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PubMed1985

Detection of Treponema pallidum in lesion exudate with a pathogen-specific monoclonal antibody.

The diagnosis of early syphilis currently requires dark-field microscopic or serologic demonstration of Treponema pallidum infection. Dark-field microscopy is not widely available and is complicated by the numerous saprophytic spirochetes which are present at oral and rectal mucosal surfaces. Serologic tests are positive in only 70 to 90% of patients with primary syphilis, and several days may be required for results to become available. We used a pathogen-specific, fluorescein-conjugated monoclonal antibody to examine lesion exudates from 61 patients for the presence of T. pallidum and compared the data with results of dark-field microscopy and serologic testing. The direct fluorescent-antibody technique revealed the presence of T. pallidum in 30 of 30 patients with early syphilis, and dark-field microscopy was positive for 29. Serologic tests were reactive for 27 of 30 patients with syphilis; in the 3 patients with nonreactive serologic tests, chancres had been present for 4, 6, and 21 days. Although 7 of 31 patients without syphilis had spiral organisms seen on dark-field microscopy, the direct fluorescent-antibody test was negative for all 31. The presence of nonpathogenic spirochetes was subsequently verified in 5 of 7 patients by using a second monoclonal antibody which reacts with nonpathogenic, as well as pathogenic, treponemes and related spirochetes. The demonstration of T. pallidum by using fluorescein-conjugated monoclonal antibodies is intrinsically specific and is as sensitive as dark-field microscopy for the diagnosis of early syphilis. This method provides a convenient, accurate means for the diagnosis of syphilis by health care providers, many of whom lack access to dark-field microscopy.

Antibodies, Bacterial↗
PubMed1988

Improved selective medium for the isolation of Treponema hyodysenteriae.

An agar medium with improved selection for Treponema hyodysenteriae was developed. Cultures of T. hyodysenteriae and T. innocens, feces from 11 clinically normal pigs, and colonic contents from 6 pigs with gross lesions consistent with swine dysentery were diluted in phosphate-buffered saline and plated on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 5% citrated bovine blood (TSA), TSA with 400 micrograms of spectinomycin per ml (TSA-S400), TSA-S400 with 25 micrograms each of colistin and vancomycin per ml, and TSA with 5% pig feces extract and five antimicrobial agents (spiramycin, rifampin, vancomycin, colistin, and spectinomycin) (BJ). Viable numbers of T. hydodysenteriae grown on BJ were virtually identical to those for TSA, TSA-S400, and TSA-S400 with colistin and vancomycin. Pure cultures of four isolates of T. hyodysenteriae and three isolates of T. innocens were sustained through six subcultures on BJ. Fecal floras were completely inhibited on BJ for 14 of 17 fecal samples from both groups of pigs. A total of 461 colonic specimens from naturally occurring cases of porcine intestinal disease were plated on TSA-S400 and BJ. T. hyodysenteriae was isolated on both TSA-S400 and BJ for 69 specimens and on BJ alone for an additional 19 specimens.

Animals↗
PubMed1989

Sensitivity and specificity of an enzyme-linked immunosorbent assay using the recombinant DNA-derived Treponema pallidum protein TmpA for serodiagnosis of syphilis and the potential use of TmpA for assessing the effect of antibiotic therapy.

The recombinant DNA-derived Treponema pallidum membrane protein TmpA, purified from Escherichia coli K-12, was used in an enzyme-linked immunosorbent assay (ELISA) to evaluate its suitability in a screening test for syphilis and to monitor the effect of antibiotic treatment. The sensitivity of the TmpA ELISA was 76% for primary syphilis, 100% for secondary syphilis, and 98% for early latent syphilis. All except 1 of 15 serum samples positive for yaws were positive in this test. A specificity of 99.6% was found by testing more than 938 donor samples. The sensitivity and specificity of the TmpA ELISA are comparable to that of the T. pallidum hemagglutination assay, and therefore the test may be useful for the diagnosis of untreated syphilis. After antibiotic treatment, the level of anti-TmpA antibodies in sera of syphilis patients dropped sharply within 1 year. Thus, TmpA might be a useful antigen for monitoring successful treatment of syphilis.

Acute Disease↗
PubMed1989

Use of an enzyme-linked immunosorbent assay for detection of Treponema hyodysenteriae infection in swine.

Discriminate analysis was used to evaluate the enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Treponema hyodysenteriae antibodies in experimentally and naturally infected swine. In trial 1, 26 pigs were randomly divided into three groups (naturally infected, n = 8; experimentally infected, n = 11; and noninfected, n = 7), and samples were collected for 10 weeks. For trial 2, 31 pigs were randomly divided into two groups (naturally infected, n = 22; and noninfected, n = 7), and samples were collected for 20 weeks. Rectal swabs for T. hyodysenteriae isolation were collected daily, and fecal samples for isolation of Salmonella spp. were collected weekly. Serum samples for ELISA evaluation were collected biweekly (trial 1) or weekly (trial 2). Results of discriminate analysis indicated that the ELISA correctly identified 90% or more of the individually infected pigs at prior probabilities of infection ranging from 60 to 90%. The test correctly identified noninfected pigs at a lower rate (61 to 92% range). The mean ELISA titers of naturally infected pigs without clinical signs were not significantly different (P less than 0.05) from the titers of both groups of experimentally infected pigs. Mean ELISA titers of naturally infected pigs without clinical signs were significantly greater than the mean titers of naturally infected pigs with clinical signs. Naturally infected pigs with clinical signs had a mean ELISA titer that was significantly greater than that of noninfected pigs and significantly less than the mean titers of the experimentally infected pigs without clinical signs and the naturally infected pigs without clinical signs.

Analysis of Variance↗
PubMed1990

Quantitative immunoassay of Treponema denticola serovar C in adult periodontitis.

Murine monoclonal antibodies specific for Treponema denticola serovar C were produced and characterized in this study. An immunoassay was then developed by using these monoclonal antibodies, and the T. denticola serovar C antigen content of subgingival plaque was quantitated for samples taken from patients with periodontitis and healthy volunteers. The human subgingival plaque samples were grouped by severity of disease and pocket depth measurements at the collection site. The T. denticola serovar C content per milligram of subgingival plaque from deep pockets (greater than 6 mm) of patients with severe periodontitis was found to be twice that of samples collected from deep pockets (4 to 6 mm) of patients with moderate periodontitis or samples collected from healthy subjects (pocket depth, less than 4 mm).

Adult↗
PubMed1990

Evaluation of the Captia enzyme immunoassays for detection of immunoglobulins G and M to Treponema pallidum in syphilis.

Two new enzyme-linked immunosorbent assays (ELISA), one for the measurement of immunoglobulin G (IgG) (Captia Syphilis-G) and one for the measurement of IgM (Captia Syphilis-M), were evaluated for detecting antibodies to Treponema pallidum. Serum samples from 169 patients, 96 with various stages of untreated syphilis, 63 with treated syphilis, and 10 who were noninfected, were investigated. All sera were also examined by traditional treponemal and cardiolipin tests and by the fluorescent treponemal antibody absorption (FTA-ABS) test for 19S(IgM). The overall sensitivity of Captia Syphilis-G was 98.3%. The IgG ELISA was very sensitive (100%) in all stages of untreated syphilis, except in primary syphilis (82%). In all diagnostic groups of syphilis, the reactivity of Captia Syphilis-M was similar to that of the 19S(IgM) FTA-ABS test, except in reinfections, in which the IgM capture ELISA was less sensitive. False-positive IgM capture ELISA results were not found in the 10 neonates born to mothers adequately treated for syphilis. However, of six serum samples containing rheumatoid factor, two were reactive in the Captia Syphilis-M test but not in the 19S(IgM) FTA-ABS test. This indicated that the specificity of the IgM capture ELISA was not absolute. All serum samples from treated patients were reactive in the IgG ELISA, but only 15 samples were reactive in the IgM capture ELISA, which appeared to be as effective as the 19S(IgM) FTA-ABS test in monitoring the effect of treatment. Simultaneous measurement of IgG and IgM antibodies for T. pallidum by the Captia immunoassays appears to be an efficient and simple method for confirming the diagnosis of syphilis as well as for indicating whether active disease is present.

Antibodies, Bacterial↗
PubMed1991

Serotyping of Canadian isolates of Treponema hyodysenteriae and description of two new serotypes.

A total of 30 isolates of Treponema hyodysenteriae collected in the Saint-Hyacinthe (Quebec, Canada) area were serotyped by agar gel double immunodiffusion by using extracted lipopolysaccharide and hyperimmune rabbit antisera. Only 17% (5 of 30) of the isolates were typed with antisera specific for each of the seven known serotypes of T. hyodysenteriae. Antisera raised against 11 untypeable local isolates were then produced and tested against each lipopolysaccharide extract. Results showed two serologically distinct groups among 21 of the 25 untypeable isolates. The isolates in each group shared identical antigens. No detectable reactions could be observed between antisera raised against these 11 isolates and the antigens extracted from 7 reference serotype strains. On the basis of these results, two new serotypes of T. hyodysenteriae, serotypes 8 and 9, are proposed. We also propose isolate FM 88-90 as the reference strain for serotype 8 and isolate FMV 89-3323 as the reference strain for serotype 9. These two new serotypes, which represented 70% of the isolates tested, seem to be the major serotypes found in the province of Quebec.

Animals↗
PubMed1991

Biochemical properties of the outer membrane of Treponema denticola.

The outer membranes (OMs) from serovars a, b, and c of Treponema denticola, originally isolated from periodontal patients, were prepared. Dialysis of the OMs against 20 mM MgCl2 yielded the aggregable (A) and the nonaggregable (NA) moieties of the OMs. The absence of muramic acid, adenosine triphosphatase, hexokinase, and nucleic acid as well as electron microscopy indicated that the OM preparations were homogeneous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the A and NA moieties of the OMs showed approximately 25 Coomassie brilliant blue R-250 stain-positive bands or 47 silver-stained polypeptides. The relative molecular masses ranged between 14 and 97 kDa. The electrophoretic polypeptide profiles of the A and NA moieties shared many similarities among serovars a, b, and c. However, they exhibited variation in the overall pattern, intensity, or location of the polypeptide stained zones. This was especially true for serovar b. Two-dimensional electrophoretic studies showed an excess of 100 silver-stained spots with isoelectric points of 4.6 to 7.0 and relative molecular masses in the 14- to 97-kDa range. The OMs contained simple proteins, glycoproteins, and lipoproteins. The NA moieties of the OMs contained 4 to 6, 10 to 12, and 4 to 6 glycopeptides as well as two, seven, and two lipoprotein bands for serovars a, b, and c, respectively. The A moieties of the OMs showed 7 to 9, 11 to 13 and 5 to 6 glycopeptides as well as four, five, and three lipoprotein bands for serovars a, b, and c, respectively. Lipopolysaccharide was detected in the OMs of the three serovars following removal of proteins with proteinase K, pronase and silver staining of sodium dodecyl sulfate-polyacrylamide gels, or removal of lipopolysaccharide from the OMs by hot phenol extraction. The 66- and 53-kDa bands were present in serovars b and c, while a band with a relative molecular mass of 45 kDa was present only in serovar c. Endotoxin-like activity was also shown in the OMs of the three serovars by the Limulus amebocyte clotting assay and the chick embryo lethality test. This is the first report on selected biochemical properties of the OM macromolecules of three known serovars of T. denticola.

Bacterial Proteins↗
PubMed1991

Use of polymerase chain reaction and rabbit infectivity testing to detect Treponema pallidum in amniotic fluid, fetal and neonatal sera, and cerebrospinal fluid.

The diagnosis of congenital syphilis continues to pose a difficult clinical challenge. Because the serodiagnosis of congenital syphilis has significant limitations, the direct detection of Treponema pallidum in suspect neonatal tissues or body fluids represents a desirable alternate diagnostic strategy. We developed and applied the polymerase chain reaction (PCR) for the detection of T. pallidum in clinical material relevant to the diagnosis of congenital syphilis but which typically contain factors inhibitory for the PCR. Four methods of specimen processing were examined to circumvent PCR inhibition; clinical materials included amniotic fluids, neonatal sera, and neonatal cerebrospinal fluids. The PCR was 100% specific for T. T. pallidum compared with the sensitive rabbit infectivity test (RIT) for all clinical materials tested. For amniotic fluids, the PCR was 100% sensitive when correlated with the RIT but had a lesser sensitivity when applied to sera or cerebrospinal fluids, which typically contain few treponemes. The combined sensitivity of the PCR for all clinical samples was 78%. Positive PCR results also were obtained among some clinical specimens for which RIT was not performed; these results correlated well with either stigmata or risk factors for congenital syphilis. The combined results suggest that the PCR can be a useful adjunct to the diagnosis and clinical management of congenital syphilis and that it will provide a valuable tool for investigations of the pathogenesis of the disorder.

Amniotic Fluid↗
PubMed1991

Detection by polymerase chain reaction of Treponema pallidum DNA in cerebrospinal fluid from neurosyphilis patients before and after antibiotic treatment.

A polymerase chain reaction with nested primer pairs based on the DNA sequence of the 39-kDa bmp gene of Treponema pallidum subsp. pallidum is described. The method allowed the detection of purified T. pallidum DNA equivalent to the amount of DNA in a single bacterium and was specific for T. pallidum subspecies. After concentration of DNA, using diatomaceous earth, it was possible to detect about 100 treponemes in 1 ml of cerebrospinal fluid. Cerebrospinal fluid samples from a total of 29 symptomatic and asymptomatic patients with neurosyphilis were tested for the presence of treponemal DNA before and at various intervals after intravenous treatment with penicillin. Prior to the penicillin treatment, we detected T. pallidum DNA in 5 of 7 patients with acute symptomatic neurosyphilis, in none of the 4 patients with chronic symptomatic neurosyphilis tested before treatment, and in 2 of 16 patients with asymptomatic neurosyphilis. Unexpectedly, T. pallidum DNA was also often detected in cerebrospinal fluid long after intervenous treatment with penicillin, sometimes up to 3 years after therapy.

Base Sequence↗
PubMed1992

Evaluation of a Treponema pallidum western immunoblot assay as a confirmatory test for syphilis.

Tests for the detection of antibodies to Treponema pallidum are recommended for the confirmation of reactive nontreponemal test results and the accurate diagnosis of syphilis. The present-day use of Western blot (immunoblot) technology for the diagnosis of retroviruses prompted the development and evaluation of a Western blot assay with whole-cell T. pallidum as the antigen. The assay detected antibodies in syphilitic serum or plasma from dilutions of specimens incubated overnight with test strips. A test was considered positive when at least three of four major antigens having molecular masses of 15.5, 17, 44.5, and 47 kDa were detected. The Western blot assay had 93.8% sensitivity and 100% specificity for clinically defined samples. The Western blot assay was compared with double-staining fluorescent treponemal antibody absorption [FTA-ABS (DS)], which had a sensitivity and a specificity of 91.7 and 92.0%, respectively. Dilution series studies of syphilis-positive specimens indicated that the Western blot assay has an endpoint of reactivity at least 3 to 4 serial dilutions greater than that for FTA-ABS (DS). Overall, the greater than 95% agreement between the Western blot assay and FTA-ABS (DS) for clinically defined specimens indicates that the sensitivity of the Western blot assay is equal to or greater than that of FTA-ABS (DS). The Western blot assay demonstrated no false-positive or equivocal reactivities for nonsyphilitic specimens, including normal specimens (both plasma and serum), biological false-positives, and specimens with elevated gamma globulin or antinuclear antibody. We conclude that the high sensitivity and specificity of the T. pallidum Western blot assay, together with its simplicity and objectivity, make it a good confirmatory test for syphilis.

Animals↗
PubMed1992

Comparison of six different culture media for isolation of Treponema hyodysenteriae.

Fecal material from pigs experimentally infected with Treponema hyodysenteriae was collected and inoculated on six different selective media to compare their abilities for recovery of T. hyodysenteriae. Additionally, various types of samples were compared to find the most appropriate for submitting material to laboratories. Isolation rates for T. hyodysenteriae were 89.7% on BJ medium, 88.3% on TSA-BJ medium, 76.6% on SVC medium, 76.6% on BA2-BJ medium, 75.2% on BA2-SVC medium, and 52.4% on TSA-S400 medium. BJ medium was the most useful, yielding good growth of T. hyodysenteriae and inhibition of the normal fecal flora. Also, undiluted fecal material kept at refrigeration temperature allowed better recovery of T. hyodysenteriae during storage than swabs.

Animals↗
PubMed1992

Comparison of the benzoyl-DL-arginine-naphthylamide (BANA) test, DNA probes, and immunological reagents for ability to detect anaerobic periodontal infections due to Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus.

Most forms of periodontal disease are associated with the presence or overgrowth of anaerobic species that could include Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus among others. These three organisms are among the few cultivable plaque species that can hydrolyze the synthetic trypsin substrate benzoyl-DL-arginine-naphthylamide (BANA). In turn, BANA hydrolysis by the plaque can be associated with periodontal morbidity and with the presence of these three BANA-positive organisms in the plaque. In this investigation, the results of the BANA test, which simultaneously detects one or more of these organisms, were compared with the detection of these organisms by (i) highly specific antibodies to P. gingivalis, T. denticola, and B. forsythus; (ii) whole genomic DNA probes to P. gingivalis and T. denticola; and (iii) culturing or microscopic procedures. The BANA test, the DNA probes, and an enzyme-linked immunosorbent assay or an indirect immunofluorescence assay procedure exhibited high sensitivities, i.e., 90 ot 96%, and high accuracies, i.e., 83 to 92%, in their ability to detect combinations of these organisms in over 200 subgingival plaque samples taken from the most periodontally diseased sites in 67 patients. This indicated that if P. gingivalis, T. denticola, and B. forsythus are appropriate marker organisms for an anaerobic periodontal infection, then the three detection methods are equally accurate in their ability to diagnose this infection. The same statement could not be made for the culturing approach, where accuracies of 50 to 62% were observed.

Bacteria, Anaerobic↗
PubMed1992

Detection of Treponema pallidum in early syphilis by DNA amplification.

By using experimentally infected rabbits as a model for early syphilis, the applicability of in vitro DNA amplification was explored for detection of Treponema pallidum. It was determined that whole blood in heparin or EDTA (but not serum), lesion exudate, and punch biopsy as well as swabs of lesions are useful specimens for examination by the polymerase chain reaction. Swabs do not require special diluents, and the specimens, whether kept at room temperature or frozen, are well suited for use in the polymerase chain reaction.

Animals↗
PubMed1992

Application of quantitative immunofluorescence to clinical serology: antibody levels of Treponema pallidum.

A previously reported method of quantitative immunofluorescence, employing a calibrated photometric system and chemically stabilized fluorescence intensity, was used to replace the subjective, visual method of endpoint determination with a quantitative, calibrated measurement of antibodies to Treponema pallidum in serum. The results of the quantitative immunofluorescence method showed a 90% correlation with the subjective determinations of the visual method.

Antibodies, Bacterial↗
PubMed1992

Growth of Porphyromonas gingivalis, Treponema denticola, T. pectinovorum, T. socranskii, and T. vincentii in a chemically defined medium.

A chemically defined medium, OMIZ (Oral Microbiology and Immunology, Zürich)-W1 was developed. Medium OMIZ-W1 supports the long-term proliferation of a wide range of oral anaerobes, including representative strains of four Treponema species and Porphyromonas gingivalis. High concentrations of ascorbic acid and ammonium ions proved to be important for the growth of these organisms. T. denticola CD-1 grew in the absence of polyamines and long-chain fatty acids, T. pectinovorum and T. socranskii required polyamines, whereas T. vincentii depended on both polyamines and lecithin for growth. Specific requirements for purines and/or pyrimidines were detected, and these requirements could be used to distinguish Haemophilus-Actinobacillus group organisms. Some strains of P. gingivalis grew without vitamin K, while others were not satisfied by menadione but required its precursor 1,4-dihydroxy-2-naphthoic acid. Protoporphyrin IX or hemin equally satisfied the porphyrin requirements of P. gingivalis and Bacteroides forsythus, whereas ferrous sulfate was more efficiently used as a source of iron than was hemin. The cellular cohesiveness of P. gingivalis increased with high concentrations of hemin in the growth medium. Prevotella intermedia, B. forsythus, and several strains of P. gingivalis were more fastidious and required a protein or serum supplement to grow in medium OMIZ-W1.

Ascorbic Acid↗