Smallpox was usually diagnosed clinically. The main criteria for clinical diagnosis of smallpox included:
· febrile prodromal phase occurring 1–4 days before the onset of rashes;
· characteristic smallpox lesions of the skin (deep, firm, round), which could be umbilicated or confluent; and
· skin lesions in same stage of development on any part of the body.
Skin lesions, such as vesicular fluid, were the specimens of choice
Electron microscope, if available, was used for direct demonstration of typical virus particles in clinical specimens. It was useful to differentiate a smallpox virus from that of the chickenpox. The Guarnieri bodies, the characteristic eosinophilic inclusion bodies, can be demonstrated in stained preparation of the clinical specimens by light microscopy.
Isolation of the virus in the laboratory is carried out by inocula-tion in the chick embryo and in cell culture. This is necessary for rapid and accurate identification of poxvirus infections.
Embryonated egg: Inoculation of vesicular fluid into theCAM of the chick embryo is a reliable method for detection and identification of variola virus. The virus produces characteristic pocks after 48–72 hours of inoculation. The variola pocks are smaller, whereas vaccinia pocks are large with necrotic centers. The monkeypox and cowpox produce well-marked hemor-rhagic lesions, whereas tenapoxvirus, molluscum contagiosum, and the Parapoxvirus do not show any growth on the CAM.
Cell culture: Human and nonhuman primate cells, such asmonkey kidney and HeLa cells, are used for isolation of virus from clinical specimens. Viruses are detected by the presence of Guarnieri bodies, the characteristic CPE produced by the vari-ola virus. Unlike the vaccinia virus, which produces the CPE rapidly within 24–48 hours, the variola virus produces the CPE very slowly.
Tenapoxvirus and parapoxviruses grow poorly, whereas mol-luscum contagiosum does not grow at all in the cell cultures.
Serological tests were useful to confirm the diagnosis of poxvi-rus infection. Indirect immunofluorescent antibody test and HI, CF, and NT tests are available for demonstration of serum antibodies that appear after first week of infection.