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Retrospective analysis of monkeypox infection.

Dubois ME, Slifka MK - Emerging Infect. Dis. (2008)

Bottom Line: Serologic cross-reactivity between orthopoxviruses is a substantial barrier to laboratory diagnosis of specific orthopoxvirus infections and epidemiologic characterization of disease outbreaks.Historically, time-consuming and labor-intensive strategies such as cross-adsorbed neutralization assays, immunofluorescence assays, and hemagglutination-inhibition assays have been used to identify orthopoxvirus infections.We used cross-adsorption to develop a simple and quantitative postadsorption ELISA for distinguishing between monkeypox and vaccinia infections.

View Article: PubMed Central - PubMed

Affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006, USA.

ABSTRACT
Serologic cross-reactivity between orthopoxviruses is a substantial barrier to laboratory diagnosis of specific orthopoxvirus infections and epidemiologic characterization of disease outbreaks. Historically, time-consuming and labor-intensive strategies such as cross-adsorbed neutralization assays, immunofluorescence assays, and hemagglutination-inhibition assays have been used to identify orthopoxvirus infections. We used cross-adsorption to develop a simple and quantitative postadsorption ELISA for distinguishing between monkeypox and vaccinia infections. Despite the difficulty of diagnosing clinically inapparent monkeypox in previously vaccinated persons, this technique exhibited 100% sensitivity and 100% specificity for identifying clinically overt monkeypox infection irrespective of vaccination history. We also describe a Western blot technique in which up to 3 diagnostic bands may be used to distinguish between vaccinia and monkeypox infection. The techniques described provide independent diagnostic tests suitable for retrospective analysis of monkeypox outbreaks.

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Diagnosis of monkeypox by postadsorption ELISA. Plasma samples were obtained from monkeypox-immune persons (2–30 months postinfection), vaccinia-immune persons (2–4 months postinfection), or uninfected orthopoxvirus-naive persons and tested on ELISA plates coated with inactivated monkeypox antigen. A) A representative monkeypox-specific ELISA with plasma samples from an unvaccinated monkeypox-infected person (MPV), a previously vaccinated (i.e., vaccinia-immune) monkeypox-infected person (VV-MPV), a vaccinia-immune person (VV), a vaccinia-immune person who was revaccinated with vaccinia (VV-VV), and an uninfected orthopoxvirus-naive person (OPV-naive). Plasma was not preadsorbed (∅, gray bars), preadsorbed with inactivated vaccinia antigen (black bars), or preadsorbed with inactivated monkeypox antigen (white bars) before ELISA on monkeypox-coated plates. Numbers above bars refer to differences in postadsorption MPV ELISA titers after adsorption with vaccinia antigen compared with adsorption with monkeypox antigen. Plasma from 1 orthopoxvirus-naive person (representative of n = 12) was not preadsorbed with viral antigen because it was seronegative (<100 ELISA units) and below our detection limit (dashed horizontal line). B) Plasma samples from monkeypox-infected persons (•, n = 13), vaccinia-immune monkeypox-infected persons (■, n = 8), vaccinia-immune persons (▲, n = 10), and revaccinated vaccinia-immune persons (▼, n = 10) were tested by postadsorption ELISA. Data show fold-differences of monkeypox antibody titers after adsorption with vaccinia antigen compared with adsorption with monkeypox antigen. Dashed horizontal line indicates a diagnostic cutoff indicative of a positive result, which was determined as a postadsorption difference score of >2.5. *Denotes results of plasma samples obtained from persons with clinically inapparent monkeypox infection.
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Figure 1: Diagnosis of monkeypox by postadsorption ELISA. Plasma samples were obtained from monkeypox-immune persons (2–30 months postinfection), vaccinia-immune persons (2–4 months postinfection), or uninfected orthopoxvirus-naive persons and tested on ELISA plates coated with inactivated monkeypox antigen. A) A representative monkeypox-specific ELISA with plasma samples from an unvaccinated monkeypox-infected person (MPV), a previously vaccinated (i.e., vaccinia-immune) monkeypox-infected person (VV-MPV), a vaccinia-immune person (VV), a vaccinia-immune person who was revaccinated with vaccinia (VV-VV), and an uninfected orthopoxvirus-naive person (OPV-naive). Plasma was not preadsorbed (∅, gray bars), preadsorbed with inactivated vaccinia antigen (black bars), or preadsorbed with inactivated monkeypox antigen (white bars) before ELISA on monkeypox-coated plates. Numbers above bars refer to differences in postadsorption MPV ELISA titers after adsorption with vaccinia antigen compared with adsorption with monkeypox antigen. Plasma from 1 orthopoxvirus-naive person (representative of n = 12) was not preadsorbed with viral antigen because it was seronegative (<100 ELISA units) and below our detection limit (dashed horizontal line). B) Plasma samples from monkeypox-infected persons (•, n = 13), vaccinia-immune monkeypox-infected persons (■, n = 8), vaccinia-immune persons (▲, n = 10), and revaccinated vaccinia-immune persons (▼, n = 10) were tested by postadsorption ELISA. Data show fold-differences of monkeypox antibody titers after adsorption with vaccinia antigen compared with adsorption with monkeypox antigen. Dashed horizontal line indicates a diagnostic cutoff indicative of a positive result, which was determined as a postadsorption difference score of >2.5. *Denotes results of plasma samples obtained from persons with clinically inapparent monkeypox infection.

Mentions: Orthopoxvirus-naive persons or persons infected with monkeypox or vaccinia were then tested to establish the diagnostic validity of this approach by using monkeypox-coated ELISA plates (Figure 1, panel A). If plasma from a representative monkeypox-immune person was preincubated with vaccinia antigen, virus-specific antibody titers decreased (from 39,904 ELISA units [EU] to 13,912 EU). However, if the sample was preincubated with an equivalent amount of monkeypox antigen before performing the ELISA, monkeypox antibody titers were further decreased, to 2,542 EU. For the data of the monkeypox-immune person shown in Figure 1, panel A, there was a 5.5-fold difference (13,912 EU divided by 2,542 EU) in the ability of monkeypox antigen to reduce monkeypox-specific antibody levels than vaccinia antigen. For persons who were vaccinia immune but still had contracted clinically apparent monkeypox disease, the difference in postadsorption ELISA titers was typically smaller (3.2-fold difference in the example shown in Figure 1, panel A), but the difference still demonstrated a clear distinction between preadsorption with monkeypox or vaccinia antigens. If a person had been infected with only vaccinia or revaccinated with vaccinia, there was essentially no difference in the ability of monkeypox antigen to deplete antiviral antibodies than an equivalent amount of vaccinia antigen on a monkeypox-coated ELISA plate. In contrast, orthopoxvirus-naive persons were seronegative by ELISA and can be easily distinguished from monkeypox-immune or vaccinia-immune persons because they score below the limits of detection by ELISA (<100 EU) without any preadsorption steps required.


Retrospective analysis of monkeypox infection.

Dubois ME, Slifka MK - Emerging Infect. Dis. (2008)

Diagnosis of monkeypox by postadsorption ELISA. Plasma samples were obtained from monkeypox-immune persons (2–30 months postinfection), vaccinia-immune persons (2–4 months postinfection), or uninfected orthopoxvirus-naive persons and tested on ELISA plates coated with inactivated monkeypox antigen. A) A representative monkeypox-specific ELISA with plasma samples from an unvaccinated monkeypox-infected person (MPV), a previously vaccinated (i.e., vaccinia-immune) monkeypox-infected person (VV-MPV), a vaccinia-immune person (VV), a vaccinia-immune person who was revaccinated with vaccinia (VV-VV), and an uninfected orthopoxvirus-naive person (OPV-naive). Plasma was not preadsorbed (∅, gray bars), preadsorbed with inactivated vaccinia antigen (black bars), or preadsorbed with inactivated monkeypox antigen (white bars) before ELISA on monkeypox-coated plates. Numbers above bars refer to differences in postadsorption MPV ELISA titers after adsorption with vaccinia antigen compared with adsorption with monkeypox antigen. Plasma from 1 orthopoxvirus-naive person (representative of n = 12) was not preadsorbed with viral antigen because it was seronegative (<100 ELISA units) and below our detection limit (dashed horizontal line). B) Plasma samples from monkeypox-infected persons (•, n = 13), vaccinia-immune monkeypox-infected persons (■, n = 8), vaccinia-immune persons (▲, n = 10), and revaccinated vaccinia-immune persons (▼, n = 10) were tested by postadsorption ELISA. Data show fold-differences of monkeypox antibody titers after adsorption with vaccinia antigen compared with adsorption with monkeypox antigen. Dashed horizontal line indicates a diagnostic cutoff indicative of a positive result, which was determined as a postadsorption difference score of >2.5. *Denotes results of plasma samples obtained from persons with clinically inapparent monkeypox infection.
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Figure 1: Diagnosis of monkeypox by postadsorption ELISA. Plasma samples were obtained from monkeypox-immune persons (2–30 months postinfection), vaccinia-immune persons (2–4 months postinfection), or uninfected orthopoxvirus-naive persons and tested on ELISA plates coated with inactivated monkeypox antigen. A) A representative monkeypox-specific ELISA with plasma samples from an unvaccinated monkeypox-infected person (MPV), a previously vaccinated (i.e., vaccinia-immune) monkeypox-infected person (VV-MPV), a vaccinia-immune person (VV), a vaccinia-immune person who was revaccinated with vaccinia (VV-VV), and an uninfected orthopoxvirus-naive person (OPV-naive). Plasma was not preadsorbed (∅, gray bars), preadsorbed with inactivated vaccinia antigen (black bars), or preadsorbed with inactivated monkeypox antigen (white bars) before ELISA on monkeypox-coated plates. Numbers above bars refer to differences in postadsorption MPV ELISA titers after adsorption with vaccinia antigen compared with adsorption with monkeypox antigen. Plasma from 1 orthopoxvirus-naive person (representative of n = 12) was not preadsorbed with viral antigen because it was seronegative (<100 ELISA units) and below our detection limit (dashed horizontal line). B) Plasma samples from monkeypox-infected persons (•, n = 13), vaccinia-immune monkeypox-infected persons (■, n = 8), vaccinia-immune persons (▲, n = 10), and revaccinated vaccinia-immune persons (▼, n = 10) were tested by postadsorption ELISA. Data show fold-differences of monkeypox antibody titers after adsorption with vaccinia antigen compared with adsorption with monkeypox antigen. Dashed horizontal line indicates a diagnostic cutoff indicative of a positive result, which was determined as a postadsorption difference score of >2.5. *Denotes results of plasma samples obtained from persons with clinically inapparent monkeypox infection.
Mentions: Orthopoxvirus-naive persons or persons infected with monkeypox or vaccinia were then tested to establish the diagnostic validity of this approach by using monkeypox-coated ELISA plates (Figure 1, panel A). If plasma from a representative monkeypox-immune person was preincubated with vaccinia antigen, virus-specific antibody titers decreased (from 39,904 ELISA units [EU] to 13,912 EU). However, if the sample was preincubated with an equivalent amount of monkeypox antigen before performing the ELISA, monkeypox antibody titers were further decreased, to 2,542 EU. For the data of the monkeypox-immune person shown in Figure 1, panel A, there was a 5.5-fold difference (13,912 EU divided by 2,542 EU) in the ability of monkeypox antigen to reduce monkeypox-specific antibody levels than vaccinia antigen. For persons who were vaccinia immune but still had contracted clinically apparent monkeypox disease, the difference in postadsorption ELISA titers was typically smaller (3.2-fold difference in the example shown in Figure 1, panel A), but the difference still demonstrated a clear distinction between preadsorption with monkeypox or vaccinia antigens. If a person had been infected with only vaccinia or revaccinated with vaccinia, there was essentially no difference in the ability of monkeypox antigen to deplete antiviral antibodies than an equivalent amount of vaccinia antigen on a monkeypox-coated ELISA plate. In contrast, orthopoxvirus-naive persons were seronegative by ELISA and can be easily distinguished from monkeypox-immune or vaccinia-immune persons because they score below the limits of detection by ELISA (<100 EU) without any preadsorption steps required.

Bottom Line: Serologic cross-reactivity between orthopoxviruses is a substantial barrier to laboratory diagnosis of specific orthopoxvirus infections and epidemiologic characterization of disease outbreaks.Historically, time-consuming and labor-intensive strategies such as cross-adsorbed neutralization assays, immunofluorescence assays, and hemagglutination-inhibition assays have been used to identify orthopoxvirus infections.We used cross-adsorption to develop a simple and quantitative postadsorption ELISA for distinguishing between monkeypox and vaccinia infections.

View Article: PubMed Central - PubMed

Affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006, USA.

ABSTRACT
Serologic cross-reactivity between orthopoxviruses is a substantial barrier to laboratory diagnosis of specific orthopoxvirus infections and epidemiologic characterization of disease outbreaks. Historically, time-consuming and labor-intensive strategies such as cross-adsorbed neutralization assays, immunofluorescence assays, and hemagglutination-inhibition assays have been used to identify orthopoxvirus infections. We used cross-adsorption to develop a simple and quantitative postadsorption ELISA for distinguishing between monkeypox and vaccinia infections. Despite the difficulty of diagnosing clinically inapparent monkeypox in previously vaccinated persons, this technique exhibited 100% sensitivity and 100% specificity for identifying clinically overt monkeypox infection irrespective of vaccination history. We also describe a Western blot technique in which up to 3 diagnostic bands may be used to distinguish between vaccinia and monkeypox infection. The techniques described provide independent diagnostic tests suitable for retrospective analysis of monkeypox outbreaks.

Show MeSH
Related in: MedlinePlus