<p>Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis</p>

Abstract

PurposeSexually transmitted infections (STIs), representing a major global health problem, are caused by different microbes, including bacteria, viruses, and protozoa. Unfortunately, infections of different sexually transmitted pathogens often present similar clinical symptoms, so it is almost impossible to distinguish them clinically. Therefore, the aim of the current study was to develop a sensitive, multitarget, and high-throughput method that can detect various agents responsible for STIs.MethodsWe developed and tested a 23-plex PCR coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) assay (sexually transmitted infection-mass spectrometry, STI-MS) that simultaneously targets 11 different agents, including 8 most common clinical pathogens related to STIs (HSV-1, HSV-2, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, and Haemophilus ducreyi) and 3 controversial microorganisms as pathogens (Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum).ResultsThe results showed that the STI-MS approach can accurately detect the expected agents, without cross-reaction with other organisms. The limit of detection of each STI-MS assay was ranged from 1.739 to 10.009 copies/reaction, using probit analyses. The verification rate for each target organism of the STI-MS ranged from a minimum of 89.3% to a maximum of 100%, using conventional assays and ultrasensitive digital PCR to confirm the STI-MS-positive results. To further evaluate the clinical performance of this assay, 241 clinical specimens (124 urethral/cervical swabs and 117 urine) were tested in parallel using the STI-MS assay and monoplex real-time PCR for each agent. The overall validation parameters of STI-MS were extremely high including sensitivity (from 85.7% to 100%), specificity (from 92.3% to 100%), PPV (from 50% to 100%), and NPV (from 99.1% to 100%) for each target.ConclusionSTI-MS is a useful high-throughput screening tool for detecting mixed infections of STIs and has great potential for application in large-scale epidemiological programs for specific microorganisms of STI.

A molecular screening assay to identify Chlamydia trachomatis and distinguish new variants of C. trachomatis from wild‐type

“…The performance of this assay was initially assessed using C. trachomatis samples ( n = 28) confirmed using the STI‐MS assay (Xiu et al ., 2019; STI‐MS is a detection method combining multiplex‐PCR with MALDI‐TOF analysis). For these 28 samples, the 23S rRNA gene was previously characterized with respect to the wild‐type, using Sanger sequencing.…”

“…The plasmids bearing the 23S rRNA C1514T, C1515T and G1523A mutations were confirmed by sequencing. Our assay was also validated using 570 urethral/genital swabs, from which DNA was extracted using the MagNA Pure LC DNA Isolation Kit 1 (Roche Diagnostics, Mannheim, Germany) and tested for sexually transmitted infections (STI) agents using the STI‐MS method (Xiu et al ., 2019). Moreover, a total of 404 archived DNA specimens extracted from first void urine (FVU) and previously confirmed as positive for C. trachomatis using the Roche Cobas ® 4800 CT/NG test (Roche Diagnostics, Indianapolis, IN, USA) were also screened using the HRM assay.…”

“…Worldwide, Chlamydia trachomatis is one of the most prevalent sexually infectious aetiological agents (Menon et al, 2015;Aung et al, 2019;Chi Wai Wong et al, 2019), causing approximately 131 million new infections each year (https://www.who.int/reproductivehealth/pub lications/stis-surveillance-2018/en/). C. trachomatis infections not only can lead to severe reproductive sequelae, including ectopic pregnancy, pelvic inflammatory disease and infertility, but also can facilitate the transmission of human immunodeficiency virus (Xiu et al, 2019;Durukan et al, 2020). However, in some populations, C. trachomatis infections are often asymptomatic, causing significant challenges (underestimations) for detection and epidemiological characterization.…”

Multiplex High-Resolution Melting Assay for Simultaneous Identification of Molecular Markers Associated with Extended-Spectrum Cephalosporins and Azithromycin Resistance in Neisseria gonorrhoeae

“…For the clinical isolates, 25 genomic DNA was extracted by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the user's guide. Moreover, DNA extraction from the clinical specimens 25,26 was performed in the MagNA Pure LC 2.0 Instrument (Roche Diagnostics GmbH, Mannheim, Germany) by using the MagNA Pure LC DNA Isolation Kit 1 (Roche Diagnostics GmbH, Mannheim, Germany).…”

“…The limit of detection of the multiplex HRM assays was determined by probit regression analysis at the 95% detection level, according to a previous study. 26 Table S1) for the porB-120 and 121, penA-G545S, opa, porA, penA-D345del, 23S-C2611T, mtrR-G45D, mtrR-H105Y, 23S-A2059G, ponA-L421P, and penA-nmA501, respectively.…”