Here we report the development and evaluation of an improved method to culture Borrelia spirochetes from peripheral blood of confirmed Lyme disease patients. Results from this study showed that by optimizing collection and transport conditions, media and growth environment, and the development of a long-term culture method together provided improved growth conditions for Borrelia. This is demonstrated by a 94% detection rate for Borrelia burgdorferi from sera of Lyme disease patients whose diagnoses were confirmed under the CDC surveillance testing guidelines. We have also found significant sequence variation in these Borrelia samples which further documents that the Borrelia grown in this culture system were not derived from laboratory contamination. The negative results from sera from healthy controls also argue against laboratory contamination as being the source of the positive cultures and further confirm the specificity of the test.
The maximum success rate for Borrelia
blood cultures as outlined in recent publications is in the 40-44% range after 8-12 weeks of culturing 10
. By refining our collection and short-term cultivation methods we were able to get similar results after just six days in culture. The highest rate of Borrelia
growth previously reported from clinical samples (up to 88%) involved cultures of skin, a tissue abundant in collagen 1
. Such studies revealed a maximal success rate similar to what we achieved in our long term, collagen supported in vitro
culture system (84% at 8 weeks and 94% at 16 weeks).
Unlike what has been reported by others, 50
we found poor results when blood specimens were collected in tubes containing EDTA. We found that the addition of BSK-H medium to the whole blood sample at the time of collection increased spirochete yield. As previously reported 44
, we also noted that allowing the serum to separate, especially when mixed with BSK-H media, for up to 24 hours from the time of the blood draw improved the culture success rate. The BSK-H medium appeared to stabilize and even draw out the spirochetes from the cellular fraction. Presence of N-acetyl glucosamine (NAG) in the BSK-H medium, a known chemoattractant for Borrelia
, may facilitate this separation and support the spirochetes during transport. Presumably, these freely suspended Borrelia
were better able to be cultivated. Interestingly, there was a subset of clinical samples that grew better in the collection tubes without additional BSK media. Because of this observation, we utilized both methods in our experiments to ensure maximal yield.
BSK-H has been the preferred media for isolating and cultivating spirochetes from human clinical samples 1
. We made four changes that include: 1. Higher concentration of rabbit serum (12% instead of 6%) incorporating the observation that cultivation of other Borrelia
strains such as Borrelia hermsii
requires a higher percentage of serum 52
. 2. Based on the previous observation that DTT added to BSK-H media helped to isolate Borrelia
spirochetes from Ixodes
, by testing different concentrations of DTT we determined that a high concentration of DTT (100 μg/ml) enhanced the growth of Borrelia
in our cultures. 3. Since Borrelia
species are known to be microaerophilic, meaning that while some oxygen is needed for viability, high oxygen concentrations can be inhibitory 53
, conditions that limit but not eliminate oxygen, such as partially shut lids, nearly full tubes and use of a CO2
incubator facilitated growth. 4. Low concentration of rifampicin (0.4-0.5 µg/ml) prevented secondary bacterial overgrowth in all but 2 out of 122 cultures used in this study. In addition, the use of two different types and sizes of tubes in the starter culture improved outcome.
The long-term culture system provided the greatest advantage in achieving a higher success rate. It was found that modified BSK-H media did not provide an efficient in vitro
culture environment after 8-10 days. We thus investigated whether matrix protein as a solid support may provide a desirable environment for growth of Borrelia
-. Several matrix proteins were tested such as fibronectin, laminin and hyaluronan, but collagen gave the best results, consistent with previously published data 38
. Interestingly, collagen is present both in skin biopsies and in the novel long-term culture presented here and may be a common reason for their growth advantage over other cultures.
The identity of the spirochetes was confirmed in all of the 68 culture-positive, CDC-positive patients enrolled in the validation study by staining positively with Bb-specific monoclonal and polyclonal immunostains. Additionally, further confirmation of the presence of Borrelia burgdorferi in these samples was accomplished by testing all clinical isolates with DNA PCR and then directly sequencing the DNA products. Again, in all cases, results were consistent with Borrelia burgdorferi.
Several measures were employed to rule out the possibility of false positives. Blood from 48 negative controls was cultivated using the above established methods and none showed any growth. Meticulous DNA analyses of the positive cultures including the use of both negative and positive controls in each PCR reaction did not reveal any evidence for contamination. To confirm that Borrelia
isolates that were cultured from the 72 CDC positive patients were unique and to further prove that the PCR products were not from an environmental contaminant, PCR products were directly sequenced at either the 16S rRNA or the CTP synthase locus or both. The results confirmed that the sequences from each positive culture sample were derived from Borrelia
. Sequence analysis within the amplified region of 16S rRNA gene showed only limited variation that is likely due to the highly conserved nature of 16S rRNA gene 55
. However, abundant sequence variations were noted at the CTP synthase locus from the positive cultures, indicating that the clinical isolates are unique and derived from wild-type Borrelia
and not from a laboratory contaminant. The PCR and sequencing results were included in this study to further validate the polyclonal and monoclonal antibody assays used for the development of this Borrelia
In summary, this report provides evidence for the value of a novel method for culturing Borrelia from human serum samples. The inclusion of key components such as modified culture media plus a unique culture environment has resulted in an improvement in the ability to cultivate this organism. This new culture method directly addresses the issue of the low numbers of Borrelia in clinical samples by amplifying their quantity through long term culture in which Borrelia were able to thrive for as long as eight months (data not shown). The versatility of this method allows for samples to be harvested from the culture at any point in time for further study, and it also serves as a source of Borrelia for a variety of direct detection techniques as well as for additional research. Finally, this unique culture method could play an important role in providing useful diagnostic information for select Lyme disease patients who might have tested negatively by other methods.