BACKGROUND:Lyme disease patients with erythema migrans are said to have post-treatment Lyme disease symptoms (PTLDS) if there is persistence of subjective symptoms for at least 6 months following antibiotic treatment and resolution of the skin lesion. The purpose of this study was to characterize PTLDS in patients with culture-confirmed early Lyme disease followed for >10 years.METHODS:Adult patients with erythema migrans with a positive skin or blood culture for Borrelia burgdorferi were enrolled in a prospective study beginning in 1991 and followed up at 6 months and annually thereafter to determine the long-term outcome of this infection. The genotype of the infecting strain of B. burgdorferi was evaluated in subjects with PTLDS.RESULTS:One hundred twenty-eight subjects with culture-confirmed early Lyme disease, of whom 55% were male, were followed for a mean ± SD of 14.98 ± 2.71 years (median = 15 years; range = 11-20 years). Fourteen (10.9%) were regarded as having possible PTLDS, but only 6 (4.7%) had PTLDS documented at their last study visit. Nine (64.3%) had only a single symptom. None of the 6 with PTLDS at their last visit was considered to be functionally impaired by the symptom(s). PTLDS was not associated with a particular genotype of B. burgdorferi.CONCLUSIONS:PTLDS may persist for >10 years in some patients with culture-confirmed early Lyme disease. Such long-standing symptoms were not associated with functional impairment or a particular strain of B. burgdorferi.

Although two-tier testing is standard practice in both the United States and Europe for the serologic diagnosis of Lyme borreliosis (LB), the test kits generally differ. The purpose of this study was to determine if the testing used in the United States will detect LB acquired in Europe and vice versa. Testing was performed on a convenience sample of archived sera from 40 LB patients from Austria and 39 from the United States, using first- and second-tier test kits from both the United States and Europe. The sensitivity of four first-tier tests from Europe and two first-tier tests from the United States was similar. Thus, two-tier testing was compared to the C6 ELISA as the first-tier test, since it is licensed in both the United States and Europe. The sensitivity of C6 two-tier testing with US assays was 9/40 (22.5 % [95 % CI 10.8-38.5 %]) for detection of LB acquired in Europe, and just 20.0 % (95 % CI 2.5-55.6 %) in the ten European patients with neurologic involvement. These results differed significantly from the sensitivity of European C6 two-tier testing that was 70.0 % (95 % CI 53.5-83.4 %) overall (p < 0.001) and 90.0 % (95 % CI 55.5-99.7 %) for the European patients with neurologic manifestations specifically (p = 0.016). In contrast, the sensitivity of European and US C6 two-tier testing was similar for detection of LB acquired in the United States. Two-tier serologic testing with the US test kits may be unsatisfactory for detection of LB acquired in Europe. First-tier testing with an assay such as the C6 ELISA should be considered as a stand-alone diagnostic strategy in such cases.

ABSTRACT Lyme disease is transmitted by the bite of certain Ixodes ticks, which can also transmit Anaplasma phagocytophilum, the cause of human granulocytic anaplasmosis (HGA). Although culture can be used to identify patients infected with A. phagocytophilum and is the microbiologic gold standard, few studies have evaluated culture-confirmed patients with HGA. We conducted a prospective study in which blood culture was used to detect HGA infection in patients with a compatible clinical illness. Early Lyme disease was defined by the presence of erythema migrans. The epidemiologic, clinical, and laboratory features of 44 patients with culture-confirmed HGA were compared with those of a convenience sample of 62 patients with early Lyme disease. Coinfected patients were excluded. Patients with HGA had more symptoms (P = 0.003) and had a higher body temperature on presentation (P < 0.001) than patients with early Lyme disease. HGA patients were also more likely to have a headache, dizziness, myalgias, abdominal pain, anorexia, leukopenia, lymphopenia, thrombocytopenia, or elevated liver enzymes. A direct correlation between the number of symptoms and the duration of illness at time of presentation (rho = 0.389, P = 0.009) was observed for HGA patients but not for patients with Lyme disease. In conclusion, although there are overlapping features, culture-confirmed HGA is a more severe illness than early 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.).

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.

A prior study in mice has shown that the timely application of topical antibiotics to the skin at the tick bite site could eradicate Borrelia burgdorferi infection. That study, however, did not evaluate antibiotic preparations that are considered suitable for use in humans. In this murine study, topical application of 2% erythromycin and 3% tetracycline preparations that are acceptable for use in humans was found to be ineffective in eliminating B. burgdorferi from the tick bite site or in preventing dissemination to other tissues. Reasons for the discrepant findings are discussed.

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.

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.

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.

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.

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.

ABSTRACT Genetic diversity among Borrelia burgdorferi isolates recovered from the skin of Lyme disease patients was assessed by ribosomal DNA (rDNA) spacer restriction fragment length polymorphism analysis, genomic restriction site polymorphism analysis, and plasmid content analysis. There was a significant association between the three rDNA spacer types, the six pulsed-field gel types, and plasmid content ( P < 0.001). The association between distinct chromosomal and plasmid markers implies a clonal origin for each genotype.

An indirect hemagglutination antibody (IHA) test was evaluated for its ability to detect borrelial antibodies in serum samples from patients with Lyme disease. The key test reagent developed for this antibody detection system was tannic acid-treated and glutaraldehyde-fixed sheep red blood cells (SRBC) containing Borrelia burgdorferi (Bb) antigens attached to the outer surface of the SRBC. In order to establish suitable cut-off titers, initial specificity and sensitivity measurements were made using sera from 100 anonymous healthy volunteers and 30 additional pre-determined samples known to be non-reactive or reactive for Lyme disease or syphilis. These results were compared with those obtained using a commercially available ELISA. At titers ≥64, the IHA test had a combined 98% specificity and 100% sensitivity for these 130 serum samples, 30 of which were known positives or negatives, whereas the ELISA was less specific (93%) and much less sensitive (80%). Subsequent testing was performed on sera from 65 patients with the erythema migrans (EM) rash and 20 patients with early disseminated (cardiac/neurologic) symptoms or with Lyme arthritis. At initial presentation, 46–48% of the EM patients had IHA reactivity, with titers ≥128, while 42% were positive in the ELISA. Follow-up testing performed on these EM patients, 8–12 days after receiving antibiotic treatment, revealed that Bb antibodies were detected best by the IHA test (83–86% reactive) relative to the ELISA (81% reactive). Bb antibodies were readily detectable on all of the serum samples from the early disseminated and late stage Lyme disease cases in both assay systems. Based on these results and because of its technical and interpretive simplicity, the IHA test should be considered as a useful and convenient alternative for the serological analysis of Bb infections.

CONTEXT:Lyme disease typically presents with a skin lesion called erythema migrans (EM), which though often distinctive in appearance may be confused with cellulitis. The first-generation cephalosporin, cephalexin monohydrate, is effective for treating bacterial cellulitis but has not been recommended or studied for treating Lyme disease because of poor in vitro activity.OBJECTIVE:To describe the outcome of patients with EM who were treated with cephalexin.PATIENTS AND METHODS:Patients presenting with EM to the Lyme Disease Diagnostic Center in Westchester, NY (May 1992-September 1997). A 2-mm punch biopsy specimen of the leading edge of the EM lesion and/or blood was cultured for Borrelia burgdorferi.RESULTS:Eleven (2.8%) of 393 study patients had been initially treated with cephalexin prior to our evaluation; 9 (82%) were originally diagnosed with cellulitis. Cephalexin was administered for 8.6 days (range, 2-21 days) prior to presentation. All 11 patients had clinical evidence of disease progression, including 8 whose rash enlarged, 2 who developed seventh-nerve palsy (1 with new EM lesions), and 1 who developed new EM lesions. Borrelia burgdorferi grew in cultures from 5 patients despite a mean of 9.8 days of treatment with cephalexin (range, 5-21 days).CONCLUSION:Cephalexin should not be used to treat early Lyme disease and should be prescribed with caution during the summer months for patients believed to have cellulitis in locations where Lyme disease is endemic.

Polymerase chain reaction is often used for detection of Borrelia burgdorferi in biological specimens. It has been suggested that polymerase chain reaction may be used as a surrogate marker of cell viability. To test this premise, B. burgdorferi cultures were treated with the antibiotic, ceftriaxone, and aliquots were cultured for cell viability and tested by polymerase chain reaction. Ceftriaxone treatment abrogated the ability to subculture B. burgdorferi by three days post-treatment. In contrast, positive polymerase chain reaction results were obtained for up to 56 days after antibiotic treatment. These findings indicate that positive polymerase chain reaction results do not provide proof of bacterial cell viability in vitro.

ABSTRACT Two hundred seventeen isolates of Borrelia burgdorferi originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of B. burgdorferi performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [ P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.

ABSTRACT This study was designed to improve the recovery of Borrelia burgdorferi from blood. With the techniques used, B. burgdorferi could be recovered from the blood of approximately 25% of patients with early Lyme disease associated with erythema migrans. Serum was a better source of culture material than whole blood. The volume of blood cultured correlated directly with yield, particularly for patients with a single erythema migrans lesion.

We investigated the appearance and evolution of immunoglobulin M (IgM) and IgG antibodies to Borrelia burgdorferi in 46 patients with culture-proven erythema migrans (EM). All patients received antimicrobial treatment and were prospectively evaluated for up to 1 year. A total of 257 serially collected serum samples were tested by commercial IgG-IgM enzyme-linked immunosorbent assay and separate IgM and IgG immunoblots (IBs). At the baseline, 33% of the patients had a positive ELISA result and 43% of the patients had a positive IgM IB result by using the criteria of the Centers for Disease Control and Prevention-Association of State and Territorial Public Health Laboratory Directors for the interpretation of IB results. Positive serology at the baseline and the rate of seroconversion correlated directly with disease duration and/or evidence of dissemination prior to treatment. At days 8 to 14 after the baseline, 91% of patients had a positive ELISA result and/or IgM IB result. Peak IgM antibody levels were seen at this time in patients with localized or disseminated disease. The most frequent IgM bands at the baseline and the peak were of 24 kDa (OspC), 41 kDa, and 37 kDa. Although 89% of the patients developed IgG antibodies as determined at a follow-up examination, only 22% were positive by the IgG IB criteria of the Centers for Disease Control and Prevention-Association of State and Territorial Public Health Laboratory Directors. The persistence of antibodies was directly related to disease duration and/or dissemination prior to treatment. Since IgM antibodies to the 24- and 41-kDa antigens remained detectable for long periods, 38% of IgM IBs were still positive at 1 year postbaseline. IgM to antigens of 39, 58, 60, 66, or 93 kDa, conversely, were most often seen in sera obtained within 1 month postbaseline. Their presence may be of assistance in confirming a recent infection with B. burgdorferi in individuals living in areas where Lyme disease is endemic.