Detection of urinary Chlamydia trachomatis, Mycoplasma genitalium and human papilloma virus in the first trimester of pregnancy by PCR method

Detection of urinary Chlamydia trachomatis, Mycoplasma genitalium and human papilloma virus in... Background: Miscarriage and preterm delivery are the most important challenges of pregnancy. Different bacterial and viral infection may cause miscarriage and preterm delivery. Among bacterial factors, Mycoplasma genitalium and Chlamydia trachomatis have the most important role and human papilloma virus (HPV ) is the leading viral factor in this regard. Methods: First void urine samples were collected from 119 pregnant women who visited health centers for routine first-trimester screening (12–14 weeks gestation). About 10 ml of the sample was centrifuged at 3000×g for 20 min and 1–2 ml of the sediment was transferred to sterile microfuges and stored at − 20 °C until analysis. DNA extraction was conducted using A101211 kits imported by Pars Tous Biotechnology Company. The following commercial kits, imported by Pars Tous Biotechnology, were used for PCR. Results: There is no significant association between urinary isolation of C. trachomatis and miscarriage (P = 0.93) and there is no significant association between urinary isolation of M. genitalium and miscarriage (P = 0.80). Regard- ing HPV, since all urine samples were PCR-negative, comparison was not possible. C. trachomatis was isolated from the urine samples of 6.72% of the pregnant women who underwent first-trimester screening in health centers using PCR. Previous studies reported a mean chlamydia isolation rate of 3% from urine specimens collected from pregnant women in general. T test showed no significant difference between the two groups (P = 0.10). Based on present study the mycoplasma isolation rate was 17.65% using PCR. Previous studies reported a mean mycoplasma isolation rate of 10% from urine specimens collected from pregnant women in general. T-test showed a significant difference between the two groups (P = 0.03). Discussion: First void urine samples in pregnant women may be an appropriate sample for detection of C. tra- chomatis and M. genitalium; however, it is not a good method for HPV isolation therefore vaginal or cervical discharge specimens should be used instead for detection of HPV. Keywords: Pregnant women, Miscarriage, Chlamydia trachomatis, Mycoplasma genitalium, Human papilloma virus, Urine sample *Correspondence: rrahimkhani@sina.tums.ac.ir Department of Lab Medical Sciences, Faculty of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 2 of 7 abortion, which have shown non-significant associations Importance [2, 3]. In the present study, we decided to investigate three Similar to mycoplasma and chlamydia, the samples major causes of STDs that also cause miscarriage in the used for detection of HPV include swab specimens sam- first trimester in a number of pregnant women. Since pled from the cervix and urine. cervical swabbing is invasive and dangerous and is not In the present study, we decided to investigate three accepted by pregnant women, first void urine specimens major causes of sexually transmitted disease (STDs) that were used in this study. Among bacterial agents, Chla- also cause miscarriage in the first trimester in a number mydia trachomatis and Mycoplasma genitalium, and of pregnant women. Since cervical swabbing is invasive among viral agents, HPV, have a major role in miscar- and dangerous and is not accepted by pregnant women, riage. These agents were surveyed in present research. early urine specimens were used in this study. Background Methods Miscarriage is one of the greatest risks threatening the Considering a precision of 7% to detect the difference life of the mother and fetus. Different bacterial, viral, and and a prevalence of 2.8% for C. trachomatis, 10% for M. parasitic factors can cause miscarriage. Statistics sug- genitalium, and 8.4% for HPV, the sample size was esti- gest that more than 90% of miscarriages are related to mated 119, 61, and 66, respectively. Finally, the largest an infectious or inflammatory factor in the placenta or sample size was considered for the study by this formula: amniotic fluid [1]. Among bacterial agents, Chlamydia trachomatis and Mycoplasma genitalium, and among 2 NZ P(1 − P) n = . viral agents, human papilloma virus (HPV), have a major d (N − 1) + Z P(1 − P) role in miscarriage. Chlamydia trachomatis is an obligate intracellu- lar, nonmotile, Gram-negative bacterium in the family In this study, urine samples were collected from 119 Chlamydiaceae. Because it is an intracellular parasite, it pregnant women who visited Almahdi Clinic Center does not grow on bacteriologic cultures, and cellular cul- in south of Tehran for routine first-trimester screening tures are used for its isolation and reproduction. Chla- (12–14  weeks gestation). Almahdi Clinic is under the mydia trachomatis is the major cause of trachoma but it supervision of the Tehran University of Medical Sciences. can also cause miscarriage, fetal anomaly, endometritis, The sampling duration time was October 2016 to August preterm delivery, and stillbirth. Since this bacterium is 2017. Approvals of the Ethics Committees of all patients not easy to culture and serological methods have a rather participating in the study were obtained (IR.TUMS. low accuracy, molecular methods like PCR are used to REC.1395-2322) The subjects first completed a question - detect it. naire and gave informed consent. Then, urine specimens Mycoplasma genitalium is a Gram-negative, pleomor- were collected. First void urine is the best urine sampling phic, nonmotile bacterium lacking a cell wall. It requires method for PCR to detect C. trachomatis, M. genitalium, special media for growth and reproduction. It is a lead- and HPV. The subjects were requested no to pass urine ing cause of miscarriage in pregnant women. Since myco- for 4  h before sampling and then their first-catch urine plasmas are difficult to grow and colonies grow after was collected in sterile containers. About 10  ml of the 3–7  days even on special media, it is rarely possible to sample was centrifuged at 3000×g for 20 min and 1–2 ml isolate the bacterium from clinical specimens; therefore, of the sediment was transferred to sterile microfuges and other diagnostic methods are used. One of these meth- stored at − 20 °C until analysis. ods is serological tests that are not very reliable and false DNA extraction was conducted using A101211 kits positive results and cross-reaction are frequent in these imported by Pars Tous Biotechnology Company. After tests. Therefore, molecular methods like PCR are used extraction, DNA samples were stored at −  70  °C until for detection. Similar to chlamydia, the samples used to PCR was done. detect mycoplasma include cervical swabs and amniotic The following commercial kits, imported by Pars Tous fluid, placenta, and urine samples. Urine is least danger - Biotechnology, were used for PCR. ous sample in pregnant women. HPV PCR kit with code number A101032. HPV is a DNA virus and causes reproductive tract This PCR amplification kit includes all necessary PCR infection in men and women. The relationship between amplification reagents, capable of amplifying a wide spec - the genome of this virus and cervical cancer was proved trum of HPV types and a house keeping gene as internal years ago; however, few studies have investigated the control. The most well-known and clinically important association between HPV infection and spontaneous HPV are detectable with this kit, including type 6, 11, Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 3 of 7 16, 18, 26, 31, 32, 33, 34, 35, 42, 45, 51, 52, 53, 55, 56, 58, Table 2 Frequency distribution of  variables, number of pregnancies and methods of contraception in pregnant 59, 61, 66, 67, 68, 69, 70, 73, PAP 155, PAP291, X4. Viral women studied DNA present in positive samples is specifically amplified by using primers complementary to viral sequences. Variables Number (%) Mycoplasma genitalium PCR kit with code number Pregnancy number A101112. This PCR amplification kit includes all neces - 1 35 (29.41) sary PCR amplification reagents with an exception of 2 47 (39.50) mineral oil. The M. genitalium PCR kit is a qualitative 3 28 (23.53) analysis test, using a DNA amplification technique for 4 7 (5.88) direct detection of M. genitalium DNA. Bacterial DNA 5 2 (1.68) present in positive samples is specifically amplified by Pregnancy prevention method using primers complementary to bacterial sequences. Natural 85 (71.43) Each kit offered Pars Tous’s optimal buffer system which Condom 12 (10.08) will enhance amplification specificity with a detection LD 12 (10.08) limit of ~ 2 copies per reaction. IUD 8 (6.72) Chlamydia trachomatis nested PCR kit with code Injection 2 (1.68) number A101122 is a qualitative analysis test, using a DNA amplification technique for direct detection of C. trachomatis DNA. Bacterial DNA present in positive The statistical analysis between urinary isolation of C. samples is specifically amplified by using primers com - trachomatis and M. genitalium and miscarriage is shown plementary to bacterial sequences. Each kit offered Pars in Tables 4 and 5, respectively. Tous’s optimal buffer system which will enhance amplifi - According to Table  4, we found no significant associa - cation specificity. tion between urinary isolation of C. trachomatis and mis- carriage (P = 0.93) and according to Table 5, we found no Results significant association between urinary isolation of M. In this study, urine samples were collected from 119 genitalium and miscarriage (P = 0.80). Regarding HPV, pregnant women with mean age 29  years old who vis- since all urine samples were PCR-negative, comparison ited health centers for routine first-trimester screening was not possible. (12–14  weeks gestation). The subjects first completed a According to the present study, chlamydia was isolated questionnaire and gave informed consent. Then, urine from the urine samples of 6.72% of the pregnant women specimens were collected and have detected C. tra- who underwent first-trimester screening in health centers chomatis, M. genitalium, and HPV with PCR method. using PCR. Previous studies reported a mean chlamydia The frequency distribution of abortion, preterm labor, isolation rate of 3% from urine specimens collected from multiple pregnancies history, number of pregnancies and pregnant women in general. T-test showed no significant methods of contraception in pregnant women studied difference between the two groups (P = 0.10). are shown in Tables 1 and 2, respectively. In our study, the mycoplasma isolation rate was 17.65% Statistical analyses showed no significant relation - using PCR. Previous studies reported a mean myco- ship between the variables assessed in the question- plasma isolation rate of 10% from urine specimens col- naire, including history of preterm delivery, number lected from pregnant women in general. T-test showed a of pregnancies, history of multiple pregnancy, contra- significant difference between the two groups (P = 0.03). ception method and urinary isolation of chlamydia and The pictures gel electrophoresis of PCR products of C. mycoplasma. trachomatis and M. genitalium is shown in Figs. 1 and 2, The number of positive M. genitalium, C. trachomatis respectively and HPV in urine samples with PCR method is shown in Figure 3 is about the DNA quality. Table 3. Table 1 Frequency distribution of abortion, preterm labor and multiple pregnancies history in pregnant women Variable Abortion history Premature delivery Multiple pregnancy Total Non One More No Yes No Yes Number (%) 88 (73.95) 25 (21.01) 6 (5.04) 116 (97.48) 3 (2.52) 116 (97.48) 3 (2.52) 119 Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 4 of 7 Table 3 The urinary isolation of  M. genitalium, C. trachomatis and HPV in pregnant women by PCR method Microorganism isolated Positive (%) Negative (%) Total M. genitalium 21 (17.65) 98 (82.35) 119 C. trachomatis 8 (6.72) 111 (93.28) 119 HPV 0 (0) 119 (100) 119 Table 4 The statistical analysis between  urinary isolation of C. trachomatis and miscarriage in pregnant women Variables Chlamydia infection Abortion Positive Negative P-value Number (%) Number (%) No 6 (75) 82 (73.87) Yes 2 (25) 29 (26.13) 0.93 Table 5 The statistical analysis between  urinary isolation of M. genitalium and miscarriage in pregnant women Fig. 2 Gel electrophoresis of PCR products of Mycoplasma genitalium (M1 control positive, M2 control negative, M3–5 urine samples) Variables Mycoplasma infection Abortion Positive Negative P-value Number (%) Number (%) No 15 (71.43) 73 (74.49) Yes 6 (28.57) 25 (25.51) 0.80 Fig. 3 DNA quality Fig. 1 Gel electrophoresis of PCR products of Chlamydia trachomatis Discussion (C1 control positive, C2 control negative, C3–5 urine samples) Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 5 of 7 Miscarriage and preterm delivery are the most impor- Poland, HPV was isolated from the vaginal specimens of tant challenges of pregnancy. Different bacterial and viral 35.14% of pregnant women using PCR [13, 14]. Another infection may cause miscarriage and preterm delivery [4]. study reported an isolation rate of 36%. A study in Brazil, Among bacterial factors, M. genitalium and C. trachoma- HPV DNA was detected in the vaginal samples of 25.3% tis have the most important role and HPV is the leading of pregnant women and 13% of non-pregnant women viral factor in this regard [1]. using PCR, which showed a significant difference [14]. All the above agents are colonized in the vaginal either The results of a study in Iran showed that of 2577 healthy symptomatically or asymptomatically. If symptomatic, women, HPV DNA was isolated from 8.4% of vaginal the person seeks treatment. What is important is a specimens using PCR [15]. woman in whom these agents are colonized in the vagina The results of a study by Ducancelle et al. showed that and the patient is asymptomatic. These women are at HPV was isolated from the 42 and 49% of the urine and increased risk of preterm delivery or miscarriage if they cervical discharge samples of 230 women, respectively. become pregnant [1, 2, 5] In this study, real time PCR was used to detect HPV. The According to a study in Samoa, 239 women aged women participating in this study were not pregnant, so 18–29  years were evaluated for C. trachomatis infection there was no risk of miscarriage. The isolation rate of the using PCR. The results showed that 36% of the women two sampling methods had a significant correlation [16]. were infected that were mostly 18–24  years. Urine sam- In a similar study, the correlation of HPV isolation from ples were used in this study [6]. A retrospective study cervical discharge specimens (45%) and urine samples on 590 women between 2010 and 2014 showed that 11 (37%) using real time PCR was about (93%) [17]. Tamim and 6.9% of the women were infected with HPV and C. et  al. assessed C. trachomatis and HPV in cervical dis- trachomatis, respectively [7]. Researchers in Switzerland charge and urine specimens (2–4  h after urinary reten- compared 146 pregnant women with preterm deliv- tion) using PCR [18]. ery with 261 women with term delivery. The results of Vanessa et  al. investigated the prevalence of myco- PCR showed a significantly higher rate of C. trachoma - plasma in the urine samples of 82 women with a his- tis infection in women with preterm delivery [8]. In the tory of miscarriage and 34 pregnant women 22  weeks USA, about 3–6% of the women below 25 years who look gestation and reported similar results [19]. Jensen et  al. healthy have C. trachomatis infection. In general, the reported that urine specimens are better than urogenital percentage of C. trachomatis infection is different in dif - swabs for detection of mycoplasma and chlamydia using ferent countries; for example, 3.9% in England, 6% in Fin- PCR [20]. land, 17% in France, 9% in Poland, and 6.5–22% in Iran. In About antibiotic treatment of genital infections with a study in 2016, C. trachomatis DNA was detected on the C. trachomatis fortunately investigations has shown endocervical swabs of 17.43% of the subjects [9]. A simi- the clinical strains of C. trachomatis with macrolide or lar study in Iran showed the colonization rate of C. tra- fluoroquinolone resistance would be uncommon, and chomatis is 2.8% in pregnant women. The rate is reported azithromycin or fluoroquinolone regimens could be rec - to be about 4.8% in the urine of pregnant women with a ommended as treatments for chlamydial infections [21]. history of miscarriage or infertility [10, 11]. Although in genital infections caused by M. genitalium Numerous studies have investigated M. genitalium. based on Anderson et al. investigation, the clinical obser- The colonization rate of mycoplasma is 12–50% in preg - vation of increasing treatment failure indicating antibi- nancy but the prevalence of mycoplasma is even higher otic resistance has been confirmed by molecular testing. in women with a history of miscarriage. A colonization Fluoroquinolone mutation screening was performed rate of about 84% was reported in a study in Malaysia. on 86 (74.8%) of the 115 samples, of which 20 (23.3%) A similar study in China showed that the colonization samples had a mutation or mutations associated with rate of Mycoplasma hominis was about 27.6 and 10% in increased resistance. This study shows the increasing women with a history of miscarriage and normal women, antibiotic resistance and the need for appropriate guide- respectively. According to a study in Iran, mycoplasma is lines to treat at-risk patients [22]. colonized in 10% of the pregnant women [10]. A similar The most common sampling method is vaginal and study showed a colonization rate of 21–45% for myco- cervical sampling to detect the above bacterial and viral plasma in women [12]. agents, which required the use of speculum. Since this As for HPV, studies have shown that the DNA of this sampling method is invasive and may cause miscarriage, virus can be detected in the vaginal swabs of 30–45% of less invasive methods should be used. In recent years, women using PCR. In another study, HPV was isolated the results of studies isolating these bacterial and viral from vaginal swabs of 5.5–65% of the subjects, depend- ing on the geographical region, using PCR. In a study in Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 6 of 7 agents from first void urine have been promising. In this All 119 women were followed up until delivery. Three method, the patient should not void for at least 4 h, and cases of preterm delivery were noted, of whom two then first-catch urine is collected a sterile container for were positive for mycoplasma and one was positive urine culture. for both mycoplasma and chlamydia. Statistical analy- In our study, first void urine was used to detect the sis showed a significant difference in preterm delivery above bacterial and viral agents that play a role in miscar- between women who were positive for one of the above riage and preterm delivery. One hundred and nineteen agents and women who were negative. pregnant women (11–14  weeks gestation) who under- In general, the aim of the present study was to iso- went first-trimester screening in health centers were late three important causes of septic abortion, i.e., recruited. After completing the questionnaire and obtain- C. trachomatis, M. genitalium, and HPV, from urine ing informed consent, the subjects were given urine col- specimens of pregnant women in the first trimester. lection containers to collect their urine. Urine samples According to the results, first void may be an appropri - were centrifuged for 20  min and DNA was extracted ate sample for detection of C. trachomatis and M. geni- from the sediment. PCR was done on the samples to talium; however, urine is not a good sample for HPV detect M. genitalium, C. trachomatis, and HPV. Accord- isolation and vaginal or cervical discharge specimens ing to the results, chlamydia was isolated from the urine should be used instead. Regarding mycoplasma, urine specimens of 6.78% of the pregnant women. Previous specimens are even better than vaginal and cervical studies reported a mean chlamydial isolation rate of 3% specimens considering the higher isolation rate. from urine specimens collected from pregnant women in general. T-test showed no significant difference between Abbreviations the two groups (P = 0.10). According to the above results, M. genitalium: Mycoplasma genitalium; C. trachomatis: Chlamydia trachomatis; the isolation rate of chlamydia from urine specimens of HPV: Human papilloma virus; STD: Sexually transmitted diseases. pregnant women was more than two times higher than Authors’ contributions vaginal and cervical discharge specimens because pre- Monireh Rahimkhani (50%), Alireza Mordadi (35%), Maryam Gilanpour (15%). vious studies showed an isolation rate of 3% for C. tra- All authors read and approved the final manuscript. chomatis from vaginal and cervical discharge specimens. Author details Therefore, urine is an appropriate sample for detection of 1 Department of Lab Medical Sciences, Faculty of Allied Medical Sciences, C. trachomatis because its collection is non-invasive and Tehran University of Medical Sciences, Tehran, Iran. Department of Epidemi- ology, Pasteur Institute, Tehran, Iran. Almahdi Kheyrieh Clinics, Tehran, Iran. offers a higher isolation rate. According to our results, the mycoplasma isolation Acknowledgements rate was 17.8% using PCR. Previous studies reported a The authors thank all staff in Almahdi Clinic, especially Dr. Mohammad Zay- andeh who had corporated in sampling from pregnant women and also We mean mycoplasma isolation rate of 10% from urine speci- appreciate the Research Vice-chancellor of the Tehran University of Medical mens collected from pregnant women in general. T-test Sciences for funding the Research Project Code Number 94-04-31-30491. showed a significant difference between the two groups Competing interests (P = 0.03). The authors declare that they have no competing interests. The 10% prevalence of mycoplasma in the pregnant women was based on cervical or vaginal specimens, Availability of data and materials Not applicable. which is an invasive sampling method in pregnant women and is not well accepted. We used urine sam- Consent for publication ples in our study and found a higher isolation rate than All of authors consent to publication. vaginal and cervical discharge specimens. Therefore, first Ethics approval and consent to participate void urine may yield better results. Approvals of the Ethics Committees of all patients participating in the study We found no significant relationship between the iso - were obtained (IR.TUMS.REC.1395-2322). The subjects first completed a ques- tionnaire and gave informed consent. lation rate of mycoplasma and chlamydia using PCR and the history of miscarriage. Since HPV was not isolated Funding from any of the urine specimens, statistical analysis was Not applicable. not possible. HPV was isolated from 8.4% of vaginal and cervical discharge specimens of pregnant women; there- Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- fore, it may be concluded that urine is not a good sam- lished maps and institutional affiliations. ple for HPV detection and cervical or vaginal specimens should be used to detect these viral agent. Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 7 of 7 Received: 10 December 2017 Accepted: 19 May 2018 12. Vosooghi S, Kheirkhah B, Karimi NA, Reza MT. A review of the role of Mycoplasma infections in humans infertility. NCMBJ. 2012;2:9–20 (Persian). 13. Medeiros LR, de Ethur ABM, Hilgert JB, Zanini RR, Berwanger O, Bozzetti MC, Mylius LC. Vertical transmission of the human papillomavirus: a systematic quantitative review. Cad Saúde Pública. 2005;21(4):1006–15. References 14. Salcedo MM, Damin AP, Agnes G, Pessini SA, El Beitune P, Alexandre CO. 1. Doyle RM, Alber DG, Jones HE, Harris K, Fitzgerald F, Peebles D, Klein N. Prevalence of human papillomavirus infection in pregnant versus non- Term and preterm labour are associated with distinct microbial commu- pregnant women in Brazil. Arch Gynecol Obstet. 2015;292(6):1273–8. nity structures in placental membranes which are independent of mode 15. Jalilvand S, Shoja Z, Nourijelyani K, Tohidi HR, Hamkar R. Meta-analysis of delivery. Placenta. 2014;35(12):1099–101. of type-specific human papillomavirus prevalence in Iranian women 2. Conde-Ferráez L, de May AAC, Carrillo-Martínez JR, Ayora-Talavera G, del with normal cytology, precancerous cervical lesions and invasive Refugio González-Losa M. Human papillomavirus infection and sponta- cervical cancer: implications for screening and vaccination. J Med Virol. neous abortion: a case–control study performed in Mexico. Eur J Obstet 2015;87(2):287–95. Gynecol Reprod Biol. 2013;170(2):468–73. 16. Ducancelle A, Legrand M, Pivert A, Veillon P, Le Guillou-Guillemette H, De 3. Skoczyński M, Goździcka-Józefiak A, Kwaśniewska A. Prevalence of Brux M, Beby-Defaux A, Agius G, Hantz S, Alain S, Catala L, Descamps P, human papillomavirus in spontaneously aborted products of conception. Postec E, Caly H, Charles-Pétillon F, Labrousse F, Lunel F, Payan C. Interest Acta Obstet Gynecol Scand. 2011;90(12):1402–5. of human papillomavirus DNA quantification and genotyping in paired 4. Rahimkhani M, Khavari-Daneshvar H, Sharifian R. Asymptomatic bacteriu- cervical and urine samples to detect cervical lesions. Arch Gynecol ria and pyuria in pregnancy. Acta Med Iran. 2008;46(5):409–12. Obstet. 2014;290(2):299–308. 5. Rahimkhani M, Mordadi A, Varmazyar S, Tavakoli A. Evaluation of urinary 17. Payan C, Ducancelle A, Aboubaker MH, Caer J, Tapia M, Chauvin A, Interleukin-8 levels in patients with spinal cord injury. Recent Pat Antiin- Peyronnet D, le Hen E, Arab Z, Legrand MC. Human papillomavirus quan- fect Drug Discov. 2015;9(2):144–9. tification in urine and cervical samples by using the Mx4000 and LightCy- 6. Walsh MS, Hope E, Isaia L, Righarts A, Niupulusu T, Temese SV, Iosefa-Siitia cler general real-time PCR systems. J Clin Microbiol. 2007;45(3):897–901. L, Auvaa L, Tapelu SA, Motu MF. Prevalence of Chlamydia trachomatis 18. Tamim H, Finan RR, Sharida HE, Rashid M, Almawi WY. Cervicovaginal infection in Samoan women aged 18 to 29 and assessment of possible coinfections with human papillomavirus and Chlamydia trachomatis. risk factors: a community-based study. Trans R Soc Trop Med Hyg. Diagn Microbiol Infect Dis. 2002;43(4):277–81. 2015;109(4):245–51. 19. Short VL, Jensen JS, Nelson DB, Murray PJ, Ness RB, Haggerty CL. Myco- 7. Depuydt CE, Verstraete L, Berth M, Beert J, Bogers J-P, Salembier G, Ver- plasma genitalium among young, urban pregnant women. Infect Dis eecken AJ, Bosmans E. Human papillomavirus positivity in women under- Obstet Gynecol. 2010. https ://doi.org/10.1155/2010/98476 0. going intrauterine insemination has a negative effect on pregnancy rates. 20. Jensen JS, Björnelius E, Dohn B, Lidbrink P. Comparison of first void Gynecol Obstet Investig. 2016;81(1):41–6. urine and urogenital swab specimens for detection of Mycoplasma 8. Baud D, Goy G, Vasilevsky S, Osterheld M-C, Roth-Kleiner M, Croxatto A, genitalium and Chlamydia trachomatis by polymerase chain reaction in Greub G. Roles of bovine Waddlia chondrophila and Chlamydia trachoma- patients attending a sexually transmitted disease clinic. Sex Transm Dis. tis in human preterm birth. N Microbes N Infect. 2015;3:41–5. 2004;31(8):499–507. 9. Ahmadi A, Khodabandehloo M, Ramazanzadeh R, Farhadifar F, Roshani 21. Deguchi T, Hatazaki K, Ito S, Kondo H, Horie K, Nakane K, Mizutani K, D, Ghaderi E, Farhangi N. The relationship between Chlamydia tra- Tsuchiya T, Yasuda M, Yokoi S, Nakano M. Macrolide and fluoroquinolone chomatis genital infection and spontaneous abortion. J Reprod Infertil. resistance is uncommon in clinical strains of Chlamydia trachomatis. J 2016;17(2):110. Infect Chemother. 2018. https ://doi.org/10.1016/j.jiac.2018.03.007 (Epub 10. Dehghan Marvast L, Aflatoonian A, Talebi A, Eley A, Pacey A. 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Detection of urinary Chlamydia trachomatis, Mycoplasma genitalium and human papilloma virus in the first trimester of pregnancy by PCR method

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Copyright © 2018 by The Author(s)
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Biomedicine; Medical Microbiology; Infectious Diseases; Drug Resistance; Microbial Genetics and Genomics; Biochemistry, general; Parasitology
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Abstract

Background: Miscarriage and preterm delivery are the most important challenges of pregnancy. Different bacterial and viral infection may cause miscarriage and preterm delivery. Among bacterial factors, Mycoplasma genitalium and Chlamydia trachomatis have the most important role and human papilloma virus (HPV ) is the leading viral factor in this regard. Methods: First void urine samples were collected from 119 pregnant women who visited health centers for routine first-trimester screening (12–14 weeks gestation). About 10 ml of the sample was centrifuged at 3000×g for 20 min and 1–2 ml of the sediment was transferred to sterile microfuges and stored at − 20 °C until analysis. DNA extraction was conducted using A101211 kits imported by Pars Tous Biotechnology Company. The following commercial kits, imported by Pars Tous Biotechnology, were used for PCR. Results: There is no significant association between urinary isolation of C. trachomatis and miscarriage (P = 0.93) and there is no significant association between urinary isolation of M. genitalium and miscarriage (P = 0.80). Regard- ing HPV, since all urine samples were PCR-negative, comparison was not possible. C. trachomatis was isolated from the urine samples of 6.72% of the pregnant women who underwent first-trimester screening in health centers using PCR. Previous studies reported a mean chlamydia isolation rate of 3% from urine specimens collected from pregnant women in general. T test showed no significant difference between the two groups (P = 0.10). Based on present study the mycoplasma isolation rate was 17.65% using PCR. Previous studies reported a mean mycoplasma isolation rate of 10% from urine specimens collected from pregnant women in general. T-test showed a significant difference between the two groups (P = 0.03). Discussion: First void urine samples in pregnant women may be an appropriate sample for detection of C. tra- chomatis and M. genitalium; however, it is not a good method for HPV isolation therefore vaginal or cervical discharge specimens should be used instead for detection of HPV. Keywords: Pregnant women, Miscarriage, Chlamydia trachomatis, Mycoplasma genitalium, Human papilloma virus, Urine sample *Correspondence: rrahimkhani@sina.tums.ac.ir Department of Lab Medical Sciences, Faculty of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 2 of 7 abortion, which have shown non-significant associations Importance [2, 3]. In the present study, we decided to investigate three Similar to mycoplasma and chlamydia, the samples major causes of STDs that also cause miscarriage in the used for detection of HPV include swab specimens sam- first trimester in a number of pregnant women. Since pled from the cervix and urine. cervical swabbing is invasive and dangerous and is not In the present study, we decided to investigate three accepted by pregnant women, first void urine specimens major causes of sexually transmitted disease (STDs) that were used in this study. Among bacterial agents, Chla- also cause miscarriage in the first trimester in a number mydia trachomatis and Mycoplasma genitalium, and of pregnant women. Since cervical swabbing is invasive among viral agents, HPV, have a major role in miscar- and dangerous and is not accepted by pregnant women, riage. These agents were surveyed in present research. early urine specimens were used in this study. Background Methods Miscarriage is one of the greatest risks threatening the Considering a precision of 7% to detect the difference life of the mother and fetus. Different bacterial, viral, and and a prevalence of 2.8% for C. trachomatis, 10% for M. parasitic factors can cause miscarriage. Statistics sug- genitalium, and 8.4% for HPV, the sample size was esti- gest that more than 90% of miscarriages are related to mated 119, 61, and 66, respectively. Finally, the largest an infectious or inflammatory factor in the placenta or sample size was considered for the study by this formula: amniotic fluid [1]. Among bacterial agents, Chlamydia trachomatis and Mycoplasma genitalium, and among 2 NZ P(1 − P) n = . viral agents, human papilloma virus (HPV), have a major d (N − 1) + Z P(1 − P) role in miscarriage. Chlamydia trachomatis is an obligate intracellu- lar, nonmotile, Gram-negative bacterium in the family In this study, urine samples were collected from 119 Chlamydiaceae. Because it is an intracellular parasite, it pregnant women who visited Almahdi Clinic Center does not grow on bacteriologic cultures, and cellular cul- in south of Tehran for routine first-trimester screening tures are used for its isolation and reproduction. Chla- (12–14  weeks gestation). Almahdi Clinic is under the mydia trachomatis is the major cause of trachoma but it supervision of the Tehran University of Medical Sciences. can also cause miscarriage, fetal anomaly, endometritis, The sampling duration time was October 2016 to August preterm delivery, and stillbirth. Since this bacterium is 2017. Approvals of the Ethics Committees of all patients not easy to culture and serological methods have a rather participating in the study were obtained (IR.TUMS. low accuracy, molecular methods like PCR are used to REC.1395-2322) The subjects first completed a question - detect it. naire and gave informed consent. Then, urine specimens Mycoplasma genitalium is a Gram-negative, pleomor- were collected. First void urine is the best urine sampling phic, nonmotile bacterium lacking a cell wall. It requires method for PCR to detect C. trachomatis, M. genitalium, special media for growth and reproduction. It is a lead- and HPV. The subjects were requested no to pass urine ing cause of miscarriage in pregnant women. Since myco- for 4  h before sampling and then their first-catch urine plasmas are difficult to grow and colonies grow after was collected in sterile containers. About 10  ml of the 3–7  days even on special media, it is rarely possible to sample was centrifuged at 3000×g for 20 min and 1–2 ml isolate the bacterium from clinical specimens; therefore, of the sediment was transferred to sterile microfuges and other diagnostic methods are used. One of these meth- stored at − 20 °C until analysis. ods is serological tests that are not very reliable and false DNA extraction was conducted using A101211 kits positive results and cross-reaction are frequent in these imported by Pars Tous Biotechnology Company. After tests. Therefore, molecular methods like PCR are used extraction, DNA samples were stored at −  70  °C until for detection. Similar to chlamydia, the samples used to PCR was done. detect mycoplasma include cervical swabs and amniotic The following commercial kits, imported by Pars Tous fluid, placenta, and urine samples. Urine is least danger - Biotechnology, were used for PCR. ous sample in pregnant women. HPV PCR kit with code number A101032. HPV is a DNA virus and causes reproductive tract This PCR amplification kit includes all necessary PCR infection in men and women. The relationship between amplification reagents, capable of amplifying a wide spec - the genome of this virus and cervical cancer was proved trum of HPV types and a house keeping gene as internal years ago; however, few studies have investigated the control. The most well-known and clinically important association between HPV infection and spontaneous HPV are detectable with this kit, including type 6, 11, Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 3 of 7 16, 18, 26, 31, 32, 33, 34, 35, 42, 45, 51, 52, 53, 55, 56, 58, Table 2 Frequency distribution of  variables, number of pregnancies and methods of contraception in pregnant 59, 61, 66, 67, 68, 69, 70, 73, PAP 155, PAP291, X4. Viral women studied DNA present in positive samples is specifically amplified by using primers complementary to viral sequences. Variables Number (%) Mycoplasma genitalium PCR kit with code number Pregnancy number A101112. This PCR amplification kit includes all neces - 1 35 (29.41) sary PCR amplification reagents with an exception of 2 47 (39.50) mineral oil. The M. genitalium PCR kit is a qualitative 3 28 (23.53) analysis test, using a DNA amplification technique for 4 7 (5.88) direct detection of M. genitalium DNA. Bacterial DNA 5 2 (1.68) present in positive samples is specifically amplified by Pregnancy prevention method using primers complementary to bacterial sequences. Natural 85 (71.43) Each kit offered Pars Tous’s optimal buffer system which Condom 12 (10.08) will enhance amplification specificity with a detection LD 12 (10.08) limit of ~ 2 copies per reaction. IUD 8 (6.72) Chlamydia trachomatis nested PCR kit with code Injection 2 (1.68) number A101122 is a qualitative analysis test, using a DNA amplification technique for direct detection of C. trachomatis DNA. Bacterial DNA present in positive The statistical analysis between urinary isolation of C. samples is specifically amplified by using primers com - trachomatis and M. genitalium and miscarriage is shown plementary to bacterial sequences. Each kit offered Pars in Tables 4 and 5, respectively. Tous’s optimal buffer system which will enhance amplifi - According to Table  4, we found no significant associa - cation specificity. tion between urinary isolation of C. trachomatis and mis- carriage (P = 0.93) and according to Table 5, we found no Results significant association between urinary isolation of M. In this study, urine samples were collected from 119 genitalium and miscarriage (P = 0.80). Regarding HPV, pregnant women with mean age 29  years old who vis- since all urine samples were PCR-negative, comparison ited health centers for routine first-trimester screening was not possible. (12–14  weeks gestation). The subjects first completed a According to the present study, chlamydia was isolated questionnaire and gave informed consent. Then, urine from the urine samples of 6.72% of the pregnant women specimens were collected and have detected C. tra- who underwent first-trimester screening in health centers chomatis, M. genitalium, and HPV with PCR method. using PCR. Previous studies reported a mean chlamydia The frequency distribution of abortion, preterm labor, isolation rate of 3% from urine specimens collected from multiple pregnancies history, number of pregnancies and pregnant women in general. T-test showed no significant methods of contraception in pregnant women studied difference between the two groups (P = 0.10). are shown in Tables 1 and 2, respectively. In our study, the mycoplasma isolation rate was 17.65% Statistical analyses showed no significant relation - using PCR. Previous studies reported a mean myco- ship between the variables assessed in the question- plasma isolation rate of 10% from urine specimens col- naire, including history of preterm delivery, number lected from pregnant women in general. T-test showed a of pregnancies, history of multiple pregnancy, contra- significant difference between the two groups (P = 0.03). ception method and urinary isolation of chlamydia and The pictures gel electrophoresis of PCR products of C. mycoplasma. trachomatis and M. genitalium is shown in Figs. 1 and 2, The number of positive M. genitalium, C. trachomatis respectively and HPV in urine samples with PCR method is shown in Figure 3 is about the DNA quality. Table 3. Table 1 Frequency distribution of abortion, preterm labor and multiple pregnancies history in pregnant women Variable Abortion history Premature delivery Multiple pregnancy Total Non One More No Yes No Yes Number (%) 88 (73.95) 25 (21.01) 6 (5.04) 116 (97.48) 3 (2.52) 116 (97.48) 3 (2.52) 119 Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 4 of 7 Table 3 The urinary isolation of  M. genitalium, C. trachomatis and HPV in pregnant women by PCR method Microorganism isolated Positive (%) Negative (%) Total M. genitalium 21 (17.65) 98 (82.35) 119 C. trachomatis 8 (6.72) 111 (93.28) 119 HPV 0 (0) 119 (100) 119 Table 4 The statistical analysis between  urinary isolation of C. trachomatis and miscarriage in pregnant women Variables Chlamydia infection Abortion Positive Negative P-value Number (%) Number (%) No 6 (75) 82 (73.87) Yes 2 (25) 29 (26.13) 0.93 Table 5 The statistical analysis between  urinary isolation of M. genitalium and miscarriage in pregnant women Fig. 2 Gel electrophoresis of PCR products of Mycoplasma genitalium (M1 control positive, M2 control negative, M3–5 urine samples) Variables Mycoplasma infection Abortion Positive Negative P-value Number (%) Number (%) No 15 (71.43) 73 (74.49) Yes 6 (28.57) 25 (25.51) 0.80 Fig. 3 DNA quality Fig. 1 Gel electrophoresis of PCR products of Chlamydia trachomatis Discussion (C1 control positive, C2 control negative, C3–5 urine samples) Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 5 of 7 Miscarriage and preterm delivery are the most impor- Poland, HPV was isolated from the vaginal specimens of tant challenges of pregnancy. Different bacterial and viral 35.14% of pregnant women using PCR [13, 14]. Another infection may cause miscarriage and preterm delivery [4]. study reported an isolation rate of 36%. A study in Brazil, Among bacterial factors, M. genitalium and C. trachoma- HPV DNA was detected in the vaginal samples of 25.3% tis have the most important role and HPV is the leading of pregnant women and 13% of non-pregnant women viral factor in this regard [1]. using PCR, which showed a significant difference [14]. All the above agents are colonized in the vaginal either The results of a study in Iran showed that of 2577 healthy symptomatically or asymptomatically. If symptomatic, women, HPV DNA was isolated from 8.4% of vaginal the person seeks treatment. What is important is a specimens using PCR [15]. woman in whom these agents are colonized in the vagina The results of a study by Ducancelle et al. showed that and the patient is asymptomatic. These women are at HPV was isolated from the 42 and 49% of the urine and increased risk of preterm delivery or miscarriage if they cervical discharge samples of 230 women, respectively. become pregnant [1, 2, 5] In this study, real time PCR was used to detect HPV. The According to a study in Samoa, 239 women aged women participating in this study were not pregnant, so 18–29  years were evaluated for C. trachomatis infection there was no risk of miscarriage. The isolation rate of the using PCR. The results showed that 36% of the women two sampling methods had a significant correlation [16]. were infected that were mostly 18–24  years. Urine sam- In a similar study, the correlation of HPV isolation from ples were used in this study [6]. A retrospective study cervical discharge specimens (45%) and urine samples on 590 women between 2010 and 2014 showed that 11 (37%) using real time PCR was about (93%) [17]. Tamim and 6.9% of the women were infected with HPV and C. et  al. assessed C. trachomatis and HPV in cervical dis- trachomatis, respectively [7]. Researchers in Switzerland charge and urine specimens (2–4  h after urinary reten- compared 146 pregnant women with preterm deliv- tion) using PCR [18]. ery with 261 women with term delivery. The results of Vanessa et  al. investigated the prevalence of myco- PCR showed a significantly higher rate of C. trachoma - plasma in the urine samples of 82 women with a his- tis infection in women with preterm delivery [8]. In the tory of miscarriage and 34 pregnant women 22  weeks USA, about 3–6% of the women below 25 years who look gestation and reported similar results [19]. Jensen et  al. healthy have C. trachomatis infection. In general, the reported that urine specimens are better than urogenital percentage of C. trachomatis infection is different in dif - swabs for detection of mycoplasma and chlamydia using ferent countries; for example, 3.9% in England, 6% in Fin- PCR [20]. land, 17% in France, 9% in Poland, and 6.5–22% in Iran. In About antibiotic treatment of genital infections with a study in 2016, C. trachomatis DNA was detected on the C. trachomatis fortunately investigations has shown endocervical swabs of 17.43% of the subjects [9]. A simi- the clinical strains of C. trachomatis with macrolide or lar study in Iran showed the colonization rate of C. tra- fluoroquinolone resistance would be uncommon, and chomatis is 2.8% in pregnant women. The rate is reported azithromycin or fluoroquinolone regimens could be rec - to be about 4.8% in the urine of pregnant women with a ommended as treatments for chlamydial infections [21]. history of miscarriage or infertility [10, 11]. Although in genital infections caused by M. genitalium Numerous studies have investigated M. genitalium. based on Anderson et al. investigation, the clinical obser- The colonization rate of mycoplasma is 12–50% in preg - vation of increasing treatment failure indicating antibi- nancy but the prevalence of mycoplasma is even higher otic resistance has been confirmed by molecular testing. in women with a history of miscarriage. A colonization Fluoroquinolone mutation screening was performed rate of about 84% was reported in a study in Malaysia. on 86 (74.8%) of the 115 samples, of which 20 (23.3%) A similar study in China showed that the colonization samples had a mutation or mutations associated with rate of Mycoplasma hominis was about 27.6 and 10% in increased resistance. This study shows the increasing women with a history of miscarriage and normal women, antibiotic resistance and the need for appropriate guide- respectively. According to a study in Iran, mycoplasma is lines to treat at-risk patients [22]. colonized in 10% of the pregnant women [10]. A similar The most common sampling method is vaginal and study showed a colonization rate of 21–45% for myco- cervical sampling to detect the above bacterial and viral plasma in women [12]. agents, which required the use of speculum. Since this As for HPV, studies have shown that the DNA of this sampling method is invasive and may cause miscarriage, virus can be detected in the vaginal swabs of 30–45% of less invasive methods should be used. In recent years, women using PCR. In another study, HPV was isolated the results of studies isolating these bacterial and viral from vaginal swabs of 5.5–65% of the subjects, depend- ing on the geographical region, using PCR. In a study in Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 6 of 7 agents from first void urine have been promising. In this All 119 women were followed up until delivery. Three method, the patient should not void for at least 4 h, and cases of preterm delivery were noted, of whom two then first-catch urine is collected a sterile container for were positive for mycoplasma and one was positive urine culture. for both mycoplasma and chlamydia. Statistical analy- In our study, first void urine was used to detect the sis showed a significant difference in preterm delivery above bacterial and viral agents that play a role in miscar- between women who were positive for one of the above riage and preterm delivery. One hundred and nineteen agents and women who were negative. pregnant women (11–14  weeks gestation) who under- In general, the aim of the present study was to iso- went first-trimester screening in health centers were late three important causes of septic abortion, i.e., recruited. After completing the questionnaire and obtain- C. trachomatis, M. genitalium, and HPV, from urine ing informed consent, the subjects were given urine col- specimens of pregnant women in the first trimester. lection containers to collect their urine. Urine samples According to the results, first void may be an appropri - were centrifuged for 20  min and DNA was extracted ate sample for detection of C. trachomatis and M. geni- from the sediment. PCR was done on the samples to talium; however, urine is not a good sample for HPV detect M. genitalium, C. trachomatis, and HPV. Accord- isolation and vaginal or cervical discharge specimens ing to the results, chlamydia was isolated from the urine should be used instead. Regarding mycoplasma, urine specimens of 6.78% of the pregnant women. Previous specimens are even better than vaginal and cervical studies reported a mean chlamydial isolation rate of 3% specimens considering the higher isolation rate. from urine specimens collected from pregnant women in general. T-test showed no significant difference between Abbreviations the two groups (P = 0.10). According to the above results, M. genitalium: Mycoplasma genitalium; C. trachomatis: Chlamydia trachomatis; the isolation rate of chlamydia from urine specimens of HPV: Human papilloma virus; STD: Sexually transmitted diseases. pregnant women was more than two times higher than Authors’ contributions vaginal and cervical discharge specimens because pre- Monireh Rahimkhani (50%), Alireza Mordadi (35%), Maryam Gilanpour (15%). vious studies showed an isolation rate of 3% for C. tra- All authors read and approved the final manuscript. chomatis from vaginal and cervical discharge specimens. Author details Therefore, urine is an appropriate sample for detection of 1 Department of Lab Medical Sciences, Faculty of Allied Medical Sciences, C. trachomatis because its collection is non-invasive and Tehran University of Medical Sciences, Tehran, Iran. Department of Epidemi- ology, Pasteur Institute, Tehran, Iran. Almahdi Kheyrieh Clinics, Tehran, Iran. offers a higher isolation rate. According to our results, the mycoplasma isolation Acknowledgements rate was 17.8% using PCR. Previous studies reported a The authors thank all staff in Almahdi Clinic, especially Dr. Mohammad Zay- andeh who had corporated in sampling from pregnant women and also We mean mycoplasma isolation rate of 10% from urine speci- appreciate the Research Vice-chancellor of the Tehran University of Medical mens collected from pregnant women in general. T-test Sciences for funding the Research Project Code Number 94-04-31-30491. showed a significant difference between the two groups Competing interests (P = 0.03). The authors declare that they have no competing interests. The 10% prevalence of mycoplasma in the pregnant women was based on cervical or vaginal specimens, Availability of data and materials Not applicable. which is an invasive sampling method in pregnant women and is not well accepted. We used urine sam- Consent for publication ples in our study and found a higher isolation rate than All of authors consent to publication. vaginal and cervical discharge specimens. Therefore, first Ethics approval and consent to participate void urine may yield better results. Approvals of the Ethics Committees of all patients participating in the study We found no significant relationship between the iso - were obtained (IR.TUMS.REC.1395-2322). The subjects first completed a ques- tionnaire and gave informed consent. lation rate of mycoplasma and chlamydia using PCR and the history of miscarriage. Since HPV was not isolated Funding from any of the urine specimens, statistical analysis was Not applicable. not possible. HPV was isolated from 8.4% of vaginal and cervical discharge specimens of pregnant women; there- Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- fore, it may be concluded that urine is not a good sam- lished maps and institutional affiliations. ple for HPV detection and cervical or vaginal specimens should be used to detect these viral agent. Rahimkhani et al. Ann Clin Microbiol Antimicrob (2018) 17:25 Page 7 of 7 Received: 10 December 2017 Accepted: 19 May 2018 12. Vosooghi S, Kheirkhah B, Karimi NA, Reza MT. A review of the role of Mycoplasma infections in humans infertility. NCMBJ. 2012;2:9–20 (Persian). 13. 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Annals of Clinical Microbiology and AntimicrobialsSpringer Journals

Published: Jun 4, 2018

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