External quality assessment program for Chlamydia trachomatis diagnostic testing by nucleic acid amplification assays

We report the results from 57 Australian diagnostic laboratories testing two external quality assessment panels using either the Roche Amplicor Chlamydia trachomatis test (R-PCR) or the Abbott LCx Chlamydia trachomatis assay (A-ligase chain reaction [LCR]). Panel samples were either normal urine spiked with Chlamydia trachomatis antigen or clinical urine specimens. There was no significant difference between laboratories or between assays in detection of C. trachomatis-positive clinical samples. Only at the lower limit of detection of the assays did the R-PCR demonstrate increased sensitivity over the A-LCR in the detection of C. trachomatis antigen. However, it was found that single-sample testing could lead to decreased test sensitivity. Detection of the presence of inhibitors of nucleic acid amplification differed between laboratories.     

Psychosocial impact of Chlamydia trachomatis testing in general practice

BACKGROUND: Urogenital Chlamydia trachomatis infections are widespread, and each year many tests are performed in general practice. AIM: First, to quantify the magnitude of stigmatization, problems related to partner, and anxiety of infertility among men and women tested for C. trachomatis in general practice. Second, to investigate the effect of a C. trachomatis test result on planned future condom use. DESIGN OF STUDY: Comparative cross-sectional study. SETTING: General practices in Aarhus County, Denmark. METHOD: Men and women tested for C. trachomatis in general practice were given a questionnaire about feelings of stigmatization, fear of partner's reaction, fear of future infertility and other psychosocial side effects related to being infected or not infected with C. trachomatis. RESULTS: A total of 277 participated in the study. The response rates were 61% (82/135) and 54% (195/365) among infected and non-infected individuals, respectively. Among the infected individuals 32% (9/28) of the men's partners and 35% (19/54) of the women's partners were upset about the test result, 9% (5/54) of the women and 11% (3/28) of the men split with their partner, 59% (32/54) of the women and 54% (15/28) of the men expressed nervousness about infertility, and 91% (19/21) of the women but only 56% (5/9) of the men said that they would use a condom more often in the future. All these figures were significantly lower for both men and women having C. trachomatis negative test results. CONCLUSION: A chlamydia test affects the individual in terms of sexuality, relation to partner, reproduction, and future contraceptive strategy. The influence is highest among women and individuals with a positive test result. These findings should be taken into account in screening programmes targeting young women and men.     

Sequences of multiple bacterial genomes and a Chlamydia trachomatis genotype from direct sequencing of DNA derived from a vaginal swab diagnostic specimen

Ultra-deep Illumina sequencing was performed on whole genome amplified DNA derived from a Chlamydia trachomatis-positive vaginal swab. Alignment of reads with reference genomes allowed robust SNP identification from the C. trachomatis chromosome and plasmid. This revealed that the C. trachomatis in the specimen was very closely related to the sequenced urogenital, serovar F, clade T1 isolate F-SW4. In addition, high genome-wide coverage was obtained for Prevotella melaninogenica, Gardnerella vaginalis, Clostridiales genomosp. BVAB3 and Mycoplasma hominis. This illustrates the potential of metagenome data to provide high resolution bacterial typing data from multiple taxa in a diagnostic specimen.     

Detection of Chlamydia trachomatis in genital specimens by the Microtrak direct specimen test

The conventional cell culture method for detection of Chlamydia trachomatis requires two to six days and is technically difficult to perform. The authors evaluated a new, relatively simple, non-culture method (MicroTrak, Syva Co., Palo Alto, CA) that requires less than one hour to complete. Two hundred fifty-one cervical and 209 male urethral specimens from three Richmond health clinics were read by direct immunofluorescence staining and compared with cell culture technics using iodine staining. Patient specimens were applied directly onto microscope slides (8 mm well) and stained with a fluorescein-labeled monoclonal antibody. Slides were examined for 10-15 minutes at X1,000 using an AO epifluorescent microscope and were considered positive if five or more typical elementary bodies were seen. The sensitivity, specificity, positive and negative predictive values for the direct smear were 89%, 97%, 85%, and 98% for males, and 93%, 96%, 85%, and 98% for females, respectively. The rapid direct specimen test appears to be a satisfactory method for detecting chlamydia in male and female genital specimens.     

Effect of differences in specimen processing and passage technique on recovery of Chlamydia trachomatis.

We have previously found that optimum recovery of Chlamydia trachomatis in microdilution plate culture required multiple blind passages. However, others have found this not to be the case for culture in vials. In the present study, the effect on recovery of the use of vials (as opposed to microdilution plates) and the effect of vortexing, sonication, or both were compared. Three different passage techniques were also evaluated. Vortexing or sonication resulted in equivalent recoveries. However, compared with vortexing alone, a combination of vortexing and sonication increased recovery from 95 (78%) to 114 (94%) of 121 positive specimens (P = 0.002). In multiple-passage experiments, the combination of vortexing and sonication, compared with vortexing only, increased the proportion of isolates recovered with no more than a single passage from 81 to 96%. Substitution of vials for microdilution plates increased recovery with only a single passage to greater than 96%, irrespective of whether sonication was employed. The most sensitive technique for single-passage technique was one using blunt scraping of cell monolayers with passage of two monolayers to one. The sensitivity of cell culture for C. trachomatis is highly dependent on the technique(s) employed. However, the combination of sonication and vortexing of clinical specimens enhanced recovery in microdilution plates, and a single blind passage did so in both microdilution plates and vials. Consideration should be given to their use for routine clinical cultures.     

Characterization of Apoptotic Activities During Chlamydia trachomatis Infection in Primary Cervical Epithelial Cells

Chlamydiae are obligate intracellular bacteria that infect human epithelial cells. It has been reported that Chlamydia trachomatis, induces apoptosis in epithelial cells, however, the molecular mechanisms responsible for host cell death especially in primary epithelial cells remained largely unknown as most of the studies are in cell line like HeLa. In this study we demonstrated that C. trachomatis induces apoptosis signaling pathway and apoptosis in primary cervical epithelial cells in a time and dose dependent manner. Live cervical epithelial cells were isolated from endocervical cells and induction was done with chlamydial EBs. Our results demonstrated that apoptosis in infected epithelial cells was associated with an increased activity of caspase 8; however, caspase 9 was activated to a lesser extent. Analysis of apoptosis pathway revealed that expression level of McL-1, Bcl-2, CASP8, and TRADD genes were found to be significantly upregulated (P?<?0.01), where as levels of Caspase 1, Caspase 10 and BRIC2 were found to be significantly downregulated (p?<?0.01). Our results showed that Chlamydia induces apoptosis and caspase activation in epithelial cells through caspase 8, with an increased expression of the McL-1, which confers a block at the mitochondrial level.     

The value of Chlamydia trachomatis antibody testing in predicting tubal factor infertility

BACKGROUND: The objective of the present study was to compare the likelihood of abnormal Chlamydia trachomatis antibody test results with that of abnormal hysterosalpingography (HSG) test results in patients with tubal factor infertility. METHODS: Anti-C. trachomatis immunoglobulin G antibodies were determined prospectively in 295 infertility patients by means of an indirect fluorescent antibody technique. In 48 of the 295 patients both HSG and laparoscopy with chromotubation were performed. The results of C. trachomatis antibody testing were compared with the results of HSG with respect to their predictive value of tubal factor infertility. Likelihood ratios for abnormal C. trachomatis antibody and HSG test results were determined in infertility patients, as assessed by laparoscopy. RESULTS: The positive likelihood ratio for C. trachomatis antibody testing was 1.8. This was comparable with the HSG, which had a positive likelihood ratio of 1.7. CONCLUSIONS: The predictive value of C. trachomatis antibody testing was equal to that of HSG, but ratios of 1.7 and 1.8 indicate a poor test, so both C. trachomatis antibody testing and HSG have a poor predictive value. C. trachomatis antibody testing causes minimal inconvenience to the patient, in contrast to HSG, and therefore should be maintained in infertility examinations.     

Detection of Chlamydia trachomatis infections in women by Amplicor PCR: comparison of diagnostic performance with urine and cervical specimens.

We used the Roche Amplicor PCR assay to compare urine and cervical swabs as sample material in the detection of Chlamydia trachomatis causing genital infections. The diagnostic performance of Amplicor PCR was compared with that of cell culture and the Gen-Probe PACE 2 assay with cervical specimens. If discrepant from other results, the specimens negative by PCR were diluted and reanalyzed to reveal PCR inhibitors. Of 666 patients, 39 (5.9%) were confirmed to have chlamydial infection. The respective sensitivity and specificity of Amplicor PCR were as follows: urine specimens, 82.0 and 99.7%; cervical specimens, 82.0 and 99.8%. Those for cell culture with cervical specimens were 84.6 and 100%. For the Gen-Probe PACE 2 assay, the sensitivity and specificity with cervical specimens were 79.5 and 100%, respectively. Without the effect of PCR inhibitors, the sensitivity of PCR with urine would have been 97.4%. Provided that the problems currently caused by inhibitors will be solved, the Amplicor PCR assay with urine specimens offers a tempting alternative for the diagnosis of C. trachomatis infection in women.     

The value of Chlamydia trachomatis antibody testing as part of routine infertility investigations

Laparoscopy is considered the gold standard for the evaluation of tubal disease but it is an invasive and costly procedure. Chlamydia trachomatis antibody testing is simple and inexpensive and causes minimal inconvenience to the patient. Using the micro-immunofluorescence technique we assessed the significance of positive serology. There was a marked association between the titre and the likelihood of tubal damage. In the group with low titres (1 in 32) there was only a 5% incidence of tubal damage; however, there was a progressive increase in the incidence of tubal damage in those with higher titres. Twenty out of 57 patients with titres higher than 1 in 32 had tubal damage (35%). The difference between the two groups was statistically significant (P < 0.0001, chi(2) test). By using C. trachomatis antibody testing more widely it may be possible to reduce the number of laparoscopies performed. It should therefore become an integral part of the fertility work-up.     

Influence of endocervical specimen adequacy on PCR and direct fluorescent-antibody staining for detection of Chlamydia trachomatis infections.

The cellular quality of the endocervical swab specimen used for the detection of Chlamydia trachomatis may dramatically impact the sensitivity of the diagnostic assay used. An evaluation of the adequacy of 319 endocervical swab specimens from women attending two inner-city sexually transmitted disease and family planning clinics, as well as five high school-based family planning clinics, was performed, and the resulting data were compared with the diagnostic results obtained by both Amplicor PCR and Microtrak direct fluorescent-antibody (DFA) staining. The swab from each patient was rolled across the open circular area of a DFA slide and then used to inoculate a transport tube for PCR (Roche), after which the swab was discarded. The slides were stained and examined by epifluorescence microscopy for the presence of C. trachomatis elementary bodies and for the presence and number of cell types to determine specimen adequacy. Cellular adequacy for a cervical swab specimen was defined as the presence of one or more columnar epithelial or metaplastic epithelial cells or the presence of more than 100 erythrocytes per high-power microscopic field. Of the 319 specimens read by DFA, 204 (63.9%) were determined to be adequate. There were 34 (10.7%) positive specimens by DFA and/or PCR. Twenty-nine (9.1%) specimens were positive by PCR, 20 (6.3%) specimens were DFA positive, and 15 (4.7%) were concordantly positive by both tests. The prevalence of chlamydia among adequate specimens was 14.2% (29/204), compared to 4.3% (5/115) for inadequate specimens (P < 0.0001). Variations in specimen quality and the sensitivity of the diagnostic assay used have a significant impact on determining the prevalence of C. trachomatis in a population.     

The immunology of Chlamydia trachomatis

Chlamydia trachomatis (C. trachomatis) is one of the most common sexually transmitted bacterial agents. What distinguishes it from other organisms is its intracellular reproductive cycle. Up to now, four antigens have been identified in the Chlamydia genus: genus-specific antigen as well as species-specific, type-specific and subspecies-specific. C. trachomatis is a powerful immunogen which stimulates the host's immunological processes. The intracellular parasitism of the bacteria is the basis for both symptomatic or asymptomatic infection as well as for chronic ones. The primary infection leads to a local inflammatory reaction due to penetration and reproduction of the bacteria in the epithelial cells and to IgA secretory antibody production. In most cases the host's reaction to the primary infection is transient and does not cause tissue damage. In the course of chronic infection or reinfection, the most important processes are those of delayed hypersensitivity, which lead to a fast and intense immunological reaction of specifically sensitized Th1 lymphocytes. This reaction leads to progressive damage of the epithelial cells and to cicatrization and fibrosis, which means irreversible complications. Interferon gamma is of special importance in the process of C. trachomatis infection. High concentrations of it inhibit the bacteria's reproductive cycle, while lower concentrations promote the development of atypical, non-contagious forms of Chlamydia of diminished metabolic activity and altered antigenicity. The chlamydial heat shock proteins are considered to be of great importance lately. Their molecular weights of 60 and 10 kDa are a powerful stimulant of immunological reactions and show significant homology (40-90%) to human and other bacterial heat shock proteins.     

Detection of Chlamydia trachomatis antigen by enzyme immunoassay: importance of confirmatory testing.

AIM--To determine when a fluorescence assay for Chlamydia trachomatis elementary bodies in the specimen buffer is of most value as a verification test for genital specimens reactive on screening enzyme immunoassay (EIA). METHOD--Genital swabs from high and medium prevalence populations were tested using EIA. Samples with absorbance values greater than the positive threshold and those within the range of 30% below this value were verified by the MicroTrak direct fluorescence assay (DFA) test. Quotients derived from the threshold value and specimen absorbances were used to establish confidence limits for the EIA. RESULTS--Of 13,283 swabs tested, 474 from the high risk group and 236 from the medium risk group were reactive on EIA and confirmed by DFA. Thirty six (5.9%) patients with confirmed reactive samples would have been missed if the kit criteria alone were followed. When confidence limits were applied to the calculated quotients, only those samples with an EIA quotient of > or = 4.0 were universally confirmed by the DFA. CONCLUSION--A scheme of testing which uses the DFA to verify EIA reactive specimens over a specified range was found to improve the sensitivity and specificity of the EIA screening test.     

Improved PCR detection of Chlamydia trachomatis by using an altered method of specimen transport and high-quality endocervical specimens.

Duplicate endocervical swabs were collected for detection of Chlamydia trachomatis by PCR (Roche Diagnostics). One swab was swirled in Specimen Transport Medium (Roche) for PCR testing and discarded. A saline aliquot from the other specimen, sent as a dry swab to the laboratory, was Papanicolaou stained to determine specimen adequacy, and the remainder was PCR tested. Significantly more (24%) PCR-positive results (118 versus 95; P < 0.001) were obtained with the dry specimens than with the swirled specimens when first tested. In addition, PCR-positive results were obtained with 107 (10.6%) of 1,007 microscopically adequate specimens but with only 3 (0.9%) of 341 inadequate specimens (P < 0.001).     

Genital infections caused by Chlamydia trachomatis in French Guiana: current status and diagnostic importance

Several laboratory aspects of Chlamydia trachomatis genital pathology were studied in order to evaluate their importance in French Guiana. Urethritis, cervicitis and sterilities were characterized by both direct methods of diagnosis and a serological study.