Method of diagnosis of lyme disease

Details Method of diagnosis of lyme disease Method of diagnosis of lyme disease

2000-07-27

2003-12-16

Zeman, Mary K. (Department: 1631)

Data processing: artificial intelligence

Knowledge processing system

C706S052000, C706S061000, C706S900000, C702S019000, C436S506000, C436S513000, C436S811000, C435S007100

Reexamination Certificate

active

06665652

ABSTRACT:

The present invention relates to an improved method for the diagnosis of diseases and, in particular, infectious diseases such as Lyme Disease. A mathematical algorithm using Bayesian analysis is applied to data derived from an immunoassay such as a Western blot, Southern blot, an immunodot or other immunoassay technique. By combining the clinician's estimate of the pretest likelihood of disease with the immunoassay results through this specific algorithm, a more accurate diagnosis can be made. This algorithm is equally valid for any disease which is diagnosed using multiple immunoassays performed in parallel and where the antigens detected by these assays produce an antibody response in controls that is independent of one another.
In the last several years, Lyme Disease has become an increasing public health concern, for example, in the Northeast and upper Midwest regions of the United States (Morb Mortal Wkly Rep. 1994;43:564-572). Lyme Disease is a multisystem infectious disease caused by the spirochete
Borrelia burgdorferi
. This spirochete is transmitted by the bite of the tick
Ixodes scapularis
, often found on white-tailed deer. Lyme Disease ordinarily begins with flu-like symptoms such as headache, chills, joint pain, nausea and fatigue. An expanding rash called erythema migrans is characteristic of this disorder in the early stages, often beginning at the site of the tick bite. “Early stage” or “early” Lyme Disease is the four (4) week period following onset of symptoms. “Late stage” or “late” Lyme Disease is the period beyond early stage. Early stage Lyme Disease is most often curable with antibiotics. However, if left untreated or undiagnosed, Lyme Disease can cause serious complications including meningitis, chronic arthritis, encephalitis, peripheral nerve damage and even cardiac abnormalities. Although often treatable, sometimes these complications may be permanent and therefore accurate and early diagnosis is imperative.
The diagnosis of Lyme Disease has, however, proved problematic. In the past, the diagnosis of Lyme Disease had been based mainly on clinical symptomatology, not laboratory tests, since culture or visualization of the spirochete
Borrelia burgdorferi
from patient specimens was infrequent, and serologic tests were just being developed. Clinicians placed emphasis on the history of a tick bite, the observation of the characteristic erythema migrans rash and the presence of typical physical signs such as arthritis or Bell's Palsy, a neurologic condition. In recent times it has been realized that only 60-80% of patients with Lyme Disease have a rash, only 33-50% recall a tick bite, and that atypical presentations of the disease are now being recognized, thus making laboratory diagnosis more important. However, currently available blood tests for Lyme Disease, for example, enzyme-linked methods such as ELISA or immunofluorescent (IFA) methods, have been known to produce both false-negative and false-positive results. False-negative results have been seen frequently in early and sometimes in late Lyme Disease. False-positive results have been seen in some normal adults and in patients with certain specific conditions such as syphilis, Epstein-Barr viral infection, and connective tissue disorders such as rheumatoid arthritis or systemic lupus erythematosus. For example, among patients seen in Lyme Disease specialty clinics in the Northeast United States, only 6-34% of those with positive serology by an ELISA method were confirmed to have active Lyme Disease. Reproducibility of results between laboratories had been poor and standardization of reagents and interpretative standards had been lacking. These serious limitations prompted the Centers for Disease Control and the Association of State and Territorial Public Health Laboratory Directors to convene the Second National Conference on Serologic Diagnosis of Lyme Disease in October, 1994. At this conference a two-step procedure for diagnosing Lyme Disease was proposed consisting of an initial screening assay by an ELISA or IFA method followed by confirmatory Western blotting for all specimens found to be positive or equivocal by the initial screening test. Negative screening tests would obviate the need for further testing. In addition, standardization of reagents and interpretive standards were proposed. This conference established the role of the Western blot test as a means of improving the specificity of Lyme serologic testing.
The Western blot technique involves separating borrelial proteins by polyacrylamide gel electrophoresis and then transferring these proteins electrophoretically (blotting) to a nitrocellulose membrane or chemically treated paper. These proteins generally will bind to the membrane or paper in a pattern identical to that in the gel. Bands of antigen (bound to the paper or membrane) can be detected visually by overlaying the blot with the patient's serum followed by an anti-immunoglobulin antibody. Both IgG and IgM type antibodies can be detected by the Western blot technique. In a typical patient with Lyme disease the initial response of the patient's body is to make IgM antibody within the first few weeks of the infection, followed shortly thereafter by an IgG antibody response. Both the number and type of bands identified in the Western blot technique are useful in distinguishing patients who have Lyme Disease from normal control patients and patients with other diseases. Western blots are also referred to as “Western immunoblots”. Interpretive standards proposed by the Centers for Disease Control and Prevention (CDC) were based on studies by Dressler et al., J. Infect. Dis. 1993; 167:392-400, Engstrom et al., and J. Clin. Microbiol. 1995; 33:419-27. According to the CDC, a positive IgM Western blot required identification of any two of the following bands: 23, 39, or 41 kilodaltons and a positive IgG Western blot required identification of any five of the following ten bands: 18, 21, 28, 30, 39, 41, 45, 58, 66 or 93 kilodaltons. Either IgG or IgM Western blots were recommended by the CDC for diagnosis of early Lyme Disease, whereas only IgG Western blotting is recommended for diagnosis of late Lyme Disease.
After the CDC proposed the above criteria, problems with both sensitivity and specificity of the test results were appreciated. A study by Aguero-Rosenfeld et al., J. Clin. Microbiol. 1996; 34:1-9 evaluated the serologic response of patients with culture-confirmed early Lyme Disease using the CDC criteria and found that only 43% had a positive IgM Western blot and only 22% had a positive IgG Western blot by these standards. If CDC guidelines are followed, then failure to confirm screening tests by IgG or IgM Western blot will likely result in undertreating some patients who have Lyme Disease. In addition, the current criteria fail to consider the influence of the pre-test likelihood of disease on the interpretation of the test results. It can be predicted that Western blot testing will yield some false positive results when applied to a population where Lyme Disease is uncommon or when typical signs and symptoms are lacking. Sivak et al., Arch. Intern. Med. 1996; 156:2105-9, recently published a Bayesian analysis of IgM Western blotting for the diagnosis of early Lyme Disease and concluded that the accuracy would be poor in patients with minimal clinical evidence for Lyme Disease. Based on the currently available technology and using CDC criteria for interpretation, patients will continue to be undertreated and overtreated for Lyme Disease.
In a presentation by Porwancher, an incorrect algorithm was employed which led to an erroneous estimation of the probability of disease. (Thirty-fifth Annual Meeting, Infectious Disease Society of America, San Francisco, Sep. 14, 1997 (Abstract 247), see Clinical Infectious Diseases August, 1997.)
Given the above background, there remains a need for improved methods for diagnosing Lyme Disease. The present invention provides a method of interpreting Western blot results by combining the pre-test likelihood of dise

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