A multiplexed real-time PCR assay for rapid detection of Chlamydia trachomatis and identification of serovar L-2, the major cause of Lymphogranuloma venereum in New York

Lymphogranuloma venereum (LGV) is caused by a rare form of Chlamydia trachomatis that is difficult to diagnose, since culture is not readily available, and since other methods are not reliable or lack sensitivity. We report here a rapid, sensitive, and specific real-time multiplex polymerase chain reaction (PCR) assay capable of detecting C. trachomatis and identifying serovar L-2 in the same reaction, directly from rectal swabs. The analytical sensitivity of the assay was 25 genome copies for C. trachomatis, and 50 genome copies for L-2. The analytical specificity was 100%, as demonstrated with a diverse range of C. trachomatis serovars and other site-specific bacterial pathogens. With the use of a rapid DNA extraction method, a blinded validation of spiked rectal swabs correctly identified 30 samples containing C. trachomatis cells, L-2 DNA, or negative samples. The multiplexed PCR assay also identified serovar L-2 in 13 of 70 rectal swab samples taken from symptomatic patients. Twelve additional samples were positive for C. trachomatis only, and omp1 sequencing determined these samples as either serovar D, E, G, J, or K. This assay represents the first real-time PCR method capable of detecting C. trachomatis DNA, and of simultaneously identifying C. trachomatis infection as serovar L-2.     

Diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae. Genitourinary infections in males by the Amplicor PCR assay of urine

The Amplicor CT/NG polymerase chain reaction (PCR) test on urine specimens from males was prospectively evaluated against established specimens and laboratory methods for diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae genitourinary infections, in patients from a remote region of Western Australia. Seventy-three males who were tested for both C. trachomatis and N. gonorrhoeae by both conventional methodology and Amplicor PCR on urine were enrolled in the study. Established testing comprised enzyme immunoassay/immunofluorescence antigen testing (EIA/IF) for C. trachomatis and microscopy and/or culture for N. gonorrhoeae on urethral swabs. Positive test results were confirmed using a set of criteria that included supplemental PCR testing and clinical history. Overall, 13.7% of patients were resolved as positive for C. trachomatis and 52.1% as positive for N. gonorrhoeae. The sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens for C. trachomatis were 80.0% (8/10) and 95.2% (60/63), compared with 60.0% (6/10) and 100.0% (63/63) for EIA/IF on urethral swabs. For N. gonorrhoeae, the sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens were both 100% (38/38 and 35/35, respectively) compared with 86.8% (33/38) and 100% (35/35) for microscopy and/or culture on urethral swabs. The results of this study indicate that the Amplicor CT/NG multiplex PCR test for C. trachomatis and N. gonorrhoeae performed on urine in males provides a highly sensitive, specific, and robust method for the diagnosis of both C. trachomatis and N. gonorrhoeae, for the early detection of both symptomatic and asymptomatic infected individuals.     

Urine-based testing for Chlamydia trachomatis using polymerase chain reaction, leucocyte esterase and urethral and cervical smears

The performance of Roche polymerase chain reaction (PCR) Amplicor to detect Chlamydia trachomatis in first-voided urine specimens from 422 males and 456 females attending two clinics for sexually transmitted infections was evaluated in comparison with cultures of urethral and cervical specimens. At the same time, the ability of leucocyte esterase (LE) in first-voided urine and the presence of leucocytes in urethral and cervical smears to identify C. trachomatis-infected individuals based on PCR and culture was determined. The prevalence of C. trachomatis infection was 10.9% in men and 7.7% in women. Sensitivity, specificity, positive predictive value and negative predictive value of Amplicor was 93.5%, 99.7%, 97.7% and 99.2% in males and 91.4%, 99.5%, 94.1% and 99.3% in females. All Chlamydia-infected men were identified by means of a combination of urethritis (4 leucocytes in the urethral smear) and/or a positive LE test in urine, although the specificity was only 42.2%. In women, the combination of urethritis and/or cervicitis and/or a positive LE test identified 85.7% of Chlamydia-infected patients with a specificity of 38.2%. It is concluded that a combination of urethral and/or cervical smears and LE testing of urine can be used as a screening test to select patients, especially males, for specific C. trachomatis testing.     

Use of the polymerase chain reaction for the detection of Chlamydia trachomatis from endocervical and urine specimens in an asymptomatic low-prevalence population of women

The Amplicor Chlamydia trachomatis test is a polymerase chain reaction (PCR)-based methodology used for the detection of a cryptic plasmid found in C. trachomatis. It was evaluated in comparison with cell culture and the Microtrak II Chlamydia enzyme immunoassay (EIA) for the detection of C. trachomatis in urogenital specimens from women. Endocervical swabs were collected from 993 women attending the women's unit at the Mount Sinai Hospital in Toronto. In addition, concomitant first void urine specimens were collected from 394 of these women for PCR testing only. As compared with culture of the endocervical specimens, PCR and EIA had a sensitivity, specificity, positive predictive value and negative predictive value of 84.6%, 99.2%, 57.9%, and 99.8% and 61.5%, 99.7%, 72.7%, and 99.5%, respectively. As compared with the secondary gold standard of a positive culture and/or a positive PCR using a primer to the major outer membrane protein the sensitivity, specificity, positive, and negative predictive values for culture were 72.2%, 100%, 100%, and 99.5%, respectively. For the Amplicor PCR and EIA the results were 88.9%, 99.7%, 84.2%, and 99.9% and 61.1%, 99.9%, 91.7%, and 99.6%, respectively. When the urine PCR was compared with the same standard, the test had a sensitivity of 91.7% and a specificity of 99.5%. Based on this study the Amplicor C. trachomatis test was found to be sensitive and specific for the detection of C. trachomatis in both endocervical and urine specimens.     

Low Prevalence of Chlamydia trachomatis by Urinary Ligase Chain Reaction in Women Patients in the Emergency Department

Recent studies suggest that women with acute urethral syndrome or abdominal pain, presenting to emergency departments (EDs), have a high prevalence of Chlamydia trachomatis. OBJECTIVES: To estimate the prevalence of C. trachomatis in women presenting to an ED and to see whether those with dysuria or abdominal pain have a higher prevalence of C. trachomatis. METHODS: The authors conducted a prospective cross-sectional study of C. trachomatis in the urine of women aged 18 to 50 years who had a urinalysis performed at a university/county ED from February through May 1998. Urine specimens were labeled for the presence of symptoms and analyzed for C. trachomatis by ligase chain reaction (LCR). Polymerase chain reaction (PCR) testing of cervical swabs for C. trachomatis was done for usual clinical indications. Difference in proportions of positive LCR tests among patients was tested with Fisher's exact test. Agreement between PCR and LCR was measured using Cohen's kappa statistic. RESULTS: Of 397 women whose urine was tested, 280 had symptoms of dysuria, abdominal pain, or both, and 117 had no symptoms. The overall prevalence of C. trachomatis by LCR was 3.8% (95% CI = 2.1% to 6.2%); and the combined PCR-LCR prevalence was 4.3% (95% CI = 2.5% to 6.8%). The presence of symptoms was not associated with a positive LCR test for C. trachomatis (p = 0.26, power = 0.8, alpha = 0.05, difference 3% vs. 12%). In the 172 patients who had both a PCR cervical swab and urine LCR, agreement was excellent (kappa = 0.67, 95% CI = 0.45 to 0.90). CONCLUSIONS: This ED had a surprisingly low prevalence of C. trachomatis. Women with symptoms were not more likely to test positive than those without.     

Ultra-rapid detection of Chlamydia trachomatis by real-time PCR in the LightCycler using SYBR green technology or 5'-nuclease probes

Chlamydia trachomatis infections are among the most common sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid based detection method. Several commercial PCR test systems are available (e.g. CobasAmplicor, Roche, Mannheim, Germany) but they require post-amplification detection by hybridization resulting in extended work-up time and possible cross-contamination. The objective of our study was to develop a routine diagnostic method for the sensitive, specific and rapid detection of C. trachomatis. The obvious choice is real-time PCR without any post-amplification procedures. The dye SYBR Green I (intercalating in dsDNA) provides a simple and fast real-time PCR in the LightCycler. Specific primer design combined with melting curve analysis allows a reliable and sensitive identification of C. trachomatis. In addition, a new commercial real-time PCR system (RealArt C. trachomatis LC PCR Reagents, artus, Hamburg, Germany) was evaluated, that combines sequence-specific primers and fluorescence-labelled (FRET) 5'-nuclease probes. An internal control integrated in this system detects false negative results and erroneous PCR conditions. All results were compared with the corresponding data from an analysis using the CobasAmplicor system (Roche). (Clin     

Serological evidence of Mycoplasma pneumoniae infection in acute exacerbation of COPD

It is a well-known fact that tubal stenosis and/or peritubal adhesion are often associated with Chlamydia trachomatis infection. Although tubal pregnancy may be attributed to this infection, there are only extremely rare cases in which the presence of C. trachomatis has been confirmed by immumo-histochemical examination on tissues isolated from patients with tubal pregnancy. We thus tried to confirm the presence of C. trachomatis infection by detecting DNA of the organism in tissues surgically isolated from patients with tubal pregnancy.Two detection methods, a ligase chain reaction (LCR) method and an immuno-histochemical staining which detects an antigen of C. trachomatis, were compared using paraffin-embedded tissue samples which were surgically isolated from patients with tubal pregnancy or hydrosalpinx. The LCR method was capable of detecting DNA of C. trachomatis in tissue samples in which the C. trachomatis-specific antigen could not be detected using immuno-histochemical staining. This was due to the fact that immuno-histochemical staining methods are applicable to the analysis of sequences the length of which range from 200 to 400 bp (base pairs), while the LCR method used here allows the analysis of sequences as small as 48 bp. This fact makes the LCR method a very convenient tool, as compared with immuno-histochemical methods, for analysis of the paraffin embedded tissue samples where by effects of formalin--used for fixation for pathologic diagnosis--the risk of fragmenting the DNA samples is relatively high. Presence of C. trachomatis DNA as detected by LCR method in surgically isolated samples from patients with tubal pregnancy supports the involvement of Chlamydia trachomatis infection in the occurrence of tubal pregnancy. Accordingly the LCR method is capable of detecting DNA of C. trachomatis in paraffin-embedded samples of tubal tissue in which presence of C. trachomatis could not be confirmed by an immuno-histochemical staining method.     

Detection of Chlamydia trachomatis by use of polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. One primer set was used, from the published sequence of the common C. trachomatis plasmid. Detection of amplified sequences was carried out by agarose gel electrophoresis. Analysis of 106 clinical samples tested by cell culture and PCR showed a sensitivity of 100% when PCR was compared with cell culture.     

VIDAS CHL检测泌尿生殖道沙眼衣原体感染

目的 探讨VIDAS CHL法用于性病患者尿道拭子标本以及男性首段尿（FCU）标本沙眼衣原体（CT)检测的可行性。方法 使用组织培养法（TC）、VIDAS CHL检测法和聚合酶链反应（PCR）平行检测男性和女性拭子标本中CT，以组织培养为金标准，对VIDAS CHL和PCR进行评价。结果 232例男性标本TC阳性49例；VIDAS CHL和PCR法用于男性尿道拭子标本的敏感性分别为95.9%、93.9%，特异性分别为95.6%、94．5％，差异无显著性；VIDAS CHL法用于男性FCU的敏感性和特异性分别为85．7％和96．7％，与拭子标本作自身配对比较，检测结果无差异。151例女性拭子标本，TC法阳性23例，VIDAS CHL和PCR法的敏感性分别为100％和95．7％，特异性分别为96.1%、93.8%，差异无显著性。结论 VIDAS CHL法用于性病患者男性和女性拭子标本的CT检测，具有很高的敏感性和特异性，用于男性患者FCU的CT检测也是可行的；对于CT阳性率较高的性病人群，VIDAS CHL可以不做阻抑证实试验。     

Application of an oligonucleotide array assay for rapid detecting and genotyping of Chlamydia trachomatis from urogenital specimens

An oligonucleotide array technology was established for rapidly detecting and genotyping Chlamydia trachomatis in urogenital infections. The VS1-VS2 region of the omp1 gene was used to design oligonucleotide probes. Eleven serovar-specific probes to serovars A, B, C, D, E, F, G, H, I, J, and K, and 3 group-specific probes to group B (B, Ba, D, E, L1, and L2), group C (A, C, H, I, J, K, and L3), and an intermediate group (F and G) were synthesized and spotted onto the nylon membrane. Two pairs of universal primers were designed for the nested polymerase chain reaction (PCR) amplification of the VS1-VS2 gene. Digoxigenin-labeled amplicons of the VS1-VS2 gene of C. trachomatis were hybridized to the membrane array. Hybridization signals were read by the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate color development. The assay developed was tested with reference strains of C. trachomatis serovars and clinical samples. The sensitivity was evaluated for 57 samples previously found to be positive for C. trachomatis by using plasmid PCR, and 98.2% (56/57) concordance was obtained. Fourteen oligonucleotide probes were optimized by trying different reaction conditions, showing specific hybridization with the corresponding reference strains, but no cross-reactions with other urogenital microorganisms. Using this procedure, a total of 59 strains were detected from 56 chlamydial samples. Eight genotypes were found, and type D, E, F, and H were the most frequently observed types (77.9%). Three cases (5.4%) had multiple infections with serovars: 1.D/E, 2.D/F, and 3.F/K. To validate the reference strains and confirm the genotype identity as determined by the oligonucleotide array technology, we sequenced all reference strains and 10 selected specimens across variable sequence VS1 and VS2. No discrepancies were found between the array typing and the genotype identity confirmed by nucleotide sequencing of the PCR product. The findings from this study indicated that the oligonucleotide array is a simple, fast, and specific assay for directly detecting and genotyping C. trachomatis from clinical samples.     

A high-resolution melting analysis for genotyping urogenital Chlamydia trachomatis

We have developed a high-resolution melting analysis (HRMA) for the genotyping of Chlamydia trachomatis and applied it specifically to the 11 sexually transmitted infection-related genotypes: D through K and L1 through L3. The variable segment 2 (VS2) was selected as the target for HRMA genotype identification. Eleven C. trachomatis genotypes were amplified by a nested real-time polymerase chain reaction (PCR) in the presence of the LCGreen saturating dye and showed no cross-reaction with 10 pathogenic bacteria or commensals from urogenital tract. The detection limit of HRMA method was 100 elementary bodies (EB)/mL. All of the 11 genotypes can be distinguished from each other by following an HRMA workflow. Genotype F, G, H, I, J, K, L2, and L3 could be directly identified from each other, whereas D, E, and L1 could be distinguished from each other by a second analysis with fewer curves or by heteroduplex formation with a known reference strain. In the validation panel of 36 C. trachomatis-positive urogenital samples genotyped by VS1-VS2 sequencing, nested real-time VS2 PCR followed by HRMA was able to discriminate between all samples correctly. This assay requires no fluorescence-labeled probes or separate post-PCR analysis and provides a simple and rapid approach for genotyping the C. trachomatis strains that are the most commonly sexually transmitted.     

Multiplex PCR: rapid DNA cycling in a conventional thermal cycler

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction. In the present study we investigated the limits to which the duration of multiplex PCR steps can be shortened using the thermal cycler Gene Amp PCR system 9600 (Perkin Elmer, Oak Brook, IL). The present multiplex PCR assay simultaneously detects five different herpes viruses (HSV-1, HSV-2, VZV, CMV, and EBV) and assesses sample suitability in a single amplification round of 40 cycles. It appears that when six target sequences are simultaneously amplified in multiplex PCR, extension time is a critical parameter. Using a PCR protocol of 0 sec at 95 degrees C, 0 sec at 60 degrees C, and 0 sec at 74 degrees C with Platinum Taq DNA polymerase (Life Technologies, Gaithersburg, MD), we were able to reduce the total cycling time of the multiplex PCR assay to as little as 55 min, without affecting the yield of PCR products or the specificity of the assay. It may be necessary to optimize each specific apparatus and template, but any such optimization would be trivial.     

微板核酸杂交-酶联显色检测性病沙眼衣原体DNA

目的 建立微板核酸杂交—ELISA方法，检测性病沙眼衣原体。方法 采用PCR、核酸杂交和ELISA三大生物技术相结合，建立微板核酸杂交ELISA法，并用此法分别同McCoy细胞培养和免疫学方法检测307份性传播疾病标本进行比较。结果 微板核酸杂交—ELISA法检测的阳性率为40．39％(122／307)，高于细胞培养的9.77％(30／307)和免疫诊断的12．05％(37／307)。结论 微板核酸杂交—ELISA技术检测性病沙眼衣原体灵敏度高、特异性好、简便快速。因此，该方法具有很强的实用价值和发展前景。     

糖原试验、聚合酶链反应和细胞培养对沙眼衣原体感染的诊断价值

目的 探讨糖原试验、聚合酶链反应（PCR）和细胞培养法检测泌尿生殖道沙眼衣原体感染的诊断价值。方法 用三种方法分别对106例女性门诊患者宫颈分泌物标本进行平行检测。结果 以细胞培养作参比。糖原试验的敏感性为80.0%，特异性为95.8%；PCR敏感性为90.0%，特异性为97.7%。结论 糖原试验对泌尿生殖道沙眼衣原体感染有一定诊断价值。     

性病病原体多重聚合酶链反应检测方法的建立和验证

目的建立同时检测淋球菌、沙眼衣原体、解脲支原体和单纯疱疹病毒-2致病微生物的多重聚合酶链反应（PCR）方法,用于四种性病的诊断和治疗监测。方法根据淋球菌外膜孔蛋白基因、沙眼衣原体基因、解脲支原体尿素酶基因和单纯疱疹病毒-2的DNA聚合酶基因序列,各设计一对特异性引物,优化反应体系和条件进行多重PCR反应。结果4对引物分别扩增出327、167、426、391bp的目的条带,并且特异性强,与其他非目的病原体基因不发生反应。结论本研究建立的多重PCR方法为相关病原体的快速检测提供方法。     

A PCR-microplate capture hybridization method to detectListeria monocytogenesin blood

In order to improve the diagnosis ofListeria monocytogenesinfection, we have developed a polymerase chain reaction (PCR)-based assay combined with microplate capture hybridization technique. The system is based on selective amplification ofL. monocytogeneswith two specific primers based on theiapgene. The amplicon produced, with digoxigenin 11-dUTP incorporated during PCR, is hybridized in streptavidin-coated microtitre plates prepared with biotinylated specific DNA probe. The method involved requires approximately 6–8 h, and its high sensitivity, rapidity and simplicity should make it valuable for diagnosis and for epidemiological studies of listeriosis.     

无症状沙眼衣原体和解脲支原体感染与输卵管妊娠的关系

目的　探讨无症状性沙眼衣原体和解脲支原体感染与输卵管妊娠的关系。方法　收集 5 0例输卵管妊娠患者的宫颈分泌物和胚胎组织作为观察组 ;另取 5 0例人工流产者宫颈分泌物和胚胎组织作为对照组。采用 PCR方法检测两组标本中沙眼衣原体和解脲支原体的感染情况。结果　输卵管妊娠组宫颈分泌物中 CT阳性率为 36 % ,UU阳性率为42 % ,CT和 UU混合阳性率为 2 2 % ,分别与对照组比较 ,差异有显著性 (P<0 .0 5 ) ;输卵管妊娠组胚胎组织中 CT阳性率为 44 % ,UU阳性率为 5 4% ,CT和 UU混合阳性率为 2 4% ,分别与对照组比较 ,差异有显著性 (P<0 .0 1)。结论　输卵管妊娠的发生与无症状沙眼衣原体和解脲支原体感染关系密切。     

Distinguishing Chlamydia species by restriction analysis of the major outer membrane protein gene.

Clinical isolates of Chlamydia pneumoniae from diverse geographic locations and strains of other Chlamydia species were typed by polymerase chain reaction (PCR) amplification of the major outer membrane protein (MOMP) gene followed by restriction fragment length polymorphism analysis of the product. Use of synthetic primers corresponding to highly conserved regions of the MOMP gene resulted in amplification of a 1070 bp product in laboratory strains and clinical isolates of C. pneumoniae, C. trachomatis and C. psittaci. PCR products were digested with restriction enzymes Alu I and Mbo I and separated by polyacrylamide gel electrophoresis. Restriction fragment patterns varied in length from 8-12 bands of 30-400 bp in size in Alu I digests, and 6-7 bands of 50-400 bp in size in Mbo I digests. Strains representing different chlamydia species were easily distinguishable by this method, as were different serovars of C. trachomatis. Strains of C. pneumoniae tested include laboratory strain TW-183 and recent clinical isolates from Atlanta, Brooklyn, Wisconsin and Norway. One combination of primers reacted with C. psittaci strains and C. pneumoniae strain TW-183, but not with other strains of C. pneumoniae tested regardless of the concentration of DNA in the sample. With use of a pan-reactive primer combination, however, restriction patterns were similar in all strains of C. pneumoniae tested. This gene typing technique can be valuable for distinguishing the three chlamydial species and potentially strains of C. pneumoniae in clinical and epidemiologic studies.     

A polymerase chain reaction (PCR) protocol for the specific detection of Chlamydia spp

The polymerase chain reaction is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. This technique was used to detect and differentiate Chlamydia trachomatis and Chlamydia psittaci in laboratory samples of infected McCoy cells. The polymerase chain reaction was shown to be both sensitive, detecting in the order of one chlamydial DNA molecule in 10(5) cells, and specific. No cross reaction (amplified product) was detected when a variety of mammalian cell and bacterial DNAs were used as template with the Chlamydia-specific oligonucleotide primers.     

Three SARS-CoV-2 PCR-negative cases of COVID-19 diagnosed using isothermal amplification methods

Coronavirus disease (COVID-19) is a viral disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 can be detected by polymerase chain reaction (PCR) and isothermal nucleic acid amplification tests, including loop-mediated isothermal amplification (LAMP) and nicking endonuclease amplification reaction (NEAR) tests. Although PCR is the most sensitive and specific method and is generally considered to be the gold standard, it is time-consuming and costly. Isothermal nucleic acid amplification tests have lower sensitivity and specificity than PCR, but are less time-consuming and costly. We encountered three cases of SARS-CoV-2 infection in which the isothermal amplification test was positive but the PCR test was negative on the day of admission; however, the PCR test was positive the next day. These cases showed that some COVID-19 patients can test negative by PCR but positive using isothermal nucleic acid amplification methods. As PCR tests have the possibility of false-negative results, tests that use isothermal amplification methods which can be performed in a shorter time and at a lower cost than PCR tests, may be able to diagnose patients who have false negative PCR results.