John Nowakowski

Vaccination against Lyme Disease with Recombinant Borrelia burgdorferi Outer-Surface Lipoprotein A with Adjuvant

BACKGROUND The risk of acquiring Lyme disease is high in areas in which the disease is endemic, and the development of a safe and effective vaccine is therefore important. METHODS We conducted a multicenter, double-blind, randomized trial involving 10,936 subjects who lived in areas of the United States in which Lyme disease is endemic. Participants received an injection of either recombinant Borrelia burgdorferi outer-surface lipoprotein A (OspA) with adjuvant or placebo at enrollment and 1 and 12 months later. In cases of suspected Lyme disease, culture of skin lesions, polymerase-chain-reaction testing, or serologic testing was done. Serologic testing was performed 12 and 20 months after study entry to detect asymptomatic infections. RESULTS In the first year, after two injections, 22 subjects in the vaccine group and 43 in the placebo group contracted definite Lyme disease (P=0.009); vaccine efficacy was 49 percent (95 percent confidence interval, 15 to 69 percent). In the second year, after the third injection, 16 vaccine recipients and 66 placebo recipients contracted definite Lyme disease (P<0.001); vaccine efficacy was 76 percent (95 percent confidence interval, 58 to 86 percent). The efficacy of the vaccine in preventing asymptomatic infection was 83 percent in the first year and 100 percent in the second year. Injection of the vaccine was associated with mild-to-moderate local or systemic reactions lasting a median of three days. CONCLUSIONS Three injections of vaccine prevented most definite cases of Lyme disease or asymptomatic B. burgdorferi infection.

PROPHYLAXIS WITH SINGLE-DOSE DOXYCYCLINE FOR THE PREVENTION OF LYME DISEASE AFTER AN IXODES SCAPULARIS TICK BITE

BACKGROUND It is unclear whether antimicrobial treatment after an Ixodes scapularis tick bite will prevent Lyme disease. METHODS In an area of New York where Lyme disease is hyperendemic we conducted a randomized, double-blind, placebo-controlled trial of treatment with a single 200-mg dose of doxycycline in 482 subjects who had removed attached I. scapularis ticks from their bodies within the previous 72 hours. At base line, three weeks, and six weeks, subjects were interviewed and examined, and serum antibody tests were performed, along with blood cultures for Borrelia burgdorferi. Entomologists confirmed the species of the ticks and classified them according to sex, stage, and degree of engorgement. RESULTS Erythema migrans developed at the site of the tick bite in a significantly smaller proportion of the subjects in the doxycycline group than of those in the placebo group (1 of 235 subjects [0.4 percent] vs. 8 of 247 subjects [3.2 percent], P<0.04). The efficacy of treatment was 87 percent (95 percent confidence interval, 25 to 98 percent). Objective extracutaneous signs of Lyme disease did not develop in any subject, and there were no asymptomatic seroconversions. Treatment with doxycycline was associated with more frequent adverse effects (in 30.1 percent of subjects, as compared with 11.1 percent of those assigned to placebo; P<0.001), primarily nausea (15.4 percent vs. 2.6 percent) and vomiting (5.8 percent vs. 1.3 percent). Erythema migrans developed more frequently after untreated bites from nymphal ticks than after bites from adult female ticks (8 of 142 bites [5.6 percent] vs. 0 of 97 bites [0 percent], P=0.02) and particularly after bites from nymphal ticks that were at least partially engorged with blood (8 of 81 bites [9.9 percent], as compared with 0 of 59 bites from unfed, or flat, nymphal ticks [0 percent]; P=0.02). CONCLUSIONS A single 200-mg dose of doxycycline given within 72 hours after an I. scapularis tick bite can prevent the development of Lyme disease.

Duration of antibiotic therapy for early Lyme disease. A randomized, double-blind, placebo-controlled trial.

Context Optimal antibiotic treatment for patients with early Lyme disease is unclear. Contribution This single-center randomized, double-blind, placebo-controlled trial found that patients with erythema migrans given any of the follwoing regimens had high response rates, defined as resolution of erythema migrans and symptoms at 30 months: 20 days of doxycycline, 83.9%; 10 days of doxycycline, 90.3%; and 10 days of doxycycline plus a single intravenous dose of ceftriaxone, 86.5%. Patients given doxycycline plus ceftriaxone more often had diarrhea than patients given doxycycline alone. Implications Oral doxycycline alone for 10 days is sufficient treatment for patients with early Lyme disease that manifests as erythema migrans. The Editors Although Lyme disease is the most common vector-borne disease in the United States (1), the appropriate duration of treatment for its most common manifestation, erythema migrans, remains unclear. A small open-label prospective study reported in 1983 found that outcome did not improve when tetracycline therapy was extended from 10 days to 20 days (2). However, most recent treatment trials have used antibiotic regimens of approximately 3 weeks (3-5), and some authorities recommend 20 to 30 days of therapy (6). This change in practice has occurred in the absence of additional prospective studies on the duration of treatment and is a source of ongoing controversy. Another uncertain issue in the management of patients with erythema migrans is whether Borrelia burgdorferi, the etiologic agent, has disseminated to the central nervous system at the time of presentation (7). If so, the outcome of therapy might be enhanced by treatment with a parenteral agent such as ceftriaxone, which readily crosses the bloodbrain barrier. To address these concerns, we conducted a placebo-controlled study comparing 10 days of doxycycline with both 10 days of doxycycline plus one dose of ceftriaxone and 20 days of doxycycline. Methods Patients at least 16 years of age who had erythema migrans and satisfied the U.S. Centers for Disease Control and Preventions surveillance definition of Lyme disease (an annular erythematous skin lesion 5 cm in diameter) (8) entered the study between 1992 and 1994. Volunteers were recruited primarily through the walk-in Lyme Disease Diagnostic Center at the Westchester Medical Center, Valhalla, New York. Exclusion criteria included pregnancy or lactation, allergy to a tetracycline or a -lactam antibiotic, receipt of antibiotic treatment for Lyme disease for more than 48 hours before enrollment, meningitis or advanced heart block, or any underlying conditions that might interfere with evaluability or follow-up. All patients gave written informed consent, and the institutional review board at New York Medical College approved the study protocol. Patients were randomly assigned to one of three treatment groups: 1) a single 2-g dose of intravenous ceftriaxone followed by 10 days of oral doxycycline capsules twice daily, then by 10 days of oral placebo capsules [cornstarch], identical in appearance to the doxycycline capsules, twice daily; 2) a placebo injection [5% dextrose] followed by 10 days of oral doxycycline, 100 mg twice daily, and then by 10 days of oral placebo twice daily; or 3) a placebo injection followed by 20 days of oral doxycycline twice daily. Since ceftriaxone is yellow, infusion bags were kept covered to maintain masking. The pharmacy dispensed study medications in accordance with a randomization schedule that was based on a computer-generated random-number code with a permuted block size of 9. Clinical staff involved in recruitment of participants or in assessing clinical outcomes were isolated from the allocation process and blinded to treatment assignment. Randomization was stratified by whether patients were symptomatic (defined as having any systemic symptoms or having multiple erythema migrans lesions) or asymptomatic (defined as having a single erythema migrans lesion and no systemic symptoms). This was done to ensure that patients who may have had dissemination of B. burgdorferi were equally represented in the three treatment groups. Evaluation Trained study personnel interviewed participants and performed physical examination at baseline and 10 days, 20 days, 3 months, 6 months, 12 months, 24 months, and 30 months after initiation of therapy. If appointments were missed, patients were interviewed by telephone; however, this occurred in fewer than 5% of patient interactions. A neurologist performed complete neurologic examination at baseline, 20 days, 3 months, 12 months, 24 months, and 30 months. At 18 months, an evaluation was conducted by telephone. Structured questionnaires using both closed- and open-ended questions were used. Symptom scores were recorded on a visual analogue scale (9). A blood sample was obtained for serologic testing (enzyme-linked immunosorbent assay) at all visits. Complete blood count was determined and serum chemistries were performed at baseline, day 10, and day 20. Electrocardiography was done at baseline and was repeated if results were abnormal. Patients were considered unevaluable if they did not adhere to study medication. Nonadherence was defined as taking fewer than 90% of prescribed capsules or not returning pill containers at the 10- or 20-day visit, receiving an intercurrent antibiotic within the first 20 days, not meeting study inclusion criteria, or not attending follow-up visits after the baseline visit. Patients who had an intercurrent episode of erythema migrans due to reinfection were considered unevaluable from that point onward. Outcome was characterized as complete response, partial response, or failure. Early treatment response was assessed at 20 days. At this time, complete response was defined as resolution of erythema migrans and associated symptoms and return to preLyme disease health status. Partial response was defined as resolution of erythema migrans but incomplete resolution or development of subjective symptoms. Failure was defined as the occurrence of any one of the following during the first 20 days: no clinical improvement by day 10; recurrence of erythema migrans; recurrence of fever attributed by the study physician to Lyme disease; development of new objective rheumatologic, cardiac, or neurologic manifestations of Lyme disease that were not present within the first 10 days; or the occurrence of meningitis, advanced heart block, or other objective manifestation of Lyme disease requiring intravenous therapy. Late response was evaluated at 3 months, 12 months, and 30 months. Complete response was defined as no recurrence of erythema migrans or associated symptoms and the continued absence of objective rheumatologic, cardiac, or neurologic manifestations of Lyme disease, with return to preLyme disease health status. Partial response was defined as no recurrence of erythema migrans and the continued absence of objective manifestations of Lyme disease, but incomplete resolution or development of subjective symptoms of uncertain cause. Failure was defined as the occurrence of objective manifestations of Lyme disease. Safety Assessment Drug safety was monitored by recording adverse events (solicited by open-ended semi-structured interview) and results of laboratory tests during treatment. Neurocognitive Evaluation At baseline, 12 months, and 30 months, a psychologist performed neurocognitive testing consisting of the Booklet Categories Test, the California Verbal Learning Test, the Wechsler Memory ScaleRevised, the Block Design Test, the Trail Making Test (Parts A and B), the Boston Naming Test, the Symbol Digit Modalities Test (written and oral), the Beck Depression Inventory, and the Symptom Checklist-90Revised. Healthy volunteers without a history of Lyme disease were recruited to serve as controls for the psychiatric interview and the neuropsychological testing. Spouses were preferred as controls since they were usually similar to the patients in age, socioeconomic level, and place of residence. Controls were 27 persons with a mean age (SD) of 42.6 15.2 years; 22% were men. Statistical Analysis Groups were compared by using one-way analysis of variance for continuous variables and the chi-square test for categorical variables (two-tailed). Data were analyzed both for participants who adhered to the study protocol and on an intention-to-treat basis. Patient blinding was evaluated at the 30-month visit by using the chi-square test and the statistic (10). For the neuropsychological tests, analysis of variance techniques were used to examine differences among treatment and control groups. For analysis of raw test scores, analysis of covariance was used, controlling for the effects of age, sex, and education. Test scores that were normalized to population standards (11-19) were analyzed by using one-way analysis of variance. For all analysis of variance models, residual plots were generated to verify adequate model fit. In the few situations in which test scores were not normally distributed or model fit was questionable, nonparametric procedures (the KruskalWallis test) or conventional transformations were applied. For categorical results, the chi-square test or the Fisher exact test was used. If the treatment variable in the global tests achieved the liberal cut-point of a P value less than 0.10, pairwise comparisons were conducted. In these instances, the Bonferroni adjustment was applied to the P value to control for multiple comparisons. Sample size was based on the estimated frequency of postLyme disease syndrome, defined as an array of subjective symptoms, such as fatigue, arthralgias, or myalgias, that may occur after infection. It was assumed that 30% of patients receiving the least effective treatment would develop postLyme disease syndrome. A more effective treatment was assumed to reduce the rate to 5%. Using an level of 0.05, two-tailed testing, and a Bonferroni multiple compari

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.

Clinical Characteristics and Treatment Outcome of Early Lyme Disease in Patients with Microbiologically Confirmed Erythema Migrans

Context Lyme disease is the most common vector-borne disease in the United States. The traditional clinical presentation, an expanding erythematous rash with partial central clearing, sometimes accompanied by systemic symptoms, was described in patients who usually had clinically manifest Lyme disease for several days. Contribution This study describes 118 patients who acquired Lyme disease while under surveillance in a vaccine trial. Fifty-nine percent of rashes were homogeneous lesions, 32% had dense central erythema, and only 9% had classic central clearing. Signs and symptoms usually resolved within 3 weeks of antibiotic treatment. Implications Early Lyme disease may present with homogeneous or dense central erythematous lesions rather than classic erythema migrans. With antibiotic treatment, the prognosis is excellent. Early Lyme disease may present with homogeneous or dense central erythematous lesions rather than classic erythema migrans. With antibiotic treatment, the prognosis is excellent. The Editors Lyme disease in the United States is caused by the tick-transmitted spirochete Borrelia burgdorferi sensu stricto (1). This infection is the most common vector-borne disease in the country (2). The illness usually presents with localized infection of the skin, erythema migrans, which is often followed days to a few weeks later by dissemination of the spirochete to multiple sites, particularly to other skin sites, the nervous system, the heart, or the joints (3). Borrelia burgdorferi has been cultured readily from skin biopsy samples of erythema migrans early in the illness (4, 5), but later culture from other sites has been difficult. As a substitute for culture, B. burgdorferi DNA may be detected by polymerase chain reaction (PCR) in most patients with erythema migrans (6, 7) and in the joint fluid of patients with Lyme arthritis (8, 9). However, PCR has had low sensitivity in samples from other sites, including cerebrospinal fluid and blood. Serodiagnosis is not sensitive during the first several weeks of infection, but patients often seroconvert during convalescence (10). The initial clinical series of patients with early Lyme disease (11) was described before identification of the causative agent. Researchers used clinical criteria for study entry, particularly the classic appearance of erythema migrans, a slowly expanding erythema with partial central clearing. In that series of 314 patients, most had systemic symptoms and nearly half also had multiple annular erythemas, suggesting dissemination of the spirochete to multiple sites. In a subsequent study, 79 patients from Westchester County, New York, with early Lyme disease had erythema migrans from which B. burgdorferi was cultured (12). Most of the patients in this series, who were often seen earlier than those in the original study, had systemic symptoms, but only 18% had multiple erythemas. In Europe, where erythema migrans more often results from infection with B. afzelii or B. garinii rather than B. burgdorferi sensu stricto, inflammation of erythema migrans is less intense and migration is slower; in addition, patients generally have fewer systemic symptoms (13). In the southeastern United States, patients have been reported to have erythema migranslike skin lesions, but laboratory evidence of B. burgdorferi infection has been lacking. This suggests that another agent, perhaps even from the Borrelia genus, may cause the infection in this geographic area (14-16). Moreover, the same tick that transmits the Lyme disease agent may transmit other infectious agents, including the agent of human granulocytic ehrlichiosis and Babesia microti, and co-infection may influence the clinical presentation of Lyme disease (17, 18). Thus, microbiological confirmation is beneficial in describing the clinical features of Lyme disease in endemic areas in the United States. The recent phase III study of SmithKline Beechams Lyme disease vaccine provided an exceptional opportunity to assess the clinical picture of early Lyme disease in a large cohort of patients who acquired the infection in major endemic locations throughout the United States (19). As part of the protocol, patients who had symptoms of Lyme disease were extensively evaluated for that infection. We describe the clinical presentation, serologic results, and treatment outcome of early Lyme disease in 118 patients with microbiologically confirmed cases of erythema migrans. Methods Patients A total of 10 936 participants 15 to 70 years of age were enrolled in a double-blind, placebo-controlled study of the efficacy and safety of an outer surface protein A Lyme disease vaccine (LYMErix, recombinant outer surface protein A, SmithKline Beecham [now GlaxoSmithKline], Collegeville, Pennsylvania) at 31 sites in 10 endemic states. The Human Investigations Review Committees at all participating centers approved the study protocol. The complete protocol, as well as all members of the Lyme Disease Vaccine Study Group, have been reported elsewhere (19). Briefly, study participants received a packet with information about Lyme disease. Participants were requested to report promptly to their clinician-investigator any symptoms that suggested infection, including onset of a new rash or flu-like illness (without predominant respiratory or gastrointestinal symptoms), arthralgias or arthritis, facial palsy, radiculopathy, or syncope. All participants who were thought to have possible Lyme disease underwent a focused history, physical examination, and laboratory testing. All clinical data were entered on an electronic data form for central analysis. Antibiotic treatment was prescribed according to recommended guidelines (20), but the actual antibiotic and the duration of treatment were at the discretion of the investigator or the patients personal physician. Of the 10 936 participants, 146 met study criteria for definite Lyme disease and 118 had an acute illness with culture-proven or PCR-confirmed erythema migrans. Two clinicians independently reviewed photographs of erythema migrans from case-patients. Laboratory Methods Serologic testing was done at the New England Medical Center exclusively by Western blot (MarDx, San Diego, California), since the standard enzyme-linked immunosorbent assay would be expected to yield positive results in patients vaccinated with outer surface protein A (21). A baseline sample was obtained at study entry, and acute and convalescent samples were obtained when the patient had symptoms suggestive of Lyme disease. The diagnosis of Lyme disease was serologically supported by IgM or IgG seroconversion, or both, between baseline and the acute phase of the illness or between the acute and convalescent phases of the illness. Samples from the same participant were always tested together in the same assay. Western blot results were interpreted according to the criteria of the Centers for Disease Control and Prevention and of the Association of State and Territorial Public Health Laboratory Directors (22). On Western blot, outer surface protein A antibodies bind to a 31-kilodalton band; this is not included in the Centers for Disease Control and Prevention criteria. Following local anesthesia, skin biopsy specimens from erythema migrans were obtained by using 2-mm punch biopsy. Half of each sample was placed directly into a 15-mL tube of BarbourStoennerKelly medium (BSK-H medium, Sigma-Aldrich, St. Louis, Missouri) with ciprofloxacin (0.4 g/mL) and rifampin (40 g/mL); the other half was placed in a 2-mL polypropylene plastic tube for PCR testing. Specimens were shipped the same day by Federal Express to New England Medical Center. On arrival at the laboratory, half of the medium was replaced with fresh medium. Skin samples for culture were placed in an incubator at 33 C and were examined weekly for 1 month by darkfield microscopy for motile spirochetes. Polymerase chain reaction assays of skin biopsy samples were performed as described elsewhere (8). Role of the Funding Source SmithKline Beecham provided data and gave the authors permission to review them, compile them, and independently present results. Dr. Parenti was employed as a research physician with SmithKline Beecham during the early phases of the study. Results During the 20-month study period, which covered two summers of Lyme disease transmission, 1917 of the 10 936 study participants were evaluated for possible Lyme disease (19). Of the 1917 patients, 146 (7.6%) met study criteria for definite Lyme disease, and 118 (6.2%) had microbiological confirmation of this infection by culture or PCR testing of erythema migrans. The mean age of these 118 patients was 51 years (range, 17 to 71 years); 53% were men, and 47% were women. Forty-seven percent were from New England, 51% were from mid-Atlantic states, and 2% were from Wisconsin, reflecting the locations of the study sites. June and July were the peak months of disease onset, which correlated with the expected peak questing period of nymphal Ixodes scapularis ticks. However, cases occurred from March through October, suggesting that adult ticks may also transmit the disease. Vaccine and placebo recipients did not differ in the size of erythema migrans, persistence of symptoms after treatment, and morphologic characteristics of the lesions. In addition, no clinical differences were noted in different geographic areas. Therefore, we present data from vaccine and placebo recipients and from different geographic areas together. Characteristics of Erythema Migrans One hundred eighteen patients had erythema migrans in which B. burgdorferi was detected by culture (88%); by PCR testing (72%); or, in most instances, by both methods (60%). Rashes, which were evaluated a median of 3 days after onset (range, 1 to 30 days), were a median of 10 cm in diameter (range, 5 to 37 cm) at the time of diagnosis. In this adult population, nearly half of the lesions were located on the groin, buttocks, or lo

Association of Specific Subtypes of Borrelia burgdorferi with Hematogenous Dissemination in Early Lyme Disease

To investigate whether genetic diversity of Borrelia burgdorferi sensu stricto may affect the occurrence of hematogenous dissemination, 104 untreated adults with erythema migrans from a Lyme disease diagnostic center in Westchester County, New York, were studied. Cultured skin isolates were classified into 3 groups by a polymerase chain reaction amplification and restriction fragment length polymorphism (RFLP) method. A highly significant association between infecting RFLP type in skin and the presence of spirochetemia was found (P<.001). The same association existed for the presence of multiple erythema migrans lesions (P=.045), providing clinical corroboration that hematogenous dissemination is related to the genetic subtype of B. burgdorferi sensu stricto. There were no significant associations between RFLP type and seropositivity or clinical symptoms and signs except for a history of fever and chills (P=.033). These results suggest that specific genetic subtypes of B. burgdorferi sensu stricto influence disease pathogenesis. Infection with different subtypes of B. burgdorferi sensu stricto may help to explain differences in the clinical presentation of patients with Lyme disease.

Borrelia burgdorferi Genotype Predicts the Capacity for Hematogenous Dissemination during Early Lyme Disease

BACKGROUND Lyme disease, the most common tickborne disease in the United States, is caused exclusively by Borrelia burgdorferi sensu stricto in North America. The present study evaluated the genotypes of >400 clinical isolates of B. burgdorferi recovered from patients from suburban New York City with early Lyme disease associated with erythema migrans; it is the largest number of borrelial strains from North America ever to be investigated. METHODS Genotyping was performed by restriction fragment-length polymorphism polymerase chain reaction analysis of the 16S-23S ribosomal RNA spacer and reverse line blot analysis of the outer surface protein C gene (ospC). For some isolates, DNA sequence analysis was also performed. RESULTS The findings showed that the 16S-23S ribosomal spacer and ospC are in strong linkage disequilibrium. Most B. burgdorferi genotypes characterized by either typing method were capable of infecting and disseminating in patients. However, a distinct subset of just 4 of the 16 ospC genotypes identified were responsible for >80% of cases of early disseminated Lyme disease. CONCLUSIONS This study identified the B. burgdorferi genotypes that pose the greatest risk of causing hematogenous dissemination in humans. This information should be considered in the future development of diagnostic assays and vaccine preparations.

Human Granulocytic Ehrlichiosis: A Case Series from a Medical Center in New York State

Human granulocytic ehrlichiosis (HGE) was first described in 1994 by Bakken and colleagues [1] in 12 patients from the midwestern United States. This report was followed by a retrospective case study [2] of 41 patients from the same geographic area. The data from these studies and from isolated case reports from several locations in the northeastern United States have been the only descriptions of the clinical and laboratory manifestations of HGE to date [2-5]. We describe the clinical and laboratory findings of 18 patients who received a diagnosis of HGE in 1994 or 1995 at a medical center in a populous area in the suburbs of New York City. Methods Patients Patients were evaluated at the Westchester County Medical Center in 1994 and 1995; one patient (patient 18) has been discussed in another report [6]. At the first visit, samples of blood were obtained for complete blood cell count, differential count, chemistry profile, buffy-coat smear for examination of morulae, polymerase chain reaction (PCR) assay for HGE, and serologic examination to test for presence of antibodies to Ehrlichia equi and E. chaffeensis. Blood samples were also collected during the first to sixth weeks of convalescence. Laboratory Testing Buffy-coat smears were stained with Wright stain, and at least 1000 granulocytes were microscopically examined for Ehrlichia morulae. The presence of antibodies was tested for by indirect immunofluorescent antibody assay using horse granulocytes infected with E. equi at an initial serum dilution of 1:80 as described elsewhere [1]. We tested for antibodies to E. chaffeensis by using immunofluorescent antibody assay with infected DH82 cells [1, 7]. Peripheral blood was tested by PCR using EDTA-anticoagulated blood and the primer pair GE9f/GE10r as described elsewhere [8]. Samples of EDTA-anticoagulated blood from two patients (patients 14 and 15) were inoculated into an HL-60 cell line for culturing as described elsewhere [9]. Statistical Analysis The two-tailed Student t-test, Pearson correlation (SPS software, Chicago, Illinois), and two-tailed Fisher exact test (EPISTAT software, Richardson, Texas) were used for data analysis. Results Eighteen patients received a diagnosis of HGE (3 in 1994 and 15 in 1995) in the Westchester County Medical Center. Most of these patients (83%) presented between June and August, Sixteen patients resided in Westchester County, and 2 resided in Putnam County; these counties are contiguous and located slightly north of New York City. Thirteen patients (72%) reported that they had been bitten by a tick: Four ticks were positively identified as Ixodes scapularis (three nymphs and one adult tick). The other nine patients who reported a tick bite described a tick whose appearance was compatible with that of I. scapularis. Symptoms appeared an average of 5.5 days after patients received the tick bite (Table 1). Two patients, neither of whom recalled being bitten by a tick, described a rash that they characterized as erythematous, pruritic, localized, and smaller than 5 cm in diameter. The described rash was not visible when these patients first visited the medical center. Patients were evaluated an average of 4.7 days after onset of symptoms (median, 3 days [range, 1 to 15 days]). Five patients (28%) required hospitalization for an average of 5 days. One patient (patient 15) was briefly admitted to an intensive care unit. None of the 18 patients died. Table 1. Demographic Characteristics, Chronology, Clinical Presentation, and Treatment of 18 Patients with Human Granulocytic Ehrlichiosis* Laboratory test results frequently showed leukopenia or thrombocytopenia and abnormal liver enzyme levels (Table 2). Lymphopenia (defined as <1.5 103 lymphocytes/mL) was seen in 8 of 13 (62%) patients, and neutropenia (defined as <2 103 neutrophils/L) was seen in 3 of 13 (23%). Lymphopenia was seen in 8 of 9 (89%) patients who presented with an illness that lasted 4 days or less and in none of 4 patients with an illness that lasted 5 days or longer (P = 0.006). Neutropenia was seen in 3 of 4 (75%) patients who had HGE for longer than 4 days but in only 1 of 9 (11%) patients who had the disease for 4 days or less (P = 0.05). Pearson correlation analysis showed that the initial neutrophil count was inversely related to the duration of symptoms before the first visit (r = 0.58[95% CI, 0.851 to 0.019];P = 0.038) and that the lymphocyte count was directly related to the duration of symptoms before the first visit (r = 0.543 [CI, 0.033 to 0.836]; P = 0.055). Patients who had HGE for longer than 4 days usually had atypical lymphocytosis of at least 10% of the differential count. No patients had elevated serum creatinine or blood urea nitrogen levels, and only one patient had a low hemoglobin level. Leukocyte and platelet counts returned to normal during observation in all patients who received treatment; these values generally returned to normal earlier than did liver enzyme levels. Table 2. Laboratory Variables during Acute and Convalescent Phases of Human Granulocytic Ehrlichiosis* Intracytoplasmic morulae were seen in 3 of 12 patients (25%). The frequency of infected granulocytes ranged from 0.3% to 6% (Table 2), and morulae were mostly seen in neutrophils (patient 15 had morulae in eosinophils). The presence of morulae correlated with age: Three of 4 patients older than 50 years of age and 0 of 8 patients younger than 50 years of age had morulae that could be detected. Results of polymerase chain reaction assay for HGE were positive in 9 of 12 patients (75%). All but 1 patient developed antibodies to E. equi. Three patients showed antibodies to E. chaffeensis in convalescent-phase specimens that had a higher titer ( 2560) to E. equi. The HGE agent grew in the HL-60 cell line from the blood of 2 patients (patients 14 and 15) who presented with HGE in the autumn of 1995. All 18 patients received doxycycline therapy, beginning an average of 13.2 days (median, 6 days [range, 1 to 78 days]) after the onset of symptoms (Table 1). Seven patients were initially treated with other antimicrobial agents. The therapy of 2 patients was changed from amoxicillin to doxycycline within 2 days of treatment. The other 4 patients, who were initially treated with amoxicillin, had recurrent fever or persistence of symptoms after 1 to 2 weeks of therapy; for these patients, therapy was changed to doxycycline. Symptoms resolved in all patients 24 to 48 hours after the start of treatment with doxycycline, which was administered for an average of 14.2 days (range, 1 to 21 days). No symptoms have been seen after follow-up as long as 1 year. Discussion We describe 18 adult patients with HGE from the northeastern United States. Symptoms in these patients developed an average of 5.5 days after a presumed or proven bite from an Ixodes tick and frequently included fever (body temperature >38.3 C), arthralgia and myalgia, and headache. Compared with the patients from the midwestern United States studied by Bakken and colleagues [1, 2], our patients were significantly younger (mean age SD, 47 16 years compared with 57 20 years; P = 0.04) and presented with a milder illness based on clinical and laboratory evaluations. Compared with the patients in our study, more midwestern patients were hospitalized (54% of midwestern patients compared with 28% in our study) and died (5% of midwestern patients compared with 0% in our study). Differences in case referral or in the threshold for diagnostic testing may explain some of these disparities. Although HGE has a nonspecific clinical presentation, abnormal results on certain routine laboratory tests considerably narrow the differential diagnosis. A diagnosis of Lyme disease may be considered in a patient with fever after a bite from I. scapularis, but cytopenia is extremely rare in that disease [10]. In contrast, approximately 80% of our patients with HGE had leukopenia or thrombocytopenia. The explanation for these abnormal hematologic values and for abnormal liver enzyme levels is not yet known. Leukopenia can be caused by lymphopenia or neutropenia, with lymphopenia in the early stages of infection, followed by lymphocytosis with atypical lymphocytes. Among the methods for specifically diagnosing HGE, microscopic detection of morulae is the quickest; for our patients, however, this method lacked sensitivity. Morulae were seen in 3 of 12 patients (25%) in our series compared with 80% of the midwestern patients. Visualization of intragranulocytic inclusions is probably an indication of massive infection and has been described as a risk factor, along with older age, for more serious illness [2]. In our series, PCR was the most sensitive diagnostic test during the acute phase. Nine of 12 (75%) of our patients had positive PCR results compared with 43% in the midwestern series [2]. As suggested by Bakken and colleagues, the lower sensitivity may have been caused by poor preservation of specimens and conditions that were suboptimal for the PCR assay. Another diagnostic approach that offers definitive evidence of infection is isolation of the organism in culture [9]. In our study, culture was done for two patients in whom morulae were seen in peripheral blood granulocytes; Ehrlichia was isolated in both cases. However, this technique is probably not easily done in most clinical laboratories, and its sensitivity has not yet been determined. Detection of antibodies to E. equi by immunofluorescent antibody assay is useful for confirming the diagnosis of HGE, but often only in retrospect. During the acute phase of HGE, the results of immunofluorescent antibody assay were positive in only 29% of our patients and 23% of the patients in the midwestern cohort. Current serologic assays to detect antibodies to Ehrlichia, however, lack standardization. Although all but one patient (94%) in our series developed antibodies to E. equi, the sensitivity and specificity of serologic testing have not been defi

Comparison of Culture-Confirmed Erythema Migrans Caused by Borrelia burgdorferi sensu stricto in New York State and by Borrelia afzelii in Slovenia

The clinical presentations of Lyme borreliosis in the United States and Europe seem to differ (1-7). For example, European patients with Lyme borreliosis may develop certain skin manifestations (acrodermatitis chronica atrophicans and borrelial lymphocytoma) that are extremely rare or nonexistent in the United States (6, 7). One explanation of such differences may be the recently appreciated variation in strains of Borrelia species between the two continents (7-9). All isolates from U.S. patients have thus far been members of the genomic group Borrelia burgdorferi sensu stricto (henceforth referred to as B. burgdorferi), whereas European isolates have included two additional genospecies, B. garinii and B. afzelii. Until now, however, no reports of clinical disparities have been based on the systematic study of large numbers of patients with culture-confirmed infection. To investigate possible differences in the manifestations of Lyme borreliosis in Europe and the United States, we compared epidemiologic, demographic, clinical, and laboratory findings in patients from New York State and patients from Slovenia who had microbiologically confirmed erythema migrans caused by B. burgdorferi and B. afzelii, respectively. Methods Patients To isolate Borrelia species from clinical specimens, we recruited patients with a clinical diagnosis of erythema migrans in two separate prospective studies. The Centers for Disease Control and Prevention surveillance definition of erythema migrans (10) was satisfied in almost all cases, although three Slovenian patients with erythema migrans lesions less than 5 cm in diameter were enrolled. Patients from the United States were seen at the Lyme Disease Diagnostic Center, Westchester Medical Center, Valhalla, New York, from June 1991 through August 1995. Slovenian patients were evaluated at the Lyme Borreliosis Outpatient Clinic, Ljubljana, Slovenia, in 1993. All patients were older than 15 years of age and gave informed consent. Patients were included in the present study if Borrelia organisms were recovered from their skin specimens. Several U.S. patients had repeated culture-confirmed episodes of erythema migrans in separate years; for each of these patients, only the first episode was included. Laboratory Methods All isolates were obtained from biopsy or needle aspiration of erythema migrans lesions (11, 12). Cultures were processed as previously reported in modified Barbour-Stoenner-Kelly medium (11) for U.S. patients and modified Kelly-Pettenkofer medium (12) for Slovenian patients. At the U.S. study site, we identified spirochetes as B. burgdorferi by using polymerase chain reaction (PCR) with specific primers directed at the ribosomal RNA genes of Borrelia species [13]. We identified the species of European isolates by using two independent methods: 1) species-specific PCR with different oligonucleotide primer sequences [14] and 2) pulsed-field gel electrophoretic separation of restriction enzyme MluI digestion fragments (15) (the pattern of large restriction fragments produced is diagnostic of the species). Because most Slovenian isolates by far were B. afzelii (12), cases of erythema migrans due to other species (including four cases due to B. burgdorferi and six due to B. garinii) were excluded. Complete blood counts were done, liver function tests were performed, and the erythrocyte sedimentation rate was measured at the time when cultures were obtained. Baseline serum specimens were tested for antibodies to B. burgdorferi. Convalescent-phase specimens were obtained after 1 week to 1 month for U.S. patients and after 2 months for Slovenian patients. In the United States, serum specimens were tested for antibodies to B. burgdorferi with a polyvalent enzyme-linked immunosorbent assay (Whittaker Bioproducts, Inc., Walkersville, Maryland) (11, 16). In Slovenia, the presence of serum IgM and IgG antibodies to B. afzelii was determined separately by immunofluorescent assay without preabsorption (17); a local skin isolate of B. afzelii was used as antigen. Titers of 1:256 or more were considered to indicate positivity. Statistical Analysis Differences in quantitative data were analyzed by using the median test, and differences in qualitative data were analyzed by using the chi-square test (with the Yates correction) or the Fisher exact test. We used Epi-Info 6 for all analyses (Statcalc, version 6.04a, Centers for Disease Control and Prevention, Atlanta, Georgia). All P values were two-tailed. Results Borrelia afzelii was recovered from the skin specimens of 85 Slovenian patients; none of these patients had been reported previously. Borrelia burgdorferi was cultured from 119 U.S. patients; 77 of these patients had been reported previously (11). Of the U.S. isolates, 30 were randomly selected for species identification by PCR, and all 30 were identified as B. burgdorferi. Thus, it is likely (although not certain) that the remainder of the isolates were also B. burgdorferi. Demographic patient data and the characteristics of erythema migrans are summarized in Table 1. All Slovenian patients and 113 of 119 U.S. patients (95%) were white. Slovenian patients were more likely than U.S. patients to recall a tick bite at the erythema migrans site (63.5% compared with 25.2%; P<0.001). Although the size of the erythema migrans lesions was similar in the two groups of patients, the median duration of erythema migrans at presentation was longer (14 days [range, 1 to 206 days] compared with 4 days [range, 1 to 39 days]; P<0.001) and central clearing was more common (68.2% compared with 35.3%; P<0.001) in Slovenian patients than in U.S. patients. Localized pain or burning at the erythema migrans site was more common in U.S. patients (34.3% compared with 18.8%; P=0.02). The occurrence of multiple erythema migrans lesions was greater in the U.S. patients than in the Slovenian patients, but the difference was not significant (13.4% compared with 7.1%; P>0.2). Table 1. Characteristics of Patients with Culture-Confirmed Erythema Migrans Patients in the United States were more likely than those in Slovenia to have systemic symptoms (68.9% compared with 50.6%; P=0.01), including fatigue (54.6% compared with 32.9%; P=0.003), myalgia (44.5% compared with 21.1%; P=0.001), fever or chills (37.8% compared with 8.2%; P<0.001), and stiff neck (38.7% compared with 8.2%; P<0.001) (Table 2). Objective findings on physical examination were also more common in U.S. patients (57.1% compared with 14.1%; P<0.001) and most frequently manifested as regional lymphadenopathy (seen in 28.6% of U.S. patients compared with 8.2% of Slovenian patients; P<0.001) or fever (body temperature 37.8 C) (seen in 15.1% of U.S. patients compared with 1.2% of Slovenian patients; P<0.001). Slovenian patients were more likely to have hepatomegaly (5.9% compared with 0; P=0.01), but U.S. patients more often had at least one abnormal result on a liver function assay (32.2% compared with 18.3%; P=0.04), were more likely to have erythrocyte sedimentation rates two or more times the upper limit of normal (25% compared with 3.7%; P<0.001), were more likely to seroconvert (84.2% compared with 10.9%; P<0.001), and were more often seropositive at presentation (35.3% compared with 22.4%; P=0.07). Table 2. Selected Clinical and Laboratory Findings in Patients with Culture-Confirmed Erythema Migrans Discussion Our findings establish what has long been believed: Some of the clinical and laboratory features of Lyme disease differ in the United States and Europe. The differences seem to result, in part, from a tendency for B. afzelii to be less virulent than B. burgdorferi. Slovenian patients with B. afzelii were significantly less likely than U.S. patients with B. burgdorferi to be systemically ill (Table 2) and to have fever (P<0.001) or regional lymphadenopathy (P<0.001) on physical examination. Although the duration of erythema migrans at presentation was longer in Slovenian patients, lesion size in Slovenian patients was similar to that in U.S. patients; this implies that B. afzelii spreads more slowly in the skin (11). Because central clearing occurs, in part, as a function of time (2), its higher frequency in Slovenian patients (68.2% compared with 35.3%; P<0.001) is probably related to the longer duration of erythema migrans at presentation. Nonetheless, in contrast to what might have been anticipated from earlier reports (1-6, 18), dissemination to multiple cutaneous sites did not occur significantly more often in U.S. patients than in Slovenian patients in our study (P=0.2). Our observations in patients in Westchester County, New York, agree with those of other studies of North American patients with erythema migrans (1, 4, 5), in which reported rates of systemic illness are often greater than 75%. In contrast, reported rates of systemic illness in Europe are often less than 35% (2, 3, 8). In our study, Slovenian patients were much less likely than U.S. patients to be seropositive (26 of 85 [30.6%] compared with 106 of 119 [89.1%]; P<0.001), principally because seroconversion was less common (7 of 64 patients [10.9%] compared with 64 of 76 patients [84.2%]; P<0.001). The seroconversion rate in our U.S. patients, however, was similar to that seen in other recent studies of U.S. patients with erythema migrans (5). Moreover, the contrasting rates of seropositivity at presentation were particularly evident when patients with longstanding erythema migrans (duration 14 days) were compared; 94% of such patients in the United States (15 of 16) but only 28% of such patients in Slovenia (13 of 47) were seropositive (P<0.001). With rare exceptions (19), European studies have reported low rates of seropositivity (20% to 50%) in patients with erythema migrans (3, 18), despite a long duration of illness (often longer than several weeks) (3). However, because convalescent-phase serologic samples from Slovenian patients were obtained at 2 months (rather th

Laboratory Diagnostic Techniques for Patients with Early Lyme Disease Associated with Erythema Migrans: A Comparison of Different Techniques

Recently, a number of refinements in diagnostic modalities for detection of Borrelia burgdorferi infection have been developed. These include large-volume blood cultures, quantitative polymerase chain reaction (PCR) techniques, and 2-stage serologic testing. In the present study, we compared 6 diagnostic modalities in 47 adult patients who had a clinical diagnosis of erythema migrans. Quantitative PCR on skin biopsy-derived material was the most sensitive diagnostic method (80.9%), followed by 2-stage serologic testing of convalescent-phase samples (66.0%), conventional nested PCR (63.8%), skin culture (51.1%), blood culture (44.7%), and serologic testing of acute-phase samples (40.4%). Results of all assays were negative for 3 patients (6.4%). We conclude that the clinical diagnosis of erythema migrans is highly accurate in an area where B. burgdorferi is endemic if it is made by experienced health care personnel, but some patients with this diagnosis may not have B. burgdorferi infection. No single diagnostic modality is suitable for detection of B. burgdorferi in every patient with erythema migrans.