Multicenter Evaluation of the Fully Automated COBAS AMPLICOR PCR Test for Detection of Chlamydia trachomatis in Urogenital Specimens

The fully automated COBAS AMPLICOR CT/NG test for the detection of Chlamydia trachomatis was evaluated in a multicenter trial. Test performance was evaluated for 2,014 endocervical swab and 1,278 urine specimens obtained from women and for 373 urethral swab and 254 urine specimens obtained from men. Culture served as the reference test. Culture-negative, COBAS AMPLICOR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the C. trachomatis major outer membrane protein gene were resolved as true positives. The overall prevalence of chlamydia was 4.3% in cervical swabs and 11.0% in urethral swabs from men. When the results for each specimen type were considered separately, the resolved sensitivities were 96.5% (83 of 86) for endocervical swab specimens, 95.1% (39 of 41) for urine specimens from women, 100.0% (41 of 41) for urethral swab specimens from men, and 94.4% (17 of 18) for urine specimens from men; the resolved specificities were 99.4% (1,912 of 1,924) for endocervical swab specimens, 99.8% (1,204 of 1,207) for urine specimens from women, 98.5% (325 of 330) for urethral swab specimens from men, and 100.0% (236 of 236) for urine specimens from men. For the subset of patients from whom both swab and urine specimens were collected, the combined results for both specimen types were used to identify all infected patients. Using these combined reslts as criteria, the resolved sensitivities for the COBAS AMPLICOR test were 82.6% (38 of 46) for endocervical swab specimens, 84.4% (38 of 45) for urine specimens from women, 84.2% (16 of 19) for urethral swab specimens from men, and 89.5% (17 of 19) for urine specimens from men. In comparison, the sensitivity of culture was only 56.5% (26 of 46) for endocervical specimens and 63.2% (12 of 19) for urethral specimens from men. The internal control provided in the COBAS AMPLICOR test revealed that 2.9% of specimens were inhibitory when they were initially tested. Nevertheless, valid results were obtained for 99.1% of specimens because 68.7% of the inhibitory specimens were not inhibitory when a second aliquot of the original sample was tested. Two additional COBAS AMPLICOR-positive specimens were detected by retesting inhibitory specimens. The COBAS AMPLICOR CT/NG test for the detection of C. trachomatis exhibited equally high sensitivities and specificities with both urogenital swab and urine specimens and, thus, is well-suited for use in screening.     

Comparison of Performances of Two Commercially Available Tests, a PCR Assay and a Ligase Chain Reaction Test, in Detection of Urogenital Chlamydia trachomatis Infection

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.     

Evaluation of the Abbott LCx Ligase Chain Reaction Assay for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine and Genital Swab Specimens from a Sexually Transmitted Disease Clinic Population

The Abbott LCx ligase chain reaction (LCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae was evaluated by using swab and urine specimens from 562 patients. C. trachomatis results by LCR were compared to those by the Gen-Probe PACE 2 assay, whereas N. gonorrhoeae results by LCR were compared to those by culture. The Gen-Probe and LCR assays were performed according to the manufacturers’ instructions. Gram-negative diplococci growing on modified Thayer-Martin medium were confirmed as N. gonorrhoeae by the GonoGen II assay. Supplemental data analysis was performed by major outer membrane protein PCR for C. trachomatis and probes for pilin gene detection for N. gonorrhoeae. A true-positive result for each pathogen was defined as a positive result for all three or two of three assays. Overall agreement among the six assays was 94.8%. C. trachomatis prevalence was 16.2%; N. gonorrhoeae prevalence was 5.5%. The overall sensitivity and specificity, respectively, for each test (after supplemental data analysis) were as follows: for C. trachomatis, Gen-Probe, 65.9 and 100%; LCR on urine, 90.1 and 100%; LCR on swab specimens, 96.7 and 100%; and for N. gonorrhoeae, culture, 80.6 and 100%; LCR on urine, 93.5 and 99.8%; and LCR on swab specimens, 96.8 and 100%. For women, the N. gonorrhoeae culture was very insensitive compared to its performance in men (58.3 versus 94.7%, respectively). For C. trachomatis, the Gen-Probe assay’s sensitivity was lower for men than for women (62.3 versus 71.1%, respectively). The sensitivity for C. trachomatis detection by LCR on urethral and cervical swab specimens was 96.2 and 97.4% for men and women, respectively. For men, swab results were slightly better than urine results for both pathogens (sensitivity for C. trachomatis in swab and urine specimens, 96.2 and 92.5%, respectively; sensitivity for N. gonorrhoeae in swab and urine specimens, 100 and 94.7%, respectively), while for women, cervical swabs were superior in sensitivity to urine samples for detecting C. trachomatis (swab, 97.4%; urine, 81.6%) and equivalent for N. gonorrhoeae (swab, 92.3%; urine, 91.6%). The LCx LCR appears to be both sensitive and specific for the detection of C. trachomatis and N. gonorrhoeae when performed on urine or genital swab samples. Swab samples had better sensitivity than urine samples for the detection of both pathogens.     

Performance of the Abbott RealTime CT/NG for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae

A multicenter clinical study was conducted to evaluate the performance characteristics of the Abbott RealTime CT/NG assay, a multiplex real-time PCR assay, for simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The specimens were collected from a total of 3,832 male and female subjects at 16 geographically diverse sites. Specimens included male and female urine samples, male urethral swabs, female endocervical swabs, and self-collected and clinician-collected vaginal swabs. Specimens were tested with the automated Abbott RealTime CT/NG assay, Aptima Combo 2 assay (Gen-Probe), ProbeTec ET CT/GC assay (Becton Dickinson), and culture for N. gonorrhoeae. The Aptima Combo 2 assay, the ProbeTec assay, and the N. gonorrhoeae culture were used as the reference assays. For each subject, a patient infected status (PIS) was determined based on the combined results from the reference assays. The overall prevalence in female subjects was 8.9% for C. trachomatis and 3.8% for N. gonorrhoeae. The overall male prevalence was 18.2% for C. trachomatis and 16.7% for N. gonorrhoeae. The overall sensitivity and specificity of the Abbott RealTime CT/NG assay were 92.4% and 99.2% for C. trachomatis and 96.9% and 99.7% for N. gonorrhoeae, respectively. In comparison, the sensitivity and specificity, respectively, for the Aptima Combo 2 assay were 94.5% and 99.0% for C. trachomatis and 96.1% and 99.5% for N. gonorrhoeae, and those for the ProbeTec ET assay were 90.3% and 99.5% for C. trachomatis and 92.0% and 97.3% for N. gonorrhoeae in this study. The Abbott RealTime CT/NG assay offers C. trachomatis and N. gonorrhoeae dual detection with high sensitivity and specificity. The automated assay provides a useful alternative nucleic acid amplification assay for clinical laboratories and clinicians.Chlamydia trachomatis and Neisseria gonorrhoeae are the two most prevalent bacterial sexually transmitted infections (STIs) reported to the Centers for Disease Control and Prevention (CDC) (2). Together these two infections accounted for over 1.5 million cases of STIs in 2008. The CDC estimates that sexually transmitted diseases (STDs) cost the health care system $1.5 billion annually. Since these infections, especially chlamydia, are most often asymptomatic, the CDC recommends yearly screening for chlamydia in all sexually active women ages 16 to 25 years. Since coinfections are common, many diagnostic test platforms assay for both organisms. Although there are several commercial assays available for performing nucleic acid amplification tests (NAATs), which are now recommended by the CDC as the test of choice, new assays and new platforms are needed by both private and public health programs.The Abbott RealTime CT/NG assay is a new real-time PCR assay for the direct, qualitative detection of the plasmid DNA of Chlamydia trachomatis and the genomic DNA of Neisseria gonorrhoeae in female endocervical or vaginal swab specimens, in male urethral swab specimens, and in male and female urine specimens from symptomatic and asymptomatic individuals. There were several sets of objectives of this study: (i) to determine the performance characteristics of the Abbott RealTime CT/NG assay compared to those of other amplified assays using specimens obtained from symptomatic and asymptomatic male and female subjects from clinical sites with high or low prevalence of Chlamydia trachomatis and/or Neisseria gonorrhoeae; (ii) to demonstrate the precision of the Abbott RealTime CT/NG assay using a precision panel consisting of combinations of high and low concentrations of Chlamydia trachomatis and Neisseria gonorrhoeae specimens; (iii) to evaluate the clinical sensitivity, clinical specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Abbott RealTime CT/NG assay in symptomatic and asymptomatic individuals from high- and low-prevalence sites; (iv) to compare the performance of the new Abbott assay with that of the Gen-Probe Aptima Combo 2 assay for female endocervical and vaginal swabs, male urethral swabs, and male and female urine specimens; (v) to compare the performance of the Abbott assay with those of the Becton Dickinson ProbeTec ET Chlamydia trachomatis and Neisseria gonorrhoeae amplified DNA assays (ProbeTec ET) for female endocervical swabs and male and female urine specimens; and (vi) to evaluate the procedure for self-collection of a vaginal swab specimen using the Abbott multi-Collect specimen collection kit.     

Diminished human immunodeficiency virus type 1 DNA yield from dried blood spots after storage in a humid incubator at 37 degrees C compared to -20 degrees C

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at −20°C. DBS were created by spotting 50-μl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at −20°C or at 37°C with ~85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at −20°C, and 398 stored at 37°C. A chi-square test showed fewer positive reactions for DBS stored at 37°C (55%) than for those stored at −20°C (78%) (P < 0.0001). Samples stored at −20°C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37°C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37°C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.     

Use of Ligase Chain Reaction with Urine versus Cervical Culture for Detection of Chlamydia trachomatis in an Asymptomatic Military Population of Pregnant and Nonpregnant Females Attending Papanicolaou Smear Clinics

Ligase chain reaction (LCR) (Abbott Laboratories, Abbott Park, Ill.) with first-catch urine specimens was used to detect Chlamydia trachomatis infections in 465 asymptomatic military women attending clinics for routine Papanicolaou smear tests. Results were compared to results of cervical culture to determine the sensitivity of the urine LCR and the possible presence of inhibitors of amplification in pregnant and nonpregnant women. Discrepant results for LCR and culture were resolved by direct fluorescent antibody staining of culture sediments, two different PCR assays, and LCR for the outer membrane protein 1 gene. The prevalence of Chlamydia in specimens by urine LCR was 7.3% compared to 5% by culture. For 434 women with matching specimens, there were 11 more specimens positive by LCR than were positive by culture, of which all but one were determined to be true positives. There were four culture-positive, LCR-negative specimens, all from nonpregnant women. The sensitivity, specificity, and positive and negative predictive values of urine LCR after discrepant results were resolved were 88.6, 99.7, 96.9, and 99.0%, respectively. The sensitivity of culture was 71.4%. From the 148 pregnant women (prevalence by LCR, 6.8%), there were no patients who were cervical culture positive and urine LCR negative to indicate the presence in pregnant women of inhibitors of LCR. Additionally, a subset of 55 of the LCR-negative frozen urine specimens from pregnant women that had been previously processed in LCR buffer were inoculated with 5 cell culture inclusion forming units of C. trachomatis each and retested by LCR; all tested positive, indicating the absence of inhibitors of LCR in urine from these pregnant women. The use of LCR testing of urine specimens from asymptomatic women, whether pregnant or not, offers a sensitive and easy method to detect C. trachomatis infection in women.     

Genotyping of Chlamydia trachomatis in Urine Specimens Will Facilitate Large Epidemiological Studies

The serovars of Chlamydia trachomatis-positive urine specimens (n = 81; as detected by PCR and ligase chain reaction) were successfully analyzed in 94% of cases by omp1 PCR-based RFLP analysis. The use of urine specimens and this simple and sensitive typing method will greatly facilitate epidemiological studies of C. trachomatis serovar distribution in asymptomatic C. trachomatis infections in both females and males.     

Performance of Three Nucleic Acid Amplification Tests for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Use of Self-Collected Vaginal Swabs Obtained via an Internet-Based Screening Program

Use of self-obtained vaginal specimens processed by nucleic acid amplification tests (NAATs) has significantly increased the utilization of nontraditional locations for Chlamydia trachomatis and Neisseria gonorrhoeae screening programs. One important emerging source of such venues includes home-based self-sampling kits available via the Internet. The objective of our study was to evaluate the performance of three commercially available NAATs (Becton-Dickinson ProbeTec SDA, Gen-Probe Aptima Combo2 TMA, and Roche Amplicor PCR) for detection of C. trachomatis and N. gonorrhoeae in vaginal samples obtained via an Internet-based screening program. From July 2004 to August 2005, 500 self-collected vaginal swabs were tested for C. trachomatis and N. gonorrhoeae by using all three NAATs. Another 500 samples were collected between August 2005 and November 2007 and tested by ProbeTec and Combo2; PCR testing was discontinued due to low specificity for N. gonorrhoeae. All tests were conducted according to the manufacturers'' procedures; the “gold standard” for an infected C. trachomatis or N. gonorrhoeae patient was defined as ≥2 positive NAAT results. Of the first 500 swabs submitted, 46 were C. trachomatis infected (9.2%) and 5 were N. gonorrhoeae infected (1.0%), and 3 of these were coinfected (0.6%). All C. trachomatis and N. gonorrhoeae Combo2-positive/ProbeTec-negative samples were confirmed as true positives by an alternative NAAT. For C. trachomatis, ProbeTec, Combo2, and PCR had sensitivities of 82.6%, 100%, and 100%, with specificities of 100%, 100%, and 99.3%, respectively. For N. gonorrhoeae, ProbeTec, Combo2, and PCR had sensitivities of 80%, 100%, and 100%, with specificities of 100%, 100%, and 98.8%, respectively. Of the total 1,000 swabs submitted, 92 were C. trachomatis infected (9.2%) and 15 were N. gonorrhoeae infected (1.5%), and 7 of these were coinfected (0.7%). There were no ProbeTec-positive/Combo2-negative samples. For C. trachomatis, ProbeTec and Combo2 had sensitivities of 81.5% and 100%, with specificities of 100% and 100%, respectively. For N. gonorrhoeae, ProbeTec and Combo2 had sensitivities of 80% and 100%, with specificities of 100% and 100%, respectively. Overall, ProbeTec had 17 C. trachomatis false-negative results (1.7%) and 3 N. gonorrhoeae false-negative results (0.3%), while Combo2 had none. Our results were consistent with the sensitivities and specificities stated by the manufacturers. NAATs perform well for detection of chlamydia and gonorrhea with self-obtained vaginal swabs shipped in a dry state to a laboratory. For 1,000 self-collected vaginal swabs tested by NAATs, the sensitivities for C. trachomatis and N. gonorrhoeae for Combo2 were 100% and 100%, while they were 81.5% and 80%, respectively, for ProbeTec. For 500 PCR samples, the C. trachomatis sensitivity was 100% and the N. gonorrhoeae sensitivity was 100%, with specificities of 99.3% and 98.8%, respectively.Each year, an estimated 3 million Chlamydia trachomatis infections occur in the United States, with over 1 million reported in 2006 surveillance reports (2). In these same reports, 358,366 cases of Neisseria gonorrhoeae infection were reported to occur in the United States (2). Since many sexually transmitted infections (STIs) are asymptomatic, especially chlamydial infections, traditional approaches to clinic-based diagnosis of STIs omit a whole segment of the population that would not ordinarily get tested. Prior to the emergence of nucleic acid amplification tests (NAATs), symptomatic persons or contacts of infected patients would be the only persons seeking care for STIs, usually at a sexually transmitted disease clinic or family planning clinic.Through the use of noninvasive urogenital samples, it is now possible to use alternative venues for screening programs outside the clinic where a clinician is not required to obtain the specimens. Utilization of self-obtained specimens for C. trachomatis and N. gonorrhoeae testing, which employ the highly sensitive and specific NAATs, has enhanced the use of noninvasive urogenital samples (9, 17). Studies have reported that self-collected vaginal specimens are acceptable and can be successfully used to diagnose STIs, especially chlamydia and gonorrhea, when they are used with NAATs (3, 14, 17, 18, 26). This can eliminate the necessity for a clinician-performed pelvic examination for women for sample collection and may represent cost savings when a woman does not require a Pap test or have symptoms (1).Expansion of alternative venues in which chlamydia testing can be performed may even be taken to Internet recruitment for home collection of vaginal swabs for mailing to a laboratory for testing (11, 13). This alternative venue may be popular among adolescents who search the Internet for health care information and information regarding STIs (20). The Internet is also used by people seeking sex partners, and these users appear to be at greater risk for STIs (22). Internet use statistics are staggering; the Pew American Life Project reports that 168 million people are on the Internet per day (http://www.pewinternet.org/reports.asp). Internet use is estimated at 83% of those aged 18 to 29 years, the prime age range in which STIs are most prevalent (http://www.pewinternet.org/reports.asp).We have successfully used an Internet-based recruitment method for home collection of self-obtained vaginal swabs for chlamydia testing (11). While such samples collected at home are not yet FDA cleared, self-obtained vaginal swabs collected in a clinic are FDA cleared for one type of NAAT (26). We therefore decided to compare the performances of the three commercially available NAATs for C. trachomatis and N. gonorrhoeae testing with vaginal samples obtained at home through an Internet screening program and mailed in a dry state (10, 17, 29) to the laboratory for testing.     

Pooling Urine Samples for Ligase Chain Reaction Screening for Genital Chlamydia trachomatis Infection in Asymptomatic Women

The accuracy of pooling urine samples for the detection of genital Chlamydia trachomatis infection by ligase chain reaction (LCR) was examined. A model was also developed to determine the number of samples to be pooled for optimal cost savings at various population prevalences. Estimated costs included technician time, laboratory consumables, and assay costs of testing pooled samples and retesting individual specimens from presumptive positive pools. Estimation of population prevalence based on the pooled LCR results was also applied. After individual urine specimens were processed, 568 specimens were pooled by 4 into 142 pools and another 520 specimens were pooled by 10 into 52 pools. For comparison, all 1,088 urine specimens were tested individually. The sample-to-cut-off ratio was lowered from 1.0 to 0.2 for pooled samples, after a pilot study which tested 148 samples pooled by 4 was conducted. The pooling algorithm was 100% (48 of 48) sensitive when samples were pooled by 4 and 98.4% (61 of 62) sensitive when samples were pooled by 10. Although 2.0% (2 of 99) of the negative pools of 4 and 7.1% (1 of 14) of the negative pools of 10 tested presumptive positive, all samples in these presumptive-positive pools were negative when retested individually, making the pooling algorithm 100% specific. In a population with 8% genital C. trachomatis prevalence, pooling by four would reduce costs by 39%. The model demonstrated that with a lower prevalence of 2%, pooling eight samples would reduce costs by 59%. Pooling urine samples for detection of C. trachomatis by LCR is sensitive, specific, and cost saving compared to testing individual samples.     

Utility of real-time PCR for diagnosis of Legionnaires' disease in routine clinical practice

The main aim of our study was to determine the added value of PCR for the diagnosis of Legionnaires’ disease (LD) in routine clinical practice. The specimens were samples submitted for routine diagnosis of pneumonia from December 2002 to November 2005. Patients were evaluated if, in addition to PCR, the results of at least one of the following diagnostic tests were available: (i) culture for Legionella spp. on buffered charcoal yeast extract agar or (ii) detection of Legionella pneumophila antigen in urine specimens. Of the 151 evaluated patients, 37 (25%) fulfilled the European Working Group on Legionella Infections criteria for a confirmed case of LD (the “gold standard”). An estimated sensitivity, specificity, and overall percent agreement of 86% (32 of 37; 95% confidence interval [CI] = 72 to 95%), 95% (107 of 112; 95% CI = 90 to 98%), and 93% (139 of 149), respectively, were found for 16S rRNA-based PCR, and corresponding values of 92% (34 of 37; 95% CI = 78 to 98%), 98% (110 of 112; 95% CI = 93 to 100%), and 97% (144 of 149), respectively, were found for the mip gene-based PCR. A total of 35 patients were diagnosed by using the urinary antigen test, and 34 were diagnosed by the 16S rRNA-based PCR. With the mip gene PCR one more case of LD (n = 36; not significant) was detected. By combining urinary antigen test and the mip gene PCR, LD was diagnosed in an additional 4 (11%) patients versus the use of the urinary antigen test alone. The addition of a L. pneumophila-specific mip gene PCR to a urinary antigen test is useful in patients with suspected LD who produce sputum and might allow the early detection of a significant number of additional patients.     

Chlamydia trachomatis serovars among strains isolated from members of rural indigenous communities and urban populations in Australia

We genotyped Chlamydia trachomatis strains from 45 women or men living in either a rural indigenous community or in urban heterosexual communities. We found six different C. trachomatis serovars: E (n = 22; 48.9%), F (n = 10; 22.2%), J/Ja (n = 5; 11.1%), D/Da (n = 4; 8.9%), G (n = 3; 6.7%), and K (n = 1; 2.2%). The distribution of C. trachomatis serovars among members of the indigenous rural and the urban Australian communities appears similar to that in other Western countries.     

Fusion of Chlamydia trachomatis-Containing Inclusions Is Inhibited at Low Temperatures and Requires Bacterial Protein Synthesis

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium with a unique developmental cycle. Within the host cell cytoplasm, it resides within a membrane-bound compartment, the inclusion. A distinguishing characteristic of the C. trachomatis life cycle is the fusion of the chlamydia-containing inclusions with each other in the host cell cytoplasm. We report that fusion of inclusions does not occur at 32°C in multiple mammalian cell lines and with three different serovars of C. trachomatis. The inhibition of fusion was inclusion specific; the fusion with sphingolipid-containing secretory vesicles and the interaction with early endosomes were unaffected by incubation at 32°C. The inhibition of fusion of the inclusions was not primarily the result of delayed maturation of the inclusion, as infectious progeny was produced in host cells incubated at 32°C, and the unfused inclusions remained competent to fuse up to 48 h postinfection. The ability to reverse the inhibition of fusion by shifting the infected cells from 32 to 37°C allowed the measurement of the rate and the time of fusion of the inclusions after entry of the bacteria. Most significantly, we demonstrate that fusion of inclusions with each other requires bacterial protein synthesis and that the required bacterial protein(s) is present, but inactive or not secreted, at 32°C.     

Induction of cross-serovar protection against genital chlamydial infection by a targeted multisubunit vaccination approach

An important consideration for antichlamydial vaccine development is the induction of cross-serovar protection, since multiple serovars (D to L) of Chlamydia trachomatis cause genital infections. We have shown previously that vaccination with C. trachomatis-derived recombinant chlamydial protease-like activity factor (rCPAF) induced significant earlier resolution of Chlamydia muridarum infection and reduced oviduct pathology. However, the vaccinated mice continued to shed chlamydiae for up to 2 weeks after challenge. In this study, C. trachomatis serovar D recombinant proteins, such as recombinant major outer membrane protein (rMOMP), recombinant inclusion membrane protein A (rIncA), and rCPAF were administered intranasally, individually or in combinations, with murine interleukin-12 (IL-12) as an adjuvant, and cross-species immunity against intravaginal C. muridarum infection was examined. Immunization with rCPAF plus IL-12 (rCPAF+IL-12), compared to immunization with rIncA+IL-12 or rMOMP+IL-12, induced the greatest antigen-specific gamma interferon production from purified CD4⁺ T cells and concurrently enhanced serum antibody production. All (100%) the animals vaccinated with rCPAF+IL-12 alone or in any combination completely resolved the infection by day 18 after challenge compared to animals vaccinated with rIncA+IL-12 (50%), rMOMP+IL-12 (33%), or phosphate-buffered saline (mock vaccinated; 0%). Moreover, oviduct pathology in mice vaccinated by any regimen that included rCPAF, but not rMOMP+IL-12 or rIncA+IL-12 alone, was markedly reduced compared to mock-immunized animals. The addition of rMOMP and/or rIncA did not significantly enhance the rCPAF+IL-12-induced effect on bacterial clearance or oviduct pathology. These results suggest a greater conservation of protective linear antigenic epitopes within CPAF than MOMP or IncA across the examined serovars and the need to identify other highly conserved antigens for use with rCPAF in a multisubunit recombinant vaccine.     

Blinded Multiplex PCR Analyses of Middle Ear and Nasopharyngeal Fluids from Chinchilla Models of Single- and Mixed-Pathogen-Induced Otitis Media

Multiplex PCR analyses for both bacterial and viral pathogens were conducted in a blinded manner on 33 archival specimens, of known culture status, procured from chinchilla models of both single- and mixed-pathogen-induced otitis media and from a pediatric patient. These specimens had been maintained at −70°C for up to 6 years. Experimental specimens evaluated included middle-ear effusions, nasopharyngeal lavage fluids and middle-ear lavage fluids from animals which were immunologically naive, sham-immunized or actively immunized with nontypeable Haemophilus influenzae antigens. Sampling times used ranged from the day of bacterial or viral challenge to 42 days after challenge. Initial PCR analyses of the 33 specimens matched the traditional culture data in 24 instances (73%), correctly identifying nontypeable H. influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, or adenovirus as the causative agent. A PCR-positive signal for the microbe(s) inoculated was also obtained in four animal model specimens (12%) which were culture negative. One of two culture-negative human effusions was also PCR positive. Thus, overall, results obtained by blinded PCR were 85% concordant with traditional culture methods or correctly indicated the specific pathogen introduced in four specimens that were sterile. In no instance was a false-positive signal obtained for any of the five etiologic agents being evaluated. We conclude that the multiplex PCR analyses are rapid and accurate methodologies when they are used to retrospectively evaluate diverse archival specimens of limited volume from experimental models of otitis media.     

Prevalence of β2-Toxigenic Clostridium perfringens in Horses with Intestinal Disorders

The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the α-toxin and the recently described β2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed for the presence of the α-, β-, β2-, and -toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the β2-toxin gene cpb2. β2-Toxigenic C. perfringens was detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded β2-toxigenic C. perfringens. No β2-toxigenic C. perfringens was found in the samples from the 58 control horses, of which only one fecal sample contained C. perfringens type A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for β2-toxigenic C. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for β2-toxigenic C. perfringens and lethal progression of the disease. No C. perfringens strains isolated in this study contained genes for the β- and -toxins and enterotoxin. The high incidence of β2-toxigenic C. perfringens in samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong evidence that β2-toxigenic C. perfringens play an important role in the pathogenesis of typhlocolitis.     

Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay

Quantification of hepatitis B virus (HBV) DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log₁₀ HBV DNA dynamic range, were thawed and stored at −70, 4, 23, 37, and 45°C (±1.5°C) for 0, 24, 72, and 120 h (±2 h) and were refrozen at −70°C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4°C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at ≤4°C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45°C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at ≤4°C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of ≥20% at 23 or 37°C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at −70 or 4°C.     

Evaluation of the Biostar Chlamydia OIA Assay with Specimens from Women Attending a Sexually Transmitted Disease Clinic

Chlamydia trachomatis infections are the most prevalent sexually transmitted diseases (STDs) in the United States. In acute-care settings such as clinics and emergency rooms, a desirable chlamydia screening assay should exhibit good sensitivity and good specificity and should provide test results while the patient is still present. The Biostar Chlamydia OIA (Biostar, Inc., Boulder, Colo.) is an optical immunoassay (OIA) that provides test results in less than 30 min and that uses a test format that allows office-based testing. This assay is performed entirely at room temperature without the need for rotators or other specialized equipment. The goal of this study was to compare the performance of the Biostar Chlamydia OIA for the detection of C. trachomatis with the performance of cell culture, direct fluorescent-antibody (DFA) assay (Syva MicroTrak; Syva Co., Palo Alto, Calif.), and PCR (Roche Amplicor Chlamydia trachomatis; Roche, Branchburg, N.J.) for the detection of C. trachomatis infections in women attending an urban STD clinic. For calculations of relative test performance (sensitivity, specificity, and positive and negative predictive values), patient specimens that yielded positive results by two or more of the four assays (cell culture, DFA assay, PCR, and OIA) were classified as “true infections.” By these criteria, 42 of 306 total specimens were classified as positive for C. trachomatis (positive prevalence, 13.7%), 11 (3.6%; 10 by PCR and 1 by DFA assay) were positive by a single assay, and 253 (82.7%) were negative by all four tests. All culture-positive specimens were also positive by at least one other assay. Among the culture-negative specimens, 14 (5%) specimens were positive by two of the three non-culture-based assays used. By using the criterion that positivity by at least two of the tests indicated a true infection, the relative sensitivities were as follows: culture and PCR, 92.9% each; Biostar Chlamydia OIA, 73.8%; and DFA assay, 59.5%.Chlamydia trachomatis infections are the most prevalent sexually transmitted diseases (STDs) in the United States and are a major preventable cause of infertility, ectopic pregnancy, and chronic pelvic pain in women (3). In addition, because the signs and symptoms of infection are often mild or even absent, laboratory testing plays a central role in efforts to control chlamydia.Optimally, tests for the detection of chlamydia should be sensitive and specific and should provide results quickly to guide patient management. Until recently, the standard for the diagnosis of C. trachomatis infection has been cell culture (8, 9). However, in most clinical settings, antigen or nonamplified nucleic acid detection tests have been preferentially used for testing for chlamydia because of their logistical advantages and lower costs, despite the observation that these tests are generally less sensitive than cell culture (8). More recently, evaluations of newer nucleic acid amplification assays for C. trachomatis diagnosis such as PCR or ligase chain reaction have shown the true sensitivity of cell culture for chlamydia detection to be 65 to 85% (15, 18). Despite their greater sensitivities, amplified nucleic acid detection tests are not used in many clinical settings, in part because of their substantially higher costs compared to those of nonamplification assays. In addition, like most currently available tests for the diagnosis of C. trachomatis infection, the nucleic acid amplification tests usually do not provide test results at the time of screening but typically require turnaround times of a day or more, a characteristic that may introduce delays in translating test results into treatment (6, 16). In some settings there are demonstrable advantages to tests that can provide results while patients are still present in clinical settings, particularly if the performance of such tests is comparable to the performance of alternate tests (6, 16).The Biostar Chlamydia OIA (Biostar, Inc., Boulder, Colo.) is an optical immunoassay (OIA) designed to rapidly detect chlamydia infection in women, providing test results in less than 30 min in a test format that allows office-based testing. To evaluate the performance of the Biostar Chlamydia OIA for the detection of C. trachomatis, we compared the performances of four assays, i.e., cell culture, an immunofluorescence antigen detection assay (Syva MicroTrak; Syva Co., Palo Alto, Calif.), PCR (Roche Amplicor Chlamydia trachomatis; Roche, Branchburg, N.J.), and the Biostar Chlamydia OIA, for the detection of C. trachomatis infections in women attending an urban STD clinic.