Abstract
Pathogenic microbes may colonize the female genital tract via sexual transmission and cause health issues like inflammation or malignancy, summarized as sexually transmitted disease (STD). A major representative of such pathogens is Trichomonas vaginalis (T.v.), whose role in the etiology of cervical cancer remains elusive. Traditional morphologic screening of cervical smears is able to detect T.v., although its identification may be complicated by look-alikes such as degenerated granulocytes and basal cells. In addition, the parasite’s endosymbiont Mycoplasma hominis (M.h.) cannot be detected in the Pap test. This investigation was aimed at designing a PCR-based method to detect specific pathogenic germs by using cervical cytology slides to overcome morphologic uncertainty and increase diagnostic accuracy. To test our molecular screening method on T.v., M.h., and HPV in archival smears, we elaborated a multiplex PCR approach based on microdissection. This assay was applied to a minute quantity of starting material which harbored or was suspected to harbor T.v.; the resulting isolated DNA was used for subsequent molecular analyses of T.v., M.h., and HPV. We clarified the diagnosis of genital T.v. infection in 88 and 1.8% of morphologically suspicious and T.v.-negative cases, respectively. We also revealed a tendency of M.h. co-infection in high-risk HPV cases. In conclusion, a microdissection-based approach to detect pathogenic microbes such as T.v., HPV, and M.h. is a molecular tool easy to implement and may help to better understand the interactivity of these germs with respect to pathogenesis.
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Adebamowo SN, Ma B, Zella D, Famooto A, Ravel J, Adebamowo C (2017) Mycoplasma hominis and Mycoplasma genitalium in the vaginal microbiota and persistent high-risk human papillomavirus infection. Front Public Health 5:140
Aslan DL, Gulbahce HE, Stelow EB, Setty S, Brown CA, McGlennen RC, Pambuccian SE (2005a) The diagnosis of Trichomonas vaginalis in liquid-based Pap tests: correlation with PCR. Diagn Cytopathol 32:341–344
Aslan DL, McKeon DM, Stelow EB et al (2005b) The diagnosis of trichomonas vaginalis in liquid-based Pap tests: morphological characteristics. Diagn Cytopathol 32:253–259
Bachmann LH, Hobbs MM, Seña AC, Sobel JD, Schwebke JR, Krieger JN, McClelland RS, Workowski KA (2011) Trichomonas vaginalis genital infections: progress and challenges. Clin Infect Dis 53(Suppl 3):S160–S172
Barykova YA, Logunov DY, Shmarov MM, Vinarov AZ, Fiev DN, Vinarova NA, Rakovskaya IV, Baker PS, Shyshynova I, Stephenson AJ, Klein EA, Naroditsky BS, Gintsburg AL, Gudkov AV (2011) Association of Mycoplasma hominis infection with prostate cancer. Oncotarget 2:289–297
Blanchard A, Yanez A, Dybvig K (1993) Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction. J Clin
Boulet GAV, Horvath CAJ, Berghmans S, Moeneclaey LM, Duys ISM, Arbyn M, Depuydt CE, Vereecken AJ, Sahebali S, Bogers JJ (2008) Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears. J Clin Pathol 61:637–641
Calderaro A, Piergianni M, Montecchini S, Buttrini M, Piccolo G, Rossi S, Arcangeletti MC, Medici MC, Chezzi C, de Conto F (2016) MALDI-TOF mass spectrometry as a potential tool for Trichomonas vaginalis identification. BMC Infect Dis 16:261
Castellsagué X, Bosch FX, Muñoz N (2002) Environmental co-factors in HPV carcinogenesis. Virus Res 89:191–199
Castle PE, Giuliano AR (2003) Chapter 4: genital tract infections, cervical inflammation, and antioxidant nutrients—assessing their roles as human papillomavirus cofactors. J Natl Cancer Inst Monogr 2003:29–34
Chen JT, Lane MA, Clark DP (1996) Inhibitors of the polymerase chain reaction in Papanicolaou stain. Removal with a simple destaining procedure. Acta Cytol 40:873–877
Cotch MF, Pastorek JG 2nd, Nugent RP et al (1997) Trichomonas vaginalis associated with low birth weight and preterm delivery. The Vaginal Infections and Prematurity Study Group. Sex Transm Dis 24:353–360
Crosetto N, Bienko M, van Oudenaarden A (2015) Spatially resolved transcriptomics and beyond. Nat Rev Genet 16:57–66
Dallo SF, Baseman JB (2000) Intracellular DNA replication and long-term survival of pathogenic mycoplasmas. Microb Pathog 29:301–309
Diaz N, Dessì D, Dessole S, Fiori PL, Rappelli P (2010) Rapid detection of coinfections by Trichomonas vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum by a new multiplex polymerase chain reaction. Diagn Microbiol Infect Dis 67:30–36
Donders GGG, Depuydt CE, Bogers J-P, Vereecken AJ (2013) Association of Trichomonas vaginalis and cytological abnormalities of the cervix in low risk women. PLoS One 8:e86266
Donné MA (1836) Animacules observes dans les matieres purulentes et le produit des secretions des organes genitaux de l’homme et de la femme. CR Acad Sci 3:e6
Fiori PL, Diaz N, Cocco AR, Rappelli P, Dessì D (2013) Association of Trichomonas vaginalis with its symbiont Mycoplasma hominis synergistically upregulates the in vitro proinflammatory response of human monocytes. Sex Transm Infect 89:449–454
Frank TS, Svoboda-Newman SM, Hsi ED (1996) Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR. Diagn Mol Pathol 5:220–224
Garrow SC, Smith DW, Harnett GB (2002) The diagnosis of chlamydia, gonorrhoea, and trichomonas infections by self obtained low vaginal swabs, in remote northern Australian clinical practice. Sex Transm Infect 78:278–281
Ghosh I, Muwonge R, Mittal S, Banerjee D, Kundu P, Mandal R, Biswas J, Basu P (2017) Association between high risk human papillomavirus infection and co-infection with Candida spp. and Trichomonas vaginalis in women with cervical premalignant and malignant lesions. J Clin Virol 87:43–48
Grodstein F, Goldman MB, Cramer DW (1993) Relation of tubal infertility to history of sexually transmitted diseases. Am J Epidemiol 137:577–584
Hansen LK, Kolle-Jorgensen P (1955) Demonstration of Trichomonas vaginalis donné by direct microscopy and stained smear; comparison. Ugeskr Laeger 117:1373–1376
Herbert A, Bergeron C, Wiener H, Schenck U, Klinkhamer P, Bulten J, Arbyn M (2007) European guidelines for quality assurance in cervical cancer screening: recommendations for cervical cytology terminology. Cytopathology 18:213–219
Ho YK, Xu WT, Too HP (2013) Direct quantification of mRNA and miRNA from cell lysates using reverse transcription real time PCR: a multidimensional analysis of the performance of reagents and workflows. PLoS One 8:e72463
Hobbs MM, Seña AC (2013) Modern diagnosis of Trichomonas vaginalis infection. Sex Transm Infect 89:434–438
Hopfe M, Deenen R, Degrandi D, Köhrer K, Henrich B (2013) Host cell responses to persistent mycoplasmas—different stages in infection of HeLa cells with Mycoplasma hominis. PLoS One 8:e54219
Kissinger P (2015) Trichomonas vaginalis: a review of epidemiologic, clinical and treatment issues. BMC Infect Dis 15:307
Krishnamurthy S (2015) Biospecimen repositories and cytopathology. Cancer Cytopathol 123:152–161
Lamont RF, Sobel JD, Akins RA, Hassan SS, Chaiworapongsa T, Kusanovic JP, Romero R (2011) The vaginal microbiome: new information about genital tract flora using molecular based techniques. BJOG 118:533–549
Mongelos P, Mendoza LP, Rodriguez-Riveros I, Castro A, Gimenez G, Araujo P, Paez M, Castro W, Basiletti J, González J, Echagüe G, Diaz V, Laspina F, Ever S, Marecos R, Deluca G, Picconi MA (2015) Distribution of human papillomavirus (HPV) genotypes and bacterial vaginosis presence in cervical samples from Paraguayan indigenous. Int J Infect Dis 39:44–49
Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, Snijders PJ, Meijer CJ, International Agency for Research on Cancer Multicenter Cervical Cancer Study Group (2003) Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348:518–527
Nathan B, Appiah J, Saunders P, Heron D, Nichols T, Brum R, Alexander S, Baraitser P, Ison C (2015) Microscopy outperformed in a comparison of five methods for detecting Trichomonas vaginalis in symptomatic women. Int J STD AIDS 26:251–256
Nayar R, Wilbur D (2015) The Bethesda system for reporting cervical cytology: definitions, criteria, and explanatory notes. Springer International Publishing
Oktay MH, Adler E, Hakima L, Grunblatt E, Pieri E, Seymour A, Khader S, Cajigas A, Suhrland M, Goswami S (2016) The application of molecular diagnostics to stained cytology smears. J Mol Diagn 18:407–415
Okuyama T, Takahashi R, Mori M, Osaka M, Kobayashi TK, Maeda S (1998) Polymerase chain reaction amplification of Trichomonas vaginalis DNA from Papanicolaou-stained smears. Diagn Cytopathol 19:437–440
Petrin D, Delgaty K, Bhatt R, Garber G (1998) Clinical and microbiological aspects of Trichomonas vaginalis. Clin Microbiol Rev 11:300–317
Rappelli P, Carta F, Delogu G, Addis MF, Dessì D, Cappuccinelli P, Fiori PL (2001) Mycoplasma hominis and Trichomonas vaginalis symbiosis: multiplicity of infection and transmissibility of M. hominis to human cells. Arch Microbiol 175:70–74
Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SSK, McCulle SL, Karlebach S, Gorle R, Russell J, Tacket CO, Brotman RM, Davis CC, Ault K, Peralta L, Forney LJ (2011) Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A 108(Suppl 1):4680–4687
Riley DE, Roberts MC, Takayama T, Krieger JN (1992) Development of a polymerase chain reaction-based diagnosis of Trichomonas vaginalis. J Clin Microbiol 30:465–472
Roeters AME, Boon ME, van Haaften M, Vernooij F, Bontekoe TR, Heintz AP (2010) Inflammatory events as detected in cervical smears and squamous intraepithelial lesions. Diagn Cytopathol 38:85–93
Rogers MB (2011) Mycoplasma and cancer: in search of the link. Oncotarget 2:271–273
Schmitt F, Barroca H (2012) Role of ancillary studies in fine-needle aspiration from selected tumors. Cancer Cytopathol 120:145–160
Sellors JW, Sankaranarayanan R (2003) Colposcopy and treatment of cervical intraepithelial neoplasia: a beginner’s manual. International Agency for Research on Cancer, Lyon
Sutcliffe S, Giovannucci E, Alderete JF et al (2006) Plasma antibodies against Trichomonas vaginalis and subsequent risk of prostate cancer. Cancer Epidemiol Biomark Prev 15:939–945
Sutton M, Sternberg M, Koumans EH, McQuillan G, Berman S, Markowitz L (2007) The prevalence of Trichomonas vaginalis infection among reproductive-age women in the United States, 2001-2004. Clin Infect Dis 45:1319–1326
Tao L, Han L, Li X, Gao Q, Pan L, Wu L, Luo Y, Wang W, Zheng Z, Guo X (2014) Prevalence and risk factors for cervical neoplasia: a cervical cancer screening program in Beijing. BMC Public Health 14:1185
Taylor-Robinson D (2017) Mollicutes in vaginal microbiology: Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum and Mycoplasma genitalium. Res Microbiol 168:875–881. https://doi.org/10.1016/j.resmic.2017.02.009
Twu O, Dessí D, Vu A et al (2014) Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses. Proc Natl Acad Sci U S A 111:8179–8184
van Beers EH, Joosse SA, Ligtenberg MJ, Fles R, Hogervorst FBL, Verhoef S, Nederlof PM (2006) A multiplex PCR predictor for aCGH success of FFPE samples. Br J Cancer 94:333–337
Van Peer G, Mestdagh P, Vandesompele J (2012) Accurate RT-qPCR gene expression analysis on cell culture lysates. Sci Rep 2:222
Vancini RG, Pereira-Neves A, Borojevic R, Benchimol M (2008) Trichomonas vaginalis harboring Mycoplasma hominis increases cytopathogenicity in vitro. Eur J Clin Microbiol Infect Dis 27:259–267
Waites KB, Katz B, Schelonka RL (2005) Mycoplasmas and ureaplasmas as neonatal pathogens. Clin Microbiol Rev 18:757–789
Walboomers JM, Jacobs MV, Manos MM et al (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189:12–19
WHO (2012) Global incidence and prevalence of selected curable sexually transmitted infections—2008. WHO, Geneva, p 2012
WHO (2014) WHO Guidelines for Screening and Treatment of Precancerous Lesions for Cervical Cancer Prevention. World Health Organization, Geneva
Zhang ZF, Graham S, Yu SZ, Marshall J, Zielezny M, Chen YX, Sun M, Tang SL, Liao CS, Xu JL, Yang XZ (1995) Trichomonas vaginalis and cervical cancer. A prospective study in China. Ann Epidemiol 5:325–332
Zhang D, Li T, Chen L, Zhang X, Zhao G, Liu Z (2017) Epidemiological investigation of the relationship between common lower genital tract infections and high-risk human papillomavirus infections among women in Beijing, China. PLoS One 12:e0178033
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This study was financed by the Institute of Pathology, Technical University Munich, Germany. We like to thank Mrs. Jutta Witmann-Pitzl for technical support.
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The Ethics Committee of the Technical University of Munich approved this study (number 184/15). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Preliminary data from this manuscript has been presented as a poster in the Annual European Urogynaecological Association (EUGA) Meeting on November 3–5, 2016, Amsterdam
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Nikas, I., Hapfelmeier, A., Mollenhauer, M. et al. Integrated morphologic and molecular analysis of Trichomonas vaginalis, Mycoplasma hominis, and human papillomavirus using cytologic smear preparations. Parasitol Res 117, 1443–1451 (2018). https://doi.org/10.1007/s00436-018-5829-3
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DOI: https://doi.org/10.1007/s00436-018-5829-3