Mycoplasma genitalium and Other Reproductive Tract Infections in Pregnant Women, Papua New Guinea, 2015–2017

Much about the range of pathogens, frequency of coinfection, and clinical effects of reproductive tract infections (RTIs) among pregnant women remains unknown. We report on RTIs (Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Treponema pallidum subspecies pallidum, bacterial vaginosis, and vulvovaginal candidiasis) and other reproductive health indicators in 699 pregnant women in Papua New Guinea during 2015–2017. We found M. genitalium, an emerging pathogen in Papua New Guinea, in 12.5% of participants. These infections showed no evidence of macrolide resistance. In total, 74.1% of pregnant women had >1 RTI; most of these infections were treatable. We detected sexually transmitted infections (excluding syphilis) in 37.7% of women. Our findings showed that syndromic management of infections is greatly inadequate. In total, 98.4% of women had never used barrier contraception. These findings will inform efforts to improve reproductive healthcare in Papua New Guinea.

Much about the range of pathogens, frequency of coinfection, and clinical effects of reproductive tract infections (RTIs) among pregnant women remains unknown. We report on RTIs (Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Treponema pallidum subspecies pallidum, bacterial vaginosis, and vulvovaginal candidiasis) and other reproductive health indicators in 699 pregnant women in Papua New Guinea during 2015-2017. We found M. genitalium, an emerging pathogen in Papua New Guinea, in 12.5% of participants. These infections showed no evidence of macrolide resistance. In total, 74.1% of pregnant women had >1 RTI; most of these infections were treatable. We detected sexually transmitted infections (excluding syphilis) in 37.7% of women. Our findings showed that syndromic management of infections is greatly inadequate. In total, 98.4% of women had never used barrier contraception. These findings will inform efforts to improve reproductive healthcare in Papua New Guinea. pregnant women, RTIs can cause miscarriage, stillbirth, preterm birth, or neonatal death, as well as serious neonatal conditions such as blindness, congenital malformations, and lifelong disability (1,8,9).
Mycoplasma genitalium is increasingly understood to be a major cause of poor sexual health and is associated with pelvic inflammatory disease, cervicitis, miscarriage, and preterm birth (10,11). Limited data exists on M. genitalium prevalence, although estimates range from <1.0% in the general adult population to 15.9% in groups at high risk, such as female commercial sex workers (12,13). In pregnant women, estimates range from 0.7% in the United Kingdom (14) to 11.9% in the Solomon Islands (15). During 2010-2019, global macrolide resistance to M. genitalium increased from 10% to >50% (16). In many regions, the prevalence of M. genitalium and its susceptibility to antimicrobial drugs is unknown.
Papua New Guinea is a country in the southwestern Pacific Ocean with >8.5 million persons (17). Poor pregnancy outcomes are common in this country. Estimates are imprecise because of weaknesses in vital registry systems, but <50% of women give birth with a skilled birth attendant (18). Ultrasound machines for gestational age assessment are largely inaccessible because of scarcity, cost, and location. The estimated prevalence of low birthweight (weight <2.5 kg) ranges from 10%-24% and preterm birth from 7%-18% (19). Papua New Guinea has a high perinatal death rate of 17 deaths/1,000 pregnancies (19). Curable STIs are common; rates of C. trachomatis, N. gonorrhoeae, T. vaginalis, and T. pallidum infections exceed those of other high-prevalence regions such as sub-Saharan Africa (1,20). However, little to no data exists on the prevalence of M. genitalium in Papua New Guinea. We evaluated the prevalence of M. genitalium and other RTIs among pregnant women attending antenatal clinics in the East New Britain (ENB) province of Papua New Guinea. We also investigated molecular markers of resistance in clinical samples from these patients. We investigated the relationships between different RTIs, factors associated with infection, and analyzed the diagnostic accuracy of syndromic management guidelines.

Study Site and Population
We studied cross-sectional baseline data from 699 pregnant women attending their first antenatal clinic. Study participants were enrolled in Healthy Mothers Healthy Babies, a prospective cohort study undertaken at 5 health facilities in 3 of the 4 districts of ENB.
The study sites included the hospitals in the 2 major urban areas and the 3 largest rural health centers of ENB. Members of the largest ethnic group, the Tolai, access all facilities; members of the second largest ethnic group, the Baining, predominantly access Kerevat Rural Hospital, the government-operated rural facility. Enrollment in the Healthy Mothers Healthy Babies cohort, and thus this study, occurred during March 2015-June 2017. Women >16 years of age who were living in the facilities' catchment area and attending clinic for the first time in the current pregnancy, regardless of gestational age, were eligible to participate. At each site, women were randomly selected through a dice roll. After the women underwent eligibility screening and provided informed consent, they completed a questionnaire administered by a trained research officer. We collected sociodemographic and clinical information through the questionnaire and patient-held medical records. We obtained urine, capillary finger prick blood, self-collected vaginal swab, and venous blood samples. We communicated all abnormal results available at the point of care, such as results of the urine dipstick and syphilis, malaria, and hemoglobin assays, to the participant and the healthcare provider.

Study Procedures
Health facility staff provided routine antenatal care, including intermittent preventive treatment in pregnancy for malaria, syndromic management for vaginal discharge (Appendix, https://wwwnc.cdc.gov/ EID/article/27/3/20-1783-App1.pdf), iron and folate supplementation, voluntary counselling and testing for HIV using Alere Determine HIV-1/2 (Abbott, https://www.abbott.com), and point-of-care syphilis testing using Alere Determine Syphilis TP (Abbott), in accordance with national guidelines (21,22). At the beginning of the study period, the participating healthcare facilities conducted syphilis testing. However, interruptions in stock supply nationally led to fewer women being tested for syphilis. The research team subsequently supplied and conducted testing for study participants. Stock interruptions of HIV testing materials also occurred; however, our research team was not qualified for voluntary counselling and testing and did not have ethics approval to conduct HIV testing.
Each participant provided 2 self-collected vaginal swab samples: 1 GeneXpert vaginal/endocervical swab (Cepheid, https://www.cepheid.com), which was placed directly into its transport medium, and 1 Copan flocked swab (Copan Diagnostics, Inc., https://www.copanusa.com), which was first used to prepare a vaginal smear on a slide for microscopy, and then placed in 1.0 mL Copan Universal Transport Medium (Copan Diagnostics, Inc.) specific for bacterial STIs. The number of vaginal swabs and smears available for diagnosis varied because of occasional reluctance to provide a swab, quality of vaginal smear, and availability of GeneXpert testing cartridges. Each woman self-collected a urine sample in a sterile container. All specimens were stored in a chilled box at 2°C-7°C for the remainder of clinic day, then stored at 2°C-7°C or −20°C until tested.

Laboratory Methods
We used the GeneXpert molecular platform (Cepheid) to test vaginal and urine specimens for C. trachomatis, N. gonorrhoeae, and T. vaginalis at the Burnet Institute/ Papua New Guinea Institute of Medical Research laboratory at St. Mary's Hospital Vunapope (Kokopo, Papua New Guinea). M. genitalilum and resistance mutations were detected by quantitative PCR (Resistance-Plus MG kit, SpeeDx Pty Ltd, https://plexpcr.com). Gram-stained vaginal smears were read by an experienced microscopist at the Melbourne Sexual Health Centre (Melbourne, Victoria, Australia) (Appendix).

Data Management and Statistical Analysis
Researchers interviewed participants and documented their responses using electronic tablets. We employed stringent data management protocols (Appendix).
The questionnaire included details about the enrollment clinic, participant characteristics at enrollment, and relevant obstetric history (Appendix). This study produced prevalence estimates of M. genitalium, C. trachomatis, N. gonorrhoeae, T. vaginalis, T. pallidum, BV, and VVC among pregnant women in Papua New Guinea. We used logistic regression to assess the association between patient characteristics and STIs, including C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium. We included all variables of interest in the univariable analysis. The multivariable model retained variables associated with the outcome at p<0.10 in the univariable analysis. We also analyzed the effectiveness of syndromic management guidelines using the standard question about current symptoms compared with an alternative question about symptoms experienced during the current pregnancy.

Ethics Considerations
All participants provided individual written, informed consent. Ethics approval was provided from the Medical Research Advisory Committee of the Papua New Guinea National Department of Health (approval no. 14 from the East New Britain Provincial Executive Committee and participating facilities. A series of community engagement meetings provided broader community support and assent for the study.

Results
We enrolled 699 pregnant women at 5 antenatal clinics in ENB.    Table 3).

Relationship between Abnormal Vaginal Discharge and Infection
We compared the infections of women with current abnormal vaginal discharge (as defined by national treatment guidelines) with those who had abnormal vaginal discharge currently or at any time in pregnancy before their first antenatal clinic visit (Table 3). A total of 98 women (14.1%; 98/697) had current symptoms (i.e., abnormal vaginal discharge) that would have prompted treatment according to syndromic management guidelines (2 women did not answer this question). An additional 37 women did not have abnormal vaginal discharge at the time of the screening but had experienced it earlier in the pregnancy. According to the national treatment guidelines, these women would not normally receive treatment.
Most STIs were asymptomatic and neither criteria (current abnormal vaginal discharge vs. current or previous abnormal vaginal discharge during this pregnancy) performed well as a marker of infection. Of those women with a detected STI, 84.1% (154/183) had no current symptoms and 77.0% (141/183) had not experienced symptoms during their current pregnancy. Conversely, 12.0% (36/301) of uninfected women had current symptoms and 17.6% (53/302) had experienced symptoms during their current pregnancy. Of those with M. genitalium infection, only 12 women (15.4%;12/78) would have been treated according to syndromic management guidelines used by Papua New Guinea.
Asking whether women had any symptoms during their current pregnancy was consistently more sensitive than asking about current symptoms as per the standard diagnostic question ( Figure 2); however, the sensitivity of both questions was <30% for all individual or collective pathogens. The alternative question was less specific for >1 current STI (82.5%  Table 4). The alternative question was best able to identify women with T. vaginalis infection (p<0.01) and VVC (p<0.01) (Appendix Table 4); however, this question still missed most infections.

Factors Associated with Curable STIs
We did not identify any factors in the univariable (Appendix Table 5) or multivariable (Table 4) analysis that were associated with an increased odds of M. genitalium infection. The univariable analysis showed that women who were younger, in their first pregnancy, employed, single or separated, had never used a modern method of contraception, or had abnormal vaginal discharge at any time in their current pregnancy were at increased risk for certain STIs, to varying degrees of statistical significance. In the multivariable analysis, primigravida women and those 16  one of the highest prevalence rates of this infection globally. We did not find evidence of macrolide resistance. The high prevalence of M. genitalium (12.5%) among pregnant women suggests an estimated 13,000 (95% CI 10,342-15,823) current cases among women of reproductive age in the province (Appendix). In addition, we provide contemporary data on RTIs in pregnant women from the New Guinea Islands region of Papua New Guinea; the most recent report on the subject is >20 years old (23). Our study indicates that >1 in 2 (53.5%) pregnant women in ENB have a treatable RTI (including BV, STI, or both) known to cause harmful sexual and reproductive health outcomes. These RTIs are not usually detectable by the syndromic management practices described in the national health guidelines of PNG. This high prevalence of poor sexual and reproductive health has major national and regional public health significance. No global surveillance system for M. genitalium currently exists (24). Different detection methods have varying levels of sensitivity, limiting scientific understanding of its epidemiology. High-income countries report rates of M. genitalium infection ranging from 0.3%-3.3% (11,13,25) in the general population, with higher estimates in certain populations (26,27). Fewer data are available from low-and middle-income countries (LMICs) but prevalence appears to be higher, ranging from 3% in the general population in Tanzania (13) to 8%-9% in Honduras and South Africa (13,28). The highest prevalence has been reported among sex workers: 16% in Kenya (29) and 26% in Uganda (30). Data on M. genitalium infection among pregnant women remains limited despite the disease's association with adverse pregnancy outcomes (26); available estimates range from 0.7%-0.9% in the United Kingdom and France (14,31) to 6.2% in Guinea-Bissau (32) and 11.9% in the Solomon Islands (15). More data on the prevalence and consequences of M. genitalium infection among pregnant women are needed.
Regional data on M. genitalium in LMICs are limited. One study from the Solomon Islands examined the effects of mass drug administration (MDA) using 1 g of oral azithromycin for eliminating ocular C. trachomatis on M. genitalium infection rates (15). Before MDA, the study found an 11.9% (95% CI 8.3%-16.6%; n = 236) prevalence of M. genitalium among pregnant women. After MDA, the prevalence remained high at 10.9% with no evidence of macrolide resistance. However, only 5 of the 28 M. genitalium-positive women in the post-MDA group had received azithromycin (15). In this study, the lack of macrolide resistance among M. genitalium infections in pregnant women warrants further exploration. Macrolide susceptibility might reflect a population's lack of exposure to this class of antimicrobial drugs. However, macrolides are used widely in Papua New Guinea (22,33) and are available without prescription (although overthe-counter macrolides are more expensive than their prescribed counterparts).
We observed a prevalence of curable STIs substantially greater than in most settings included in the 2016 global estimates of curable STIs (3). The 32.1% observed prevalence of >1 current STI diagnosable by GeneXpert is less than the 42.7% reported in a study of antenatal clinics from 3 mainland provinces of Papua New Guinea in 2014 (20), but similar to the 33.7% prevalence among pregnant women in Madang Province in 2012 (34). We found a 19.1% prevalence of C. trachomatis infection among pregnant women, consistent with reports from other provinces (22.9% in the Eastern Highlands, Hela, and Central provinces [20] and 20.0% in the Milne Bay province [35]) and the neighboring Solomon Islands (20.3%) (36)  14.2%) (34,(36)(37)(38) in the world. In addition, 2 studies from South Africa also report very high rates of N. gonorrhoeae: 10.1% among patients in a primary care setting (39) and 6.4% among pregnant women (40). Risk factors for different STIs identified in this study (e.g., primigravida, age 16-24 years, employment, being single or separated) could have several explanations. Younger women in their first pregnancy might have had less interaction with reproductive health services. Also, employed women might have more mobility, which increases risk for STI acquisition. We did not identify any risk factors for M. genitalium infection, although younger women (16-24 years of age) were at increased risk for >1 of the curable current STIs. Risk factors for STIs in pregnancy reported elsewhere in Papua New Guinea include having >1 lifetime sexual partner, low education level of the woman or her partner, rural location, history of miscarriage or stillbirth, and low socioeconomic status (20,34).
This study also provides data on BV and VVC; 57.6% of participants had >1 of these infections. VVC can cause extreme discomfort and increase a woman's risk for postpartum breast candidiasis, which can affect breastfeeding, but VVC is treatable with antimicrobial drugs (41). We found a 37.5% prevalence of VVC, higher than the 23% prevalence reported in Papua New Guinea in 1991 (42). Comparisons with other LMICs are difficult because of the limited amount of contemporary data (41,43). We found a 1-in-4 prevalence of BV among pregnant women, higher than the 17.6% prevalence previously reported in Papua New Guinea (35), but in keeping with recent global estimates of 23%-29% (5). However, our results might underestimate the true prevalence because diagnosis was limited to only women with a Nugent score of 7-10.
In Papua New Guinea, syndromic management of RTIs is common because access to diagnostic services is limited. We confirm previous reports from Papua New Guinea and elsewhere (28,37) that syndromic management is an inadequate tool to effectively treat RTIs. This approach missed 78.2% of M. genitalium infections and 3 of 4 RTIs. Alternative approaches are essential to effectively prevent, detect, and treat RTIs in a cost-effective, feasible manner in resource-constrained settings. Although condoms are available, their use is limited by gender disparities, stigma, and financial barriers (23). Improved access to affordable, accurate point-ofcare diagnostics would lead to more accessible and appropriate treatment, resulting in improved sexual and reproductive health; the widespread implementation of GeneXpert for tuberculosis diagnosis (44) might also increase access to STI diagnosis in Papua New Guinea.
The main limitation of this study is the facilitybased recruitment of participants because results might not represent women who do not attend any antenatal clinic. However, routinely collected provincial data for 2015-2017 estimated that 73%-85% of pregnant women attended >1 appointment at an antenatal clinic (45,46). The number of women who had a pointof-care syphilis test was lower than other tests. These results did not differentiate between active or latent infection; we also were unable to exclude exposure to yaws, which is endemic to Papua New Guinea (47). Yaws and syphilis are caused by different subspecies of T. pallidum and cannot be distinguished by this test alone. Prevalence of yaws varies widely within Papua New Guinea; estimates for ENB are unavailable, although neighboring New Ireland Province has a 1.8% prevalence of active yaws according to a populationwide survey (48).
In conclusion, we provide data on M. genitalium prevalence and antimicrobial resistance markers in Papua New Guinea, revealing a high prevalence of infection underrecognized by syndromic management guidelines. This data contributes to the understanding of the global prevalence of this infection among pregnant women. We found that STIs were common among pregnant women; 37.7% of participants had >1 STI at the time of the study. This study also highlights the high prevalence of BV and VVC and confirms that current antenatal screening practices with syndromic management is inadequate. This high prevalence of disease negatively affects sexual and reproductive health. Urgent action towards ensuring access to affordable prevention, diagnosis, and treatment of RTIs in communities in Papua New Guinea and similar settings is essential. This action will be crucial to achieving the sustainable development goal of ensuring universal access to sexual and reproductive healthcare services by 2030 (49). Expanding treatment access will contribute to improved sexual and reproductive health outcomes for women in Papua New Guinea.