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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 infection by Western blot analysis. Plasma samples from unvaccinated monkeypox-infected (2–30 months postinfection) (MPV), vaccinia-immune monkeypox-infected (2–6 months postinfection) (VV-MPV), primary vaccinia-immune (2–4 months postimmunization) (VV), long-term vaccinia-immune (>20 years postimmunization) (VV long-term), and orthopoxvirus-naive (OPV) persons were analyzed by Western blot after adsorption with vaccinia-infected BSC40 cell lysate to reduce cross-reactive antibodies as described in Figure 2. Immunoreactivity to diagnostic protein bands of ≈39 kDa, 124 kDa, and 148 kDa was assessed by A) unblinded analysts with knowledge of subject medical history (n = 2) and B) blinded analysts who did not have knowledge of subject medical history (n = 4). Findings of each analyst were averaged for each person and percentages shown represent a composite of all data points.
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Figure 3: Diagnosis of monkeypox infection by Western blot analysis. Plasma samples from unvaccinated monkeypox-infected (2–30 months postinfection) (MPV), vaccinia-immune monkeypox-infected (2–6 months postinfection) (VV-MPV), primary vaccinia-immune (2–4 months postimmunization) (VV), long-term vaccinia-immune (>20 years postimmunization) (VV long-term), and orthopoxvirus-naive (OPV) persons were analyzed by Western blot after adsorption with vaccinia-infected BSC40 cell lysate to reduce cross-reactive antibodies as described in Figure 2. Immunoreactivity to diagnostic protein bands of ≈39 kDa, 124 kDa, and 148 kDa was assessed by A) unblinded analysts with knowledge of subject medical history (n = 2) and B) blinded analysts who did not have knowledge of subject medical history (n = 4). Findings of each analyst were averaged for each person and percentages shown represent a composite of all data points.

Mentions: Unblinded analysis of the Western blots was first performed by 2 independent analysts who had access to patients’ medical histories (Figure 3, panel A). Reactivity to the 39-kDa band resulted in 100% sensitivity for identifying primary monkeypox infection and 75% sensitivity for identifying secondary monkeypox infection. However, 20%–30% of the negative control samples (vaccinia primary, vaccinia long-term, and orthopoxvirus naive) also reacted with this band, which resulted in a specificity of only 70%–80% (76% overall specificity). The 124-kDa band showed 96% sensitivity for identifying primary monkeypox infection but only 31% sensitivity for identifying secondary monkeypox infection. However, this protein was more specific than the 39-kDa protein; 0%–10% of negative controls showed reactivity (97% overall specificity). The 148-kDa band showed low diagnostic sensitivity; only 37%–46% of plasma samples from monkeypox-immune persons reacted with it in Western blot analysis. Conversely, the 148-kDa band showed high specificity with 0%–5% of negative control plasma that were positive (i.e., overall specificity >97%). Other bands were also identified as potential diagnostic indicators of monkeypox infection, such as the 98-kDa and 18-kDa bands (Figure 2, panels B and C; online Technical Appendix, http://www.cdc.gov/EID/content/14/4/592-Techapp.pdf). However, further analysis showed that the 98-kDa band had low diagnostic potential (48% sensitivity and 72% specificity), as did the 18-kDa band shown in Figure 2, panel B (60% sensitivity and 66% specificity).


Retrospective analysis of monkeypox infection.

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

Diagnosis of monkeypox infection by Western blot analysis. Plasma samples from unvaccinated monkeypox-infected (2–30 months postinfection) (MPV), vaccinia-immune monkeypox-infected (2–6 months postinfection) (VV-MPV), primary vaccinia-immune (2–4 months postimmunization) (VV), long-term vaccinia-immune (>20 years postimmunization) (VV long-term), and orthopoxvirus-naive (OPV) persons were analyzed by Western blot after adsorption with vaccinia-infected BSC40 cell lysate to reduce cross-reactive antibodies as described in Figure 2. Immunoreactivity to diagnostic protein bands of ≈39 kDa, 124 kDa, and 148 kDa was assessed by A) unblinded analysts with knowledge of subject medical history (n = 2) and B) blinded analysts who did not have knowledge of subject medical history (n = 4). Findings of each analyst were averaged for each person and percentages shown represent a composite of all data points.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2570942&req=5

Figure 3: Diagnosis of monkeypox infection by Western blot analysis. Plasma samples from unvaccinated monkeypox-infected (2–30 months postinfection) (MPV), vaccinia-immune monkeypox-infected (2–6 months postinfection) (VV-MPV), primary vaccinia-immune (2–4 months postimmunization) (VV), long-term vaccinia-immune (>20 years postimmunization) (VV long-term), and orthopoxvirus-naive (OPV) persons were analyzed by Western blot after adsorption with vaccinia-infected BSC40 cell lysate to reduce cross-reactive antibodies as described in Figure 2. Immunoreactivity to diagnostic protein bands of ≈39 kDa, 124 kDa, and 148 kDa was assessed by A) unblinded analysts with knowledge of subject medical history (n = 2) and B) blinded analysts who did not have knowledge of subject medical history (n = 4). Findings of each analyst were averaged for each person and percentages shown represent a composite of all data points.
Mentions: Unblinded analysis of the Western blots was first performed by 2 independent analysts who had access to patients’ medical histories (Figure 3, panel A). Reactivity to the 39-kDa band resulted in 100% sensitivity for identifying primary monkeypox infection and 75% sensitivity for identifying secondary monkeypox infection. However, 20%–30% of the negative control samples (vaccinia primary, vaccinia long-term, and orthopoxvirus naive) also reacted with this band, which resulted in a specificity of only 70%–80% (76% overall specificity). The 124-kDa band showed 96% sensitivity for identifying primary monkeypox infection but only 31% sensitivity for identifying secondary monkeypox infection. However, this protein was more specific than the 39-kDa protein; 0%–10% of negative controls showed reactivity (97% overall specificity). The 148-kDa band showed low diagnostic sensitivity; only 37%–46% of plasma samples from monkeypox-immune persons reacted with it in Western blot analysis. Conversely, the 148-kDa band showed high specificity with 0%–5% of negative control plasma that were positive (i.e., overall specificity >97%). Other bands were also identified as potential diagnostic indicators of monkeypox infection, such as the 98-kDa and 18-kDa bands (Figure 2, panels B and C; online Technical Appendix, http://www.cdc.gov/EID/content/14/4/592-Techapp.pdf). However, further analysis showed that the 98-kDa band had low diagnostic potential (48% sensitivity and 72% specificity), as did the 18-kDa band shown in Figure 2, panel B (60% sensitivity and 66% specificity).

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