Susan Bittker

The clinical spectrum of early lyme borreliosis in patients with culture-confirmed erythema migrans

BACKGROUND The diagnosis of erythema migrans (EM), the characteristic rash of early Lyme borreliosis, is based primarily on its clinical appearance since it often occurs prior to the development of a specific antibody response. Other skin disorders, however, may be confused with EM. METHODS Between June 1991 and September 1993, a prospective study was conducted at the Lyme Disease Diagnostic Center of the Westchester County Medical Center to isolate Borrelia burgdorferi systematically from patients with Em, and to characterize the clinical manifestations of patients with culture-documented infection. Skin biopsies and/or needle aspirates of the advancing margin of primary lesions, and blood specimens from adult patients were cultured for B burgdorferi in modified Barbour-Stoenner-Kelly medium at 33 degrees C. RESULTS B burgdorferi was recovered from 79 patients (49 [62%] males) ranging in age from 16 to 76 years old (mean, 43 +/- 14 years old). Maximum EM diameter (mean, 16 +/- 10 cm; range, 6-73 cm) was a function of EM duration (mean 6.7 +/- 6.4 days; range, 1-39 days) (correlation coefficient = 0.7; P < 0.001). Twenty (25%) patients had noted a tick bite at the site of the primary lesion a mean of 10 days (range, 1-27 days) before onset. Multiple EM lesions (range, 2-70) were present in 14 (18%) patients. Systemic symptoms were present at the time of culture in 54 patients (68%) including fatigue (54%), arthralgia (44%), myalgia (44%), headache, (42%), fever and/or chills (39%), stiff neck (35%), and anorexia (26%). Thirty-three patients (42%) had at least one objective finding on physical examination in addition to EM, including 18 (23%) with localized lymphadenopathy, 13 (16%) with fever (t > or = 37.8 degrees C), seven (9%) with tender neck flexion, six (8%) with joint tenderness, and 1 each with joint swelling, nuchal rigidity, and facial nerve palsy. No patient had new electrocardiogram evidence of atrioventricular block. Liver function assays were abnormally elevated in 37% of patients. Thirty-four percent of patients were seropositive by enzyme-linked immunosorbent assay at presentation. Most others rapidly seroconverted so that 69 of 78 evaluable patients (88%) were seropositive at some point during the first month after diagnosis. CONCLUSIONS We describe the largest group of culture-positive patients with EM from the United States to date. Although systemic symptoms were present in most patients, objective evidence of advanced disease was uncommon. Our patients with culture-confirmed EM were less sick than those described in the days before culture confirmation was possible. The ability to isolate B burgdorferi from lesional skin of large numbers of patients with EM should make culture-positive patients the standard by which to define manifestations of early Lyme borreliosis associated with this rash. Microbiologic documentation of Lyme borreliosis will help delineate the manifestations of this illness, and should form the framework for research directed at pathophysiology, diagnosis, treatment, and prevention.

Improving the Yield of Blood Cultures from Patients with Early Lyme Disease

ABSTRACT Approximately 45% of untreated United States patients with early Lyme disease associated with erythema migrans have a positive blood culture based on microscopic detection of Borrelia burgdorferi in Barbour-Stoenner-Kelly medium after 2 to 12 weeks of incubation. In this study we demonstrate that the yield of blood cultures can be significantly increased to 70.8% by the use of a combined culture-quantitative PCR technique and that among those patients found to have a positive blood culture, positivity was detected in over 90% within just 7 days of incubation. Patients with multiple erythema migrans were almost uniformly culture positive by this technique.Approximately 45% of untreated United States patients with early Lyme disease associated with erythema migrans have a positive blood culture based on microscopic detection of Borrelia burgdorferi in Barbour-Stoenner-Kelly medium after 2 to 12 weeks of incubation. In this study we demonstrate that the yield of blood cultures can be significantly increased to 70.8% by the use of a combined culture-quantitative PCR technique and that among those patients found to have a positive blood culture, positivity was detected in over 90% within just 7 days of incubation. Patients with multiple erythema migrans were almost uniformly culture positive by this technique.

Microbiologic Evaluation of Patients from Missouri with Erythema Migrans

BACKGROUND Borrelia lonestari infects Amblyomma americanum, the tick species that is the most common cause of tick bites in southeast and south-central United States, and this spirochete has been detected in an erythema migrans (EM)-like skin rash in 1 patient. Therefore, B. lonestari is considered to be a leading candidate for the etiologic agent of EM in this region. METHODS Skin biopsy specimens obtained from patients from the Cape Girardeau area of Missouri who had EM-like lesions were cultured in Barbour-Stoenner-Kelly medium and evaluated by polymerase chain reaction (PCR) targeting multiple genes. Serum specimens were tested by enzyme-linked immunosorbent assay for antibodies against sonicated whole-cell Borrelia burgdorferi. Results were compared with those obtained over the same period for patients from New York State who had EM. RESULTS B. lonestari was not detected by PCR in any of 31 skin biopsy specimens collected from 30 Missouri patients. None of 19 cultures of Missouri skin samples that were suitable for evaluation were positive for B. burgdorferi, compared with 89 (63%) of 142 cultures of samples collected from New York State patients (P<.001). None of the 25 evaluable Missouri patients were seropositive for antibodies against B. burgdorferi, compared with 107 (75%) of 143 New York State patients (P<.001). CONCLUSIONS Neither B. lonestari nor B. burgdorferi is likely to be the cause of EM-like skin lesions in patients from the Cape Girardeau area of Missouri. The etiology of this condition remains unknown.

Yield of Large-Volume Blood Cultures in Patients with Early Lyme Disease

To improve yield, 6 3-mL plasma cultures (18 mL total) were established for adult patients with early Lyme disease associated with erythema migrans. Borrelia burgdorferi was recovered from the blood of 22 (44.0%) of 50 evaluable patients. The recovery rate per plasma culture and the frequency of positive results for plasma cultures for individual patients were consistent with a level of spirochetemia of approximately 0.1 cultivable cell/mL of whole blood. Our findings suggest that, if further improvements in the yield of blood cultures are possible, they probably will depend on enhancing the sensitivity of the culture method rather than increasing the volume of material cultured.

Differentiation of Reinfection from Relapse in Recurrent Lyme Disease

BACKGROUND Erythema migrans is the most common manifestation of Lyme disease. Recurrences are not uncommon, and although they are usually attributed to reinfection rather than relapse of the original infection, this remains somewhat controversial. We used molecular typing of Borrelia burgdorferi isolates obtained from patients with culture-confirmed episodes of erythema migrans to distinguish between relapse and reinfection. METHODS We determined the genotype of the gene encoding outer-surface protein C (ospC) of B. burgdorferi strains detected in cultures of skin or blood specimens obtained from patients with consecutive episodes of erythema migrans. After polymerase-chain-reaction amplification, ospC genotyping was performed by means of reverse line-blot analysis or DNA sequencing of the nearly full-length gene. Most strains were further analyzed by determining the genotype according to the 16S-23S ribosomal RNA intergenic spacer type, multilocus sequence typing, or both. Patients received standard courses of antibiotics for erythema migrans. RESULTS B. burgdorferi isolates obtained from 17 patients who received a diagnosis of erythema migrans between 1991 and 2011 and who had 22 paired episodes of this lesion (initial and second episodes) were available for testing. The ospC genotype was found to be different at each initial and second episode. Apparently identical genotypes were identified on more than one occasion in only one patient, at the first and third episodes, 5 years apart, but different genotypes were identified at the second and fourth episodes. CONCLUSIONS None of the 22 paired consecutive episodes of erythema migrans were associated with the same strain of B. burgdorferi on culture. Our data show that repeat episodes of erythema migrans in appropriately treated patients were due to reinfection and not relapse. (Funded by the National Institutes of Health and the William and Sylvia Silberstein Foundation.).

Failure to isolate borrelia burgdorferi after antimicrobial therapy in culture-documented Lyme borreliosis associated with erythema migrans: Report of a prospective study

BACKGROUND Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, has occasionally been isolated from tissues or body fluids of patients after antimicrobial treatment. A prospective study of patients with Lyme borreliosis associated with erythema migrans (EM) was initiated in Westchester County, New York, to determine: (1) the clinical and laboratory parameters associated with culture positivity, and (2) the microbiologic response to treatment. METHODS Skin biopsies were performed in patients with EM and cultured for B. burgdorferi in modified Barbour-Stoenner-Kelly medium at 33 degrees C. Subsequent biopsies for culture were performed adjacent to the original biopsy site for culture-positive patients after the completion of antimicrobial therapy. RESULTS Initial biopsy cultures were performed for 44 patients; 6 were unevaluable due to culture contamination with other bacteria. Cultures were positive in 21 of 29 patients prior to treatment (72%), but in none of 9 patients during treatment (p < 0.001). The only other identified factor associated with successful recovery of B. burgdorferi was shorter duration of EM. When patients who had received prior antimicrobial therapy were excluded, the mean duration of the EM lesion for those with positive cultures was 5.0 +/- 5.2 days compared with 14.6 +/- 9.9 days for those with negative cultures (p < 0.01). B. burgdorferi could not be reisolated from any of 18 evaluable subsequent biopsies of skin from 13 culture-positive patients 4 to 209 days after completion of a course of antimicrobial therapy. Five patients had negative subsequent biopsy cultures on two separate occasions 3 to 5 months apart. CONCLUSIONS After brief courses of antibiotics, B. burgdorferi appears to be rapidly eliminated from the skin at EM sites. The ability to recover B. burgdorferi from skin biopsy cultures of untreated patients with EM lesions wanes with increasing duration of EM, suggesting that this organism may also be spontaneously cleared from skin over time.

A Decline in C6 Antibody Titer Occurs in Successfully Treated Patients with Culture-Confirmed Early Localized or Early Disseminated Lyme Borreliosis

ABSTRACT C6, a Borrelia burgdorferi-derived peptide, is used as the antigen in the C6-Lyme disease diagnostic test. We assessed retrospectively whether a fourfold decrease or a decrease to a negative value in anti-C6 antibody titer is positively correlated with a positive response to treatment in a sample of culture-confirmed patients with either early localized (single erythema migrans [EM]; n = 93) or early disseminated (multiple EM; n = 27) disease. All of these patients had been treated with antibiotics and were free of disease within 6 to 12 months of follow-up. Results show that a serum specimen taken at this time was either C6 negative or had a ≥4-fold decrease in C6 antibody titer with respect to a specimen taken at baseline (or during the early convalescent period if the baseline specimen was C6 negative) for all of the multiple-EM patients (P < 0.0001) and in 89% of the single-EM patients (P < 0.0001). These results indicate that a decline in anti-C6 antibody titer coincides with effective antimicrobial therapy in patients with early localized or early disseminated Lyme borreliosis.

Variations in Barbour-Stoenner-Kelly Culture Medium Modulate Infectivity and Pathogenicity of Borrelia burgdorferi Clinical Isolates

ABSTRACT The effects of variations in Barbour-Stoenner-Kelly (BSK) medium on the infectivity and pathogenicity of Borrelia burgdorferi clinical isolates were assessed by retrospective and prospective studies using a murine model of Lyme borreliosis. Thirty of 35 (86%) mice infected with any of six virulent B. burgdorferi clinical isolates grown in a BSK-H medium developed clinically apparent arthritis. By contrast, arthritis was observed in only 25 of 60 (42%) mice inoculated with two of these B. burgdorferi strains grown in a different lot of BSK-H medium (P < 0.001). In a prospective study, mice inoculated with a B. burgdorferi clinical isolate grown in a BSK medium prepared in-house produced significantly greater disease than those injected with the same isolate cultured in BSK-H medium (P < 0.05). The attenuated pathogenicity is not due to the loss of plasmids during in vitro cultivation. The data suggest that variations in BSK medium have a significant impact on the infectivity and pathogenicity of B. burgdorferi clinical isolates.

Comparison of five diagnostic modalities for direct detection of Borrelia burgdorferi in patients with early Lyme disease.

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 5 direct diagnostic methods (culture and nested polymerase chain reaction [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. Results of one or more of 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 5 assays were performed invariably included either a positive blood or a 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 4 (6.1%) of the 66 patients evaluated.

Blood Cultures for Patients with Extracutaneous Manifestations of Lyme Disease in the United States

Spirochetemia in US patients with extracutaneous manifestations of Lyme disease is not well documented. In this study, blood culture results were positive for 5 (19.2%; 95% confidence interval, 6.6%-39.4%) of 26 untreated adult patients with extracutaneous manifestations but only for patients with clinical evidence for a short duration of infection.