LETTER
The ability to cultivate Borreliella (Borrelia) burgdorferi is a cornerstone of Lyme disease research. For blood, plasma is the preferred source of culture material (1–3). Studies in the United States have used EDTA as the anticoagulant (1, 2, 4–6). Our study demonstrated that using sodium citrate as the anticoagulant improved the yield of B. burgdorferi culture from plasma samples. (This study has been registered at ClinicalTrials.gov under registration no. NCT00001539.)
Severe combined immunodeficient (SCID) mice were injected intradermally with 106 bacteria (low-passage-number B. burgdorferi strain B31). Spirochetemic mouse blood (25 μl), collected at day 8 (7), was used to spike 1 ml of human blood in EDTA or citrate tubes. Quantitative reverse transcription-PCR (RT-PCR) (8) indicated a level of 1 ×103 to 2 ×103 B. burgdorferi bacteria/ml of mouse blood. After 3 or 12 h in room temperature, plasma was separated by centrifugation (260 × g for 15 min) and 0.2 ml inoculated into 4 ml of Barbour-Stoenner-Kelly (BSK) H medium, containing 6% rabbit serum. Cultures were incubated at 34°C and examined by dark-field microscopy. Blood samples were acquired from three healthy volunteers (ClinicalTrials.gov registration no. NCT00001539). Animal experiments followed institutional guidelines of the University of Maryland.
Results are shown in Fig. 1. Comparison of the time to event curves by the log rank (Mantel-Cox) test showed a significant difference, with a P value of <0.0001. Overall, the best results were obtained by using citrate and processing the samples within a 3-h interval, with a median time to positive result of 5 days, followed by using citrate and processing the samples within 12 h (median time to positive result, 7 days), using EDTA and processing the samples within 3 h (10 days), and using EDTA and processing the samples within 12 h (28 days).
FIG 1.
B. burgdorferi growth in BSK-H media from EDTA or citrate plasma samples. (A) Kaplan-Meier time to event curve for positive B. burgdorferi cultures from EDTA or citrate plasma samples. Comparisons of the curves by the log rank (Mantel-Cox) test showed a significant difference, with a P value of <0.0001. (B) Table showing the results from the 3 separate experiments. Experiment 1 compared EDTA and citrate samples processed after a 12-h interval. Experiment 2 examined samples processed within a 3-h interval. Experiment 3 examined both intervals. The columns show the number of positive tested samples/total number of tested samples. Culture vials were examined until positive results were obtained and/or until day 28. ND, not done.
The underlying mechanism of the deleterious effect of EDTA on B. burgdorferi is unknown, but the effect may be caused by the chelation of divalent cations, such as Mg2+ and Ca2+, as well as metals like manganese and zinc, which are critical for bacterial cell division and metabolism. In comparison, sodium citrate has milder calcium and magnesium chelation properties (9). Growth of B. burgdorferi was inhibited when bacteria were cultivated in the presence of EDTA in concentrations above 0.5 mmol/liter (10). The concentration of EDTA was 1.8 mg per milliliter (about 4.45 mmol/liter) of blood in the BD Vacutainer lavender-top blood collection tubes.
While there was no direct comparison using blood samples from patients with untreated acute Lyme disease, the study design emulates the clinical situation. We used spirochetemic murine blood to spike human blood samples, as this more closely replicates hematogenously disseminated B. burgdorferi, compared with cultured B. burgdorferi. The ratio of plasma to media and the 3-h interval were chosen to reproduce the protocol established at the New York Medical College (5), while the 12-h interval mimics the time to receipt of samples mailed to a laboratory or other causes of delay in processing.
In conclusion, when an anticoagulant is required for B. burgdorferi culture, citrate is superior to EDTA, and its use will likely improve the sensitivity of the procedure. The time to inoculation in culture media is also important, and, whenever possible, blood samples should be processed within 3 h of collection. Studies comparing samples from patients with acute Lyme disease are needed to corroborate these findings.
ACKNOWLEDGMENTS
We thank Gary Wormser, New York Medical College, for comments on the manuscript.
This research was supported by the Intramural Research Program of the NIH, NIAID (A.R.M., S.-P.T., C.D.W., M.A.L.). This work was also partially supported by NIH grant numbers AI-080615 and AI-116620 (to U.P.) and AI-065359 (to A.G.B.).
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
All of us report that we have no potential conflicts of interest.
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