The Natural Course of Chlamydia Trachomatis, Neisseria Gonorrhoeae

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Article The Natural Course of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium in Pregnant and Post-Delivery Women in Pemba Island, Tanzania

Naomi C. A. Juliana 1,†, Abdulla Mbaruk Omar 2,†, Jolein Pleijster 3, Fahad Aftab 2,4, Nina B. Uijldert 3, Said M. Ali 2, Sander Ouburg 3, Sunil Sazawal 4, Servaas A. Morré 1,3, Saikat Deb 2,4,† and Elena Ambrosino 1,*,†

1 Department of Genetics and Cell Biology, Institute for Public Health Genomics (IPHG), Faculty of Health, Medicine & Life Sciences, Research School GROW (School for Oncology & Developmental Biology), University of Maastricht, 6229 ER Maastricht, The Netherlands; [email protected] (N.C.A.J.); [email protected] (S.A.M.) 2 Public Health Laboratory-Ivo de Carneri, Chake, Pemba Island, Tanzania; [email protected] (A.M.O.); [email protected] (F.A.); [email protected] (S.M.A.); [email protected] (S.D.) 3 Laboratory of Immunogenetics, Department of Medical Microbiology and Infection Control, Amsterdam UMC, Vrije Universiteit, 1105 AZ Amsterdam, The Netherlands; [email protected] (J.P.); [email protected] (N.B.U.); [email protected] (S.O.) Citation: Juliana, N.C.A.; 4 Centre for Public Health Kinetics, New Delhi 110024, India; [email protected] Omar, A.M.; Pleijster, J.; Aftab, F.; * Correspondence: [email protected] Uijldert, N.B.; Ali, S.M.; Ouburg, S.; † Shared authorship. Sazawal, S.; Morré, S.A.; Deb, S.; et al. The Natural Course of Chlamydia Abstract: This study aimed to determine the persistence of Chlamydia trachomatis (CT), Neisseria trachomatis, Neisseria gonorrhoeae, gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) infections during Trichomonas vaginalis Mycoplasma , and pregnancy and after delivery in vaginal swabs of women from Pemba Island, Tanzania. In the context genitalium in Pregnant and of an earlier biobanking effort, vaginal swabs were collected at two timepoints during pregnancy Post-Delivery Women in Pemba and once post-delivery. Detection of CT, NG, TV, and MG was performed by PCR using validated Island, Tanzania. Microorganisms 2021, 9, 1180. https://doi.org/10.3390/ detection kits in samples from 441 pregnant women aged 16–48 years old. Among those, 202 samples microorganisms9061180 were matched during pregnancy and 38 at the second timepoint of the pregnancy and post-delivery CT infection persistence during pregnancy was 100% (n = 11) after an average of eight weeks, that Academic Editor: Jacob Bornstein of TV infection 82% (n = 11) after ten weeks, and that of MG infection 75% (n = 4) after ten weeks. Post-delivery (after approximately 22 weeks) infection persistence was 100% for CT (n = 1) and 20% Received: 20 April 2021 for TV (n = 5). NG was only detected at the last collection timepoint, its persistence rate could not Accepted: 24 May 2021 be determined. These results show persistence and clearance of curable infections during and after Published: 30 May 2021 pregnancy. Analysis of biobanked samples is a valuable approach in the investigation of the natural history of curable pathogens. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: pregnancy; genital infections; Tanzania; sub-Saharan Africa; sexual and reproductive published maps and institutional afﬁl- health; Chlamydia trachomatis; Neisseria gonorrhoeae; Trichomonas vaginalis; Mycoplasma genitalium iations.

1. Introduction Copyright: © 2021 by the authors. Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma Licensee MDPI, Basel, Switzerland. genitalium are common curable sexually transmitted infections (STIs) that are mostly This article is an open access article asymptomatic and have a high burden in sub-Saharan Africa [1,2]. In 2016, the esti- distributed under the terms and C. trachomatis conditions of the Creative Commons mated prevalence among women between 15 and 49 years of age was 5% for Attribution (CC BY) license (https:// infection, 1.9% for N. gonorrhoeae infection and 12% for T. vaginalis infections in the African creativecommons.org/licenses/by/ region [3]. For M. genitalium, no recent regional data for sub-Saharan Africa are available, 4.0/). but prevalence among reproductive-age women in South Africa has been reported between

Microorganisms 2021, 9, 1180. https://doi.org/10.3390/microorganisms9061180 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 1180 2 of 12

1.6 and 11% [4]. These STIs can cause acute urogenital problems, such as inflammation of the cervix (cervicitis), but their often asymptomatic presentation (as is often the case for C. trachomatis and N. gonorrhoeae infections) is the reason why most cases remain undiagnosed, thus contributing to long-term sequelae such as pelvic inflammatory disease [3,5]. Dur- ing pregnancy and delivery, vertical transmission might occur, possibly causing adverse neonatal outcomes [3]. In recent years, infections with these four pathogens have been, in turn, associated with, among others, premature delivery, low birthweight and neonatal sepsis [5–12]. Despite the availability of effective antibiotic treatments, the burden of STIs remains high, including among pregnant women, in most African countries [2,13]. Fortunately, natural protective immunity to these infections does occur in some women, but studies on their natural history, especially in pregnancy, are limited due to the standard care available once the pathogen is diagnosed [14–16]. Notwithstanding, one study observed that 44% of 140 women with asymptomatic C. trachomatis infection spontaneously cleared it over the course of 1 to 14 days during pregnancy [17]. Information about the natural history of C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium infections during pregnancy is scant. However, data exist on non-pregnant women [14,18–21]. A previous study on asymptomatic non-pregnant women showed the resolution rate of C. trachomatis to be 11–45% [14]. The follow-up intervals differed by weeks, months and even years [14]. For instance, C. trachomatis cleared in 45% of non-pregnant Dutch women in a year, and in 94% of non-pregnant Colombian women after a 4-year follow-up [22,23]. Studies showed that asymptomatic N. gonorrhoeae infection can persist as long as five months in non-pregnant women, and that close to 25% of patients clear the infection within that time period [21]. For M. genitalium infection in non-pregnant women, the median clearance time is about 2 months; one study showed that 93% of 119 women involved in the study cleared it within 12 months [18]. Finally, prediction studies suggest that the average duration of untreated T. vaginalis infection is 3 to 5 years in non-pregnant women [19,20]. The infection persistence rate differs between pregnant and non-pregnant women, possibly due to the difference in the immune response as a result of hormonal regulation [24,25]. For some infections, pregnant women are more susceptible, and at times more severely affected than non-pregnant women [25]. Inflammatory responses that are important to clear a pathogen can have negative consequences on the outcome of the pregnancy; pregnant women are biased towards an anti-inflammatory phenotype that is less aggressive towards disease pathogenesis, in a bid to protect viability of the pregnancy [24,25]. Designing longitudinal studies to investigate the persistence of curable infections is ethically challenging and for prospective studies unacceptable, but evidence on the natural history of infections is paramount, particularly among the most vulnerable hosts, as is the case for pregnant women; more so in communities with a high burden of STIs and related pregnancy complications, as is the case for Tanzania [7,26–28]. In the eastern part of the country, on the Island of Pemba, a biobanking effort was established in 2014, and follow-up studies have been set up to investigate contributors to maternal mortality and morbidity [29,30]. The present study retrospectively analyzed previously collected vaginal swabs to determine the persistence of C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium infections during pregnancy and after delivery. Information on the natural history of curable genital infections in pregnancy provides insight into pathophysiology and offers useful scientific guidance to public health efforts in infection screening and control. Furthermore, the generated data will provide insight on the progression of untreated infections during pregnancy among undiagnosed women and support the need for perina- tal screening in order to improve maternal and child health globally.

2. Materials and Methods Vaginal samples and health data were collected by healthcare ofﬁcials in the context of an established biobanking effort (AMANHI) that started in 2014 [29]. The overall aim of the biobank was to collect detailed epidemiological and biological data of pregnant women Microorganisms 2021, 9, 1180 3 of 12

and their newborns in order to improve advancing knowledge on key pregnancy and birth outcomes. As previously reported, vaginal swabs were collected under the supervision of a healthcare worker in healthcare facilities and stored in 1 mL eNAT buffer (Copan, Italy) at −20 ◦C at the Public Health Laboratory Ivo de Carneri on Pemba Island [31]. The participants were informed in their native language about the study and its procedures [29]. All of the included women gave consent to participate in the biobanking effort and the further research linked to it, and study ethics approval was obtained from the Zanzibar Medical Research and Ethics Committee (ZAMREC) [29]. Samples were transported in dry ice to the Netherlands and stored at −20 ◦C. In total, 686 vaginal samples from 441 women were included for analysis in this study. Of these, 385 samples were collected at first timepoint during pregnancy (between 8 and 20 gestational age (GA) weeks), 257 samples at second timepoint during pregnancy (between 20 and 40 GA weeks), and 44 samples post- delivery (between 42 and 60 days after birth). All vaginal samples were retrospectively tested for the presence of C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium and the positive results were immediately shared with the stakeholders in Tanzania so the participants could be informed of their health status for further action. Details on sample processing and on the genital infections molecular assays have been previously described elsewhere [31–35]. In short, DNA of C. trachomatis, N. gonorrhoeae, and T. vaginalis were detected by their respective CE-IVD-certified Presto C. trachomatis, N. gonorrhoeae (Goffin Molecular Diagnostics, Houten, The Netherlands) and Presto T. vaginalis (Goffin Molecular Diagnostics, Houten, The Netherlands) tests and Real-Time polymerase chain reaction (PCR) with ABI Taqman 7500 (Applied Biosystems, Foster City, CA, USA) as per the manufacturer’s instructions [33,34]. Moreover, a M. genitalium assay, as described in Muller et al., was used on the LightCycler 480 II PCR machine (Roche Diagnostics, Basel, Switzerland) [31–35]. Samples were marked positive if the crossing point value (Cp) was between 11 and 38 samples or if samples had a Cp value of 39–40 with an S-shape amplification curve. Due to logistical and practical constraints, not all women were consecutively sampled at all timepoints. Longitudinal analysis was performed on vaginal samples collected from the same women at more than one timepoint. Persistence and clearance of infection were investigated if samples of women who tested positive at a specific timepoint were also collected at later timepoints. Infection persistence was defined as two or more consecutively sampled vaginal swabs testing positive for the same genital pathogen using the PCR tests mentioned above. Clearance was defined by a subsequent negative result on a sample which had previously tested positive for the same pathogen, again using the PCR tests mentioned above. Demographic and health data such as maternal age, parity, gravidity, school attendance years, religion, previous diagnosis with human immunodeficiency virus (HIV), current symptoms of urinary tract infection and antibiotics use were self-reported via a questionnaire at each sample collection visit. Even though the questionnaire was filled at each visit by all women included in the study, it is important to note that not every questionnaire item might have been answered. Therefore, the total number of responders at a specific demographic and health questionnaire item was explicitly mentioned whenever the total number of available demographic and health data did not match the total number of samples at a specific timepoint. The frequency and descriptive analysis of the demographic and health data retrieved from the biobank questionnaire were analyzed using IBM SPSS statistical software v. 26 (SPSS Inc., Chicago, IL, USA). Chi-square test with Yates continuity correction was used to determine whether the point prevalence of different infections significantly differed at each collection timepoint. A p-value of less than 0.05 was considered statistically significant.

3. Results 3.1. Population Characteristics Vaginal samples of 441 pregnant women from Pemba Island were included in this study. The mean maternal age at ﬁrst sampling was 28.3 years (range 16–48), the mean Microorganisms 2021, 9, x 4 of 12

3. Results Microorganisms 2021, 9, 1180 3.1. Population Characteristics 4 of 12 Vaginal samples of 441 pregnant women from Pemba Island were included in this study. The mean maternal age at first sampling was 28.3 years (range 16–48), the mean parity 3.5 3.5 ( (0–10),0–10), and and 60 60 (14%) (14%) women women were were primigravida. primigravida. Mean Mean gravidity gravidity was was4.6 (n 4.6 = 436;(n = range 436; range 1–16), 1–16), and only and one only woman one woman self-reported self-reported having having HIV infection HIV infection (n = 433; (n 0.23%).= 433; The0.23%). mean The school mean attendance school attendance was two years was two (range years 0–5 (range). A significant 0–5). A significantmajority of majoritysubjects identifiedof subjects as identified (n = 436) asMuslim (n = 436) Shirazi Muslim (99.8%), Shirazi while (99.8%), one woman while identified one woman as Christian identified- Shirazi.as Christian-Shirazi.

3.2. Sexually Sexually Transmitted Transmitted Infections Prevalence and Symptomatology InIn this this cohort, cohort, the the STI STI burden burden was 12.2% among the 385 vaginal samples tested at the firstfirst timepoint.timepoint. Twenty-fiveTwenty-five (6.5%) (6.5%) vaginal vaginal samples samples tested tested positive positive for forT. vaginalisT. vaginalisinfection, infec- tionseventeen, seventeen (4.4%) (4.4%) for C. trachomatisfor C. trachomatisinfection, infection five (1.3%), five for (1.3%)M. genitalium for M. genitaliuminfection andinfection none and(0%) none for N. (0%) gonorrhoeae for N. gonorrhoeaeinfection infection (Figure1a). (Figure At this 1a) timepoint,. At this timepoint, twelve of twelve the 325 of women the 325 women(3.7%) who (3.7%) filled who out filled the questionnaire out the questionnaire item reported item reported that they that had they urinary had tract urinary infection tract infectionsymptoms. symptoms. The vaginal The sample vaginal of sample one of of them one tested of them positive tested for positiveC. trachomatis for C. trachomatisinfection, infection,and the vaginal and the sample vaginal of anothersample of for anoT. vaginalisther for T.infection. vaginalis One infection woman. One (of thewoman 357 women (of the 357who women filled in who the filled questionnaire in the questionnaire item) reported item) having reported urinary having tract urinary infection tract symptoms infection symptomsand admitted and to admitted taking antibiotics to taking (unknown antibiotics indication). (unknown Eightindication). other women Eight other also reported women alsoantibiotics reported use. antibiotics None of use. the nineNone women of the whonine reportedwomen who antibiotics reported usage antibiotics (2.5%) testedusage (2.5%)positive tested for a positive genitalinfection. for a genital infection.

(a). First collection timepoint (b). Second collection timepoint (c). Post-delivery collection timepoint

Figure 1. Venn diagramsdiagrams with with point point prevalence prevalence and and combined combined prevalence prevalence of Chlamydia of Chlamydia trachomatis trachomatis(CT), (CT),Neisseria Neisseria gonorrhoeae gonor- (NG),rhoeae Trichomonas(NG), Trichomonas vaginalis vaginalis(TV) and (TV)Mycoplasma and Mycoplasma genitalium genitalium(MG) genital (MG) infectionsgenital infections during andduring after and pregnancy. after pregnancy. The number The number represents the amount (n) of samples with a detected microorganism. Above is the individual point prevalence represents the amount (n) of samples with a detected microorganism. Above is the individual point prevalence per genital per genital infection and below each diagram is the total number of tested samples and the combined point prevalence of infection and below each diagram is the total number of tested samples and the combined point prevalence of genital genital infections. (a) the sexually transmitted infection (STI) burden was 12.2% among the 385 vaginal samples tested at infections.the first timepoint (a) the sexually. (b) 16.7% transmitted among the infection 257 vaginal (STI) samples burden wastested 12.2% positive among for theat least 385 vaginalone STI samples at the first tested timepoint at the first. (c) timepoint.The total STI (b )burden 16.7% amongpost-delivery the 257 was vaginal 11.4% samples among tested44 samples positive tested. for at least one STI at the first timepoint. (c) The total STI burden post-delivery was 11.4% among 44 samples tested. At the second timepoint, the STI burden was 16.7% in the 257 vaginal samples tested. NineteenAt the of secondthe 257 timepoint,(7.4%) vaginal the STI samples burden tested was positive 16.7% in for the T. 257 vaginalis vaginal infection samples, fifteen tested. Nineteen of the 257 (7.4%) vaginal samples tested positive for T. vaginalis infection, fifteen (5.8%) for C. trachomatis infection, eight (3.1%) for M. genitalium infection and one (0.4%) (5.8%) for C. trachomatis infection, eight (3.1%) for M. genitalium infection and one (0.4%) for for N. gonorrhoeae infection (Figure 1b). One respondent out of 255 (0.4%) for the question- N. gonorrhoeae infection (Figure1b). One respondent out of 255 (0.4%) for the questionnaire naire item reported urinary tract infection symptoms. Her vaginal sample was negative item reported urinary tract infection symptoms. Her vaginal sample was negative for all for all tested infections. Nine of the 248 respondents (3.6%) self-reported antibiotics use tested infections. Nine of the 248 respondents (3.6%) self-reported antibiotics use between between the first and second timepoint in pregnancy. The vaginal sample of one of them the first and second timepoint in pregnancy. The vaginal sample of one of them was positive was positive for T. vaginalis infection. The type and exact time of antibiotics use was not for T. vaginalis infection. The type and exact time of antibiotics use was not available. available. Of the 44 vaginal samples tested post-delivery, three (6.8%) were positive for T. vaginalis infection, one (2.3%) for C. trachomatis infection, one (2.3%) for N. gonorrhoeae infection, and none (0%) for M. genitalium infection (Figure1c). The total STI burden was 11.4%. Post-delivery, none of the 37 respondents reported symptoms of urinary tract infections Microorganisms 2021, 9, x 5 of 12

Of the 44 vaginal samples tested post-delivery, three (6.8%) were positive for T. Microorganisms 2021, 9, 1180vaginalis infection, one (2.3%) for C. trachomatis infection, one (2.3%) for N. gonorrhoeae 5 of 12 infection, and none (0%) for M. genitalium infection (Figure 1c). The total STI burden was 11.4%. Post-delivery, none of the 37 respondents reported symptoms of urinary tract infections (100%), and five women self-reported antibiotic use (13.5%) (of which none (100%), and ﬁve women self-reported antibiotic use (13.5%) (of which none tested positive tested positive for a genital infection). for a genital infection). Prevalence of different infections at each timepoint did not significantly differ. Prevalence of different infections at each timepoint did not signiﬁcantly differ.

3.3. Sexually3.3. SexuallyTransmitted Transmitted Infections Infections Persistence Persistence and Clearance and Clearance during Pregnancy during Pregnancy In total, In202 total, women 202 women were weresampled sampled at first at firstand and second second timepoints. timepoints. Among Among them, them, eleven eleven (5.4%)(5.4%) tested tested positive positive for for C. C.trachomatis trachomatis infectioninfection at the at thefirst first timepoint timepoint,, eleven eleven (5.4%) (5.4%) for for T. vaginalisT. vaginalis infection,infection, four (3.1%) four (3.1%) for M. forgenitaliumM. genitalium infection,infection, and none and for none N. gonorrhoeae for N. gonorrhoeae infectioninfection (Figure (Figure2). Of the2). 202 Of theparticipants, 202 participants, persistence persistence of C. trachomatis of C. trachomatis infectioninfection was was 100% (ntotal = 11; average time between sampling collection was 8+2 weeks(+days)+2), 82% for(+days) T. 100% (ntotal = 11; average time between sampling collection was 8 weeks ), 82% vaginalis infection (ntotal = 11; average time between sampling collection was 9+6 weeks) and +6 for T. vaginalis infection (ntotal = 11; average time between sampling collection was 9 75% for M. genitalium infection (ntotal = 4; average time between sampling collection was weeks) and 75% for M. genitalium infection (ntotal = 4; average time between sampling 9+6 weeks)collection (Figure 2). was Clearance 9+6 weeks) of T. (Figure vaginalis2). Clearanceinfection during of T. vaginalis pregnancyinfection (n = 2) duringhappened pregnancy within 13(n+0= and 2) happened 21+5 weeks, within respectively 13+0 and (Figure 21+5 weeks, 2). Where respectively clearance (Figure of M.2). genitalium Where clearance infectionof occurredM. genitalium duringinfection pregnancy occurred (n = 1), during it was pregnancy within 12 (n+6 =weeks 1), it was(Figure within 2). The 12+6 weeks vaginal (Figuresamples2). of The seven vaginal women samples tested of sevenpositive women for a testedgenital positive infection for at a genitalthe second infection at timepoint,the while second their timepoint, samples while at the their first samplestimepoint at were the first negative timepoint (Figure were 2). negative (Figure2).

Figure 2. Schematic overview of the persistence and clearance of CT, NG, TV and MG genital infections of positive vaginal Figure 2. Schematic overview of the persistence and clearance of CT, NG, TV and MG genital infections of positive vaginal swabs collected during pregnancy (n = 33 infected women out of 202 tested women). Horizontal labels indicate the time of swabs collected during pregnancy (n = 33 infected women out of 202 tested women). Horizontal labels indicate the time of collectioncollection in gestational in gestational age in weeks. age in weeks.Each dot Each represents dot represents a vaginal a vaginalsample positive sample positivefor C. trachomatis for C. trachomatis (in red), T.(in vaginalis red), T. vaginalis (in yellow),(in yellow),N. gonorrhoeaeN. gonorrhoeae (in green),(in and green), M. genitalium and M. genitalium (in blue).(in Open blue). dots Open represent dots represent cleared infections. cleared infections. The thin Thegrey thin grey arrows, witharrows, the withinterval the between interval betweentesting in testing weeks inon weeks top, connect on top, a connect positive a positivesample with sample matching with matching coloured colouredopen dot open dot and it refersand to it cleared refers to infections. cleared infections. The seven The dots seven in the dots seco innd the collection second collection timepoint timepoint period indicate period samples indicate with samples de novo with de novo infectionsinfections (as samples (as samplesfrom the fromsame the women same collected women collected earlier were earlier not were positive not positivefor the same for the pathogens). same pathogens). All infections All infections detected detectedat first timepoint at ﬁrst timepoint did persist did if persistno thin if grey no thin arrow grey was arrow connected was connected to them. to them.

Nine womenNine womenself-reported self-reported symptoms symptoms of urinary of urinarytract infection tract infection at the first at thetimepoint first timepoint during pregnancy,during pregnancy, but only but one only of them one oftested them positive tested positivefor C. trachomatis for C. trachomatis infection,infection, and and remainedremained so at the second so at the timepoint second (despite timepoint being (despite asymptomatic being asymptomatic at second testing). at second None testing). of theseNone women of these reported women antibiotic reported use antibiotic during use pregnancy. during pregnancy. Furthermore, Furthermore, six women six women reportedreported use of antibiotics use of antibiotics at the first at thetimepoin first timepoint,t, none of nonethese oftested these positive tested positivefor a genital for a genital infectioninfection at first timepoint; at first timepoint; one subsequently one subsequently tested positive tested positivefor C. trachomatis for C. trachomatis infectioninfection at at the secondthe timepoint. second timepoint. At the second At the timepoin second timepoint,t, six women six womenreported reported antibiotics antibiotics use, with use, with only oneonly testing one positive testing positive for T. vaginalis for T. vaginalis infectioninfection at the atsame the sametimepoint. timepoint. No information No information on on the indicationthe indication and type and typefor antibiotics for antibiotics used usedwas available. was available.

3.4. Sexually Transmitted Infections Persistence and Clearance after Pregnancy Thirty-eight women were sampled at the second timepoint during pregnancy and post-delivery. Of these women, one of the vaginal samples (2.6%) was positive for C. Microorganisms 2021, 9, 1180 trachomatis infection, five (13.2%) for T. vaginalis infection, and none for M. genitalium or6 of 12 N. gonorrhoeae infections at the second timepoint (Figure 3). Twenty-two weeks after the second timepoint, the persistence rate post-delivery was 100% for C. trachomatis (ntotal = 1) and 20% for T. vaginalis infections (ntotal = 5) (Figure 3). Four women had post-delivery (n = 1) and 20% for T. vaginalis infections (n = 5) (Figure3). Four women had samplestotal that tested negative for T. vaginalis after an averagetotal of 18+6 (range 15+1–22+0) weeks post-delivery samples that tested negative for T. vaginalis after an average of 18+6 (range of having tested positive (Figure 3). In the group of 38 women, one vaginal sample with 15+1–22+0) weeks of having tested positive (Figure3). In the group of 38 women, one N. gonorrhoeae and another with T. vaginalis were newly detected post-delivery (Figure 3). vaginal sample with N. gonorrhoeae and another with T. vaginalis were newly detected None of these 38 women self-reported symptoms of urinary tract infection. Six women post-delivery (Figure3). None of these 38 women self-reported symptoms of urinary self-reported use of antibiotics at the second timepoint during pregnancy, and no genital tract infection. Six women self-reported use of antibiotics at the second timepoint during infection was detected at second timepoint during pregnancy and post-delivery in their pregnancy, and no genital infection was detected at second timepoint during pregnancy vaginal samples. and post-delivery in their vaginal samples.

Figure 3. Schematic overview of the persistence and clearance of CT, NG, TV and MG genital infections of positive vaginal Figureswabs 3. Schematic collected overview during pregnancy of the persistence and post-delivery and clearance (n = of 8 CT infected, NG, TV women and MG out genital of 38 tested infections women). of positive Horizontal vaginal labels swabs collected during pregnancy and post-delivery (n = 8 infected women out of 38 tested women). Horizontal labels indicate the time of collection in gestational age in weeks. Each dot represents a vaginal sample positive for C. trachomatis indicate the time of collection in gestational age in weeks. Each dot represents a vaginal sample positive for C. trachomatis (in red), T. vaginalis (in yellow), N. gonorrhoeae (in green), and M. genitalium (in blue). M. genitalium was not detected at the (in red), T. vaginalis (in yellow), N. gonorrhoeae (in green), and M. genitalium (in blue). M. genitalium was not detected at the secondsecond timepoint timepoint in inthis this sub-cohort. sub-cohort. Open Open dots dots represent clearedcleared infections. infections. The The thin thin grey grey arrows, arrows, with with the intervalthe interval between betweentesting testing in weeks in weeks on top, on connect top, connect a positive a positive sample withsample a matching with a matching coloured coloured open dot andopen it dot refers and to it cleared refers infections.to cleared The infections.two dots The (T. two vaginalis dots (T.n = vaginalis 1) and N. (n gonorrhoeae= 1) and N.( ngonorrhoeae= 1)) in the (n post-delivery = 1)) in the post-delivery collection timepoint collection period timepoint indicate period samples indicatewith samples de novo infectionswith de novo (assamples infections from (asthe samples same womenfrom the collected same women at the secondcollected timepoint at the second were not timepoint positive were for the not same positivepathogens). for the Allsame infections pathogens). detected All atinfections second timepoint detected didat second persist iftimepoint no thin grey did arrowpersist was if no connected thin grey to them.arrow was connected to them. 4. Discussion 4. Discussion The study observed the persistence of genital infections during pregnancy (C. tra- chomatisThe study, T. vaginalis observedand theM. persistence genitalium) andof genital post-delivery infections (C. trachomatisduring pregnancyand T. vaginalis (C. ). trachomatisDuring, pregnancy,T. vaginalisT. and vaginalis M. genitaliuminfection clearance) and post-delivery (26 and 33 weeks(C. trachomatis after first and collection) T. vaginalisoccurred). During in two pregnancy, (18%) of the T. vaginalis cases, and infectionM. genitalium clearanceinfection (26 and clearance 33 weeks (27 after weeks first after collection)first collection) occurred in in one two case (18%) (25%); of C.the trachomatis cases, andinfection M. genitalium did not infection clear in anyclearance of the (27 eleven weekscases. after Pre- first and collection) post-delivery, in oneT. case vaginalis (25%);infection C. trachomatis cleared infection in one (80%) did not case clear (an averagein any of of the18 eleven weeks aftercases. second Pre- and collection), post-delivery, while T. there vaginalis was noinfection clearance cleared of C. in trachomatis one (80%)infection case (an 22average weeks of after 18 theweeks second after collection. second collection), For N. gonorrhoeae while ,there the persistence was no clearance of infection of couldC. trachomatisnot be determined. infection 22 weeks after the second collection. For N. gonorrhoeae, the persistenceTo ourof infection knowledge, could this not is thebe determined. first study to investigate the natural history of T. vaginalis andTo M.our genitalium knowledge,during this pregnancy is the first and study post-delivery. to investigate In a non-pregnantthe natural history cohort ofof 119T. M. vaginalisgenitalium and M.-infected genitalium sex during workers pregnancy in Kenya and (prevalence post-delivery. 14%), In 45% a non-pregnant spontaneously cohort cleared of 119the M. infection genitalium over-infected the course sex workers of three months,in Kenya whereas (prevalence in this 14%), study 45% (a spontaneously much smaller M. clearedgenitalium the infectioninfected over cohort the (n course= 4)),that of three wasthe months, case for whereas just 25% in over this the study course (a ofmuch almost smaller13 weeks M. genitalium [18]. However, infected the cohort intervals (n = 4)), between that was sample the case collection for just points 25% over were the not course uniform of almostin this 13 study. weeks The [18]. sample However, of the womanthe intervals who cleared betweenM. sample genitalium collectioninfection points was collectedwere not almostuniform 13 in weeks this study. after theThe first sample pregnancy of the woman collection who timepoint, cleared M. while genitalium there was infection a shorter interval between initial detection and the second collection timepoint (8–10 weeks) for the other three women with persistent infection. As expected, the wider the time interval between collection timepoints, the greater the chance of clearance [17,18]. Existing results from a non-pregnant adolescent (14–17 years) cohort in the United States of America indicate that untreated T. vaginalis infection can persist for up to 12 weeks, and that individuals can be asymptomatic for the entire duration of infection [20]. Mod- MicroorganismsMicroorganismsMicroorganisms 2021 2021 2021, ,9 ,9, 9,x ,x x 77 7 of ofof 12 1212 Microorganisms 2021, 9, x 7 of 12

waswaswas collected collectedcollected almost almostalmost 13 1313 weeks weeksweeks after afterafter the thethe first firstfirst pregnancy pregnancypregnancy collection collectioncollection timepoint, timepoint,timepoint, while whilewhile there therethere waswaswaswas a collected a ashorter shortershorter interval almost intervalinterval 13between betweenbetween weeks initial after initialinitial the detection detectiondetection first pregnancy and andand the thethe second secondcollectionsecond collection collectioncollection timepoint, timepoint timepointtimepoint while (8–10 there (8–10(8–10 weeks)wasweeks)weeks) a shorter for forfor the thethe interval other otherother three threebetweenthree women womenwomen initial with withwith detection persis persispersistent tentandtent infection. theinfection.infection. second As As Ascollection expected, expected,expected, timepoint the thethe wider widerwider (8–10 the thethe timeweeks)timetime interval intervalinterval for the between betweenbetween other three collection collectioncollection women timepoints, timepoints,timepoints, with persis the thethetent greater greatergreater infection. the thethe chance chancechance As expected, of ofof clearance clearanceclearance the [17,18].wider [17,18].[17,18]. the time ExistingintervalExistingExisting betweenresults resultsresults from from fromcollection a a anon-pregnant non-pregnantnon-pregnant timepoints, adoles adoles adolesthe greatercentcentcent (14–17 (14–17 (14–17the chance years) years)years) of cohort cohort cohortclearance in inin the the the[17,18]. United UnitedUnited Microorganisms 2021, 9, 1180 7 of 12 StatesStatesStatesExisting of ofof America AmericaAmerica results indicate indicateindicate from athat thatnon-pregnantthat untreated untreateduntreated T. adoles T.T. vaginalis vaginalisvaginaliscent (14–17infection infectioninfection years) can cancan cohortpersist persistpersist infor for forthe up up upUnited to toto 12 1212 weeks,Statesweeks,weeks, ofand andand America that thatthat individuals individualsindividuals indicate thatcan cancan be untreated bebe asymptomatic asymptomaticasymptomatic T. vaginalis for forfor the the theinfection entire entireentire duration durationdurationcan persist of ofof infection infectioninfectionfor up to[20]. [20].[20]. 12 Modellingweeks,ModellingModelling and based thatbasedbased individuals on onon data datadata from fromfrom can non-pregnantbe non-pregnantnon-pregnant asymptomatic women womenwomen for the show showshow entire that thatthat duration T. 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T. T.T. vaginalis and vaginalisvaginalis withmore infection theinfectioncomparableinfection longer also alsoalso persistence withcleared clearedcleared the more longer ratemoremore observedoften oftenpersistenceoften in inin women in womenwomen the rate modelling whose observed whosewhose samples samplessamples [in15 ].theT. weremodellingvaginaliswerewere collected collectedcollectedinfection [15]. at T.atat also larger vaginalislargerlarger cleared time timetime infection more intervals intervalsintervals often also (Figures in(Figures (Figurescleared women 2 more2 2 whose and andand often 3). 3). samples3). 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C.C. trachomatis trachomatispregnancy,trachomatis infection infectionrespectively.infection for forfor up upup to toto 13 1313 weeksweeksweeksFurthermore,Furthermore, during during during pregnancy. pregnancy. pregnancy. this this study study This This This shows C. showsC. C. trachomatis trachomatis trachomatis a apersistence persistence persistence persistence persistence of ofC.C. (100%)trachomatis (100%) (100%) trachomatis differs differs differs infection infectionfrom from from that that forthat forreported upreported reported upto 13 to byweeks13byby Sheffield weeks SheffieldSheffield during during et et et pregnancy.al. al.al. (pregnant pregnancy. (pregnant(pregnant This cohort) cohort)cohort) ThisC. trachomatisC. and andand trachomatis othe otheothe persistencerr rnon-pregnant non-pregnantnon-pregnantpersistence (100%) data data (100%)data differs (retrieved (retrieved(retrieved differs from fromthat fromfrom reported Golden thatGoldenGolden re- etbyportedetet al. Sheffieldal.al. and and byand Sheffield Geisler GeisleretGeisler al. (pregnant et etetet al.al.) al.)al.) (pregnant (Figure (Figurecohort)(Figure 4) and cohort)4)4) [1 [1 [1othe4,17,36].,4,17,36].,4,17,36]., andr non-pregnant other where wherewhere non-pregnant spontaneous spontaneousspontaneous data (retrieved data resolution resolutionresolution (retrieved from Golden of fromofof C. C.C. trachomatisetGoldentrachomatistrachomatis al. and et al.occurred Geisler occurredoccurred and Geisler etin in in al.)44% 44%44% et(Figure of of al.)of 1521 15211521 (Figure 4)pregnant pregnantpregnant [14,17,36].,4)[ 14 North-American North-AmericanNorth-American,17 ,36where]., where spontaneous spontaneouswomen womenwomen in inresolutionin the thethe resolution course coursecourse of of ofofC. of3 3 3 monthstrachomatisC.monthsmonths trachomatis [17]. [17].[17]. occurred The TheTheoccurred discrepancy discrepancydiscrepancy in 44% in 44% of can can of1521can 1521be bebe pregnant accounted accountedaccounted pregnant North-American for North-Americanforfor the thethe lower lowerlower prevalence prevalenceprevalence women women in of of inofthe C. C.theC. trachomatiscourse trachomatistrachomatis course of of3 inmonths3inin monthsthe thethe present presentpresent [17]. [17 ].The study study Thestudy discrepancy discrepancy (5.4%) (5.4%)(5.4%) compared comparedcompared can can be be accountedto accountedtoto Shef ShefSheffieldfieldfield for foret et et theal. the al.al. (9%) lower (9%) lower(9%) and andandprevalence prevalence the thethe limited limitedlimited of of C.number C. numbernumber trachomatis trachomatis of ofof C. C.C. trachomatisinintrachomatistrachomatis the the present present positive positivepositive study study samples (5.4%) samples (5.4%)samples compared compared at atat the thethe first firstfirst to to timepoint timepointShef Sheffieldtimepointfield et( et n((n nal. al.= = =8) (9%)8) (9%)8) in inin this and this andthis study thestudy thestudy limited limited compared comparedcompared number number to toto other otherof ofother C.C. studiestrachomatistrachomatisstudiesstudies (Figure (Figure(Figure positivepositive 4) 4)4) [17]. [17].[17]. samples samples at at the the first first timepoint timepoint ( (nn = 8) 8) in in this this study study compared to other studiesstudies (Figure (Figure 44))[ [17].17].

100100100 100 909090 90 808080 80 707070 trachomatis trachomatis

trachomatis 70 . . . 606060 C trachomatis C C

. 60 C 505050 50 161616 non-pregnancy non-pregnancy non-pregnancy studies studies studies infection

infection 40 infection 4040 16 non-pregnancy studies

infection 40 PregnancyPregnancyPregnancy studies studies studies 303030 Pregnancy studies 30 202020 20 1010 Persistence (%) of Persistence 10 Persistence (%) of Persistence Persistence (%) of Persistence 10 Persistence (%) of Persistence 000 0 000 200 200 200 400 400 400 600 600 600 800 800 800 1000 1000 1000 1200 1200 1200 0 200 400 600 800 1000 1200 TimeTimeTime (days) (days) (days) Time (days)

FigureFigureFigureFigure 4. 4.4. 4. Urogenital UrogenitalUrogenital Urogenital C. C.C. C. trachomatis trachomatistrachomatis trachomatis presence presencepresence presence in inin in women womenwomen women during duringduring during follow-up. follow-up.follow-up. follow-up. Percentages PercentagesPercentages Percentages of ofof of C. C.C. C. trachomatis trachomatistrachomatis trachomatis infected infectedinfected infected women womenwomen women are areare are Figure 4. Urogenital C. trachomatis presence in women during follow-up. Percentages of C. trachomatis infected women are describeddescribeddescribeddescribed by byby by Golden GoldenGolden Golden et etet et al. al.al. al. and andand Geisler GeislerGeisler et etet al. al. al.■■ ■are are are compared compared compared with with with two two two pregnancy pregnancy pregnancy studies studies studies (Sheffield (Sheffield (Sheffield (Shefﬁeld et et et al. al. al. ● ●● andand and and the thethe the present presentpresent present described by Golden et al. and Geisler et al.■ are compared with two pregnancy studies (Sheffield et al. ● and the present studystudystudystudy ● ●●)[) )[14,17,36].) 14 [14,17,36].[14,17,36].,17,36]. The TheThe curve curvecurve shows showsshows the thethe trend trendtrend as asas calculatedcalc calccalculatedulatedulated by byby the thethe sixteen sixteen sixteen non-pregnant non-pregnantnon-pregnant studies.studies. studies.studies. study ●) [14,17,36]. The curve shows the trend as calculated by the sixteen non-pregnant studies. DysbioticDysbioticDysbioticDysbiotic vaginal vaginalvaginalvaginal microbiota microbiota microbiotamicrobiota has has hashas been been beenbeen linked linked linkedlinked with with withwith increased increased increasedincreased susceptibility susceptibility susceptibilitysusceptibility to to toto genital genital genitalgenital infections [37,38]. It will be of further interest to investigate how host genetic determinant infectionsinfectionsinfectionsDysbiotic [37,38]. [37,38].[37,38]. vaginal It ItIt will willwill microbiota be bebe of ofof further furtherfurther has interest interestbeeninterest linked to toto investigate investigateinvestigate with increased how howhow host host hostsusceptibility genetic geneticgenetic determinant determinantdeterminant to genital of infections inﬂuence clearance of C. trachomatis or other genital pathogens, in particular ofinfectionsofof infections infectionsinfections [37,38]. influence influenceinfluence It will clearance clearanceclearance be of further of ofof C. C.C. interesttrachomatis trachomatistrachomatis to investigate or oror other otherother genital genital genitalhow host pathogens, pathogens,pathogens, genetic in determinant inin particular particularparticular during pregnancy [24,25,37–43]. It was hypothesized that the persistence level of geni- duringofduringduring infections pregnancy pregnancypregnancy influence [24,25,37–43]. [24,25,37–43].[24,25,37–43]. clearance ofIt ItIt was C. waswas trachomatis hypoth hypothhypothesizedesizedesized or other that thatthat genitalthe thethe persistence persistencepersistence pathogens, level levellevel in particularof ofof genital genitalgenital tal infections would be higher among pregnant women, since the women’s innate and infectionsduringinfectionsinfections pregnancy would wouldwould be [24,25,37–43].bebe higher higherhigher among amongamong It was pregna pregnapregna hypothntntnt esized women, women,women, that since since sincethe persistence the thethe women’s women’swomen’s level innate innate innateof genital and andand infectionsacquired immunewould be systems higher are among altered pregna [44] tont preventwomen, the since rejection the women’s of a semi-allogeneic innate and foetus, while protecting both mother and foetus from infection [44]. The macrophages in the maternal decidual epithelium (part of innate immunity) have the ability to remodel tissue, supress maternal immune response and present antigens [45,46]. While changes in

acquired immunity also occur in pregnancy, there is a decrease in circulating levels of pro- inﬂammatory cytokines and interleukin-10, the amount of regulatory T-cells is increased in the decidua, maternal immunity shifts towards a T helper (Th) 2 cells phenotype, and Th2 cells outnumber Th1 cells in the decidua [47–49]. The mechanisms of immune cell Microorganisms 2021, 9, 1180 8 of 12

interactions in pregnancy have been extensively reviewed elsewhere and are out of the scope of this paper [24,44,50–53]. Nevertheless, because of these immunological changes, data about infection persistence and clearance observed in non-pregnant women are not fully representative of pregnant women [54,55]. Our findings show that the clearance might be lower in pregnant women than in non-pregnant cohorts (Figure4). As discussed, due to the low number of samples tested, more pregnancy-related information is warranted to confirm this trend (Figure4). The presence of C. trachomatis DNA, but not M. genitalium, has been detected in chorionic villi from spontaneous miscarriage [56]. Thus, it is not only important to further investigate the interplay between genital infections and host immunity during pregnancy, but also the burden genital infections potentially have on mother and foetus (such as intra-uterine infections, low birthweight, preterm birth, and respiratory infections) [44]. The drawbacks of this study are the relatively small sample size and the limited longitudinal sampling, which, combined with the prevalence of infections, have provided results of a limited power, therefore cautious extrapolation is recommended. Furthermore, information about general antibiotic use in Pemba is limited and for this study has been obtained via a questionnaire, which lacked details on the indication for, and type of antibiotic used; samples from women using antibiotics were not excluded from the analysis. The relationship between antibiotic use in the preceding months (not directly related to an STI) and lower prevalence of STIs is still debatable even though it might have an effect on C. trachomatis prevalence also in countries with low per capita antibiotics consumption [57,58]. However, it should be noted that a large part of background antibiotics use are not first-line treatments for STIs, such as C. trachomatis (azithromycin), M. genitalium (doxycycline or azithromycin), or N. gonorrhoeae (ceftriaxone), and might not have an impact on their course as they are infrequently used for incidental treatment [57]. In relation to this study, detailed information on genital symptoms was also missing. Only one woman self-reported urinary tract infection symptoms, which are often hard to differ- entiate from genital ones [31,59]. Lastly, there was no differentiation between persistence of infections and re-infection. It is therefore possible that some women achieved clearance from a specified pathogen, and were re-infected before the subsequent collection timepoint. To mitigate against this phenomenon, pathogen genotyping testing should be considered, especially in a larger cohort analysis [18]. Bacterial load and co-infections with other bur- densome pathogens, for example HIV, vaginal dysbiosis and chronic inflammation should also be considered to better understand the clinical implications of these infections [18]. However, in this cohort the number of women who self-reported diagnosis of HIV was very low. Current understanding of the natural history of C. trachomatis, T. vaginalis, N. gonorrhoeae and M. genitalium is hampered by a lack of robust scientific studies; it is mostly limited to that provided by low sensitivity and specificity studies (using culture-based methods) in the pre-antibiotic era [16,17,20]. Logically, with current antibiotic treatment possibilities, ethical constraints are major reasons for the lack of studies investigating the natural history of curable pathogens. Nonetheless, and as shown with this retrospective approach, information about the natural history of infection in a specific population can still be obtained with the help of existing biobank samples and database. Retrieving this information remains important, as evidence like the one provided here is essential to public health officials when evaluating the burden and influence of genital infections on maternal and neonatal health. Currently, most genital screening is not implemented in antenatal care in a standard way, or only implemented early in pregnancy [60,61]. Furthermore, the management of these STIs is in most countries syndromic, while most infected women are asymptomatic undiagnosed carriers, a situation that puts them and the foetus at risk [62]. Thus, the observation that there is not always natural clearance of these curable pathogens during the pregnancy, and the possibility of de novo infections, shows remaining challenges in detecting and treating C. trachomatis, T. vaginalis, N. gonorrhoeae and M. genitalium during pregnancy [60,61,63,64]. The urgency of better screening practices is ever Microorganisms 2021, 9, 1180 9 of 12

more pertinent, particularly as new resistant pathogen strains arise, as is the case for N. gonorrhoeae, C. trachomatis, T. vaginalis, and M. genitalium [65–69]. Similarly, understanding of the natural progression of these infections would be beneﬁcial for vaccine design against these microorganisms [16,64].

5. Conclusions This study reports the persistence rate of C. trachomatis (100%), T. vaginalis (82%) and M. genitalium (75%) infections during pregnancy and C. trachomatis (100%) and T. vaginalis (20%) before and post-delivery in vaginal samples from pregnant women living in Pemba Island, Tanzania. The persistence of N. gonorrhoeae during pregnancy could not be determined because of low prevalence of infection. The detection and persistence of these curable genital infection stresses on the importance of screening all women during the pre and postnatal periods, where treatment will decrease sequelae. Investigating the clearance and persistence rate of these infections will improve the knowledge of the pathophysiology of these genital infections during pregnancy and post-delivery. With a retrospective approach, larger biobank cohorts can build on this data and identify factors associated with duration or clearance of infection during pregnancy. Such evidence will support effective management and screening programs, as well as innovative biomedical approaches targeting them.

Author Contributions: Conceived the idea: S.D., S.A.M., E.A.; methodology, N.C.A.J., J.P., N.B.U.; formal analysis, N.C.A.J., S.O.; data and biospecimen collections, A.M.O., F.A., S.D.; resources, S.M.A., S.S., S.D.; writing—original draft, N.C.A.J.; writing—review and editing, N.C.A.J., S.A.M., E.A., supervision: S.A.M., S.D., E.A.; project administration, S.S., S.D.; funding acquisition: S.S., S.A.M., E.A. All authors have read and agreed to the published version of the manuscript. Funding: This study was in part subsidized by the Otto Kranendonk Fund from the Netherlands Society for Tropical Medicine and International Health (NVTG). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the Zanzibar Medical Research and Ethics Committee (ZAMREC), 24 March 2014, amended 4 October 2016. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data are available from the authors with the permission of the Biobank governing body/local institution and the Principal Investigator of the site. Acknowledgments: To all the women who participated in the biobank effort, and the staff at the biobank in Pemba Island, Tanzania. Further thanks to Sonja N.H. Puljhun, Monique M. Verveer and Roel Heijmans for their technical assistance in the laboratory in the Netherlands. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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