Specimen Collection, Transport, and Processing
Peripheral blood is the specimen of choice for direct detection of borreliae that cause relapsing fever. Borrelia burgdorferi can be visualized and cultured, although serology is considered the best means to diagnose Lyme disease. Specimens submitted for stain or culture include blood, biopsy specimens, and body fluids including joint and cerebrospinal fluids. Body fluids should be transported without any preservatives. Tissue biopsy specimens should be placed in sterile saline to prevent drying.
Direct Detection Methods
Relapsing Fever. Clinical laboratories rely on direct observation of the organism in peripheral blood from patients for diagnosis. Organisms can be found in 70% of cases when blood specimens from febrile patients are examined. The organisms can be seen directly in wet preparations of peripheral blood (mixed with equal parts of sterile, nonbacteriostatic saline) under dark- or bright field illumination, in which the spirochetes move rapidly, often pushing the red blood cells around. The organisms may also be visualized by staining thick and thin films with Wright’s or Giemsa stains using procedures similar to those used to detect malaria.
Lyme Disease. B. burgdorferi may be visualized in tissue sections stained with Warthin-Starry silver stain. In general, the number of spirochetes in blood of patients with Lyme borreliosis is below the lower limits of microscopic detection. Polymerase chain reaction (PCR) has become important in diagnosing Lyme disease. PCR has detected B. burgdorferi DNA in clinical specimens from patients with early and late clinical manifestations; optimal specimens include urine, synovial tissue, synovial fluid, and skin biopsies from patients with EM. Laboratories have used a variety of molecular methods to increase sensitivity and specificity and decrease turnaround time for diagnosing Lyme borreliosis. PCR has confirmed EM with an overall sensitivity and specificity of 68% and 100%, respectively. The ability to detect spirochetes in blood or plasma by PCR is dependent on the stage of illness (from 40% of patients with secondary EM to only 9.5% of patients with primary EM); PCR also does relatively well in detecting B. burgdorferi sensu lato in synovial fluids. In contrast, variable results using PCR have been achieved in cerebrospinal fluid (CSF) specimens obtained from patients with peripheral or central nervous system involvement with Lyme borreliosis; overall sensitivity is only in the range of 20%.
Cultivation
Although the organisms that cause relapsing fever can be cultured in nutritionally rich media under microaerobic conditions, the procedures are cumbersome and unreliable and are used primarily as research tools. Similarly, the culture of B. burgdorferi may be attempted, although the yield is low. The best specimens for culture in untreated patients include the peripheral area of the EM ring lesion or synovial tissue. CSF and blood or plasma (greater than 9 mL) in general are of low diagnostic yield by culture or PCR. This seems to correlate with the duration of the neurologic disease—in other words, positive results decrease as the duration of the disease increases. To cultivate the organism, the plasma, spinal fluid sediment, or macerated tissue biopsy is inoculated into a tube of modified Kelly’s medium (BSK II, BSK-H, or Preac-Mursic) and incubated at 30° to 34° C for up to 12 weeks under microaerophilic conditions. Blind subcultures (0.1 mL) are performed weekly from the lower portion of the broth to fresh media, and the cultures are examined by dark-field microscopy or by fluorescence microscopy after staining with acridine orange for the presence of spirochetes. Because of the long incubation time and low sensitivity associated with cultivation, cultivation is often confined to reference or research laboratories.
Serodiagnosis
Relapsing Fever. Serologic tests for relapsing fever have not demonstrated reproducible or reliable data for diagnosis because of the many antigenic shifts Borrelia organ isms undergo during the course of disease. Protein heterogeneity in strains of different species is quite variable. For example, the OspC protein has 21 major recognized antigenic types. In addition, patients may exhibit increased titers to Proteus OX K antigens (up to 1 : 80), but other cross-reacting antibodies are rare. Certain reference laboratories, such as those at the Centers for Disease Control and Prevention (CDC), may perform special serologic procedures on sera from selected patients.
Lyme Disease. Despite its inadequacies, serology continues to be the standard for the diagnosis of Lyme disease. B. burgdorferi has numerous immunogenic lipids, proteins, lipoproteins, and carbohydrate antigens on its surface and outer membrane. The earliest antibody response and development of IgM is in response to the OspC membrane protein, the flagellar antigens (FlaA and FlaB), or the fibronectin binding protein (BBK32). The IgM levels peak within several weeks but may be detectable for several months. The IgG response devel ops slowly during the first several weeks of disease and increases with antibody responses to Osp17 (decorin binding protein) and additional proteins including p39 (BmpA) and p58. The late-stage infection demonstrates IgG antibodies to numerous antigens.
Numerous serologic tests are commercially available; however, these tests have not yet been standardized, and their performance characteristics vary greatly. The most common of these tests are the indirect immunofluorescence assay (IFA), the enzyme-linked immunosorbent assay (ELISA), and Western blot. Measuring antibody by enzyme-linked immunosorbent assay (ELISA) is the primary screening method because it is quick, reproducible, and relatively inexpensive. However, false-positive rates are high, mainly as a result of cross-reactivity. The specificity of IFA may be improved by adsorption of serum with Treponema phagedenis sonicate (IFA-ABS). Patients with syphilis, HIV infection, leptospirosis, mononucleosis, parvovirus infection, rheumatoid arthritis, and other autoimmune diseases commonly show positive results. Capture EIAs have been developed to avoid false positive reactions with rheumatoid factor. In addition, this may be overcome by pretreatment of the patient’s sera with anti-IgG. For the United States, the CDC recommends a two-step approach to the serologic diagnosis of Lyme disease. The first step is to use a sensitive screening test such as an ELISA or IFA; if this test is positive or equivocal, the result must be confirmed by immunoblot ting (Figure 1). In certain clinical situations, results of serologic tests must be interpreted with caution. For example, patients with Lyme arthritis frequently remain antibody-positive despite treatment but do not necessarily have persistent infection. Conversely, patients with a localized EM may be seronegative. Because of these limitations and others, the Food and Drug Administration (FDA) and the American College of Physicians have published guidelines regarding the use of laboratory tests for Lyme disease diagnosis. Of paramount importance is the clinician’s determination before ordering serologic tests of the pretest probability of Lyme disease based on clinical symptoms and the incidence of Lyme disease in the population represented by the patient.
Fig1. Two-step serodiagnostic procedure. *Note: If neuroborreliosis is suspected, a paired sera and CSF specimen is recommended for testing. Any disease of short duration. For Lyme disease, it is recommended that a follow-up serologic test be performed at a later date. (Modified from Versalovic J: Manual of clinical microbiology, ed 10, Washington, DC, 2011, ASM Press.)
Molecular Diagnostics
A variety of molecular methods have been developed for the diagnosis of borreliae infections. These methods have been used on blood, body fluids, and tissue specimens. Nucleic acid based methods are typically not avail able in routine clinical laboratories.
