Serologic Testing for Lyme Disease

[Guest guest]

Serologic Testing for Lyme Disease

To the Editor: We were surprised by the results presented by Dr Schutzer and

colleagues1 reporting on an immune complex diagnostic method for Lyme

disease; this method was first described by Schutzer et al almost 10 years

ago.2 Our experience with the immune complex approach differs substantially

from theirs.

In February 1999, Dr Schutzer and Dr Coyle participated in a blinded study

(referred to as a pilot study in their article) with the Centers for Disease

Control and Prevention (CDC), in which the immune complex approach was

compared with the nationally recommended 2-tier serologic testing.3 The CDC

provided 60 blinded and coded samples to each participating group: 25

samples from 13 patients with early, culture-confirmed Lyme disease, 25

samples from control subjects with no history of Lyme disease, and 5

repeated samples from each of these 2 sample groups.

The comparative findings of this evaluation (Table 1) were decoded, returned

to the participants, and presented during a working group meeting in the

same month. In that study, the rate of true-positive immune complex test

results in samples from patients with early Lyme disease was 52%, compared

with a rate of 60% by 2-tier testing; this differs markedly from the 95%

rate reported by Schutzer et al.1 Similarly, in the pilot study the rate of

true-negative results for the immune complex test was only 76% and the

reproducibility was 60%, in contrast to the true-negative rate of 99% and

reproducibility of 100% reported by Schutzer et al.

These discrepancies may have been caused by sample selection bias. For

example, Schutzer et al present results for only 10 of the 60 test samples

used in the CDC pilot study. The authors state that these 10 samples (from 7

patients, not 10), were chosen for the report because they were from

patients who had positive cultures for Borrelia burgdorferi but were

serologically negative by 2-tier testing. In the pilot study, only 5 of

these samples (not 6) from 3 patients were positive by immune complex

testing. Schutzer et al also do not report the results of 7 additional

samples from culture-positive patients in the pilot study whose results were

negative by immune complex testing but positive by 2-tier serologic testing.

Results of the pilot study suggest that the immune complex approach is no

better than 2-tier serologic testing in any performance category. A

definitive determination of the accuracy of immune complex test performance

awaits evaluation with a blinded and unbiased sample set that represents the

entire target patient population.

E. Schriefer, PhD

T. Dennis, MD, MPH

Duane J. Gubler, ScD

B. , MD

Barbara J. B. , PhD

May C. Chu, PhD

Centers for Disease Control and Prevention

Fort , Colo

1. Schutzer SE, Coyle PK, Reid P, Holland B. Borrelia burgdorferi-specific

immune complexes in acute Lyme disease. JAMA. 1999;282:1942-1946. ABSTRACT

| FULL TEXT | PDF | MEDLINE

2. Schutzer SE, Coyle PK, Belman AL, Golightly MG, Drulle J. Sequestration

of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme

disease. Lancet. 1990;335:312-315. MEDLINE

3. Centers for Disease Control and Prevention. Recommendations for test

performance and interpretation from the Second National Conference on

Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep.

1995;44:590-591. MEDLINE

In Reply: In their letter, Dr Schriefer and colleagues compare a relatively

large study to a smaller pilot study that uses a different method and then

compare the immune complex data from the pilot study to free antibody data

in their laboratory. They contrast our 95% true-positive rate of Borrelia

burgdorferi-specific immune complex assay in more than 158 patients with

Lyme disease who were stratified into groups with a 52% true-positive rate

in their pilot study of 13 patients, also stratified into groups.

As they suggest, methodological differences may account for the disparities.

The pilot study examined only enzyme-linked immunosorbent assay (ELISA)

immune complex using whole Bb antigens. No immunoblots were used. After the

pilot studies were performed, the ELISA preparation was found to be

deficient in OspC, a major early expressed Bb antigen. This alone would

prompt us to repeat the study. Also, in comparison to the larger study, the

smaller pilot groups may be more subject to sampling error and not be

adequate for statistical analysis by groups. Pilot samples obtained after

antibiotic therapy should be expected to yield different results.

The immune complex assay measures something different than free antibody.

Even if the percentages of positivity were identical, the immune complex

results would give the added benefit of indicating an active vs a past

infection.1, 2 Therefore, we believe it is useful to couple the immune

complex assay to an ELISA or blot test.

Schriefer et al present the immune complex data from the pilot study by

combining IgM and IgG results. These are, however, distinct assays. When

separated, IgM immune complex results were positive in 8 (62%) of 13

patients with Lyme disease and 0 (0%) of 25 controls, and detected 2 (67%)

of 3 Lyme disease patients who had negative results in the 2-tiered assays

(Table 2). This implies, for the IgM immune complex assay, a 100%

true-negative rate and a 62% true-positive rate, which was possibly lowered

by the lack of OspC. The latter rate increased if a 2-SD cutoff point

(similar to many commercial assays) was used, with 10 (77%) of 13 patients

with Lyme disease having positive results and 2 of 13, borderline results

(92% combined total). The 92% rate, which includes the borderline results,

follows the interim criteria for the first-tier test3 referred to by

Schriefer et al. There was no reduction in specificity with respect to

healthy controls; 1 patient with syphilis (4%) had a positive result. The

IgG immune complex test had unacceptable results as performed under the

conditions of the pilot study and would require recombinant or immunoblot

modifications.

We thank Schriefer et al for pointing out that 10 seronegative samples (from

7 patients) were incorrectly labeled " patients " in some sections. We

understand the concern of using only a subset of samples from the pilot

study. Our intent was to illustrate both the existence of apparent

seronegativity in early documented disease and the potential of the immune

complex assay to identify some of these patients. We felt that

methodological differences between the 2 studies precluded combined

analysis. If combined, however, the true-positive rate of the immune complex

assays, as defined by Schriefer et al, is 94%.

It is difficult to respond to the issue of the true-positive rate of the

test, as performed in the laboratory of Schriefer et al, in the absence of a

defined cutoff point for a positive test result and information on how the

cutoff point for the first tier was computed (eg, proprietary to the

manufacturer of the Biomerieux VIDAS machine used for their assay). Without

this information, no direct comparisons can be made.

We endorse and plan further collaborative evaluations in this serious and

costly disease.

E. Schutzer, MD

Bart Holland, PhD

Reid, BS

University of Medicine and Dentistry of New Jersey

Newark

P. K. Coyle, MD

State University of New York

Stony Brook

1. Brunner M, Stein S, PD, Sigal LH. Immunoglobulin M capture assay

for serologic confirmation of early Lyme disease: analysis of immune

complexes with biotinylated Borrelia burgdorferi sonicate enhanced with

flagellin peptide epitope. J Clin Microbiol. 1998;36:1074-1080. MEDLINE

2. Zhong W, Oschmann P, Wellensiek HJ. Detection and preliminary

characterization of circulating immune complexes in patients with Lyme

disease. Med Microbiol Immunol (Berl). 1997;186:153-158. MEDLINE

3. Centers for Disease Control and Prevention. Recommendations for test

performance and interpretation from the Second National Conference on

Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep.

1995;44:590-591. MEDLINE

Letters Information

Guidelines for Letters

Letters Section Editors: J. Lurie, MD, PhD, Contributing Editor;

Phil B. Fontanarosa, MD, Executive Deputy Editor.

http://jama.ama-assn.org/issues/v284n6/full/jlt0809-3.html