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PubMed1992

[Evaluation of chlamydial IgM antibodies for clinical diagnosis].

Chlamydia trachomatis (C. trachomatis), C. psittaci, and C. pneumoniae are now well established as pathogens of respiratory infections including pneumonia. Serum samples from 223 infants and children with pneumonia, 31 patients with adult inclusion conjunctivitis, 16 parents of babies with neonatal inclusion conjunctivitis and others were tested for IgM antibodies to Chlamydiae. Diagnostic kits for chlamydial IgM antibodies (SeroELISA and IPAzyme) have been also evaluated for their diagnostic value. It was found that detection of specific IgM antibodies with SeroELISA has a diagnostic value in chlamydial pneumonias.

Adolescent↗
PubMed2002

Detection and differentiation of chlamydiae by fluorescence in situ hybridization.

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and PARACHLAMYDIA: The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.

Amoeba↗
PubMed1998

Co-cultivation of conjunctival epithelial cells and Chlamydia trachomatis: electron microscopic findings.

This study used primary culture of rabbit conjunctival epithelial cells to investigate the infection process of chlamydia. The epithelial cells isolated from conjunctiva of rabbit were initially cultured for three weeks. After attaining confluence they were infected with Chlamydia trachomatis (C. trachomatis) serotype D, and after co-cultivation for 24, 48, and 96 hours, electron microscopic study was performed. An inclusion body, a characteristic finding of chlamydial infection, was observed in the vicinity of the nucleus after 24 hours of co-cultivation. It contained a large number of elementary and reticulate bodies and their intermediate forms. Infectious particles known as elementary bodies were noted in the inclusion as 20 to 30 microns sized round bodies with an electron dense core. Reticulate bodies were also noted; they too were round but somewhat pleomorphic and larger than elementary bodies. Some reticulate bodies multiplied actively by means of binary fission. In this study, we observed the characteristic changes of C. trachomatis-infected cells; this in-vitro system might provide a suitable model for the study of some aspects of the pathogenesis of ocular chlamydia infection.

Animals↗
PubMed1997

Nested polymerase chain reaction strategy simultaneously targeting DNA sequences of multiple bacterial species in inflammatory joint diseases. I. Screening of synovial fluid samples of patients with spondyloarthropathies and other arthritides.

OBJECTIVE: Bacteria play a crucial pathogenetic role in Lyme arthritis (LA), reactive arthritis (ReA), other forms of spondyloarthropathy (SpA), and possibly in undifferentiated oligoarthritis (uOligo). Polymerase chain reaction (PCR) technology has been applied to detect bacterial DNA of individual microbes in synovial fluid (SF) of patients with arthritides. We screened for DNA sequences of 8 bacterial species simultaneously in SF of patients with inflammatory joint disease. METHODS: We examined 104 SF samples of 96 patients with ReA (n = 13), undifferentiated SpA (uSpA, n = 10), uOligo (n = 50), juvenile chronic arthritis (JCA, n = 13), and rheumatoid arthritis (RA, n = 10). A nested PCR approach was developed to detect DNA sequences of 8 bacteria: Chlamydia trachomatis, C. pneumoniae, Yersinia enterocolitica, Salmonella enteritidis, Campylobacter jejuni, Shigella flexneri, Klebsiella pneumoniae, and Borrelia burgdorferi. The detection limit was determined at 10 bacterial/sample. Serology and lymphocyte proliferation assay were done in parallel in most patients. RESULTS: In 12 cases bacterial DNA of B. burgdorferi (n = 7), C. trachomatis (n = 2), C. jejuni (n = 2), and C. pneumoniae (n = 1) was detected in patients with uOligo (n = 9) and JCA (n = 3), while no evidence of bacterial DNA was found in patients with ReA, uSpA, and RA. Shigella flexneri DNA was detected in 4 cases, but the significance of this finding remains uncertain due to the high sequence homology of this species with Escherichia coli. DNA of Y. enterocolitica, S. enteritidis, or K. pneumoniae was not found. A positive serologic response was found in 7/9 PCR positive patients. In 11/96 cases antibodies to 2 or more bacteria were found in parallel (11.5%). Antigen specific lymphocyte proliferation was observed in 5/9 PCR positive patients. CONCLUSION: Bacterial DNA was detected in peripheral joint of patients with uOligo and JCA, but not in ReA, uSpA, or RA in this study. The detection of bacterial DNA in synovial material by PCR technology gives useful diagnostic information, especially when antibodies against several microbes are present or antibodies are not detectable. Failure to detect bacterial DNA in patients with ReA and uSpA with longstanding disease suggests that in later stages autoimmune mechanisms may operate.

Adolescent↗
PubMed1989

Enzyme immunoassay to determine exposure to Chlamydia pneumoniae (strain TWAR).

Recent studies suggest that a group of Chlamydia strains known as TWAR, which are now proposed to be a new species called Chlamydia pneumoniae, may be a frequent cause of respiratory disease in the United States and many other countries. Current serotesting methods do not allow rapid screening of large numbers of samples to distinguish C. trachomatis exposure from C. pneumoniae exposure. We developed an enzyme immunoassay to decrease cross-reactivity between immunoglobulin G antibodies reactive with C. trachomatis and C. pneumoniae. Elementary bodies of C. trachomatis or C. pneumoniae were treated with a detergent-chelating solution to decrease the reactivity of the common lipopolysaccharide antigens. Sera from four groups of patients, totaling 143 persons, were tested by this assay. The prevalences of titers of greater than or equal to 128 to C. trachomatis and C. pneumoniae, respectively, were as follows: (i) for 23 women seropositive for C. trachomatis by the microimmunofluorescence test, 21 (91%) and 18 (78%); (ii) for 50 adult blood donors, 13 (26%) and 39 (78%); (iii) for 40 sexually transmitted disease clinic patients, 20 (50%) and 32 (80%); (iv) for 30 healthy children 5 to 7 years old, 0 (0%) and 8 (27%). Western blots (immunoblots) of each antigen corroborated the differential reactivity of C. trachomatis-positive, C. pneumoniae-negative and C. trachomatis-negative, C. pneumoniae-positive serum samples. Western blots of serum samples from rabbits immunized with either C. trachomatis or C. pneumoniae elementary bodies revealed at least two protein bands (30 and 80 kilodaltons) which appeared to represent unique C. pneumoniae antigens.

Adult↗
PubMed2004

Inactivation of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae by ozone.

AIMS: To clarify the inhibitory effects of ozone on Chlamydia trachomatis and C. pneumoniae. METHODS AND RESULTS: Cell culture was performed using HeLa229 cells for C. trachomatis, and Human Line cells for C. pneumoniae. C. trachomatis strain D/UW-3/Cx and C. pneumoniae strain AR-39 were used. Ozone water was generated by an ozone water dispenser and diluted to desired concentration just before each experiment. Preinoculation minimum cidal concentration (MCC) and postinoculation MCC methods were employed. In preinoculation MCC, chlamydial strains were treated with serially diluted ozone water followed by inoculation to cells. In postinoculation method, chlamydial strains were inoculated to cells and incubated for 24 h. Then infected cells were treated with ozone water, followed by additional incubation for 48 h. Complete inactivation was obtained in preinoculation MCC method at 0.5 ppm of ozone water for 30 s, or 4 ppm for 5 s. CONCLUSION: Ozone at a concentration of 4 ppm was enough for immediate inactivation of both C. trachomatis and C. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: Ozone water at 4 ppm should be applicable for prevention of C. trachomatis urogenital infections.

Cell Line↗
PubMed2003

In vitro inhibitory effects of tea polyphenols on the proliferation of Chlamydia trachomatis and Chlamydia pneumoniae.

In vitro inhibitory effects of tea polyphenols on Chlamydia trachomatis and C. pneumoniae were investigated. A product of tea polyphenols, Polyphenon 70S was used. Chlamydial strains used were C. trachomatis D/UW-3/Cx and L(2)/434/Bu, and C. pneumoniae AR-39 and AC-43 strains. HeLa229 cells and HL cells were used for cultivation of C. trachomatis and C. pneumoniae, respectively. In the post-inoculation method, no inclusions of C. trachomatis were observed at 0.5 mg/ml of Polyphenon 70S. However, the toxicity of Polyphenon 70S was noted in HeLa229 cells and HL cells at a concentration of 0.25 mg/ml. In the pre-inoculation method, no toxic effects of Polyphenon 70S on the cells were noted. Complete inhibition of C. trachomatis D and L(2) was noted at concentrations of 1.6 and 0.4 mg/ml, respectively. With C. pneumoniae strains, the end points were 0.8 and 1.6 mg/ml for AR-39 and AC-43, respectively. Our findings encouraged the application of tea polyphenols for topical usage.

Chlamydia trachomatis↗
PubMed2006

Development of a rapid real-time PCR assay for detection and quantification of four familiar species of Chlamydiaceae.

Chlamydiae are one of the causative agents of various diseases in animals and human beings, which include abortion, pneumonia, gastroenteritis, encephalomyelitis, conjunctivitis, arthritis and sexually transmitted diseases. Much work has been carried out to attempt to develop an efficient pathogen detection strategy. Here, we presented a Chlamydiaceae-specific 23S rRNA-based real-time PCR assay for simultaneous detection and quantification of four members of Chlamydiaceae family, C. trachomatis, C. psittaci, C. pneumoniae and C. pecorum, using SYBR Green and Lightcycler. The assay was characterized using plasmid constructs of the bacteria and verified on standard strains of all four species of the Chlamydiaceae and a large cohort of clinical samples collected from human and animals by comparison with fluorescence immunohistochemistry method. The results showed that the present real-time PCR assay was of high specificity and sensitivity. It was capable of detecting as few as 250fg of chlamydial DNA (equivalent to 10(-1)IFU) and was applicable to both liquid cultures and clinical samples. This assay may therefore offer a rapid, economic and reliable means for screening of the chlamydiaceae pathogens.

Animals↗
PubMed1991

[Isolation and identification of Chlamydia spp. from effusion of otitis media].

Three chlamydial strains isolated from patients of otitis media with effusion were studied by comparing reactivity to monoclonal antibody (MAb) and polyclonal antibody (PAb) produced against one clinical isolate (named Mk), which was first isolated by Dr Mukai (Mukai Microbiological Research Laboratory, Yamato-shi, Kanagawa prefecture). Commercially supplied antibody (Microtrak (Syva), Culture-set (Ortho diagnostic system)) was also used. To isolate the Chlamydia spp, the yolk sacs of eggs were immediately inoculated with sample effusions (0.2 to 0.4 ml per sac) as soon as the samples were received. The eggs were observed every day for a period of 12 days thereafter for signs of life or death. One to two blind passages were first done in the eggs and then in HeLa 229 cells. The reactivity was examined by both micro-IF tests, among various strains of Chlamydia (C. trachomatis: L2. C. pneumoniae, C. psittaci: Budgerigar, Izawa, Meningopneumonitis (MP)) and by immunoblot analysis. Chlamydia spp were isolated in two of the twenty-nine sample effusions (6.9%). These isolates were then tested for reactivity to MAb and PAb. It was found that MAb reacted with MP and Mk, but not with Budgerigar, Izawa and C. pneumoniae. The antibody of Culture-set reacted with C. trachomatis C. pneumoniae and C. psittaci. No reactivity was observed in Mk by MicroTrak. Immunoblot analysis revealed that MAb reacted with about 95 KDa protein of Mk, the two clinically isolated Chlamydia spp and MP. By using PAb from rabbits, similar blotting patterns were observed in Mk, the clinical isolates and MP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Immunoreactivity of the 60 kDa cysteine-rich proteins of Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae expressed in Escherichia coli.

The 60 kDa cysteine-rich proteins (CrPs) of Chlamydia are developmentally regulated outer envelope proteins synthesized late in the chlamydial growth cycle. These proteins, found only on the extracellular infectious elementary bodies, elicit major antibody responses in chlamydial infection. We have cloned and expressed in Escherichia coli the complete 60 kDa CrP genes from Chlamydia trachomatis, C. psittaci and C. pneumoniae. The recombinant products were expressed as either 'native' proteins or as fusions with the bacteriophage T7 gene 10 protein. Electron microscopy showed that recombinant proteins were produced as insoluble inclusions within the E. coli host cells. The recombinant 60 kDa CrPs were purified and used to raise high titre polyclonal antisera. In immunoblot analysis these antisera reacted with the 60 kDa CrPs from purified elementary bodies of all three chlamydial species in a genus-specific manner. Further molecular analysis allowed the genus-specific cross-reacting epitopes to be localized by using overlapping synthetic peptides covering the C. trachomatis 60 kDa CrP. Immunogold labelling experiments, using purified infectious elementary bodies from the three chlamydial species indicated that the 60 kDa CrPs are not surface accessible to antibody binding.

Amino Acid Sequence↗
PubMed1996

A new primer pair for detection of Chlamydia pneumoniae by polymerase chain reaction.

In order to improve the detection and identification of Chlamydia pneumoniae, new primers for polymerase chain reaction (PCR) were designed based on the DNA base sequence within the 53-kDa protein gene, which is specific for C. pneumoniae. The primers permitted the identification of 24 C. pneumoniae strains collected from different geographical locations, but no reaction was observed with C. trachomatis, C. psittaci nor C. pecorum. The primers were unable to amplify the DNA of bacteria commonly related to respiratory tract infections. The positive amplification was achieved with only 9 EBs/assay. Therefore, the new primers seem to be useful in the diagnosis of C. pneumoniae infections.

Base Sequence↗
PubMed1996

Development of a simplified polymerase chain reaction-enzyme immunoassay for the detection of Chlamydia pneumoniae.

The 16S rRNA genes of two Chlamydia pneumoniae and two C. psittaci strains of different serovars were sequenced then compared to previously reported Chlamydia 16S rRNA gene sequences. Chlamydia pneumoniae-specific regions were identified and specific primers for nested PCR were synthesized. Nested PCR reactions were performed, in a single tube, by varying the annealing temperature of the amplification cycles. The initial thermal cycles were selected to allow annealing and extension of only the outer primer pair, whilst in later cycles a temperature that allowed inner primer annealing was employed. The inner primers were labelled, one with biotin and the other with fluorescein and consequently the dual labelled amplicon could be immobilized onto antibiotin-coated microtitre plates and detected colorimetrically via an antifluorescein-enzyme conjugate. The assay was found to be sensitive and specific. No cross reactions were observed with C. trachomatis, C. psittaci or other common respiratory pathogens.

Chlamydophila pneumoniae↗
PubMed2002

Biological properties and cell tropism of Chp2, a bacteriophage of the obligate intracellular bacterium Chlamydophila abortus.

A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.

Animals↗
PubMed1992

Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.

Animals↗
PubMedDate not supplied

[Inborn deficiency of complement C4A and C4B isotypes in persons infected with Chlamydia].

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio to detect the inherited deficiency of the isotypes. The frequency of deficiency in healthy persons blood donors was equal for C4A and C4B (0.14 for each isotype), i.e. 14% of total number (22) donors, or 28% totally. These results agree with the literary data, on which the frequency of deficiency of C4A is 0.14, and of C4B is 0.11-0.16. The inherent deficiencies of C4A and C4B for persons infected by Chlamydia were studied. For this purpose the patients (35 persons) with in blood antibodies (IgG or IgM) to Chlamydia (C. trachomatis, C. psittaci and C. pneumoniae) were investigated. The frequencies of deficiency of C4A and C4B were 0.29 and 0.46 respectively. Thus, the number of the undeficiency patients was only 25%, while among healthy persons 70-75% of individuals not having deficiencies of isotypes C4 were observed. The deficiencies of isotypes of C4 at this pathology is detected for the first time. The obtained data suggest the existence of the predisposition to the development of diseases stipulated by Chlamydia in persons with inherent deficiency of C4 component of complement.

Adult↗
PubMedDate not supplied

Modern diagnostics of Chlamydia trachomatis infections.

Chlamydia trachomatis (C. trachomatis) is the most common agent of sexually transmitted infections. The clinical spectrum of the disease ranges from urethritis to infertility in women and to trachoma. Intracellular localisation of the pathogen creates a challenge for routine diagnostics. In this review possible diagnostic tests have been presented, varying from classic cell culture analysis and serodiagnostics (Enzyme-linked Immunoassays, Indirect Immunofluorescence) to the most sophisticated nucleic acid analyses (hybridisation, Polymerase Chain Reaction, Transcription Mediated Amplification, Ligase Chain Reaction), Advantages and disadvantages of the leading tests are discussed. Possible reasons of false positive as well as false negative results of genetic testing are presented.

Chlamydia Infections↗
PubMed2005

Seroprevalence of Chalmydia trachomatis infection amongst patients with pelvic inflammatory diseases and infertility.

Chlamydia trachomatis (C. trachomatis) is a common cause of pelvic inflammatory disease in female population. This infection can be diagnosed both by culture and serology. The present study evaluated the seroprevalence of chlamydial infection in patients with pelvic inflammatory disease (ND) and infertility and in control population of healthy normal females. The seroprevalence was found to be 82.7% in patients and 32% in controls by Enzyme Linked Immuno Sorbent Assay (ELISA) for any one or more class of antichlamydial antibodies (IgG/IgM/ IgA). The study demonstrates the importance of serology to monitor trends of chlamydial infections in women with PID and infertility.

Adult↗
PubMed1988

[Detection of Chlamydia trachomatis by several methods in the uterine cervix of pregnant women].

Most pregnant women who have Chlamydia trachomatis (C. trachomatis) in the uterine cervix are asymptomatic. Several ways of detecting C.trachomatis were tested on 331 pregnant women, as well as 146 female patients attending our STD clinic as a control. 1) The detection rates for C.trachomatis in the cervix of pregnant women were 5.1% using the cell culture method, 2.4% with Micro Trak, and 2.2% employing Chlamydiazyme. These rates were higher in those patients visiting the STD clinic. 2) In pregnant women, the positive rate of Chlamydiazyme was 66.7% in the cell culture-positive cervical specimens, whereas Micro Trak was positive in 33.3%. 3) The antibody-positive rate was 84.6% in cases with PID caused by C.trachomatis. The antibody was found in only 17.7% of the pregnant women. Additionally, no significant correlation was noted between the antibody titer and C.trachomatis colonization in specimens obtained from the cervix of pregnant women. Although Micro Trak, Chlamydiazyme and possibly the microplate immunofluorescence antibody technique can be substituted for a cell culture method for detecting C.trachomatis in cases of symptomatic infection, these tests are not considered to be useful for screening Chlamydia-positive pregnant women.

Antibodies, Bacterial↗