Erythema migrans-like rash illness at a camp in North Carolina: a new tick-borne disease?

BACKGROUND: Borrelia burgdorferi, the causative agent of Lyme disease, has never been isolated from a patient thought to have acquired Lyme disease in any southeastern state. OBJECTIVE: To investigate 14 cases of an erythema migrans (EM)-like rash illness that occurred during 2 summers at an outdoor camp in central North Carolina in an effort to determine the etiologic, epidemiological, and clinical aspects of this illness. METHODS: Using active surveillance, we identified cases of clinically diagnosed EM in residents and staff of the camp. We collected clinical and demographic information; history of exposure to ticks; acute and convalescent serum antibodies to B. burgdorferi, Rickettsia rickettsii, and Ehrlichia chaffeensis; and cultures for spirochetes from biopsy specimens of skin lesions. Serum samples from a group of residents and staff who did not develop rashes were tested for the same antibodies. We speciated ticks removed from people and collected from vegetation. RESULTS: We identified 14 cases of EM-like rash illness during the 2 summers. Of the 14 case-patients, 10 had associated mild systemic symptoms and 1 had documented fever. All 14 case-patients had removed attached ticks, and 8 remembered having removed a tick from the site where the rash developed a median of 12 days earlier (range, 2-21 days). One tick removed from the site where a rash later developed was identified as Amblyomma americanum, the Lone Star tick; 97% of ticks collected from vegetation and 95% of ticks removed from people were A. americanum. No spirochetes were isolated from skin biopsy specimens. Paired serum samples from 13 case-patients did not show diagnostic antibody responses to B. burgdorferi or other tick-borne pathogens. CONCLUSIONS: This investigation suggests the existence of a new tick-associated rash illness. We suspect that the disease agent is carried by A. americanum ticks. In the southern United States, EM-like rash illness should no longer be considered definitive evidence of early Lyme disease.

Ability of experimentally infected chickens to infect ticks with the Lyme disease spirochete, Borrelia burgdorferi.

Chickens were used as a laboratory model to determine the conditions affecting the ability of birds to infect ticks with Lyme disease spirochetes. Chicks (Gallus gallus) were exposed to 12 nymphal Ixodes scapularis at one week or three weeks of age. Xenodiagnostic larval ticks fed these birds at weekly intervals thereafter. Chicks exposed to infected nymphs at one week of age infected 87% of larvae at three weeks of age, but only infected 3% of larvae at four weeks and 0% of larvae at five weeks. Chicks exposed to nymphs at three weeks of age infected only 12% of larvae at four weeks, and 0% thereafter. Thus, experimentally infected chicks can infect larval ticks, but only for a brief interval after exposure. Young chicks are more infectious than older chickens. The immune response of infected chicks was rapid and directed against diverse antigens.

Evaluation of a two-test serodiagnostic method for community assessment of Lyme disease in an endemic area.

Epidemiological methods are needed to evaluate community exposure to Borrelia burgdorferi, the causative agent of Lyme disease (LD). For LD serodiagnosis, the Centers for Disease Control and Prevention (CDC) recommends a 2-test approach that involves enzyme immunoassay (EIA) testing and Western immunoblotting (WB) of EIA-equivocal and EIA-positive specimens. The specificity of this approach was evaluated among residents of a LD-endemic community and was compared with WB alone and with a simplified 2-test approach (WB of equivocal EIA only). Participants reporting no previous diagnosis of LD were recruited during a community-wide serosurvey on Block Island, Rhode Island. Of 80 eligible participants, 20 had received LD vaccine. Seven (35%) of 20 vaccinees and 22 (37%) of 60 nonvaccinees reported nonspecific symptoms compatible with LD in the previous year. In this highly LD-endemic community, the overall specificity of the CDC-recommended approach was highest (100%), followed by WB alone (98.7%), then the simplified approach (95%).

An interstate outbreak of tick-borne relapsing fever among vacationers at a Rocky Mountain cabin.

In July 1995, an outbreak of acute febrile illness affected 11 (48%) of 23 family members from Nebraska and Kansas who had vacationed at a Colorado cabin in June. Similar symptoms were identified among five (17%) of 30 additional persons from Nebraska, Kansas, Florida, and Texas who had vacationed at the same cabin. Symptoms suggested tick-borne relapsing fever (TBRF). Although no spirochetes were detected in available blood smears from five case-patients, Borrelia hermsii was cultured from the blood of one case-patient and two chipmunks trapped near the cabin. Case-patients were more likely than non-ill cabin visitors to have slept on the floor (odds ratio [OR] = 28.0, 95% confidence interval [CI] = 3.0-258) or in the top bunk bed (OR = 5.2, 95% CI = 1.1-25.1). Tick-borne relapsing fever should considered in the differential diagnosis of fever in patients who have stayed overnight in mountain cabins in the western United States.

Solitary erythema migrans in Georgia and South Carolina.

OBJECTIVE: To evaluate the incidence of Borrelia burgdorferi infection in humans with erythema migrans (EM) in 2 southeastern states. DESIGN: Prospective case series. SETTING: Family medicine practice at academic center. PATIENTS: Twenty-three patients with solitary EM lesions meeting Centers for Disease Control and Prevention (CDC) criteria for Lyme disease. INTERVENTIONS: Patients underwent clinical and serologic evaluation for evidence of B burgdorferi infection. All lesions underwent photography, biopsy, culture and histopathologic and polymerase chain reaction analysis for B burgdorferi infection. Patients were treated with doxycycline hyclate and followed up clinically and serologically. MAIN OUTCOME MEASURES: Disappearance of EM lesions and associated clinical symptoms in response to antibiotic therapy; short-term and follow-up serologic assays for diagnostic antibody; growth of spirochetes from tissue biopsy specimens in Barbour-Stoenner-Kelly II media; special histopathologic stains of tissue for spirochetes; and polymerase chain reaction assays of tissue biopsy specimens for established DNA sequences of B burgdorferi. RESULTS: The EM lesions ranged from 5 to 20 cm (average, 9.6 cm). Five patients (22%) had mild systemic symptoms. All lesions and associated symptoms resolved with antibiotic therapy. Overall, 7 patients (30%) had some evidence of B burgdorferi infection. Cultures from 1 patient (4%) yielded spirochetes, characterized as Borrelia garinii, a European strain not known to occur in the United States; 3 patients (13%) demonstrated spirochetallike forms on special histologic stains; 5 patients (22%) had positive polymerase chain reaction findings with primers for flagellin DNA sequences; and 2 patients (9%) were seropositive for B burgdorferi infection using recommended 2-step CDC methods. No late clinical sequelae were observed after treatment. CONCLUSIONS: The EM lesions we observed are consistent with early Lyme disease occurring elsewhere, but laboratory confirmation of B burgdorferi infection is lacking in at least 16 cases (70%) analyzed using available methods. Genetically variable strains of B burgdorferi, alternative Borrelia species, or novel, uncharacterized infectious agents may account for most of the observed EM lesions.

Murine typhus: updated roles of multiple urban components and a second typhuslike rickettsia.

Studies using serologic and polymerase chain reaction-(PCR) facilitated analysis of field samples from southern Texas indicate the presence of Rickettsia typhi and ELB agent infected cat fleas, Ctenocephalides felis (Bouché), and the first observation of ELB infected vertebrates (opossums). The ELB agent is a recently described typhus-like rickettsia that is not distinguished from R. typhi or R. prowazekii by currently available serologic reagents. Restriction digests of PCR products from 399 fleas revealed an ELB agent infection rate of 3.8% and a R. typhi infection rate of 0.8%. Three of nine tested opossums (Didelphis virginiana) were shown to harbor ELB agent infections. No R. typhi infected rats, Rattus norvegicus, or rat-fleas, Xenopsylla cheopis Rothschild, were detected among surveyed samples. The persistence of this murine typhus disease focus appears to be better accounted for by the presence of infected cat fleas, opossums, and other non-rat hosts found in close association with human populations. Involvement of the ELB agent in the biology of murine typhus is suggested by its prevalence among suspected vectors and reservoir hosts.

Metabolism of juvenile hormone during adult development of Dermacentor variabilis (Acari: Ixodidae).

Juvenile hormone (JH)-I and -III were used as model substrates to study the in vitro metabolism of JH in the hemolymph and body homogenates of the American dog tick, Dermacentor variabilis (Say). Ester hydrolysis was the principal pathway of JH metabolism in hemolymph and homogenates. JH also was converted into JH-diol primarily by body homogenates, indicating the presence of JH epoxide hydrolase activity. JH epoxide hydrolase activity, alpha-naphthyl acetate esterase activity, and protein concentration per milligram wet weight were significantly lower (t test, alpha = 0.05) in homogenates of partially fed, virgin and replete, mated females of D. variabilis compared with unfed, virgin females. The decline in these factors was probably because of the influx of water into the tissues caused by the blood meal. In addition, the epoxide hydrolase and alpha-naphthyl acetate esterase activity per milligram tissue protein decreased significantly during this time. Mating of fed females rather than feeding alone caused a significant decline in the tissue JH esterase activity per milligram wet weight but not per milligram protein. The JH esterase activity per milligram protein was significantly higher in partially fed, virgin and replete, mated females compared with unfed females, indicating that feeding may actually increase JH esterase activity on a protein basis. JH-III was metabolized 1.4 times faster than JH-I by the hemolymph of partially fed, virgin females. The inhibitors O,O-diisopropyl phosphorofluoridate and octylthio-1,1,1-trifluoropropan-2-one at 10(-4) M inhibited JH and alpha-naphthyl acetate esterase activity in hemolymph and body homogenate.

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group.

AIMS: To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores. METHODS AND RESULTS: Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis, e.g. lack of beta-haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis. PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis, but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an AluI restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different. CONCLUSIONS: Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.

Cross-reactivity to Borrelia burgdorferi proteins in serum samples from residents of a tropical country nonendemic for Lyme disease.

Reports of Lyme disease from areas where the disease is not endemic have increased. Eighty-six human serum samples from Papua New Guinea (nonendemic for Lyme disease) were examined for the presence of IgG antibodies that recognize Borrelia burgdorferi antigens, using the currently recommended two-tiered system of analysis (sensitive ELISA with Western blot). The percentage of positive tests dropped from 50% to 10% when individual negative controls were included in the two-tiered analysis. Positive serum samples failed to inhibit the growth of B. burgdorferi in culture and did not yield positive reactions in the fluorescent treponemal antibody-absorption test. These characteristics, together with atypical Western blot antigen recognition patterns and the absence of known vectors, provide evidence that seropositive results for these persons are not the result of exposure to B. burgdorferi. Individual negative controls may minimize false-positive results for serologic tests for Lyme disease, and these tests must be interpreted in the context of clinical and epidemiologic data.

Detection of polymerase chain reaction-amplified malarial DNA in infected blood and individual mosquitoes.

Chelex treatment of Plasmodium falciparum and P. berghei infected tissues, in lieu of organic extraction, was followed directly by polymerase chain reaction amplification of primed circumsporozoite gene sequences. The amplified DNA products were detected in stained gels and hybridization blots of extracts from individual infected mosquitoes and dissected mosquito tissues as well as small volumes of infected blood. Parasite development, within the mosquito midgut and salivary gland, was also monitored as a function of time post infectious blood meal. The temporal presence of amplifiable circumsporozoite gene sequences in the infected mosquito midgut lumen, midgut endothelium, and salivary glands corresponded directly to the visual identification of ookinetes, oocysts, and salivary gland sporozoites, respectively.

Rickettsia felis: a new species of pathogenic rickettsia isolated from cat fleas.

A flea-borne rickettsia, previously referred to as ELB, has been implicated as a cause of human illness. Using sequence data obtained from a fragment of the citrate synthase gene, we compared ELB, Rickettsia australis, R. rickettsii, and R. akari with the louse-borne R. prowazekii. We tallied 24 base pair differences between ELB and R. prowazekii and 25 between R. rickettsii and R. prowazekii; there were 30 base pair differences between R. australis and R. prowazekii and 29 between R. akari and R. prowazekii. We observed 32 differences between Rickettsia typhi and ELB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses of ELB, with typing sera against R. typhi indicate that ELB surface antigens are more closely related to the flea-borne R. typhi than to the mite-borne R. akari. On the basis of the results of citrate synthase gene sequence comparisons, as well as previous comparisons with 16S rRNA and 17-kDa-protein gene segments, we found that ELB is sufficiently genetically distinct from other rickettsiae to be designated a new species, Rickettsia felis.

Molecular identification of rickettsia-like microorganisms associated with colonized cat fleas (Ctenocephalides felis).

Cat fleas (Ctenocephalides felis) from eight commercial flea colonies from various regions of the USA were examined by selective PCR amplification, and subsequent restriction digest analysis and Southern hybridization of PCR products, for the presence of a rickettsia-like organism (ELB agent). These flea colonies were either started with fleas from one supplier (EL Labs), in which ELB agent was first identified, or were started with fleas from stray cats and dogs and later came into contact with ELB-infected fleas. Infection rates in the colonies ranged from 43% to 93%. The successful propagation of ELB agent in these colonies may be due to efficient trans-stadial and transovarial transmission. While ELB agent has recently been identified in blood from human murine typhus cases, attempts to infect mammalian cells and SCID mice with flea isolates were unsuccessful.

Identification of a novel rickettsial infection in a patient diagnosed with murine typhus.

Identification of ELB agent-infected fleas and rodents within several foci of murine typhus in the United States has prompted a retrospective investigation for this agent among human murine typhus patients. This agent is a recently described rickettsia which is indistinguishable from Rickettsia typhi with currently available serologic reagents. Molecular analysis of the 17-kDa antigen gene and the citrate synthase gene has discriminated this bacterium from other typhus group and spotted fever group rickettsiae. Current sequencing of its 16S ribosomal DNA gene indicates a homology of 98.5% with R. typhi and 99.5% with R. rickettsii. Through a combination of restriction fragment length polymorphism and Southern hybridization analysis of rickettsia-specific PCR products, one of five tested patient blood samples was shown to be infected with ELB while R. typhi infections were confirmed in the remaining samples. This is the first reported observation of a human infection by the ELB agent and underscores the utility of PCR-facilitated diagnosis and discrimination of these closely related rickettsial infections.

Serodiagnosis of Louse-Borne relapsing fever with glycerophosphodiester phosphodiesterase (GlpQ) from Borrelia recurrentis.

Human louse-borne relapsing fever occurs in sporadic outbreaks in central and eastern Africa that are characterized by significant morbidity and mortality. Isolates of the causative agent, Borrelia recurrentis, were obtained from the blood of four patients during a recent epidemic of the disease in southern Sudan. The glpQ gene, encoding glycerophosphodiester phosphodiesterase, from these isolates was sequenced and compared with the glpQ sequences obtained from other relapsing-fever spirochetes. Previously we showed that GlpQ of Borrelia hermsii is an immunogenic protein with utility as a serological test antigen for discriminating tick-borne relapsing fever from Lyme disease. In the present work, we cloned and expressed the glpQ gene from B. recurrentis and used recombinant GlpQ in serological tests. Acute- and convalescent-phase serum samples obtained from 42 patients with louse-borne relapsing fever were tested with an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) that used whole cells of B. recurrentis and with immunoblotting to whole-cell lysates of the spirochete and Escherichia coli producing recombinant GlpQ. The geometric mean titers of the acute- and convalescent-phase serum samples measured by IFA were 1:83 and 1:575, respectively. The immunoblot analysis identified a high level of reactivity and seroconversion to GlpQ, and the assay was more sensitive than the whole-cell IFA and ELISA using purified, recombinant histidine-tagged GlpQ. Serum antibodies to GlpQ and other antigens persisted for 27 years in one patient. We conclude that assessment of anti-GlpQ antibodies will allow serological confirmation of louse-borne relapsing fever and determination of disease prevalence.

Epidemiologic and diagnostic studies of patients with suspected early Lyme disease, Missouri, 1990-1993.

A retrospective case-control study investigated 45 Missouri outpatients with annular rashes meeting a surveillance case definition for erythema migrans and with onset in 1990-1991. Risk factors included being male, living near a body of water, and hunting. Twenty patients (44%) associated their rash with the bite of a tick; of these, 5 described an adult Amblyomma americanum. A typical rash was described as expanding over time and measuring 8 cm in diameter at 4 days after onset. Mild constitutional symptoms were common but fever was uncommon. Serologic tests failed to incriminate Borrelia burgdorferi or selected other arthropodborne pathogens. Skin specimens from suspected erythema migrans lesions of 23 Missouri patients sampled prospectively in 1991-1993 were culture-negative for B. burgdorferi. Thus, tick bite-associated annular rashes in Missouri remain idiopathic. Possible causes include infection with a novel A. americanum-transmitted pathogen and an atypical toxic or immunologic reaction to tick-associated proteins.

Mouse model for exoerythrocytic stages of Plasmodium falciparum malaria parasite.

Research on the exoerythrocytic (EE) stages of human malaria parasites has been hindered because of the lack of an easily available suitable animal model. We report here an approach to produce mature EE-stage Plasmodium falciparum parasites by using severe combined immunodeficient (scid) mice with transplanted human hepatocytes. Transplantation of human hepatocytes into scid mice (scid hu-hep), their subsequent intravenous infection with P. falciparum sporozoites, and the development of mature liver-stage merozoites was achieved. Immunofluorescent staining of scid hu-hep kidney tissue sections demonstrated the presence of circumsporozoite protein (early during infection), merozoite surface antigen 1, and liver schizont antigen 1. The scid hu-hep model can serve as a source of human malaria liver-stage parasites, decreasing the need for nonhuman primates. Use of this model will facilitate characterization of EE-stage antigens and the assessment of stage-specific chemotherapeutic agents and candidate vaccines.

Typhus and typhuslike rickettsiae associated with opossums and their fleas in Los Angeles County, California.

The recent discovery of cat fleas (Ctenocephalides felis) infected with a typhuslike rickettsia (designated the ELB agent) raises the question of whether similar rickettsial infections exist in wild cat flea populations. We verified the presence of the ELB agent and Rickettsia typhi in urban and suburban areas of Los Angeles, Calif. Opossums trapped in close proximity to the residences of human murine typhus cases in Los Angeles county and other areas within the city of Los Angeles were tested for the presence of typhus group rickettsiae by the polymerase chain reaction (PCR). The presence of rickettsiae in the spleen tissues of three opossums (n = 9) and in 66 opossum fleas (n = 205) was determined by PCR and was verified by dot blot and Southern transfer hybridization. Further analysis of the amplified PCR products generated by a series of primer pairs derived from either the 17-kDa antigen gene or the citrate synthase gene revealed that both R. typhi and the ELB agent were present in the tested samples. Dual infection was not noted in the samples; however, the fleas were infected with either R. typhi or the ELB agent. The presence of the ELB agent in the cat flea population may have implications for public health. Whether this agent is responsible for the mild cases of human murine typhus in urban and suburban areas of Los Angeles or in other endemic foci remains to be determined.

Emergence of Lyme disease in Hunterdon County, New Jersey, 1993: a case-control study of risk factors and evaluation of reporting patterns.

Reported cases of Lyme disease in Hunterdon County, New Jersey, increased almost 200% from 75 (67/100,000 population) in 1992 to 216 (193/100,000 population) in 1993. For evaluation of risk factors for Lyme disease and for determination of the cause of this increase, a case-control study was conducted, and the reporting practices of physicians' offices were evaluated. For cases reported in 1993, age and sex distribution, month of disease onset, and proportion of cases with erythema migrans rash were within expected limits. Analysis of age-matched case-control data showed that rural residence; clearing periresidential brush during spring and summer months; and the presence of rock walls, woods, deer, or a bird feeder on residential property were associated with incident Lyme disease. A review of physician reporting patterns suggested that the increase in reported cases in 1993 was due to improved reporting as well as to an increase in the numbers of patients diagnosed with Lyme disease. In addition, substantial underreporting of Lyme disease by physicians' offices was found.

Clinical and epidemiological features of early Lyme disease and human granulocytic ehrlichiosis in Wisconsin.

To compare clinical features and assess risk factors for human granulocytic ehrlichiosis (HGE) and early Lyme disease, we enrolled patients in a case-control study during the 1996 and 1997 tick seasons. Clinical and demographic characteristics were assessed for patients with laboratory-confirmed cases of HGE or Lyme disease, and risk factors were compared with those of matched control subjects. We identified 83 persons with Lyme disease, 27 with HGE, and 11 with apparent coinfection. Unsuspected Ehrlichia infection was identified in 8 (13%) of 60 patients with Lyme disease. Patients with HGE were older and more likely to have fever, chills, or dyspnea than were those with Lyme disease only. Most patients with apparent coinfection did not have hematologic abnormalities. In the risk factor analysis, tickborne illness was independently associated with rural residence and camping. The clinical spectrum of HGE overlaps that of Lyme disease, and physicians in areas of endemicity should consider both diseases in treating patients with a compatible rash or febrile illness.