Serologic detection of a rare case of Q fever in New York City having hepatic and unusual renal complications.

We describe the case of a 46-year-old resident of New York City with a one-year history of frequent urination and 3 weeks of undulating fevers. He also had liver and bone marrow abnormalities where a non-culturable Gram-negative rod was identified. Q fever was suspected and confirmed based on highly elevated phase I and II serum IgM/IgG antibodies against Coxiella burnetii.

Efficacy of an evernimicin (SCH27899) in vitro and in an animal model of Lyme disease.

The MICs of evernimicin at which 90% of Borrelia burgdorferi patient isolates were inhibited ranged from 0.1 to 0.5 microg/ml. Evernimicin was as effective as ceftriaxone against B. burgdorferi in a murine model of experimental Lyme disease. As assessed by culturing the urinary bladders of infected C3H mice, no live Borrelia isolates were recoverable following antibiotic treatment.

An indirect hemagglutination antibody test to detect antibodies to Borrelia burgdorferi in patients with Lyme disease.

An indirect hemagglutination antibody (IHA) test was evaluated for its ability to detect borrelial antibodies in serum samples from patients with Lyme disease. The key test reagent developed for this antibody detection system was tannic acid-treated and glutaraldehyde-fixed sheep red blood cells (SRBC) containing Borrelia burgdorferi (Bb) antigens attached to the outer surface of the SRBC. In order to establish suitable cut-off titers, initial specificity and sensitivity measurements were made using sera from 100 anonymous healthy volunteers and 30 additional pre-determined samples known to be non-reactive or reactive for Lyme disease or syphilis. These results were compared with those obtained using a commercially available ELISA. At titers >/=64, the IHA test had a combined 98% specificity and 100% sensitivity for these 130 serum samples, 30 of which were known positives or negatives, whereas the ELISA was less specific (93%) and much less sensitive (80%). Subsequent testing was performed on sera from 65 patients with the erythema migrans (EM) rash and 20 patients with early disseminated (cardiac/neurologic) symptoms or with Lyme arthritis. At initial presentation, 46-48% of the EM patients had IHA reactivity, with titers >/=128, while 42% were positive in the ELISA. Follow-up testing performed on these EM patients, 8-12 days after receiving antibiotic treatment, revealed that Bb antibodies were detected best by the IHA test (83-86% reactive) relative to the ELISA (81% reactive). Bb antibodies were readily detectable on all of the serum samples from the early disseminated and late stage Lyme disease cases in both assay systems. Based on these results and because of its technical and interpretive simplicity, the IHA test should be considered as a useful and convenient alternative for the serological analysis of Bb infections.

Activity of sera from patients with Lyme disease against Borrelia burgdorferi.

Sera from patients with Lyme disease were evaluated for their ability to kill Borrelia burgdorferi in vivo and in vitro. Separate groups of C3H mice received sera from seropositive humans with early- or late-stage Lyme disease or from seronegative controls. Eighteen to 24 hours after passive transfer of sera, the mice were challenged with 100,000 low-passage B. burgdorferi strain B31 or CA287 organisms. Sera from subjects with late-stage Lyme disease protected the mice against infection after challenge with B. burgdorferi, but sera from subjects with early-stage Lyme disease were not protective. Late-stage sera also inhibited the growth of B. burgdorferi in microcultures on Barbour-Stoenner-Kelly media better than early-stage sera. Immunoblot analysis revealed that the protective properties of late-stage sera were associated with a response of antibodies to multiple proteins. This response included strong reactivity with the outer-surface proteins A and B, which was lacking in early-stage sera.

Sensitivity and specificity of the borreliacidal-antibody test during early Lyme disease: a "gold standard"?

The serodiagnosis of early Lyme disease has been plagued with problems of sensitivity and specificity. We found that the flow-cytometric borreliacidal-antibody test had a sensitivity of 72% for the detection of patients with early Lyme disease. By contrast, the sensitivity of the enzyme immunofluorescence assay was 28%. The enhanced sensitivity of the borreliacidal-antibody test was due to the use of Borrelia burgdorferi 50772, which lacks OspA and OspB. When B. burgdorferi 297, which expresses both OspA and OspB, was used, the sensitivity of the borreliacidal-antibody test was 15%. Our results also showed that the borreliacidal-antibody test was specific. No borreliacidal activity was detected in normal sera or in sera from patients with mononucleosis, rheumatoid factor, or syphilis. These results demonstrate that the flow-cytometric borreliacidal-antibody test may be the laboratory "gold standard" for the serodiagnosis of Lyme disease.

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.

Impact of the saponin adjuvant QS-21 and aluminium hydroxide on the immunogenicity of recombinant OspA and OspB of Borrelia burgdorferi.

The impact of the adjuvants QS-21 and aluminium hydroxide (alum) on the immunogenicity of recombinant outer surface proteins A (OspA) and B (OspB) of Borrelia burgdorferi was investigated. Both non-acylated OspA and OspB derived from strain B31 were expressed in Escherichia coli and purified by reversible citraconylation and anion-exchange chromatography. Antisera to OspA or OspB were prepared in mice with antigens formulated with QS-21 or alum, and evaluated for specific immunoglobulin G isotypes, agglutination and borreliacidal activity. QS-21 significantly enhanced IgG2a and IgG2b antibody responses to OspA and OspB, and IgG1 response to OspA when compared with the formulation containing antigen alone. In contrast, alum significantly inhibited the induction of IgG2a and IgG2b responses to OspA. Alum had no significant effect on IgG1 response to OspA, or IgG2a and IgG2b responses to OspB, but significantly enhanced IgG1 antibody response to OspB. Antisera to OspA or OspB formulated by QS-21 possessed higher titres of agglutinating antibody than antisera to OspA or OspB alone. Borreliacidal activity was eight- to 64-fold higher in antisera to OspA formulated with QS-21 than in antisera to OspA formulated with or without alum. These antisera were highly borreliacidal to New York strain B31, a California isolate CA-2-87, German isolate Fr, and Swedish isolate G25. Antisera to OspB formulated with QS-21 were highly borreliacidal to strains B31 and Fr, but not to CA-2-87 and G25. Antisera to OspB formulated with alum were borreliacidal only to B31. Thus, OspA was superior to OspB and QS-21 superior to alum at eliciting functional antibody responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Sustained cellular immune responses to Borrelia burgdorferi: lack of correlation with clinical presentation and serology.

Fifty-one patients with erythema migrans were followed up prospectively with serial clinical evaluations, serologic determinations for antiborrelial antibodies, and lymphocyte stimulation responses to Borrelia burgdorferi antigens to determine (i) the factors associated with sustained cellular immune responses and (ii) whether lymphocyte stimulation is a good indicator of prior exposure to B. burgdorferi in patients treated early after erythema migrans. Positive lymphocyte stimulation responses ( > 2 standard deviations above normal control values) were found in 15 (29%) of 51 patients 3 months after treatment for erythema migrans and in 8 (18%) of 44 patients 1 year posttreatment. Heightened lymphocyte responses were not associated with the number or duration of erythema migrans lesions prior to treatment, the mean size of the largest erythema migrans lesion, or the number of symptoms at the time of presentation. The development of Jarisch-Herxheimer reaction, choice of antibiotic, and clinical outcome also were not associated with a positive lymphoproliferation assay result. Changes in the lymphocyte stimulation indices between the two time points assessed (3 months and 1 year posttreatment) also did not correlate with the above variables. When serologic results and lymphoproliferative responses were evaluated as categorical or continuous variables, there were no correlations between values. One year after treatment for early Lyme disease, lymphocyte reactivity is not a good indicator of prior infection with B. burgdorferi.

A murine IgG1 monoclonal antibody that binds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi.

A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi, has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii. The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA as well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.

Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions.

Current laboratory diagnosis of Lyme disease relies on tests for the detection of antibodies to Borrelia burgdorferi, the etiologic agent of the disease. These tests are often unreliable because of a lack of sensitivity and specificity and test-to-test variability. The purpose of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) amplification for detection of B. burgdorferi in skin biopsy specimens. Forty-six 2-mm skin biopsy samples were obtained from 44 patients with a clinical diagnosis of erythema migrans, 9 of whom were receiving antibiotic therapy at the time of biopsy. Specimens were ground in BSK medium with separate aliquots taken for culture and PCR. Of the specimens from the untreated group, 57% (21 of 37) were positive by culture and 22% (8 of 37) were culture negative; 22% (8 of 37) of the cultures were uninformative because of contamination. By comparison, 22 (59%) of 37 specimens were positive by PCR amplification. Of 21 culture-positive samples, 13 (62%) were also positive by PCR analysis. Thus, the sensitivity of the PCR was 59 to 62%, based on either a clinical or cultural diagnosis of untreated Lyme disease. None of the nine specimens from antibiotic-treated patients grew in culture, whereas two of the nine were positive by PCR analysis. Given the complexity and time required for culture, PCR is a promising technique for the diagnosis of early Lyme disease.

Passive immunization protects guinea pigs from lethal Toxoplasma infection.

The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti-Toxoplasma immunoglobulins or immune cells that had been enriched for B cells prior to transfer. Cells residing in the spleen, lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 x 10(7) sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cell-mediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.

Immune response to the Lyme spirochete Borrelia burgdorferi affected by ethanol consumption.

Rats fed excessive amounts of ethanol developed marked hematologic and immunologic changes. These included a reversal of the normal lymphocyte to granulocyte ratio in the peripheral blood, lower spleen and lymph node weights and a greatly reduced capacity to express normal cell mediated immune functions, based on poor lymphocyte reactivity in vivo, and in vitro to T and B cell mitogens and borrelial antigens shortly after primary immunization with the bacterial spirochete, Borrelia burgdorferi. Further evidence for impaired immune function caused by ethanol was based on little or no antibody response against Borrelia in rats following in vivo sensitization with B. burgdorferi incorporated in complete Freund's adjuvant. These findings provide substantial direct evidence strengthening the notion that high levels of ethanol ingestion adversely affect the host immune system and can interfere with the immune response to microorganisms.

Antiborrelial activity of serum from rats injected with the Lyme disease spirochete.

The functional properties of humoral factors generated in rats immunized against Borrelia burgdorferi were investigated. After Lewis strain rats were injected intraperitoneally with live B. burgdorferi or in the footpad with dead borreliae incorporated in complete Freund's adjuvant, they produced high-titered antisera. At a dilution of less than or equal to 1:10, sera from immunized or infected rats but not control sera inhibited growth of B. burgdorferi in vitro. Neutralization of growth of three different strains of B. burgdorferi by posttreatment sera was dose-dependent and was detected equally well by direct microscopic counts or by measuring incorporation of tritiated adenine into newly synthesized nucleic acids. These findings provide direct evidence that infection or immunization with the Lyme disease spirochete induces the formation of serum factors capable of preventing the growth of B. burgdorferi in vitro.

Immunization and protection against malaria during murine pregnancy.

Normal and immune mice were evaluated for their ability to resist infection to the rodent malaria parasite, Plasmodium yoelii, during pregnancy. Parasitemia levels were slightly higher and time-to-death shorter in the nonimmunized pregnant group infected with virulent parasites relative to virgin controls. Subinoculation experiments revealed that numerous virulent organisms were present in the placentas of unprotected gravida but were absent from the fetal livers of their conceptuses. It was also found that mice preimmunized with irradiated P. yoelii survived a usually lethal challenge infection during mid-gestation and delivered healthy newborns. Associated with this protection against transplacental spread of parasites was the additional key finding that placental macrophages were as effective as peritoneal exudate cells in phagocytosing parasite derived material in vitro. This murine malaria-pregnancy model should provide new insights on the various factors (virulence, immunogenicity) of microbial infections affecting the fetal-maternal relationship, as well as on the expression of immune effector mechanisms and immunoregulation, during the reproductive process.

Survival of Borrelia burgdorferi in human blood stored under blood banking conditions.

Hematogenous dissemination of organisms occurs in many spirochetal diseases, including Lyme disease and syphilis. Although syphilis has been transmitted by transfusion, in the vast majority of cases, only fresh blood products were involved, in part because Treponema pallidum survives poorly when refrigerated in citrated blood. Because of the rising incidence of Lyme disease in certain areas, whether its causative agent, Borrelia burgdorferi, could survive under blood banking conditions was studied. Dilutions of stock cultures of two strains of B. burgdorferi were inoculated into samples of citrated red cells (RBCs). Viable spirochetes were recovered from RBCs inoculated with 10(6) organisms per mL, after refrigeration for as long as 6 weeks. It is concluded that B. burgdorferi may survive storage under blood banking conditions and that transfusion-related Lyme disease is theoretically possible.

Reversible defect in cAMP metabolism in lymphocytes in malaria infection.

Peripheral blood lymphocytes were observed to have a defect in adenosine 3',5'-monophosphate (cAMP) metabolism during acute malaria infection which reversed once parasites were eliminated from the host circulation. The defect was characterized by decreased intracellular cAMP levels in lymphocytes and by hyporesponsiveness to adenosine or forskolin stimulation of cAMP production. These biochemical changes appeared to correlate functionally with a reduction in the proliferative response of lymphocytes to concanavalin A. A defect in the second messenger role of cAMP in immune effector cells may underlie immunosuppression in malaria infection.

Isolation of Borrelia burgdorferi from the blood of seven patients with Lyme disease.

PURPOSE: Borrelia burgdorferi, the etiologic agent of Lyme disease, has rarely been successfully cultured from blood. We report on seven patients from Westchester County, New York, with B. burgdorferi bacteremia diagnosed between April 1987 and August 1987. PATIENTS AND METHODS: One hundred thirty-two attempts to isolate spirochetes were made on blood specimens obtained from 104 patients. Twenty-two of these specimens were obtained from nine patients who had recently been bitten by Ixodes ticks but who were asymptomatic. Heparinized blood or serum specimens (0.2 to 0.4 mL) were inoculated onto 6 mL of modified Barbour-Stoenner-Kelly medium. Lyme serology was performed by enzyme-linked immunosorbent polyvalent, IgM, and IgG assays, fluorescent immunoassay, and microhemagglutination. RESULTS: Four of the seven patients had erythema migrans, two had facial nerve palsy, and one had a flu-like syndrome without rash. These patients represented 21% (four of 19) of all patients with the characteristic skin lesion who had blood cultures for B. burgdorferi, and 40% (two of five) of all those with facial nerve palsy. Serologic testing was frequently nonreactive; two patients had no detectable antibody on multiple sera by five different assays. All patients improved with antibiotic treatment, and had negative subsequent blood cultures, but five of seven had persistent complaints after completion of therapy. CONCLUSION: Culturing blood for B. burgdorferi may be useful in confirming the diagnosis of Lyme disease in selected patients. Use of spirochete blood cultures may facilitate a better understanding of the pathogenesis and natural history of Lyme disease.

Thymocyte-dependent immunity to toxoplasmosis in the normal and immunocompromised guinea-pig host.

Guinea-pigs made T-cell deficient by thymectomy and irradiation, and protected with syngeneic bone marrow cells (TXB) have a greatly reduced capacity to express normal cell-mediated immune functions, based on their poor responses to T-cell mitogens, prolonged acceptance of skin allografts, and susceptibility to the lethal effects of graft-versus-host disease. Further evidence for impaired T-cell activity in TXB guinea pigs was based on their inability to be fully sensitized to mycobacterial antigens, and increased susceptibility to an intradermally induced infection with the intracellular protozoan parasite, Toxoplasma gondii (RH strain). After challenge at multiple sites with 10(6) or 10(5) parasites, toxoplasmosis in thymus-intact, fully immunocompetent guinea pigs is a self-limiting and survivable infection, whereas the disease takes an acutely lethal course in the majority of TXB guinea-pigs. The latter also had more parasites disseminating to various tissues sites than their euthymic counterparts. The reduced capacity of TXB guinea-pigs to respond to mycobacterial products, and to generate anti-Toxoplasma immunity can be restored by an intravenous infusion of normal syngeneic thymocytes. These findings provide substantial direct evidence strengthening the concept that protection against toxoplasmosis is heavily dependent upon an intact T-cell component of the host's immune response.

Protection against experimental toxoplasmosis by adoptive immunotherapy.

The role of humoral and cell-mediated immunity against toxoplasmosis in experimentally infected guinea pigs was examined by using a syngeneic passive transfer system. Serum or spleen and lymph node cells from guinea pigs immune to infection with the RH strain of Toxoplasma gondii conferred partial protection against symptomatic disease in recipient guinea pigs. This result was based on the reduced dissemination or growth of T. gondii parasites from the primary inoculation site to various selected organ sites of the recipients of immune serum or cells. Similar levels of partial protection against disseminated toxoplasmosis occurred in animals infused with cell suspensions enriched for immune T cells, whereas treatment of immune cells with a monoclonal anti-guinea pig T cell antibody plus complement abolished their ability to transfer resistance. These findings provide substantial direct evidence implicating both cellular and humoral components of the immune response as important effector mechanisms in host resistance to toxoplasmosis.