Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease.

AIMS: 16S rDNA sequences of Borrelia burgdorferi sensu lato were aligned with the 16S rDNA sequences of Borrelia hermsii, Borrelia turicatae, and Borrelia lonestari in order to identify primers that might be used to more specifically identify agents of human Lyme disease in ticks in human skin samples. METHODS AND RESULTS: Standard polymerase chain reaction (PCR), using an oligonucleotide sequence, designated TEC1, was shown, in combination with a previously developed primer (LD2) to amplify strains of B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, but not the non-Lyme causing B. hermsii or B. turicatae. This primer pair, designated Bbsl, was successfully used to amplify B. burgdorferi sensu lato from skin biopsies of patients with Lyme disease symptoms as well as from Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis ticks. CONCLUSIONS: The primer set Bbsl allows for the rapid detection and differentiation of B. burgdorferi sensu lato from non-Lyme disease-causing Borrelia species in ticks and human tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR primer set, Bbsl, will greatly facilitate detection of the causative agents of Lyme disease in infected ticks and human skin samples assisting in epidemiological studies, and potentially allowing for a more rapid diagnosis of the disease in patients.

Host utilization and seasonal occurrence of Dermacentor species (Acari:Ixodidae) in Missouri, USA.

A total of 3,235 Dermacentor variabilis (Say) specimens were collected from birds, mammals, and by dragging vegetation, and 2,683 D. albipictus (Packard) ticks were collected from deer from 1993 to 1996. Peak seasonal occurrence of adult D. variabilis was from May through July with a precipitous decrease in August. Nymphal D. variabilis populations peaked in June. Peak activity of larvae was bimodal, with one activity peak during late summer (September) and a second peak in winter or early spring. The raccoon, Procyon lotor (L.), was the principal host of adults followed by the Virginia opossum, Didelphis virginiana Kerr. Rodents and the eastern cottontail rabbit, Sylvilagus floridanus (J. A. Allen), were the primary hosts of nymphs. The marsh rice rat, Oryzomys palustris (Harlan), was the principal host of larvae followed by the pine vole, Microtus pinetorum (Le Conte), and white-footed mouse, Peromyscus leucopus (Rafinesque). All stages of D. albipictus were found only on white-tailed deer, Odocoileus virginianus (Zimmermann). Numbers of adult and nymphal D. albipictus peaked in November, whereas larvae peaked in September.

First isolation and cultivation of Borrelia burgdorferi sensu lato from Missouri.

Five Borrelia burgdorferi sensu lato isolates from Missouri are described. This represents the first report and characterization of such isolates from that state. The isolates were obtained from either Ixodes dentatus or Amblyomma americanum ticks that had been feeding on cottontail rabbits (Sylvilagus floridanus) from a farm in Bollinger County, Mo., where a human case of Lyme disease had been reported. All isolates were screened immunologically by indirect immunofluorescence by using monoclonal antibodies to B. burgdorferi-specific outer surface protein A (OspA) (antibodies H3TS and H5332), B. burgdorferi-specific OspB (antibody H6831), Borrelia (genus)-specific antiflagellin (antibody H9724), and Borrelia hermsii-specific antibody (antibody H9826). Analysis of the isolates also involved a comparison of their protein profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the isolates were analyzed by PCR with six pairs of primers known to amplify selected DNA target sequences specifically found in the reference strain B. burgdorferi B-31. Although some genetic variability was detected among the five isolates as well as between them and the B-31 strain, enough similarities were found to classify them as B. burgdorferi sensu lato.

Some factors affecting infestation of white-tailed deer by blacklegged ticks and winter ticks (Acari:Ixodidae) in southeastern Missouri.

A total of 5,669 ticks of 4 species was collected from 515 hunter killed, white-tailed deer. Odocoileus virginianus (Zimmerman), in southeastern Missouri from 1993 through 1995. The American dog tick, Dermacentor variabilis (Say) (4 adults), the lone star tick, Amblyomma americanum (L.) (57 adults, 2 nymphs), the blacklegged tick, Ixodes scapularis (Say) (3,120 adults), and the winter tick, Dermacentor albipictus (Packard) (2.059 adults, 436 nymphs, 1 larva) were collected. Patterns of adult D. albipictus and I. scapularies infesting deer were analyzed with respect to upland versus lowland habitat, county, and host sex. Prevalence and intensity of infestation by D. albipictus were higher on bucks than does, and a higher infestation prevalence was recorded for this tick on deer from upland than from lowland habitats. Mean intensities for D. albipictus were not significantly different between counties. Prevalence and mean intensity of infestation for I. scapularis were significantly higher on deer from uplands than lowlands and on bucks than does; mean intensities also differed between counties for this tick. Because adjacent populations, as well as the sex of the host, can differ in infestation rates, differences between local populations of I. scapularis should be recognized to optimize tick surveys and population models.

Azithromycin compared with amoxicillin in the treatment of erythema migrans. A double-blind, randomized, controlled trial.

OBJECTIVE: To determine whether azithromycin or amoxicillin is more efficacious for the treatment of erythema migrans skin lesions, which are characteristic of Lyme disease. DESIGN: Randomized, double-blind, double-dummy, multicenter study. Acute manifestations and sequelae were assessed using a standardized format. Baseline clinical characteristics and response were correlated with serologic results. Patients were followed for 180 days. SETTING: 12 outpatient centers in eight states. PATIENTS: 246 adult patients with erythema migrans lesions at least 5 cm in diameter were enrolled and were stratified by the presence of flu-like symptoms (such as fever, chills, headache, malaise, fatigue, arthralgias, and myalgias) before randomization. INTERVENTION: Oral treatment with either amoxicillin, 500 mg three times daily for 20 days, or azithromycin, 500 mg once daily for 7 days. Patients who received azithromycin also received a dummy placebo so that the dosing schedules were identical. RESULTS: Of 217 evaluable patients, those treated with amoxicillin were significantly more likely than those treated with azithromycin to achieve complete resolution of disease at day 20, the end of therapy (88% compared with 76%; P=0.024). More azithromycin recipients (16%) than amoxicillin recipients (4%) had relapse (P=0.005). A partial response at day 20 was highly predictive of relapse (27% of partial responders had relapse compared with 6% of complete responders; P<0.001). For patients treated with azithromycin, development of an antibody response increased the possibility of achieving a complete response (81% of seropositive patients achieved a complete response compared with 60% of seronegative patients; P=0.043). Patients with multiple erythema migrans lesions were more likely than patients with single erythema migrans lesions (P<0.001) to have a positive antibody titer at baseline (63% compared with 17% for IgM; 39% compared with 16% for IgG). Fifty-seven percent of patients who had relapse were seronegative at the time of relapse. CONCLUSIONS: A 20-day course of amoxicillin was found to be an effective regimen for erythema migrans. Most patients were seronegative for Borrelia burgdorferi at the time of presentation with erythema migrans (65%) and at the time of relapse (57%).

Lyme and/or Lyme-like disease in Missouri.

Missouri patients who fulfill the strict CDC surveillance definition for Lyme disease have been reported in significant numbers since 1987, although there are no viable Missouri human cultures of Borrelia burgdorferi. The Missouri erythema migrans rashes are indistinguishable from those in other areas, and the clinical syndrome appears similar to Lyme disease nationally. The authors suspect atypical B. burgdorferi, and/or other Borrelia spirochetes of causing this clinical borreliosis syndrome.

Erythema migrans. Rash as key to early diagnosis of Lyme disease.

The presence of erythema migrans offers physicians the best opportunity for diagnosis and treatment of Lyme disease. Serologic testing has poor sensitivity in patients with early disease. Therefore, aggressive antibiotic treatment initiated solely on the basis of clinical findings is appropriate. A number of recently studied regimens have shown success against the infection.