Post by nyxie63 on Aug 24, 2008 5:43:53 GMT -8
Evaluation of a Western Blot Method for the Detection of Yersinia Antibodies: Evidence of Serological Cross-Reactivity between Yersinia Outer Membrane Proteins and Borrelia burgdorferi
cvi.asm.org/cgi/content/full/12/11/1269
Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens.
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The ability to differentiate between IgG, IgA, and IgM antibodies compared to IgG and IgM detection by the CF method is of significant importance in the diagnosis of yersinia-associated complications. Antibodies against Yersinia Yops develop after infection and often persist at high levels in cases of reactive arthritis (7, 26). IgA antibodies have been shown to persist for 14 to 16 months following the onset of infection, with peak levels correlating directly with the severity of arthritis. This is in contrast to the persistence of IgA antibodies for only 5 months in cases of yersiniosis without subsequent complications (5). In cases of chronic enteritis, IgA antibodies develop against YopE (23 kDa), YopD (35 kDa), and YopB (41 kDa). IgA antibodies against YopD develop in 90% of reactive arthritis cases (7, 26, 27). IgG antibodies develop against all outer membrane proteins of Yersinia; but they develop more frequently against YopE (23 kDa), YopB (41 kDa), YopD (35 kDa), and YopH (51 kDa) (8, 11, 21). IgG antibodies can persist longer in cases of reactive arthritis, but not as consistently as IgA antibodies. IgM antibodies persist for only 1 to 3 months following the onset of infection and are not as useful for the diagnosis of reactive arthritis (5). As observed in this study, both the Western blot assays and the ELISAs allow the differentiation of specific antibody isotypes, including the specific detection of IgA antibodies, which are the most important for the diagnosis of reactive arthritis. The CF assay detects only IgG and IgM antibodies and does not differentiate between specific antibody isotypes.
Cross-reactivity was observed with all assays evaluated. The CF assay exhibited the lowest amount of cross-reactivity with other organisms among the methods evaluated, although this may be attributed to the poor sensitivity of the assay. The highest cross-reactivity for the CF assay was shown with C.pneumoniae, with three of eight C. pneumoniae-positive samples testing positive for Yersinia. Cross-reactivity was also observed with Brucella, F. tularensis, and TSI by the CF assay. One sample from each category for B. henselae, Brucella, C.pneumoniae, and R. rickettsii showed cross-reactivity by Western blot assay. Of the methods evaluated, the ELISA exhibited the highest cross-reactivity with other organisms. All organisms and antibodies tested, excluding C. burnetti and M.pneumoniae, showed cross-reactivity by the ELISA. This could be attributed to the lower specificity of the ELISA. Previous studies have reported cross-reactivity between Yersinia, Brucella (2,17-19), R. rickettsii (2, 23), and TSI (1, 2, 13, 24). Cross-reactivity between F. tularensis and Brucella spp. has been reported (9), indicating a possible explanation for the cross-reactivity observed between F. tularensis and Yersinia by the CF assays and the ELISAs. However, it is possible that some of the samples showing cross-reactivity with Yersinia are actually true positives. There are no known reports of cross-reactivity between Yersinia, B. henselae, and C. pneumoniae.
cvi.asm.org/cgi/content/full/12/11/1269
Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens.
snip
The ability to differentiate between IgG, IgA, and IgM antibodies compared to IgG and IgM detection by the CF method is of significant importance in the diagnosis of yersinia-associated complications. Antibodies against Yersinia Yops develop after infection and often persist at high levels in cases of reactive arthritis (7, 26). IgA antibodies have been shown to persist for 14 to 16 months following the onset of infection, with peak levels correlating directly with the severity of arthritis. This is in contrast to the persistence of IgA antibodies for only 5 months in cases of yersiniosis without subsequent complications (5). In cases of chronic enteritis, IgA antibodies develop against YopE (23 kDa), YopD (35 kDa), and YopB (41 kDa). IgA antibodies against YopD develop in 90% of reactive arthritis cases (7, 26, 27). IgG antibodies develop against all outer membrane proteins of Yersinia; but they develop more frequently against YopE (23 kDa), YopB (41 kDa), YopD (35 kDa), and YopH (51 kDa) (8, 11, 21). IgG antibodies can persist longer in cases of reactive arthritis, but not as consistently as IgA antibodies. IgM antibodies persist for only 1 to 3 months following the onset of infection and are not as useful for the diagnosis of reactive arthritis (5). As observed in this study, both the Western blot assays and the ELISAs allow the differentiation of specific antibody isotypes, including the specific detection of IgA antibodies, which are the most important for the diagnosis of reactive arthritis. The CF assay detects only IgG and IgM antibodies and does not differentiate between specific antibody isotypes.
Cross-reactivity was observed with all assays evaluated. The CF assay exhibited the lowest amount of cross-reactivity with other organisms among the methods evaluated, although this may be attributed to the poor sensitivity of the assay. The highest cross-reactivity for the CF assay was shown with C.pneumoniae, with three of eight C. pneumoniae-positive samples testing positive for Yersinia. Cross-reactivity was also observed with Brucella, F. tularensis, and TSI by the CF assay. One sample from each category for B. henselae, Brucella, C.pneumoniae, and R. rickettsii showed cross-reactivity by Western blot assay. Of the methods evaluated, the ELISA exhibited the highest cross-reactivity with other organisms. All organisms and antibodies tested, excluding C. burnetti and M.pneumoniae, showed cross-reactivity by the ELISA. This could be attributed to the lower specificity of the ELISA. Previous studies have reported cross-reactivity between Yersinia, Brucella (2,17-19), R. rickettsii (2, 23), and TSI (1, 2, 13, 24). Cross-reactivity between F. tularensis and Brucella spp. has been reported (9), indicating a possible explanation for the cross-reactivity observed between F. tularensis and Yersinia by the CF assays and the ELISAs. However, it is possible that some of the samples showing cross-reactivity with Yersinia are actually true positives. There are no known reports of cross-reactivity between Yersinia, B. henselae, and C. pneumoniae.
