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Evaluation of a Novel Chlamydia trachomatis Microsphere Suspension Assay for Detection and Genotyping of the Different Serovars in Clinical Samples

2011; Elsevier BV; Volume: 13; Issue: 2 Linguagem: Inglês

10.1016/j.jmoldx.2010.11.017

ISSN

1943-7811

Autores

Koen D. Quint, D.T. Geraets, Henk A.M. van den Munckhof, Maurits N.C. de Koning, Vitaly Smelov, Willem J. G. Melchers, Henry J.C. de Vries, Servaas A. Morré, Chris J.M. Meijer, Dirk C.J.G. van Alewijk, Leen‐Jan van Doorn, Wim Quint,

Tópico(s)

Cervical Cancer and HPV Research

Resumo

A novel Chlamydia trachomatis (Ct) microsphere suspension (MS) assay was evaluated for identification of the different serovars, using the same PCR primer set established for the Ct Detection and genoTyping assay. Both assays can detect and identify all 14 major serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) and one genovariant of serovar J. The probe specificity for the Ct-MS assay was determined using 14 Ct reference strains and 1 clinical isolate from a genovariant of serovar J. Also, the Ct-MS assay and the Ct detection and genoTyping assay were compared in 712 Ct-positive clinical samples. The Ct-MS assay showed a highly specific reaction for all probes with the amplicons of the reference strains, giving a very low background median fluorescence intensity signal (median fluorescence intensity ≤ 10). An excellent overall agreement in the Ct detection (kappa = 0.947, 95% confidence interval, 0.89 to 0.999; McNemar's test, P = 1.000) and the Ct genotyping (kappa = 0.993, 95% confidence interval, 0.977 to 1.000; McNemar's test, P = 0.053) was observed between the Ct detection and genoTyping (DT) assay and the Ct-MS assay. In conclusion, the novel Ct-MS assay permits simultaneous detection and genotyping of Ct serovars, making the Ct-MS assay an excellent high throughput method. A novel Chlamydia trachomatis (Ct) microsphere suspension (MS) assay was evaluated for identification of the different serovars, using the same PCR primer set established for the Ct Detection and genoTyping assay. Both assays can detect and identify all 14 major serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) and one genovariant of serovar J. The probe specificity for the Ct-MS assay was determined using 14 Ct reference strains and 1 clinical isolate from a genovariant of serovar J. Also, the Ct-MS assay and the Ct detection and genoTyping assay were compared in 712 Ct-positive clinical samples. The Ct-MS assay showed a highly specific reaction for all probes with the amplicons of the reference strains, giving a very low background median fluorescence intensity signal (median fluorescence intensity ≤ 10). An excellent overall agreement in the Ct detection (kappa = 0.947, 95% confidence interval, 0.89 to 0.999; McNemar's test, P = 1.000) and the Ct genotyping (kappa = 0.993, 95% confidence interval, 0.977 to 1.000; McNemar's test, P = 0.053) was observed between the Ct detection and genoTyping (DT) assay and the Ct-MS assay. In conclusion, the novel Ct-MS assay permits simultaneous detection and genotyping of Ct serovars, making the Ct-MS assay an excellent high throughput method. Chlamydia trachomatis (Ct) is considered a global, public health problem, with an approximate 90 million new cases worldwide annually.1Xiong L. Kong F. Zhou H. Gilbert G.L. Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis.J Clin Microbiol. 2006; 44: 1413-1418Crossref PubMed Scopus (42) Google Scholar Ct can be divided in 3 serogroups (serogroup B, C, and intermediate) and 19 different serovars (A, B, Ba, C, D, Da, E, F, G, Ga, H, I, Ia, J, K, L1, L2, L2a, and L3) based on their reactivity with specific monoclonal antibodies targeting the major outer membrane protein.2Wang S.P. Kuo C.C. Barnes R.C. Stephens R.S. Grayston J.T. Immunotyping of Chlamydia trachomatis with monoclonal antibodies.J Infect Dis. 1985; 152: 791-800Crossref PubMed Scopus (140) Google Scholar, 3Kuo C.C. Wang S.P. Holmes K.K. Grayston J.T. Immunotypes of Chlamydia trachomatis isolates in Seattle.Washington, Infect Immun. 1983; 41: 865-868PubMed Google Scholar The majority of anogenital Ct infections are caused by serovars D–K and may lead to infertility in both sexes.4Westrom L. Joesoef R. Reynolds G. Hagdu A. Thompson S.E. Pelvic inflammatory disease and fertility A cohort study of 1,844 women with laparoscopically verified disease and 657 control women with normal laparoscopic results.Sex Transm Dis. 1992; 19: 185-192Crossref PubMed Scopus (651) Google Scholar, 5Cengiz T. Aydoganli L. Baykam M. Mungan N.A. Tuncbilek E. Dincer M. Yakupoglu K. Akalin Z. Chlamydial infections and male infertility.Int Urol Nephrol. 1997; 29: 687-693Crossref PubMed Scopus (28) Google Scholar Besides an infection in the anogenital region, some Ct serovars (A to C) can infect the conjunctiva leading to a follicular conjunctivitis and trachoma, which is still the major cause of blindness in developing countries.6Baral K. Osaki S. Shreshta B. Panta C.R. Boulter A. Pang F. Cevallos V. Schachter J. Lietman T. Reliability of clinical diagnosis in identifying infectious trachoma in a low-prevalence area of Nepal.Bull World Health Organ. 1999; 77: 461-466PubMed Google Scholar, 7Bobo L.D. Novak N. Munoz B. Hsieh Y.H. Quinn T.C. West S. Severe disease in children with trachoma is associated with persistent Chlamydia trachomatis infection.J Infect Dis. 1997; 176: 1524-1530Crossref PubMed Scopus (64) Google Scholar Finally, some Ct serovars (L1–L3) invade to the submucosa of the proctum and the lymph nodes, causing the destructive disease lymphogranuloma venereum.8Sturm P.D. Moodley P. Govender K. Bohlken L. Vanmali T. Sturm A.W. Molecular diagnosis of lymphogranuloma venereum in patients with genital ulcer disease.J Clin Microbiol. 2005; 43: 2973-2975Crossref PubMed Scopus (20) Google Scholar, 9O'Farrell N. Morison L. Moodley P. Pillay K. Vanmali T. Quigley M. Sturm A.W. Genital ulcers and concomitant complaints in men attending a sexually transmitted infections clinic: implications for sexually transmitted infections management.Sex Transm Dis. 2008; 35: 545-549Crossref PubMed Scopus (19) Google Scholar Clinically, differentiation between a lymphogranuloma venereum Ct infection and an A–K serovar Ct infection is necessary because a lymphogranuloma venereum infection needs prolonged antibiotic treatment.10de Vries H.J. Smelov V. Middelburg J.G. Pleijster J. Speksnijder A.G. Morre S.A. Delayed microbial cure of lymphogranuloma venereum proctitis with doxycycline treatment.Clin Infect Dis. 2009; 48: e53-e56Crossref PubMed Scopus (66) Google Scholar Nowadays, several in-house reverse line blots have been developed for Ct genotyping, often based on amplification of the Omp1 gene, encoding for the major outer membrane protein.11Molano M. Meijer C.J. Morre S.A. Pol R. van den Brule A.J. Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens.J Clin Microbiol. 2004; 42: 2935-2939Crossref PubMed Scopus (54) Google Scholar, 12Zheng H.P. Jiang L.F. Fang D.Y. Xue Y.H. Wu Y.A. Huang J.M. Ou Z.Y. Application of an oligonucleotide array assay for rapid detecting and genotyping of Chlamydia trachomatis from urogenital specimens.Diagn Microbiol Infect Dis. 2007; 57: 1-6Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 13Quint K. Porras C. Safaeian M. Gonzalez P. Hildesheim A. Quint W. van Doorn L.J. Silva S. Melchers W. Schiffman M. Rodriguez A.C. Wacholder S. Freer E. Cortes B. Herrero R. Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens.J Clin Microbiol. 2007; 45: 3986-3991Crossref PubMed Scopus (23) Google Scholar, 14Quint K.D. van Doorn L.J. Kleter B. de Koning M.N. van den Munckhof H.A. Morre S.A. ter Harmsel B. Weiderpass E. Harbers G. Melchers W.J. Quint W.G. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.J Mol Diagn. 2007; 9: 631-638Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Previously, we evaluated a commercially available Ct-detection and genoTyping (DT) assay, comprising Ct-detection by a DNA enzyme immunoassay (DEIA) and Ct-genotyping by a reverse hybridization assay (RHA) using the same amplicons.14Quint K.D. van Doorn L.J. Kleter B. de Koning M.N. van den Munckhof H.A. Morre S.A. ter Harmsel B. Weiderpass E. Harbers G. Melchers W.J. Quint W.G. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.J Mol Diagn. 2007; 9: 631-638Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar An excellent agreement between the Ct-DT DEIA and other Ct detection assays [ie, Hybrid Capture 2 (Qiagen, Valencia, CA) and COBAS TaqMan tests (Roche Molecular Systems, Branchburg, NJ)] was observed.13Quint K. Porras C. Safaeian M. Gonzalez P. Hildesheim A. Quint W. van Doorn L.J. Silva S. Melchers W. Schiffman M. Rodriguez A.C. Wacholder S. Freer E. Cortes B. Herrero R. Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens.J Clin Microbiol. 2007; 45: 3986-3991Crossref PubMed Scopus (23) Google Scholar, 14Quint K.D. van Doorn L.J. Kleter B. de Koning M.N. van den Munckhof H.A. Morre S.A. ter Harmsel B. Weiderpass E. Harbers G. Melchers W.J. Quint W.G. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.J Mol Diagn. 2007; 9: 631-638Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Also, Ct genotyping with the Ct-DT RHA is a sensitive alternative for Omp1 sequencing.15Quint K.D. Bom R.J. Bruisten S.M. van Doorn L.J. Nassir Hajipour N. Melchers W.J. de Vries H.J. Morre S.A. Quint W.G. Comparison of three genotyping methods to identify Chlamydia trachomatis genotypes in positive men and women.Mol Cell Probes. 2010; 24: 266-270Crossref PubMed Scopus (30) Google Scholar This algorithm of amplification, detection, and genotyping is a relatively fast method, easy to perform, and particularly suitable for study populations with a low Ct prevalence. At this moment, Ct genotyping with the Ct-DT RHA was performed in several epidemiological studies.16Bax C.J. Schachter J. Prevalence of multiple site or double chlamydia trachomatis serovar infections and the prevalence of serovar G/Ga in urogenital vs rectal specimens in male and female patients attending a sexual transmitted disease clinic or out-patient departmetn of obstetrics and gynecology. International Chlamydia Symposim, San Fransisco, CA2010: 441-444Google Scholar, 17Porras C. Safaeian M. Gonzalez P. Hildesheim A. Silva S. Schiffman M. Rodriguez A.C. Wacholder S. Freer E. Quint K. Bratti C. Espinoza A. Cortes B. Herrero R. Epidemiology of genital Chlamydia trachomatis infection among young women in Costa Rica.Sex Transm Dis. 2008; 35: 461-468Crossref PubMed Scopus (18) Google Scholar Recently, a new Ct-microsphere suspension (MS) assay was developed that combines Ct detection and Ct genotyping, using the same amplification step as in the Ct-DT assay. The Ct-MS assay utilizes xMAP Microsphere Suspension Array technology (Luminex Corp, Austin, TX) that demonstrates great potential for large epidemiological studies in microbial and viral subtyping due to the high throughput scale.18Cowan L.S. Diem L. Brake M.C. Crawford J.T. Transfer of a Mycobacterium tuberculosis genotyping method Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system.J Clin Microbiol. 2004; 42: 474-477Crossref PubMed Scopus (124) Google Scholar, 19Huang C.T. Wong W.W. Li L.H. Chiang C.C. Chen B.D. Li S.Y. Genotyping of Chlamydia trachomatis by microsphere suspension array.J Clin Microbiol. 2008; 46: 1126-1128Crossref PubMed Scopus (12) Google Scholar, 20Geraets D.T. Heideman D.A. de Koning M.N. Snijders P.J. van Alewijk D.C. Meijer C.J. van Doorn L.J. Quint W.G. High-throughput genotyping of high-risk HPV by the digene HPV Genotyping LQ Test using GP5+/6+-PCR and xMAP technology.J Clin Virol. 2009; 46: S21-S26Abstract Full Text PDF PubMed Scopus (27) Google Scholar In the present study, we evaluated the novel Ct-MS assay in a large number of clinical samples. Ct serovars reference strains (A/Sa-1, B/TW-5, C/UW-1, D/IC-CAL-8, E/DK-20, F/MRC-301, G/IOL-238, H/UW-4, I/UW-12, J/UW-36, K/UW-31, L1/440-L, L2/434-B, and L3/404-L) and one clinical sample containing a genovariant of serovar J (referred as J*) were selected. All strains were previously sequenced to confirm presence of the correct Ct serovar strain.14Quint K.D. van Doorn L.J. Kleter B. de Koning M.N. van den Munckhof H.A. Morre S.A. ter Harmsel B. Weiderpass E. Harbers G. Melchers W.J. Quint W.G. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.J Mol Diagn. 2007; 9: 631-638Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar The DNA isolation was performed using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany), including pretreatment with proteinase K, according the manufacturer's instruction. The DNA isolates (stored at −80°C) from 712 clinical samples were selected. The samples were determined as Ct positive in previous studies with different Ct detection assays [ie, an in-house based real-time PCR assay, the Aptima Combo 2 assay, or the PACE2 assay (Gen-Probe Inc., San Diego, CA)].15Quint K.D. Bom R.J. Bruisten S.M. van Doorn L.J. Nassir Hajipour N. Melchers W.J. de Vries H.J. Morre S.A. Quint W.G. Comparison of three genotyping methods to identify Chlamydia trachomatis genotypes in positive men and women.Mol Cell Probes. 2010; 24: 266-270Crossref PubMed Scopus (30) Google Scholar, 16Bax C.J. Schachter J. Prevalence of multiple site or double chlamydia trachomatis serovar infections and the prevalence of serovar G/Ga in urogenital vs rectal specimens in male and female patients attending a sexual transmitted disease clinic or out-patient departmetn of obstetrics and gynecology. International Chlamydia Symposim, San Fransisco, CA2010: 441-444Google Scholar, 21Smelov V. Quint K.D. Pleijster J. Savelkoul P.H. Shalepo K. Shipitsyna E. Domeika M. Gorelov A. Savicheva A. Quint W.G. de Vries H.J. Ouburg S. Morre S.A. Chlamydia trachomatis serovar distributions in Russian men and women: a comparison with Dutch serovar distributions.Drugs Today (Barc). 2009; 45: 33-38PubMed Google Scholar In short, 172 samples were collected from a Russian cohort (January 2006 to 2008), including 83 male urethral samples and 89 cervical samples. Those samples were all determined Ct positive with an in-house nucleic acid amplification test, described elsewhere.22Shipitsyna E. Zolotoverkhaya E. Agne-Stadling I. Krysanova A. Savicheva A. Sokolovsky E. Domeika M. Unemo M. First evaluation of six nucleic acid amplification tests widely used in the diagnosis of Chlamydia trachomatis in Russia.J Eur Acad Dermatol Venereol. 2009; 23: 268-276Crossref PubMed Scopus (32) Google Scholar Another 50 samples were selected between January 2007 and 2009, from an outpatient sexually transmitted disease clinic (Amsterdam), consisting of 4 first void urines, 25 rectal swabs, 14 cervical swabs, 3 urethral swabs, and 4 vaginal swabs. The 25 rectal swabs were collected from men who have sex with other men. All 50 samples were determined Ct positive with the Aptima Combo 2 assay (Gen-Probe Inc.). The remaining 490 samples were selected from a second outpatient sexually transmitted disease clinic (the Hague) in 2008, including 245 cervical swabs, 98 rectal swabs, 98 first void urines, 10 pharyngeal samples, and 39 urethral samples. All 490 samples were determined Ct positive with the PACE 2 assay (Gen-Probe Inc.). In total, the 712 DNA isolates could be divided into 17.6% urethral samples, 49.4% cervical/vaginal samples, 17.3% rectal samples, 14.3% first void urines and 1.4% pharyngeal samples. All DNA isolates were transported on dry ice to DDL Diagnostic Laboratory and stored at −80°C for a maximum of 3 years. The Ct-DT assay and the Ct-MS assay are comprised of a general PCR amplification step, which uses a Ct multiplex broad-spectrum PCR primer mixture, targeting both the Omp1 VD2 region and the endogenous plasmid as previously described.14Quint K.D. van Doorn L.J. Kleter B. de Koning M.N. van den Munckhof H.A. Morre S.A. ter Harmsel B. Weiderpass E. Harbers G. Melchers W.J. Quint W.G. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.J Mol Diagn. 2007; 9: 631-638Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 23Quint K.D. de Koning M.N. Geraets D.T. Quint W.G. Pirog E.C. Comprehensive analysis of human papilloma virus and Chlamydia trachomatis in in-situ and invasive cervical adenocarcinoma.Gynecol Oncol. 2009; 114: 390-394Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar The Ct PCR primer set is designed to amplify all of the known serovars available in GenBank. Briefly, the multiplex primer set amplifies an 89 bp and a 160/157 bp fragment from the endogenous plasmid and the variable region 2 of the Omp1 gene. The PCR mixture consists of 10 μL of isolated DNA, 2.5 mmol/L MgCl2, 1 × GeneAmp PCR buffer II, 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), 0.2 mmol/L deoxynucleoside triphosphates (GE Healthcare, Buckinghamshire, UK) and 15 pmol of each primer (Eurogentec S.A., Seraing, Belgium) in a total volume of 50 μL. The PCR program includes a 9-minute preheating step at 94°C for AmlipTaq Gold activation, followed by 40 cycles of amplification (30 seconds at 94°C, 45 seconds at 60°C, and 45 seconds at 72°C) and a final 5-minute elongation step at 72°C. All PCR reactions were carried out in parallel with negative controls (water) and positive controls consisting of Ct serovar L2/434-B reference strain DNA. The primer specificity was previously determined for the other species: Chlamydophila psittaci, Chlamydophila pneumoniae, Chlamydia muridarum, Enterococcus faecalis, E. coli, Gardnerella vaginalis, Klebsiella pneumoniae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Streptococcus pyogenes, and Staphylococcus aureus. No DNA from the other species was amplified, indicating a Ct specific PCR.14Quint K.D. van Doorn L.J. Kleter B. de Koning M.N. van den Munckhof H.A. Morre S.A. ter Harmsel B. Weiderpass E. Harbers G. Melchers W.J. Quint W.G. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.J Mol Diagn. 2007; 9: 631-638Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar The Ct-DT detection and genotyping step was performed according to the manufacturer's instructions (Labo Bio-medical Products BV, Rijswijk, The Netherlands) and as previously described.14Quint K.D. van Doorn L.J. Kleter B. de Koning M.N. van den Munckhof H.A. Morre S.A. ter Harmsel B. Weiderpass E. Harbers G. Melchers W.J. Quint W.G. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.J Mol Diagn. 2007; 9: 631-638Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Briefly, the detection of Ct amplicons was performed by a DEIA. The PCR products were captured on streptavidin-coated microtiter plates, denaturated by alkaline treatment, and detected by a cocktail of digoxinenin-labeled probes. The DEIA provides an optical density value at 450 nm. Each DEIA run contained separate positive, borderline positive and negative controls and a PCR-positive control. Samples yielding optical density values equal to or higher than the borderline are considered positive. Amplicons from the Ct-DT DEIA-positive samples can be further genotyped by the Ct-DT RHA. The biotin-labeled PCR amplicons hybridize to specific probes on a nitrocellulose strip. The strip contains a probe for the endogenous plasmid, probes for the three different Ct serogroups (B, C, and I), and probes for the 14 major serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). One extra probe was added to detect a genovariant of serovar J. The Ct-MS assay (Labo Bio-medical Products BV, Rijswijk, The Netherlands) permitted simultaneous detection and identification of multiple Ct serovars in a single hybridization step. The Ct-MS assay contained a probe to detect the endogenous plasmid, probes to detect the 3 serogroups and 15 probes to detect the 14 major serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) and the genovariant J*. The probes were immobilized on color-coded Luminex beads (Luminex Corp). The Ct-MS genotyping kit was performed in the Luminex 100 IS system, according to the manufacturer's instruction. In short, 3B buffer (Labo Bio-medical Product BV) was added to the Ct bead mix to minimize background signals. Subsequently, 4 μL of Ct PCR products were added. Next, heat denaturation, hybridization under stringent temperature conditions (50°C), washing and incubation with streptavidin-conjugated R-phycoerythrin detection conjugate were followed by read-out according to the specified instrument settings, resulting in median fluorescence intensity (MFI) levels for the Ct endogenous plasmid probe, the Ct serogroup probes, and the Ct serovar probes for each specimen. The Ct-MS read-out was determined as Ct positive when the probe for the endogenous plasmid and/or the probes for genotyping were positive. The results with the Ct-MS assay were generated without knowledge of the serovar results obtained with the Ct-DT assay. The PCR product of a clinical sample with serovar strain L2 was performed in a 10-fold dilution series. The results of the dilution series were analyzed by 3% agarose gel electrophoresis, the Ct-DT detection step (DEIA), the Ct-DT genotyping step (RHA), and the Ct-MS assay. Only a semiquantitive result can be reported because no exact copy number of amplicons was known per dilution. A total of 10 μL of PCR product was mixed with 2 μL ethidium bromide, and gel electrophoresis was performed on a 3% agarose gel for 225 to 300 volt-hours in 0.5X Tris borate-EDTA buffer. Briefly, amplified DNA fragments were excised from the 3% Tris-borate-EDTA agarose gels and purified with the QIAquick gel extraction kit (Qiagen, Hilden, Germany). Purified amplicons were directly sequenced using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems). The sequenced products were subsequently analyzed using the ABI 3100-Avant Genetic Analyzer (Applied Biosystems). The resulting DNA sequences were analyzed with the Vector NTI Advance 9.0 software (Invitrogen, Breda, The Netherlands) and compared with C. trachomatis serovars present in GenBank. The overall risk of contamination with exogenous nucleic acid in our laboratory is limited to an acceptable level. Contamination is prevented by a set of measures, including a unidirectional workflow, physically separate areas for reagent preparation, nucleic acid extraction, and handling of postamplification products, decontamination of surfaces, and strict regulations concerning housekeeping, clothing, etc. The effectiveness of these measures is monitored on a continuous basis using blank controls and swabbing of critical surfaces. The actual risk of contamination is monitored by including blank controls in nucleic acid isolation and PCR runs. During the current study, no indications for contamination were observed. Personnel involved are competent to perform the work, according to the requirements of ISO9001. In the current study, two qualified individuals performed the Ct-DT assay and the Ct-MS assay. For analytical comparison of the Ct-MS assay with the Ct-DT DEIA/RHA, two threshold levels for positivity (30 MFI and 100 MFI) are presented in this study. The Ct-MS assay kit insert recommended a standard MFI threshold of 100 MFI. However, due to the low background, a lower threshold of 30 MFI was also evaluated to demonstrate the potentially higher sensitivity of the Ct-MS assay. The level of agreement between the Ct-DT DEIA/RHA and Ct-MS assay was determined using a Cohen's kappa, and a two-tailed McNemar's test was performed to investigate the differences between both assays. The level of statistical significance was set at P < 0.05. All analyses were performed in SPSS version 17.0 (Gorinchem, The Netherlands). The specificity of the Ct-MS assay was evaluated with PCR products derived from a panel of 15 Ct reference strains. The endogenous plasmid probe showed a positive result for all reference strains. The probe for serogroup B showed a specific reaction with the amplicons from the reference strains B, D, E, L1, and L2, whereas the intermediate serogroup gave a specific reaction with the amplicons from serovars F and G. The serogroup C probe showed a specific reaction for the serovars A, C, H, I, J, J*, K, and L3. Amplicons of the different genotypes demonstrated probe specific hybridization with MFI values between 400 and 7129, whereas the background read-out varied between 0 and 10 (Table 1). No cross-reaction with untargeted serovars was observed for any of the probes. As with the Ct-DT RHA, differentiation between serovar J* and serovar L3 was possible by pattern recognition because a probe specific for serovar L3 was added.Table 1Type Specificity of the Ct-MS Assay for 15 Ct Reference StrainsChlamydia trachomatis genotypes/serovarsGroup BGroup IGroup CProbeBDEL1L2FGACHIJJvarKL3Conj857485727444879586408273855582277858823486948224923891878668Ct-CP⁎Probe for endogenous plasmid.262424962511274229162635266625812376241426632409193125392465Serogroup probes Ct-gB1764329019922480178611101111110 Ct-gI722223363288823232222 Ct-gC222222235743176301741073204251420732702Serovar probes Ct-B/Ba525022131111011111 Ct-D/Da119801111211141111 Ct-E111876111111111111 Ct-L1112267921221112112 Ct-L2/L2a433630303354333234 Ct-F233234712222223322 Ct-G/Ga333234136033333232 Ct-A222222256452322222 Ct-C111211131221112214 Ct-H111112221197911111 Ct-I/Ia222222344227694233 Ct-J222223234221732252 Ct-J/L32222222323437542400 Ct-K222222232222224472 Ct-L3221121212121227129Analytical type specificity of the Ct-MS assay. Indicated are the MFIs read outs of the Ct-DT PCR amplicons generated from Ct reference strains (listed on top) in relation to the bead-bound capture probes (listed on the left). The conjugate control (conj.) serves as the positive control for correct incubation with the detection conjugate for each separate serovar.Bold numbers indicate a positive MFI value. Probe for endogenous plasmid. Open table in a new tab Analytical type specificity of the Ct-MS assay. Indicated are the MFIs read outs of the Ct-DT PCR amplicons generated from Ct reference strains (listed on top) in relation to the bead-bound capture probes (listed on the left). The conjugate control (conj.) serves as the positive control for correct incubation with the detection conjugate for each separate serovar. Bold numbers indicate a positive MFI value. The results of the serovar L2 amplicons 10-fold dilution series is reported in Table 2. No differences in the limit of detection (LOD) for the endogenous plasmid was observed between the Ct-DT DEIA (LOD: 10−3 dilution), Ct-DT RHA (LOD: 10−3 dilution), and the Ct-MS assay at a 30 MFI threshold (LOD: 10−3 dilution). However, the agarose gel (LOD: 10−1 dilution) was less sensitive. On genomic level (ie, serovar and serogroup), an LOD was observed for the Ct-MS assay (threshold, 30 MFI) at a 10−2 dilution, whereas the agarose gel shows an LOD at a 10−1 dilution. For the Ct-DT RHA, the lowest LOD was observed (LOD: 10−3 dilution).Table 2Dilution Series of Serovar L2 AmpliconsSample dilution3% agarose gelCt-DT detectionCt-DT genotyping (RHA)Ct-MS assay (MFI value)Plasmid (89 bp)Omp1 (160 bp)(DEIA)PlasmidGroup BSerovar L2PlasmidGroup BSerovar L2Undiluted++++++615516432782L2 10−1++++++3958387905L2 10−2−−++++113041110L2 10−3−−++++11338L2 10−4−−−−−−913L2 10−5−−−−−−202Results were observed with a 3% agarose gel, the Ct-DT detection (DEIA) and genotyping (RHA) step and the Ct-MS assay (threshold 30 MFI).Bold numbers indicate a positive MFI value. Open table in a new tab Results were observed with a 3% agarose gel, the Ct-DT detection (DEIA) and genotyping (RHA) step and the Ct-MS assay (threshold 30 MFI). Bold numbers indicate a positive MFI value. First a comparison was made with the Ct-DT DEIA for Ct detection because the Ct-MS assay combines Ct detection and Ct genotyping. In total, 712 clinical samples were analyzed with the Ct-DT assay and the Ct-MS assay. Overall, 670 samples were Ct positive with both the Ct-DT DEIA and Ct-MS assays and 38 samples were negative with both assays (Table 3). Four samples showed a discordant result, of which 2 were only positive with the Ct-MS assay and 2 were only positive with the Ct-DT DEIA. An excellent agreement between the Ct-DT DEIA and the Ct-MS assay was observed (kappa = 0.947; 95% CI = 0.895 to 0.999; McNemar's test, P = 1.000) (Table 3). No difference in Ct detection was observed when the MFI threshold was lowered from 100 MFI to 30 MFI.Table 3Comparison between the Ct-DT DEIA Assay and the Ct-MS Assay for Ct Detection (n = 712)Ct-MS assayCt-DT assay+−DEIA+6702DEIA−238Kappa = 0.947; 95% confidence interval = 0.895 to 0.999; McNemar's test, P = 1.000. Open table in a new tab Kappa = 0.947; 95% confidence interval = 0.895 to 0.999; McNemar's test, P = 1.000. In the 712 clinical samples, the probe specific genotyping results revealed a complete concordance between both assays in respectively 690 (96.9%)

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