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Confirmation of active Lyme disease
Monitoring Lyme disease treatment
PCR testing should be limited to patients with a positive, or at least an equivocal, serologic test for antibody to Borrelia burgdorferi.
Lyme disease is a multisystem and multistage infection caused by 3 species of tick-borne spirochetes in the Borrelia burgdorferi sensu lato genogroup. These spirochetes include Borrelia burgdorferi sensu stricto (North America and Western Europe), Borrelia afzelii (Central and Western Europe and Russia), and Borrelia garinii (Europe, Russia, and northern Asia). Endemic areas for Lyme disease in the United States correspond with the distribution of 2 tick species, Ixodes dammini (Northeastern and Upper Midwestern US) and Ixodes pacificus (West Coast US). In Europe, Ixodes ricinus transmits the spirochete.
Lyme disease exhibits a variety of symptoms that may be confused with immune and inflammatory disorders. Inflammation around the tick bite causes skin lesions. Erythema chronicum migrans (ECM), a unique expanding skin lesion with central clearing resulting in a ring-like appearance, is the first stage of the disease. Any of the following clinical manifestations may be present in patients with Lyme disease: arthritis, neurological disease, cardiac disease, or skin lesions. Neurologic and cardiac symptoms may appear with stage 2 and arthritic symptoms with stage 3 of Lyme disease. In some cases, a definitive distinction between stages is not always seen. Further, secondary symptoms may occur even though the patient does not recall a tick bite or a rash.
Early antibiotic treatment of Lyme disease can resolve clinical symptoms and prevent progression of the disease to later stages. Treatment with penicillin, tetracycline, erythromycin, chloramphenicol, or ceftriaxone is considered appropriate therapy.
Serology is currently the diagnostic method of choice for Lyme disease. The Second National Conference on the Serologic Diagnosis of Lyme Disease (1994) recommended that laboratories use a 2-test approach for the serologic diagnosis of Lyme disease. Accordingly, specimens are first tested by the more sensitive EIA or enzyme-linked immunosorbent assay (ELISA). A Western blot (WB) assay is used to confirm positive Lyme EIA or ELISA results due to the presence of IgG- or IgM-class antibodies. WB identifies the specific proteins to which the patient's antibodies bind. Although there are no proteins that specifically diagnose Borrelia burgdorferi infection, the number of proteins recognized in the WB assay is correlated with diagnosis.
Since serology may not be positive until 2 to 4 weeks after onset of ECM, direct detection of Borrelia burgdorferi-specific target DNA sequences using PCR is a promising adjunct to existing diagnostic tests. PCR has shown utility for detection of Borrelia burgdorferi DNA from skin biopsies of ECM lesions, and from synovial and cerebrospinal fluid in late-stage disease. Borrelia burgdorferi DNA can also, rarely, be detected from blood, but is not the test of choice from this source.
A positive result indicates the presence of DNA from Borrelia burgdorferi, the agent of Lyme disease.
A negative result indicates the absence of detectable DNA from Borrelia burgdorferi in the specimen. Due to the diagnostic sensitivity limitations of the PCR assay, a negative result does not preclude the presence of the organism or active Lyme disease.
Serologic tests are recommended for diagnosis of Lyme disease. PCR may play an adjunctive role, but may not detect Borrelia burgdorferi DNA from blood in cases of active or chronic disease. A negative result does not rule-out Lyme disease, since inhibitory substances may be present in the specimen and the assay has limited diagnostic sensitivity when testing certain types of specimens. If clinical features of illness are highly indicative of Lyme neuroborreliosis, serologic testing on cerebrospinal fluid is warranted. Patients with active infection due to Borrelia afzelii or Borrelia garinii may have positive results from this PCR test, which will be reported as atypical gene sequence and prompt additional testing. PCR test results should be used as an aid in diagnosis and not considered diagnostic by themselves. These results should be correlated with serologic and epidemiologic data and clinical presentation of the patient. Concurrent infections with multiple tick-borne pathogens, including Ehrlichia chaffeensis/Anaplasma phagocytophilum and Babesia microti have been reported in United States.
The following validation data supports the use of this assay for clinical testing.
Accuracy/Diagnostic Sensitivity and Specificity:
Results from this real-time PCR assay on the LightCycler (LC PCR) directed to the plasminogen-binding protein (pbp) were compared to those generated using conventional PCR (target ospA gene) for synovial fluid (82), whole blood (22), and cerebrospinal fluid (CSF) (85). Using the conventional PCR as the gold standard, the diagnostic sensitivity and specificity for detection of Borrelia burgdorferi was as follows: synovial fluid (98.1%; 100%), whole blood (100%; 100%), and CSF (80%; 100%).
In the original spiking studies, fresh and paraffin tissue, synovial fluid, CSF, and whole blood were spiked with Borrelia plasmid at an approximate concentration of 100 targets/mcL. At 100 targets/mcL, 100% of fresh and paraffin tissue, synovial fluid, and CSF were positive and 92% of whole blood samples were positive. In additional spiking studies, CSF (40), fresh tissue (60), and whole blood (40) negative samples were tested by spiking half with positive-control plasmid at the limit of detection (25-50 targets/mcL). The samples were extracted and tested in a blinded fashion; 97% of spiked CSF, 100% of spiked tissue, and 91% of spiked whole blood were positive and 100% of the nonspiked specimens were negative.
Analytical Sensitivity/Limit of Detection (LoD):
The lower limit of detection (LoD) is approximately 1,000 genomic targets/mcL whole blood.
No PCR signal was obtained from the extracts of 22 bacterial, viral, parasitic, and fungal isolates that can cause symptoms similar to Lyme disease including; Rickettsia rickettsii, Rickettsia typhi, Ehrlichia canis, Babesia microti, Plasmodium falciparum, Plasmodium vivax, Bartonella henselae, Bartonella quintana, Herpes simplex virus, and Toxoplasma gondii.
Inter-assay precision was 100% and intra-assay precision was 100%.
Although the reference range is "Negative" for this assay, it may detect low-grade asymptomatic bacteremia from individuals exposed to Lyme-endemic areas. However, this assay is only to be used for patients with a clinical history and symptoms consistent with Lyme, and must be interpreted in the context of serologic tests, which are the gold standard for diagnosis of Lyme disease. This test is not used to screen asymptomatic patients.
This is a qualitative assay, and the results are reported as negative or positive for targeted Borrelia burgdorferi.
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2. Nocton JJ, Bloom BJ, Rutledge BJ, et al: Detection of Borrelia burgdorferi DNA by polymerase chain reaction in cerebrospinal fluid with Lyme neuroborreliosis. J Infect Dis 1996;174:623-627
3. Nocton JJ, Dressler F, Rutledge BJ, et al: Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis. N Engl J Med 1994;330:229-234
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6. Babady NE, Sloan LM, Vetter EA, et al: Percent positive rate of Lyme real-time polymerase chain reaction in blood, cerebrospinal fluid, synovial fluid, and tissue. Diagn Microbiol Infect Dis 2008;62(4):464-466