Lab Tests Lack Sensitivity
By Lorraine Johnson, J.D., MBA, Executive Director
California Lyme Disease Association
National Institute of Allergies and Infectious Diseasesi :Lyme disease can be difficult to diagnose, especially in the later stages of infection when an individual's antibodies can fall to very low levels. National Institute of Allergies and Infectious Diseases (National Institute of Health), NIAID Collaboration Yields New Test for Lyme Disease. NIH News Advisory, (June 18, 2001).
The Food and Drug Administration:A negative result indicates only that there was no serologic evidence of infection with B. burgdorferi. It should not be used as the basis for excluding B. burgdorferi as the cause of illness, especially if the blood was collected within 2 weeks of when symptoms began. A negative result should not be used as the sole basis for excluding B. burgdorferi as the cause of illness…. Many patients with active or recent infectionsi do not have detectable anti-Bb in a single specimen. This happens because such antibodies often develop after manifestations of early infection or because detectable anti-Bb may diminish or never develop in patients treated with antibioticsi . Food & Drug Administration, Lyme disease test kits: potential for misdiagnosis. FDA Medical Bulletin, 1999, Summer, Final Issue.
Lange, R. and S. Seyyedi (2002). “Evidence of a Lyme borreliosis infection from the viewpoint of laboratory medicine.” Int J Med Microbiol 291 Suppl 33: 120-4.
More than 20 years after the first description of the pathogen Borrelia burgdorferi, the diagnosis of Lyme borreliosis is based primarily on clinical criteria. Multiple differential diagnoses have to be considered and laboratory confirmation based on various test procedures is required until now. For screening assays the immunofluorescence assay (IFA), enzyme linked immunoassay (EIA), or indirect hemagglutination assay (IHA) are frequently in use. During the last ten years a stepwise diagnostic procedure in USA and Europe has been recommended. The use of a serological screening assay as the first diagnostic step, followed by a confirmatory assay only in the case of a positive or borderline result of the screening assay is now “state of the art.” The most frequently used method is a sensitive enzyme linked immunoassay followed by a sensitive and specific immunoblot technique. However, the quality of these indirect serological tests and, most importantly, the interpretation criteria of the commercial tests vary dramatically. The choice of Borrelia strains, antigen preparation, production conditions and test procedures vary widely. The immunoblot, used as a confirmatory assay, must meet the highest quality standards but in practice under routine laboratory conditions, these levels are often not reached. The major reason for this "shortcoming" is that Lyme borreliosis is not a sexually transmitted disease and no official approval has been required by the commercial suppliers so far. Another increasingly important aspect is the introduction of molecular biological assays into laboratory routines. Numerous authors published many articles about the usefulness of polymerase chain reaction as a diagnostic tool for the detection of borreliae in human specimens. However, a routine method has not yet been established. Furthermore, no standardized method for DNA isolation from different human specimens has been developed so far. For this reason PCRi is not accepted as a routine method and the application should be restricted to a few laboratories with experience in the field of Lyme borreliosis. In summary, it is important to inform clinicians about the limitations of the tests used and it would be very helpful to stage a European or worldwide conference to establish accepted rules for the diagnosis of Lyme borreliosis. Aguero-Rosenfeld, M. E., J. Nowakowski, et al. (1993). "Serodiagnosis in early Lyme disease." J Clin Microbiol 31(12): 3090-5.Using a commercially available enzyme-linked immunosorbent assay (ELISA) and an immunoblot assay (IB), we tested sera from 100 patients with erythema migrans (EM) seen in 1991 at the Westchester County Medical Center Lyme Disease Diagnostic Center. Convalescent-phase sera were available from 59 patients. Fifty-five patients had EM of <7 days’ duration, 31 had EM of 7 to 14 days’duration, and 14 had EM of >14 days’ duration. During the acute phase of infection, 35 patients had a positive ELISA result and 43 had a positive IB result by the recently published criteria of Dressler et al…. for interpretation of IB in patients with Lyme disease. A greater sensitivity of IB was observed in patients with EM of <7 days’ duration, as follows: 14 of 55 (25%) for IB versus 7 of 55 (13%) for ELISA (P = 0.144). Sera of all 14 patients with EM of >14 days’ duration were reactive by both tests, as follows: 13 positive and 1 equivocal by ELISA and 12 positive and 2 indeterminate by the IB. The band reactivity most frequently observed in the IB was to the 41- and 25-kDa antigens, the latter being the most frequent band observed in immunoglobulin M blots. Seroconversion was observed in 74 and 64% of evaluable patients by ELISA and IB, respectively, despite the use of antibiotic therapy. | TABLE 4. ELISA and IB results for 59 patients with EM during acute and convalescent phases |
| Test and acute-phase result | No. of patients | No. (%) of serum specimens in convalescent phase |
| ELISA | | Negative | ELISA equivocal or IB indeterminate | Positive |
| Negative Equivocal Positive | 31
7
21 | 8 (26)
0
0 | 7 (23)
2 (29)
0 | 16 (51)
5 (71)
21 (100) |
| IB | | | | |
| Negative Indeterminate Positive | 22
11
26 | 7 (32)
1 (9)
0 | 6 (27)
4 (36)
6 (23) | 9 (41)
6 (55)
20 (77) |
Coulter, P., C. Lema, et al. (2005). “Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease.” J. Clin. Microbiology 43(10): 5080-84.Lyme disease is usually diagnosed and treated based on clinical manifestations. However, laboratory testing is useful for patients with confusing presentations and for validation of disease in clinical studies. Although cultivation of Borrelia burgdorferi is definitive, prior investigations have shown that no single test is optimal for Lyme disease diagnosis. We applied high-volume blood culture, skin biopsy culture, PCR, and serodiagnosis to a cohort of patients with suspected Lyme disease acquired in Maryland and southern Pennsylvania. The study was performed to confirm the relative utility of culture and to identify laboratory-testing algorithms that will supplement clinical diagnosis. Overall, 30 of 86 patients (35%) were culture positive, whereas an additional 15 of 84 (18%) were seropositive only (51% total sero- and culture positive), and PCR on skin biopsy identified 4 additional patients who were neither culture nor seropositive. Among 49 laboratory test-positive patients, the highest sensitivity (100%) for diagnosis was obtained when culture, skin PCR, and serologic tests were used, although serologic testing with skin PCR was almost as sensitive (92%). Plasma PCR was infrequently positive and provided no additional diagnostic value. Although culture is definitive and has a relatively high sensitivity, the results required a mean of 3.5 weeks to recovery. The combination of acute-phase serologyi and skin PCR was 75% sensitive, offering a practical and relatively rapid alternative for confirming clinical impression. The full battery of tests could be useful for patients with confusing clinical signs or for providing strong laboratory support for clinical studies of Lyme disease.Excerpt: | TABLE 1. Agreement of laboratory diagnostic tests with initial clinical assessment for Lyme disease |
| Laboratory diagnostic test (no. tested) | Initial clinical assessment for Lyme disease [number for which test result agreed (%)] |
| | Probable | Probable or possible |
| Initial serologyi (80)Any serology (80) Skin biopsy culture (46) Plasma culture (79) Any culture (82)Any serology or any culture (82) Skin biopsy PCR (23) Plasma PCR (55)Any serology, any culture, or any PCR (82) | 32 (40)21 (26)10 (22)10 (13) 3 (4) 14 (17)9 (39)27 (49)18 (22) | 40 (50)55 (69)19 (41)46 (58)54 (66)63 (77)11 (48)15 (27)67 (82) |
Probable Lyme disease is EM or history of EM and a characteristic clinical syndrome with headache, VIIth cranial nerve palsy, or arthritis; possible Lyme disease is tick exposure, rash not typical for EM, or summertime fever without identifiable source.
Marangoni, A., M. Sparacino, et al. (2005). “Comparative evaluation of three different ELISA methods for the diagnosis of early culture-confirmed Lyme disease in Italy.” J Med Microbiol 54(Pt 4): 361-7. In this study the raising and development of the immunei response to Borrelia burgdorferi infection in 45 Italian patients suffering from culture-confirmed Lyme borreliosis erythema migrans was investigated. A total of 95 serially collected serum samples were tested by using three different commercial ELISAs: recomWell Borrelia (Mikrogen), Enzygnost Borreliosis (DADE Behring) and Quick ELISA C6 Borrelia (Immunetics). The sensitivities of the ELISAs were as follows: Enzygnost Borreliosis IgM, 70.5 %; Quick ELISA C6 Borrelia, 62.1 %; recomWell Borrelia IgM, 55.7 %; recomWell Borrelia IgGi , 57.9 %; and Enzygnost Borreliosis IgG, 36.8 %. In order to compare the specificity values of the three ELISAs, a panel of sera obtained from blood donors (210 samples coming from a non-endemic area and 24 samples from an endemic area) was tested, as well as sera from patients suffering from some of the most common biological conditions that could result in false-positive reactivity in Lyme disease serology (n = 40). RecomWell Borrelia IgG and recomWell Borrelia IgM were the most specific (97.1 % and 98.9 %, respectively), followed by Quick ELISA C6 Borrelia (96.7 %). Enzygnost Borreliosis IgG and IgM achieved 90.1 % and 92.3 % specificity, respectively. Sera that gave discrepant results when tested by the three ELISAs were further analysed by Western blotting. Sivak, S. L., M. E. Aguero-Rosenfeld, et al. (1996). “Accuracy of IgM immunoblotting to confirm the clinical diagnosis of early Lyme disease.” Arch Intern Med 156(18): 2105-9.BACKGROUND: A 2-test approach for the serologic diagnosis of Lyme disease has recently been proposed. A positive or equivocal result on a first-stage test (e.g., an enzyme immunoassay) is followed by a Western immunoblot test. For a sample to be considered seropositive for Lyme disease, the immunoblot result must be positive. OBJECTIVES: To assess the accuracy of IgM immunoblotting for detection of early Lyme disease and to establish interpretative criteria for a commercially available immunoblot assay. METHODS: Serum samples from 44 patients with erythema migrans were tested by an IgM immunoblot assay. All patients were culture-positive for Borrelia burgdorferi. Serum samples from 2 different control groups were also tested. Interpretative criteria were developed using receiver operating characteristic curves. RESULTS: The presence of any 2 IgM bands was found to be the optimal criterion for a positive test result, and in patients with illness of less than 7 days' duration, this was significantly more sensitive than the criterion of any 2 of the 3 specific bands defined by the Centers for Disease Control and Prevention/Association of State and Territorial Public Health Laboratory Directors Lyme Disease Workgroup (P < .05). Specificity of the criterion of any 2 bands was 100% for 1 group of controls but only 96% for the more clinically relevant control group; this small difference had a large impact on the positive predictive value in populations at low risk for Lyme disease. CONCLUSIONS: Using a commercially available immunoblot test kit, the presence of any 2 IgM bands is proposed as a positive result. The predictive value of a positive IgM immunoblot result, however, is poor in patients with minimal clinical evidence for Lyme disease. California Lyme Disease Association – PO Box 1423 – Ukiah, CA 95482 – www.lymedisease.org - 2007
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Louise CFSi i ,CPNi i +/Bb+(Lyme) Cholestyramine 1-2 pks @ HS for Porphyriai i +fattyEndotoxins HS PRN, Wheldon CAPi i 6/07,all supps, Doxyi i 200QD, Roxi 300BID, Tinidazole 500 BIDx20day Pulses, VitD3-10,000IU,Iodoral25mg,SAM-e100mgQD+B-vits, Pyruvate3.75Gm at 1PM