Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR

Visual DNA microarrays, based on gold label silver stain (GLSS) and coupled with multiplex asymmetrical PCR, were developed for simultaneous, sensitive and specific detection of Ureaplasma urealyticum and Chlamydia trachomatis. 5'-end-amino-modified oligonucleotides, which were immobilized on glass surface, acted as capturing probes that were designed to bind complementary biotinylated targets DNA. The gold-conjugated streptavidins were introduced to the microarray for specific binding to biotin. The black image of microarray spots, resulting from the precipitation of silver onto nanogold particles bound to streptavidins, were used to detect biotinylated targets DNA visually or with a visible light scanner. Multiplex asymmetrical PCR of U. urealyticum, C. trachomatis and Bacillus subtilis (used as positive control) was performed to prepare abundant biotinylated single-stranded targets DNA, which affected detection efficiency and sensitivity of hybridization on microarray. Plenty of clinical samples of U. urealyticum and C. trachomatis from infected patients were tested using home-made DNA microarrays. For its high sensitivity, good specificity, simplicity, cheapness and speed, the present visual gene-detecting technique has potential applications in clinical fields.     

Association of Chlamydia trachomatis,Neisseria gonorrhoeae,Mycoplasma genitalium and Ureaplasma species infection and organism load with cervicitis in north Indian population

Cervicitis is predominantly caused by Neisseria gonorrhoeae and Chlamydia trachomatis, which accounts for almost half of all the cases of cervicitis. The role of newer organisms like Mycoplasma genitalium and Ureaplasma sp. and association of bacterial load with cervicitis are also not well established. So the study aimed to determine the relative frequency of these organisms and their load in association with cervicitis cases from north India. A case–control study involving 300 women was conducted using quantitative real-time PCR from endocervical swabs for identification of organisms and quantification of bacterial load. Among 150 cervicitis cases, C. trachomatis, N. gonorrhoeae, M. genitalium and Ureaplasma parvum were detected in 5 (3·3%), 10 (6·6%), 37(24·6%) and 47 (31·3%) respectively. Old age (<0·001, chi-squared test) and irregular menstrual cycles (<0·001, chi-squared test) were significantly associated with cervicitis. M genitalium was the only organism to be associated significantly with cervicitis with regard to age (<0·031) and symptoms like discharge (P < 0·033, chi-squared test) and dysuria (P < 0·044, chi-squared test) in multivariate analysis. Our finding suggests that the bacterial load of these organisms is not significantly associated with cervicitis. However, we found significant association of M. genitalium infection with clinical characteristics of cervicitis cases.     

Comparison of various PCR methods for the detection of Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma hominis in women with impaired reproductive function

The comparative evaluation of the PCR test "Polimik" (Research and Production Firm "Litekh", Moscow) and the PCR test of the Novosibirsk Institute of Bioorganic Chemistry (NIBC) was carried out. The results obtained with the use of the PCR test "Polimik" and the PCR test of the NIBC of the detection of C. trachomatis and M. hominis coincided in 97.8% and 97.4% of cases. For U. urealyticum, the coincidence of the results of both PCR tests was 81.2%. Among women who visited gynecologists for reproductive function disturbances, the use of the PCR tests made it possible to detect C. trachomatis in 19 (5.5%) out of 343 cases, U. urealyticum in 96 (39.0%) out of 246 cases and M. hominis in 25 (16.9%) out of 148 cases. The results of the investigation revealed that the occurrence of C. trachomatis infection in Novosibirsk was comparable with that in other regions of the world among the low-risk groups of the population. The detection frequency of M. hominis and U. urealyticum with the use of the PCR tests showed that the occurrence of infections caused by these causative agents coincided with the data obtained in other countries.     

Determination of the number of tuf genes in Chlamydia trachomatis and Neisseria gonorrhoeae

Restriction endonuclease fragments of DNA from Neisseria gonorrhoeae and Chlamydia trachomatis (mouse pneumonitis biovar) were hybridized to probes from the N-terminal and C-terminal portions of the Escherichia coli tufA gene. In common with other Gram-negative bacteria, the genome of N. gonorrhoeae was found to contain two homologous sequences (presumptive tuf genes). The C. trachomatis genome contained a single tuf sequence.     

生殖道解脲支原体和沙眼衣原体的感染与输卵管妊娠关系探究

目的 探讨分析生殖道解脲支原体和沙眼衣原体的感染与输卵管妊娠的关系。方法 选取新疆医科大学第二附属医院2018年1月—6月妇科住院经腹腔镜手术证实输卵管妊娠并行患侧输卵管切除的患者作为研究组,同期腹腔镜下行妇科良性疾病输卵管或附件切除术者为对照组,分别采集两组宫颈管分泌物、输卵管组织进行解脲支原体培养以及沙眼衣原体检测,并作宫颈分泌物解脲支原体药敏试验。结果 输卵管妊娠组的宫颈分泌物及输卵管组织中解脲衣原体检出率（34.3%、30.2%）及沙眼衣原体的检出率（18.6%、16.2%）明显高于对照组宫颈分泌物及输卵管中解脲衣原体检出率（16.2%、6.9%）及沙眼衣原体的检出率（4.6%、2.3%）,对照组与实验组两组比较差异具有统计学意义（χ2=3.909、4.074、7.679、4.962,Ps＜0.05）。本研究22例宫颈解脲支原体感染中,解脲支原体对交沙霉素、强力霉素、美满霉素、阿奇霉素、克拉霉素的敏感率均高于80%。结论 输卵管妊娠与生殖道解脲支原体及沙眼衣原体感染有密切关系,临床工作中应尽早筛查和诊断,并根据药敏试验进行预防及合理治疗用药,减少输卵管妊娠的发生。     

Modelling of an association of Chlamydia trachomatis and Neisseria gonorrhoeae in in ovo experiments

In experiments in ovo mixed chlamydial and gonococcal infection has been obtained by the successive infection of developing chick embryos with C. trachomatis and N. gonorrhoeae into the yolk sack. The competitive interrelations between the associated microorganisms with respect to their pathogenicity characteristics for chick embryos have not been established. This simulator is intended for use in the primary selection of etiotropic chemical preparations capable of producing combined effect on C. trachomatis and N. gonorrhoeae.     

Structures of glyceraldehyde 3‐phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co‐infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng‐Ct co‐infections. Development of a safe, effective, and inexpensive dual therapy for Ng‐Ct co‐infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X‐ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high‐throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.     

The first performance report for the Bio-Rad Dx CT/NG/MG assay for simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in urogenital samples

We evaluated the clinical performance of the Bio-Rad Dx CT/NG/MG assay for the detection of Chlamydia trachomatis, Mycoplasma genitalium and Neisseria gonorrhoeae in urogenital samples in comparison with the Roche COBAS? TaqMan? CT assay for C. trachomatis and an in-house TaqMan PCR assay for M. genitalium. Swab specimens were cultured for N. gonorrhoeae. In this prospective study, urogenital samples were obtained from symptomatic and asymptomatic patients attending the sexually transmitted disease clinic of Bordeaux, France, from January to April 2010. A total of 658 clinical specimens (259 male and 180 female urines, 191 vaginal, 21 endocervical and 7 urethral swabs) from 453 patients were analyzed. The prevalence of C. trachomatis and M. genitalium infections was 8.1% (21/260) and 1.9% (5/260) in men and 10.4% (20/193) and 2.1% (4/193) in women, respectively. The Bio-Rad Dx CT/NG/MG clinical sensitivity was 100% for C. trachomatis and M. genitalium in men and women. In male urine, the clinical specificity was 99.6% for C. trachomatis and 100% for M. genitalium. In women, the specificity was 99.5% for swabs and 100% for urines for detecting C. trachomatis and M. genitalium. All seven N. gonorrhoeae PCR-positive samples were also positive by culture. Patients were co-infected in 5/57 cases (8.8%), with C. trachomatis and M. genitalium in three cases, and C. trachomatis and N. gonorrhoeae in two cases. In conclusion, the Bio-Rad Dx CT/NG/MG assay can be recommended for the simultaneous detection of C. trachomatis, M. genitalium and N. gonorrhoeae in urogenital specimens of symptomatic and asymptomatic individuals.     

Development of real-time PCR for the differential detection and quantification of Ureaplasma urealyticum and Ureaplasma parvum

Ureaplasma parvum and Ureaplasma urealyticum are recently recognized species of the genus Ureaplasma. In humans, Ureaplasma spp. can be found on mucosal surfaces, primarily in the respiratory and urogenital tracts. They have been implicated in various human diseases such as nongonococcal urethritis, intrauterine infections in association with adverse pregnancy outcome and fetal morbidity, and pneumonitis in immunocompromised hosts. We have developed two quantitative real-time PCR assays to differentially detect U. parvum and U. urealyticum. Based upon the sequence information of the urease gene (ureB), we designed two TaqMan primer and probe combinations specific for U. parvum and U. urealyticum, respectively. The assays did not react with nucleic acid preparations from 16 bacterial species commonly encountered in relevant clinical specimens, including seven urease-producing species. Each assay had a detection limit of approximately five copies per reaction of the respective gene target. The results suggest that these assays are both sensitive and specific for U. parvum and U. urealyticum. Further investigation of both assays using clinical specimens is appropriate.     

男性尿道炎尿道多形核白细胞与沙眼衣原体和淋病奈瑟菌感染临床相关性分析

目的：沙眼衣原体和淋病奈瑟氏球菌是引起泌尿生殖系统尿道炎疾病常见的致病菌。临床化验一般采用美蓝染色检查男性尿道多形核白细胞或病原微生物阳性可提示尿道炎症状,指导临床的早期治疗。在缺乏临床症状体征和尿道多形核白细胞的情况下,临床的治疗将是不同的。本研究采用美兰染色检查男性尿道炎患者多形核白细胞数量、沙眼衣原体抗原和淋病奈瑟氏球菌培养,评价非淋菌性尿道炎和淋病的临床意义。方法：3,000例性病患者的尿道分泌物进行美兰涂片染色镜检,衣原体抗原检测和淋病奈瑟氏球菌染色镜检和培养。结果：3,000例性病患者中,387例患者（12.9%）沙眼衣原体抗原阳性。 在沙眼衣原体患者中,242例（62.5%）≥5个多形核白细胞, 59例（15.2%）为1~4个多形核白细胞,86例（22.2%）为0个多形核白细胞,其中36例患者（9.3%）无症状,141例患者（36.4%）无体征。415例（13.8%）淋病奈瑟氏球菌阳性,在淋病患者中,397例（95.7%）≥5个多形核白细胞, 10例（2.4%）1~4个多形核白细胞, 8例（1.9%）为0个多形核白细胞,其中5例（1.2%）患者无症状, 46例(11.1%)无体征。86例沙眼衣原体和淋病奈瑟氏球菌合并感染的患者中,76例阳性患者≥5个多形核白细胞,5例阳性患者为1~4个多形核白细胞, 5例阳性患者无多形核白细胞。结论：本研究分析了尿道炎患者尿道多形核白细胞,沙眼衣原体和淋球菌感染的相互之间的关系,男性尿道炎患者尿道多形核白细胞数量与沙眼衣原体感染和淋球菌感染存在明显的差异（P<0.001）。86例（22.2%）沙眼衣原体感染和8例（1.2%）淋病患者的尿道中无多形核白细胞。因此,加强临床与实验室诊断可提高男性尿道炎的诊断和控制性病的传播。     

慢性阴道炎患者生殖道解脲脲原体和沙眼衣原体感染分析

目的通过荧光定量聚合酶链反应(PCR)技术检测慢性阴道炎患者阴道内解脲脲原体(Uu)和沙眼衣原体(Ct)感染状况,用以指导临床正确治疗。方法应用荧光定量聚合酶链反应对380例慢性阴道炎患者的阴道分泌物进行解脲脲原体和沙眼衣原体PCR检测。结果解脲脲原体阳性率为42.9%,其阳性标本的平均拷贝数为3.4×105copies/ml,沙眼衣原体阳性率为16.6%,其阳性标本的平均拷贝数为5.8×104copies/ml,解脲脲原体和沙眼衣原体合并感染的阳性率为12.6%。结论PCR技术具有简便、快捷、准确的优点,是目前快速诊断慢性阴道炎患者Uu、Ct感染状况的可靠诊断方法之一。     

衣原体和淋球菌检测技术

从理论和应用方面介绍衣原体和淋球菌的各种检测技术,比较了各种检测技术的优缺点以及检测的敏感性、特异性,并展望了今后的应用前景。     

An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1, HBV, and HCV

We describe a novel microarray-based approach for simultaneous identification and quantification of human immunodeficiency virus type 1 (HIV-1) and hepatitis B and C viruses (HBV and HCV) in donor plasma specimens. The method is based on multiplex real-time RT-PCR performed within the microarray hydrogel pads. Double-stranded amplification products are simultaneously detected using nonspecific SYBR Green I dye due to the reaction run in separate pads bearing 5'-immobilized specific primers. Both the sensitivity and specificity of the assay, based on 132 blood specimens analyzed, were 100% (56, 26, and 8 specimens were seropositive to HBV HCV and HIV-1, respectively; 22 were positive to both HIV-1 and HCV and 2 positive to all three viruses; 18 samples were pathogen-negative). The dynamic range of the quantitative analysis covered a six-order interval ranging from 100 to 106 genome equivalents per assay. The 95% detection limits were 14 gEq for HIV-1, 10 gEq (1.7 IU) for HBV, and 15 gEq (7.5 IU) for HCV per assay. The proposed approach is considered to be versatile and could be adapted for simultaneous identification and quantification of numerous genetic targets.     

Evaluation of the Versant CT/GC DNA 1.0 Assay (kPCR) for the Detection of Extra-Genital Chlamydia trachomatis and Neisseria gonorrhoeae Infections

Screening for extra-genital Chlamydia trachomatis and Neisseria gonorrhoeae infections is a crucial component for sexually transmitted diseases management, even if at present days no commercial methods have been approved for use on pharyngeal and rectal specimens by the US FDA or have received the conformity CE marking. Here we report the analytical sensitivities of the Versant CT/GC 1.0 assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) on rectal and pharyngeal swabs, and an evaluation about the suitability for this assay with two widely used swab collection devices (E-Swab and eNAT, Copan, Brescia, Italy). The limits of detection for rectal and pharyngeal specimens with the Versant assay were 10 copies/ml and 1.0 copies/ml, for C. trachomatis and N. gonorrhoeae, respectively. False positive results due to the presence of non-gonococcal Neisseria species were excluded when clinical rectal and pharyngeal samples containing organisms identified as N. meningitidis, N. sicca, N. flavescens and N. subflava were tested. Due to its sensitivity and specificity, the Versant assay represents a good choice for the diagnosis of chlamydial and/or gonococcal infections not only in genito-urinary samples, but also on rectal and pharyngeal swabs.     

388例非淋菌性尿道炎患者Uu、CT感染情况分析

目的了解延安市非淋菌性尿道炎(NGU)患者解脲支原体(Uu)和沙眼衣原体(CT)感染状况及其特点。方法采用实时荧光聚合酶链式反应技术(FQ-PCR)对388例临床确诊的NGU患者的泌尿生殖道分泌物同时作Uu DNA和CT DNA检测。结果 388例NGU患者阳性检出率为62.11%,其中Uu、CT和混合感染的阳性检出率分别为23.20%、21.13%和17.78%。不同性别在不同病原体感染中有所差异,总体上女性感染率高于男性,但差异无显著性(P0.05);Uu单一感染中,女性感染率远高于男性,差异有极显著性(P0.01);CT单一感染中,男性感染率高于女性,差异有极显著性(P0.01);不同年龄感染情况显示,感染者主要集中在21~40岁年龄段,其中31~40岁年龄段阳性病例数最多。结论本地区NGU患者Uu的感染率稍高于CT,女性在性活跃期感染率较高。     

Nucleic acid spot hybridization for detection of Chlamydia trachomatis

Abstract The TMAO reductase activity of Escherichia coli grown anaerobically in the presence or absence of TMAO was analysed on linear sucrose gradients and on non-denaturing polyacrylamide gels. The results, together with those obtained by analysis of some other properties of TMAO reductase, showed that there are significant differences between the enzyme synthesized in the absence of TMAO ("constitutive" enzyme) and that synthesized in its presence ("inducible" enzyme).
A similar study of a tor mutant specifically altered in the structural gene for TMAO reductase, showed that the enzymes synthesized under the 2 growth conditions are probably 2 distinct enzymes encoded by different genes.