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Evaluation of a new particle gel immunoassay for determination of antibodies against Treponema pallidum.

A new particle gel immunoassay (DiaMed AG, Cressier sur Morat, Switzerland) with three recombinant Treponema pallidum antigens was evaluated with serum samples from patients with syphilis (n = 124) and patients without syphilis (n = 490). It proved to be a simple, rapid (20 min), and useful test with sensitivity, specificity, and positive and negative predictive values of 91.9, 99.8, 99.2, and 98%, respectively.

Antibodies, Bacterial↗

Whole-blood counting immunoassay as a short-turnaround test for detection of hepatitis B surface antigen, anti-hepatitis C virus antibodies, and anti-Treponema pallidum antibodies.

Whole-blood samples were used for a counting immunoassay (CIA) with the aim of developing a short- turnaround test. After optimization of the CIA, hepatitis B surface antigen (HBsAg), anti-hepatitis C virus antibodies (anti-HCV), and anti-Treponema pallidum antibodies (anti-TP) were detected as efficiently as by an enzyme immunoassay (EIA) with serum samples. The correlations between whole-blood CIA and serum EIA were 99.8, 97.1, and 99.4% for HBsAg, anti-HCV, and anti-TP, respectively. Whole-blood CIA may be of value when rapid screening of many samples is required.

Antibodies, Bacterial↗

Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola.

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. In this study, we initially designed the primers for LAMP assays to detect these bacteria and evaluated the specificity and sensitivity of these assays. The specificities of the primers for these bacteria were examined using various oral bacteria and various reaction times. The lower detection limits of the 60-min LAMP reaction without loop primers were 1 microg/tube for P. gingivalis, 10 fg/tube for T. forsythia, and 1 ng/tube for T. denticola. Addition of the loop primers for each bacterium improved the detection specificities and sensitivities by several magnitudes. Furthermore, LAMP assays were applied to the rapid detection of these periodontal pathogens in clinical specimens, and the results were compared with those of conventional PCR detection. The results of the LAMP assays corresponded to those of conventional PCR assays. These results indicate that the LAMP assay is an extremely rapid, highly sensitive, specific method. This method is very useful for the rapid detection of periodontopathic bacteria and the diagnosis of periodontal disease.

Bacteroidaceae↗

Molecular subtyping of Treponema pallidum from North and South Carolina.

Patients from five clinics in North and South Carolina who had lesions suggestive of primary or secondary syphilis were evaluated using molecular techniques that allow the differentiation of Treponema pallidum strains on the basis of two variable genes, tpr and arp. Lesion samples were screened for the presence of T. pallidum DNA using PCR for polA, which represents a segment of the polymerase I gene that is unique to the spirochete. Twenty-seven of 154 lesion samples were found to contain T. pallidum, 23 of which had typeable DNA. Seven molecular subtypes were found (10f, 12f, 13f, 14f, 14g, 15f, and 16f); one to four subtypes were identified at each clinic. Subtype 14f was found in 52% of the typeable specimens and was distributed in four of the five clinics. Subtype 16f was found in 22% of specimens and was concentrated at one clinic. Further data are needed to define the role of this technique in examining the epidemiology of syphilis.

Adult↗

Reactivity of antibodies from syphilis patients to a protein array representing the Treponema pallidum proteome.

To identify antigens important in the human immune response to syphilis, the serum antibody reactivity of syphilitic patients was examined with 908 of the 1,039 proteins in the proteome of Treponema pallidum subsp. pallidum using a protein array enzyme-linked immunosorbent assay. Thirty-four proteins exhibited significant reactivity when assayed with human sera from patients in the early latent stage of syphilis. A subset of antigens identified were further scrutinized for antibody reactivity at primary, secondary, and latent disease stages, and the results demonstrate that the humoral immune response to individual T. pallidum proteins develops at different rates during the time course of infection.

Antibodies, Bacterial↗

Microtitration agglutination for detection of Treponema hyodysenteriae antibody.

A microtitration agglutination test for the detection of Treponema hyodysenteriae antibody in swine and rabbit sera is described. The following methods provided the best test results: antigen produced from the spirochete after a culturing period of 36 to 44 h at 38 degrees C, washed antigen inactivated with 0.01% Merthiolate at 4 degrees C for 24 to 36 h, sera heated at 56 degrees C for 30 min, a diluent of phosphate-buffered saline (0.01 M, pH 7.2), and test results read macroscopically after 18 to 24 h of incubation at 38 degrees C. The test enabled detection of antibody against pathogenic T. hyodysenteriae with a high level of consistency and sensitivity. Sera against nonpathogenic T. hyodysenteriae produced low agglutinating titers (less than or equal to 1:8) when reacted against antigen from pathogenic isolates. Inactivated antigen remained stable for 7 to 10 days. Specificity of the reaction in the agglutination test was shown by absorption studies.

Agglutination Tests↗

Detection of Treponema pallidum in the vitreous by PCR.

BACKGROUND: Ocular involvement of syphilis still poses a clinical challenge due to the chameleonic behaviour of the disease. As the serodiagnosis has significant limitations, the direct detection of Treponema pallidum (TP) in the vitreous represents a desirable diagnostic tool. METHODS: Real-time polymerase chain reaction (PCR) for the detection of TP was applied in diagnostic vitrectomies of two patients with acute chorioretinitis. Qualitative verification of TP by real-time PCR and melting point analysis according to a modified protocol was ruled out. Patients underwent complete ophthalmological examination with fundus photographs, fluorescein angiography, serological examination, antibiotic treatment and follow-up. RESULTS: In two cases of acute chorioretinitis of unknown origin, real-time PCR of vitreous specimens of both patients provided evidence of TP and was 100% specific. Initial diagnosis of presumed viral retinitis was ruled out by PCR of vitreous specimen. Patients were treated with systemic antibiotics and showed prompt improvement in visual function and resolution of fundus lesions. CONCLUSIONS: With real-time PCR, detection of TP in the vitreous was possible and delivered a sensitive, quick and inexpensive answer to a disease rather difficult to assess. In cases of acute chorioretinitis, the use of PCR-based assays of vitreous specimens in the diagnostic evaluation of patients is advisable. Although syphilitic chorioretinitis is a rare disease, PCR should include search for TP, as diagnostic dilemmas prolong definitive treatment in a sight-threatening disease.

Adult↗

Modification of the system of screening for antisyphilis antibodies in a blood transfusion centre, featuring a miniaturisation of the Treponema pallidum haemagglutination assay.

A modified, miniaturised version of the Fujizoki Treponema pallidum haemagglutination assay (TPHA) is described. The method is fast, sensitive, specific, and very economical. During a test period of one year, use of the method gave rise to a 67% increase in confirmed positives among new donor sera and a 112.5% increase among the antenatal sera over the numbers that would have been detected using a cardiolipin test alone. With the new donor sera, the rate of confirmed positives detected during the test period was 150% greater than that of 1980, when a cardiolipin test alone was used (0.05 greater than p greater than 0.02 (chi 2 test)).

Antibodies, Bacterial↗

Syphilis screening in the Blood Transfusion Service: a report of four years' experience with the Treponema pallidum haemagglutination assay and the subsequent development of a rapid "spin" method.

A comparison of cardiolipin and a modified Treponema pallidum haemagglutination assay (TPHA) method over a four year period confirmed the superior sensitivity and specificity of TPHA. In 86,495 new donor sera 19 (0.02%) confirmed positive results were detected by TPHA, 10 of which did not react by the cardiolipin test. In 150,789 antenatal samples 49 confirmed positive results were found by TPHA, 30 of which did not react by cardiolipin. No cardiolipin positive, TPHA negative samples were confirmed as positive by the absorbed fluorescence treponemal antibody test, and overall 78% of cardiolipin reactions gave false biological positive results. Cardiolipin tests were continued only because of their speed. A further modification ("spin") of the TPHA has now been developed which is rapid, sensitive, and inexpensive, and in testing 21,807 sera, gave results equivalent to those of the previous "settle" method. Serious consideration should be given to dispensing with cardiolipin tests.

Blood Donors↗

Evaluation of the equivocal test results of Treponema pallidum haemagglutination assay.

Two hundred and eighty Rapid Plasma Reagin (RPR) positive sera with an emphasis on cases with negative and borderline positive Treponema pallidum haemagglutination assay (TPHA) results were selected. Modified TPHA (M-TPHA) and fluorescent treponemal antibody absorption (FTA-abs) tests were used for comparison. One hundred and twenty five samples were TPHA negative, of which 78 and 69 cases were also negative by M-TPHA and FTA-abs, respectively. Eighty one sera negative by TPHA at a titre of 1/80 and positive at 1/40, considered to be negative according to the manufacturer's instructions, were also negative by M-TPHA (n = 11) and by FTA-abs (n = 1). Fifty borderline positive TPHA specimens gave one negative result by both M-TPHA and FTA-abs. The remaining 24 sera were positive by all three tests. Because of the high percentage of TPHA negative results among the positive RPR sera which became reactive when rechecked by the FTA-abs, it is concluded that as a confirmatory test the TPHA should be used not instead of but in addition to the FTA-abs.

Antibodies, Bacterial↗

New evidence for the non-infectivity of Treponema pallidum for mice.

We have recently shown that syphilitic rabbits are resistant to challenge with Listeria monocytogenes. This resistance was thought to reflect stimulation of cell-mediated immunity by active infection with Treponema pallidum. We now report data which show that the growth of Listeria was not suppressed in mice inoculated with T. pallidum. Re-inoculation with T, pallidum or with a large dose of an avirulent treponeme also failed to suppress the growth of Listeria. These results contrast with those obtained in rabbits and provide additional evidence that T. pallidum is not infective for the mouse.

Animals↗

Attachment of virulent Treponema pallidum to human mononuclear phagocytes.

The predominant phagocyte in established human syphilitic lesions is the mononuclear phagocyte. As these leucocytes may be important in immunity to Treponema pallidum, the initial interaction between human phagocytes and pathogenic T. pallidum (Nichols strain) was studied in vitro. Motile, virulent T. pallidum attach to the surface membrane of phagocytes but are not ingested by these cells. Heated, non-motile treponemes are not cell-associated but are observed free in the extracellular medium. Attachment is polar, at one or both terminal portions of the treponeme, and is neither serum- nor complement-dependent. Ingestion of virulent treponemes was not observed by phase-contrast or by electron microscopy in the presence of normal human or rabbit serum and complement. Techniques were chosen to preserve both the fragile surface constituents of the treponeme and the phagocytic function of mononuclear cells. Unfixed preparations were observed by phase-contrast microscopical examination during incubation to differentiate motile from non-motile organisms and fixed preparations were used for quantitation of attachment. This model should be useful for studying humoral and cell-mediated immunity in syphilis.

Cell Adhesion↗

Treponema pallidum immune adherence test for serodiagnosis of syphilis. 1: An improved method of the TPIA test.

Clinical investigations using the Treponema pallidum immune adherence (TPIA) test as reported by Nelson (1953) were performed only by Olansky et al in 1954. In this paper an improved method of the TPIA test is described. The test was simple to perform and was highly specific for antitreponemal antibody. Furthermore, as it was possible to use heated T. pallidum as antigen the antigen suspension could be preserved for long periods. The TPIA test for syphilis was evaluated by examining the sera from 40 healthy subjects and from 166 patients with syphilis.

Complement System Proteins↗

Treponema pallidum immune adherence test for serodiagnosis of syphilis. 2: Comparison with glass plate, TPHA, and FTA-ABS tests.

Using sera from 340 patients with syphilis the Treponema pallidum immune adherence (TPIA) test was compared with the glass plate, T. pallidum haemagglutination (TPHA), and fluorescent treponemal antibody-absorbed (FTA-ABS) tests. The results of the TPIA test agreed with those of the glass plate, TPHA, and FTA-ABS tests in 65%, 82%, and 73% of cases respectively. In the quantitative TPIA test no significant correlation with the other tests was observed, and it is, therefore, concluded that the TPIA test has highly individual characteristics. From gel filtration the particular feature of the test was its high sensitivity to the IgM antibody. The TPIA test thus appears to be suitable for estimating antibody in the early stages of the disease.

Chromatography, Gel↗

Use of CB hamsters in the study of Treponema pertenue.

The CB/Ss LAK strain of inbred hamster was used as a model for studies of infection with Treponema pertenue and of acquired resistance to it. When infected, this strain developed cutaneous lesions which lasted for six to seven months, even in the presence of peak titres of antitreponemal antibody. The rate of appearance and resolution of these lesions varied with the size of the inoculum. The infected hamsters' inguinal lymph nodes increased significantly in weight and teemed with treponemes for several weeks. Animals infected for eight or 10 weeks obtained quick resolution of their lesions by treatment with penicillin and were thereafter resistant to reinfection.

Animals↗

Cultivation of Treponema pallidum in subcutaneous chambers implanted in golden hamsters.

Treponema pallidum was successfully grown in subcutaneous chambers in golden hamsters. Abnormally long forms of T. pallidum were found in these chambers. The cause of this abnormality may possibly be due to their being coated with antibody which could inhibit cell division or render the organism avirulent or both. T. pallidum was successfully passaged between hamsters but with a possible diminution in virulence.

Animals↗