性病衣原体三种检测方法的比较研究

目的通过实验探讨一种简便、快速、准确而又经济的性病衣原体检测方法。方法对所选择的临床标本同时用ＭｃＣｏｙ细胞分离培养、ＭｃＡｂ荧光染色和ＰＣＲ法进行沙眼衣原体的检测，并用统计学方法分析结果。结果４４份临床标本中有３０份用３种方法检测均为阴性，７份为阳性，余７份结果不尽相同。三种方法检出率依次为２０．４５％、２５．００％、２９．５５％，用多组配对计数资料的ｘ２检验表明差别有显著意义。以培养法为标准，ＭｃＡｂ荧光法的符合率（９０．９１％）高于ＰＣＲ法（８６．３６％），而阳性率低于ＰＣＲ法。结论ＭｃＡｂ荧光法和ＰＣＲ法可为一种简便、快速而经济的判断衣原体感染的方法，但在进行临床诊断时，宜以一种以上方法同时检测，并结合临床表现进行评判为宜。     

不同引物对PCR检测沙眼衣原体结果的影响

为了解不同引物对ＰＣＲ检测泌尿生殖道沙眼衣原体（ＣＴ）感染的影响,作者以直接免疫荧光法（ＤＩＦＡ）为标准,选用引物不同的市售ＰＣＲ试剂盒检测了２０９例泌尿生殖道可疑ＣＴ感染标本。结果引物１ＰＣＲ阳性２９例,引物２ＰＣＲ阳性２８例;在ＤＩＦＡ阳性２５例中,引物１ＰＣＲ阳性２４例,引物２ＰＣＲ阳性２３例;引物１ＰＣＲ敏感性为９６．０％,特异性为９７．８％;引物２ＰＣＲ敏感性为９２．０％,特异性为９８．     

PCR 与 DIFA 检测沙眼衣原体的比较研究

用所建立的沙眼衣原体质粒引物聚合酶链反应技术（ＰＣＲ）检测１１６例泌尿生殖道沙眼衣原体，并与直接免疫荧光法（ＤＩＦＡ）进行比较。结果ＰＣＲ阳性３８例，ＤＩＦＡ阳性的３２例中有３１例ＰＣＲ阳性。ＰＣＲ敏感性为９６．９％，特异性为９１．７％。ＰＣＲ阳性者于治疗结束１～２周后复诊２１例，其中４例ＰＣＲ仍然阳性。结果显示，ＰＣＲ可快速、敏感、特异地检测泌尿生殖道沙眼衣原体ＤＮＡ，然而对治疗后短期内复诊患者，似乎不宜仅以ＰＣＲ阳性结果作为重复治疗的依据。     

16SrRNA基因PCR加反相杂交技术检测细菌DNA

目的探讨聚合酶链反应（ＰＣＲ）加反相杂交技术在细菌ＤＮＡ检测中的应用。方法以１６ＳｒＲＮＡ基因为靶序列，设计引物及寡核苷酸探针，采用ＰＣＲ法加反相杂交检测标准菌株及临床标本细菌ＤＮＡ。结果对２０株不同标准菌株进行ＰＣＲ扩增，均出现３７１ｂｐ长度的ＤＮＡ片段，敏感性试验可检测出１０－１２ｇ的细菌ＤＮＡ，与人基因组及病毒无交叉反应；２２份血培养阳性标本及４份脑脊液培养阳性标本均扩增出３７１ｂｐ长度ＤＮＡ条带，反相杂交区分革兰阳性／阴性细菌与培养结果相符。结论１６ＳｒＲＮＡ基因ＰＣＲ加反相杂交技术检测细菌ＤＮＡ，具有敏感、快速、准确的特点，为细菌感染的临床诊断提供了科学的依据。     

聚合酶链反应检测新生儿衣原体感染

目的用聚合酶链反应检测新生儿衣原体感染状况。方法参照文献报道的沙眼衣原体及肺炎衣原体基因序列,分别设计一对引物,采用ＰＣＲ法检测住院新生儿衣原体感染。结果因呼吸道感染住院的１２３名新生儿的咽拭子标本,沙眼衣原体及肺炎衣原体感染率分别为１３．８％和４．９％。结论ＰＣＲ检测沙眼衣原体及肺炎衣原体感染具有特异、敏感、快速的特点,可为临床诊断提供科学依据。     

PCR与DIFA检测沙眼衣原体的比较研究

用所建立的沙眼衣原体质粒引物聚合酶链反应技术检测１１６例泌尿生殖道沙眼衣原体，并与直接免疫荧光法进行比较。结果ＰＣＲ阳性３８例，ＤＩＦＡ阳性的３２例中有３１例ＰＣＲ阳性。ＣＰＲ敏感性为９６．９％，特异性为９．１７％。ＰＣＲ阳性者于治疗结束１－２财后复诊２１例，其中４例ＰＣＲ仍然阳性。     

多重聚合酶链反应检测乙型肝炎和丙型肝炎病毒

在同一反应体系中同时快速检测乙型肝炎病毒（ＨＢＶ）和丙型肝炎病毒（ＨＣＶ），探讨多重聚合酶链反应（ＰＣＲ）在临床中的应用价值。方法自行设计了ＨＢＶ和ＨＣＶ各一对引物，应用多重ＰＣＲ对临床标本进行检测。结果５例ＨＢｓＡｇ和ＨＣＶ抗体均阳性者，其ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性；２０例单ＨＢｓＡｇ阳性标本，１１例单ＨＢＶＤＮＡ阳性，５例ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性；２０份单ＨＣＶ抗体阳性者，ＨＣＶＲＮＡ均阳性，３例合并ＨＢＶＤＮＡ阳性；１０例慢性非甲非乙型肝炎（ＮＡＮＢ）抗ＨＣＶ阴性肝炎标本８份ＨＣＶＲＮＡ阳性，１例ＨＢＶＤＮＡ和ＨＣＶＲＮＡ阳性。结论多重ＰＣＲ一次反应即可完成多次常规ＰＣＲ检测，是一种值得临床应用的方法。     

应用PCR技术检测HLA—B27

应用多聚酶链反应（ＰＣＲ）技术检测了８１例广东地区汉族健康人群和４８例类风湿关节炎病人的ＨＬＡ－Ｂ２７的频率分布，阳性率分别为４．９３％和６．２５％。同时对血清学方法检测ＨＬＡ－Ｂ２７为阳性的８５例和阴性的１６例强直性脊柱炎病人的标本应用ＰＣＲ技术进行检测，发现血清学方法检测为阳生的标本中８４例为阳性，符合率为９８．７％；阴性标本中６例为阳性，误判率为３７．４％。通过引进一对内对照引物，避免假阴性     

肺炎衣原体套式聚合酶链式反应检测研究

目的 探讨肺炎衣原体（Ｃｐｎ）的套式聚合酶链式反应（ｎＰＣＲ）检测技术。方法 根据Ｃａｍｐｂｅｌｌ克隆的ＣｐｎＤＮＡ序列设计套式引物，并建立ｎＰＣＲ反应体系。结果 Ｃｐｎ经ｎＰＣＲ扩增出现３７８ｂｐ的阳性条带，而沙眼衣原体和鹦鹉热衣原体以及其他用作特异性试验的微生物均不能扩增。３例阳性标本经ＤＮＡ测序与Ｃｐｎ（ＣＷＬ－２９株）序列完全一致。Ｃｐｎ ｎＰＣＲ之灵敏度比传统ＰＣＲ高１００倍。各种呼吸系     

聚合酶链反应对脑脊液标本中结核杆菌DNA重复序列的检测

采用聚台酶链反应（ＰＣＲ）对３４例结核性脑膜炎（结脑）患者脑脊液（ＣＳＦ）进行检测，其中５份结核杆菌培养阳性的标本，ＰＣＲ检测均阳性；２９例ＣＳＦ结核杆菌培养阴性的标本，１９例ＰＣＲ检测阳性，总的阳性率为７０．５％。而作为对照的２０例非结脑患者的ＣＳＦ标本则无阳性。利用育法微量结核杆菌与对照菌测试，可以反证本方法的准确性，其符合率为１００％，提示本方法可以用于临床标本中结核杆菌ＤＮＡ的快速检测，不失为一快速，灵敏而又特异的诊断手段。关键词     

Evaluation of diagnostic efficacy of PCR methods for Chlamydia trachomatis infection in genital and urine specimens of symptomatic men and women in India

In India, given the scarce availability of sensitive and specific methods, Chlamydia trachomatis genital infections may lead to severe clinical complications when left undiagnosed or underdiagnosed. The present study was conducted to evaluate the diagnostic efficiency and feasibility of polymerase chain reaction (PCR) assays using genital and urine specimens from men and women in India. Genital swabs and urine specimens collected from 143 patients attending the sexually transmitted disease (STD) clinic, Government General Hospital, Chennai, were tested by culture and a plasmid based PCR. Culture was positive in 27 (18.9%) patients. PCR gave positive results for 46 (32.2%) cases using genital specimens, and the positivity rate in urine was 25.2%. Once the discordant results between culture and PCR had been resolved by using a major outer membrane protein PCR, the overall sensitivity, specificity, and positive and negative predictive values for the plasmid PCR in genital specimens were 100%, 98%, 95.7%, and 100%, respectively. Corresponding values for urine PCR were 81.8%, 100%, 100%, and 92.5%, respectively. The prevalence of confirmed C. trachomatis infection was 30.8% in this STD population. The results confirmed the need to use sensitive and specific molecular assays like PCR to prevent underdiagnosis of genital chlamydial infections and to facilitate better clinical management of this infection in India.     

Comparison of polymerase chain reaction and enzyme immunoassay (Chlamydiazyme) in detection of Chlamydia trachomatis from male urethritis]

A method using polymerase chain reaction (PCR) was compared to an enzyme immunoassay (Chlamydiazyme) for detection of Chlamydia trachomatis by testing a reference strain and clinical specimens. Two oligonucleotides based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used as primers for the PCR. A DNA fragment of 242 bp specific for C. trachomatis was amplified by the PCR, when DNA of greater than or equal to 10(2) C. trachomatis was used as template for the PCR. A chlamydial antigen was detected by Chlamydiazyme, when greater than or equal to 2.6 x 10(3) C. trachomatis were applied for the enzyme immunoassay. The PCR method was 26 times more sensitive than Chlamydiazyme in detection of C. trachomatis. The PCR method and Chlamydiazyme were carried out to examine 74 urethral swabs obtained from male patients with urethritis for detection of C. trachomatis. In 45 of 74 specimens, the DNA fragment of C. trachomatis was amplified by the PCR, and in 41 of 74, the chlamydial antigen was detected by Chlamydiazyme. The detection rate of the PCR method (60.8%) was higher than that of Chlamydiazyme (55.4%). The positive coincidence rate of the PCR method to Chlamydiazyme was 100% (41/41) and negative coincidence rate was 87.9% (29/33). The overall coincidence rate between the two methods was high (94.6%). Thus, the PCR method was more sensitive than Chlamydiazyme for detection of C. trachomatis and specific for diagnosis of chlamydial urethritis.     

The laboratory diagnosis of Chlamydia trachomatis infections.

Lower genital tract infections with Chlamydia trachomatis are predominantly asymptomatic in men and women. Diagnostic technology has provided several approaches to the diagnosis of C trachomatis. Outside of cells, Chlamydia can die or degrade without optimal storage and transportation. Because some of the other assays perform better on certain specimen types, it is important for laboratories to recognize these differences and provide advice to physicians and nurses collecting patient specimens, with the objective of diagnosing lower genital tract infections to prevent transmission and upper tract damage. Most invasive specimens, such as cervical or urethral swabs, may be collected for culture, antigen or nucleic acid detection. Noninvasive samples such as first-void urine and vaginal swabs can be easily collected by the patient; these samples must be tested by more sensitive nucleic acid amplification tests. These newer investigative strategies should enable implementation of screening programs to identify and treat partners. Serology has not been particularly useful for the diagnosis of acute C trachomatis infections in adults. Presently, it appears that antibiotic-resistant C trachomatis is not a clinical problem. Laboratories providing C trachomatis diagnosis require participation in continuous quality improvement programs.     

Usefulness of real-time PCR in detecting Chlamydia trachomatis and Neisseria gonorrhoeae in endocervical swabs and first-voided urine specimens

We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detecting N. gonorrhoeae was 100% in all 3 specimentypes. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.     

Vaginal tampons as specimen collection device for the molecular diagnosis of non-ulcerative sexually transmitted infections in antenatal clinic attendees

Self-inserted vaginal tampons for the molecular diagnosis of non-ulcerative STIs were evaluated. Cervical and vaginal swabs, tampons and urines were collected from 185 first-time antenatal clinic attendees. Cultures and nucleic acid amplification assays (NAA) were performed. The sensitivity of PCR on tampons for Trichomonas vaginalis was with 94% (CI 85-98%) significantly higher (P<0.001) than culture (50%, CI 38-62%) or urine (53%, CI 41-65%). Neisseria gonorrhoeae culture had a sensitivity of 64% (CI 36-86%), strand displacement assay (SDA) had a sensitivity of 79% (CI 49-94%) using tampon specimens, 57% (CI 30-81%) using endocervical swabs and 43% (CI 19-70%) using urines. There was no difference in sensitivity of SDA for Chlamydia trachomatis using tampon specimens, urine or endocervical swabs. The specificity approached 100% for all assays on all specimens. NAA on tampons for the detection of T. vaginalis, N. gonorrhoeae and C. trachomatis identified more infections than assays on swabs or urines. This reached statistical significance for T. vaginalis only.     

Simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis by PCR in genitourinary specimens from men and women attending an STD clinic

Neisseria gonorrhoeae and Chlamydia trachomatis are the two most common bacterial sexually transmitted infections that manifest primarily as urethritis in males and endocervicitis in females, though the infection may be asymptomatic especially in women. Since complications may occur in untreated symptomatic and asymptomatic infected individuals, early diagnosis and treatment of infected individuals is required to prevent severe sequelae and spread of these diseases. Recently molecular amplification assays like Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) have been found to be highly sensitive and specific methods for detection of N. gonorrhoeae and C. trachonmatis not only in urethral and cervical specimens but also in urine. The objective of this study was to screen male and female Sexually Transmitted Disease (STD) clinic attenders, with and without symptoms suggestive of urethritis and cervicitis for presence of N. gonorrhoeae and C. trachomatis using a multiplex PCR based assay, to compare its performance with culture for N. gonorrhoeae and Direct Fluorescent Antibody (DFA) staining for C. trachomatis and also to compare the efficacy of PCR test performed on urine and genital swab specimens collected from this high risk group. Genital specimens and urine was collected from STD clinic attenders. N. gonorrhoeae and C. trachomatis was detected in genital specimens by culture and DFA respectively. Multiplex PCR was used to detect N. gonorrhoeae and C. trachomatis infection in both genital and urine specimens. Among men with urethritis, N. gonorrhoeae was detected in 70% by culture and 77% by PCR, while C. trachomatis as detected in 7.5% by DFA and 17.5% by PCR. Among females with endocervicitis, N. gonorrhoeae was detected in 7.7% by culture and 30.7% by PCR, while C. trachomatis was detected in 7.7% by DFA and in 15.4% by PCR. None of the asymptomatic males were positive for N. gonorrhoeae and C. trachomatis by conventional methods, while 43.9% were positive for N. gonorrhoeae and 7.5% for C. trachomatis by PCR. Fifty per cent of asymptomatic women were positive for C. trachomatis by PCR alone. We encountered PCR positive but culture/DFA negative results and also PCR negative but culture/DFA positive results. In view of this a single PCR test cannot be used for diagnosis and treatment of N. gonorrhoeae and C. trachomatis infection unless confirmed by a second test.     

Screening for Chlamydia trachomatis infection using the BDProbeTec ET Chlamydia trachomatis amplified DNA assay on urine in a GUM clinic setting: a simple, fast and cost-effective alternative

This study compared the BDProbeTec ET Chlamydia trachomatis amplified DNA assay on urine specimens with culture of genital swabs for the detection of C. trachomatis in patients attending the Department of Genitourinary Medicine (GUM), Cardiff Royal Infirmary. Almost twice as many patients tested positive by BDProbeTec ET than by culture. A similar difference was found for both males and females. The case notes of those patients positive by BDProbeTec ET alone were analysed and a significantly greater number were found to have risk indicators for C. trachomatis infection when compared with age and sex comparable controls, providing clinical validation of our findings. The BDProbeTec ET assay was easy to use, more importantly, the test format features an internal control integral with every sample. The cost per true positive was calculated as comparable with culture. We conclude that the BDProbeTec ET assay is a superior alternative to culture for identifying patients infected with C. trachomatis in the GUM clinic setting.     

Which test is best for chlamydia?

Nucleic acid amplification tests are now the tests of choice for diagnosing Chlamydia trachomatis infection. For the first time there are diagnostic tests for Chlamydia trachomatis that are more sensitive than tissue culture. Another major advantage is that they can be used with first-catch urine specimens and vaginal swabs. It is thus possible to test for genital chlamydial infection without using invasive specimen collection methods.     

Detection of Neisseria gonorrhoeae from male patients with urethritis by polymerase chain reaction]

A polymerase chain reaction (PCR) procedure was developed for detection of Neisseria gonorrhoeae. Two oligonucleotides based on sequences within a 16S ribosomal RNA gene from N. gonorrhoeae were used as extension primers for the PCR. A single DNA fragment of 206 bp was amplified, when N. gonorrhoeae DNA was template for the PCR. No amplified product was detected in Chlamydia trachomatis DNA, Ureaplasma urealyticum DNA or other bacterial DNAs. The DNA fragment of 206 bp was detected on agarose gel electrophoresis, when DNA of greater than or equal to 6.5 N. gonorrhoeae per PCR was used as template DNA for the PCR. The culture and the PCR were carried out for detection of N. gonorrhoeae in 67 urethral swabs obtained from male patients with urethritis. In 27 of 28 specimens in which N. gonorrhoeae was isolated and identified by the culture, 206 bp DNA fragment was amplified by the PCR, but in one specimen no DNA fragment was detected. In 2 of 39 culture-negative specimens, 206 pb DNA fragment was detected and in the remaining specimens, PCR was negative for N. gonorrhoeae. The overall detection coincidence rate between the culture and the PCR was 95.5% (64/67). Thus, the PCR procedure developed in this study was sensitive and specific for detection of N. gonorrhoeae and could be applied for diagnosis of gonococcal urethritis.     

Detection of Chlamydia trachomatis from urethral swab in male urethritis by polymerase chain reaction]

A polymerase chain reaction (PCR) with Chlamydia trachomatis-specific primers was applied for detection of C. trachomatis from urethral swab in male urethritis. The results were compared with those of culture method for detection of C. trachomatis. Of 18 clinical specimens tested in this study, inclusion bodies of C. trachomatis were detected in 11 specimens by the culture method. For PCR, sample DNA was prepared from transport medium in which urethral smear was suspended and two oligonucleotides based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used as extension primers. In 12 of the 18 specimens, 242bp DNA fragment was amplified by PCR and demonstrated to be the DNA fragment of C. trachomatis by Southern blot hybridization. No DNA of 242bp was amplified by PCR from five specimens in which any inclusion bodies of C. trachomatis were observed or from a specimen in which one inclusion body per cover slip was detected by culture method. C. trachomatis DNA of 242bp was amplified from all specimens in which 14 and more inclusion bodies per cover slip were detected by culture method. In two specimens concluded s negative by culture method, amplified C. trachomatis DNA were detected by PCR. Thus, the PCR would be a more simple and sensitive method for detection of C. trachomatis, compared with the culture method.