Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: Diagn Microbiol Infect Dis. 2012 May 8;73(3):243–245. doi: 10.1016/j.diagmicrobio.2012.03.026

Comparison of Five Diagnostic Modalities for Direct Detection of Borrelia burgdorferi in Patients with Early Lyme Disease

Dionysios Liveris 1, Ira Schwartz 1,2, Donna McKenna 2, John Nowakowski 2, Robert Nadelman 2, Joseph DeMarco 1, Radha Iyer 1, Susan Bittker 2, Denise Cooper 2, Diane Holmgren 2, Gary P Wormser 2,*
PMCID: PMC3377843  NIHMSID: NIHMS368616  PMID: 22571973

Abstract

Lyme disease, the most commonly reported tick-borne infection in North America, is caused by infection with the spirochete Borrelia burgdorferi. Although an accurate clinical diagnosis can often be made based on the presence of erythema migrans, in research studies microbiologic or molecular microbiologic confirmation of the diagnosis may be required. In this study, we evaluated the sensitivity of five direct diagnostic methods (culture and nested PCR of a 2 mm skin biopsy specimen, nested PCR and quantitative PCR (qPCR) performed on the same 1 mL aliquot of plasma and a novel qPCR-blood culture method) in 66 untreated adult patients with erythema migrans. One or more these tests were positive in 93.9% of the patients. Culture was more sensitive than PCR for both skin and blood, but the difference was only statistically significant for blood samples (p< 0.005). Blood culture was significantly more likely to be positive in patients with multiple erythema migrans skin lesions compared to those with a single lesion (p=0.001). Positive test results among the 48 patients for whom all five assays were performed invariably included either a positive blood or skin culture.

The results of this study demonstrate that direct detection methods such as PCR and culture are highly sensitive in untreated adult patients with erythema migrans. This enabled microbiologic or molecular microbiologic confirmation of the diagnosis of B. burgdorferi infection in all but four (6.1%) of the 66 patients evaluated.


Lyme disease, caused by Borrelia burgdorferi, is the most commonly reported tick-borne infection in North America (Bacon et al. 2008). Erythema migrans (EM) is the most common clinical manifestation (Dandache and Nadelman 2008). For most patients with EM-like skin lesions in clinical practice the diagnosis can be made by visual inspection. However, in research studies, microbiologic or molecular microbiologic confirmation of the diagnosis may be required and a number of diagnostic modalities have been used successfully in this setting including polymerase chain reaction (PCR) and culture of both skin biopsy specimens and blood (Aguero-Rosenfeld et al. 2005; Wang et al. 2010).

We have recently reported that the sensitivity of blood cultures in patients with EM can be significantly improved by testing aliquots of culture medium after short time periods of incubation for the presence of B. burgdorferi DNA by a quantitative PCR (qPCR) method (hereafter called qPCR-blood culture) (Liveris et al. 2011a). In addition, we have optimized the sensitivity of PCR on blood specimens directly by sampling a 1 mL aliquot of plasma and by using both qPCR and nested PCR (nPCR) assays (Liveris et al. 2011b).

In this study, we compare the sensitivity of five diagnostic modalities in untreated adult patients with EM.

Methods

Patients

Plasma samples in EDTA-blood collection tubes were obtained from adult patients with a clinical diagnosis of EM that had been established at the Lyme Disease Diagnostic Center (Valhalla, NY) in the years 2005, 2006 and 2007. All eligible patients with EM were invited to participate in this study. Samples were excluded from this analysis if the patients had recently been treated with a beta-lactam or tetracycline antibiotic before the plasma sample was obtained. The protocol was approved by the Institutional Review Board of New York Medical College and written informed consent was obtained from all participants.

Blood cultures using quantitative PCR on culture aliquots

Cultivation of 9 mL plasma specimens was performed as described previously (Liveris et al. 2011a). Briefly, (three 3-mL aliquots of plasma were inoculated into separate flasks containing 60 mL of antibiotic-free Barbour-Stoenner-Kelly (BSK) medium. Cultures were incubated at 32°C – 33°C for 8–12 weeks (12 weeks in 2005 and 2006 and 8 weeks in 2007), and were examined for spirochetes by fluorescence microscopy at 2 weeks and thereafter at 2–4 week intervals. A 10-μL aliquot of culture material was mixed with 10 μL of an acridine orange staining solution (100 μL in PBS [pH 7.41]), placed on a slide, overlaid with a coverslip and examined at 400x magnification. A minimum of 20 high-power fields were viewed for the presence of spirochetes. Isolation of borrelial DNA from culture aliquots after the cultures had been incubated for time periods ranging from 1 day to 21 days and application of real time qPCR to this DNA, were performed as described previously (Liveris et al. 2011a).

Skin biopsy and culture

Skin biopsy specimens (2 mm in diameter) were obtained from the advancing border of primary EM lesions, as described elsewhere (Nowakowski et al. 2001). Biopsy specimens were placed into transport medium, which consisted of modified BSK medium (this preparation of BSK lacks rabbit serum and bovine serum albumin) plus rifampin 40 μg/mL, for later laboratory processing. Tissue specimens were then transferred to a micro tissue grinder (Spectrum Medical Industries), which contained 0.4 mL of modified BSK medium without rifampin, and were ground; 0.2–0.3 mL of this suspension was added to a 7-mL screw-cap tube that contained 6 mL of complete BSK medium (with rabbit serum and 35% BSA solution, but devoid of antibiotics). The screw-cap tube was tightly capped and incubated at 33°C for the duration of the culturing period. The remaining suspension plus the skin fragment itself were returned to the transport medium and sent for PCR studies. Cultures were examined by means of fluorescence microscopy at 2 weeks and, thereafter, at 2-week intervals for up to 8 weeks, as previously reported (Nowakowski et al. 2001).

PCR of plasma aliquots

Plasma was separated from whole blood by centrifugation at 260Xg for 15 minutes. A 1 mL aliquot of plasma was then centrifuged at 14,000Xg for 10 minutes, pellets were resuspended in 100 μL of IsoQuick sample buffer, DNA was extracted by means of a commercial nucleic acid extraction kit (IsoQuick; Orca Research, Bothell, WA) as described (Schwartz et al. 1993) and suspended in 50 μL of distilled water, RNA grade (Fisher Scientific). Two microliters of DNA suspension was used for PCR amplification. Nested PCR was performed as previously described (Liveris et al., 2002; Barbour et al. 1996). Real-time qPCR was performed in an ABI Prism 7900HT Sequence Detection System (Taqman) using two flaB-specific primers FL-571F and FL-662R, as described (Liveris et al. 2011b).

Statistics

Categorical variables were compared by the Fisher’s exact test (two-tailed). A p value <0.05 was considered to be significant.

Results

The 66 patients in this study were adults between the ages of 19 and 84 years, 36 (54.5%) of whom were male. Fifty-two (78.8%) of the 66 patients had all five direct diagnostic tests performed namely culture and nPCR of a 2 mm skin biopsy specimen, nPCR and qPCR done on the same 1 mL aliquot of plasma and qPCR-blood culture. Some of the results have been previously reported (Liveris et al. 2011a; Liveris et al. 2011B).

Culture was more sensitive than PCR for both skin and plasma, but the difference was only statistically significant for plasma using the qPCR-blood culture method (p< 0.005). It should be noted that the conventional culture method was used for skin biopsy samples in which a culture was determined to be positive solely based on microscopic detection of spirochetes in culture medium after periods of incubation of up to 8 weeks (Table 1). PCR and culture were both more sensitive on plasma samples of patients with multiple skin lesions compared to those with a single lesion. A statistically significant difference was observed, however, for only the comparison of qPCR-blood culture of patients with multiple EM skin lesion compared to patients with a single lesion (22/23 with multiple EM skin lesions were positive versus 24/42 with a single EM skin lesion, p=0.001). Only four patients (6.1%) were negative on all of the tests that were performed, three of whom had a single EM skin lesion. Of the 62 patients (93.9%) with at least one positive test result, all but 7 (11.3%) had positive test results on more than one of the tests performed. For six of the seven with just a single positive test all five assays were performed.

Table 1.

Comparison of PCR and Culture Tests in Adult Patients with EM

Sample Tested Method of Testing P value
All EM Patients n=66
nPCR Standard culture
Skin 23/54* (42.6%) 34/55 (61.8%) 0.06
nPCR qPCR Either nPCR or qPCR qPCR-blood culture
Plasma 26/64 (40.6%) 22/65 (33.8%) 29/65 (44.6%) 46/65 (70.8%) 0.004**
Multiple EM Patients n=23
nPCR Standard culture
Skin 7/19 (36.8%) 9/20 (45.0%) 0.75
nPCR qPCR Either nPCR or qPCR qPCR-blood culture
Plasma 11/23 (47.8%) 9/23 (39.1%) 14/23 (60.9%) 22/23 (95.7%) 0.01**
Single EM Patients n=43
nPCR Standard culture
Skin 16/35 (45.7%) 25/35 (71.4%) 0.051
nPCR qPCR Either nPCR or qPCR qPCR-blood culture
Plasma 15/41 (36.6%) 13/42 (31.0%) 15/42 (35.7%) 24/42 (57.1%) 0.08**
*

Number positive/number tested

**

For the comparison of the frequency of a positive qPCR-blood culture with the frequency of a positive test result for either nPCR or qPCR directly on plasma

Fifty-two patients had all five assays performed, 48 (92.3%) of whom had at least one positive test result (Table 2). Positive test results among these 48 patients invariably included either a positive qPCR-blood culture or a positive skin culture. Although it could be argued that the 4 patients with negative assays were misdiagnosed and did not actually have active B. burgdorferi infection, this would seem unlikely, since 3 were found to be seropositive for antibodies to B. burgdorferi on acute or convalescent phase testing (data not shown).

Table 2.

Frequency of Positive Test Results for the 52 Patients with EM for Whom All Five Assays were Performed.

Positive on at least one assay 48 (92.3%)
Positive qPCR-blood culture 39 (75.0%)
Positive nPCR plasma 20 (38.5%)
Positive qPCR plasma 16 (30.8%)
Positive nPCR of skin 22 (42.3%)
Positive culture of skin 32 (61.5%)
Positive qPCR-blood culture or positive skin culture 48 (92.3%)
Positive on exactly 5 assays 4 (7.7%)
Positive on exactly 4 assays 8 (15.4%)
Positive on exactly 3 assays 13 (25.0%)
Positive on exactly 2 assays 15 (28.8%)
Positive on exactly 1 assay 8 (15.4%)

Discussion

Numerous published studies have reported on the utility of culture or PCR for detection of B. burgdorferi in clinical specimens (Aguero-Rosenfeld et al. 2005; Wang et al. 2010). In general, cultivation of skin biopsies obtained from EM lesions of untreated patients resulted in >50% positivity; the highest reported culture yield in US patients was 86% in a study that employed 4 mm skin biopsies (Berger et al. 1992). In two earlier published series from our group, culture of 2 mm skin biopsies was positive in 57% (Schwartz et al. 1992) and 54% (Liveris et al. 2002) of patients, consistent with the 61.5% yield obtained in the present study. PCR of skin biopsy specimens has typically resulted in a higher positivity rate than culture; the median sensitivity in 17 published studies was 69% (Wang et al. 2010). We have previously reported positive PCR results in 59% and 64% of skin biopsy specimens (Schwartz et al. 1992; Liveris et al. 2002), a rate essentially identical to that obtained with culture. In the current study, only 42.6% of skin biopsy specimens were positive by nested PCR. The reason for this discrepancy is not clear, but is not likely to be due to PCR inhibitors present in the processed specimens, as the procedures were identical to those employed in our earlier studies that yielded the higher PCR positive rates. Furthermore, samples were processed within 12–24 hours of collection and were kept refrigerated until processing, making degradation of DNA unlikely.

Twenty-nine of 65 (44.6%) plasma samples were positive by a PCR method (either nested or quantitative PCR). This is similar to a previous report by Jones et al. (Jones et al. 2006) and higher than that reported in several other studies (Aguero-Rosenfeld et al. 2005; Goodman et al. 1995). This value is also consistent with the positivity rate obtained by culture using the conventional end-point of microscopic detection of spirochetes in the culture medium (Nowakowski et al. 2001). Of note, the novel qPCR-blood culture technique used in this study, in which a positive culture was determined based on a positive qPCR assay of culture aliquots rather than microscopic detection, resulted in 70.8% rate of positive blood cultures overall, which increased to 95.7% (22/23) in the subgroup of patients with multiple EM skin lesions.

Direct detection methods such as PCR and culture have the advantage of high specificity and can assist in classifying and characterizing the strains of B. burgdorferi causing the infection. As shown here and as in discussed in more detail elsewhere (Liveris et al. 2011a), the qPCR-culture method of plasma was able to identify evidence of borrelial DNA in plasma of all but one of the patients with multiple EM skin lesions further reinforcing the concept that secondary EM skin lesions arise by hematogenous dissemination.

The results of this study demonstrate that direct detection methods such as PCR and culture are highly sensitive in untreated adult patients with EM allowing microbiologic or molecular microbiologic confirmation of the diagnosis of B. burgdorferi infection in 94% of the 66 patients evaluated. The most efficient way to identify B. burgdorferi infected patients was through the use of the qPCR-blood culture of plasma in combination with skin culture.

Acknowledgments

This work was supported by NIH grant AR41511 to IS.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP. Diagnosis of Lyme borreliosis. Clin Microbiol Rev. 2005;18:484–509. doi: 10.1128/CMR.18.3.484-509.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bacon RM, Kugeler KJ, Mead PS. Surveillance for Lyme disease--United States, 1992-2006. MMWR Surveill Summ. 2008;57:1–9. [PubMed] [Google Scholar]
  3. Berger BW, Johnson RC, Kodner C, Coleman L. Cultivation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin. J Clin Microbiol. 1992;30:359–361. doi: 10.1128/jcm.30.2.359-361.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dandache P, Nadelman RB. Erythema migrans. Infect Dis Clin North Am. 2008;22:235–60. doi: 10.1016/j.idc.2007.12.012. [DOI] [PubMed] [Google Scholar]
  5. Goodman JL, Bradley JF, Ross AE, Goellner P, Lagus A, Vitale B, Berger BW, Luger S, Johnson RC. Bloodstream invasion in early Lyme disease: results from a prospective, controlled, blinded study using the polymerase chain reaction. Am J Med. 1995;99:6–12. doi: 10.1016/s0002-9343(99)80097-7. [DOI] [PubMed] [Google Scholar]
  6. Jones KL, Glickstein LJ, Damle N, Sikand VK, McHugh G, Steere AC. Borrelia burgdorferi genetic markers and disseminated disease in patients with early Lyme disease. J Clin Microbiol. 2006;44:4407–4413. doi: 10.1128/JCM.01077-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Liveris D, Schwartz I, Bittker S, Cooper D, Iyer R, Cox ME, Wormser GP. Improving the yield of blood cultures from patients with early Lyme disease. J Clin Microbiol. 2011a;49:2166–2168. doi: 10.1128/JCM.00350-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Liveris D, Schwartz I, McKenna D, Nowakowski J, Nadelman RB, Demarco J, Iyer R, Cox ME, Holmgren D, Wormser GP. Quantitation of cell-associated borrelial DNA in the blood of Lyme disease patients with erythema migrans. Eur J Clin Microbiol Infect Dis. 2011b Aug 16; doi: 10.1007/s10096-011-1376-x. 2011. [Epub ahead of print] [DOI] [PubMed] [Google Scholar]
  9. Liveris D, Wang G, Girao G, Byrne DW, Nowakowski J, McKenna D, Nadelman RB, Wormser GP, Schwartz I. Quantitative detection of Borrelia burgdorferi in 2-millimeter skin samples of erythema migrans lesions: correlation of results with clinical and laboratory findings. J Clin Microbiol. 2002;40:1249–1253. doi: 10.1128/JCM.40.4.1249-1253.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nowakowski J, Schwartz I, Liveris D, Wang G, Aguero-Rosenfeld ME, Girao G, McKenna D, Nadelman RB, Cavaliere LF, Wormser GP. Laboratory diagnostic techniques for patients with early Lyme disease associated with erythema migrans: a comparison of different techniques. Clin Infect Dis. 2001;33:2023–2027. doi: 10.1086/324490. [DOI] [PubMed] [Google Scholar]
  11. Schwartz I, Bittker S, Bowen SL, Cooper D, Pavia C, Wormser GP. Polymerase chain reaction amplification of culture supernatants for rapid detection of Borrelia burgdorferi. Eur J Clin Microbiol Infect Dis. 1993;12:879–882. doi: 10.1007/BF02000415. [DOI] [PubMed] [Google Scholar]
  12. Schwartz I, Wormser GP, Schwartz JJ, Cooper D, Weissensee P, Gazumyan A, Zimmermann E, Goldberg NS, Bittker S, Campbell GL, Pavia CS. Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions. J Clin Microbiol. 1992;30:3082–3088. doi: 10.1128/jcm.30.12.3082-3088.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Wang G, Aguero-Rosenfeld ME, Wormser GP, Schwartz I. Detection of Borrelia burgdorferi. In: Samuels DS, Radolf JD, editors. Borrelia: Molecular Biology, Host Interaction and Pathogenesis. Caister Academic Press; Norfolk, UK: 2010. pp. 443–466. [Google Scholar]

RESOURCES