JCM Accepts, published online ahead of print on 24 December 2014 J. Clin. Microbiol. doi:10.1128/jcm.03370-14 Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 3 4 5 6 7 8 9 10 11 12 13 Assessment of co-infection of Sexually Transmitted pathogen microbes by performing the molecular kit Anyplex TM II STI-7 B. Berçot 1,2,3,, R. Amarsy 1,2,3, A. Goubard 4, C. Aparicio 5, H.U. Loeung 5, C. Segouin 6, D. Gueret 1, H. Jacquier 1,2,3, F. Meunier 1, F. Mougari 1,2,3, E. Cambau 1,2,3. 1 APHP, Lariboisière Fernand Widal Hospital, Laboratory of Bacteriology, F-75010 Paris, France, Associated Laboratory for the National Reference Centre for gonococci, F-75010 Paris, France, 2 INSERM, IAME, UMR 1137, F-75018 Paris, France, 3 University Paris Diderot, IAME, UMR 1137, Sorbonne Paris Cité, F-75018 Paris, France, 4 Institut Fournier, National Reference Centre for gonococci, F-75014 Paris, France, 5 Policlinic PASS, APHP, Lariboisière Hospital, F-75010 Paris, France, 6 Public Health and Health Economics Department, AP-HP, Lariboisière Hospital, F-75010 Paris, France. 14 15 16 17 18 19 20 21 22 Correspondence: Mailing address: Béatrice Berçot, Service de Bactériologie-Virologie, Groupe Hospitalier Saint Louis-Lariboisière-Fernand Widal, 2, rue Ambroise-Paré, Paris 75010, France. Phone: 33-1-49-95-65-54 / Fax: 33-1-49-95-85-37 E-mail: beatrice.bercot@lrb.aphp.fr Running title: Evaluation of the Anyplex TM II STI-7 Keywords: STD, STI, Chlamydiae trachomatis, Neisseria gonorrhoeae, Ureaplasma parvum Real time PCR, Anyplex II STI-7 23 1
24 25 26 27 28 Abstract Anyplex STI-7 is a new molecular kit detecting seven sexually transmitted pathogens. Among 202 subjects screened for genital infection, 143 (70.4%) were diagnosed with at least one pathogen, in concordance with reference methods. In addition, it demonstrated co-infections, such as that of Ureaplasma parvum and Chlamydia trachomatis in young women. 29 2
30 31 32 33 34 35 36 37 38 39 40 41 42 Text Since consequences of genital infections through sexually transmitted diseases (STD) can be as severe as sterility and disseminated infections, molecular assays detecting bacterial pathogens such as Chlamydiae trachomatis and Neisseria gonorrhoeae were shown to be relevant not only in patent infections but also in pauci-symptomatic sexually active women (1, 2). Several real time PCR assays commercially available enable this double detection (3). Worldwide, Trichomonas vaginalis is the first pathogen responsible of STD, but it remains poorly diagnosed as well as Mollicutes (Ureaplasmas and Mycoplasmas) which are difficult to cultivate (4-6). These latter bacteria are involved in early pregnancy loss, stillbirth, preterm birth and neonatal morbidities as in male infertility (6-9), although they are also commonly identified in the vaginal flora of 40-80% of healthy women depending on the population (6, 7). Among them, U parvum is a pathogen ignored. The Anyplex TM II STI-7 kit (STI-7, Seegene, Eurobio) is a multiplex real-time PCR assay 43 relying on a newly developed TOCE TM technology (Tagging Oligonucleotide Cleavage and 44 45 46 47 48 49 50 51 52 53 Extension). This assay is marketed to detect simultaneously seven microorganisms involved in sexually transmitted infections: C. trachomatis (CT), N. gonorrhoeae (NG), T. vaginalis (TV), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and U. parvum (UP). Our study aimed to evaluate its performances for diagnosing and screening for STD in our hospital cohort. Results were compared to validated methods. In addition, we seek for the co-existence of pathogens, which is usually undetected. Two or more pathogens were highly prevalent especially in sexually active young women showing or not infection symptoms. Two hundred thirteen specimens from 202 patients were tested from January to June 2012: 94 vaginal swabs, 46 cervical swabs, 61 first void urines, and 12 pelvic aspirated fluids. Cervical and urethral swabs were discharged in 3 ml of M4-RT medium (Abbott) and other 3
54 55 56 57 58 59 60 61 samples were put into sterile tubes with no additives and transported to the lab within 12 hours. All specimens were submitted to the Abbott RealTime CT/NG assay (Abb CT/NG ) used routinely in our lab for detection of CT and NG. Samples were processed and interpreted as recommended by the manufacturer using the Abbott m2000 apparatus. Results were given as positive, negative or equivocal results ; these latter being retested and reclassified positive or negative. For the STI-7 assay, DNA was extracted from 190 μl of the sample (M4-RT medium, urines or fluid) by using the EZ1 extractor with the EZ1 DNA tissue kit (Qiagen, France). Then, real time PCR was done as recommended by manufacturer s guidelines using 3 μl of DNA extract with the STI-7 62 detection assay on CFX96 TM Bio-Rad Thermocycler associated with the IVD software. An 63 64 65 66 67 68 69 70 71 72 73 74 75 76 internal control (IC) was included in each reaction and results were given as positive or negative. Cultivation of UU, UP and MH was performed in Mycoplasma Duo suspension medium tube (Biorad, France) and Mycoplasma MYCO agar (BioMérieux, France) and that of NG onto PolyViteX Chocolate VCAT agar (BioMérieux, France) incubated in 5% CO2 atmosphere, respectively. Detection of T. vaginalis was done by microscopic examination. STI-7 results were compared with other methods (Abb CT/NG test for CT and NG, culture for UU/UP/MH, microscopic examination for TV) taken as references and STI-7 performances (sensitivity, specificity, positive and negative predictive values) are presented in Table 1. Concordance with Abb CT/NG was 93.4% for CT and 97.2% for NG detection. These values seem acceptable with regard to new CDC guidelines, which reported a concordance > 90% between tests for all the studies performed since 2002 (2). The discrepancies observed between the two assays were investigated by analysing clinical data of the patients, and the result of a third technique (Xpert CT/NG, Cepheid) (Table 2). Nine and six discrepancies were observed for CT and NG detection, respectively. In six cases, the patients were considered as infected by the 4
77 78 79 80 81 82 83 84 85 86 87 88 89 90 clinician and treated. In the other cases, we noticed some difficulties to conclude especially for the asymptomatic women. In most of these cases, the cycle threshold appeared late. Among the 202 subjects included, 184 (91%) were females, 6 have a history of hepatitis B and one was HIV-positive. Median age was 26.6 years (range, 15-56 years). The detection was positive for at least one pathogen in 143 patients (70.7 %). Prevalence of each pathogen was detailed in Table 3. One, two or three pathogens were found in 67 (33%), 55 (27%) and 21 (10%) patients, respectively. For all pathogens, including CT, NG and TV that are well recognized as genital pathogenic micro-organisms, the prevalence of each pathogen was higher in co-infection than alone: 60% for UU, 61% for NG, 74% for UU, 82% for CT, 96% for MG, and 100% for TV and MH). Moreover, the presence of U. parvum was significantly correlated with the presence of C. trachomatis, which corroborates that was recently described (10). It was shown indeed that U. parvum can favour the persistence of CT in Hela cells by inhibiting the action of interferon- (11) and consequently that the coinfection U. parvum / C. trachomatis could promote the establishment of a chronic infection to C trachomatis. 91 92 93 94 95 96 97 98 99 Finally, we compared the prevalence of each pathogen with regard to clinical signs of infection. We observed that for all pathogens, except NG, the prevalence was higher in paucisymptomatic sexually active young women consulting for SDT screening at Free anonymous HIV testing Centre than in patients consulting at the Hospital or hospitalized for patent infections (urethritis, salpingitis, leucorrhea, arthritis, peritonitis, ) (Table 3). This confirms that they are at high risk of STD and genital infections and that screening for all pathogens may be worthwhile in addition to CT and NG, which are already recommended. The association of U. parvum and C. trachomatis was also significantly higher (p< 0.001) in these young women at risk of STD (Table 3). 5
100 101 102 103 Our study reports the first comparison of the STI-7 method to the Abb CT/NG, which is widely widespread and used in the world. The STI-7 assay has the interest to detect simultaneously several STD pathogens by sensitive and specific manner and at lower cost when the other kit detects only N. gonorrhoeae and C. trachomatis. 104 105 106 ACKNOWLEDGMENTS We thank Eurobio for providing 25% of the kits for this study. 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 REFERENCES 1. Bignell C, Unemo M. 2013. European STI Guidelines Editorial Board. 2012 European guideline on the diagnosis and treatment of gonorrhoea in adults. Int. J. STD. AIDS. 24:85-92. 2. Papp JR, Schachter J, Gaydos CA, Van Der Pol B. 2014. Recommendations for the Laboratory-Based Detection of Chlamydia trachomatis and Neisseria gonorrhoeae. MMWR Recomm. Rep. 63(RR-02):1-19. 3. Tabrizi SN, Unemo M, Limnios AE, Hogan TR, Hjelmevoll SO, Garland SM, Tapsall J. 2011. Evaluation of six Commercial Nucleic Acid Amplification Tests for Detection of Neisseria gonorrhoeae and Other Neisseria species. J. Clin. Microbiol. 49:3610-3615. 4. Schwebke JR, Hook EW 3rd. 2003. High rates of Trichomonas vaginalis among men attending a sexually transmitted diseases clinic: implications for screening and urethritis management. J. Infect. Dis. 188:465 468. 5. Ross JD, Jensen JS. 2006. Mycoplasma genitalium as a sexually transmitted infection: implications for screening, testing, and treatment. Sex. Transm. Infect. 82:269 271. 6. Gwee A, Curtis N. 2014. Ureaplasma--Are you sitting comfortably? J. Infect. 68:S19-23. 6
124 125 126 127 128 129 130 131 132 133 134 135 7. Waites KB, Katz B, Schelonka RL. 2005. Mycoplasmas and Ureaplasmas as Neonatal Pathogens. Clin. Microbiol. Rev. 18:757-789. 8. Viscardi RM. 2010. Ureaplasma species: role in diseases of prematurity. Clin. Perinatol. 37:393 409. 9. Capoccia R, Greub G, Baud D. 2013. Ureaplasma urealyticum, Mycoplasma hominis and adverse pregnancy outcomes. Curr. Opin. Infect. Dis. 26:231-240. 10. Yamazaki T, Matsumoto M, Matsuo J, Abe K, Minami K and Yamaguchi H. 2012. Frequency of Chlamydia trachomatis in Ureaplasma-positive healthy women attending their first prenatal visit in a community hospital in Sapporo, Japan. BMC Infect. Dis. 12:82. 11. Yamazaki T, Matsuo J, Nakamura S, Oguri S, Yamaguchi H. 2014. Effect of Ureaplasma parvum co-incubation on Chlamydia trachomatis maturation in human epithelial HeLa cells treated with interferon. J. Infect. Chemother. 20:460-464. 136 7
Table 1: Performances of molecular detection by the Anyplex STI-7 compared (vs.) for N. gonorrhoeae (NG) and C. trachomatis (CT) detection to results of the Abbott RT CT/NG assays and, for U. urealyticum (UU), U. parvum (UP) and M. hominis (MH) to the results of cultures on 202 patients. NG STI7 vs NG Abbott CT STI7 vs CT Abbott UU or UP vs culture a MH vs culture Sensitivity (95%IC) 90 (86-94) 85.1 (80.4-89.9) 83.3 (74.9-91.8) 70 (59.7-80.3) Specificity (95%IC) 98.4 (NA) 97.8 (NA) 76.5 (66.9-86.1) 97 (NA) PPV (95%IC) 90 (85.9-94.1) 95.5 (92.6-98.3) 62.5 (51.3-73.7) 77.8 (68.2-87.3) NPV (95%IC) 98.4 (NA) 92.5 (88.9-96.1) 90.7 (84-97.4) 95.5 (NA) Concordance (95%IC) 97.2 (94.9-99.5) 93.4 (90-96.8) 78.7 (69.2-88.1) 93.4 (87.7-99.1) Kappa (95%IC) 88.4 (84-92.8) 85.1 (80.3-90) 55 (43.5-66.4) 69.9 (59.4-80.5) PPV: Predictive Positive Value; PNV: Predictive Negative Value; NA, non applicable, is indicated when statistical analysis cannot be performed. a Results of molecular method from UU and UP are compared together to those of cultures because UU and UP cannot be differentiated by cultures.
Table 2: Characteristics of subjects and detail of the results showing discrepancy between Abb CT/NG and STI-7 assays for detection for CT or for NG Sample STI7 assay Abbott assay Cepheid assay Clinical data Associated Conclusions (cycle threshold) (cycle threshold) (cycle threshold) pathogen Discrepant results for CT Urine NEG POS (34) POS (31) Leucorrhoea UU a, TV a, NG a Infected Urine NEG POS (28) POS (29) Leucorrhoea Ureaplama sp b Infected Vaginal swabs NEG POS (38) NEG Asymptomatic NG a Uncertain Vaginal swabs NEG POS (40) NEG Asymptomatic none Uncertain Vaginal swabs NEG POS (>42; 37.65) c POS (36) Asymptomatic UP and MH a Uncertain Pelvic aspirate fluid POS NEG d POS (38) Salpingitis none Infected Vaginal swabs NEG NEG (>42; 42.07) NEG Asymptomatic NG a Non infected Vaginal swabs POS NEG POS (31) Asymptomatic UP a Uncertain Vaginal swabs POS NEG POS (34) Asymptomatic UP a Uncertain Discrepant results for NG Urine NEG d POS (38) POS (34/33) e Fitz Hugh Curtis syndrome Urine NEG POS (38) POS (38/39) e Left ankle monoarthritis G. vaginalis, C. albicans, U sp b Infected POS: positive; NEG: negative; a pathogen found in PCR only; b pathogen found in culture; c for CT Abbott results, the two values of Cycle Threshold were indicated when results were given first as equivocal results ; d Test found positive on another sample from the same patient e For NG Cepheid result, the first and the second value of Cycle Threshold indicated correspond to the NG2 and the NG4 targets, respectively. Any CT is available for the Anyplex method. none Infected Vaginal swabs NEG POS (33) NEG (44/0) e Asymptomatic CT Uncertain Vaginal swabs POS NEG NEG Asymptomatic CT Uncertain Vaginal swabs POS NEG (43.6) POS (36/34) e Asymptomatic UP/MH/CT Infected Vaginal swabs POS NEG NEG Asymptomatic CT Uncertain
Table 3. Comparison of the frequency of pathogens or Mollicutes detection in the symptomatic or paucisymptomatic population. Symptomatic patient (n=109) Paucisymptomatic patient (n= 93) p value UP (n=87) 31 56 <0.001 Coinfection UP/CT 6 25 <0.001 UU (n=34) 11 23 0.005 MH (n=25) 11 14 0.286 MG (n=7) 2 5 0.17 TV (n=7) 2 5 0.17 NG (n=28) 18 10 0.23 CT (n=61) 22 39 <0.001 Statistical analyses were performed by a X 2 test for UU and UP and Fisher test for MH and MG.