Vijay K. Sikand

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.

Atovaquone and azithromycin for the treatment of babesiosis

BACKGROUND Babesiosis is a tick-borne, malaria-like illness known to be enzootic in southern New England. A course of clindamycin and quinine is the standard treatment, but this regimen frequently causes adverse reactions and occasionally fails. A promising alternative treatment is atovaquone plus azithromycin. METHODS We conducted a prospective, nonblinded, randomized trial of the two regimens in 58 subjects with non-life-threatening babesiosis on Nantucket, on Block Island, and in southern Connecticut. The subjects were assigned to receive either atovaquone (750 mg every 12 hours) and azithromycin (500 mg on day 1 and 250 mg per day thereafter) for seven days (40 subjects) or clindamycin (600 mg every 8 hours) and quinine (650 mg every 8 hours) for seven days (18 subjects). RESULTS Adverse effects were reported by 15 percent of the subjects who received atovaquone and azithromycin, as compared with 72 percent of those who received clindamycin and quinine (P<0.001). The most common adverse effects with atovaquone and azithromycin were diarrhea and rash (each in 8 percent of the subjects); with clindamycin and quinine the most common adverse effects were tinnitus (39 percent), diarrhea (33 percent), and decreased hearing (28 percent). Symptoms had resolved three months after the start of therapy in 65 percent of those who received atovaquone and azithromycin and 73 percent of those who received clindamycin and quinine (P=0.66), and after six months no patient in either group had symptoms. Three months after the completion of the assigned regimen, no parasites could be seen on microscopy, and no Babesia microti DNA was detected in the blood of any subject. CONCLUSIONS For the treatment of babesiosis, a regimen of atovaquone and azithromycin is as effective as a regimen of clindamycin and quinine and is associated with fewer adverse reactions.

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

Persistent Parasitemia after Acute Babesiosis

BACKGROUND Babesiosis, a zoonosis caused by the protozoan Babesia microti, is usually not treated when the symptoms are mild, because the parasitemia appears to be transient. However, the microscopical methods used to diagnose this infection are insensitive, and few infected people have been followed longitudinally. We compared the duration of parasitemia in people who had received specific antibabesial therapy with that in silently infected people who had not been treated. METHODS Forty-six babesia-infected subjects were identified from 1991 through 1996 in a prospective, community-based study designed to detect episodes of illness and of seroconversion among the residents of southeastern Connecticut and Block Island, Rhode Island. Subjects with acute babesial illness were monitored every 3 months for up to 27 months by means of thin blood smears, Bab. microti polymerase-chain-reaction assays, serologic tests, and questionnaires. RESULTS Babesial DNA persisted in the blood for a mean of 82 days in 24 infected subjects without specific symptoms who received no specific therapy. Babesial DNA persisted for 16 days in 22 acutely ill subjects who received clindamycin and quinine therapy (P=0.03), of whom 9 had side effects from the treatment. Among the subjects who did not receive specific therapy, symptoms of babesiosis persisted for a mean of 114 days in five subjects with babesial DNA present for 3 or more months and for only 15 days in seven others in whom the DNA was detectable for less than 3 months (P<0.05); one subject had recrudescent disease after two years. CONCLUSIONS When left untreated, silent babesial infection may persist for months or even years. Although treatment with clindamycin and quinine reduces the duration of parasitemia, infection may still persist and recrudesce and side effects are common. Improved treatments are needed.

Disease-Specific Diagnosis of Coinfecting Tickborne Zoonoses: Babesiosis, Human Granulocytic Ehrlichiosis, and Lyme Disease

To determine whether a unique group of clinical and laboratory manifestations characterize certain major deer tick-transmitted human pathogens in North America, we compared the symptoms, short-term complications, and laboratory test results of New England residents who became ill due to > or =1 of these pathogens. Patients completed a uniformly structured questionnaire and submitted blood samples for serologic and polymerase chain reaction (PCR) testing after developing symptoms of Lyme disease, human babesiosis, or human granulocytic ehrlichiosis (HGE). Complete blood count with thin blood smear, PCR, and immunoglobulin M antibody tests helped differentiate the acute manifestations of these diseases. Physicians should consider use of tests designed to diagnose babesiosis and HGE in patients with Lyme disease who experience a prolonged flulike illness that fails to respond to appropriate antiborrelial therapy.

Prospective Study of Serologic Tests for Lyme Disease

BACKGROUND Tests to determine serum antibody levels-the 2-tier sonicate immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and Western blot method or the IgG of the variable major protein-like sequence-expressed (VlsE) sixth invariant region (C6) peptide ELISA method-are the major tests available for support of the diagnosis of Lyme disease. However, these tests have not been assessed prospectively. METHODS We used these tests prospectively to determine serologic responses in 134 patients with various manifestations of Lyme disease, 89 patients with other illnesses (with or without a history of Lyme disease), and 136 healthy subjects from areas of endemicity and areas in which the infection was not endemic. RESULTS With 2-tier tests and the C6 peptide ELISA, only approximately one-third of 76 patients with erythema migrans had results that were positive for IgM or IgG seroreactivity with Borrelia burgdorferi in acute-phase samples. During convalescence, 3-4 weeks later, almost two-thirds of patients had seroreactivity with the spirochete B. burgdorferi. The frequencies of seroreactivity were significantly greater among patients with spirochetal dissemination than they were among those who lacked evidence of disseminated disease. Of the 44 patients with Lyme disease who had neurologic, heart, or joint involvement, all had positive C6 peptide ELISA results, 42 had IgG responses with 2-tier tests, and 2 patients with facial palsy had only IgM responses. However, among the control groups, the IgG Western blot was slightly more specific than the C6 peptide ELISA. The differences between the 2 test systems (2-tier testing and C6 peptide ELISA) with respect to sensitivity and specificity were not statistically significant. CONCLUSIONS Except in patients with erythema migrans, both test systems were sensitive for support of the diagnosis of Lyme disease. However, with current methods, 2-tier testing was associated with slightly better specificity.

Asymptomatic Infection with Borrelia burgdorferi

The natural history of asymptomatic seroconversion to Borrelia burgdorferi has been unclear. We report here, on the basis of a post hoc assessment, the frequency and outcome of asymptomatic seroconversion to B. burgdorferi in participants of a large Lyme disease vaccine trial. We show that infection with B. burgdorferi may be asymptomatic but that asymptomatic infection is unusual in the United States.

Burden and viability of Borrelia burgdorferi in skin and joints of patients with erythema migrans or lyme arthritis

OBJECTIVE To determine the burden and viability of Borrelia burgdorferi in the skin and joints of patients with Lyme disease. METHODS Standard and quantitative polymerase chain reaction (PCR) techniques were used to detect B burgdorferi DNA in skin samples from 90 patients with erythema migrans (EM) and in synovial fluid (SF) from 63 patients with Lyme arthritis (LA) and in synovial tissue from 9 patients. Quantitative PCR determinations of B burgdorferi DNA, messenger RNA (mRNA), and ribosomal RNA (rRNA) were made in 10 skin samples from EM patients and 11 SF samples from LA patients. RESULTS Skin lesions in most patients with EM had positive PCR results for B burgdorferi DNA. In the majority of patients with LA, a late disease manifestation, PCR results in pretreatment SF samples were positive. In patients with antibiotic-refractory arthritis, positive PCR results persisted for as long as 11 months, but positive results in samples taken during the postantibiotic period did not correlate with relapse or with the subsequent duration of arthritis, and at synovectomy, all results of PCR of synovial tissue were negative. B burgdorferi mRNA, a marker of spirochetal viability, was detected in 8 of 10 skin samples from EM patients, but in none of 11 SF samples from LA patients, even when obtained prior to antibiotic administration. Moreover, the median ratio of spirochetal rRNA to DNA, a measure of ribosomal activity, was 160 in the 10 EM skin samples, but only 0.15 in the 3 LA SF samples with positive results. CONCLUSION B burgdorferi in the skin lesions of EM patients were active and viable, whereas those in the SF of LA patients were moribund or dead at any time point. Thus, detection of B burgdorferi DNA in SF is not a reliable test of active joint infection in Lyme disease.

Prospective Study of Coinfection in Patients with Erythema Migrans

The frequency of coinfection with Borrelia burgdorferi and either Anaplasma phagocytophila or Babesia microti among patients with erythema migrans, the initial skin lesion of Lyme disease, was assessed in 2 mainland locations in Rhode Island and Connecticut in a 4-year prospective study. Of the 93 patients with culture-proven erythema migrans, 2 (2%) patients had coinfection with A. phagocytophila and 2 (2%) had coinfection with B. microti. We concluded that the frequency of coinfection with these agents was low among patients with erythema migrans in the study areas.

Borrelia burgdorferi Genetic Markers and Disseminated Disease in Patients with Early Lyme Disease

ABSTRACT Three genetic markers of Borrelia burgdorferi have been associated with disseminated disease: the OspC type, the 16S-23S rRNA intergenic spacer type (RST), and vlsE. Here, we modified previous methods so as to identify the three markers by PCR and restriction fragment length polymorphism in parallel, analyzed B. burgdorferi isolates from erythema migrans (EM) skin lesions in 91 patients, and correlated the results with evidence of dissemination. OspC type A was found approximately twice as frequently in patients with disseminated disease, whereas type K was identified approximately twice as often in those without evidence of dissemination, but these trends were not statistically significant. The remaining seven types identified were found nearly equally in patients with or without evidence of dissemination. RST 1 strains were significantly associated with dissemination (P = 0.03), whereas RST 2 and RST 3 strains tended to have an inverse association with this outcome. The vlsE gene was identified in all 91 cases, using primer sets specific for an N-terminal sequence of B. burgdorferi strain B31 (vlsEB31) or strain 297 (vlsE297), but neither marker was associated with dissemination. Specific combinations of the three genetic markers usually occurred together. OspC type A was always found with RST 1 and vlsEB31, type K was always identified with RST 2 and more often with vlsE297, and types E and I were almost always found with RST 3 and equally often with vlsEB31 and vlsE297. We conclude that B. burgdorferi strains vary in their capacity to disseminate, but almost all strains isolated from EM lesions sometimes caused disseminated disease.