Testing for Lyme disease in humans

by Nathan Wei, MD, FACP, FACR

Nathan Wei is a nationally known board-certified rheumatologist and author of the Second Opinion Arthritis Treatment Kit. It's available exclusively at this website... not available in stores.

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From the Massachusetts State Health Department...

Lyme disease is an infection caused by the corkscrew-shaped bacteria Borrelia burgdorferi that are transmitted to humans by the bite of infected deer ticks (Ixodes scapularis) in the northeastern United States and western black-legged ticks (Ixodes pacificus) on the Pacific Coast.

The infection can cause serious long-term musculoskeletal, cardiac, and/or neurologic problems if not recognized and treated early.

Lyme disease is the most commonly reported vector-borne disease in the United States, accounting for more than 95% of all reported cases. A table of reported Lyme disease cases nationwide by state for 1990-2000 can be found at http://www.cdc.gov/ncidod/dvbid/lyme/ldcases90-99.htm. In 2000,

The reported incidence of Lyme disease is greatest in the Northeast, mid-Atlantic, and upper-Midwest regions of the United States.

While a diagnosis of Lyme disease is based on clinical signs and symptoms, laboratory testing may also be done. Laboratory confirmation of infection with B. burgdorferi is established when a laboratory isolates the spirochete from tissue or body fluid, detects diagnostic levels of IgM or IgG antibodies to the spirochete in serum or CSF, or detects a significant change in antibody levels in paired acute and convalescent serum samples. Serological assays remain the most common and practical method. Since the immune response to spirochetes is relatively slow, serological tests often remain negative for several weeks after exposure. The CDC recommends that, initially, serum specimens be tested by a sensitive test such as an enzyme immunoassay (EIA) or immunofluorescent assay (IFA). Samples with positive or equivocal results from these tests should be re-tested using a standardized Western blot procedure. The two-step algorithm, as opposed to using a single test, increases the specificity of laboratory testing.

From the National Institutes of Health and the National Institute of Allergy and Infectious Diseases (NIAID)...

All would agree that the successful management and treatment of any infectious disease – including Lyme disease – rests mainly upon early and accurate diagnosis. However, since the tests now being used for the diagnosis of Lyme disease are not as sensitive and specific as one would like, their results must be viewed with some reservations. Obviously, the only way one can be absolutely sure that a disease is in fact caused by a particular infectious agent is to isolate and culture that agent from the blood or tissues of those suspected of having the disease. In most cases, it is not possible to isolate and culture Borrelia burgdorferi, the spirochete that causes Lyme disease, from patients suspected of having Lyme disease. Therefore, one is forced to rely upon indirect serological tests (e.g., Western blot and ELISA assays) that detect the presence of serum antibodies specific for the antigens against B. burgdorferi. The presence of such antibodies, which are likely to persist for long periods of time even after infection has been eliminated after successful antibiotic therapy, is presumptive and does not necessarily prove that an individual is actively infected. To be sure, the polymerase chain reaction (PCR) is an extremely sensitive laboratory test that is capable of detecting very few molecules of bacterial DNA. However, the numbers of Borrelia likely to be present -- if at all -- in patients suspected of having Lyme disease are too small to generate sufficient amounts of bacterial DNA to be detected by this procedure. Western blot and ELISA assays are the two most widely used serological tests for the diagnosis of Lyme disease. Although these tests have been improved considerably during the past few years, they are not quantitative and do not enable one to assess the severity of an infection. Furthermore, they have problems related to standardization and reproducibility of the results obtained, not only within the same laboratory but also between different laboratories. Both tests can yield a high number of false-positive – or false-negative – results, depending on the particular patient population considered. For example, if these tests are done on populations where the likelihood of having Lyme disease is low, the probability of obtaining a false-positive result is greater than that of obtaining an unequivocal true positive result. Although an individual may be positive for both tests, others may be positive for one – but not the other – test, and individuals with clinical symptoms associated with Lyme disease may be negative for both tests. Neither the Western blot nor the ELISA tests are sufficiently quantitative to enable one to monitor and evaluate the efficacy of antibiotic therapy during the course of treatment. Because of these and other considerations, some have expressed the view that Lyme disease is either under or over diagnosed and treated, and that current estimates of the incidence of this disease in the United States are inaccurate. Consequently, a history of having had a deer tick bite, followed by the characteristic "bulls eye" lesion (erythema migrans rash) with flu-like symptoms is considered to be the most reliable diagnostic indicator of Lyme disease; such a history is sufficient to justify antibiotic therapy in the absence of further serological tests, since only about 30 percent of such individuals would usually be seropositive by Western blot and ELISA assays.

The development of a rapid, sensitive and specific diagnostic test to distinguish those who were infected in the past but are no longer infected from those who are now actively infected would be a significant advance for many infectious diseases, including Lyme disease. Such a test is needed, especially for patients with those symptoms (e.g., fatigue, muscular or neurological aches and pains, cognitive difficulties) that are usually associated with chronic Lyme disease, but which are not unique for Lyme disease and thus could be attributed to a variety of other illnesses or medical conditions. The availability of a test that is both sensitive and indicative of active infection with B. burgdorferi also would enable one to identify those patients who would benefit from antibiotic therapy, as well as to judge the effectiveness of such therapy on the resolution of infection. Since the genome of B. burgdorferi has now been sequenced, the application of novel approaches, such as microarray technology and proteomics, to address this issue holds much promise, and NIAID is now encouraging and supporting basic research in that regard. NIAID also is supporting research to improve the sensitivity and specificity of existing diagnostic methods, as well as to identify virulence-associated antigens generated during the course of infection. The latter would have great potential for use in the development of new diagnostic procedures and may also serve as indicators of active infection.

Until better tests are available, the diagnosis of Lyme disease must be based on characteristic clinical findings in which the results of laboratory tests play a supportive role. Consequently, primary care physicians in endemic areas must be familiar with all aspects of this disease and understand both the characteristics and limitations of laboratory tests commonly used for the diagnosis of Lyme disease. The American College of Physicians has published a position paper evaluating the limitations and performance of various laboratory tests now being used for the diagnosis of Lyme disease. Several mechanisms also are available to instruct both the medical community and the general public on the prevention, diagnosis, and treatment of Lyme disease. These include NIH- and CDC-sponsored Web sites and publications, as well as Web sites, publications, and newsletters developed by local public health agencies as well as patient advocate organizations.

Both the CDC and the Infectious Disease Society of America (IDSA) have published specific guidelines, which are updated periodically, on the diagnosis and treatment of Lyme disease. Treatment indications for previously untreated acute or chronic disease are relatively straightforward and standards of care exist. The most current standard of care guidelines have been prepared and published by the IDSA. Treatment standards for individuals who remain symptomatic after completing a generally accepted standard course of antibiotic therapy for either acute or chronic Lyme disease have not been defined. In the absence of a consensus on the treatment of such individuals, NIAID initiated both intramural and extramural clinical treatment trials on Chronic Lyme. The intramural clinical study of Lyme disease is assembling a well-characterized cohort of patients with chronic Lyme disease and relevant controls. Multiple research projects are being developed based on the findings derived from these studies. Ongoing research on the development of new or improved diagnostic tests for Lyme disease include: a new PCR and a RT-PCR assay for Borrelia; evaluation of a new culture medium for the isolation of Borrelia from blood and/or tissue specimens; research on the basis for variability of the Western blot and ELISA assays, as well as the use of well-defined and specific recombinant protein antigens in such assays; collaborative studies on the application of a newly developed ELISA test; research on the application of the Western blot assay for use with cerebrospinal fluid; and evaluation of the use of specific immune complexes testing for the diagnosis and treatment of chronic Lyme disease.

The National Institute of Allergy and Infectious Diseases (NIAID), in collaboration with the CDC and the Office of Rare Diseases, sponsored a conference on the laboratory diagnosis of Lyme disease in August 1998. At this conference, the strengths and weaknesses of all laboratory tests routinely used for the diagnosis of Lyme disease were evaluated and examined in great detail. Many recommendations were made as to how diagnosis could be improved and some of these now are in the process of being implemented. NIAID, in collaboration with the CDC, plays a major role in encouraging the development of novel approaches to improve the diagnosis of Lyme disease in the presence of co-infecting agents (e.g., Babesia microti, and Ehrlichia species) that could comlicate or interfere with diagnosis, as well as in individuals previously immunized with the recently licensed LYMErix(R) vaccine for Lyme disease. A significant development is the use of a synthetic peptide, composed of 26 amino acid residues derived from a variable surface antigen (VlsE) of B. burgdorferi, in a new, rapid, and extremely sensitive ELISA test for the diagnosis of Lyme disease. Since this test does not detect antibodies specific for Osp-A, it can be used even in those who have been immunized with the recently licensed Osp-A-based LYMErix(R) vaccine.

In 1996, the National Institute of Allergy and Infectious Diseases (NIAID) awarded a five-year contract to the New England Medical Center (NEMC), Boston, MA, to study how chronic Lyme disease develops and how to improve its treatment. The proposed study included an evaluation of old and new tests used to diagnose and manage Lyme disease and other tick-borne diseases. The investigators included this evaluation because diagnostic tests for Lyme disease have been controversial due to problems with their ability to detect evidence of the Lyme bacterium and to distinguish it from closely related bacteria; the lack of standardized materials used to perform the tests; and variation within and among laboratories performing the tests.

In the February 15 issue of the American Journal of Medicine, Mark S. Klempner, M.D., of NEMC and his scientific collaborators in New York, Connecticut and Washington, D.C., report their findings about the reliability of two Lyme disease tests: an IgG Western blot blood test and the Lyme urine antigen test, or LUAT. The IgG Western blot is a licensed test used to screen blood samples for antibodies to the Lyme bacterium, Borrelia burgdorferi. The LUAT detects proteins derived from the bacterium in urine samples. Although the LUAT has not been approved by the Food and Drug Administration as a valid diagnostic test for Lyme disease, it is widely used, and the NIAID Lyme Disease Advisory Panel asked that it be further evaluated.

To perform the IgG Western blot, the investigators collected blood samples from 21 patients who met the case definition of Lyme disease established by the Centers for Disease Control and Prevention (CDC). All patients had been treated for acute Lyme disease symptoms with recommended antibiotics, but they had continued to suffer symptoms of fatigue, muscle and joint pains, or neurocognitive problems for at least six months thereafter. These 21 blood samples were analyzed and compared with those from 10 healthy volunteers who had no history or symptoms of Lyme disease.

Testing was performed at NEMC, a laboratory recognized by CDC as qualified to do so. In accordance with current recommendations, the IgG test was considered positive if it revealed 5 or more of the 10 bands (indicative of antibodies) deemed significant for Lyme disease.

Duplicate fractions of blood from the 21 patients with chronic Lyme disease symptoms were tested at separate times. The laboratory performing the test was not aware of the results from the previous test. On initial testing, two-thirds (14/21) of the patient samples tested positive for Lyme disease by standard IgG Western blot criteria; the remainder tested negative. The same results were obtained when the duplicate fractions of the same 21 samples were tested. All samples from the same 10 healthy volunteers tested negative.

The LUAT was performed by the test’s manufacturer (IgeneX, Palo Alto, CA) on urine samples taken from 10 healthy volunteers who had no history or symptoms of Lyme disease. Each urine sample was divided into five equal fractions, or replicates, so that a total of 50 individual LUATs were performed. The LUAT results, unlike the highly reproducible IgG Western blot results, varied markedly. At least one urine fraction from each of the 10 samples examined tested false-positive. Two urine samples consistently showed false-positive results. Replicates for the eight remaining samples examined were a mixture of positive and negative values, making it impossible to conclude that they were positive or negative.

The study results indicate that the IgG Western blot is a highly reproducible test for detecting antibodies to the Lyme disease bacterium. These antibodies indicate prior exposure to B. burgdorferi. In contrast, the variable results obtained with the LUAT indicate that it is not a reliable test for evaluating patients with active or suspected Lyme disease.

Although not part of the published report, the investigators also examined the reliability of standard tests for three other tick-borne diseases -- tick-borne encephalitis, babesiosis and ehrlichiosis -- and found all three to be highly reproducible when conducted in experienced laboratories.

Reference: MS Klempner et al. Intralaboratory reliability of serologic and urine testing for Lyme disease. American Journal of Medicine 110(3): 217-19 (2001).

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