Ability of new APTIMA CT and APTIMA GC assays to detect Chlamydia trachomatis and Neisseria gonorrhoeae in male urine and urethral swabs.

A clinical evaluation was conducted in six North American centers to determine the ability of APTIMA CT (ACT) and APTIMA GC (AGC) nucleic acid amplification assays to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections in 1,322 men by testing their urethral swabs and first-catch urine (FCU). The results obtained with ACT and AGC assays were compared to an infected patient status determined by testing the specimens with the APTIMA Combo 2 and the BD ProbeTec energy transfer multiplex assays. Symptoms did not influence the values. Positive and negative agreements of the ACT and AGC assays for individual specimens were high, with each comparator assay ranging between 94.3 and 100% for positives and 93.9 and 99.4% for negatives. The ACT and AGC assays performed on noninvasive specimens such as FCU effectively identified C. trachomatis or N. gonorrhoeae infections in symptomatic and asymptomatic men and should be suitable for screening male populations.

Chlamydia trachomatis diagnostics.

Nucleic acid amplification (NAA) assays for the diagnosis of Chlamydia trachomatis infections started to appear in the peer reviewed literature about 12 years ago and during that period we have seen an incredible effort put into the development and evaluation of commercially developed NAA kits to diagnose and treat infections.

Direct detection of vanA and vanB genes in clinical specimens for rapid identification of vancomycin resistant enterococci (VRE) using multiplex PCR.

Surveillance for vancomycin resistant enterococci (VRE) by culture can be labour intensive and time consuming. We have developed a multiplex polymerase chain reaction (MPCR) which can be performed directly on the clinical specimen. The assay allows sensitive detection of enterococci with vanA - and vanB -mediated resistance to vancomycin. DNA was purified from stool and rectal specimens using the XTRAX(TM)DNA Extraction Kit (Gull Labs). Multiplex PCR amplified vanA and vanB targets were detected using a microtiter plate EIA. Two-hundred specimens were tested by routine culture and MPCR. Culture identified 44 VRE isolates and MPCR detected 38 of the 44 culture positives. Multiplex PCR detected three additional positive VRE specimens missed by culture for a sensitivity and specificity of 86.4 and 98.1%, respectively. When the presence of PCR inhibitors was addressed in the six culture positive/MPCR negative specimens, four additional VRE positive specimens were detected. Performing MPCR on the original specimens and on a 1:10 dilution of all specimens to minimize the effect of inhibitors gave a sensitivity and specificity of 95.5 and 98.1%, respectively. Multiplex PCR with confirmation by microtiter plate hybridization could be completed in 8 h compared with 24-48 h required for culture.

Nucleic acid tests for the diagnosis of sexually transmitted diseases.

Nucleic acid (NA) assays have been developed and commercialized for many sexually transmitted diseases (STDs). Solid phase, liquid phase or in situ hybridization of nucleic acids without amplification procedures have been successfully used for diagnosing Chlamydia trachomatis, Neisseria gonorrhoeae and human papillomaviruses. Tests which use amplification procedures have provided better sensitivity and specificity than traditional tests. With special temperatures and enzymes, the new tests are designed to amplify either the target nucleic acid or the probe after annealing to the target. A third approach uses signal amplification. This article discusses the technology, specimen requirements and the current status of NA assay performance for diagnosing STDs and HIV by traditional and non-invasive clinical specimens.

Molecular Diagnosis of Chlamydia trachomatis Infections by Probe Hybridization, PCR, LCR,TMA, and Q-ß Replicase.

The use of cell cultures for the laboratory diagnosis of Chlamydia trachomatis infections was popularized during the 1970s and 80s (1-4). The techniques required live organisms and were restricted to specialized laboratories. During the 1980s the detection of chlamydia-specific antigens was extensively used and compared to cell culture, provided a less stringent transportation of clinical specimens. Both direct fluorescent antibodies (DFA) and enzyme immunoassay (EIA) systems were commercialized (5-7) and algorithms for confirming false positives were popularized (8,9). The first nucleic acid detection assay for C.trachomatis was evaluated during this time frame with comparisons to culture and antigen detection (10-14). In the early 1990s, the following amplified nucleic acid assays detecting C. trachomatis gene fragments were developed: polymerase chain reaction (PCR), ligase-chain reaction (LCR), Q-ß replicase-amplified hybridization (QBRAH), transcription-mediated amplification (TMA), and nucleic acid sequence-based amplification (NASBA). Extensive evaluations of PCR and LCR have shown, through discordant analysis and expansion of the reference standard for positives, that these amplified assays are 20-30% more sensitive than culture, antigen, or nonamplified nucleic-acid detection methods (15-29). This range in sensitivity (for PCR and/or LCR) is influenced by the rate of inhibitors of amplification found in clinical specimens.

Predictors of positivity for hepatitis B and the derivation of a selective screening rule in a Canadian sexually transmitted disease clinic.

OBJECTIVES: To determine the prevalence of hepatitis B surface antibody (anti-HBs) and antigenemia (HBsAg), the risk factors for seropositivity and the effectiveness of a selective serologic screening rule among sexually transmitted diseases (STD) clinic attendees. STUDY DESIGN: Clients in the Hamilton STD Clinic were surveyed from October 1992 to July 1993 on sociodemographic, past medical, and behavioural data, were tested for several STDs and were offered serological testing and vaccination against hepatitis B. Predictors of seropositivity were determined by single variable analysis. A selective serologic screening rule was derived using logistic regression modelling. RESULTS: The seroprevalence of anti-HBs was 6.8% (21/310) in the 310 of 385 clients (80.5%) who agreed to be tested and interviewed. There were no HBsAg carriers. Five independent risk factors were identified by logistic regression: (1) age greater than 35 years; (2) birth outside Canada and histories of; (3) syphilis; (4) gonorrhoea; or (5) injection drug use. If clients with at least one of these predictors had been tested, 34.5% would have been selected for serologic testing and 85.7% of all positives would have been detected. The screening rule was more effective for men than for women. CONCLUSION: In this low prevalence setting, selecting STD clinic clients based on the presence of any one of five risk predictors appears to be an effective strategy for hepatitis B serologic screening in the context of a Canadian vaccination program.

Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis infected women.

In a comparison of commercial ligase chain reaction (LCR; Abbott) and polymerase chain reaction (PCR; Roche) assays, measuring plasmid genes of Chlamydia trachomatis, some specimens were found to be negative by either or both assays but positive in traditional culture or antigen detection tests. Of 767 women, 35 were found to be infected by cervical or urine testing. Twenty three specimens from 16 women may have contained inhibitors in six cervical swabs (CS) and 15 first void urines (FVU). By performing dilution and 'spiking' experiments on five FVU, inhibitors of PCR, LCR or both, which disappeared by dilution, were demonstrated. Confirmatory assays were used which amplified segments of the major outer membrane gene by PCR or LCR. When comparisons of assays were made on a single specimen type, the sensitivities of the amplification assays, compared to an expanded reference standard, were as follows: on CS, PCR was 93.8% (30/32) and LCR was 96.9% (31/32); on FVU, PCR was 76.6% (23/30) and LCR was 93.3% (28/30). When a combined calculation was made to determine the ability of the assays to detect patients infected in the cervix or urethra by testing FVU, the sensitivities dropped to 71.4% (25/35) for PCR and 80.0% (28/35) for LCR: CS sensitivity was 88.6% (31/35) for both amplified tests. There were two CS and five FVU false-positives by PCR which reduced to one CS and three FVU in the combined analysis. There were no false-positives by LCR. Inhibitors and low levels of chlamydial plasmid nucleic acids may have contributed to lower than expected sensitivities, suggesting a possible need for internal positive controls, especially for PCR, when testing urine. More studies with multiple sampling and more than one amplification assay are needed to confirm these findings and to identify and remove inhibitors of amplification assays.

Ability of commercial ligase chain reaction and PCR assays to diagnose Chlamydia trachomatis infections in men by testing first-void urine.

A total of 287 men (37.6% with symptoms of urethritis) attending a hospital-based sexually transmitted disease clinic had urethral swabs tested by culture and by direct fluorescent-antibody assay. First-void urine (FVU) was tested for Chlamydia trachomatis by commercially available ligase chain reaction (LCR) and PCR assays. By using an expanded reference standard, 35 men (12.2%) were found to be positive. By performing LCR and PCR, the infection prevalence was found to be approximately twice (11.5 and 12.2%, respectively) that determined swab testing. The sensitivity values were 94.3% for LCR and 100% for PCR. One of the two positive specimens missed by LCR contained inhibitors. PCR produced five false-positive results and LCR produced one.

How can industry, academia, public health authorities, and the sexually transmitted diseases diagnostics initiative work together to help control sexually transmitted diseases in developing countries?

PIP: More than 300 million new cases of gonorrhea, chlamydia, syphilis, and chancroid will develop in 1997, with 85% occurring in developing countries. While diagnostic tests for sexually transmitted diseases (STDs) are sensitive and specific, their expense has led the World Health Organization to promote syndromic management of STDs. This approach, however, can lead to overtreatment with expensive drugs and may result in development of antibiotic-resistant strains of infection. Also, gonococcal and chlamydial infections are often asymptomatic in women. Because the presence of an STD facilitates HIV transmission, STD treatment is an important strategy in HIV/AIDS prevention and control. Since 1990, the STD Diagnostics Initiative (SDI) has sought to identify sensitive, specific, simple, stable, and inexpensive means of diagnosing STDs. Since 1994, 8 research proposals have received a total of $850,000 for a 3-year period. The efficient diagnostic tests sought by the SDI would encourage greater expenditures on STD treatment. The SDI believes that collaboration with industry should remove most of the constraints to product development and market penetration that exist in developing countries. Incentives to achieve the goals of the SDI include a million dollar prize offered by the Rockefeller Foundation for development of a rapid, sensitive, specific, simple, stable, and inexpensive assay. SDI can provide research funds, controlled access to pedigreed clinical specimens, and guidelines for effective evaluations. Industry has the market and the challenge to join with SDI in this effort.^ieng

Detection of Chlamydia trachomatis antigens in male urethral swabs and urines with a microparticle enzyme immunoassay.

BACKGROUND AND OBJECTIVES: More information is needed on the natural history of Chlamydia trachomatis urethral infections in men. Newer assays for detecting antigens in male first void urine and urethral swabs identify patients for control programs. A new microparticle enzyme immunoassay from Abbott Laboratories called IMX Select Chlamydia was evaluated and compared with culture and an expanded gold standard for sensitivity and specificity. STUDY DESIGN: Paired samples of first void urine and two urethral swabs were tested from 230 men, 73% of whom had symptoms of urethritis. Both specimen types were tested with IMX Select, the other swab was cultured, and a part of the first void urine was tested by Chlamydiazyme enzyme immunoassay. Performance calculations were made against urethral culture and an expanded gold standard that included direct fluorescent staining of discordant specimens by Microtrak. RESULTS: Compared with urethral swab culture, the IMX Select test performed on urethral swabs and first void urine had sensitivities of 93.8% and 81.3%, and specificities of 95.2% and 95.7%, respectively. Calculations of sensitivity and specificity based on the expanded gold standard were: IMX Select on urethral swabs, 88.5% and 99.4%; IMX Select on first void urine, 80.8% and 100%; Chlamydiazyme after blocking confirmation on first void urine, 73.1% and 100%; culture on urethral swabs, 61.5% and 100%. CONCLUSION: This IMX Select Chlamydia enzyme immunoassay, which generates laboratory results within 2 hours, performed better than culture and an established enzyme immunoassay on male urethral swabs. The experimental first void urine protocol showed promise for noninvasive male testing.

Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine.

Genitourinary infection with Chlamydia trachomatis is a common and potentially serious sexually transmitted disease. Diagnosis of C trachomatis infection in women typically relies on culture of endocervical swabs, an invasive and expensive procedure. The ligase chain reaction (LCR) is an in-vitro nucleic acid amplification technique that exponentially amplifies selected DNA sequences. We have compared an LCR-based assay to detect C trachomatis plasmid DNA in first void urine with culture of endocervical swabs for matched specimens from 1937 women from four geographic regions. Discordant specimen pairs were further tested by direct fluorescent antibody staining for elementary bodies and an alternative LCR assay based on the chlamydial outer membrane protein gene. An "expanded gold standard" was defined to include all culture-positive as well as culture-negative, confirmed LCR-positive women. The sensitivity and specificity of the LCR assay with first void urine samples compared with the expanded gold standard were 93.8% and 99.9%, respectively; the corresponding values for culture were 65.0% and 100%, respectively. Thus, an automated LCR assay of readily obtained urine samples showed a detection rate for infected women almost 30% greater than that of endocervical swab culture. The LCR assay was highly effective for the detection of C trachomatis in urine from women with or without signs or symptoms of chlamydial genitourinary tract infection.

Diagnosis of Chlamydia trachomatis urethral infection in symptomatic and asymptomatic men by testing first-void urine in a ligase chain reaction assay.

A multicenter study compared ligase chain reaction (LCR) of Chlamydia trachomatis plasmid DNA with culture of urethral swab specimens from 542 men (study A); a second study (B) compared LCR of first-void urine (FVU) with urethral swab cultures from 1043 men. Discordant results were resolved with direct fluorescent antibody staining of sediments from the FVU or urethral culture specimen and with a second LCR directed against a fragment of the major outer membrane protein gene. Test performance was calculated on the basis of an expanded reference standard. The LCR plasmid assay had a sensitivity of 98.0% in study A and 93.5% in study B; specificity was 99.8%-100%. The sensitivity of culturing urethral swabs from all study sites was 68.2% (range by sites, 40.0%-84.6%). The presence or absence of urethral symptoms did not alter the results. Use of this LCR test should allow more meaningful investigation and treatment of C. trachomatis infections in men.

Diagnosis of Chlamydia trachomatis infections in men and women by testing first-void urine by ligase chain reaction.

From April to September 1993, 305 men and 447 women in Hamilton, Canada, consented to the collection of a urethral or cervical swab, respectively, for culture and 20 ml of first-void urine (FVU) for testing by the enzyme immunoassay Chlamydiazyme and by ligase chain reaction (LCR) in the form of a kit from Abbott Laboratories called LCx Chlamydia trachomatis. Evaluation of test performance with each specimen was calculated on the basis of an expanded "gold standard" of a patient found to be positive by culture or by a confirmed nonculture test. By using this expanded standard, the prevalence of infection was determined to be 6% (27/447) for the women and 18.4% (56/305) for the men. LCR testing of FVU in both studies was the most sensitive approach (96%). The performance of Chlamydiazyme was as follows: cervical swab, 78.3% sensitivity; female FVU, 37% sensitivity; and male FVU, 67.9% sensitivity. Culture was the least sensitive approach to diagnosis: female cervix, 55.6%; and male urethra, 37.5%. LCR testing of FVU from men or women diagnosed the greatest number of genitourinary tract infections with no false positives.

Role of confirmatory PCRs in determining performance of Chlamydia Amplicor PCR with endocervical specimens from women with a low prevalence of infection.

The role of confirmatory PCR assays for determining the performance of Chlamydia Amplicor PCR for endocervical specimens from women with a low prevalence of infection was evaluated. An endocervical swab was collected from 770 women and tested by culture or direct fluorescent antibody (DFA) staining. A second swab was tested by Chlamydia Amplicor PCR (Roche Molecular Systems, Branchburg, N.J.). Discordant results were resolved by three confirmatory PCRs: one targeting the plasmid by using different primers and two directed to the major outer membrane protein (MOMP) gene. Of the 30 swabs that were positive by culture or DFA (3.9%), 27 were positive by Amplicor PCR. An additional five swabs were positive by Amplicor PCR but negative by culture or DFA. Both plasmid and MOMP confirmatory PCRs identified the five culture-DFA negatives and the three Amplicor negatives as true positives. The three specimens originally classified as negative by Amplicor PCR were positive on repeat Amplicor testing. After resolution of the discordant results by confirmatory PCR testing, the sensitivity of the initial Amplicor PCR was 91.4% (32 of 35 specimens), changing to 100% after storage and repeat testing. The specificity of Amplicor PCR was 100% (735 of 735 specimens). Our results demonstrated that plasmid and MOMP confirmatory PCRs worked equally well in resolving false-positive and false-negative Amplicor PCR results. Some specimens may contain inhibitors of Amplicor PCR which may disappear with time.

Leukocyte esterase urine strips for the screening of men with urethritis--use in developing countries.

BACKGROUND AND OBJECTIVES: The leukocyte esterase (LE) strip is a useful tool for the screening of men with urethritis. In developing countries, where laboratory facilities are limited, and sexually transmitted diseases endemic, simple and inexpensive diagnostic tests which perform well, would be of great value. METHODS: Men presenting with urethritis to a referral clinic for sexually transmitted diseases in Nairobi, Kenya participated in this cohort analytical study. First-void urine was collected for LE dipstick testing as part of the diagnostic work-up. The results of the dipstick measurement were compared with the laboratory detection of Chlamydia trachomatis and Neisseria gonorrhoeae. RESULTS: Of 200 men with symptoms of urethritis, 33 (17%) had a pathogen detected from the urethra or the urine. Chlamydia was detected in urine by PCR in 22 (11%), and gonorrhoea was cultured from the urethra in 11 (6%). Esterase activity (trace or greater) had a sensitivity of 76%, a specificity of 80%, a positive predictive value of 42% and a negative predictive value of 94% for the presence of chlamydia or gonorrhoea. CONCLUSIONS: The use of the LE dipstick for the screening of men with symptomatic urethritis can improve diagnostic accuracy and reduce the amount of empiric antimicrobial therapy. The low detection rate of chlamydia in these men with a clinical diagnosis of nongonococcal urethritis needs further study.

Interlaboratory agreement study of a double set of PCR plasmid primers for detection of Chlamydia trachomatis in a variety of genitourinary specimens.

We conducted a tricenter interlaboratory agreement study to assess the agreement of PCR results obtained for detection of Chlamydia trachomatis in genitourinary specimens. A total of 120 specimens (49 positive and 71 negative), including 20 first-void urine samples, 50 endocervical and 50 urethral swabs (40 males), were coded and sent from a reference laboratory (laboratory A) to two other laboratories. Laboratories B and C were provided with a standardized protocol and reagent package including two sets of plasmid PCR primers (KL1-KL2 and T1-T2) and were asked to test each specimen with the first set of primers (KL1-KL2) and to confirm positives with the second set of primers (T1-T2). Laboratory B identified 47 of 49 positives and 69 of 70 negatives (one specimen dried up on shipping) following the initial PCR, for an accuracy of 97.5% (116 of 119), and 47 of 49 positives and 70 of 70 negatives after confirmatory testing of the positives, for an accuracy of 98.3% (117 of 119). Laboratory C identified 42 of 49 positives and 70 of 70 negatives for the initial PCR, for an accuracy of 94.1% (112 of 119), and 39 of 42 positives and 70 of 70 negatives for the confirmatory PCR, for an accuracy of 91.6% (109 of 119). The overall accuracy of PCR testing was 96.6% (345 of 357). The kappa agreement statistics for agreement between pairs of laboratories after confirmation of positives were 0.97 for laboratories A and B, 0.83 for laboratories B and C, and 0.83 for laboratories A and C. Use of the confirmatory PCR improved the specificity and overall accuracy of results for individual laboratories but reduced slightly the results obtained for agreement between laboratories. These results demonstrate that when standardized reagents and protocols are used, PCR results are highly reproducible and excellent agreement between laboratories is obtainable.