An enzootic transmission cycle of Lyme borreliosis spirochetes in the southeastern United States.

Lyme borreliosis, or Lyme disease (LD), is a tick-borne zoonotic infection of biomedical significance, caused by Borrelia burgdorferi sensu lato (s.l.) spirochetes and transmitted by Ixodes species ticks. It usually circulates among wildlife vertebrate reservoirs and vector ticks but may infect humans, causing multisystem problems. In far western and northern North America, the host reservoirs, tick vectors, and genospecies of Borrelia are well known but not so in the southern U.S., where there is controversy as to the presence of "true" LD. Here we report the presence of the LD spirochete B. burgdorferi sensu stricto (s.s.) and Borrelia bissettii, three main reservoir hosts, and two enzootic tick vectors in the southeastern U.S. The two enzootic tick vectors, Ixodes affinis and Ixodes minor, rarely bite humans but are more important than the human biting "bridge" vector, Ixodes scapularis, in maintaining the enzootic spirochete cycle in nature. We also report extraordinary longevities and infections in the reservoir rodents Peromyscus gossypinus, Sigmodon hispidus, and Neotoma floridana.

White grain mycetoma caused by a Cylindrocarpon sp. in India.

We describe a case of white grain eumycetoma of the foot of an Indian male caused by a slow-growing, poorly sporulating fungus that does not match any known agent of this infection. Histologic examination of a biopsy tissue specimen showed oval, lobular, white granules composed of hyaline, septate hyphae, and thick-walled chlamydospores. Culture of granules from a draining sinus yielded compact, very-slow-growing, poorly sporulating colonies producing a strong reddish brown pigment that diffused into the medium. The fungus was identified as a Cylindrocarpon sp. based on the development of rare cylindrical conidia borne from solitary phialides lacking collarettes, in addition to chlamydospores formed singly or in short chains.

Isolation, cultivation, and characterization of Borrelia burgdorferi from rodents and ticks in the Charleston area of South Carolina.

Twenty-eight Borrelia burgdorferi isolates from the Charleston, S.C., area are described. This represents the first report and characterization of the Lyme disease spirochete from that state. The isolates were obtained from December 1994 through December 1995 from the tick Ixodes scapularis, collected from vegetation, and from the rodents Peromyscus gossypinus (cotton mouse), Neotoma floridana (eastern wood rat), and Sigmodon hispidus (cotton rat). All isolates were screened immunologically by indirect immunofluorescence with monoclonal antibodies to B. burgdorferi-specific outer surface protein A (OspA) (antibodies H5332 and H3TS) and B. burgdorferi-specific OspB (antibodies H6831 and H614), a Borrelia (genus)-specific antiflagellin antibody (H9724), Borrelia hermsii-specific antibodies (H9826 and H4825), and two polyclonal antibodies (one to Borrelia species and another to B. burgdorferi). Six of the isolates were analyzed by exposing Western blots to monoclonal antibodies H5332, H3TS, H6831, and H9724. All isolates were also analyzed by PCR with five pairs of primers known to amplify selected DNA target sequences specifically reported to be present in the reference strain, B. burgdorferi B-31. The protein profiles of six of the isolates (two from ticks, one from a cotton mouse, two from wood rats, and one from a cotton rat) also were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the 28 Charleston isolates are B. burgdorferi sensu stricto based on their similarities to the B. burgdorferi B-31 reference strain.

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.

Immunohistochemical evaluation of bcl-2 oncoprotein in oral dysplasia and carcinoma.

The proto-oncogene bcl-2 is associated with follicular lymphoma involving translocation t(14;18)(q32;q21) and is also overexpressed in various neoplasms. We report deregulation of bcl-2 expression during progression from oral epithelial dysplasia to squamous cell carcinoma. Immunohistochemical analysis with monoclonal antibodies to bcl-2 oncoprotein in formalin-fixed paraffin-embedded tissue sections revealed that severe epithelial dysplasias had a higher percentage of immunoreactivity than did mild and moderate dysplasias and squamous cell carcinomas. Expression of this oncoprotein was directly proportional to the degree of epithelial dysplasia, and nondysplastic basal cells contiguous to neoplastic lesions also expressed bcl-2. These findings, along with down-regulation of bcl-2 in differentiating carcinomas, suggest a role for this oncoprotein in relatively early stages of oral tumor progression. Differentiating neoplastic cells with marginal or no bcl-2 reactivity showed heterogeneous cell labeling of varying intensity for differentiation-associated cytokeratin (CK13), indicating their inverse topographic relationship.

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.

Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue.

Embalmed tissues are adequate for the detection of JC virus in lesions of progressive multifocal leukoencephalopathy (PML) by immunohistologic and molecular methods. JC virus was readily detected in embalmed brain tissue using immunohistochemistry (IHC), in situ hybridization (ISH), and the polymerase chain reaction (PCR). Two brains were removed from bodies that had been embalmed at least 24 h prior to autopsy. They were subsequently post fixed in 10% buffered formalin for 10-14 days before dissection and molecular studies were performed. Though these techniques are not novel, their use in embalmed tissues is. Routine embalming should not eliminate these diagnostic procedures from consideration.

p16 (CDKN2/MTS1) gene deletions are rare in prostatic carcinomas in the United States and Japan.

PURPOSE: The incidence of clinically apparent prostatic carcinoma is much higher in the United States than in Japan. Alterations in the p16 tumor suppressor gene have been identified in various tumor types, including cultured prostatic carcinoma cell lines. We studied the possible deletions of either exon 2 or 3 of this gene in primary clinical prostatic carcinomas from Japan and the United States. MATERIALS AND METHODS: Genomic DNA was extracted from 36 formalin-fixed, paraffin-embedded clinical prostatic carcinomas from Japan and 27 carcinomas from the United States. Exons 2 and 3 of the p16 gene were amplified using comparative multiplex polymerase chain reactions (PCR) and then analyzed for possible deletions of either exon. RESULTS: Two out of 36 (5.6%) carcinomas from Japan clearly demonstrated deletion of p16 exon 2, but this deletion was not detected in any of the 27 carcinomas from the United States. CONCLUSIONS: Although slightly higher in Japan than in the United States, the frequency of p16 exon deletions in clinical prostatic carcinomas is very low, and probably is not important in the development of this neoplasm.

Unusual strain of Borrelia burgdorferi isolated from Ixodes dentatus in central Georgia.

A new, unusual spirochete was cultured in Barbour-Stoenner-Kelly (BSK II) medium from the midgut and other tissues of the tick Ixodes dentatus. The tick was collected from leaf litter in an oak-pine wood lot in Bibb County approximately 7.2 km from Macon in central Georgia during February 1993. Characterization by indirect immunofluorescence using 5 murine monoclonal antibodies, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole spirochetal lysates, and by polymerase chain reaction assay for several known DNA target sequences indicates that the spirochete is Borrelia burgdorferi sensu lato. It is genetically different from the B-31 reference strain of B. burgdorferi sensu stricto that is typical of strains causing Lyme borreliosis in North America. Range of infectivity and pathogenesis of the Bibb County isolate (BC-1) are unknown but being investigated. The BC-1 strain is the first B. burgdorferi isolate from I. dentatus in the southeastern United States (I. dentatus is not the common vector for Lyme borreliosis in humans). Additionally, the collection site was approximately 322 km from the Atlantic coast, far distant from where most B. burgdorferi isolates have been obtained.

Rhinosporidiosis: three domestic cases.

Three cases of rhinosporidiosis in Americans who had not traveled abroad are reported. We believe this is the largest cluster of indigenous cases reported in the United States. The three patients had lived in rural northeast Georgia all of their lives. One had a polypoid conjunctival lesion, and the two others had nasal polyps. In each case, the diagnosis was made by demonstrating morphologically distinctive fungal elements in histopathologic sections. Clinically, rhinosporidiosis had not been suspected.

Opportunistic infections of the central nervous system in children with HIV infection: report of 9 autopsy cases and review of literature.

Central nervous system (CNS) abnormalities attributed to direct effects of HIV infection are seen in most of children with acquired immunodeficiency syndrome (AIDS). Secondary CNS infections with opportunistic and common pathogens are infrequent in this age group. We report 9 cases of opportunistic infection of the CNS found among 65 autopsy cases of pediatric AIDS. These included 4 cases of cytomegalovirus (CMV) infection, 1 of which was associated with aspergillosis, and 2 cases of candidiasis, 1 of which coexisted with Mycobacterium avium intracellulare (MAI) infection. There were also 2 cases of leptomeningitis, 1 due to Mycobacterium tuberculosis (MTB) and the other to Cryptococcus neoformans. In 1 child progressive multifocal leukoencephalopathy (PML) coexisted with mycotic encephalitis caused by an Aspergillus sp.

Natural occurrence and characterization of the Lyme disease spirochete, Borrelia burgdorferi, in cotton rats (Sigmodon hispidus) from Georgia and Florida.

This is the first report of natural infection by Borrelia burgdorferi in the cotton rat Sigmodon hispidus. Nine B. burgdorferi isolates were obtained from ear tissues, urinary bladders, or both, by culturing tissues in BSKII medium. The rat from which the SI-3 isolate was cultured was from the same site (Sapelo Island, Georgia) as an infected cotton mouse Peromyscus gossypinus and Ixodes scapularis tick reported previously. The 8 B. burgdorferi isolates from rats in Florida included 1 (AI-1) from Amelia Island, 1 (FD-1) from Faver-Dykes State Park, and 6 (MI-3 through MI-8) from Merritt Island. The distance between Sapelo Island and Merritt Island is approximately 400 km. All B. burgdorferi isolates were characterized by indirect immunofluorescence using monoclonal antibodies to OspA (H3TS, H5332) and OspB (H5TS, H6831), polymerase chain reaction detection of specific B. burgdorferi B-31 DNA target sequences (ospA, fla, and a random chromosomal sequence), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of spirochetal proteins. The phenotypic and genotypic characteristics of the isolates are discussed, as well as the probable importance of the cotton rat as a reservoir for B. burgdorferi in the southern United States.

Osteomyelitis caused by Neosartorya pseudofischeri.

The first case of osteomyelitis caused by Neosartorya pseudofischeri is reported. The patient, a 77-year-old male with a history of silicosis and tuberculosis, on X-ray examination revealed lytic lesions of L2 and L3 vertebrae suspicious for metastatic lesions. Histologic examination of biopsy specimens from vertebral bodies showed short, distorted, extra- and intracellular, hyaline hyphal fragments. The culture from the biopsy tissue produced numerous, evanescent asci containing eight ellipsoidal ascospores with two distinctive equatorial bands ca. 1 micron wide. When examined by a scanning electron microscope, ascospores exhibited a convex surface ornamented with raised flaps of tissue, in shape resembling triangular projections or long ridge lines. The conidial state (anamorph) was identified as Aspergillus thermomutatus on the basis of conidial columns which were smaller and less tightly packed as well as of a lighter shade of green than those observed in Aspergillus fumigatus. On the basis of the morphologic features of the ascospores, the teleomorph was identified as N. pseudofischeri.

Histogenesis of vascular tumors in the Proteus syndrome.

Proteus syndrome (PS) is a congenital disorder manifesting with severe deformities, the salient features being gigantism and vascular tumors. The disorder is poorly understood, and there has been much discrepancy in the terminology regarding the vascular tumors in PS. The purpose of this study was to elucidate the histogenesis of these tumors by correlating microscopic observations with immunohistologic information. The value of immunoperoxidase studies in the pathologic evaluation of PS was also assessed. Fourteen formalin-fixed, paraffin-embedded tissue specimens obtained from vascular tumors of six children with PS were stained with Ulex europaeus agglutinin I (UEA-I) lectin and the following immunohistochemical reagents: anti-factor VIII-related antigen (FVIII-RAg) and anti-CD34. The tumors showed varied proportions of vascular, lipomatous, and fibrous tissue components consistent with vascular hamartomas. The predominant vascular channels of the tumors were morphologically consistent with lymphatic vessels. Immunostaining of the endothelium of these vessels was most consistently positive with UEA-I lectin. Although a color reaction product was present in small vessels and some larger blood vessels, anti-CD34 immunostaining spared the lumens of lymphatic channels. In addition, a striking population of dendritic spindle cells was noted with the anti-CD34 but was unnoticed with the other reagents. We concluded that the vascular tumors of PS are primarily lymphatic hamartomas. The spindle cells noted with anti-CD34 immunostaining may relate to angiogenesis and need further delineation.

Detection of Epstein-Barr virus and cytomegalovirus in patients with chronic fatigue.

Patients with chronic fatigue as a major complaint frequently present with recurrent sore throat, and on physical examination they have hyperemia and lymphoid hyperplasia of the pharyngeal area. Pharyngeal scrapings were obtained from 41 such patients and analyzed for Epstein-Barr virus or cytomegalovirus DNA by colorimetric in situ hybridization. Results were compared with healthy control subjects matched for age and sex. Epstein-Barr virus-DNA was detected more frequently in male patients, 5/9 (55.6%), than controls, 0/6 (0%), but there was no difference in frequency in female patients, 4/32 (12.5%), than control subjects, 1/29 (3.4%). Cytomegalovirus-DNA was detected infrequently in patients and controls, 13% versus 22% respectively. The presence of EBV-DNA did not correlate with antibody titers nor with the complaint of sore throat. Four of the five males who had positive EBV-DNA in the pharyngeal smears have now recovered.

Isolation and transmission of the Lyme disease spirochete from the southeastern United States.

The isolation of the Lyme disease spirochete (Borrelia burgdorferi) from the southeastern United States is reported. Three isolates, two from cotton mice (Peromyscus gossypinus) and one from the black-legged tick (Ixodes scapularis), were recovered from Sapelo Island, Georgia, in July and September 1991. The spirochetes were characterized by indirect fluorescent antibody assay using a battery of five monoclonal antibodies, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of whole cell lysates, and by the polymerase chain reaction (PCR) assay using primers for three DNA target sequences found in B. burgdorferi reference strain B-31. Transmission experiments indicate that the three Georgia isolates can infect experimentally inoculated hamsters and mice. Tick transmission of one of the isolates has been attempted so far; I. scapularis transmitted isolate SI-1 from hamsters to mice, but the lone-star tick, Amblyomma americanum, did not.

Detection of Borrelia burgdorferi in laboratory-reared Ixodes dammini (Acari: Ixodidae) fed on experimentally inoculated white-tailed deer.

Larvae and nymphs of Ixodes dammini Spielman, Piesman, Clifford & Corwin from a laboratory colony were fed on two white-tailed deer, Odocoileus virginianus (Zimmerman) inoculated with either the SH2-82 or JD-1 strains of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner. Ticks were exposed to one deer 43 and 69 d after inoculation of the spirochete and to a second deer 35 and 61 d after inoculation. Polymerase chain reaction assays amplified the 158 bp OspA DNA target sequence in 11.1% (n = 9) of fed larvae and 3.3% (n = 30) of nymphs from the deer inoculated with the SH2-82 strain, and 22.7% (n = 22) of larvae and 0% (n = 21) of nymphs from a second deer inoculated with the JD-1 strain of B. burgdorferi. One of three females derived from nymphs fed on one of the inoculated deer showed presence of B. burgdorferi DNA, but none of four males was positive. Experimentally inoculated deer can serve as a source of at least two geographic strains of B. burgdorferi to I. dammini larvae and nymphs for at least several weeks.