The Centers for Disease Control and Prevention launched a syphilis eradication effort in , but by , infection rates in the United States had climbed to their highest levels since before the eradication program began.
Norris said he ultimately hopes to modify the system to make it more efficient by eliminating the need for tissue cells altogether. A simplified process might make it easier to develop new ways to detect, treat or prevent the infection, he says.
The new process for growing T. This article has been republished from materials provided by McGovern Medical School. Note: material may have been edited for length and content. For further information, please contact the cited source.
Reference: Edmondson, D. However, after days, further growth of the spiral form ceased and spirochetes tended to convert into different morphological forms data not shown as described previously 39 -[42]. In addition, several solid BSK-based media 46 and PMR-agar media 43 were similarly tested but likewise were not capable of maintaining spirochetal growth. We thus explored other variables: media and tubes ml in 15 ml glass tubes and 1ml Despite altering these culture conditions, there was no improvement in growth.
In summary, while the addition of serum, a reducing agent and rifampicin had a positive effect on the short-term Borrelia cultures both by increasing yield and rate of initial growth, additional parameters needed to be optimized to improve long-term Borrelia cultures. In infected mammals, Borrelia are most often found to be sequestered in collagen and especially collagen type I 47 A collagen matrix has been shown to support growth of Borrelia burgdorferi B31 and strains in vitro 38 To study this further we initiated an experiment in which different amounts of Bb B31 cells 1, 10, , were spiked into 10 ml of whole peripheral blood from healthy donors and after a short term culture of 6 days in mBSK-H the culture was transferred to long-term cultures set up in Coplin jars with collagen-coated slides in a total of 45 ml of mBSK-H media.
After 14 days, an accumulation of Borrelia growth was observed on all collagen coated slides. These data confirm that under favorable conditions human blood containing as few as one Borrelia B31 spirochete can be cultured in mBSK-H with a collagen source, which supports the original hypothesis that a matrix-supported environment is beneficial for their growth. Magnification: X. To explore whether a collagen source would be beneficial to growth of wild type Borrelia , 10 cultures from serologically positive Lyme disease patients that were negative at day 6 in culture were converted into long-term cultures.
After 4 and 8 weeks spirochetal growth was identified in four of these ten cultures and those spirochetal structures stained positively with the polyclonal anti- Borrelia antibody. Examination of the slides showed individual spirochetes as well as small and large aggregates of spirochetes. To test whether a long-term culture can be set up directly, serum from seropositive Lyme disease patients was seeded into 45 ml of mBSK-H media in a Coplin jar containing two collagen slides.
All attempts to establish a long-term Borrelia culture from patient sera in this manner were not successful data not shown. This confirmed the need to seed a long-term culture from a short-term starter culture.
The serum sample was cultured with our culture method for 8 weeks and the obtained collagen-coated slides were immunostained with polyclonal fluorescent antibodies against Borrelia burgdorferi. Panels A-D represent 4 different fields of one collagen-coated slide showing individual spirochetes and small and large spirochetal aggregates that stained positively for Borrelia burgdorferi antigen green staining.
This study enrolled 72 Lyme disease patients whose diagnosis was confirmed by a typical clinical history and a positive result on a 2-tiered serological test following CDC testing guidelines. It was required that each donor had not been exposed to antibiotics for a minimum of 4 weeks prior to blood draw as recommended in previous studies 33 Short-term cultures were established in mBSK-H as described and after six days these cultures were used to seed collagen-supported long-term cultures.
The samples were cultured with our novel method for 6 days, 8 weeks and 16 weeks and the cultures were confirmed to be Borrelia burgdorferi species by immunocytochemical and PCR methodologies see Material and Methods. The confidence interval values are depicted by bars. The samples were cultured with our novel method for 8 weeks and immunostained with polyclonal fluorescent antibodies against Borrelia burgdorferi green staining.
Panels A-I: Nine representative samples, x. The samples were cultured with our novel method for 8 weeks and immunostained with monoclonal fluorescent antibodies against Borrelia burgdorferi green staining. Borrelia -specific PCR products were amplified from each of the antibody-positive samples confirming that each sample contained Borrelia. Significant sequence variations were identified among 51 samples sequenced at the CTP synthase gene locus indicating that samples were derived from independent sources and not from laboratory contamination.
Limited sequence variation was also noted at the well-conserved 16S rDNA site data not shown. Numbers at nodes indicate bootstrap support values replicates.
To ensure the specificity of this culture method and any subsequent detection system, serum was collected from 48 healthy controls with no history of tick bites or of Lyme disease and was processed using the above short-term and long-term culture methods.
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.
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.
By refining our collection and short-term cultivation methods we were able to get similar results after just six days in culture. 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 51 , 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. 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 CO 2 incubator facilitated growth.
Low concentration of rifampicin 0. 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 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. 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 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 culture. 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. The authors thank Dr. Joseph Burrascano, Jr. Alan MacDonald and Dr. Parkash Gill for helpful discussion. National Center for Biotechnology Information , U.
Int J Med Sci. Published online Feb Eva Sapi 1. Find articles by Eva Sapi. Namrata Pabbati 1. Akshita Datar 1. Ellen M Davies 1. Amy Rattelle 1. Bruce A Kuo 1. Author information Article notes Copyright and License information Disclaimer. Ph: Fax: Email: ude. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors have declared that no competing interests exist. Received Dec 12; Accepted Feb Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. This article has been cited by other articles in PMC. Abstract In this report we present a method to cultivate Borrelia spirochetes from human serum samples with high efficiency.
Keywords: Clinical microbiology, Vector-borne diseases, Lyme disease, Spirochetes. Introduction It has long been an elusive goal to develop a successful in vitro culture method for wild-type, pathogenic Borrelia. Blood collection Full informed consent was obtained from each study participant and HIPAA-compliant privacy measures were maintained. Table 2 Description of the different culture environments for short-term cultures.
Open in a separate window. The lack of continuous in vitro culture severely restricts studies of the organism? A system involving co-culture of T. However, repeated attempts to expand the yield or longevity of growth using refinement of growth conditions, subculture, medium exchange, or other tactics have not been successful.
In this project, novel approaches will be utilized to further define the growth characteristics and requirements and gene expression patterns of T. In the first Aim, recently developed methods for computational analysis of microscopy images and assessment of cell wall synthesis and DNA synthesis will be employed to gain a better understanding of the growth characteristics of T.
In addition, co-incubation with tissue explants, as successfully applied to Borrelia burgdorferi studies, will be examined as a means to provide a? The resulting information will be invaluable in defining the poorly understood promoters and regulatory elements of this organism, including the potential identification of regulatory RNA species.
In addition, comparison of transcription profiles from in vivo and in vitro grown organisms may provide fruitful directions for the in vitro growth studies of Aim 1.
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