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          Abstract

          Background: Sexually transmitted diseases (STD) are a major cause of infertility, long-term disability, ectopic pregnancy, and premature birth. Therefore, the development of fast and low-cost laboratory STD diagnostic screening methods will contribute to reducing STD-induced reproductive tract damage and improve women's health worldwide. In this study, we evaluated a novel multiplex real-time PCR melting curve assay method for the simultaneous detection of 9 STD pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum , and herpes simplex virus.

          Methods: The analytical performance of the method, including its limit of detection (LOD), specificity, repeatability, and effect on different DNA extraction kits were evaluated. Additionally, we obtained 1,328 clinical specimens from 3 hospitals to detect the 9 STD pathogens using multiplex real-time PCR melting curve and Sanger sequencing, to evaluate the sensitivity, specificity, and consistency of the assay method.

          Results: The results showed that the analytical sensitivity of the novel multiplex real-time PCR melting curve assay is very excellent, with LOD of DNA corresponding to <200 copies/μL for the DNA of the 9 STDs and 1.00 × 10 4 color change unit /ml for those of UU and UP. Additionally, this assay demonstrated excellent analytical specificity, excellent repeatability, and its results had no effect of different DNA extraction kits. The performance, in terms of sensitivity (91.06–100%) and specificity (99.14–100%), was remarkable, since the consistency between it and Sanger sequencing was more than 0.85 in the clinic.

          Conclusion: The novel multiplex real-time PCR melting curve assay method has high sensitivity and specificity, relatively low cost, and simple to use for the simultaneous detection of 9 STD pathogens in genitourinary secretions.

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          Most cited references15

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          Sexually transmitted diseases treatment guidelines, 2010.

          These guidelines for the treatment of persons who have or are at risk for sexually transmitted diseases (STDs) were updated by CDC after consultation with a group of professionals knowledgeable in the field of STDs who met in Atlanta on April 18-30, 2009. The information in this report updates the 2006 Guidelines for Treatment of Sexually Transmitted Diseases (MMWR 2006;55[No. RR-11]). Included in these updated guidelines is new information regarding 1) the expanded diagnostic evaluation for cervicitis and trichomoniasis; 2) new treatment recommendations for bacterial vaginosis and genital warts; 3) the clinical efficacy of azithromycin for chlamydial infections in pregnancy; 4) the role of Mycoplasma genitalium and trichomoniasis in urethritis/cervicitis and treatment-related implications; 5) lymphogranuloma venereum proctocolitis among men who have sex with men; 6) the criteria for spinal fluid examination to evaluate for neurosyphilis; 7) the emergence of azithromycin-resistant Treponema pallidum; 8) the increasing prevalence of antimicrobial-resistant Neisseria gonorrhoeae; 9) the sexual transmission of hepatitis C; 10) diagnostic evaluation after sexual assault; and 11) STD prevention approaches.
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            Multiplex polymerase chain reaction: a practical approach.

            Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features. Copyright 2002 Wiley-Liss, Inc.
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              Re-evaluating the treatment of nongonococcal urethritis: emphasizing emerging pathogens--a randomized clinical trial.

              Nongonococcal urethritis (NGU) is a common chlamydia-associated syndrome in men; however, Trichomonas vaginalis and Mycoplasma genitalium are associated with its etiology and should be considered in approaches to therapy. We sought to determine whether the addition of tinidazole, an anti-trichomonal agent, to the treatment regimen would result in higher cure rates than those achieved with treatment with doxycycline or azithromycin alone. A secondary aim was to compare the efficacy of doxycycline therapy and with that of azithromycin therapy. Randomized, controlled, double-blinded phase IIB trial of men with NGU. Participants were randomized to receive doxycycline plus or minus tinidazole or azithromycin plus or minus tinidazole and were observed for up to 45 days. The prevalences of Chlamydia trachomatis, M. genitalium, and T. vaginalis were 43%, 31%, and 13%, respectively. No pathogens were identified in 29% of participants. Clinical cure rates at the first follow-up visit were 74.5% (111 of 149 patients) for doxycycline-containing regimens and 68.6% (107 of 156 patients) for azithromycin-containing regimens. By the final visit, cure rates were 49% (73 of 149 patients) for doxycycline-containing regimens and 43.6% (68 of 156 patients) for azithromycin-containing regimens. There were no significant differences in clinical response rates among the treatment arms. However, the chlamydia clearance rate was 94.8% (55 of 58 patients) for the doxycycline arm and 77.4% (41 of 53 patients) for the azithromycin arm (P = .011), and the M. genitalium clearance rate was 30.8% (12 of 39 patients) for the doxycycline arm and 66.7% (30 of 45 patients) for the azithromycin arm (P = .002). Addition of tinidazole to the treatment regimen did not result in higher cure rates but effectively eradicated trichomonas. Clinical cure rates were not significantly different between patients treated with doxycycline and those treated with azithromycin; however, doxycycline had significantly better efficacy against Chlamydia, whereas azithromycin was superior to doxycycline for the treatment of M. genitalium.
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                Author and article information

                Contributors
                Journal
                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Infect. Microbiol.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                2235-2988
                12 November 2019
                2019
                : 9
                : 382
                Affiliations
                [1] 1Department of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University , Ganzhou, China
                [2] 2Department of Precision Medicine Center, First Affiliated Hospital of Gannan Medical University , Ganzhou, China
                [3] 3Department of Medical Laboratory, The Fourth Affiliated Hospital of Nanchang University , Nanchang, China
                [4] 4Department of Laboratory Medicine Center, Nanfang Hospital, Southern Medical University , Guangzhou, China
                [5] 5Department of Obstetrics and Gynecology, First Affiliated Hospital of Gannan Medical University , Ganzhou, China
                [6] 6Department of Dermatology, First Affiliated Hospital of Gannan Medical University , Ganzhou, China
                [7] 7Department of Cardiac and Thoracic Surgery, First Affiliated Hospital of Gannan Medical University , Ganzhou, China
                [8] 8Jiangxi Shiningmed Medical Technology Ltd. , Ganzhou, China
                Author notes

                Edited by: Margaret E. Bauer, School of Medicine, Indiana University Bloomington, United States

                Reviewed by: Michael Marceau, Université Lille Nord de France, France; Subash C. Sonkar, Florida International University, United States

                *Correspondence: Shao Huang huangshao@ 123456shiningmed.com

                This article was submitted to Clinical Microbiology, a section of the journal Frontiers in Cellular and Infection Microbiology

                †These authors have contributed equally to this work

                Article
                10.3389/fcimb.2019.00382
                6861374
                31781517
                ec426522-b442-4eeb-b00b-f909667dfb12
                Copyright © 2019 Hu, Xu, Jiang, Deng, Gu, Xie, Ji, Wang, Li, Tian, Song, Huang, Zheng and Zhong.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 10 May 2019
                : 25 October 2019
                Page count
                Figures: 0, Tables: 6, Equations: 0, References: 15, Pages: 8, Words: 5352
                Funding
                Funded by: National Natural Science Foundation of China 10.13039/501100001809
                Categories
                Cellular and Infection Microbiology
                Methods

                Infectious disease & Microbiology
                multiplex,polymerase chain reaction,sanger sequencing,sexually transmitted diseases,pathogen

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