Serum samples were obtained from 84 of 86 enrolled patients, including acute-phase samples from all, and convalescent-phase samples from 47 were obtained at a median of day 29 after the acute-phase sample. At least one serologic result was available for 80 of the 82 patients who had plasma cultures performed, for 47 who had skin cultures, for 57 patients who had plasma PCR, and for 23 who had skin PCR. Among 23 patients who had skin PCR, plasma/skin culture and serologic results were available for 22, and culture results only were available for one other patient. Combining acute and convalescent serologic results, 36 of 84 (43%) were seropositive. Among acute-phase samples, 21 (25%) were seropositive, including 8 with IgG and 17 with IgM by immunoblotting. Among the 47 convalescent-phase sera, 25 (53%) were seropositive (6 IgG and 20 IgM). Only 47 of 84 (56%) patients returned for serologic testing, suggesting a potential bias for patients with clear features of Lyme disease, such as EM. However, the proportions of patients with and without EM who returned for convalescent serology were similar (P = 0.61; χ2 test). Serologic results among culture- and PCR-positive individual patients follow.
Plasma, skin, and CSF cultures.
Plasma cultures from 82 patients were prepared, including triplicate cultures for 70 and duplicate cultures for 12. Plasma was not received for four additional enrolled patients. B. burgdorferi was cultivated from 22 patient plasma specimens (27%), including 8 for whom all three flasks were positive, 2 for whom both of the two inoculated flasks were positive, 4 for whom two of three cultures were positive, 1 for whom one of two cultures was positive, and 7 for whom one of three cultures was positive. Of the remaining 60 patients, 182 flasks had no growth throughout the 8 weeks, and 8 flasks were discarded for heavy bacterial contamination, including all 3 flasks for one patient only. The time to recovery from plasma ranged from 7 to 49 days, with a mean time to culture detection of 24 days. The mean interval before processing and cultivation was similar between culture-positive and culture-negative patients (1.06 days ± 1.11 standard deviation [SD] versus 1.02 days ± 0.99 SD; P = 0.90) and between culture-positive samples and culture-negative, seropositive patients (1.06 days ± 1.11 SD versus 1.32 days ± 1.07 SD; P = 0.44). Among the 22 patients with positive plasma cultures, all had either localized or disseminated EM skin lesions, and a description was available for 20. Of these, 12 were described as typical (central erythema, central clearing, or homogeneous), 7 were described as atypical (blue, vesicle, punctum, or size of <5 cm), and for one patient there were multiple lesions.
The average symptom duration among plasma culture-positive patients was 6 days (median, 4 days), whereas for those who were plasma culture negative, this interval was 64 days (median, 10 days). Among the 79 patients who had both serologic testing and plasma culture results available, 17 (22%) were both seropositive and culture positive, 18 (23%) were seropositive but culture negative, 4 (5%) were seronegative and culture positive, and 40 (51%) were negative for both.
Skin biopsies were obtained for culture from 47 patients; 15 (32%) cultures grew B. burgdorferi. Mean time to recovery of B. burgdorferi from skin biopsies was 25 days, with a range of 14 to 56 days. Among these, 9 (19%) were both seropositive and culture positive, 6 (13%) were seronegative and culture positive, 16 (34%) were seropositive and culture negative, and 16 (34%) were negative for both. Among 49 patients for whom the character of a skin lesion was reported, B. burgdorferi was cultured from skin biopsy of 7 of 28 (25%) with typical EM, 5 of 17 (29%) with atypical EM, and all 3 (100%) with multiple EM. Of these, other laboratory evidence of Lyme disease was obtained for 23 (30% skin culture positive) with typical EM, 11 (45% skin culture positive) with atypical EM, and all 3 with multiple EM. Of the three CSF specimens, one grew B. burgdorferi, one culture had no growth, and one culture was contaminated and unable to be analyzed.
Overall, when plasma and skin culture results were combined for the 86 patients, 30 (35%) were either plasma or skin culture positive, including 8 from both plasma and skin, 14 from plasma only, 7 from skin only, and 1 from both skin and CSF. Of the 46 patients with both plasma and skin cultures attempted, 25 (54%) were positive in either, while 8 were positive in both, 10 were positive only in plasma, and 7 were positive only in skin. The remaining 21 (46%) were negative in both skin and plasma. However, among the 84 patients with both serologic and culture results available, 21 (25%) were both seropositive and culture positive, 7 (8%) were seronegative but culture positive, 15 (18%) were seropositive and culture negative, and 41 (49%) were both seronegative and culture negative.
All positive plasma and skin cultures were confirmed to contain B. burgdorferi DNA by PCR. By quantitative PCR, of 57 plasma samples tested, only 2 had sufficient B. burgdorferi DNA present for detection (2.1 × 105 and 2.4 × 105 borreliae/ml); both patients were seropositive but neither had B. burgdorferi isolated from plasma. Thus, of 57 plasmas tested, B. burgdorferi DNA was detected in 2 of 33 (6%) culture-positive or seropositive patients and in none of 16 from patients who were culture positive only; one plasma tested by PCR was contaminated and could not be cultured.
Of the 47 skin biopsies obtained, sufficient residual material was available to conduct quantitative PCR on 23. Among these, 9 were seronegative and culture negative, 4 were B. burgdorferi skin culture positive, 10 were B. burgdorferi plasma culture positive (3 had B. burgdorferi isolated from both skin and plasma), 13 were seropositive, and 14 (61%) were positive by either culture or serology. Of the 23 tested, 10 were positive by skin biopsy PCR, including all 4 who were skin biopsy culture positive, 5 who were seropositive, 5 who were plasma culture positive, and 6 who were either skin/plasma culture positive or seropositive. Compared to the combined culture and serology results, 6 (26%) were both skin PCR and sero/culture positive, 8 (35%) were skin PCR negative but sero/culture positive, and 5 (22%) were negative for all three tests. Of interest, 4 (17%) patients with B. burgdorferi DNA detected in skin were both seronegative and skin/plasma culture negative. All four of these skin biopsies were confirmed to contain B. burgdorferi genomic DNA based upon separate amplification of ospA.
Comparisons with qualitative clinical assessments.
Qualitative clinical assessments were made prospectively for 82 patients, of whom 25 were classified as unlikely, 32 as possible, and 25 as probable to have Lyme disease. Overall, initial serologic tests agreed with possible or probable clinical Lyme disease diagnosis in only 50% (40/80) of cases, increasing to 69% (55/80) when follow-up serologic tests were included (Table ). Agreement of individual tests with initial possible or probable clinical Lyme disease diagnosis was otherwise poor, including skin culture (41% [19/46 cases]) and skin PCR (48% [11/23 cases]), except for plasma culture (59% [46/79 cases]). The combinations of tests that agreed best with initial possible or probable clinical Lyme disease diagnosis were serology, any culture, or skin PCR at 82% (67/82 patients), serology or skin PCR at 75% (60/80 patients), and plasma or skin culture at 66% (54/82 patients).
Agreement of laboratory diagnostic tests with initial clinical assessment for Lyme disease
Figure shows a comparison of results using various methodologies. Since no single method was able to identify all patients with objective laboratory evidence of Lyme disease, we sought to identify a profile of diagnostic tests that would provide the highest possible sensitivity. Thus, we defined a reference Lyme disease patient population based upon one or more of the accepted diagnostic tests (culture, serology per CDC/Association of State and Territorial Public Health Laboratory Directors criteria [2
], and PCR of skin or plasma confirmed by targeting two B. burgdorferi
genes). Not all tests were obtained on all patients; thus, denominators varied for purposes of calculating proportions and were based upon the number of patients who had the test and who were either seropositive, plasma or skin culture positive, or skin biopsy PCR positive.
FIG. 1. Number of patients tested by each method who had laboratory evidence of Lyme disease and who were positive (black bars) or negative (gray bars) by each test or test combination compared with the total number of enrolled subjects for whom each test was (more ...)
Of the applied methods, plasma PCR tests were performed on 57 patients, of whom 33 were positive by either culture or serology and 2 were positive only by skin biopsy PCR. Among these, the plasma PCR proved to be the least sensitive diagnostic procedure, identifying only 6% (2 of 35) of patients with laboratory evidence of Lyme disease, and was never the sole positive test. The most sensitive diagnostic test remained application of acute and convalescent serology (47 patients), the frequent reference for Lyme disease laboratory diagnosis, which was 77% (36/47; 95% confidence interval [CI], 62 to 88%) sensitive, followed by cumulative plasma and skin culture at 60% (29/48; 95% CI, 45 to 74%), skin biopsy PCR at 56% (10/18; 95% CI, 31 to 79%), plasma culture alone at 47% (22/47; 95% CI, 32 to 62%), and skin culture alone at 42% (15/36; 95% CI, 26 to 60%). With the exception of plasma PCR, any of the described methods when combined with one or more methods increased the positive percentage rate. The most sensitive approach proved to be the combination of acute and convalescent serology with skin biopsy PCR (100% of 18 patients; 95% CI, 82 to 100%), whereas acute and convalescent serology coupled with skin and plasma culture was nearly as sensitive, detecting 92% (44/48; 95% CI, 80 to 98%) of all patients. The sensitivity of acute-phase serology coupled with skin PCR, both which can be analyzed within days of presentation, yields a sensitivity rate of 78% (14/18; 95% CI, 52 to 94%), a marked but not significant improvement over the 45% sensitivity of acute-phase serology alone and unhelpful in the absence of rash.
When patients with any laboratory evidence of Lyme disease were divided into disease phase groups (33 early localized, 12 early disseminated, 3 late), no differences in the proportion of positive tests or combinations described above were found (χ2 test), although too few patients with late Lyme disease were available for critical analysis.