With the microhematocrit technique, in which the cellular elements at the interface between plasma and packed erythrocytes are examined, the limit of detection might be as low as 10³ spirochetes (71).
...  By either phase-contrast or dark-field microscopy of live organisms or standard light microscopy of stained, fixed organisms, B. burgdorferi can usually be distinguished from other borreliae by its looser and more irregular coiling.
... Tinctorially, borreliae are gram negative. However, the Gram stain is not nearly as sensitive as Giemsa (39, 40) and silver (31, 58, 61) stains for demonstrating the organisms. Acridine orange was used to detect spirochetes in phagocytic cells (29) and in the CSF of a patient with Bannwarth's syndrome (36). This dye was also used to stain what appeared to be spirochetes in the urine of field mice (35). Warthin-Starry and modified Dieterle silver stains have been used to reveal the spirochetes in a variety of biopsy and autopsy materials that have been Formalin fixed and embedded in paraffin (31, 61, 62, 67, 99, 123, 130, 146). According to Duray and Johnson, a modified Dieterle stain is easier to perform than the Warthin-Starry stain (61). In various reports, silver stains have been used successfully to detect spirochetes in <1 to 100% of ECM lesion biopsies (31, 32, 63, 67, 130); for most investigators the success has been about 40 to 50%. The spirochetes are most easily found if the biopsy is taken from the advancing edge of the ECM lesion and the papillary dermis is examined. Usually fewer spirochetes are located in the center of the lesion and in the epidermis. A "positive control" slide prepared from B. burgdorferi cells suspended in an agar block should be included when biopsy material is examined by silver stains (50).
In exceptional cases, spirochetes have also been detected in synovial tissue biopsies with either the standard or modified Dieterle silver stain (63, 98). When seen, the numbers of spirochetes present were very low. The borreliae were seen within and close to small vessels displaying microangiopathic changes (98). A modification of the Steiner silver stain is reported to further improve the sensitivity of histologic detection (58). In this method the tissues are treated with amylase after they have been fixed in Formalin and before the immersion in silver nitrate. When compared with the Warthin-Starry stain, this method has been reported to provide greater contrast between the spirochetes and the background tissues. Using this modified Steiner stain (Bosma-Steiner), de Koning et al. reported 100% sensitivity in demonstrating spirochetes in skin biopsies of ECM patients and in synovial biopsies of Lyme arthritis patients (58).

Polyclonal antibodies have been used successfully in immunohistologic studies to demonstrate spirochetes in tissues (35, 102). However, with monoclonal antibodies not only are spirochete structures demonstrated, but also the particular type of spirochete can be determined (18, 20, 21). Using a monoclonal antibody to a borrelial flagellar antigen, Park et al. demonstrated spirochetes in a frozen section of skin biopsy from a patient with ECM (133). Monoclonal antibodies were also used by MacDonald and Miranda to reveal spirochetes in touch preparations of unfixed human brain tissues from an autopsy specimen (115), by Magnarelli et al. to identify B. burgdorferi in the kidney tissues of a dog with renal disease (118), and by Burgess et al. to show borreliae in organs of an infected cow (47). Direct and indirect immunofluorescence assays with antiborrelial antibodies have been used to determine the prevalence of infected ticks in different geographic areas (7, 43). Although this approach has proved useful in field studies, laboratory experiments with ticks have shown that some borreliae in the ticks may either not react at all with certain monoclonal antibodies or react more weakly than they usually do with polyclonal antisera (43, 110). This phenomenon suggests that antigenic variation occurs.

In Vitro Cultivation. Koch's postulates are partially fulfilled by isolating the offending organism from the affected patient; this has been done in several cases of Lyme borreliosis as described below. The culture medium is complex and expensive and has a short shelf life. Only a minority of cultures from definite cases of Lyme borreliosis yield spirochetes. Under these circumstances, B. burgdorferi cultivation can hardly be considered the diagnostic method of choice, but this approach remains the only way to confirm a diagnosis. Recovery of B. burgdorferi from a patient indicates an active or latent disease state and not simply an inconsequential colonization.

We first recovered a spirochete from I. dammini ticks by using Stoenner's version of Kelly's medium (39). This, or a closely related formulation, was then used to recover identical spirochetes from the blood, skin, and CSF of patients
with Lyme disease (26, 161). By additional modifications of Stoenner-Kelly medium to improve the buffering capacity and make preparation easier (BSK medium), we were able to isolate a borrelia from I. ricinus ticks of Europe and to grow B. burgdorferi from a single organism (15). This culture capability allowed us to clone the newly isolated spirochetes by limiting dilution. For details of the current medium formulation used in our laboratory, BSK II, see reference
11. Kanamycin and 5-fluorouracil have been added to BSK medium for the selective isolation of the spirochetes from ticks (96). Others have used neomycin, gentamicin, rifampin, or kanamycin alone to reduce contamination (9, 42, 43, 130, 161). We currently use rifampin (50 pug/ml) and phosphomycin (100 pg/ml) to prevent the growth of other bacteria (A. Barbour and A. MacDonald, unpublished results). ...

B. burgdorferi is grown at temperatures between 30 and 37ºC in the laboratory. [see also http://lymerick.net/Borrelia-growth-optimum.html]. At temperatures above 38ºC, borrelial growth slows substantially (11). Most investigators use temperatures of 32 to 34ºC. The cap or lid of the culture vessel is usually tight or sealed to prevent loss of carbon dioxide from the medium. The generation time is 8 to 24 h, and culture-adapted strains achieve cell densities of about 108 spirochetes per ml (11). The microaerophilic character of the borrelia is indicated by its preference for the bottom portion of the culture medium during initial growth (11, 92). Addition of low concentrations (0.1 to 0.2%) of agarose to further thicken the medium improves the recovery of B. burgdorferi from animal fluids and tissues (4, 6, 94, 95).
 ... Our early studies showed that B. burgdorferi would grow as a lawn on BSK medium containing 0.8% agarose (11). Kurtti and colleagues subsequently grew these organisms as isolated colonies by increasing the agarose concentration to 1.3% and doubling the amount of gelatin (109). The plates were incubated for 2 to 3 weeks in a candle jar. Using this solid medium, these investigators demonstrated growth of at least two different colony types of B. burgdorferi.
... B. burgdorferi can survive in citrated blood stored at 4ºC for 25 days (G. Baronton and I. Saint-Girons, Abstr. Int. Conf. Lyme Dis., abstr. no. 55, 16 Sept. 1987), but most cultures have been inoculated soon after blood collection. Citrated or heparinized blood is lightly centrifuged to separate the plasma from the cellular blood elements. The plasma is then centrifuged at a higher force, and the plasma pellet is suspended in growth medium. The plasma pellet should contain platelets as borrelia are usually found in the platelet-rich fraction of blood. Cells in the CSF have also been concentrated by centrifugation to improve the odds of recovery (136, 138, 139).

The frequency of recovery of borrelia from skin biopsies of ECM lesions has ranged from about 6 to 45% (8, 32, 139, 140, 160). Most biopsies yielding positive cultures have been taken from the expanding edge of the ECM lesion where histologic stains have shown that the spirochetes are in highest numbers. B. burgdorferi has also been isolated from skin biopsies of patients with acrodermatitis chronica atrophicans of several years duration (9, 130) and patients with lymphocytoma (86). A skin biopsy was stored frozen at -80'C for 2 years before successful recovery of borreliae in culture medium (130). The etiologic agent has been isolated only infrequently from affected joints (140, 153); synovial fluid cultures of humans and dogs with Lyme arthritis have usually been negative (83, 104, 160). Nevertheless, the ameliorating effect of antibiotics on Lyme arthritis strongly suggests that viable organisms are required for disease progression (64, 83, 159).

B. burgdorferi has been isolated from Ixodes spp. ticks (2, 7, 15, 39, 43, 96, 161). The culture success rate is close to prevalence of infection in the tick population as established by immunofluorescent detection. Although organisms have been isolated from whole ticks ground up and inoculated into culture medium, the midgut is the site most likely to contain cultivable spirochetes (39, 40, 43). Dissection of the midgut out of the tick reduces the chance of contamination of the cultures (15, 39). Preparing serial dilutions of the inoculated medium can also reduce the chance of contamination; enough spirochetes are usually present in the tick to permit up to 1,000-fold dilutions of the original inoculum (15). In some studies, antibiotics have been added to the medium to prevent overgrowth of bacteria colonizing the exterior and interior of the tick (43, 96).

Once in culture the borreliae may undergo change in one or more traits. Alteration in both the size of outer membrane proteins and the reactivity of these proteins with monoclonal antibodies has been seen after an isolate has been passaged as few as 10 to 20 times (70 to 140 generations) in the laboratory (20, 151, 176).

Cell-Mediated Immunity
Several studies have been carried out on the role of cell-mediated immunity in Lyme disease and, specifically, the interaction between B. burgdorferi cells and the cellular elements of the immune system. Although much of this work
does not have immediate relevance for clinical microbiology, a brief consideration of pertinent immunologic findings might provide useful background for understanding the disease and future developments.
The B. burgdorferi cells associate with macrophages and polymorphonuclear leukocytes even in the absence of immune serum (29, 135). Phagocytosis has been demonstrated under these conditions, but it is still unclear whether the association between the borreliae and the macrophages in this situation is entirely due to uptake of the spirochetes into the phagocyte. The spirochetes that are taken up may be those already damaged during the centrifugation and washing steps of cell preparation. Moreover, the finding that killed B. burgdorferi cells adhere to T- and B-cell lymphocytes suggests that the spirochete has binding sites on its surface for eucaryotic cell ligands (68). The addition of specific antiserum significantly increases the association of the spirochetes with macrophages and other phagocytic cells; this appears to be due to phagocytosis of the borreliae
(29, 135). The borreliae appear to be killed once inside a phagocytic cell (29). [killing rate not 100% PMID: 8423346] Uptake appears not to be mediated by heat-labile plasma components such as complement (29, 135).

Investigations of cell-mediated immunity showed a specific response by T cells of Lyme borreliosis patients to B. burgdorferi antigens (57, 124, 132, 152). This response has been demonstrated for T cells obtained from the peripheral blood, the diseased synovium, and the CSF. The specific proliferative response of T cells from either CSF or synovial fluid is greater than that of peripheral blood T cells obtained at the same time from a Lyme disease patient (132, 152). The greatest reactivity is at the site of the localized infection, be it the nervous system or a joint. Antigen-specific T-cell responses have also been demonstrated in the synovial fluids of patients with postinfectious Reiter's syndrome (66). T-cell clones that respond specifically to B. burgdorferi antigens have been recovered from the CSF of a patient with Lyme borreliosis (124).  
Patient's T cells were significantly stimulated by whole cells as well as soluble components of the spirochete (57, 132, 152). T-cell blastogenesis was more vigorous in the presence of whole borreliae than when a sonicated supernatant was used (R. J. Dattwyler, D. J. Volkman, J. Thomas, P. A. Falldorf, and M. G. Golightly, Ann. N.Y. Acad. Sci., in press). The active response to whole cells of B. burgdorferi indicates that patients are responding to cell surface components. The stimulation index of T-cell responsiveness to whole borreliae has been used at one institution to confirm the diagnosis of Lyme borreliosis (57). Some patients demonstrate a significant cell-mediated immune response to the borreliae when they have only borderline or slightly elevated antibody titers to the organisms (57). [probably due to formation of immune complexes that traps many/most/all formed antibodies which are becoming bound to antigen only whenever there are many Borreliae currently active]. Family members of Lyme disease patients have higher stimulation indices than unrelated controls, indicating either a hereditary predisposition or shared exposure to the infectious agent (152). The T-cell responses of patients show less apparent cross-reactivities than the corresponding antibody responses to relapsing fever borreliae antigens (152).

In affected tissues and organs, there is usually a prominent lymphocytic infiltration. Among T cells, there are more helper/inducer cells than cytotoxic/suppressor cells (57, 127, 152); this is also the case in joint fluids in rheumatoid arthritis patients (72). In the skin, large numbers of dendritic Langerhans cell have also been seen (37). In biopsies of ECM lesions, HLA-DR markers have been found on keratinocytes; patients acrodermatitis chronica atrophicans have both HLA-DR and HLA-DQ markers displayed on keratinocytes in their chronic skin lesions (172).

Infected humans produce IgM, IgG (14, 54, 82, 116, 161, 168, 178), and IgE antibodies (30) that recognize B. burgdorferi antigens. There does not seem to be a significant specific IgA response (82). The bulk of the IgG-reactive antibodies are of the IgG1 and IgG3 subclasses (B. Vandvik, Abstr. Int. Conf. Lyme Dis., abstr. no. 43, 16 Sept. 1987; K. E. Hechemy, H. L. Harris, and M. J. Duerr, Abstr. Int. Conf. Lyme Dis., abstr. no. 18, 14 Sept. 1987).

In the IFA [indirect immunofluorescent antibody], whole borrelial cells are invariably used. The cells are dried on the slide with or without yolk sac material. Our laboratory uses washed sheep erythrocytes mixed with the borreliae to evenly distribute the spirochetes on the smear and to provide a convenient reference point for microscope focusing (14, 21). Once dried on the slide, the spirochetes are fixed with methanol or acetone; some investigators freeze the slides without the organic solvent fixation step.

While many laboratories use the original Shelter Island, N.Y., isolate of B. burgdorferi, strain B31 (ATCC 35210), several other isolates are also used. This practice of using different strains may have little consequence for testing
within North America, where strains are very similar to one another in their antigenic makeup (12, 20, 43). On the other hand, European strains are more heterogeneous in the types of major outer membrane proteins they possess (Fig. 3) (19, 155, 176; B. Wilske, V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer, Ann. N.Y. Acad. Sci., in press). The differences between strains are not great enough to completely invalidate use of one strain, even a North American one, like B31, in all geographic areas. This strain has been used successfully in Europe for serologic testing (2). Nevertheless, a one- or two-tube difference between the reactivity of a particular immune serum against one strain versus another could, in some instances, mean that a sample could be called falsely negative. There is also the question whether continuous passage of the test strain could result in loss of certain critical antigens for IFA and ELISA. Changes in the major outer membrane proteins OspA and OspB (Fig. 3) have been noted during serial in vitro cultivation (20, 33, 151, l51a, 176; Wilske et al., in press).
 
False-positive IFA and ELISA reactions can occur in patients with syphilis or relapsing fever, two other spirochetal diseases (89, 116, 117). Lyme borreliosis patients occasionally have reactive fluorescent treponemal antibody absorption and treponemal agglutination tests for syphilis (89, 117, 128); in these as well as treponemal antibody negative cases, reagin antibody-assays, such as the Venereal Disease Research Laboratory and rapid plasma reagin tests, are negative. As demonstrated by IFA, syphilis, yaws, and pinta patients have had high titers of antibodies that react with B. burgdorferi (14, 117, 126). The immunofluorescence
reactions of sera from syphilis patients are qualitatively different from those seen with sera from Lyme disease patients. The cross-reactive antibodies of syphilis patients do not bind to the outer membrane blebs of the spirochete,
and, consequently, the stained spirochetes appear thinner and less irregular than fixed organisms bound by antibodies recognizing outer membrane antigens (14). In serologic tests, some Lyme disease patients have equivalent titers to B.
burgdorferi and B. hermsii, a relapsing fever agent in North America (117). Patients with tick-borne and louse-borne relapsing fever have cross-reactive antibodies to B. burgdorferi (117). Although this is a potential problem, the clinical
presentation and the epidemiologic features of the case would usually allow discrimination between Lyme borreliosis and relapsing fever. There is much less cross-reactivity between B. burgdorferi and the leptospires (14, 117). Only a
few serovars of Leptospira interrogans seem to be weakly cross-reactive with B. burgdorferi (118).
Some patients with rheumatic diseases, such as rheumatoid arthritis and systemic lupus erythematosus, which the physician may be trying to distinguish from Lyme borreliosis, have false-positive reactions in B. burgdorferi serologic
tests (14, 53, 116, 144). These reactions appear due to the nonspecific sticking of rheumatoid factor aggregates or immune complexes to the borrelial antigens. One indicator of a false-positive IFA reaction among rheumatic disease patients is the beaded appearance of the spirochetes (128). This type of staining reaction is also seen in false-positive fluorescent treponemal antibody-absorption test results of patients with systemic lupus erythematosus and other autoimmune diseases (125).
IgM antibody determinations tend to be less specific than those for IgG antibodies, but may be useful in early disease or when reactivation or reinfection is suspected (54, 126). Patients with infectious mononucleosis often have falsepositive IgM tests (126, 161, 168). Magnarelli and Johnson found that 5 of 16 patients with Rocky Mountain spotted fever and three of 7 patients with rheumatoid arthritis had a positive IgM-specific ELISA for B. burgdorferi (116).
While patients with second- or third-stage Lyme borreliosis almost always have elevated IgG titers, those with early disease often have serum antibody titers below the diagnostic threshold for 6 weeks or more after onset (2, 54). Only about 50 to 60% of patients with early disease, i.e., ECM, have diagnostic titers as measured by either IFA or ELISA (2, 14, 54, 126). Antibiotic therapy of first-stage disease may blunt the immunoglobulin response to the point that diagnostic thresholds are never reached (161). In cases of reinfection, the antibody titers to B. burgdorferi may show a fourfold rise from the previous convalescent value (137).
In patients with a neurologic disorder attributable to Lyme borreliosis, the antiborrelia antibody concentrations in the CSF are usually higher than could be accounted for by leakage of circulating antibodies into the CSF. Any CSF titer above 5 is probably significant (168). However, antibodies may be present in the CSF as a consequence of disturbance of the blood-brain barrier. One indication of nervous system involvement is the presence of oligoclonal  immunoglobulin peaks in the CSF but not in the serum (82, 84, 129, 143). To further establish that the antiborrelia antibodies were produced intrathecally, a comparison of serum antibodies and CSF antibodies can be carried out. CSF/serum-specific antibody ratios can be adjusted by using factors that take
into account the total IgG, IgM, or albumin concentrations in the CSF and serum. The resultant indices serve to identify those patients with antibody produced locally in the central nervous system (82, 84, 85, 105, 168, 170, 178). Calculations of such an index may be needed, for example, to accurately diagnose the disease in a patient who has a neurologic disorder resembling multiple sclerosis and an elevated titer to B. burgdorferi in the serum.

Western blot (immunoblot) assays have been performed on a research basis to determine to which protein antigens patients are responding with antibody (12, 14, 51, 53, 74, 176, 178; Wilske et al., in press). These studies have confirmed the finding of IFA and ELISA studies that there is a delay in production of detectable amounts of antibody to the borreliae. Once antibody production begins, it is usually in the form of IgM antibody to flagellin (18), a 41,000-dalton (41-kilodalton [kDa]) protein that is the predominant component of the flagella (53, 74). With time, both IgM and IgG antibodies to a variety of other antigens appear; these include proteins with apparent molecular weights of 15,000, 27,000, 55,000, 60,000, 66,000, and 83,000 (12, 14, 53, 177, 178). The 66-kDa protein appears to be another protein associated with the outer membrane (20, 51). The more chronic and complicated the disease, the greater the number of antigens to which the patients respond. Almost all patients with Lyme disease of more than a few weeks duration have IgG antibody to the 41-kDa flagellin protein (14, 51, 53, 74). Some patients have IgE antibodies to the 41-kDa protein (30).
Other abundant proteins of the B. burgdorferi cell are the major surface-exposed proteins OspA and OspB (87, 88). In most North American strains, the apparent molecular weights of these proteins are 31,000 and 34,000, respectively (Fig. 3) (20, 21).  These proteins appear to be highly immunogenic in experimental animals that have been injected with whole organisms (20, 21; unpublished observations). Paradoxically, humans with Lyme borreliosis develop antibody against OspA and OspB, if they develop them at all, only late in the course of the disease (14, 51, 53). Sera from patients with other spirochetal disease have shown cross-reactions in Western blots to the 41- and 60-kDa proteins of B. burgdorferi (14, 74, 176). Considering the known antigenic relatedness between the flagella of the different Borrelia spp. (18), one might expect some degree of cross-reactivity to the flagellin protein. Epitopes of the 60-kDa protein antigen appear to be conserved among various spirochetes (75a, 176).
Qualitative as well as quantitative differences may be seen in Western blots and other immunoblotlike assays that use serum and CSF obtained from the same patient with neurologic involvement (129, 178). To date, these differences have not been noted when paired serum and synovial fluid specimens from patients with arthritis have been examined (53; unpublished observations). The B-cell response to B. burgdorferi has been demonstrated by studies of immunoglobulin synthesis and specificity on an individual B-cell level (111).
The finding of almost universal responsiveness to the 41-kDa flagellar protein has been used by investigators as a point of departure for studies of subunit components of B. burgdorferi. Coleman and Benach used purified flagellin protein eluted from sodium dodecyl sulfate-polyacrylamide electrophoresis gels (51); the protein presumably was denatured during purification. These investigators found that an ELISA based on a cruder but undenatured "flagellin-enriched" fraction was more sensitive than an ELISA that used purified flagellin eluted from a gel. Hansen et al. isolated whole flagella through mild detergent disruption of the cells and subsequent density gradient ultracentrifugation (76); flagella remain intact by this method (18). This group used the isolated flagella as the antigen in ELISA testing and found improved sensitivity in serologic tests of patients with early disease when compared with a standard ELISA (76). The heightened sensitivity was due in part to lowering of the cutoff point between positive and negative reactions. The assay of Hansen and co-workers appeared to provide greater discrimination between patients with Lyme borreliosis and those either without disease or with nonspirochetal disorders.
The outer membrane OspA and OspB proteins are other isolated components of the borrelial cell that have been examined by Coleman and Benach for use in immunoassays (51). This study, which used eluted proteins in an ELISA, confirmed the Western blot analyses that showed antibodies against these antigens appearing later in the course of the disease. Although these outer membrane proteins may not be useful for immunodiagnosis of early disease, they could have a role as components of a very specific assay in secondary or tertiary disease in humans. A patient with Lyme arthritis had antibodies that bound to recombinant OspA and OspB proteins (88), and, thus, it is likely that patients are responding to the proteins themselves and not carbohydrate or glycolipid moieties that might be associated with them.
The Western blot analysis has been proposed as a practical clinical laboratory test for Lyme borreliosis (74). The advantage to this procedure is that the response to individual components can be examined. Grodzicki and Steere found
the Western blot to be the most sensitive test in early Lyme borreliosis (74). Kirsch et al. used the Western blot to diagnose Lyme disease in a patient with a fatal illness (102). Almost all immunodiagnostic assays reported on have used culture-grown borreliae that were washed and centrifuged at least twice before use in the assay. Whether or not loosely associated spirochetal antigens, such as a slime layer, could be dislodged from the cell surface during antigen preparation is not known. Neubert and colleagues used borreliae obtained directly from the blood of an infected mouse for their IFA; however, the Borrelia species used in the test was not B. burgdorferi (130). Another area that has been little investigated is whether there are important nonproteinaceous antigens of B. burgdorferi.

TYPING STRAINS
As the number of isolates of B. burgdorferi from different human and animal sources and from different parts of the world increases, greater attention is being paid to strain distinctions. Several options are available, including poly-acrylamide gel electrophoresis profiles of cellular proteins, reactivities of monoclonal antibodies, and plasmid analysis. The initial isolates of B. burgdorferi from the United States were almost identical in their polyacrylamide gel electrophoresis profiles (12, 20, 21, 43). They all had major proteins of 31 (OpsA) and 41 (flagellin) kDa. A large majority had an abundant 34-kDa surface protein, OspB, but some isolates either lacked this protein or had an OspB with a slightly different electrophoretic migration (12, 20). As more isolates from Europe were examined, differences in the OspA and OspB proteins were noted (19, 155, 176; Wilske et al., in press). The OspA-like proteins varied from approximately 30 to 33 kDa in apparent size. OspB-like proteins also varied; some European  strains had no major protein that could be considered the equivalent of OspB. Some strains, especially those from regions of Germany, Austria, and Scandinavia, lacked even an OspA-like protein. Instead, they had a major protein of about 22 kDa. This protein has been designated "pC" by Wilske et al. until its surface localization can be confirmed (176; in press). A single United States strain with a major surface protein of about the same size as pC has been isolated from a tick in California (33).
When antisera prepared against whole cells of different strains have been compared by IFA, too few differences in the reactivities of the various isolates have been seen to justify a serologic typing scheme based on use of antisera against whole cells (7). Polyclonal antibodies to isolated cell components, such as OspA and pC protein, offer better discrimination between strains (Wilske et al., in press), as do monoclonal antibodies. The monoclonal antibodies are directed against single epitopes in one protein, usually either OspA- or OspB-like proteins (20, 21; Wilske et al., in press). Using criteria of polyacrylamide gel electrophoresis profiles, polyclonal antisera reactivities, and monoclonal antibody binding, Wilske et al. (in press) identified seven distinct types of B. burgdorferi among a panel of European strains.
Another way to characterize B. burgdorferi isolates is to analyze their plasmid content; both circular and linear plasmids have been identified (13, 16, 90, 151a). A relatively simple extraction procedure can be used to enrich for plasmids in the DNA preparation (13, 16). The plasmid species are then separated on low-percent agarose gels. Analyses have shown considerable heterogeneity in plasmid profiles among strains, even those from North America (13). Plasmids either undergo rearrangement or are lost from the cell during serial in vitro cultivation (13, 90, 151a). DNA hybridization of whole chromosomal DNA has shown that B. burgdorferi is a distinct species in the genus Borrelia (90, 92, 93, 148) and that strains within the species differ in the amount of DNA relatedness. These differences may not be great enough, however, to use genomic DNA hybridization as a routine typing procedure for B. burgdorferi. Its most appropriate use is still as a tool for determining whether an unknown arthropod-associated spirochete is a member of the genus Borrelia and to what species it is most closely related. Use of DNA probes for specific genes, such as the ospA gene, may offer more advantages for distinguishing between strains within the species (19).

LABORATORY SAFETY
B. burgdorferi is a blood-borne pathogen of humans and domestic animals that can cause significant and prolonged disease. It may be confused with a variety of other chronic, noninfectious disorders. B. burgdorferi, like the relapsing fever borreliae, has been considered a biocontainment level 2 [Wiki] organism, and it is appropriate to continue to treat it as such. Although there have been no documented examples of laboratory-acquired Lyme borreliosis in humans, there clearly has not been enough experience with the organism to be complacent about its risk to laboratory workers. The most likely routes of infection would be through a break in the skin, the conjunctiva, and the oral mucosa; experience with the closely related relapsing fever borreliae indicates
that infection can occur through these routes (17). Infected blood and cultures pose the greatest potential risk, but animal and laboratory workers may also be infected through contact with urine of infected animals (35, 46) and through handling live ticks. There is probably little chance of infection through aerosolization or contact with spirochetes that have dried on animate or inanimate materials.

In six patients, a skin biopsy specimen was taken at the site of a previous erythematous skin lesion 1 to 6 months after disappearance of the lesion. Four of them presented with early disseminated Lyme borreliosis. In one additional patient with early disseminated Lyme borreliosis, the site of a previous tick bite was biopsied. None of these patients had been treated with antibiotics before presentation.
The cultures of the skin biopsy specimens of the seven patients showed growth of Borrelia species. By rRNA gene restriction analysis and genospecies-specific PCR, six isolates were classified as Borrelia garinii and one as Borrelia group VS461 [Borrelia afzelii].

Excerpt on procedure:
... A 4-mm-diameter biopsy was taken with a biopsy punch (Disposable Biopsy Punch; Stiefel, Wachtersbach, Germany) after the skin was treated with either 1% iodine (wt/vol) or 0.5% chlorhexidine in 70% (vol/vol) alcohol.
... Culture method for B. burgdorferi. Skin biopsy specimens were transferred to 5.5 ml of modified Kelly's medium (28) in 7-ml Duran borosilicate glass disposable culture tubes (Schott, Mainz, Germany). Prior to use, the tubes were rinsed 10 times with distilled water and subsequently sterilized with heated air at 180°C for 2 h. Rifampin (final concentration of 50 mg/liter) and fosfomycin (final concentration of 100 mg/liter) were added to the medium to inhibit the growth of contaminating bacteria. Preliminary experiments had shown that these concentrations of antibiotics did not inhibit the growth of B. burgdorferi. For culturing of CSF specimens, approximately 2 to 4 ml of CSF was centrifuged at 5,000 x g for 20 min. The pellet was transferred to 5.5 ml of modified Kelly's medium without antimicrobial agents. Incubation at 33°C was done for 8 weeks. The presence of spirochetes was determined by dark-field microscopy weekly. A culture was considered positive when motile spirochetes were seen. All isolates were preserved at - 70°C with 50% glycerolpeptone for cryoprotection.

... 57 consecutive patients referred to our hospital. All these patients were examined by a dermatologist (Toni Ramselaar or Irina Cairo) and met the Centers for Disease Control case definition criteria for EM. The recovery rate of B. burgdorferi from skin biopsy specimens from patients with EM was 86% (36).
... Four of them (patients 1, 3, 4, and 5) had neuroborreliosis, as shown by an increase of the leucocyte count and protein level in the CSF. Patient 2 had clinical symptoms of arthritis and cardiac involvement.
... Three of the four patients with neuroborreliosis had also antibodies against B. burgdorferi in the CSF; one patient (patient 4), who only had immunoglobulin M (IgM) antibodies against B. burgdorferi in her serum, had no CSF antibodies against B. burgdorferi.
... Two other patients were included in the study, since they reported previous erythematous lesions and had antibodies against B. burgdorferi in their sera. One of them (patient 6) [sampled 60 days after disapperance of erythema IgM+IgG serum antibody positive] had mild myalgia at the time of skin biopsy, whereas the other patient (patient 7) [sampled 180 days after disappearance of erythema, IgM negastive, IgG positive] was asymptomatic.
...None of the cultures became contaminated with other microorganisms. From patient 7, a culture from a second skin biopsy specimen taken at a site without a previous skin lesion remained negative.
... It was concluded that the strains from patients 4 and 6 were B. garinii and the strain from patient 7 was group VS461and that an additional typing method was necessary for the other four strains. Therefore, isolates were also genotyped by genospecies-specific PCR (18).  Using the primer pair specific for B. garinii, a 527-bp fragment was amplified from the strains recovered from patients 1 through 6 (Fig. 4). A 591-bp fragment was amplified from the isolate from patient 7 by the primer pair specific for group VS461. Therefore, we conclude that the strains from patients 1 through 6 were B. garinii and the strain from patient 7 belonged to group VS461 [~Borrelia afzelii].

Detection of antibodies to B. burgdorferi. Antibodies to B. burgdorferi in serum and CSF samples were detected by flagellum-enzyme-linked immunosorbent assay (ELISA) (Dakopatts A/S, Glostrup, Denmark) (13).
Table 1: One patient (#4) with radiculopathy and CSF pleocytosis of  207 and Sp-protein 0.69 g7l was IgM positive, but IgG negative for Borrelia antibodies in serum at time of biopsy 32 days after disappearance of the skin lesion;  all  the others were IgG serum antibody positive.
... Interestingly, at 42 days after the onset of disease one of the seven patients (patient 4) had developed only IgM but no IgG antibodies against B. burgdorferi. This is unusual, although such patients were also described by Hansen and Lebech (14), who reported that 44 of 187 patients with neuroborreliosis had only IgM antibodies against B. burgdorferi; the range of their disease duration was 6 to 54 days, with a median of 19 days.
We cultured B. burgdorferi from CSF samples from two of four patients included in this study (50%), but CSF cultures from 13 other patients with neuroborreliosis seen in our hospital remained negative. Our total recovery rate from CSF specimens from patients with neuroborreliosis was therefore 3 of 17 (18%), which is similar to the recovery rate reported in the above-cited studies. The rather high recovery rate from CSF specimens from the four patients included in this study could possibly be explained by the recent onset of neuroborreliosis in them; also, Karlsson et al. reported that CSF cultures were most successful for patients with a recent onset of neurological symptoms (15).
Usually, patients with EM are treated with an antibiotic, since such treatment is generally effective in preventing the onset of early disseminated or chronic LB (4). Our patients had no antibiotic treatment because their skin lesions had not been recognized as EM by themselves or by their general physicians. Whether antibiotic treatment always leads to elimination of B. burgdorferi from the skin has been studied by culturing posttreatment skin biopsy specimens taken from healthy-looking skin from the site from which B. burgdorferi had been cultured before treatment (8, 23). In total, 26 patients were involved in these studies, and cultures were negative in all cases. However, B. burgdorferi has been cultured from skin biopsy specimens obtained at the sites of previous erythematous lesions from three patients after antibiotic therapy (27).
In vitro studies showed that human skin fibroblasts can protect B. burgdorferi from antibiotic treatment (11). Invasion of spirochetes into fibroblasts has been documented and could serve as a possible mechanism by which spirochetes
can escape from antimicrobial actions of antibiotics (16). Intracellular persistence of spirochetes could also lead to a decrease of the inflammatory skin reaction and to fading of the erythema. Alternatively, a down-regulation of the cellular
immune response, which has been documented for the related microorganism Treponema pallidum (10), could explain the disappearance of the erythema during persistence of microorganisms in the skin.

Both serum and whole blood were collected from 25 patients who presented with signs and symptoms of persistent Lyme disease and whose disease was confirmed clinically and by serological methods. ...
One-milliliter quantities of whole blood were added to 10-ml tubes containing 5.0 ml of MPM medium and to 10-ml tubes containing 5.0 ml of BSK-H medium (complete medium; Sigma, St. Louis, Mo.).
The MPM tubes were incubated for 4 weeks at 30°C, and the BSK-H tubes were incubated for 4 weeks at 35°C. Aliquots of a control culture of B. burgdorferi 2591 were added to replicates containing either MPM or BSK-H medium and incubated similarly. All tubes were periodically sampled, and the slides were stained with acridine orange (AO) stain. A terminal PCR targeting the OspA gene (1) was performed on all patient cultures and controls. None of the patient samples showed growth of B. burgdorferi. The control culture in BSK-H grew luxuriantly, as evidenced by the appearance of spirochetes in the AO stain and a positive PCR result. The control organism failed to grow in MPM medium.

Comments:

These authors do not state exactly how they diagnosed their patients to have persistent Lyme symptomatology, if the blood specimens were collected during current flare activity and if the blood samples were cultured immediately after blood draw or there had been an interval between sampling and culture (sending samples by mail for instance, time without media, freezing, cooling of samples ... conditions that may affect the time to succefull culture?). 
 
Borrelia spirochetes can only be found in the blood for at very short time window of only about 24 hours in the beginning of a new flare, for instance by simple microscopy using the microhematocrit technique as described by Barbour above already in 1988 (and by others before improving diagnosis of RF Borrelia); phase contract microscopy of the buffy-coat fraction (WMV Febr. 6, 2008 by Marie Kroun) and darkfield microscopy after double centrifugation in Dualdur reagent (WMV Oct. 16, 2008 by Bela Bozsik), shows two illustrative examples of spirochetes detected in blood by simple microscopy from the same patient with persistent borreliosis, the samples were taken 8 months apart and no antibiotic treatment was given to the patient in between the samples, because first the patient was trying to persuade her GP to ask the microbiology department for attempting culture for Borrelia and that took time and then she was waiting for several months after the first microscopy for the answer from the microbiologist, if they would try to culture her blood and had to abstain from taking antibiotic treatment because that would have ruined the option of getting confirmation of persistent borrelia infection! - culture was unfortunately denied, but in the meantime the patient had - as she usually does during summer time - experienced a sponataneusly improved period; symptoms of persistent Borrelia infection usually flares more and for longer time (a few weeks to 3-4 months) during spring and autumn, corresponding to the blood-meal seeking activity tick seasons out in nature! - thus she waited until the next big autumn flare hit in order to get re-microscopy confirmation by dr. Bozsik!

It is thus very important to time the bloodsampling for microscopy or culture according to the individual patients CURRENT FLARE PATTERN, it is far the best to sample blood for culture within the first day of a new flare, since the chance of finding visible spirochetes in the blood outside this narrow time window can be calculated to be only around 3-10%, when not taking the cyclical relapse pattern into consideration for optimal sampling; i.e. when the patient experiences a monthly (i.e. flares comes at about 4 weeks intervals, 1/30 days ~ 3% chance) versus a weekly (i.e. flares comes at 8-10 days intervals, 1/10 ~ 10% chance) - this easily explains why many researchers who have NOT taken the cyclicity pattern into consideration when sampling, has so low luck in finding spirochetes in blood from patients with long term persistent Lyme borrelia infection!

Whenever the patient has mounted a detectable amount of ANTIBODY no matter if it is being measured by conventional ELISA or by Blot methods - even the new variants (Line blot) that are based on  recombinant selected diagnostic relevant Borrelia antigens from more strains - the finding of a CURRENTLY SEROPOSITIVE STATUS indicates at the same time that there must be low or no level of Borrelia ANTIGEN circulating in the blood!
- hence it is quite unlikely to get a positive culture (or microscopy or PCR) of Borrelia from blood from currently SEROPOSITIVE individuals.
Patients from whose blood one will attempt to culture Borrelia must optimally be selected from the group of patients displaying A CURRENT (WEEKLY) RELAPSING PATTERN and sampling must preferentially be done on DAY ONE of a new flare, and selectively attempted in those patients, who are currently seronegative - or who recently turned from higher positive level to current lower positive or seronegative indicating that more of the formed antibodies are now again being used up quickly by binding to proliferating and in blood circulating ANTIGEN - THIS IS THE TIME TO HAVE A LOOK; therefore the patient must be urged to keep a very detailed daily symptom log like the Excel symptom-diary (Marie Kroun Leicester 2007, UK powerpoint) - so cyclicity can be overviewed and detected fast and sampling can be planned to start of the next expected flare start!

Only 4 weeks is a rather short time considering the slow growth rate of Borrelia and knowledge was present aldready in 2001, that under adverse conditions for growth of the spirochete form the spirochetes quickly turn into their alternate forms (cysts, spheroblasts, L-forms granules, blebs - many names associated with these structures, see 100 years pictorial) that it may take between 9 days and 4 weeks or longer for the "cyst" form to return to (produce several new baby-) spirochetes again, according to 1998-Brorson! ...  already during skin transmission experiments done back in the 1950'ies showed that a certain maturation time of 8-10 weeks of the clinically affected skin material was needed before successful transfer of the infection could occur [Pashoud JM. Hautarzt 1957;8:197-211, 1958; 9:153-165, 1958; 9:263-269 - 1958;9: 311-315.]

Finally why did the authors culture at different temperatures in MPM and BSK? ---when comparing two media for culture, all other conditions should have been kept equal; a difference in time to culture Borrelia, with lower chance of positive culture in MPM at suboptimal temperature compared to in BSK at a more optimal temperature!

These authors chose to attempt culture in WHOLE BLOOD despite Barbour already in 1988 had recommended incubation of the pellet residue (after of double-centrifugation of blood then serum, like dr. Bozsik does!) re-suspended in culture medium; my own culture experiment support Barbours suggestion, since there in my first attempt was quicker growth in sample from buffy-coat fraction with large amount of serum, but not quick growth in full blood sample not in serum fraction taken from way above the buffy-coat layer all from the same blood sample!
 

Culture of B. burgdorferi sensu lato involves incubating a specimen in Barbour-Stoenner-Kelly medium (BSK) (or modifications of BSK) and detecting the presence of characteristic spirochetes by dark-field microscopy or by fluorescent microscopy with  acridine orange or a specific fluorescent antibody (FA).

Some microbiologists consider Borrelia culture to be too expensive and tedious to be practical for many clinical laboratories. In reality, isolation of B. burgdorferi sensu lato is no more difficult and is probably easier than recovering and identifying other fastidious microorganisms such as Legionella or Mycobacteria spp. from clinical specimens. BSK is commercially available*, and the only specialized equipment required is a fluorescent microscope or a light microscope equipped with a dark-field condenser.**  Time to detection of a positive specimen can be within a clinically relevant time frame, provided cultures are examined for the presence of spirochetes at frequent intervals, especially during the first 2 weeks of incubation. Of 90 specimens that were submitted to Marshfield Laboratories from 1991 to 1997 and that turned out to be culture positive, 74 (82%) were identified as positive for Borrelia within the first week of incubation and 32 (35%) were identified within the first 3 days, with the longest time to detection of a positive culture being 16 days (Fig. 1).

*) SigmaAldrich sells ready to use BSK-H medium (after thawing) with a guaranteed durability of 9 months in frozen condition, and in small amounts of 100 ml, thus the laboratory will not risk loosing money on buying a large amount of BSK-H medium that are not used up before the expiration date.

**) also needed is an incubator in order to keep a constant optimal temperature for growth of Borrelia around 35ºC - otherwise it may take very long time to get growth of Borrelia! 

Borrelial infection may manifest with a wide range of clinical signs, and in many cases, microbiological findings are essential for a proper diagnosis. This study included 48 patients with a working clinical diagnosis of Lyme neuroborreliosis, 45 patients with a working clinical diagnosis of suspected Lyme neuroborreliosis, and a control group comprising 42 patients with tick-borne encephalitis and 21 neurosurgical patients. The aim of the study was to analyze and compare findings of two PCR methods and Borrelia burgdorferi sensu lato culture results by examination of prospectively collected cerebrospinal fluid (CSF) and blood specimens from patients with clinical features of Lyme neuroborreliosis. Borrelial DNA was detected with at least one of the PCR approaches in 16/135 (11.9%) blood samples and 24/156 (15.4%) CSF samples. Using MseI restriction of PCR products of the amplified rrf-rrl region, we identified the majority of strains as Borrelia afzelii. Borreliae were isolated from 1/135 (0.7%) blood samples and from 5/156 (3.2%) CSF specimens. Using MluI restriction for characterization of isolated strains, Borrelia garinii was identified in all CSF isolates. Our study revealed that different approaches for direct demonstration of borrelial infection give distinct results, that there is an urgent need for standardization of the methods for direct detection of borrelial infection, and that the design of studies evaluating the validation of such methods should include appropriate control group(s) to enable assessment of both sensitivity and specificity.
 
Material and Methods:
Blood (5 ml) and CSF (1 ml) samples were obtained at initial examination from all patients with a working clinical diagnosis of Lyme neuroborreliosis, suspected Lyme neuroborreliosis, and TBE.
...
Isolation of Borrelia strains from blood and CSF.
One milliliter of CSF, obtained by lumbar puncture, was immediately inoculated into a tube with 5 ml of MKP medium and promptly transported to the laboratory (16). Five milliliters of blood obtained by venipuncture was placed in a tube containing sodium citrate and centrifuged at 100 X g for 5 min, and the supernatant was inoculated into two or more tubes of MKP medium (16, 21, 23). Samples were cultivated at the Institute of Microbiology and Immunology of the Faculty of Medicine Ljubljana at 33°C as described previously (16) and were examined once a week for the presence of spirochetes by using dark-field microscopy. Samples were considered negative if no growth was detected after 9 weeks of incubation for CSF and 12 weeks for blood (16).

[MK: rather short culture observation time! .. and 2 degree lower temp. than what most other report to be the optimal temp. for culture]

PCR targeting ospA detected the presence of borrelial DNA in 14/135 (10.4%) blood samples and in 19/156 (12.2%) CSF specimens. Blood samples were positive in 7/48 (14.6%) patients with a working clinical diagnosis of Lyme neuroborreliosis, in 5/45 (11.1%) patients with suspected Lyme neuroborreliosis, and in 2/42 (4.8%) of patients with TBE (nonsignificant differences). The corresponding results for CSF samples were 10/48 (20.8%), 7/45 (15.6%), and 2/42 (4.8%), respectively. PCR targeting ospA found no borrelial DNA in the CSF samples of the 21 patients in the neurosurgical control group (Table 1 and Fig. 1). ...
Using PCR targeting the rrf-rrl region, 14/135 (10.4%) blood samples and 19/156 (12.2%) CSF samples tested positive. Blood samples were positive in 5/48 (10.4%) patients with a working clinical diagnosis of Lyme neuroborreliosis, in 7/45 (15.6%) patients with suspected Lyme neuroborreliosis and in 2/42 (4.8%) patients with TBE (nonsignificant differences). The corresponding findings for CSF were 10/48 (20.8%), 8/45 (17.8%) and 1/42 (2.4%), respectively (Table 1). Absence of borrelial DNA in the CSF of the neurosurgical patients was established with PCR targeting the rrf-rrl region.
...
The simultaneous presence of borrelial DNA in both blood and CSF was established in only three patients—in two with working clinical diagnosis of Lyme neuroborreliosis and in one with suspected Lyme neuroborreliosis.
...
With MseI restriction of the PCR product of the amplified intergenic rrf-rrl region, we were able to characterize 13/14 (92.9%) DNA-positive blood samples and 18/19 (94.7%) DNA-positive CSF samples; the majority of strains were found to be B. afzelii (Table 2).

Borreliae were isolated from 1/135 (0.7%) blood samples and from 5/156 (3.2%) CSF specimens. Four out of the five CSF isolates were from patients with a clinical diagnosis of Lyme neuroborreliosis; the fifth was from a patient with suspected Lyme neuroborreliosis. Borreliae were not isolated from CSF samples of patients without signs of borrelial infection (control group of TBE and neurosurgical patients).
...
We were able to identify to the species level all five strains isolated from CSF and the single strain isolated from blood (Table 3). Using pulsed-field gel electrophoresis after MluI restriction, four of the CSF isolates (all from patients with a working clinical diagnosis of Lyme neuroborreliosis) were found to be B. garinii (three typed as Mlg2, one as Mlg3); the fifth CSF isolate (from a patient with suspected Lyme neuroborreliosis) was B. afzelii (typed as Mla1). The strain isolated from the blood of a patient with suspected Lyme neuroborreliosis was also found to be B. afzelii (typed Mla1). In only half of the culture-positive CSF and blood specimens were we able to demonstrate the presence of borrelial DNA directly in the corresponding samples with at least one of the two PCR approaches (Table 3).
...
The main advantage of targeting the rrf-rrl region rather than ospA is the possibility of identifying the borrelial species. In this way, we were able to characterize all but two (31/33 [94%]) PCR-positive blood and CSF samples (Table 2). The two specimens that were not characterized had an unusual restriction pattern that will be investigated further using sequence analysis.
Overall, B. afzelii was the species most frequently identified, although it is not the principal causative agent of Lyme neuroborreliosis in Europe (23).
Low sensitivity of borrelia detection in the blood of patients with (suspected) Lyme neuroborreliosis (Table 1) could have been the consequence of a transitional spirochetemia, which in the majority of patients most probably ended at the time of central nervous system involvement, and/or the result of low numbers of spirochetes in the blood. With regard to the method itself, possible inhibitors in host blood can diminish positive findings (1). Although we predicted a low proportion of PCR-positive blood samples, we were surprised to find an almost equal CSF-positive rate (Table 1). According to literature reports, the sensitivity of PCR in CSF ranges from 12% to 100% (median, 23%) and depends upon numerous factors, including method, origin of samples, and the differing approaches used in individual studies (1)

Several significant differences were established when comparing PCR results for blood and CSF samples from patients in the Lyme neuroborreliosis group (and to a lesser extent in those constituting the suspected Lyme neuroborreliosis group) with findings for the control group(s), and it was somewhat surprising that borrelial DNA was detected in some of the patients with TBE. We emphasize that all contamination precautions were strictly followed, that all routine negative controls gave negative results, and that the quality of the samples was monitored. To determine the specificity of the methods, we also used CSF samples of neurosurgical patients who had no signs of borrelial infection and no known (recent) exposure to ticks; the results of all tests were negative, indicating high specificity of the utilized methods.
...
In Slovenia, approximately 30% of Ixodes ricinus ticks are infected with Borrelia burgdorferi sensu lato (data based on tick culture) and 0.3% to 1.2% with TBE virus (15; N. Knap, unpublished results) ... It is also quite possible that not all borrelia infections, even those that can be demonstrated in CSF and/or blood by PCR, result in an illness.

Our findings emphasize the importance of appropriate design of studies evaluating the validity of PCR approaches for the detection of borrelia infection; the design should include not only the assessment of sensitivity but also an appropriate control group for determination of specificity. Interpretation of the results of published studies that have used differing PCR approaches for the demonstration of borrelial DNA would be much safer with the inclusion of an appropriate control group that would enable assessment of both specificity and sensitivity.

Cultivation of blood samples gave an extremely low yield; we were able to isolate borreliae from only 1 out of 135 blood samples. We had expected to isolate more borrelia strains, although, in Europe, the recovery rate has generally been low (<10% in patients with early Lyme borreliosis manifested by erythema migrans) using volumes of blood similar to those in the present study (1, 19). The explanations for the low recovery rate could be the same as those for the low proportion of positive PCR findings in blood (4, 12, 13).

The isolation of the etiological agent from CSF samples is the most reliable method for diagnosis of borrelial central nervous system infection in patients with suspected Lyme neuroborreliosis and also provides live organisms that can be further characterized (23). However, this is a low-yield procedure that takes several weeks. In patients with proven Lyme neuroborreliosis, the reported recovery rate from CSF is <10% (23). In the present study, isolation from CSF was successful in 3.7% (5/135) of samples, but the isolation rate in the group of patients with a working clinical diagnosis of Lyme neuroborreliosis was 8.3% (4/48).

We were able to characterize all the isolated borrelia strains (Table 3). Using MluI restriction for borrelia characterization, B. garinii was identified in all CSF isolates of patients with Lyme neuroborreliosis, whereas in a patient with suspected Lyme neuroborreliosis who did not have pleocytosis, the isolated species was B. afzelii. These findings corroborate the results of previous studies on the predominance of B. garinii strains isolated from CSF samples of patients with Lyme neuroborreliosis (3, 16, 23) and on the uncertain value of B. afzelii as the causative agent of Lyme neuroborreliosis (23). However, PCR performed directly on CSF samples revealed a predominance of B. afzelii (Table 2).
Comparison of the results of cultivation and the two PCR methods revealed that several culture-positive samples were PCR negative and vice versa. An explanation for the finding that an individual CSF sample may be positive only with cultivation but not with PCR could be the presence of a low number of borrelia cells in CSF; this may not allow the detection of borrelial DNA by PCR but would still allow proliferation of borrelia during cultivation. In patients with CSF samples positive with PCR only, negative culture results could be explained by the influence of the immune response and also by the fact that the borreliae must adapt to an artificial culture medium, which probably limits the isolation rate, whereas their DNA, not only from living but also from dead cells, can be detected with PCR.

[MK:  why did these authors use MKP meidum instead of BSK-H? - and why culture at 33 degree C instead of at 35 degree C, which others have reported the optimal temperature for growth of Borreliae? - how about CO₂ and O₂ concentration, how about pH?   ...which is not mentioned in the text thus apparently not taken into consideration?
- could sub-optimal conditions for growth perhaps explain the relatively low positive culture rate compared to they got an expectable rate of positive PCRs in blood and CSF, considering the authors did apparently not take cyclical relapse pattern into account when sampling for culture?
... if - at it seems from my studies - Borrelia spirochetes only wanders from the tissues to the blood stream at intervals of 8-10 days, and Borrelia within 1-24 hours under unfavourable growth conditions for the spirochete form changes a "cyst" form, that take at least 9 days to revert back to spirochete form, and that reversal may perhaps not happen ever, unless the microbes are given the very best conditions for survival of the spirochete form, i.e. optimal growth medium, optimal temperature at 35 degree C, optimal pH, optimal CO₂ and O₂ concentration ...  not taking care of all this could result in low recovery rate of Borrelia by culture!
 
When findings show that Borrelia afzelii is often detectable in the CSF and may cause symptoms of neuroborreliosis with same exact degree of disease / severity of debilitating symptoms as patients infected which Borrelia garinii
- since in ref. 23 the only significant difference in the patients symptoms was the presence of "typical radicular pain" in  only 65% of Borrelia garinii patients and abscence of this symptom in the Borrelia afzelii neuroborreliosis group
- i.e. that particular sypmtom, which is usually put very much weight on in the clinical diagnosis of "typical neuroborreliosis", but which can not be a requirement for the diagnosis of neuroborreliosis, because certainly even not all Borrelia garinii patients had it!
- and in the Borrelia afzelii group often was abscence of the laboratory signs considered "typical" and normally considered required for the diagnosis of Lyme neuroborreliosis, i.e. pleocytosis, increased protein and seropositive Borrelia index status ...
- is NOT fulfilled for all patients in which Borrelia burgdorferi sensu lato can be detected
- has the time then not come NOW, to change the "common view" on the definition of Lyme neuroborreliosis definition? 
- and accept that this particular picture only seems partially true for Borrelia garinii, but certainly is not true for most cases of Borrelia afzelii ...

Since patients with Borrelia afzelii infection may be just as sick and may have Borrelia afzelii in their CSF and or blood - whether they express the usual laboratory signs of "certain neuroborreliosis" or not, it should be clear to all that the patients infected with Borrelia afzelii also deserve to get the correct diagnosis and treatment for their Borrelia infection!
It seems that Borrelia afzelii is apparently less immunogenic / more stealth in its nature than Borrelia garinii, so correct diagnosis is not made by the conventional test methods, are not made early at the time when there is great chance of cure by short term antibiotic treatment - because the patients are not diagnosed and treated as early they have a worse long term prognosis and are not cured by conventional treatment for Borrelia, resulting in long term debilitating sickness, and inability of patients to work etc. etc ... the impact on the society of undiagnosed Borrelia afzelii infection with or without sign of neuroborreliosis is probably much more common than hitherto beleived, because usual tests failed to give the correct diagnosis, a much larger proportion of chronic illness could be due to undetected and untreated Borrelia afzelii infection, compared to typical Borrelia garinii neuroborreliosis infection, that usually raise a quick and high immune response in its victims ... leading to fast diagnosis and treatment and good chance of cure by early treatment!? .... WE NEED ACCESS TO DIRECT DETECTION METHODS, must find what is the OPTIMAL CULTURE CONDITIONS! - by systematic comparison of all known methods and media!]

Several pathogens of humans and domestic animals depend on hematophagous arthropods to transmit them from one vertebrate reservoir host to another and maintain them in an environment. These pathogens use antigenic variation to prolong their circulation in the blood and thus increase the likelihood of transmission. By convergent evolution, bacterial and protozoal vector-borne pathogens have acquired similar genetic mechanisms for successful antigenic variation. Borrelia spp. and Anaplasma marginale (among bacteria) and African trypanosomes, Plasmodium falciparum, and Babesia bovis (among parasites) are examples of pathogens using these mechanisms. Antigenic variation poses a challenge in the development of vaccines against vector-borne pathogens

Results: Compared with Slovenian patients, U.S. patients had erythema migrans for a briefer duration (median duration, 4 days compared with 14 days; P , 0.001) but were more likely to have systemic symptoms (P 5 0.01), abnormal findings on physical examination (P , 0.001), and seroreactivity (P , 0.001). Central clearing of erythema migrans lesions was more likely in Slovenian patients (P ,0.001).
Conclusions: Erythema migrans caused by B. afzelii in Slovenia and erythema migrans caused by B. burgdorferi in New York have distinct clinical presentations. Caution should be used when clinical and laboratory experience from one side of the Atlantic is applied to patients on the other.