A multi-centre evaluation of nine rapid, point-of-care syphilis tests using archived sera.

OBJECTIVES: To evaluate nine rapid syphilis tests at eight geographically diverse laboratory sites for their performance and operational characteristics. METHODS: Tests were compared "head to head" using locally assembled panels of 100 archived (50 positive and 50 negative) sera at each site using as reference standards the Treponema pallidum haemagglutination or the T pallidum particle agglutination test. In addition inter-site variation, result stability, test reproducibility and test operational characteristics were assessed. RESULTS: All nine tests gave good performance relative to the reference standard with sensitivities ranging from 84.5-97.7% and specificities from 84.5-98%. Result stability was variable if result reading was delayed past the recommended period. All the tests were found to be easy to use, especially the lateral flow tests. CONCLUSIONS: All the tests evaluated have acceptable performance characteristics and could make an impact on the control of syphilis. Tests that can use whole blood and do not require refrigeration were selected for further evaluation in field settings.

Use of PCR in the diagnosis of early syphilis in the United Kingdom.

OBJECTIVES: To evaluate a Treponema pallidum polymerase chain reaction (PCR) test in the laboratory diagnosis of early syphilis in the United Kingdom. SUBJECTS AND SETTING: Men and women attending genitourinary medicine clinics in England. METHODS: A trial PCR service was offered for the analysis of swabs of ano-genital or oral ulcers suspected to be syphilitic in origin. Clinical details, results of treponemal serology, and other relevant laboratory tests carried out by the sending laboratories were obtained retrospectively by questionnaire. RESULTS: Data from 98 patients, representing 100 episodes of ulceration, were analysed. The majority of patients (70) attended clinics in the Greater Manchester area. Eighty six patients were male and 58 were men who have sex with men (MSM), of whom 24 were HIV positive. PCR results agreed with the clinical diagnosis for 95 patients; samples from 26 patients were PCR positive and serologically diagnosed as primary (18) or secondary (8) syphilis, whereas 70 patients had PCR negative samples and were not diagnosed as having active syphilis. These data include two HIV positive patients who were PCR positive 12 and 21 days before their treponemal seroconversion. One positive PCR result was not supported by positive treponemal serology (this patient coincidentally received a 10 day course of co-amoxiclav 1 week after sampling). Three patients had negative PCR results but positive syphilis serology. The sensitivity, specificity, positive and negative predictive value for primary syphilis were 94.7%, 98.6%, 94.7%, and 98.6%, respectively, and for secondary syphilis these were 80.0%, 98.6%, 88.9%, and 97.2%, respectively. CONCLUSION: PCR is a sensitive and specific test for T pallidum, and an important adjunct to dark ground microscopy and treponemal serology in diagnosing infectious syphilis in the United Kingdom.

Evaluation of the specificities of five DNA amplification methods for the detection of Neisseria gonorrhoeae.

The intragenus specificities of five molecular diagnostic methods for Neisseria gonorrhoeae were determined. Three assays were considered suboptimal. Molecular detection of N. gonorrhoeae from sites where other Neisseria spp. commonly occur or from any site in low-prevalence settings should be confirmed by a test targeting a different genetic locus.

Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae.

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.

The major outer membrane protein of Chlamydia psittaci functions as a porin-like ion channel.

The major outer membrane protein (MOMP) of Chlamydia species shares several biochemical properties with classical porin proteins. Secondary structure analysis by circular dichroism now reveals that MOMP purified from Chlamydia psittaci has a predominantly beta-sheet content (62%), which is also typical of bacterial porins. Can MOMP form functional ion channels? To directly test the "porin channel" hypothesis at the molecular level, the MOMP was reconstituted into planar lipid bilayers, where it gave rise to multibarreled channels, probably trimers, which were modified by an anti-MOMP monoclonal antibody. These observations are consistent with the well-characterized homo-oligomeric nature of MOMP previously revealed by biochemical analysis and with the triple-barreled behavior of other porins. MOMP channels were weakly anion selective (PCl/PK approximately 2) and permeable to ATP. They may therefore be a route by which Chlamydia can take advantage of host nucleoside triphosphates and explain why some anti-MOMP antibodies neutralize infection. These findings have broad implications on the search for an effective chlamydial vaccine to control the significant human and animal diseases caused by these organisms.

Molecular cloning and characterization of the genes coding for the highly immunogenic cluster of 90-kilodalton envelope proteins from the Chlamydia psittaci subtype that causes abortion in sheep.

Proteins present in the outer membrane of chlamydiae that are involved in mucosal epithelial cell infection must clearly be identified and characterized if we are to understand and modify the pathogenic mechanisms utilized by these organisms. We have identified and isolated a family of four genes encoding putative outer membrane proteins (POMPs), a group of proteins of approximately 90 kDa present in the outer membrane of the subtype of Chlamydia psittaci that causes ovine enzootic abortion (strain S26/3). These proteins, although minor components, are major immunogens, as shown by the immunoblotting of chlamydial outer membrane complexes with postabortion sheep sera, and are therefore potential diagnostic and/or protective antigen candidates. Immunoblotting of the expressed amino- and carboxy-terminal halves of one of the POMPs with postabortion sheep sera showed that the major humoral immune response appeared to be directed solely against the amino-terminal half. This result, in combination with the positive immunofluorescence staining of S26/3-infected cells using POMP-specific (specific to the amino-terminal half of the proteins) monoclonal antibodies, suggests the probable surface localization of the POMPs and, more specifically, the surface exposure of the amino-terminal half of these proteins. The four pomp genes are highly homologous, sharing 82 to 100% similarity with each other (two of the genes are identical). Genes with strong and weak homologies were also detected in C. psittaci avian and feline pneumonitis strains, respectively. No pomp homologs were found in strains of C. trachomatis and C. pneumoniae, but this does not preclude their existence. The absence of homology with various subtypes of C. pecorum, which complicate the diagnosis of the ovine abortion subtype, indicates the possible suitability of the these 90-kDa proteins as serodiagnostic antigens.

Cloning and expression of the 75 kDa DnaK-like protein of Chlamydia psittaci and the evaluation of the recombinant protein by immunoblotting and indirect ELISA.

The 75 kDa dnaK-like gene of Chlamydia psittaci ovine abortion strain S26/3 was isolated from an EMBL 3 chlamydial DNA library. A 7 kb DNA fragment containing the gene was subcloned into Bluescribe (M13+) plasmid and used to transform competent E. coli. These cells were found to express a cytoplasmic protein of 75 kDa. Monospecific antibodies against the protein prepared by antibody elution reacted with the native 75 kDa protein. Recombinant clones did not adhere to McCoy cell monolayers in cell adhesion studies. The 75 kDa protein purified by ion-exchange chromatography was used in immunoblotting and indirect enzyme-linked immunosorbant assay (ELISA) studies using sera previously screened for chlamydial antibodies by an indirect ELISA incorporating solubilised chlamydial elementary bodies and by microimmunofluorescence. Immunoblotting identified 6/11 sera from infected ewes that had either typical placental lesions or had been found positive on examination of stained placental smears and 1/11 sera from ewes that had no typical placental lesions. The ELISA gave positive reactions with 29 of 65 known positive sera and 15 of the 76 negative sera. It is concluded that the 75 kDa DnaK-like protein is unsuitable as an antigen for antibody detection but its potential as a component for a sub-unit vaccine against ovine enzootic abortion warrants further study.

Identification of a multigene family coding for the 90 kDa proteins of the ovine abortion subtype of Chlamydia psittaci.

While attempting to identify genes and their corresponding antigens that could be used to improve the current methods of diagnosing Chlamydia psittaci infection which causes enzootic abortion in ewes, two candidate clones were isolated from a lambda gt 11 genomic DNA expression library of ovine abortion subtype (strain S26/3) C. psittaci. These clones contained fragments of a gene coding for a group of three chlamydial proteins of approximately 90 kDa which appeared as major immunogens by immunoblotting experiments, indicating their potential as diagnostic or possibly protective antigens. Southern blotting of S26/3 genomic DNA using the two clones as probes identified a family of three or four genes. These represent the first example of protein gene duplication reported in Chlamydia.

Detection of Chlamydia in formalin-fixed and paraffin-embedded avian tissue by in situ hybridization. A comparison between in situ hybridization and peroxidase-antiperoxidase labelling.

In situ hybridization, (ISH) using a digoxigenin-antisense RNA-probe to detect chlamydial rRNA was applied to post mortem tissue of birds. The technique was optimized and validated using tissue from experimentally-infected chicken embryos. Tissue sections were also tested by immunohistochemistry (peroxidase-antiperoxidase reaction, PAP) for the presence of chlamydial antigen using a genus specific monoclonal antibody. In the chicken embryo tissue, ISH and PAP were comparably sensitive and specific (100% and 100%, respectively). ISH and PAP in general were correlated to microscopic lesions. For further comparison, ISH with PAP was applied retrospectively to tissues of 82 birds from which Chlamydia had been isolated, or which were suggestive of chlamydiosis. Using in situ hybridization 47 of 82 birds were found to be positive, and as were 23 of 82 birds with PAP. None of the ISH-only positive cases were found to be strongly positive. On the other hand, cases which were found positive with the ISH were also positive with other methods (PAP and isolation of Chlamydiae from chicken embryos). There was no close correlation between the positive cells and histological lesions. In spite of the higher sensitivity and specificity of the ISH, this technique is not suitable for routine diagnostic investigations. ISH is expensive, laborious, and time consuming.

Pathogenesis of Chlamydia psittaci infection in sheep: detection of the organism in a serial study of the lymph node.

Sixteen sheep were inoculated subcutaneously over the left prefemoral lymph node with an abortifacient strain of Chlamydia psittaci. Groups of four animals were killed after 3, 6, 12 and 18 days. Four of eight sheep which received a control inoculum were killed on day 6 and four on day 18. The left and right prefemoral lymph nodes were removed and weighed and portions taken from each for examination by the polymerase chain reaction (PCR), by culture, and by histopathological and immunohistochemical methods. The left prefemoral lymph nodes enlarged after the injection of C. psittaci, with the group mean weight on day 6 being the greatest and that on day 18 being normal. Examination by "nested" PCR showed samples from these nodes to be positive, except for one animal killed on day 3 and one on day 12. Live organisms, however, were not cultured from any of the samples collected. C. psittaci antigen was detected immunohistochemically in three of four nodes on day 3, in each of four on day 6, and in two of four on both days 12 and 18. Nodes from the contralateral side remained normal, as did those from unchallenged control sheep, and no antigen or DNA was detected in them.

Analyses of the genomes of chlamydial isolates from ruminants and pigs support the adoption of the new species Chlamydia pecorum.

Analysis of the genomic DNAs of chlamydial isolates from sheep, cattle, and pigs was performed by Southern blot hybridization and by restriction endonuclease (RE) profiling of DNA amplified by PCR. The hybridization probes were derived from whole genomic DNA, the major outer membrane protein (MOMP) gene, the 16S rRNA gene, and an avian Chlamydia psittaci isolate plasmid. The PCR analysis used targets in the MOMP gene, the 16S rRNA gene, and the 60-kDa cysteine-rich protein gene. Together, the results showed that although there was considerable heterogeneity in the DNA sequence in the MOMP gene region, all the isolates had the same underlying total genomic RE profiles and yielded identical RE profiles for the rRNA and 60-kDa-protein gene regions. Most of the isolates were found to hybridize with the plasmid probe. Comparison of the MOMP sequence of one of the isolates (P787) with that of a known Chlamydia pecorum strain together with the results of the RE analyses allowed the conclusion that the isolates should all be classified within this new species.

Identification of a region of the alcelaphine herpesvirus-1 genome associated with virulence for rabbits.

The gammaherpesvirus Alcelaphine Herpesvirus 1 (AHV-1) causes the fatal lymphoproliferative disease known as malignant catarrhal fever (MCF), in susceptible hosts. The virulent C500 isolate of AHV-1 became attenuated for the laboratory model, the rabbit, as a result of serial passage in cells of bovine origin. This work describes the identification of a region of the central unique sequence of the C500 genome, located close to the terminal repeat units of the molecule, which is altered on attenuation. The virulent C500 genome contains two copies of a sequence of approximately 2 kbp, contained within a 7 kbp region of the unique DNA located adjacent to the terminal repeats at the left end of the molecule. In the genome of the attenuated virus, there are also two copies of the 2 kbp sequence but they are located at the ends of the attenuated genome unique region, adjacent to the terminally repeated sequences. One open reading frame (ORF), designated putative polypeptide 5, was altered on attenuation such that the 3' sequence was lost. The location of this ORF, coupled with the loss of its 3' sequence, suggests that this ORF may encode a gene involved in the virulent mechanisms of this virus, in a manner similar to that of the transforming proteins of Herpesvirus saimiri (HSV).

Electrophoretic analysis of the major outer membrane protein of Chlamydia psittaci reveals multimers which are recognized by protective monoclonal antibodies.

Purified major outer membrane protein, detergent solubilized and reduced with dithiothreitol but not heated, gave an apparent molecular weight in sodium dodecyl sulfate (SDS)-polyacrylamide gels almost three times that observed for the heat-denatured SDS-treated peptide. This is similar to the behavior of porin trimers from gram-negative bacteria. Two protective monoclonal antibodies showed strong binding to the proposed trimer but not to denatured, monomeric major outer membrane protein.

Detection of bovine herpesvirus 1 in clinical samples by the polymerase chain reaction.

A PCR assay with primers selected from the gl gene and flanking a 468 bp DNA fragment was tested on clinical samples. Of 27 samples (nasal swabs, lung, lymph nodes, tracheal mucosa) collected from 16 different outbreaks in Scotland, 18 were positive by PCR and 13 by virus isolation. Some samples of isolated DNA had to be diluted by a factor 50-100 to obtain a positive PCR result. PCR assay could detect the BHV-1 positive samples collected from different European regions, namely from Slovakia, Italy, England, Scotland as well as the IPV sample.

Development and evaluation of an indirect ELISA to detect antibodies to abortion strains of Chlamydia psittaci in sheep sera.

A novel indirect enzyme-linked immunosorbent assay (ELISA) for antibodies against abortion strains of Chlamydia psittaci (C. psittaci) has been developed. The antigen used was chlamydial elementary bodies treated sequentially with N-lauroyl sarcosine and n-octyl-beta-D-glucopyranoside and finally solubilized with N-lauroyl sarcosine and dithiothreitol. Treating the antigen with sodium periodate after coating of the plates increased the specificity for antibodies to abortion strains. The test was evaluated initially with sera from experimentally infected sheep and an uninfected control group. These sheep were monitored for lambing performance and infection status. When used in conjunction with the indirect micro-immunofluorescence test (MIF), the ELISA was able to identify as negative all twenty-five sera from ewes that had no typical placental lesions and identified as positive twenty of twenty-one sera from infected ewes that had either typical placental lesions or had been found positive by isolation of chlamydia in cell culture. The combination of ELISA and MIF was also able to discriminate correctly groups of sera from six flocks with a history of infection from four known uninfected flocks.

Rapid detection of bovine herpesvirus 1 (BHV 1) using the polymerase chain reaction.

A polymerase chain reaction (PCR) assay based on primers from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size was amplified using DNA from herpesviruses isolated from reindeer, red deer and goats. The PCR assay was able to detect virus in nasal swabs up to 14 days after experimental infection of cattle and there was a good correlation when PCR was compared with virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful.

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis.

A polymerase chain reaction-based assay capable of detecting a broad range of pestiviruses from pigs, cattle, or sheep was developed. Of six sets of primers selected from different parts of the pestivirus genome, the best results were provided by a pair from the highly conserved 5' non-coding region which gave amplification with all 129 isolates tested. This panel consisted of 33 isolates from pigs, 79 from cattle, and 17 from sheep. Differentiation between the viruses was achieved by cutting the PCR-amplified products with the restriction endonucleases AvaI and Bg1I. Using this procedure it was possible to distinguish at least 3 genogroups; group 1 (HCV) contained 32 of the pig isolates, group II (BVDV) contained all the cattle isolates tested plus 6 sheep isolates and group III (BDV) contained 11 sheep isolates and 1 pig isolate.

Typing Chlamydia psittaci--a review of methods and recent findings.

When the present chlamydial classification was established it was recognized that a wide variety of types were contained within the arbitrary designation Chlamydia psittaci. Early workers relied mostly on observations of growth characteristics to differentiate the types of C. psittaci isolated from a wide range of different hosts. The differences between isolates were confirmed serologically using a variety of tests of which the most sensitive was the micro-immunofluorescence (MIF) test which was able to recognize nine immunotypes among the mammalian isolates alone. This approach has recently been improved by the use of monoclonal antibodies in the MIF test which has confirmed most of the mammalian immunotypes and divided the avian strains into four groups. Studies on the nucleic acid of C. psittaci isolates show clear differences in the size distribution of DNA fragments produced by restriction endonuclease digestion of the genomes of the various types. Most importantly, studies of DNA/DNA homologies showed that at least four of the types identified by biological, serological and restriction endonuclease tests were sufficiently different to be considered separate species. Most recently, attention has been focused on DNA sequence comparisons of C. psittaci genes amplified by the polymerase chain reaction (PCR). The usual target has been the major outer membrane protein gene for which much sequence information is now available. The combination of PCR and MIF with monoclonals has provided a set of practical techniques with which all chlamydial isolates can be detected and typed with relative ease. It is likely that these developments will lead to the reclassification of the genus and, hopefully, a rapid increase of our understanding of the diseases caused by C. psittaci.

A liquid-hybridization method for typing the Vp4 and Vp7 genes of bovine rotaviruses.

A simple liquid-hybridization assay was developed which allows assessment of the degree of hybridization between the two serotype-determining genes of the bovine rotavirus strain UK and the homologous genes of the isolate under test. 32P-labelled transcription probes were produced from cloned complementary DNA (cDNA) copies of UK gene segments 4 and 8 and hybridized to double stranded RNA (dsRNA) extracted from rotavirus-positive field samples. Subsequent treatment with ribonuclease A (RNase A), separation of the RNase A-resistant hybrid fragments by polyacrylamide gel electrophoresis (PAGE) and autoradiography yielded a specific, reproducible banding pattern for each isolate. A total of 74 field samples was tested by both the hybridization assay and by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies (Mabs). The results obtained were in excellent agreement and confirmed that serotype G6 rotaviruses predominated. Hybridization of these G6 viruses with the gene 4 probe suggested that viruses with Vp4s related to that of UK rotavirus are also common. The hybridization assay was more sensitive than the ELISA.