie, most dense on trunk)	Often begins on face and spreads in centrifugal pattern (although cases have been reported with centripetal pattern of spread)
Lesions on palms and soles	Common	Almost never occur	Common
Lymphadenopathy	Rare	Rare	Common (up to 90%)
Timing for occurrence of lesions	Generally emerge over 1-2 days and then progress at same rate	Occur in "crops" and may be at different stages of maturation at any given point in time	Lesions usually progress at same rate but may occur in crops (in about 20% of patients)
Evolution of lesions	Progress over several days from macules (day 1), to papules (day 2), to vesicles (days 3-5), to pustules (days 7 to about 14), to scabs (day 14 to about 20)	Progress quickly over about 24 hr from macules to papules to vesicles, then to crusted lesions	Progress in pattern similar to smallpox
Sensation associated with lesions	May be painful and only become pruritic during scabbing stage	Often intensely pruritic; not usually painful unless superimposed bacterial infection occurs	May be painful (although often milder than smallpox)
Depth of lesions	Extend deep into dermis and often cause pitted scarring	Superficial and generally do not cause scarring	Generally superficial (although pitted scarring can occur)
Duration of illness	14-21 days	4-7 days	14-21 days
Severity	Patients often appear toxic and case-fatality rate may be as high as 50%	Patients often do not appear severely ill and illness is rarely fatal	Illness can vary in severity but often is mild and self-limited
Epidemiology	Cases can be expected to occur in all age-groups; illness may be somewhat milder in adults over age 30 who were vaccinated as young children	Most cases occur in children; adults likely to be immune	Cases can be expected to occur in all age-groups; illness may be milder in people who have received smallpox vaccination
Illness may be milder in patients with partial immunity; fever may be less common and fewer lesions may occur with more rapid healing. †Although the prodrome may be milder in patients with chickenpox, a review of 932 chickenpox cases demonstrated that 7% to 17% of unvaccinated patients with chickenpox may meet the smallpox febrile prodrome criteria as put forth by CDC (see References: Moore 2004). Adapted from CDC: Acute, generalized vesicular or pustular rash illness protocol and Giulio 2004 (see References).*

Diagnostic Issues

Criteria for Determining the Likelihood of Smallpox

The likelihood of a smallpox diagnosis determines the appropriate laboratory testing and handling of specimens. CDC has developed criteria for determining the risk of smallpox (see References: CDC: Acute, generalized vesicular or pustular rash illness protocol).

High risk for smallpox (when all three of the following features are present):

Febrile prodrome (occurring 1 to 4 days before rash onset) with fever greater than 102°F and at least one of the following:

Prostration
Headache
Backache
Chills
Vomiting
Severe abdominal pain

Classic smallpox lesions:

Deeply embedded in the dermis
Firm/hard
Round
Well-circumscribed
May be umbilicated
May be discrete, semiconfluent, or confluent

Lesions in the same stage of development (ie, on any one area of the body, all of the lesions are at the same stage [all lesions are papules or vesicles or pustules])

Moderate risk for smallpox:

Febrile prodrome (as outlined above under "High risk for smallpox") and at least one major smallpox criteria (classic smallpox lesions as described above or lesions in the same stage of development) or
Febrile prodrome and at least four of the five minor criteria:

Centrifugal distribution (lesions are more numerous on the face and distal extremities)
First lesions appeared on the oral mucosa/palate, face, or forearms
Patient appears toxic or moribund
Slow evolution of lesions from macules to papules to pustules over several days
Lesions on the palms and soles

Low risk for smallpox:

No viral prodrome or
Febrile prodrome and fewer than four of the five minor criteria outlined above (under "Moderate risk for smallpox")

A recent study indicated that physicians in the United States may be poorly prepared to diagnose smallpox. Only 36% of 178 physicians correctly answered 3 of 4 questions regarding smallpox and chickenpox differential diagnosis. In addition, only 17% indicated that they felt "comfortable" diagnosing smallpox and 95% thought physicians needed more training in smallpox diagnosis (see References: Woods 2005). Similarly, baseline knowledge assessment of 631 physicians in 30 internal medicine residency programs in 16 states and Washington, DC, showed that only 50.7% of participating physicians could correctly diagnose smallpox (see References: Cosgrove 2005).

An algorithm developed by the CDC after the 2001 bioterrorism attack to rapidly evaluate patients for smallpox may prove useful. A recent prospective, multicenter study used the algorithm to classify 26,747 cases of rash or rashlike illness at emergency departments and inpatient units of 12 acute-care hospitals in six states. Eighty-nine patients presenting with acute generalized vesicular or pustular rash were determined to be eligible for the study, and 73 were enrolled. Physicians or study staff classified none of the 73 as being at high risk, 72 as low risk, and 1 as moderate risk of having smallpox. The discharge diagnosis for 55 of the 73 patients was varicella illness. Use of the algorithm did not result in misclassification of any patients as high risk for smallpox (see References: Hutchins 2008).

Laboratory Diagnosis

Specimen Collection and Handling

Collection

If a patient is defined as high risk for smallpox (see Criteria for Determining the Likelihood of Smallpox above), physicians should immediately contact their local or state health department for further instructions before collecting specimens.
CDC has recently outlined procedures for collecting specimens from patients who may have smallpox (see References: CDC: Smallpox response plan and guidelines: Guide D).
Only recently vaccinated (ie, within the past 3 years) personnel wearing appropriate barrier protection (ie, gloves, gown, shoe covers) should be involved in specimen collection.
If unvaccinated personnel must collect specimens, they should wear fit-tested N95 respirators and appropriate barrier protection. They also should have no contraindications to vaccination in case the diagnosis of smallpox is confirmed and vaccination is immediately required.

The following table outlines collection of laboratory specimens for the diagnosis of smallpox (variola) and smallpox vaccine (vaccinia)–associated infections.

Collection of Laboratory Specimens for the Diagnosis of Smallpox (Variola) and Smallpox Vaccine Virus Infection (Vaccinia)
Sample	Specimen Collection*
Vesicles or pustules	—Sanitize the patient's skin with an alcohol wipe and allow to dry. —Use scalpel (or 26-gauge needle) to open and remove top of vesicle or pustule; place skin of vesicle top into a 1.5- to 2-mL screw-capped or plastic tube, let dry. —Scrape base of vesicle or pustule with blunt edge of scalpel or with wooden end of applicator stick or swab and smear scrapings onto glass or plastic light microscope slide. Allow to air dry for 10 min. —Take another slide and touch it repetitively to open lesion using progressive movement of slide to make a touch prep. Allow to air dry 10 min. —Store dried slides in plastic slide holders, using a different holder for each patient. —If slide is not available, swab base of lesion with polyester or cotton swab, place in screw-capped plastic vial, break off applicator handle, and screw on lid (do not add transport medium to vial). —If available, touch shiny side of 3 electron microscope grids to unroofed base of lesion and air dry; place in gridbox. Use varying degrees of pressure (minimal, light, and moderately firm) in application of each grid to unroofed lesion. —Biopsy two vesicles with 3.5- or 4-mm punch biopsy kit; place one biopsy in formalin and one in 1.5- to 2-mL screw-capped container without added fluid. —Draw 10 cc blood into plastic marble-topped tube or plastic yellow-topped serum separator tube; if plastic tubes not available, use equivalent glass tubes and package with styrofoam protector (Note: central line sample may be needed if peripheral blood draw is difficult because of sloughing skin in dense rash area). —Swab or brush posterior tonsillar tissue and package in 1.5- to 2-mL tube, as above (do not add transport medium). —Draw 5 cc blood into plastic purple-topped tube, gently shake tube to mix contents (if plastic not available, use glass as described above)
Scab lesions	—Sanitize patient's skin with alcohol wipe and allow to dry. —Use 26-gauge needle to pry off at least four scabs. —Place two scabs in each of two screw-capped plastic 1.5- to 2-mL vials. —Obtain two biopsy specimens with a 3.5- or 4-mm punch biopsy kit; place one in formalin and one in 1.5- to 2-mL screw-capped container. —Draw 10 cc blood into plastic marble-topped tube or plastic yellow-topped serum separator tube as described above. —Collect swab of tonsillar tissue as described above. —Draw 5 cc blood into plastic purple-topped tube as described above.
Nondermatologic specimens	Collect as appropriate, such as cerebrospinal fluid for post–vaccinia vaccine encephalitis.
Autopsy specimens	—Ship frozen portions of skin-containing lesions, liver, spleen, lung, lymph nodes, and/or kidney. —Collect formalin-fixed tissue from skin-containing lesions, liver, spleen, lung, lymph nodes, and/or kidney; package separately from frozen fresh tissue. —Use plastic vials, bottles, or slide holders as primary container for all specimens.
*Check with local LRN laboratory before collecting specimens. Specific collection recommendations change and may vary depending on local policies. Adapted from CDC: Smallpox response plan and guidelines: Guide D; CDC: Specimen collection of smallpox (vaccinia) vaccine virus; CDC: Current expectations for laboratory testing and adverse smallpox vaccine reactions; CDC: Laboratory Information > Specimen Selection; CDC: Smallpox and vaccinia laboratory testing: a national training initiative (see References).

Handling and shipping

Storage and shipping conditions (see References CDC: Smallpox response plan and guidelines: Guide D; CDC: Specimen collection of smallpox [vaccinia] vaccine virus; CDC: Current expectations for laboratory testing and adverse smallpox vaccine reactions; CDC: Laboratory Information > Specimen Selection; CDC: Smallpox and vaccinia laboratory testing: a national training initiative):

For patients with high risk of smallpox, contact public health authorities before collecting, processing, or shipping specimens.
Keep all specimens away from direct sunlight.
For formalin-fixed specimens, electron microscope grids, and touch preparations, store and ship at room temperature.
For whole blood and other specimens shipped within 24 hours of collection, store and ship refrigerated (4^ºC). (Note: Spin, separate, and freeze serum onsite if shipping is to be delayed.)
For serum and fresh biopsy material and other material potentially containing infectious particles if shipped more than 24 hours after collection, store and ship on dry ice (–20^ºC to –70^ºC).
Seal vials with parafilm to avoid pH changes from dry-ice vapors.

Packaging:

Package one sample per container.

Additional shipping information:

Guidelines have been published for packing and shipping of infectious substances, diagnostic specimens, and biological agents from suspected bioterrorism (see References: ASM: Sentinel laboratory guidelines for suspected agents of bioterrorism: packing and shipping infectious substances, diagnostic specimens, and biological agents). Variola virus is classified under WHO risk group 4. In general, specimens or culture isolates that are reasonably suspected to contain variola virus must be transported as "infectious substances." International Air Transport Association (IATA) rules require training of all individuals involved in the transport of dangerous goods, including infectious substances.

Laboratory Response Network (LRN)

The Laboratory Response Network (LRN) has been developed in the United States to coordinate clinical diagnostic testing for bioterrorism events (see References: CDC: Facts about the Laboratory Response Network). The LRN consists of three levels of laboratory response: sentinel, reference, and national laboratory activities, plus a fourth level specific to smallpox. Each response level has access to standardized protocols to test for smallpox and other agents of bioterrorism.

LRN sentinel laboratories (formerly level A) include most clinical laboratories with at least BSL-2 containment. These laboratories function as laboratory first-responders that "recognize, rule-out, and refer."
LRN reference laboratories (formerly levels B and C) are mostly state or local public health laboratories with BSL-3 containment facilities that have been given access to nonpublic testing protocols and reagents. These laboratories function to "rule-in and refer."
LRN national laboratories (formerly level D) have BSL-4 containment facilities; currently, the only laboratories so designated are at the CDC and the US Army Medical Research Institute of Infectious Diseases (USAMRIID). National laboratories function to "confirm, validate, and archive" bioterrorism agents.
LRN variola surge capacity laboratories are facilities selected by the CDC to conduct enhanced variola virus identification tests and handling procedures (see References: CDC. Acute, generalized vesicular or pustular rash illness testing protocol in the United States).

The LRN can be accessed by contacting local or state public health laboratories (see References: Besser 2003; CDC: Laboratory Response Network for bioterrorism; Morse 2003). The CDC has established algorithms for laboratory evaluation of patients with acute, generalized vesicular or pustular rash illnesses, based primarily on likelihood of smallpox (see References: CDC. Acute, generalized vesicular or pustular rash illness testing protocol in the United States).

High risk for smallpox (see Criteria for Determining the Likelihood of Smallpox): Contact public health authorities immediately, before collecting specimens. Photograph patient's clinical presentation for uploading to public health departments and CDC (use digital camera or scan film image). Initial testing will be performed by LRN variola surge capacity laboratories in parallel with the CDC. Electron microscopy (EM) can be performed at the local facility, assuming BSL-3 containment is used for preparation of the grid. Variola virus should be ruled out prior to other testing, and results should not be released without CDC confirmation. The Federal Bureau of Investigation may initiate chain-of-custody for high-risk specimens.
Low or moderate risk for smallpox (see Criteria for Determining the Likelihood of Smallpox): Testing may be conducted at LRN laboratories and/or clinical laboratories with at least BSL-2 facilities. Testing for suspected cases of monkeypox or adverse vaccine reactions should be conducted at LRN laboratories. If varicella-zoster virus (VZV) diagnosis is questionable, laboratory testing should begin as soon as possible. Testing options include Tsank smear; direct fluorescent antibody test for VZV and herpes simplex virus (HSV); polymerase chain reaction (PCR) for VZV, HSV, and enterovirus; EM; and viral culture. If all results are negative, nonvariola orthopox infection, such as a vaccine-related reaction or monkeypox infection should be considered. Contact the local LRN laboratory for testing. If nonvariola orthopox tests also are negative, reevaluate the patient and assess the need for additional dermatologic and histologic testing. If upon reevaluation the risk for smallpox is upgraded to high, switch to the high-risk protocol and contact local public health authorities.
Environmental samples: Testing of environmental samples is performed only at LRN reference laboratories under direction of public health or public safety authorities.
It is likely that laboratories will receive specimens from patients with possible orthopoxvirus infections without being notified of risk level for smallpox. Theoretically, properly practiced universal precautions should protect the laboratory worker and community from accidental exposure.
Laboratory safety practices associated with variola virus and other potential agents of bioterrorism have been reviewed elsewhere (see References: Sewell 2003; CDC: Laboratory Information > Microbiology Biosafety).
Vaccination is not recommended for LRN sentinel laboratory personnel (see References: CDC: Laboratory Information / Biosafety / Vaccines). If a sample containing suspect smallpox virus is handled, vaccination within 3 days postexposure is considered effective.
Variola major virus (smallpox virus), variola minor virus (alastrim), and monkeypox virus are classified as select agents and therefore are regulated under 42 CFR part 73, which was published as an Interim Final Rule in the Federal Register on December 13, 2002 (see References: HHS: Possession, use, and transfer of select agents and toxins). As specified in the Public Health Security and Bioterrorism Preparedness and Response Act of 2002, 42 CFR part 73 provides requirements for laboratories that handle select agents (including registration, security risk assessments, safety plans, security plans, emergency response plans, training, transfers, record keeping, inspections, and notifications). These new requirements went into effect on February 7, 2003, and override earlier government requirements regarding possession and transfer of select agents. For more information about CDC’s Select Agent Program, see References: CDC: Select agent program. In addition, CDC has published additional guidelines for enhancing laboratory security for laboratories working with select agents (see References: CDC: Laboratory security and emergency response guidance for laboratories working with select agents).

Tests for Detection and Identification of Variola Virus

Culture on egg chorioallantoic membrane (CA): This is the classical method for identification of poxviruses and was used extensively before the eradication of smallpox. Poxviruses grow on CA, and each species forms characteristic pock lesions under defined temperature conditions (see References: Fenner 1988: Chapter 2). CA requires BSL-4 isolation.
Direct examination of vesicle or pustular material: As one of the largest viruses known, variola virus and aggregations of virus (Guarnieri bodies) may be seen in the cytoplasm of Giemsa-, hematoxylin-eosin (H-E)-, or silver-stained preparations viewed by light microscopy. Guarnieri bodies appear reddish purple with Giemsa stain and hematoxylino-eosinophilic with Bouin-fixed H-E, they contain Feulgen-positive material, and they show no evidence of fat or polysaccharide on histochemical examination (see References: Kato 1959). Direct examination was used in the past in outbreak settings and would likely be useful if smallpox were reintroduced. It is not considered reliable today as an identification or detection tool because of difficulty of interpretation (see References: Fenner 1988: Chapter 2).
Tissue culture: Growth in cultured cells has been used for quantitative culture of variola virus, and attempts have been


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