Chromosome-wide distribution of haplotype blocks and the role of recombination hot spots.

Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.

Lyme disease: disparity between culture and polymerase chain reaction detection of Borrelia burgdorferi after exposure to ceftriaxone in vitro.

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

Genetic diversity of Borrelia burgdorferi in lyme disease patients as determined by culture versus direct PCR with clinical specimens.

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

Comparison of culture-confirmed erythema migrans caused by Borrelia burgdorferi sensu stricto in New York State and by Borrelia afzelii in Slovenia.

BACKGROUND: The clinical manifestations of Lyme borreliosis in North America and Europe seem to differ, but a systematic comparison has never been done. OBJECTIVE: To compare European and U.S. patients with culture-confirmed erythema migrans. DESIGN: Prospective, clinical cohort study. SETTING: University medical centers in Westchester County, New York, and Ljubljana, Slovenia. PATIENTS: 119 U.S. patients with Borrelia burgdorferi sensu stricto infection and 85 Slovenian patients with B. afzelii infection. MEASUREMENTS: Interview, physical examination, and laboratory assays. RESULTS: Compared with Slovenian patients, U.S. patients had erythema migrans for a briefer duration (median duration, 4 days compared with 14 days; P < 0.001) but were more likely to have systemic symptoms (P = 0.01), abnormal findings on physical examination (P < 0.001), and seroreactivity (P < 0.001). Central clearing of erythema migrans lesions was more likely in Slovenian patients (P < 0.001). CONCLUSIONS: Erythema migrans caused by B. afzelii in Slovenia and erythema migrans caused by B. burgdorferi in New York have distinct clinical presentations. Caution should be used when clinical and laboratory experience from one side of the Atlantic is applied to patients on the other.

Geographic risk for lyme disease and human granulocytic ehrlichiosis in southern New York state.

Ixodes scapularis, the tick vector of Lyme disease and human granulocytic ehrlichiosis (HGE), is prevalent in much of southern New York state. The distribution of this species has increased, as have reported cases of both Lyme disease and HGE. The unreliability of case reports, however, demonstrates the need for tick and pathogen surveillance in order to accurately define areas of high risk. In this study, a total of 89,550 m2 at 34 study sites was drag sampled in 1995 and a total of 51,540 m2 at 40 sites was sampled in 1996 to determine tick and pathogen distribution in southern New York state. I. scapularis was collected from 90% of the sites sampled, and regionally, a 2.5-fold increase in nymphal abundance occurred from 1995 to 1996. I. scapularis individuals from all sites were infected with Borrelia burgdorferi in 1995, while an examination of ticks for both B. burgdorferi and the agent of HGE in 1996 confirmed that these organisms were present in all counties; the average coinfection rate was 1.9%. No correlation was found between estimated risk and reported cases of Lyme disease. The geographic disparity of risk observed among sites in this study underscores the need for vector and pathogen surveillance on a regional level. An entomologic risk index can help identify sites for targeted tick control efforts.

Clinical and laboratory spectrum of culture-proven human granulocytic ehrlichiosis: comparison with culture-negative cases.

We describe the clinical and laboratory manifestations of human granulocytic ehrlichiosis (HGE) in eight patients for whom cultures were positive for the HGE agent and compare them with 15 patients for whom cultures were negative but who fulfilled a modified New York State Surveillance definition for HGE. Polymerase chain reaction analysis was positive in 8 (100%) of 8 culture-positive cases vs. 3 (20%) of 15 culture-negative cases (P < .001), morulae were detected in 7 (100%) of 7 culture-positive cases in which tests were performed vs. 0 of 15 culture-negative cases (P < .001), and a fourfold change in antibody titer was demonstrated in 6 (75%) of 8 culture-positive cases vs. 9 (69%) of 13 culture-negative cases (P = not significant). Patients for whom cultures were positive had higher mean oral temperatures +/- SD at presentation than did patients for whom cultures were negative (38.6 degrees C +/- 0.7 degree C vs. 37.2 degrees C +/- 0.8 degree C, respectively; P = .002). Other symptoms and signs were not significantly different between the two groups. Multivariate analysis revealed that the lymphocyte count at presentation was significantly lower in culture-positive cases than in culture-negative cases. Clinical response to treatment was similar in the two groups. Culture confirmation of HGE is the gold standard for defining the sensitivity and specificity of other diagnostic tests presently being developed.

Prevalence of tick-borne pathogens in Ixodes scapularis in a rural New Jersey County.

To assess the potential risk for other tick-borne diseases, we collected 100 adult Ixodes scapularis in Hunterdon County, a rapidly developing rural county in Lyme disease endemic western New Jersey. We tested the ticks by polymerase chain reaction for Borrelia burgdorferi, Babesia microti, and the rickettsial agent of human granulocytic ehrlichiosis (HGE). Fifty-five ticks were infected with at least one of the three pathogens: 43 with B. burgdorferi, five with B. microti, and 17 with the HGE agent. Ten ticks were coinfected with two of the pathogens. The results suggest that county residents are at considerable risk for infection by a tick-borne pathogen after an I. scapularis bite.

Deer ticks (Ixodes scapularis) and the agents of Lyme disease and human granulocytic ehrlichiosis in a New York City park.

Rodent trapping and drag sampling in Van Cortlandt Park, New York City, yielded all stages of Ixodes scapularis, the deer tick vector of Lyme disease and human granulocytic ehrlichiosis (HGE). Polymerase chain reaction analyses of the ticks showed Borrelia burgdorferi and the Ehrlichia sp. that causes HGE.

Inhibition of efficient polymerase chain reaction amplification of Borrelia burgdorferi DNA in blood-fed ticks.

Polymerase chain reaction (PCR) analysis is a widely used method for detection of Borrelia burgdorferi DNA in biological specimens, including ticks. Studies have demonstrated that substances present in mammalian blood can inhibit PCR amplification. This would limit the utility of PCR for determination of B. burgdorferi infection in engorged ticks that have taken a blood meal from a human or other animal host. To systematically assess the potential for such inhibition, nymphal Ixodes scapularis, which had acquired B. burgdorferi as larvae, were fed on rats. These engorged ticks were lysed in standard PCR lysis buffer and aliquots were subjected to PCR analysis; 0 of 56 were PCR positive. An equivalent cohort of unfed (unengorged) ticks had an infection rate of 19% (11 of 57) as determined by identical PCR analysis (P = 0.0006, by Fisher's exact test). When lysates from the engorged ticks were spiked with the 500 prelysed B. burgdorferi, none of the samples yielded a positive PCR signal, indicating the presence of inhibitory substances. Consistent with this observation, PCR amplification of the original engorged tick lysates after extraction with a DNA extraction kit, resulted in detection of B. burgdorferi DNA in 13 specimens (23%). Furthermore, when 500 prelysed B. burgdorferi were added to the treated extracts, all samples (56 of 56) were PCR positive. Thus, extraction resulted in removal of inhibitors of PCR amplification present in unprocessed engorged tick lysates. Furthermore, additional titration experiments showed that some inhibitory substances may also be present in unfed ticks, although this inhibition does not completely prevent detection of B. burgdorferi DNA in unprocessed lysates. This study clearly demonstrates that inhibitors of PCR amplification are present in engorged ticks and prevent accurate determination of B. burgdorferi infection rates by this method unless steps are taken to remove such inhibitors.

Cellular changes and induction of apoptosis in human promyelocytic HL-60 cells infected with the agent of human granulocytic ehrlichiosis (HGE).

Human granulocytic ehrlichiosis (HGE) is an emerging and occasionally fatal human infectious disease whose pathogenesis is largely unknown. Goodman et al. (1) recently described the successful cultivation of the HGE infectious agent in human promyelocytic HL-60 leukemic cells. It was reported in the same study that infectivity invariably led to host cell death, although the mechanism by which HGE infection triggers cellular self-destruction is as yet undetermined. In this communication, we show that in vitro passage of HGE pathogen-infected blood elicits a significantly dysfunctional G1-to-S transition. Moreover, we provide evidence that the cytopathic properties of the HGE pathogen are attributed to its ability to induce apoptosis in host HL-60 cells. Determination of specific protein expression changes by Western blot analysis showed that HGE infection resulted in reduced expression of PCNA and pRB, both of which play a role in cell cycling. Moreover, the steady state level of bcl-2, which protects eukaryotic cells against apoptosis, is suppressed by exposure to the HGE agent. These results suggest that this pathogen HGE induces apoptosis in HL-60 cells by a mechanism involving the shut-off of multiple cell cycle and apoptosis regulatory events.

Positive Lyme disease serology in patients with clinical and laboratory evidence of human granulocytic ehrlichiosis.

In 10 consecutive patients with an acute febrile illness, human granulocytic ehrlichiosis was confirmed with specific polymerase chain reaction studies, serologic conversion, or both. Although no patients had the clinical features most suggestive of early Lyme disease (eg, erythema migrans or cranial nerve palsy), tests for antibody to Borrelia burgdorferi produced a reaction in most patients. In 6 of 7 patients (86%) with evaluable results, enzyme-linked immunosorbent assay yielded positive or equivocal findings, and an immunoblot technique yielded positive findings in 60% to 90% of patients, depending on the criteria used for interpretation. Inasmuch as approximately 25% of nymphal Ixodes scapularis ticks in Westchester County, New York, are infected with B burgdorferi, the probability that at least 9 of these patients were coinfected with B burgdorferi and human granulocytic ehrlichiosis by the same tick bite is estimated to be .00003. These observations suggest that serodiagnosis is insufficient to establish the presence of coinfection with B burgdorferi.

Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis.

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.