Evaluation of the immunoglobulin G avidity test for diagnosis of toxoplasmic lymphadenopathy.

Toxoplasmic lymphadenopathy (TL) is the most common clinical manifestation of acute acquired toxoplasma infection in normal individuals. The diagnosis is established by serologic methods and lymph node biopsy. Recently, tests for avidity of toxoplasma immunoglobulin G (IgG) antibodies have been introduced to help discriminate between recently acquired and distant infection with the parasite. We studied an avidity test to define the usefulness of this method and to determine the evolution of the IgG avidity in TL. Seventy-three consecutive patients diagnosed as having TL were studied. IgG avidity test titers were noted to be time dependent from the clinical onset of lymphadenopathy. Low IgG avidity test results were observed in patients who had developed lymphadenopathy from <1 month to 17 months prior to the sampling of sera, emphasizing that low IgG avidity test results are not reliable for diagnosis of recently acquired infection. In contrast, high IgG avidity test results were observed only in patients who had developed lymphadenopathy at least 4 months earlier. Thus, a high IgG avidity test result in an individual who has recent onset of lymphadenopathy (e.g., within 2 to 3 months of sera sampling) suggests a cause other than toxoplasmosis. In such cases, further workup is warranted in order to determine the cause of the lymphadenopathy.

Effect of moxifloxacin on secretion of cytokines by human monocytes stimulated with lipopolysaccharide.

OBJECTIVE: To determine the effect of moxifloxacin on secretion of cytokines by human monocytes stimulated with lipopolysaccharide (LPS) or Pansorbin. METHODS: Monocytes obtained from 10 healthy volunteer donors were stimulated with LPS or Pansorbin and exposed or not to different concentrations of the fluoroquinolone antibiotic moxifloxacin. At 3, 6 and 24 h, the amounts of interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-10, IL-12 (p70) and tumour necrosis factor-alpha (TNF-alpha) were measured in the supernatants of the monocyte cultures using enzyme-linked immunosorbent assay. RESULTS: Stimulation of human monocytes with either LPS or Pansorbin resulted in a significant increase in secretion of each of the cytokines examined. Treatment of LPS-stimulated monocytes with moxifloxacin significantly inhibited (P < 0.01) secretion of IL-1alpha by monocytes of each of 10 human donors; the secretion of TNF-alpha was significantly inhibited (P < 0.01) in monocytes from six of 10 donors. In general there was a trend towards inhibition of secretion of IL-6, IL-10 and IL-12 (p70), but the inhibitory effect was not statistically significant. Secretion of cytokines by Pansorbin-stimulated monocytes was not significantly inhibited by moxifloxacin. CONCLUSIONS: Moxifloxacin has immunomodulatory activity through its capacity to alter the secretion of IL-1alpha and TNF-alpha by human monocytes.

Infectious complications among 620 consecutive heart transplant patients at Stanford University Medical Center.

A total of 1073 infectious episodes (IEs) that occurred in 620 consecutive heart transplantation patients at Stanford Medical Center between 16 December 1980 and 30 June 1996 were reviewed. Infectious complications were a major cause of morbidity and mortality, second only to rejection as the cause of early deaths and the most common cause of late deaths. Of the IEs, 468 (43.6%) were caused by bacteria, 447 (41.7%) by viruses, 109 (10.2%) by fungi, 43 (4.0%) by Pneumocystis carinii, and 6 (0.6%) by protozoa. The largest number of IEs occurred in the lungs (301 [28.1%]). A significant reduction in the incidence of IEs and a delay in presentation after transplantation were observed; these were most likely related to the introduction of new chemoprophylactic regimens during the study period and prevention of significant disease caused by cytomegalovirus.

Simple and efficient method for measuring anti-toxoplasma immunoglobulin antibodies in human sera using complement-mediated lysis of transgenic tachyzoites expressing beta-galactosidase.

A simple and efficient method using transgenic Toxoplasma gondii tachyzoites expressing beta-galactosidase was developed for detection of specific antibodies against the parasite in sera of patients. The titers obtained with the new test were similar to those obtained with the Sabin-Feldman dye test run in parallel. Although significant changes in endpoint titers were not observed when sera drawn sequentially at 2- to 3-week intervals were tested with both procedures, apparent differences in antibody affinity were observed with the new test which were not perceptible with the Sabin-Feldman dye test. Like the Sabin-Feldman dye test, the new test is based on complement lysis of tachyzoites, but it is much easier to perform and the reaction is read colorimetrically instead of visually.

Effect of testing for IgG avidity in the diagnosis of Toxoplasma gondii infection in pregnant women: experience in a US reference laboratory.

The usefulness of testing for IgG avidity in association with Toxoplasma gondii was evaluated in a US reference laboratory. European investigators have reported that high-avidity IgG toxoplasma antibodies exclude acute infection in the preceding 3 months. In this US study, 125 serum samples taken from 125 pregnant women in the first trimester were chosen retrospectively, because either the IgM or differential agglutination (AC/HS) test in the Toxoplasma serologic profile suggested or was equivocal for a recently acquired infection. Of 93 (74.4%) serum samples with either positive or equivocal results in the IgM ELISA, 52 (55.9%) had high-avidity antibodies, which suggests that the infection probably was acquired before gestation. Of 87 (69.6%) serum samples with an acute or equivocal result in the AC/HS test, 35 (40.2%) had high-avidity antibodies. Forty women were given spiramycin, to prevent congenital transmission, and 7 (17.5%) had high-avidity antibodies. These findings highlight the value of testing a single serum sample obtained in the first trimester of pregnancy for IgG avidity.

Confirmatory serologic testing for acute toxoplasmosis and rate of induced abortions among women reported to have positive Toxoplasma immunoglobulin M antibody titers.

OBJECTIVE: Results obtained with commercial testing kits for immunoglobulin M Toxoplasma antibodies may be inaccurate or may be inaccurately interpreted, which may influence whether a woman decides to terminate the pregnancy. This study was undertaken to determine whether confirmatory testing at a reference laboratory and communication of the results and an expert interpretation to the patient's physician would affect the rate of induced abortions among pregnant women with positive results of testing for immunoglobulin M Toxoplasma antibodies in outside laboratories. STUDY DESIGN: This was a retrospective cohort study of 811 consecutive pregnant women for whom the toxoplasma serologic profile was performed at a reference laboratory. Almost all the patients had been informed by their physicians that a result of a test for immunoglobulin M Toxoplasma antibodies performed in an outside laboratory was positive. Women were separated into those with a toxoplasma serologic profile result suggestive of a recently acquired infection (group 1) and those with a result suggestive of an infection acquired in the more distant past (group 2). Physician reports of induced abortions were used to determine rates of induced abortion in groups 1 and 2. RESULTS: Of the 811 women 321 (39.6%) were considered likely to have a recent infection (group 1) and 490 (60.4%) were considered likely to have a past infection (group 2). Physicians reported pregnancy outcomes for 433 (53.4%) of 811 women (65.1% and 45.7% in groups 1 and 2, respectively). Whereas 36 of 209 women in group 1 (17.2%) terminated the pregnancy, only 1 of 224 women in group 2 (0.4%) chose abortion (P <.001). CONCLUSION: Confirmatory serologic testing in a reference laboratory and communication of the results and their correct interpretation by an expert to the patient's physician decreased the rate of unnecessary abortions by approximately 50% among women for whom positive immunoglobulin M Toxoplasma test results had been reported by outside laboratories.

Activity of gatifloxacin alone or in combination with pyrimethamine or gamma interferon against Toxoplasma gondii.

The activity of gatifloxacin against Toxoplasma gondii, either alone or in combination with pyrimethamine or gamma interferon (IFN-gamma), was examined in vitro and in vivo. In vitro, gatifloxacin significantly inhibited intracellular replication of tachyzoites of the RH strain with a 50% inhibitory concentration of 0.21 microg/ml at 48 h after addition of the drug to the cultures. Toxicity for host cells was not observed at this concentration. A synergistic effect (combination indices < 0.5) was demonstrated in vitro following 48 h of treatment with the combination of gatifloxacin and pyrimethamine (1:1 ratio). Doses of gatifloxacin of 100 and 200 mg/kg of body weight/day administered orally to mice for 10 days resulted in significant (P values of 0.056 and <0.0001, respectively) prolongation in time to death following infection with a lethal inoculum of tachyzoites. A dose of 400 mg/kg resulted in 20% survival (P = 0.0001). Mortality was 100% in untreated control mice and in mice treated with 25 or 50 mg/kg/day. Treatment of infected mice with a combination of gatifloxacin at 200 mg/kg/day and pyrimethamine at 12.5 mg/kg/day resulted in 85% survival, whereas 100 and 80% of mice treated with gatifloxacin alone or pyrimethamine alone, respectively, died (P < 0.0001). Moreover, a gatifloxacin dose of 200 mg/kg/day administered orally for 10 days plus 2 microg of recombinant murine IFN-gamma/day administered intraperitoneally for 10 days resulted in significant survival compared with IFN-gamma alone (P < 0.0001) or gatifloxacin alone (P < 0.007).

Strain typing of Toxoplasma gondii: comparison of antigen-coding and housekeeping genes.

Molecular characterization of Toxoplasma gondii isolates is central for understanding differences in disease transmission and manifestations. Only 3 subgroups (lineages) have been discerned with subtle within-lineage variation, permitting low-resolution classification of isolates. Because proteins, coding sequences, and especially antigen-coding genes have been used extensively in previous studies, we focused on sequence variation in introns of housekeeping genes, which may be more informative for phylogenetic analysis because they evolve under lower selection. We compared sequence variation in introns of 5 housekeeping genes with 2 antigen-coding genes. Introns of housekeeping genes were slightly more polymorphic than coding and noncoding regions of antigen-coding genes and only the former showed intralineage variation. Intragenic linkage disequilibrium was complete, but intergenic linkage, although highly significant, was incomplete, suggesting that genes are partially uncoupled. Six of 7 substitutions found within the region coding for the tachyzoite surface antigen, SAG2, were nonsynonymous, indicating that diversifying selection acts on this locus. Typing isolates on the basis of housekeeping and antigen-coding genes was consistent, but the phylogenetic relationships among the resulting groups was inconsistent. A cougar isolate typed as lineage II using a restriction fragment length polymorphism assay possessed multiple unique polymorphisms, suggesting that it represents a new lineage. We concluded that introns of housekeeping genes are preferred markers for phylogenetic study, and that multilocus genotyping is preferred for typing parasites, especially from feral or unstudied environments.

Human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL SCID) models of toxoplasmosis.

Toxoplasmosis is a potentially fatal opportunistic infection of immunocompromised hosts. Improved animal models of toxoplasmosis are needed to more nearly approximate conditions that occur in immunocompromised humans. The development of models of toxoplasmosis using human peripheral blood lymphocytes (hu-PBL) transplanted into severe combined immunodeficiency (SCID) mice is described here. Transplantation of hu-PBL into SCID mice without prior conditioning of the mice resulted in detectable differences in quantitative histological scores of brain inflammation due to Toxoplasma gondii infection, but did not alter mortality when compared to SCID mouse controls. The lack of detectable differences in survival were due to inadequate engraftment of hu-PBL, as assessed by flow cytometry. Unconditioned hu-PBL SCID mice had low titre T. gondii-specific antibody detectable after infection. When pretransplantation conditioning with irradiation and antiasialo GM 1 (n-glucolyl neuraminic acid) antibody was used, prolonged hu-PBL engraftment was observed in SCID mice, which was associated with worsened histopathology and usually impaired survival when compared with SCID mouse controls. When pretransplantation conditioning with irradiation, antiasialo GM antibody and polyethylene glycol-conjugated IL-2 was used, prolonged hu-PBL engraftment was also documented, but this did not affect survival from T. gondii infection when compared with similarly conditioned SCID mouse controls. The latter conditioning protocol resulted in hu-PBL SCID mice producing high titre T. gondii-specific antibody after infection. Conditioned hu-PBL SCID mice had evidence of increased T. gondii-induced inflammatory scores when compared with conditioned SCID mice. These models show promise for the study of the pathogenesis of toxoplasmosis and conditioned hu-PBL SCID mice may have applications for the evaluation of novel therapies for toxoplasmosis in immunocompromised humans.

Detection of immunoglobulin M antibodies to P35 antigen of Toxoplasma gondii for serodiagnosis of recently acquired infection in pregnant women.

We examined the efficiency of detection of immunoglobulin M (IgM) antibodies to a 35-kDa antigen (P35) of Toxoplasma gondii for serodiagnosis of acute infection in pregnant women. A double-sandwich enzyme-linked immunosorbent assay (ELISA) with recombinant P35 antigen (P35-IgM-ELISA) was used for this purpose. On the basis of the clinical history and the combination of results from the toxoplasma serological profile (Sabin-Feldman dye test, conventional IgM and IgA ELISAs, and the differential agglutination test), the patients were classified into three groups: group I, status suggestive of recently acquired infection; group II, status suggestive of infection acquired in the distant past; group III, status suggestive of persisting IgM antibodies. Eighteen (90.0%) of 20 serum samples from group I patients were positive by the P35-IgM-ELISA, whereas none of the 33 serum samples from group II patients were positive. Only 4 (25.0%) of 16 serum samples from group III patients were positive by the P35-IgM-ELISA, whereas all these serum samples were positive by the conventional IgM ELISA. These results indicate that demonstration of IgM antibodies against P35 by the P35-IgM-ELISA is more specific for the acute stage of the infection than demonstration of IgM antibodies by the ELISA that uses a whole-lysate antigen preparation. Studies with sera obtained from four pregnant women who seroconverted (IgG and IgM antibodies) during pregnancy revealed that two of them became negative by the P35-IgM-ELISA between 4 and 6 months after seroconversion, whereas the conventional IgM ELISA titers remained highly positive. The P35-IgM-ELISA appears to be useful for differentiating recently acquired infection from those acquired in the distant past in pregnant women.

Protection against lipopolysaccharide-induced death by fluoroquinolones.

Because fluoroquinolones have an immunomodulatory effect on cytokine production by lipopolysaccharide (LPS)-treated human monocytes, we examined the effect of fluoroquinolones on the survival of mice injected with a lethal dose of LPS. Trovafloxacin (100 mg/kg), ciprofloxacin (250 mg/kg), and tosufloxacin (100 mg/kg) protected 75% (P = 0.0001), 25% (P = 0.002), and 50% (P = 0.002), respectively, of mice against death. The fluoroquinolones significantly reduced serum levels of interleukin-6 and tumor necrosis factor alpha in LPS-treated mice. The protective effects of fluoroquinolones in LPS-induced shock in mice may also occur in humans.

Ketolide ABT-773 is active against Toxoplasma gondii.

ABT-773 is active in vitro and in vivo against Toxoplasma gondii. It inhibited replication of RH strain tachyzoites in human foreskin fibroblasts. Mice infected intraperitoneally with tachyzoites and treated orally with 25, 50 or 100 mg/kg/day of ABT-773 for 10 days had 20% (P: = 0.016), 50% (P: = 0.003) and 100% (P: = 0.001) survival, respectively. Remarkable and highly significant survival was also noted in mice infected orally with strain C56 cysts and treated with ABT-773. Thus, ABT-773 may be useful for therapy of human toxoplasmosis.

Serodiagnosis of recently acquired Toxoplasma gondii infection using an enzyme-linked immunosorbent assay with a combination of recombinant antigens.

An enzyme-linked immunosorbent assay (ELISA) using four recombinant antigens of Toxoplasma gondii (rP22, rP25, rP29, and rP35) was used in an attempt to differentiate pregnant women with toxoplasma serologic profiles (TSPs) indicative of recently acquired infections (acute profile) from those with TSPs indicative of infections acquired in the distant past (chronic profile). In general, immunoglobulin G antibodies in sera from women with the acute profile reacted more strongly with the recombinant antigens than did those in sera from women with the chronic profile. However, reactivities differed significantly between antigens that reacted with a single serum and between sera that reacted with a single antigen. Because of these variations, we employed a combination of the four antigens in an ELISA (Comb-ELISA) and evaluated its ability to distinguish pregnant women with the acute profile from those with the chronic profile. Eighteen of 20 (90%) sera from acute-profile women were positive in the Comb-ELISA, whereas 69 of 70 (98.6%) sera from the chronic-profile women were negative. Thus, the Comb-ELISA may be useful for diagnosis of toxoplasmosis in pregnant women and for differentiation between recently acquired infections and infections acquired in the more distant past.

Effect of quinupristin/dalfopristin on production of cytokines by human monocytes.

The effect of the novel streptogramin antibiotic quinupristin/dalfopristin (synercid) on cytokine production in vitro was examined in monocytes obtained from healthy human volunteers and stimulated with either lipopolysaccharide or heat-killed Staphylococcus aureus (Pansorbin). Synercid at concentrations that are achievable in humans (1, 5, and 10 microgram/mL) significantly suppressed production of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha in a concentration-dependent manner. Thus, synercid possesses significant immunomodulatory activity, in addition to its antimicrobial activity.

Decreased resistance of B cell-deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN-gamma, TNF-alpha, and inducible nitric oxide synthase.

The role of B cells in resistance against Toxoplasma gondii was studied using B cell-deficient (muMT) mice. Following peroral infection with 10 cysts of the ME49 strain, all muMT mice survived the acute stage of the infection but died between 3 and 4 wk after infection. In contrast, all control mice were alive at 8 wk after infection. At the stage during which muMT animals succumbed to the infection, parasite replication and pathology were most evident in their brains; small numbers of tachyzoites were also detectable in their lungs. Significantly greater numbers of T. gondii cysts and areas of inflammation associated with tachyzoites were observed in brains of muMT than in control mice. Large areas of necrosis associated with numerous tachyzoites were observed only in brains of muMT mice. Anti-T. gondii IgG Abs were detected only in sera of control mice, whereas similar levels of IFN-gamma were detected in sera of both strains of mice. Amounts of mRNA for IFN-gamma, IL-10, and inducible NO synthase in the brain did not differ between infected muMT and control mice. Expression of mRNA for TNF-alpha was increased in brains of muMT mice. Administration of polyclonal rabbit anti-T. gondii IgG Ab prevented early mortality and pathology associated with tachyzoites in the brain in the infected muMT mice. These results indicate that B cells play an important role, most likely through their production of specific Abs, in resistance to persistent active (tachyzoite) infection with T. gondii in mice, especially in the brain and lung.

Serodiagnosis of recently acquired Toxoplasma gondii infection with a recombinant antigen.

A portion of a cDNA encoding a 35-kDa antigen from Toxoplasma gondii was cloned into the CKS expression vector and expressed in Escherichia coli. By using the enzyme-linked immunosorbent assay (ELISA), the recombinant protein (rP35 antigen) was examined for reactivity with immunoglobulin G (IgG) antibodies in the sera of pregnant women. Of these women, 41 had a toxoplasma serologic profile suggestive of recently acquired T. gondii infection (Sabin-Feldman dye test [DT] titers from 1:256 to 1:32,000, positive IgM ELISA titers from 2.3 to 9.7, positive IgA ELISA from 1 to >28, and acute patterns in the differential agglutination [AC/HS] test) (group I), and 50 women had a toxoplasma serologic profile suggestive of infection acquired in the distant past (low DT titers from 1:16 to 1:512, negative IgM ELISA titers from 0 to 0.8, and chronic patterns in the AC/HS test) (group II). The classification of acute or chronic profile was based on the individual's clinical history as well as the combination of the results of the toxoplasma serological profile. An additional group (group III) was composed of sera from 50 women who were seronegative for T. gondii antibodies in the DT. The results revealed that whereas 85.3% of women in group I had IgG antibodies that reacted with the rP35 antigen, only 8% of women in group II had IgG antibodies that reacted with the same antigen. In immunoblots, the rP35 antigen was recognized by IgG antibodies in a pool of sera from individuals with a toxoplasma serologic profile compatible with acute infection but not in a pool of sera from individuals with a serologic profile characteristic of a chronic infection. These results reveal that IgG antibodies against the P35 antigen are produced during the acute stage of the infection but are uncommon in the latent or chronic phase of the infection. Thus, the rP35 antigen may be a useful serologic marker to differentiate between recently acquired infection and that acquired in the more distant past.

Use of the polymerase chain reaction for diagnosis of ocular toxoplasmosis.

OBJECTIVE: To report a cohort of patients in whom polymerase chain reaction (PCR) was performed on vitreous samples and to place in perspective the current role of PCR in the diagnosis of ocular toxoplasmosis. DESIGN: Noncomparative case series. PARTICIPANTS: Fifteen patients in whom toxoplasmic retinochoroiditis was considered in the differential diagnosis and in whom the clinical presentation was not diagnostic and/or response to treatment was inadequate. INTERVENTION: Examination of vitreous fluid by PCR and of serum for the presence of Toxoplasma-specific antibodies. MAIN OUTCOME MEASURES: Presence of Toxoplasma gondii DNA, serologic test results, clinical findings, treatment, and outcome. RESULTS: In 7 of 15 patients, vitreous fluid examination results by PCR were positive for the presence of T. gondii DNA. Five of these seven patients had serologic test results consistent with Toxoplasma infection acquired in the distant past; the other two patients had serologic test results consistent with retinochoroiditis in the setting of acute toxoplasmosis. The PCR results influenced the management of these patients in six of the seven positive cases. In the eight patients in whom vitreous examination results were negative by PCR, either Toxoplasma serology was negative (6), the retinal lesions were caused by cytomegalovirus (1), or, on further consideration, the eye signs were not consistent with those of toxoplasmic retinochoroiditis (1). CONCLUSION: In patients in whom toxoplasmosis is considered in the differential diagnosis but in whom the presentation is atypical, PCR was frequently a useful diagnostic aid.

Quinupristin-dalfopristin is active against Toxoplasma gondii.

Synercid and each of its components (quinupristin and dalfopristin) were examined for their activities against Toxoplasma gondii. In vitro, intracellular replication of tachyzoites was inhibited by synercid and each of its two components. The 50% inhibitory concentrations of synercid, quinupristin, and dalfopristin were 1.6, 2.7, and 6.3 microg/ml, respectively. Thus, synercid was markedly more active than its components. Treatment of acutely infected mice with 100 or 200 mg of synercid per kg of body weight per day administered intraperitoneally for 10 days resulted in survival of 50% (P = 0.0002) and 100% (P < 0.0001) of infected mice, respectively, whereas all control mice died by day 18. In contrast, treatment with 200 mg of either quinupristin and dalfopristin per kg per day alone resulted in only 20% survival; treatment with 50 mg of either drug per kg per day resulted only in the prolongation of time to death. These results suggest that synercid may be useful for treatment of toxoplasmosis in humans.

Anti-Toxoplasma gondii activities and structure-activity relationships of novel fluoroquinolones related to trovafloxacin.

Eleven novel fluoroquinolones closely related to trovafloxacin were evaluated for their in vitro activity against Toxoplasma gondii, and their structure-activity relationships were examined. The 50% inhibitory concentration (IC50) of trovafloxacin against T. gondii was 2.93 microM; the IC50 of the 11 analogs ranged from 0.53 to 14. 09 microM. Six analogs had IC50s lower than that of trovafloxacin. Examination of the structure-activity relationships of the compounds revealed that addition of a -CH3 at C-5 of the 1,8-naphthyridone ring, at C-2 of the azabicyclohexane ring, or on the -NH2 at the 6 position of the azabicyclohexane ring resulted in a four- to sixfold increase in activity. Moreover, replacement of 2,4-difluorophenyl by cyclopropyl at N-1 of the 1,8-naphthyridone ring increased activity twofold, and moving the -NH2 one atom further away from the azabicyclohexane ring decreased activity. There was no difference between the naphthyridone and quinolone analogs. These results indicate that structure-activity studies of compounds related to drugs active against T. gondii may be useful in producing compounds with more potent activities against the parasite.