Duplex PCR system for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical specimens.
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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 Gram9s 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.Keywords:
Neisseria gonorrhoeae
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A sensitive and specific system for detection of amplified Chlamydia trachomatis DNA from cervical specimens by fluorometric quantitation in an enzyme immunoassay (EIA) format (polymerase chain reaction [PCR]-EIA) is described. The primers selected for PCR-amplified DNA were from the 15 serovars of C. trachomatis and two strains of Chlamydia pneumoniae (TWAR). One strain of Chlamydia psittaci (Borg) was not amplified. One hundred four previously cultured cervical specimens were evaluated. Forty-six culture-positive specimens containing from 1+ to 4+ inclusion bodies were all positive by PCR-EIA. Of 58 culture-negative specimens, 2 were repeatedly positive and were nonreactive with control probes. This assay system represents a sensitive and specific combination of technologies for the quantitative detection of C. trachomatis DNA directly from a body fluid.
Chlamydia psittaci
Chlamydophila pneumoniae
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Chlamydophila pneumoniae
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The diagnostic value of the polymerase chain reaction (PCR) for detection of Chlamydia trachomatis in comparison with that of the culture technique was established in a follow-up study of 32 patients (81 samples) who were treated for a C. trachomatis infection. The PCR was performed with two different sets of primers, a genus-specific primer set directed against the rRNA genes and a C. trachomatis-specific set directed against the common endogenous plasmid. After treatment with doxycycline, all patients became culture negative after 1 week. Results for the detection of C. trachomatis by the PCR were in complete agreement with the results by the culture method of detection, except for one culture-negative sample, which was found to be positive by the PCR. The results indicated that 1 week after treatment, no residual chlamydial DNA was found in the samples. Therefore, the PCR can be used for monitoring infections by chlamydiae.
Chlamydiae
Primer (cosmetics)
Chlamydia psittaci
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From January 1, 2014, to May 31, 2015, 452 individuals received extragenital nucleic acid amplification-based Neisseria gonorrhoeae and Chlamydia trachomatis testing through public health venues. Seventy-four individuals (16%) tested positive for Neisseria gonorrhoeae and/or Chlamydia trachomatis at an extragenital site and 40 (54%) would not have been effectively diagnosed and treated in the absence of extragenital testing.
Neisseria gonorrhoeae
Chlamydial infection
Gonococcal infection
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A polymerase chain reaction (PCR) was developed for Chlamydia trachomatis in which a 380 base pair DNA fragment was amplified. Amplification occurred with the DNA from the 15 serovars but not with that from other Chlamydia spp or with DNA from a variety of other organisms. Chlamydial DNA (10(-16) g) could be detected and the PCR seemed to be able to detect single organisms. Urethral swabs were obtained from 37 men with acute non-gonococcal urethritis (NGU), 18 (49%) of whom were positive for C trachomatis by MicroTrak. As a result of clinical re-examinations 65 urethral swabs were available for analysis by the PCR. In comparison with MicroTrak, PCR had a sensitivity of 95%, a specificity of 94%, a positive predictive value of 86% and a negative predictive value of 98%. The PCR was apparently less sensitive (82%) in tests on urine samples. Overall, however, values of sensitivity and specificity of the PCR compared favourably with those of MicroTrak. The PCR for C trachomatis is likely to be a valuable technique for research, but problems of DNA contamination suggest that it should not be recommended for routine diagnosis.
Non-gonococcal urethritis
Neisseria gonorrhoeae
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A polymerase chain reaction (PCR) for the detection of Chlamydia trachomatis was developed and evaluated. Two primer-probe sets were designed; one detected a specific sequence of the plasmid, and the other detected the gene encoding the major outer membrane protein. Both sets reacted species specifically and amplified sequences from all human serovars. A simple protocol was used for sample pretreatment. The PCR was optimized by addition of tetramethylammonium chloride and bovine serum albumin. The results of the PCR with the plasmid primer-probe set were compared with those of culture and the Chlamydiazyme and Gen-Probe PACE 2 tests for urogenital specimens from 220 patients. The rates of prevalence of infection with C. trachomatis were 22.7, 16.4, 15.0, and 14.5%, respectively. The sensitivities of the Chlamydiazyme and Gen-Probe PACE 2 assays compared with culture were 66.7 and 61.1%, respectively, and their sensitivities compared with PCR were 60.0 and 60.0%, respectively. The sensitivity of culture compared with PCR was 70.0%. Forty-eight of the 50 specimens positive by PCR with the plasmid primer-probe set could be confirmed by PCR with the major outer membrane protein primer-probe set or culture. It is concluded that the PCR is the most sensitive technique for laboratory detection of C. trachomatis.
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The prevalence of Chlamydia infection in 95 sex partners was determined by both polymerase chain reaction (PCR) and cell culture. Thirty-three (18%) of 186 specimens were positive by culture and 61 (33%) were positive by PCR-EIA. PCR was positive in 75% (21/28) of male partners of PCR-positive women compared with culture, which was positive in only 45% (9/19) of male partners of culture-positive women (P = .053). For female partners of infected men, the difference was less marked. PCR was positive in 58% (21/36) of female partners of infected men versus culture, which was positive in 56% (15/36) offemale partners of culture-positive men. The correlation of PCR between partners and sequence analysis of Chlamydia DNA showing the same sequence from sex partners of 7 couples support the accuracy of the assay. These data suggest that PCR is more sensitive than culture for detection of Chlamydia trachomatis , particularly for male partners of infected women.
Chlamydia trachomatis infection
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A DNA probe assay (PACE; Gen-Probe, San Diego, Calif.) was compared with a culture reference method for the detection of Chlamydia trachomatis. Using stock isolates of each of the 15 serovars (A to K, Ba, L1, L2, and L3) of C. trachomatis, the lower limit of sensitivity for the DNA probe ranged between 1,086 inclusion-forming units (IFU) for serovar E (Bour) to 2,930 IFU for serovar L1 (440), with the only exception being serovar C (TW-3), with which 99 IFU was detected. There was no cross-reactivity with Chlamydia psittaci (Texas turkey) and Chlamydia pneumoniae (TWAR-183). Bacterial and fungal isolates representing 14 species of normal vaginal flora as well as Neisseria gonorrhoeae gave negative results with the DNA probe when tested at a level of 1.5 X 10(7) CFU/ml. In addition, the DNA probe, a direct fluorescent-antibody stain (DFA) (MicroTrak; Syva Corp., Palo Alto, Calif.), and an enzyme-linked immunosorbent assay (Chlamydiazyme; Abbott Laboratories, North Chicago, Ill.) were compared with culture for the detection of C. trachomatis, using 196 clinical cervical samples. Of the 196 samples, 20 (10%) were culture positive. Of the 176 culture-negative samples, 1 was not evaluated by DNA probe and 4, because of a lack of cellular material, were not evaluated by DFA. The sensitivities of the DNA probe, DFA, and enzyme-linked immunosorbent assay were 60, 75, and 85%, respectively, and specificities were 95, 99, and 97%, respectively. Of the false-positive direct results, there was only one specimen with which more than one direct method was positive, and with this specimen all three direct methods were positive. The majority of false-negative results by the direct methods were from specimens which by the culture method gave <100 IFU per culture.
Neisseria gonorrhoeae
Chlamydia psittaci
Lymphogranuloma venereum
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To investigate the value of polymerase chain reaction (PCR) for follow-up patients infected by Chlamydia trachomatis.Follow-up specimens were collected from 30 patients. Chlamydia trachomatis positive were detected by PCR and direct fluorescence assay test (DFA) in the 30 patients before therapy. 15 patients were treated with minocycline (100 mg twice daily) for 10 days, and 15 patients were treated with 1.0 g of azithromycine as a single oral dose.After 1-2 weeks of antimicrobial therapy, all patients had negative DFA for Chlamydia trachomatis, but 9 had positive Chlamydia trachomatis DNA as detected by PCR.The 9 specimens were not confirmed to livae viable organisms of Chlamydia trachomatis. The debris of nonviable Chlamydia trachomatis DNA was excluded from urinogenital tract at about one month.
Minocycline
Chlamydia trachomatis infection
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