Pooling cervical swabs and testing by ligase chain reaction are accurate and cost-saving strategies for diagnosis of Chlamydia trachomatis

Specimen pooling to achieve efficiency when testing urine specimens for Chlamydia trachomatis nucleic acids has been suggested. We pooled endocervical swabs from 1,288 women and also tested individual swabs by ligase chain reaction (LCR). Out of 53 positive specimens, pools of 4 or 8 specimens missed two positives, providing 96.2% accuracy compared to individual test results. Dilution and positive-control spiking experiments showed that negative specimens with inhibitors of LCR in the pool reduced the signal. Conversely, two extra positives, detected only through pooling, were negative by individual testing but became positive after storage, suggesting that fresh positive specimens with labile inhibitors may be positive in a pool because of dilution of inhibitors. For this population of women with a 4% prevalence of C. trachomatis infection, substantial savings in cost of reagents (55 to 63%) and technologist time (50 to 63%) made pooling strategies a desirable alternative to individual testing.     

Pooling nasopharyngeal/throat swab specimens to increase testing capacity for influenza viruses by PCR

Real-time PCR methodology can be applied to rapidly and accurately detect influenza viruses. During times of surge testing or enhanced pandemic surveillance, public health laboratories (PHLs) may experience overwhelming demand for testing, even while the prevalence of positive specimens remains low. To improve laboratory capacity and testing efficiency during surges, we evaluated whether nasopharyngeal (NP)/throat swab specimens can be pooled and tested for the presence of the 2009 H1N1 influenza virus without a reduction in sensitivity. Pools of 10 specimens were extracted and concentrated upon elution on the MagNA Pure LC instrument, and real-time PCR was performed on the Applied Biosystems 7500 Fast platform, using the CDC swine influenza virus real-time RT-PCR detection panel (rRT-PCR swine flu panel). Specimens in positive pools were singly re-extracted and retested by PCR to identify individual positive samples. Initial studies showed that spiking a pool of nine negative specimens (100 μl each) or 900 μl of virus transport medium with 100 μl of a positive clinical specimen caused no loss of sensitivity by rRT-PCR testing. Pools containing either multiple positive specimens or specimens positive for other respiratory viruses also showed no negative effect on crossing threshold (C(T)) values. To test the robustness of the pooling protocol, a panel of 50 blinded samples was sent to three PHLs and tested in five pools of 10. All PHLs correctly identified the positive specimens. This study demonstrates the feasibility of using a pooling strategy to increase capacity and conserve resources during surge testing and periods of enhanced influenza surveillance when the prevalence is low.     

Duplex PCR system for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical specimens.

AIMS--To evaluate the use of a duplex polymerase chain reaction (PCR) assay for the simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical samples. METHODS--Genital swab specimens were obtained from both China (203 swabs) and Hong Kong (202 swabs). N gonorrhoeae and C trachomatis were detected in each specimen with a number of tests including enzyme immunoassays (IDEIA) and PCR assays using both single and double primer pairs. The primer pair for N gonorrhoeae was derived from the cppB gene on its cryptic plasmid and the PCR product was 390 base pairs long. For C trachomatis, the PCR product was 473 base pairs long, resulting from amplification of a sequence in the common 7.4 kilobase plasmid present in all serovars. For N gonorrhoeae, PCR results were also compared with those obtained by culture and Gram's smear of the discharges. RESULTS--For the 203 specimens collected in China, similar numbers of positive results (177) were obtained by both Gonozyme and duplex PCR for the detection of N gonorrhoeae. No discrepant results were found among the cultured specimens when Gonozyme and duplex PCR were compared. C trachomatis was detected in 47 specimens by duplex PCR, but was detected in only 28 by IDEIA. Of the 202 Hong Kong specimens, 46 were positive for N gonorrhoeae, detected by both Gonozyme and duplex PCR; 34 were positive for C trachomatis, 25 of which were detected by IDEIA and the remainder by duplex PCR. CONCLUSIONS--The duplex PCR assay is a satisfactory diagnostic tool for the simultaneous detection of N gonorrhoeae and C trachomatis in clinical swab samples. Further evaluation is suggested.     

Solid-phase C1q-directed bacterial capture followed by PCR for detection of Chlamydia trachomatis in clinical specimens.

An antigen capture system based on the binding of bacteria to solid-phase immobilized complement C1q followed by PCR for detection of Chlamydia trachomatis in clinical samples was developed and clinically evaluated. Comparison of C1q-directed antigen capture PCR with cell culture and direct PCR on 71 consecutive clinical specimens revealed an identical sensitivity. In this group, all 11 cell culture-positive samples were positive by direct PCR and C1q-directed antigen capture PCR. In addition, two samples found negative by cell culture were found positive by both direct PCR and C1q-directed antigen capture PCR. To further assess the sensitivity of C1q-directed antigen capture PCR, 20 clinical samples with one to five inclusions in cell culture and 20 clinical samples with 6 to 20 inclusions in cell culture were tested. Results obtained showed sensitivities of 95 and 90% for clinical samples with 6 to 20 and 1 to 5 inclusions in cell culture, respectively. Using C1q-coated solid phases, C1q-binding Chlamydia particles can be concentrated from large volumes with concomitant removal of inhibitors of PCR, allowing the use of large volumes of clinical samples for clinical testing. Since C1q has been shown to bind to a range of gram-negative bacteria, the newly developed technique has utility for a broad range of bacteria.     

Pooling of urine specimens for detection of asymptomatic Chlamydia trachomatis infections by PCR in a low-prevalence population: cost-saving strategy for epidemiological studies and screening programs

Pooling, in groups of five, of urine specimens from asymptomatically infected men in a population with 4% prevalence, as determined by case finding, is 100% sensitive and specific and results in a 60.5% reduction in the number of tests needed. Pooling of urine specimens in groups of 10 for the estimation of population-based prevalence is 96.1% sensitive and 100% specific and saves 90% of the test costs.     

PCR assessment of Chlamydia trachomatis infection of semen specimens processed for artificial insemination

In order to ascertain the microbiological quality of stored semen specimens processed for artificial insemination by a donor (AID), we developed a PCR assay targeting the chlamydial plasmid to detect Chlamydia trachomatis in semen. The lower limit of detection of this assay corresponded to 2.5 to 5 elementary bodies per microl of semen. A total of 669 cryopreserved ejaculates from 97 asymptomatic donors were tested for C. trachomatis infection. Twelve ejaculates, originating from four donors, were found to be positive, indicating a 4% prevalence of C. trachomatis infection among the donor population studied. Cross-contamination between the cryopreserved specimens in the storage container was studied by typing using sequence analysis of PCR-amplified omp1 genes of the strains. Two donors were infected with serovar E, one was infected with serovar F, and one was infected with serovar K. For two donors, the duration of C. trachomatis positivity could be assessed. One donor donated C. trachomatis-positive semen for at least 4 successive months, and the other did so for at least 16 months. The occurrence of C. trachomatis infection in cryopreserved donor semen indicates that ejaculates from donors not tested for a C. trachomatis infection just prior to donation should be tested for infection by a direct test such as the PCR described here. Direct testing of semen specimens will detect not only donors with an active infection but also C. trachomatis-infected ejaculates already stored and will thus improve the microbiological quality of AID, since discrepancies in the presence of C. trachomatis in urine and semen specimens have been reported.     

Replicate PCR testing and probit analysis for detection and quantitation of Chlamydia pneumoniae in clinical specimens

Nucleic acid amplification of clinical specimens with low target concentration has variable sensitivity. We examined whether testing multiple aliquots of extracted DNA increased the sensitivity and reproducibility of Chlamydia pneumoniae detection by PCR. Nested and non-nested C. pneumoniae PCR assays were compared using 10 replicates of 16 serial dilutions of C. pneumoniae ATCC VR-1310. The proportion positive versus the C. pneumoniae concentration was modeled by probit regression analysis. To validate the model, 10 replicates of 26 previously positive patient specimens of peripheral blood mononuclear cells (PBMC), sputum, or nasopharyngeal swabs (NPS) were tested. The proportion of replicates that were positive varied with the concentration of C. pneumoniae in the sample. At concentrations above 5 infection-forming units (IFU)/ml, both nested and non-nested PCR assay sensitivities were 90% or greater. The nested PCR was more sensitive (median detection, 0.35 versus 0.61 IFU/ml; relative median detection, 0.58; 95% confidence interval, 0.31 to 0.99; P = 0.04). In clinical specimens, replicate PCR detected 15 of 26 (nested) versus 1 of 26 (non-nested, P < 0.001). For PBMC specimens, testing 1, 3, or 5 replicates detected 3, 5, or 9 of 10 positive specimens, respectively. Median C. pneumoniae concentrations were estimated at 0.07 IFU/ml for PBMC and at <0.03 IFU/ml for NPS specimens. We conclude that performing 5 or 10 replicates considerably increased the sensitivity and reproducibility of C. pneumoniae PCR and enabled quantitation for clinical specimens. Due to sampling variability, PCR tests done without replication may miss a large proportion of positive specimens, particularly for specimens with small amounts of target C. pneumoniae DNA present.     

Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Genitourinary specimens.

We developed a multiplex PCR (M-PCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. M-PCR employed C. trachomatis-specific primers KL1-KL2 and N. gonorrhoeae-specific primers HO1-HO3 and produced products of 241 and 390 bp, respectively. PCR products were easily detected by agarose gel electrophoresis and confirmed by Southern hybridization using labelled oligonucleotide probes. M-PCR had a sensitivity of 10 fg of C. trachomatis and N. gonorrhoeae DNA (equivalent to 1 to 2 genome copies). M-PCR detected the presence of C. trachomatis and N. gonorrhoeae DNA in 15 male urethral and 12 female endocervical specimens, 3 of which were positive for C. trachomatis, 18 of which were positive for N. gonorrhoeae and 6 of which were positive for both organisms. M-PCR was evaluated further by testing 200 male first void urine (FVU) specimens, of which 18 were positive by C. trachomatis PCR and Chlamydiazyme and 4 were positive by C. trachomatis PCR but negative by Chlamydiazyme. All 22 FVU specimens were positive by a confirmatory PCR using a second plasmid target and were positive by M-PCR. Ten of 11 men with cultures that were positive for N. gonorrhoeae had FVU specimens that were positive by both N. gonorrhoeae PCR and M-PCR. Two other men with negative N. gonorrhoeae urethral cultures had FVU specimens that were positive by N. gonorrhoeae PCR, by two confirmatory N. gonorrhoeae PCR assays using 165 rRNA and cytosine methyltransferase primers, and by M-PCR. The sensitivity of M-PCR for detecting C. trachomatis was 100% (22 of 22 specimens), compared with 81.8% (18 of 22 specimens) for enzyme immunoassay. Sensitivity of M-PCR for N. gonorrhoeae was 92.3% (12 of 13 specimens) compared with 84.6% (11 of 13 specimens) for urethral culture. The specificity of M-PCR was 100% for both C. trachomatis (178 of 13 specimens) and N. gonorrhoeae (187 of 187 specimens). M-PCR testing of FVU specimens provided a sensitive and noninvasive method for detecting C. trachomatis and N. gonorrhoeae infection in men.     

实时荧光定量PCR检测沙眼衣原体的研究

目的采用TaqMan探针建立检测沙眼衣原体的实时荧光定量PCR（real-time PCR）方法。方法根据沙眼衣原体外膜蛋白A的基因（ompA）序列设计引物和探针,以克隆的ompA部分基因片段作DNA模板,建立实时荧光定量检测方法。结果建立的荧光定量PCR检测方法的最低检出限为5 copies/反应,检测线性范围100～107线性关系良好（r2=0.997）,比巢式PCR敏感100倍;且与鹦鹉热衣原体、淋球菌、解脲脲原体、大肠杆菌等病原菌DNA以及人基因组DNA均无交叉反应,表明该方法具有良好的特异性。以巢式PCR作参比,建立的荧光定量PCR法检测沙眼衣原体的阳性符合率为100.00%,阴性符合率为95.09%,总符合率为96.81%。结论建立的检测沙眼衣原体实时荧光定量PCR具有特异性强和敏感性高的特点,可快速检测样本中微量沙眼衣原体DNA,适用于对沙眼衣原体进行大规模筛选。     

Quality assessment of conjunctival specimens for detection of Chlamydia trachomatis by PCR in children with active trachoma

One component of control programmes to eliminate trachoma is the treatment of Chlamydia trachomatis infection. A diagnosis of trachoma is based on clinical grounds, but the signs of active trachoma do not always correlate with the presence of C. trachomatis. During a therapeutic trial, the level of C. trachomatis infection in children with active trachoma in Guinea and Pakistan was assessed using a qualitative commercially available PCR that targeted the C. trachomatis plasmid. The influence of the quality of specimens on the efficiency of the PCR was investigated using two quantitative real-time PCRs targeting the specific omp1 gene of C. trachomatis and human chromosomal DNA, respectively. C. trachomatis was detected in c. 23% of children (aged 1-10 years) who presented with clinically active trachoma. Controls showed that PCR-related problems did not influence this detection rate. For 14% of the positive samples, C. trachomatis was detected in only one eye, with a significantly lower mean load of bacteria. These results suggest that epidemiological and therapeutic surveys should be conducted by sampling and testing both eyes. Moreover, the high variability of the cell load observed in the conjunctival swabs suggests that the effectiveness of swabbing may be questionable.     

荧光定量PCR检测沙眼衣原体及临床应用

目的：用荧光定量聚合酶链反应（FQ－PCR）检测沙眼衣原体的含量,探讨FQ－PCR在衣原体性尿道炎诊断中的价值。方法：应用荧光探针标记引物的荧光定量聚合酶链反应对172例疑为沙眼衣原体感染患者标本进行检测,并与常规PCR（电泳－EB染色）进行比较。结果：FQ－PCR阳性率为19．8％,常规PCR法为20．9％,两法符合率为63．9％。女性宫颈分泌物标本FQ－PCR阳性为29．2％,常规PCR法为16．7％;男性分泌物FQ－PCR阳性率为16．9％,常规PCR法为13．8％。FQ－PCR特异性较常规PCR法高。结论;FQ－PCR在扩增中实时在线检测,并选择理想的标准曲线做出较精确的临值分析,具有较高的特异性和敏感性,对病原体的诊断有一定的临床意义。     

Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay.

To determine that susceptibility of AMPLICOR Chlamydia trachomatis PCR to inhibitory factors possibly present in cervical specimens, we obtained cervical specimens from 200 gynecology patients attending our outpatient clinic. The prevalence of C. trachomatis infection was 4.1%, as determined by cell culture. All AMPLICOR specimens were tested in one procedure as described by the manufacturer, and after the specimen was spiked with C. trachomatis, several other pretreatment protocols were used. Complete inhibition of the PCR was observed in 38 (19%) cervical specimens. Heat treatment at 95 degrees C, freeze-thawing, or 10-fold dilution of the samples reduced the initial inhibition to 9, 16, or 9%, respectively. A combination of heat treatment and 10-fold dilution reduced the inhibition to 4% of the samples. A second specimen type (swabs inoculated in 0.2 M sucrose phosphate buffer [2SP]) was also evaluated. A 10-fold dilution of the spiked 2SP specimen resulted in an inhibition rate of 6%, which was comparable to that obtained by centrifugation of the 2SP specimen prior to processing. Furthermore, it was shown that the inhibition was not correlated with blood contamination. Processing the specimens on the day of collection or the day after resulted in a higher inhibition rate than did delayed processing (27.6 versus 15.5%, respectively). An inverse correlation was found between the concentration of C. trachomatis added to the sample and the rate of inhibition observed. The inhibition was partly correlated with the pH of the cervical mucosa. Decreased inhibition was found at pH values of > or = 7.5. The effects of blood, pH, and delay in processing were all evaluated by using the AMPLICOR specimen. We conclude that the susceptibility of AMPLICOR C. trachomatis PCR to inhibiting factors in cervical specimens can be significantly reduced if the pretreatment procedure includes heat treatment or the use of 2SP transport medium. Also, a 10-fold dilution of the clinical specimen followed by heat treatment will largely prevent the inhibition of this PCR.     

Diagnosis of genital Chlamydia trachomatis infections in asymptomatic males by testing urine by PCR.

An enzyme-linked immunosorbent assay (EIA) (MikroTrak; Syva) was compared with PCR (Amplicor; Roche) for detection of Chlamydia trachomatis in first-void urine (FVU) from 184 men attending a skin and venereal disease clinic. The prevalence of C. trachomatis in the population studied was 18.5%. Discrepant results between Syva EIA and Roche PCR were retested by using major outer membrane protein primer-based PCR. After retesting, the sensitivity, the specificity, and the positive and negative predictive values for the Syva EIA were 85.3, 100, 100, and 77.5%, respectively, and those for the Roche PCR 100, 100, 100, and 100%, respectively. It was concluded that PCR provides a highly sensitive and specific noninvasive screening method for genital chlamydial infection in asymptomatic men.     

Detection of Chlamydia trachomatis in male and female urine specimens by using the amplified Chlamydia trachomatis test.

The amplified Chlamydia trachomatis test (AMP-CT; Gen-Probe), a new diagnostic test for the detection of Chlamydia trachomatis, was evaluated with urine specimens from 1,000 patients visiting the outpatient department for sexually transmitted diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands, by comparing the results to those of cell culture. From February 1996 to July 1996, urine samples for the AMP-CT test and urethral swabs for cell culture were collected from 544 men, while cervical swabs from 456 women were also taken for cell culture. Positive test results were obtained for 130 (13%) of the patients. AMP-CT test and cell culture results were discordant for 70 (7%) specimens. Analysis of the samples with discordant results was performed by an in-house PCR. After resolution of the discordant results, the sensitivity, specificity, and positive and negative predictive values of the AMP-CT test were 84.3, 98.8, 89.6, and 98%, respectively, for samples from females and 100, 99.2, 93.1, and 100%, respectively, for samples from males, while for cell culture these values were 72.5, 99.2, 92.5, and 98%, respectively, for samples from females and 57.4, 99.0, 86.1, and 95.4%, respectively, for samples from males. We conclude that the AMP-CT test is a fast and reliable test for the detection of C. trachomatis in urine specimens from females and, in particular, males.     

Effect of polybrene on isolation of Chlamydia trachomatis from clinical specimens.

Polybrene treatment of McCoy cells was used to increase the infectivity of Chlamydia trachomatis in vitro. The isolation rate for C. trachomatis in 500 specimens from males and females attending a sexually transmitted disease clinic and other clinics increased by 6.8% with the Polybrene treatment. The data also suggested that this treatment facilitated the diagnosis of low grade or latent infections.     

Direct detection of Chlamydia trachomatis in clinical specimens by a dot-immunobinding technique using monoclonal antibody

A dot-immunobinding technique (DIBT) has been developed to permit detection of Chlamydia trachomatis organisms or antigen in clinical specimens. This method was evaluated for the rapid diagnosis of chlamydia infections using monoclonal antibody. The membrane antigen extracted from reticulate bodies was used for the production of species-specific monoclonal antibodies by an in vitro immunization procedure. The DIBT involved spotting clinical specimen directly onto a nitrocellulose membrane followed by reaction with monoclonal antibody and a biotin-avidin-peroxidase indicator system. Specimens were tested for the presence of chlamydia by the cell culture method. Of these, 361 positives and 317 negatives were selected for detection of antigen using the DIBT method. Of 678 clinical specimens that were evaluated by DIBT, 654 (96.7%) gave identical results to the cell culture method, whereas 24 (3.5%) were positive by the DIBT but culture negative. The overall sensitivity was 100% with a specificity of 92.4%. The test could detect as little as 75 pg of chlamydial antigen.     

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).     

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by ligase chain reaction-based assays with clinical specimens from various sites: implications for diagnostic testing and screening.

Ligase chain reaction (LCR)-based tests for the diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae infections in men and women attending a sexually transmitted disease clinic were evaluated. LCR testing of urethral swab and urine specimens from men and cervical swab and urine specimens from women was compared with culture of male urethral swabs and female cervical and urethral swabs, respectively. An expanded "gold standard" was defined as a positive culture or at least one specimen confirmed to be positive by LCR testing. The prevalence of C. trachomatis infection as detected by cell culture was 7.0% among 614 men and 5.0% among 602 women. By LCR, these values increased to 11.4 and 9.9% with urethral swabs and urine, respectively, for men and 9.6 and 9.1% with cervical swabs and urine, respectively, for women. Relative to the expanded gold standard, the sensitivity of cell culture with male urethral swabs or female cervical swabs was 57.3 and 45.5%, respectively, compared with corresponding values of 93.3 and 87.9% for LCR. The sensitivity of LCR with urine specimens was 77.3 and 78.8% for men and women, respectively. The prevalence of N. gonorrhoeae infection as detected by culture was 5.9% among 220 men and 2.9% among 383 women. The corresponding values were 8.2 and 5.5%, respectively, by LCR testing of swabs. Prevalence values by LCR testing of urine were 7.3% for men and 2.9% for women. The sensitivity of culture was 72.2% for men and 50.0% for women. The sensitivities of LCR were 100% with male urethral swabs, 95.4% with female cervical swabs, 88.9% with male urine, and 50.0% with female urine. These results indicate that the LCR-based assays represent a major improvement in C. trachomatis and N. gonorrhoeae diagnostics. The sensitivity of testing of urethral or cervical swabs by LCR was markedly greater than that by culture. The sensitivity of testing female or male urine specimens was equal to or greater than that of culturing cervical or urethral specimens. LCR testing of urine specimens may prove useful for screening for C. trachomatis.     

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay.

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.