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
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
—Distinguishing Features Between Smallpox and Chickenpox
—Monkeypox
Diagnostic Issues
—Criteria for Determining the Likelihood of Smallpox
—Specimen Collection and Handling
Laboratory Diagnosis
—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
—New Vaccinia Vaccines
—Recommendations for Use of Vaccinia Vaccines
—Vaccination Schedule
—Dosage and Route of Administration
—Local Reaction to Vaccination
—Contraindications and Precautions
—Adverse Reactions
—Treatment of Adverse Reactions with Vaccinia Immune Globulin
—Use of Vaccine for Postexposure Prophylaxis
—Use of Vaccine During a Smallpox Emergency
Infection Control
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 (approximately the size of a bacterial spore)
• Enveloped
• Dumbell-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 to differentiate them (see References: National Center for Biotechnology Information; LeDuc 2001).
• 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

Environmental survival of variola virus:

• 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)

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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 is an extremely valuable clue to the diagnosis and 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.

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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). 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/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).

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). 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).

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: Interim 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.
• The observed secondary attack rates among susceptible close contacts have varied from 37% to more than 70% (see References: Rao 1968, Arnt 1972, Heiner 1971).
• 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

• 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 (see References: Johns Hopkins Center for Civilian Biodefense Studies). 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 (see References: WHO 2001).
• 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).

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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 and WHO (see References: CDC: Smallpox: rash illness images; WHO: Smallpox: rash illness slideset).

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 are shown in the table below.

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.

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.

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). No clear congenital syndrome has been associated with smallpox infection in utero.

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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.

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Differential Diagnosis

Differential Diagnosis of the Rash illness

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

Distinguishing Features Between Smallpox and Chickenpox

Smallpox and chickenpox cause somewhat similar illnesses, although smallpox generally is much more severe. However, smallpox in partially immune patients may be mild and may resemble chickenpox.

Early in the clinical course, smallpox may be mistaken for chickenpox if the clinical suspicion for smallpox is low. Distinguishing features of the two illnesses are outlined in the table below.

Monkeypox

• Human monkeypox is caused by monkeypox virus, which (like variola virus) is in the Orthopoxvirus genus.
• 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 lympadenopathy, which generally is not a feature of either chickenpox or smallpox (see References: Arita 1985, Breman 1980, Jezek 1987).
• The illness occurs naturally only in Western and Central Africa. Animal reservoirs include several squirrel species and forest-dwelling primates (see References: Khodakevich 1988).
• The first human case was recognized in 1970; since then sporadic cases and outbreaks have been recognized, although the illness appears to be relatively uncommon.
• 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.

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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: 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")

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: Interim 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.

Handling and Shipping

Storage and shipping conditions:

• Store and ship at room temperature for samples in formalin, 4^oC for electron microscope grids and serum, and -20^oC to -70^oC (dry ice) for fresh biopsy material.
• Other samples should be stored and shipped at 4^oC if shipped within 24 hours of collection and at -20^o C to -70^o C (dry ice) if held for longer periods of time.
• Seal vials with parafilm to avoid pH changes from dry-ice vapors.
• If there will be a delay in shipping, spin serum to separate from clot, then store and ship at 4^oC.

Packaging:

• Package one sample per container.

Transport:

• Contact CDC to obtain specific instructions for transporting specimens.

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Laboratory Diagnosis

Laboratory Response Network (LRN)

The LRN has been developed in the United States to coordinate clinical diagnostic testing for bioterrorism events (see References: CDC: Emergency response; CDC: Biological and chemical terrorism; Gilchrist 2001). The network is organized into four laboratory levels (A, B, C, and D). Level D laboratories must meet BSL-4 criteria; currently, the only laboratories so designated are at the CDC and the US Army Medical Research Institute of Infectious Diseases (USAMRIID). The LRN can be accessed by contacting local or state public health laboratories.

• All testing of samples from patients at high risk for smallpox should be performed by Level D laboratories only (see Criteria for Determining the Likelihood of Smallpox above and see References: CDC: Biosafety in microbiological and biomedical laboratories).
• Testing for rash illness (such as varicella-zoster virus testing) on specimens from patients not at high risk for smallpox (see Criteria for Determining the Likelihood of Smallpox above) can be performed at other laboratory levels that have at least BSL-2 containment facilities. If smallpox cannot be ruled out through other testing, the local or state health department should be contacted for further instructions.
• Level C laboratories are expected in the near future to have the ability to conduct testing for smallpox on inactivated samples using such methods as polymerase chain reaction (PCR) and electron microscopy.

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).
• Direct examination of vesicle or pustular material: As one of the largest viruses known, variola virus may be seen in the cytoplasm of Giemsa- or silver-stained cells viewed by light microscopy. This was used in the past in outbreak settings and is not significant today per se but remains important because poxviruses may be inadvertently discovered in the laboratory (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 made to characterize its cytopathic effect for identification purposes (see References: Marennikova 1974, Fenner 1988). As with direct examination by light microscopy, this is important because current tissue culture methods may result in inadvertent discovery.
• Electron microscopy: Negative staining is used to visualize the characteristic large brick shape and fine structure detail of poxviruses. Electron microscopy by itself is insufficient for definitive identification of variola virus, but can be useful to distinguish poxviruses that infect humans (eg, variola, vaccinia, cowpox, monkeypox) from varicella-zoster virus.
• PCR-based methods: These have been developed to detect and identify poxviruses (see References: Ropp 1995).
• DNA probe: A method for identifying orthopoxviruses to the species level, using an oligonucleotide microchip, has been described (see References: Lapa 2002).
• Serology: Classical methods such as complement fixation and gel precipitation commonly were used in the past. Experimental enzyme-linked immunoassays are currently being evaluated (see References: LeDuc 2001).
• Strain identification: A restriction fragment length polymorphism assay (RFLP) has been developed by CDC using polymorphisms found on 45 variola strains from 1939 to the 1970s (see References: Le Duc 2001).
• Antiviral susceptibility testing: USAMRIID is developing animal models for evaluating antiviral drugs; this testing is being conducted at the CDC BL-4 facility (see References: LeDuc 2001).

Rapid Tests for Diagnosis of VZV and HSV

Laboratories that have at least BSL-2 containment facilities can perform rapid tests for diagnosis of rash illness in patients not considered at high risk for smallpox (see Criteria for Determining the Likelihood of Smallpox above). The most likely alternative agents are varicella-zoster virus (VZV) and herpes simplex virus (HSV); available rapid tests for these two agents include the following:

• Cytology smears: Tzanck preparations stained with Giemsa or Papanicolaou stain are rapid, inexpensive, and 80% sensitive by one study (see References: Cohen 1994, Oranje 1986, Gershon 1999).
• Direct fluorescent antibody (DFA): An assay by Chemicon (see References: Chemicon, Inc) detects VZV and HSV simultaneously. A recent study shows a sensitivity of 80% and specificity of 98.3% for HSV with same-day turnaround. A shell vial direct immunoperoxidase assay had a sensitivity of 87.6% and a specificity of 100%, and a turnaround time of 1 to 2 days. With VZV-positive samples, the DFA had a correlation of 87.1% with a cytospin DFA method (see References: Chan 2001).
• Standard PCR methods: PCR has been shown to be more sensitive than immunofluorescence for detection of VZV (see References: Bezold 2001) and significantly more sensitive than electron microscopy (see References: Jain 2001). PCR has been shown to detect HSV and VZV effectively from Tzanck smears and vesicle fluid but less effectively from fixed-tissue specimens (see References: Nahass 1995). FDA-approved methods are not yet available.
• "Real time" PCR assays: Rapid PCR assays using TaqMan and LightCycler technologies have been developed for HSV and VZV. These assays appear to have very high sensitivity and specificity. In some studies, assay sensitivity is higher than culture sensitivity (see References: Espy 2000, Hawrami 1999, Ryncarz 1999, Aldea 2002, Koenig 2001, Nicoll 2001). FDA-approved methods are not yet available.

Testing in Areas With Confirmed Smallpox

Once smallpox is confirmed in a geographic area, additional cases can be diagnosed clinically (see References: CDC: Interim smallpox response plan and guidelines: Guide A).

In such situations, laboratory resources will be used for specimen testing in the following cases:

• Those in which clinical presentation is unclear
• Those that will provide information about a potential source of exposure
• Those that will facilitate law enforcement activities or case detection

Inadvertent Discovery of Variola Virus in a Laboratory Specimen

Variola virus and other poxviruses grow readily on many cell lines such as Vero, HeLa, SF, and MRC-5. Accidental discovery of variola virus by a clinical virologist would constitute a danger to the laboratorians and could precipitate unintentional release to the community. The following features of variola virus in cell culture have been described in the older literature (see References: Fenner 1988: Chapter 2; Marennikova 1974; Ono 1968):

• In human cell lines, variola virus tends to form "hyperplastic foci" as cells are pushed together by growing cells around them.
• Within 24 to 48 hours, giant multinucleated cells form.
• When stained, there may be a circular arrangement of nuclei around an eosinophilic part of the cytoplasm, often containing inclusions.
• Within 72 to 96 hours, the number of giant cells increases, as does degeneration of the cell layer.
• Variola virus can cause numerous inclusion bodies (Guarnieri bodies) in the cytoplasm of infected cells, which can be viewed after staining by Giemsa, modified silver stain, or other stains. Interpretation was difficult during times of smallpox occurrence and would be more difficult today.

If an unusual cytopathic effect is observed on any cell culture, especially involving giant cells, laboratory personnel should determine the suspected diagnosis for the patient before proceeding with identification. If the patient is at high risk for smallpox or at moderate risk for smallpox without alternate diagnoses, then the cell culture should be sealed, stored securely, and the local or state health department contacted for further instructions. Staining of cells suspected of harboring poxvirus is not recommended.

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Treatment

Treatment for smallpox largely consisted of general supportive measures:

• Adequate fluid intake (difficult because of the enanthem)
• Alleviation of pain and fever
• Keeping the skin lesions clean to prevent bacterial superinfection

No specific antiviral treatment of demonstrated effectiveness was available in the pre-eradication era.

In recent years, 274 antiviral compounds have been screened for therapeutic activity against variola virus and other orthopoxviruses (see References: Le Duc 2001). Cidofovir as well as 27 other compounds have demonstrated activity against orthopoxviruses, including variola. In advanced clinical testing for other viral infections, cidofovir, adefovir dipivoxil, cyclic cidofovir, and ribavirin have shown significant in vitro activity (see References: Franz 1997). All promising compounds will be further evaluated in animal models.

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Smallpox Vaccination

Historical Perspective

• The first efforts at smallpox vaccination involved a process called variolation, which was the deliberate cutaneous inoculation of variola virus via infectious material obtained from smallpox pustules of a patient with active disease (see References: Fenner1988: Chapter 6). Variolation was practiced as early as 1000 AD in China and gradually spread around the globe.
• Variolation generally resulted in a severe localized reaction, a generalized rash, and constitutional symptoms. The case-fatality rate following variolation was much lower than that following natural smallpox (about 0.5% to 2% and 20% to 30%, respectively) and, therefore, this practice was widely implemented.
• The variola virus used in variolation was not attenuated, and one of the disadvantages of this practice was that smallpox could be spread to susceptible contacts of a person infected via variolation.
• In the late 1700s, Edward Jenner successfully used cowpox virus to vaccinate people against smallpox. Because this practice was safer and relatively effective, it rapidly gained wide acceptance and replaced variolation as the primary method of conferring protection against smallpox.
• Over time, vaccinia virus gradually replaced cowpox virus as the agent used in smallpox vaccine. Vaccinia virus is genetically distinct from cowpox virus, although its origin remains unknown. It may have been derived from cowpox virus initially and modified over time through serial passage in laboratory cultures, or it may represent another orthopoxvirus that is now extinct in nature.

Dryvax Vaccinia Vaccine

• The vaccinia vaccine that has been available in the United States since the 1970s is a lyophilized preparation of infectious vaccinia virus (Dryvax, manufactured by Wyeth Laboratories, Lancaster, Pennsylvania).
• Existing Dryvax supplies were produced in the 1970s and have been maintained in lyophilized storage since that time; evaluation has shown that the vaccine is still potent, and it is estimated that there are about 15.4 million doses of Dryvax vaccine currently available (see References: LeDuc 2001).
• Published data indicate that the current Dryvax vaccine may be diluted 1:5 without significant loss of vaccine potency (see References: Frey 2002: Dose-related effects of smallpox vaccine; Frey 2002: Clinical responses to undiluted and diluted smallpox vaccine). Currently the diluent is prepackaged at a volume of 1.25 mL, affording one-step reconstitution-dilution at a 1:5 dilution with a sufficient margin of excess virus. The aim of diluting vaccine stocks is to substantially extend current supplies, and all existing product is being diluted to the 1:5 ratio. This will bring the existing Dryvax supply to about 77 million doses.
• Diluting the Dryvax vaccine 1:10 (ie, to a titer of 10^7.0 plaque-forming units [pfu] per milliliter, or approximately 10,000 pfu/dose) was also capable of eliciting adequate vaccination responses (Frey 2002, as above); however, widespread use of the 1:10 dilution under field conditions would be problematic, as there would be only a narrow margin of excess virus in the vaccine which, with any mishandling, could lead to many failures. Therefore, the vaccine will not be diluted at the 1:10 ratio.
• CDC is the only vaccine distributor for civilians; current smallpox vaccines are only available under an Investigational New Drug (IND) protocol from CDC's Drug Service [call 404-639-3670] (see References: CDC: Drug Service). The vaccine is only made available for laboratory and healthcare workers who are at risk of exposure to vaccinia viruses and to military personnel. It is not available for the general public.
• An additional 85 million doses of smallpox vaccine (prepared using the same strain as Dryvax but stored frozen) recently have been located by Aventis and donated to the US government. These vaccine supplies will be held in reserve for use on an as-needed basis.

Information about the efficacy of Dryvax can be found in the 2001 statement on smallpox vaccine from ACIP (see References: CDC: Vaccinia [smallpox] vaccine):

• Successful primary vaccination is demonstrated by occurrence of a pustular or vesicular skin lesion at the site of vaccination after about 7 days. Successful revaccination (ie, in a person who has received at least one prior dose of vaccine) is indicated by palpable inflammation at the site 6 to 8 days after vaccination (a pustule may or may not be present with revaccination).
• The presence of a localized skin reaction to vaccination correlates with the development of neutralizing antibody, which appears about 10 days after primary vaccination and about 7 days after revaccination.
• Neutralizing or hemagglutination inhibition antibody titers of 1:10 or higher will develop in 95% of primary vaccinees. Although the level of antibody required for protection against vaccinia infection has never been fully evaluated in the field, available data suggest that titers of 1:10 or greater will confer protection for most vaccinated persons.
• The duration of immunity has never been adequately measured. Epidemiologic studies suggest that protection against smallpox persists for 5 to 10 years after primary vaccination. Neutralizing antibody titers of 1:10 or higher are found in 75% of persons up to 10 years after receiving two doses of vaccine and up to 30 years after receiving three doses.
• It has been estimated that fewer than 20% of persons vaccinated before the early 1970s (when routine vaccination was discontinued in the United States) have immunologic protection today (see References: Henderson 1999: Smallpox as a biological weapon). It is not clear whether a remote history of receiving at least one dose of smallpox vaccine will modulate disease severity in the event that infection occurs.

New Vaccinia Vaccines

• In September 2000, CDC entered into an agreement with OraVax (since renamed Acambis, Cambridge, Massachusetts) to produce a new cell culture-derived vaccinia vaccine.
• In October 2001, the federal government contracted with Acambis and Acambis-Baxter for at least 209 million doses of smallpox vaccine produced in cell culture; these doses are expected to be available by the end of 2002 (see References: CDC: Supplemental recommendations of the ACIP).
• Because the new vaccine has a different method of production, studies to assess vaccine efficacy are needed. Clinical trials are currently under way and should be completed by 2003. Supplies of cell culture-derived vaccine will be made available as needed under an IND protocol from CDC.
• The vaccine produced by Acambis (ACAM1000) and the vaccine produced by Baxter (ACAM2000) both contain 100 doses per vial, should be stored at 2° to 8°C, and are reconstituted with 0.25 mL of diluent (see References: CDC: Interim smallpox response plan and guidelines: Annex 3).

Recommendations for Use of Vaccinia Vaccines

• Routine smallpox vaccination in the United States stopped in 1972 for children and in 1976 for healthcare workers. Prior to 1972, smallpox vaccine was recommended for all children in the United States at 1 year of age.
• Vaccinia vaccination currently is recommended for laboratory workers and certain healthcare workers:
  • Those who directly handle vaccinia virus cultures, contaminated dressings or other infectious material, recombinant vaccinia viruses, or other orthopoxviruses that infect humans (eg, monkeypox)
  • Those who handle animals contaminated or infected with vaccinia virus, recombinant vaccinia viruses, or other orthopoxviruses that infect humans
• The best strategies for pre- and post-exposure smallpox vaccine deployment for both the general public and potentially high-risk populations recently have been subjects of intense national debate (see References: Fauci 2002, Bicknell 2002). The current CDC strategy for use of the vaccine in outbreak control is outlined under Use of Smallpox Vaccine During a Smallpox Emergency below. In addition, the ACIP made the following recommendations on June 20, 2002 (see References: CDC: Supplemental recommendations of the ACIP: use of smallpox [vaccinia] vaccine):
  • Under current circumstances, with no confirmed smallpox, and the risk of an attack assessed as low, vaccination of the general population is not recommended, as the potential benefits of vaccination do not outweigh the risk of vaccine complications.
  • Smallpox vaccination is recommended for persons pre-designated by the appropriate bioterrorism and public health authorities to conduct investigation and follow-up of initial smallpox cases that would necessitate direct patients contact.
  • Smallpox vaccination is recommended for selected personnel in facilities pre-designated to serve as referral centers to provide care for the initial cases of smallpox. These facilities would be pre-designated by the appropriate bioterrorism and public health authorities, and personnel within these facilities would be designated by the hospital.
• Vaccinia vaccine should never be used for therapeutic purposes. There is no evidence that it has any value in the treatment of any condition, and use is reserved for prevention of infections caused by human orthopoxviruses.

Vaccination Schedule

• Vaccination consists of a single dose followed by a booster every 10 years.
• Revaccination every 3 years (see References: CDC: Vaccinia [smallpox] vaccine) should be considered for persons who work with:
  • Nonhighly attenuated vaccinia viruses
  • Recombinant viruses developed from nonhighly attenuated vaccinia viruses
  • Nonvariola orthopoxviruses such as monkeypox

Dosage and Route of Administration

The vaccine is administered using a droplet of the vaccine applied to a bifurcated needle in the following manner (see References: Fenner 1988: Chapter 11; CDC: Interim smallpox response plan and guidelines: Guide B):

• Remove the rubber stopper from the vaccine vial and place it in a sterile container.
• Insert a sterile bifurcated needle into the ampule of reconstituted vaccine and withdraw the needle perpendicular to the floor.
• Use the outer aspect of the upper right arm over the insertion of the deltoid muscle as the standard vaccination site.
• Clean the vaccination site only if it is grossly contaminated. If cleaning is necessary, clean the site with alcohol and let dry completely.
• Hold the needle at a right angle to the skin of the upper arm with the wrist of the vaccinator against the vaccinee's forearm.
• Make 15 perpendicular strokes of the needle rapidly in an area about 5 mm in diameter. The strokes should be sufficiently vigorous so that a trace of blood appears at the vaccination site after 15 to 30 seconds. If blood does not appear, repeat the procedure.
• Do not redip the needle into the vaccine vial if the needle has touched the skin.
• Wipe the excess vaccine and blood from the site with gauze and discard in a hazardous waste receptacle.
• Cover the site with a loose, nonocclusive dressing to prevent autoinoculation of another body site (historically, this was the most common complication of vaccination in the United States).
• If the vaccine is to be stored for subsequent use, recap the vial with the sterile rubber stopper and store the capped vial at 2°C to 8°C.

Local Reaction to Vaccination

Upon primary vaccination, all recipients experience a local reaction to the vaccine. A typical reaction occurs in the following sequence:

• About 3 days after vaccination, a red papule appears at the vaccination site.
• By day 5, the papule becomes vesicular.
• By day 7, it becomes a typical "jennerian" pustule (whitish, umbilicated, multilocular, containing turbid fluid, and surrounded by an erythematous areola that may continue to expand for 3 more days).
• The pustule eventually dries, leaving a dark crust that normally falls off after about 3 weeks.
• Regional lymphadenopathy and fever are common.

Cutaneous reactions to subsequent vaccinations are weaker and manifest a range of the local reactions above. For a revaccination to be considered successful, palpable inflammation or a pustule must be present. With revaccination, the less intense the "jennerian" pustule, the greater the likelihood of some degree of residual immunity. No reaction to revaccination indicates inadequate technique or insufficient virus in the inoculation.

Contraindications and Precautions

Vaccinia vaccine for pre-exposure use is contraindicated for:

• Persons with a history of eczema (even if the condition is mild or not presently active) or other significant exfoliative skin conditions, because the risk of eczema vaccinatum is increased in this group
• Persons with conditions causing immunodeficiency (eg, HIV infection, leukemia, lymphoma, generalized malignancy, agammaglobulinemia or other hereditary immunodeficiency, or therapy with alkylating agents, antimetabolites, radiation, or large doses of corticosteroids)
• Persons with household contacts who are immunodeficient or who have a history of eczema
• Pregnant women
• Persons with hypersensitivity reactions to vaccine components, including polymyxin B sulfate, streptomycin sulfate, chlortetracycline hydrochloride, and neomycin sulfate

If routine pre-exposure smallpox vaccination were reintroduced, a recent study estimated that up to 25% of the US population would be excluded from vaccination, either because of a direct contraindication or the potential to expose high-risk household members (see References: Kemper 2002).

Adverse Reactions

Serious adverse reactions to smallpox vaccination can occur; these are outlined in the following table. Not all of these reactions can be successfully treated with vaccinia immune globulin (VIG). For images of smallpox vaccine reactions, see References: CDC: Smallpox: vaccine reaction images).

A pre-exposure smallpox vaccination campaign for the US general public aged 1 to 65 could result in as many as 4,600 serious adverse events and 285 deaths (excluding high-risk persons and their contacts) (see References: Kemper 2002).