On 2015 Nov 16, Raphael Stricker commented:

Response to the Study of Fallon et al.

Jyotsna S. Shah PhD, Nick S. Harris PhD

IGeneX Reference Laboratory, Palo Alto, CA

November 15, 2015

The analysis of Lyme disease serologic testing by Fallon et al. [1] reported that “Specialty Lab B” had an overall Borrelia burgdorferi test specificity of 42.5%. We therefore asked Dr. Fallon to send us the key for the study samples that were tested by “Specialty Lab B”, and we reviewed the data for all positive and negative samples.

In the study by Fallon et al., it is notable that two-tier testing endorsed by the Centers for Disease Control and Prevention (CDC) had a sensitivity of only 48.6% for patients with persistent symptoms following Lyme disease treatment, similar to the low sensitivity described in the medical literature [2]. In contrast, if both the IgM and IgG Western blot (WB) results are considered positive, 33 of 37 patients (89%) with persistent Lyme disease symptoms were positive by “Specialty Lab B” interpretation.

“Specialty Lab B” includes bands 31 kDa (Osp A) and 34 kDa (Osp B) in its interpretation of WB results [3]. Antibodies to these proteins are present later in the disease [4] as well as in patients vaccinated with the now-defunct Osp A vaccine. In addition, “Specialty Lab B” uses two strains of B. burgdorferi (B31 and 297) to make WB strips. In strain 297, the 39 kDa antigen is well expressed [5], whereas in strain B31 expression of the 39 kDa antigen can vary between 4-50% [4, 6]. These factors may explain the higher WB sensitivity of “Specialty Lab B”.

According to the revised interpretive criteria of “Specialty Lab B”, a WB is considered positive if two of the following six bands are reactive with patient serum: 23-25 kDa (Osp C), 31 kDa (Osp A), 34 kDa (Osp B), 39 kDa (BmpA), 41 kDa (Flagellin) and 83-93 kDa. However at position 31 kDa on the WB, a non-specific protein co-migrates with Osp A [3]. Therefore both the IgM and IgG WBs are reported as indeterminate if only bands 31 kDa and 41 kDa are present. Likewise, it is known that some viral antibodies cross-react with the 83-93 kDa B. burgorferi antigen. Therefore the IgM WB is reported as negative if only bands 41 kDa and 83-93 kDa are present, and an indeterminate IgM WB is reported if only bands 31 kDa and 83-93 kDa are present. Thus “Specialty Lab B” offers the option to retest using a recombinant Osp A antigen WB to clarify whether a positive band at 31kDa indeed represents reactivity to B. burgdorferi or not.

“Specialty Lab B” reported a significantly higher number of “healthy” controls (23/40) as positive compared to other labs. In contrast, only one “healthy” control was positive by two-tier test criteria. Of the 23 positive “healthy” controls, three had insufficient samples for retesting and therefore were excluded from further analysis. Three control samples with bands 41 kDa and 83-93 kDa on IgM WB were considered negative. The remaining 17 samples were tested with recombinant antigen WBs. Thirteen, including two positive by CDC two-tier criteria, had B. burgdorferi-specific antibodies. Two sera with a band at 31 kDa on WB were considered negative as they did not test positive on the Osp A recombinant antigen WB. Therefore 13/37 healthy controls had B. burgdorferi specific antibodies, and only two had a false-positive WB. Thus the performance of the WB by “Specialty Lab B”, when using revised interpretation criteria and recombinant antigen WB, demonstrates a specificity of 91.7%.

According to the study by Fallon et al. [1], the sensitivity of Lyme disease antibody testing was 48.6% in the best laboratories using CDC two-tier criteria. In an effort to improve that sensitivity, “Specialty Lab B” uses additional band criteria to raise its combined IgM and IgG WB sensitivity to 89% [3]. Considering the significant underdiagnosis of Lyme disease based on poor two-tier test sensitivity, it is important to improve this sensitivity so that clinical cases of Lyme disease will not be missed.

Lyme serology continues to be problematic and we continue to look for ways to improve sensitivity and specificity. In particular, the specificity of the tests is problematic because people living or traveling to endemic areas can have antibodies without disease [7], and antibodies of other diseases can cross-react with B. burgdorferi antigens. Therefore analysis of clinical history and symptoms is essential for the accurate diagnosis of Lyme disease. We agree with the conclusions drawn by Fallon et al. [1] that interlaboratory variability is considerable and remains a problem in Lyme disease testing.

References

1.Fallon BA, Pavlicova M, Coffino SW, Brenner C. A comparison of Lyme disease serologic test results from four laboratories in patients with persistent symptoms after antibiotic treatment. Clin Infect Dis. 2014; 59:1705-10.

2.Stricker RB, Johnson L. Lyme disease diagnosis and treatment: Lessons from the AIDS epidemic. Minerva Med 2010;101:419-25.

3.Shah JS, Du Cruz I, Narciso W, Lo W, Harris NS. Improved sensitivity of Lyme disease Western blots prepared with a mixture of Borrelia burgdorferi strains 297 and B31. Chronic Dis Int. 2014;1:7.

4.Ma B, Christen B, Leung D, Vigo-Pelfrey C. Serodiagnosis of Lyme borreliosis by Western immunoblot: reactivity of various significant antibodies against Borrelia burgdorferi. J Clin Microbiol.1992; 30: 370–376.

5.Engstrom, SM, Shoop E, Johnson RC. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol. 1995; 33: 419–427.

6.Aguero-Rosenfeld ME, Nowakowski J, Bittker S, Cooper D, Nadelman RB, et al. Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans. J Clin Microbiol. 1996; 34: 1–9.

7.Steere AC, Sikand VK, Schoen RT, Nowakowski J. Asymptomatic infection with Borrelia burgdorferi. Clin Infect Dis. 2003; 37: 528–532.

Disclosure: RBS is a member of the International Lyme and Associated Diseases Society (ILADS) and a director of LymeDisease.org. He has no financial or other conflicts to declare. JSS and NSH are members of ILADS, and they have financial interests in IGeneX Reference Laboratory.

On 2015 Nov 16, Raphael Stricker commented:

Response to the Study of Fallon et al.

Jyotsna S. Shah PhD, Nick S. Harris PhD

IGeneX Reference Laboratory, Palo Alto, CA

November 15, 2015

The analysis of Lyme disease serologic testing by Fallon et al. [1] reported that “Specialty Lab B” had an overall Borrelia burgdorferi test specificity of 42.5%. We therefore asked Dr. Fallon to send us the key for the study samples that were tested by “Specialty Lab B”, and we reviewed the data for all positive and negative samples.

In the study by Fallon et al., it is notable that two-tier testing endorsed by the Centers for Disease Control and Prevention (CDC) had a sensitivity of only 48.6% for patients with persistent symptoms following Lyme disease treatment, similar to the low sensitivity described in the medical literature [2]. In contrast, if both the IgM and IgG Western blot (WB) results are considered positive, 33 of 37 patients (89%) with persistent Lyme disease symptoms were positive by “Specialty Lab B” interpretation.

“Specialty Lab B” includes bands 31 kDa (Osp A) and 34 kDa (Osp B) in its interpretation of WB results [3]. Antibodies to these proteins are present later in the disease [4] as well as in patients vaccinated with the now-defunct Osp A vaccine. In addition, “Specialty Lab B” uses two strains of B. burgdorferi (B31 and 297) to make WB strips. In strain 297, the 39 kDa antigen is well expressed [5], whereas in strain B31 expression of the 39 kDa antigen can vary between 4-50% [4, 6]. These factors may explain the higher WB sensitivity of “Specialty Lab B”.

According to the revised interpretive criteria of “Specialty Lab B”, a WB is considered positive if two of the following six bands are reactive with patient serum: 23-25 kDa (Osp C), 31 kDa (Osp A), 34 kDa (Osp B), 39 kDa (BmpA), 41 kDa (Flagellin) and 83-93 kDa. However at position 31 kDa on the WB, a non-specific protein co-migrates with Osp A [3]. Therefore both the IgM and IgG WBs are reported as indeterminate if only bands 31 kDa and 41 kDa are present. Likewise, it is known that some viral antibodies cross-react with the 83-93 kDa B. burgorferi antigen. Therefore the IgM WB is reported as negative if only bands 41 kDa and 83-93 kDa are present, and an indeterminate IgM WB is reported if only bands 31 kDa and 83-93 kDa are present. Thus “Specialty Lab B” offers the option to retest using a recombinant Osp A antigen WB to clarify whether a positive band at 31kDa indeed represents reactivity to B. burgdorferi or not.

“Specialty Lab B” reported a significantly higher number of “healthy” controls (23/40) as positive compared to other labs. In contrast, only one “healthy” control was positive by two-tier test criteria. Of the 23 positive “healthy” controls, three had insufficient samples for retesting and therefore were excluded from further analysis. Three control samples with bands 41 kDa and 83-93 kDa on IgM WB were considered negative. The remaining 17 samples were tested with recombinant antigen WBs. Thirteen, including two positive by CDC two-tier criteria, had B. burgdorferi-specific antibodies. Two sera with a band at 31 kDa on WB were considered negative as they did not test positive on the Osp A recombinant antigen WB. Therefore 13/37 healthy controls had B. burgdorferi specific antibodies, and only two had a false-positive WB. Thus the performance of the WB by “Specialty Lab B”, when using revised interpretation criteria and recombinant antigen WB, demonstrates a specificity of 91.7%.

According to the study by Fallon et al. [1], the sensitivity of Lyme disease antibody testing was 48.6% in the best laboratories using CDC two-tier criteria. In an effort to improve that sensitivity, “Specialty Lab B” uses additional band criteria to raise its combined IgM and IgG WB sensitivity to 89% [3]. Considering the significant underdiagnosis of Lyme disease based on poor two-tier test sensitivity, it is important to improve this sensitivity so that clinical cases of Lyme disease will not be missed.

Lyme serology continues to be problematic and we continue to look for ways to improve sensitivity and specificity. In particular, the specificity of the tests is problematic because people living or traveling to endemic areas can have antibodies without disease [7], and antibodies of other diseases can cross-react with B. burgdorferi antigens. Therefore analysis of clinical history and symptoms is essential for the accurate diagnosis of Lyme disease. We agree with the conclusions drawn by Fallon et al. [1] that interlaboratory variability is considerable and remains a problem in Lyme disease testing.

References

1.Fallon BA, Pavlicova M, Coffino SW, Brenner C. A comparison of Lyme disease serologic test results from four laboratories in patients with persistent symptoms after antibiotic treatment. Clin Infect Dis. 2014; 59:1705-10.

2.Stricker RB, Johnson L. Lyme disease diagnosis and treatment: Lessons from the AIDS epidemic. Minerva Med 2010;101:419-25.

3.Shah JS, Du Cruz I, Narciso W, Lo W, Harris NS. Improved sensitivity of Lyme disease Western blots prepared with a mixture of Borrelia burgdorferi strains 297 and B31. Chronic Dis Int. 2014;1:7.

4.Ma B, Christen B, Leung D, Vigo-Pelfrey C. Serodiagnosis of Lyme borreliosis by Western immunoblot: reactivity of various significant antibodies against Borrelia burgdorferi. J Clin Microbiol.1992; 30: 370–376.

5.Engstrom, SM, Shoop E, Johnson RC. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol. 1995; 33: 419–427.

6.Aguero-Rosenfeld ME, Nowakowski J, Bittker S, Cooper D, Nadelman RB, et al. Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans. J Clin Microbiol. 1996; 34: 1–9.

7.Steere AC, Sikand VK, Schoen RT, Nowakowski J. Asymptomatic infection with Borrelia burgdorferi. Clin Infect Dis. 2003; 37: 528–532.

Disclosure: RBS is a member of the International Lyme and Associated Diseases Society (ILADS) and a director of LymeDisease.org. He has no financial or other conflicts to declare. JSS and NSH are members of ILADS, and they have financial interests in IGeneX Reference Laboratory.