Aims:To evaluate the sensitivity of the Roche Cobas, Roche Amplicor plate kit, ligase chain reaction (LCR), and an in house polymerase chain reaction (PCR) by titration of purified elementary bodies (EB) and also to test 245 urethral and endocervical specimens for Chlamydia trachomatis by the four assays as well as conventional culture. Study design: EB titrations were run in duplicate in each commercial assay and six times in the in house PCR. Clinical samples were aliquoted and tested by each assay and were considered positive if C trachomatis was detected by two or more separate tests or if the sample was either culture or immunofluorescence positive. Major outer membrane protein (MOMP) specific primers were used as a confirmatory assay for the in house PCR. Results: The in house PCR, Roche Cobas Amplicor, LCR, and Amplicor plate kit gave detection limits of approximately 1, 1-2, 2, and 2-4 EBs respectively. By the criteria described above for definition of a C trachomatis positive result in clinical samples we identified 23 true positives among the 245 clinical specimens. The in house PCR detected all 23 giving a sensitivity of 100% and a specificity of 98%. The Roche Cobas Amplicor, Roche Amplicor plate kit, and LCR detected 21, 19, and 19 of these respectively giving sensitivities of 87.5%, 82%, and 82% respectively and specificities of 99.5%, 99%, and 100% respectively. The culture gave a sensitivity of 78% and specificity of 100%. Conclusion: All four amplification assays had a greater sensitivity than the culture used routinely in this laboratory. The in house plasmid PCR had the greatest sensitivity and when combined with confirmation by immunofluorescence detected the greatest number of positives. This increased sensitivity is likely to have been achieved by the use of a DNA purification step and of nested primers in the amplification stage and their combined use in routine diagnostic assays for chlamydia might increase the frequency of C trachomatis detections. However, this assay is much less user friendly than the two semiautomated commercial assays investigated in this study. (Sex Transm Inf 1998;74:289-293)
Aims-To evaluate a commercial polymerase chain reaction (PCR) kit for the detection of Chlamydia trachomatis. Methods-Two hundred and fifty seven genital specimens, which had been submitted in 2SP medium for chlamydial isolation and subsequently stored at -700C, were retrospectively examined by a commercial PCR kit which detects chlamydial plasmid DNA. Culture negative, PCR positive specimens were examined by immunofluorescence and an in-house major outer membrane protein (MOMP)-PCR. Results-All 49 specimens which were culture positive were also PCR positive. Another 14 specimens were also PCR positive. After resolution of these results by immunofluorescence and a PCR assay for MOMP the sensitivity for PCR was 98-4% and that of culture 79%. The specificities were 99 5% and 100%, respectively. Conclusions-This kit, which is highly sensitive and specific, is straightforward to use and has a built-in safeguard against cross contamination. The role of this test in the examination of routine genital specimens from patients with uncomplicated chlamydial infection is questionable due to its expense. It may have a place in the investigation of trachoma or infertility, however, where it has been shown that DNA can be detected when culture is unsuccessful.
Our aim was to determine the number of chlamydial infections detected by Cobas Amplicor CT/NG multiplex polymerase chain reaction (PCR) testing of genital and first-voided urine (FVU) specimens compared with routine culture. Two hundred and eighty-six female and 276 male patients attending the Genito-Urinary Medicine (GUM) Unit at Edinburgh Royal Infirmary were included in the study. Case notes were analysed retrospectively to determine how many infected patients would not have been treated had diagnosis relied on routine culture. Polymerase chain reaction on FVU from women had a sensitivity, specificity, positive and negative predictive value of 91%, 100%, 100% and 99.1%: corresponding values for genital PCR and culture were 96%, 100%, 100%, 99.6% and 65%, 100%, 100%, 96.7% respectively. PCR on FVU from men had a sensitivity, specificity, positive and negative predictive value of 96%, 99.1%, 92.6% and 99.5%: corresponding values for genital PCR and culture were 89%, 99.5%, 95.8%, 98.6% and 48%, 100%, 100%, 94.3% respectively. In both men and women genital PCR and urine PCR were significantly more sensitive than culture. PCR almost doubled the number of patients detected by culture (49 vs 27). Of the 22 cases detected only by PCR 8 would not have received treatment on the basis of clinic treatment policy.
Tetracycline resistant Neisseria gonorrhoeae (TRNG) contain a 25.2 MDa TetM plasmid encoding a 68 KDa cytoplasmic protein which confers high-level tetracycline resistance. The aim of this study was to subtype all TRNG isolated in Scotland between 1992 and 1998. Subtyping was performed by a polymerase chain reaction (PCR) assay which characterizes the TetM plasmid as either the Dutch variant (443 base pair product) or the American variant (777 base pair product). Of the 78 TRNG isolates, 35 were the American variant and 43 were the Dutch variant. TRNG were distributed amongst 30 serovar/auxotype classes, the most common being 1A6/NR (11.5%), 1A6/P (14.1%) and 1B4/NR (14.1%). The country where infection was acquired was known for 36 of the 46 TRNG strains isolated between 1996 and 1998. All infections acquired in Asia and South America were the Dutch variant whereas all infections acquired in Africa were the American variant. A penicillinase plasmid was present in 66% (23/35) of the American variant TRNG compared with 51% (22/43) of the Dutch variant: the 3.2 MDa penicillinase plasmid was found in 87% of the American variant TRNG whereas the 4.4 MDa penicillinase plasmid was found in 68% of the Dutch variant TRNG. We conclude that subtyping of TRNG by PCR is a useful tool in studying the epidemiology of gonococcal infection due to plasmid-mediated resistant isolates.
We examined the hypothesis that the sac-4 gene of Neisseria gonorrhoeae varies with gonococcal subtype and that this could account for an earlier report that sac-4 increased the likelihood of co-infection with Chlamydia trachomatis. A polymerase chain reaction (PCR) assay was used to determine the prevalence of sac-4 in 435 gonococcal isolates. The prevalence of sac-4 was analysed in relation to chlamydial co-infection, serovar, auxotype, gender and sexual orientation. Although the prevalence of sac-4 was higher in isolates from patients with chlamydial co-infection (55%) than in those without chlamydial co-infection (42%) the difference was not significant (P<0.05). Statistically significant differences in association with sac-4 were, however, shown between various serovars and auxotypes. Dual classification based on auxotype/serovar (A/S) classes showed highly significant differences in sac-4 prevalence between groups: 95% in NR/1B18 and 8% in P/1B2 (P<0.001). Sac-4 was also significantly less common (P<0.05) in isolates from homosexual men (35%) than from heterosexual men (49%) or women (49.5%). Sac-4 appears to have an epidemiological association with gonococcal auxotype and serovar rather than a direct association with chlamydial co-infection.
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