Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth: A cross-sectional survey

RESEARCH ARTICLE Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth: A cross-sectional survey Lola Madrid 1,2 *, Rosauro Varo 1,2 , Sonia Maculuve 1 , Tacilta Nhampossa 1 , Carmen Muñoz- Almagro 3,4,5 , Enrique J. Caldero ´n 4,6 , Cristina Esteva 3 , Carla Carrilho 7,8 , Mamudo Ismail 7,8 , Begoña Vieites 9 , Vicente Friaza 4,6 , Marı ´a del Carmen Lozano-Dominguez 10 , Clara Mene ´ndez 1,2,4 , Quique Bassat 1,2,11,12 1Centro de Investigac¸ão em Sau ´de de Manhic¸a (CISM), Maputo, Mozambique, 2ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clı´nic - Universitat de Barcelona, Barcelona, Spain, 3Molecular Microbiology Department, Institut de Recerca Sant Joan de De ´u, University Hospital Sant Joan de De ´u, Barcelona, Spain, 4CIBER de Epidemiologı ´a y Salud Pu ´blica CIBERESP, Instituto de Salud Carlos III, Madrid, Spain, 5School of Medicine. Universitat Internacional de Catalunya, Barcelona, Spain, 6Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocı ´o/CSIC/Universidad de Sevilla, Seville, Spain, 7Department of Pathology, Maputo Central Hospital, Maputo, Mozambique, 8Faculty of Medicine, Eduardo Mondlane University, Maputo, Mozambique, 9Department of Microbiology, Hospital Universitario Virgen del Rocı ´o, Seville, Spain, 10 Department of Pathology, Hospital Universitario Virgen del Rocı ´o, Seville, Spain, 11 ICREA, Barcelona, Spain, 12 Pediatric Infectious Diseases Unit, Pediatrics Department, Hospital Sant Joan de De ´u (University of Barcelona), Barcelona, Spain *[email protected] Abstract Background Congenital cytomegalovirus (cCMV) infection is the most prevalent congenital infection acquired worldwide, with higher incidence in developing countries and among HIV-exposed children. Less is known regarding vertical transmission of parvovirus B19 (B19V) and enterovirus (EV). We aimed to assess the prevalence of CMV, B19V and EV vertical transmission and compare results of screening of congenital CMV obtained from two different specimens in a semirural Mozambican maternity. Methods A cross sectional study was conducted among pregnant mothers attending Manhic¸a District Hospital upon delivery. Information on maternal risk factors was ascertained. Dried umbilical cord (DUC) samples were collected in filter paper for CMV, B19V and EV detection by realtime polymerase chain reaction (RT-PCR), and nasopharyngeal aspirates (NPA) to test for CMV by RT-PCR. Maternal blood samples and placental biopsy samples were also obtained to investigate CMV maternal serology, HIV status and immunopathology. Results From September 2014 to January 2015, 118 mothers/newborn pairs were recruited. Prevalence of maternal HIV infection was 31.4% (37/118). CMV RT-PCR was positive in 3/115 PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 1 / 17 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Madrid L, Varo R, Maculuve S, Nhampossa T, Muñoz-Almagro C, Caldero ´n EJ, et al. (2018) Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth: A cross-sectional survey. PLoS ONE 13(3): e0194186. https://doi.org/10.1371/journal. pone.0194186 Editor: Soren Gantt, University of British Columbia, CANADA Received: December 10, 2017 Accepted: February 26, 2018 Published: March 14, 2018 Copyright: ©2018 Madrid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data are originally from the PIPAC study and are restricted since it contains sensitive patient information. To request access to the data, please contact the Internal Scientific Committee and Local Ethical Committee of Centro de Investigac¸ão de Manhic¸a (+258 823041570) and from National Ethical Committee of Mozambique (+258 214308114/21427131). The authorisation of these two ethics committees is required for data access. The authors of the PIPAC (2.6%) of DUC samples and in 3/96 (6.3%) of NPA samples obtained from neonates. The concordance of the RT-PCR assay through DUC with their correspondent NPA sample was moderate (Kappa = 0.42 and p<0.001. No differences on cCMV prevalence were found among HIV-exposed and unexposed. All (100%) mothers were seropositive for CMV IgG. RT-PCR of EV and B19V in DUC were both negative in all screened cases. No histological specific findings were found in placental tissues. No risk factors associated to vertical transmission of these viral infections were found. Conclusions This study indicates the significant occurrence of vertical transmission of CMV in southern Mozambique. Larger studies are needed to evaluate the true burden, clinical relevance and consequences of congenital infections with such pathogens in resource-constrained settings. Background Despite the impressive reduction in child mortality in the last decades, neonatal mortality has declined more slowly and now accounts for nearly half (45%) of all under-5 child deaths[1]. Congenital and perinatal infections are well-known causes of neonatal morbidity and mortality and stillbirths in high-income countries (HIC)[2–4]. However, estimates of the global burden of congenital infections and attributable stillbirths, neonatal disease, disability or deaths due to mother-to-child transmission (MTCT) of these infections in low-income countries (LIC) are limited on account of a generalized scarcity of data[5]. Despite the positive impact in terms of health outcomes shown by the introduction of screening and treatment policies for several pregnancy-relevant infections such as human immunodeficiency virus (HIV), syphilis or malaria[6,7]; pathogens that may also be vertically transmitted beyond these infections are rarely the focus of clinical practice and research[8,9]. Several viruses, including among others cytomegalovirus (CMV), parvovirus B19V (B19V) and enterovirus (EV), may cause mild and self-limiting clinical manifestations among infected pregnant women, but more severe or even life-threatening disease in their offsprings. The Zika virus epidemic of 2016 in Latin America has contributed to highlight the emerging threat that maternal viral infections may carry for the health of the foetus and newborn[10]. Despite being the most prevalent congenital infection worldwide, congenital CMV infection (cCMV) remains largely neglected in the developed and developing world[11]. Although the global prevalence of cCMV has been reported to vary from approximately 0.2% to 2% (mean 0.65%), most of these studies have been conducted in high-income regions of Europe, USA or Japan were prevalence of cCMV ranges between 0.6–0.7%[12–14]. Data from LIC varies substantially, with some estimates peaking at 6–14%[15–17]. Higher overall rates of cCMV are found in countries with higher maternal CMV seroprevalence[12,13,18] and among infants exposed to HIV during pregnancy. Indeed, maternal HIV infection is thought to significantly increase (from 2.3% to 10.3%) the prevalence of cCMV in those HIV-exposed infants compared to those born to HIV-negative mothers, both in industrialized countries and also in LIC[19–23]. Furthermore, CMV infection seems to play a role as a cofactor for HIV disease progression in HIV/CMV co-infected newborns[24]. Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 2 / 17 study can be contacted for questions regarding the data at: [email protected]. Funding: Quique Bassat had during the duration of the study a fellowship from the program Miguel Servet of the ISCIII (Plan Nacional de I+D+I 2008- 2011, grant number: CP11/00269). Lola Madrid had a fellowship from the program Rı ´o Hortega of the ISCIII (CM13/00260). Rosauro Varo has a fellowship from the program Rio Hortega of the ISCIII (CD16/00024). CISM receives financial support from the Spanish Agency for International Cooperation. ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya. SM, TN, CMA, EJC, CE, CC, MI, BV, VF, CLD and CM have nothing to declare. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Different methods have been evaluated for cCMV screening based on saliva, urine and blood specimens, being saliva and urine the generally considered most appropriate samples [25]. Virus isolation from saliva or urine in rapid culture has been traditionally considered the standard method for identification of infants with cCMV but such methods appear unfeasible to perform for large screening efforts [26]. In contrast, real-time polymerase chain reaction (RT-PCR), also considered as gold standard [26–28], allows large numbers of specimens to be screened at a relatively low cost. However, RT-PCR based methods applied to dried-blood- spot (DBS), tested in countries where DBS are routinely collected for newborn metabolic screening, have shown a low sensitivity as a screening methodology[26]. Dried umbilical cord (DUC) sample-based PCR assays have demonstrated utility for diagnosis of cCMV, as part of retrospective investigations of the underlying aetiology of hearing impairment[29,30]. Other samples such as nasopharyngeal aspirates (NPA) which are easily obtained and simple to store have been used to investigate respiratory pathogens of public health importance[31,32]. However, to our knowledge, NPAs have not been evaluated as a screening methodology for cCMV diagnosis, although the Child Health and Mortality Prevention Surveillance Network (CHAMPS) aiming to know cause of death through innovative techniques such as the minimally invasive tissue sampling (MITS), proposes NPA as a standard specimen for diagnosis of cCMV[33,34]. Knowledge gaps regarding the burden of other congenital infections associated to fatal foetal outcomes, such as B19V and EV, remain significant. B19V can cause a variety of foetal complications including spontaneous abortion, non-immune hydrops foetalis or intrauterine foetal death[35]. The epidemiology of B19V infection in pregnancy has been well studied in industrialized countries whereby prevalence has been estimated to vary from 1 to 5% in pregnant women with transmission rates to the foetus ranging between 17–33%[36]. However, the burden of this infection during pregnancy in LIC has been rarely documented and studies investigating in these settings active B19V infection in newborns have not been conducted. Enteroviruses, which include coxsackieviruses and echoviruses, cause about one billion infections every year worldwide but their consequences during pregnancy have been seldom described[37]. Transplacental transmission of EV has been associated to stillbirths, nonimmune hydrops foetalis and also severe neonatal infections, although the epidemiology and characterization of neonatal outcomes is not well documented[37]. Although data in LIC remain insufficient, higher burden of congenital infections may be assumed in regions like Southern Mozambique where HIV is highly prevalent and effective vaccines against pathogens such as rubella are partially or even not implemented[15,38–41]. This is a pilot study exploring congenital acquisition of CMV, B19V and EV determined at birth. We additionally aimed to compare the results of two simple screening methodologies using RT-PCR for cCMV investigation, using DUC and NPA specimens obtained from the newborns. Methods Study site The study was conducted in Manhic¸a, a semi-rural site in Southern Mozambique. The Manhic¸a Health Research Centre (CISM) runs a Demographic Surveillance System (DSS) in the area linked to a Hospital Morbidity Surveillance System (HMSS) ongoing at the Manhic¸a District Hospital (MDH) including all admitted children. A detailed description of MDH, CISM and the study area can be found elsewhere[42]. MDH is the referral hospital for the Manhic¸a district, covering a population of circa 183,000 inhabitants. The MDH includes adult and paediatric wards, together with a maternity, where an average of 3500–4000 deliveries takes place Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 3 / 17 annually. Around 85% of all deliveries are institutionalized (A. Nhacolo, personal communication). It also includes an outpatient department and an antenatal care (ANC) clinic where pregnant women are routinely followed. As part of the National policy, all pregnant women are invited to attend ANC clinic during their pregnancy, where HIV testing and syphilis screening are routinely offered. Malaria transmission of moderate intensity is perennial with some seasonality and, intermittent preventive treatment during pregnancy (IPTp) for malaria prevention is recommended[42]. HIV prevalence in Manhic¸a district is amongst the highest in the world, with rates estimated at around 29% at the ANC clinic [40]. In 2013, MDH introduced WHO-recommended Option B+ for the prevention of mother-to-child HIV transmission[43], which is offered to mothers free of charge. No proactive strategies to screen for risk factors of neonatal sepsis or to prevent it are currently implemented in Mozambique. Study design and population This observational pilot study was conducted at the delivery wards of MDH, between September 15 th 2014 and January 15 th 2015, running continuously during working hours (8:00–16:00) and working days. We recruited pregnant women upon delivery (regardless of gestational age (GA) and their offspring. Participants were eligible for inclusion if they were >18 years old and able and willing to participate in the study and to provide informed consent after an explanation of the study. As this was a pilot study, in order to obtain a more representative sample of the study population, only the first three women seen every day were approached for recruitment. Definitions Gestational age was defined using fundal height, measured from the top of the mother’s uterus to the top of the mother’s pubic symphysis and assessed by a nurse specialist in maternal child health. A preterm baby was defined as that with a gestational age at birth of <37 weeks and a stillbirth case as an intrauterine death occurring after 28 weeks of GA. Low-birth weight was defined as weight at birth <2.500 grams[44]. Microcephaly was defined following the WHO growth standards[45]. Congenital CMV, B19V or EV infections were defined as detection of viral DNA/RNA by RT-PCR in dried cord umbilical samples obtained from neonates at birth [33]. Although cCMV diagnosis through NPA has not been validated, we also explored prevalence of CMV through positive RT-PCR in NPA specimens. Positive HIV status was defined according to national guidelines, which required for mothers two positive rapid testing (an initial discriminatory diagnostic test (Determine 1 ) and a confirmatory test(Unigold 1 ); and for children 18 months of age a positive rapid test in addition to a confirmatory positive PCR test. Study procedures A placental biopsy and two drops of umbilical cord blood collected in filter paper (Whatman 903 1 , Florham Park, NJ) were obtained immediately after delivery in order to determine DNA/RNA of CMV, B19V and EV and assess placental histopathology. In addition, a NPA was collected from neonates within the first two hours from birth, using a bulb aspiration kit conveniently pre-filled with sterile saline (M-PRO NPAK nasopharyngeal aspiration kit 1 ). At least 1ml of NPA specimen mixed with sterile saline was collected and stored with the objective of screening for CMV DNA. A blood sample was collected from mothers for assessing anti- CMV antibodies. Maternal HIV status was determined and recorded if not previously registered in antenatal source documents. Other screening test results routinely performed at the Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 4 / 17 ANC clinic, such as syphilis screening (using Rapid Plasma Reagin) or haemoglobin determination were also recorded. DNA extraction and real time -PCR for viral amplification. CMV and B19V DNA and EV RNA were extracted from 1 drop of DUC (around 50 ul) and CMV from 400 ul of NPA specimen by using the NucliSENS 1 easyMag 1 (bioMe ´rieux, Marcy l’Etoile, France) instrument according to the manufacturer’s procedure and eluted in 25 μl of elution buffer for DUC and 50 ul for NPA. Samples were tested for the presence of CMV DNA, B19V DNA and EV RNA with three different real-time PCR, CMV Q- PCR alert Kit 1 , Elitech Group Molecular Diagnostics, RealStar 1 Parvovirus B19V kit 1 , Altona Diagnostics and and a published inhouse real-time RT-PCR assay for EV detection [46]. Positive results with cycle threshold values above 40 were classified as negative. Appropriate positive and negative controls were included in all the experiments. Samples were considered as valid when a clear positive or negative result was obtained. Seroprevalence of CMV among pregnant women. Serum collected from the mothers was tested for the presence of anti-CMV immunoglobuline G (IgG) antibodies (AB) using the electrochemiluminescence immunoassay “ECLIA” intended for use on Elecsys and cobas immunoassay analyzers, according to the manufacturer’s instructions. Positive, negative, and cut-off controls were included in all runs, and positive samples were retested to confirm initial positive result. Placental biopsy. A placental biopsy was obtained with a scalpel blade and immediately placed into 10% buffered formalin for transport to the laboratory. Each placental sample was embedded in paraffin wax, sectioned in 5μm tissue pieces and stained with standard haematoxylin–eosin for histopathologic examination. Different histopathological parameters were assessed including a macroscopic description (weight, oedema, haemorrhage, infarct and calcifications) and a microscopic description (inflammation and presence of microorganisms). Additional sections were saved for histochemical staining (PAS, Grocott and Gram) performed in an automated stainer (BenchMark Special Stains- Ventana Roche), following commercial recommendations. An immunohistochemical assay was performed on formalinefixed paraffin-embedded tissue sections according to standard procedures, using the Ventana- Roche automated immunostainer system (BenchMark XT-Ventana Roche 1 ). Antigen retrieval was performed by heat-induced epitope retrieval and B19V (Master Diagnostica 1 , Granada, SP) and CMV (Cell Marque 1 , Rocklin, CA, USA) antibodies were applied according to manufacturing instructions. Infant follow-up. Clinical examination of all newborns, including the evaluation of head circumference and Apgar score at minute 1, 5 and 10, was performed at birth. Dubowitz score for postnatal GA determination was done in live neonates at least 12h after birth[47]. Maternal and child HIV status were registered. All participants were followed using the HMSS in order to check any hospital admissions to MDH during the first six months after birth. No specific imaging or hearing screening was performed in these children. Statistical analysis All data were prospectively collected using standardized questionnaires, which were double entered in specific study databases, created using Openclinica software. Discrepancies were solved after comparison with the original source documents by a senior data clerk, and in close collaboration with the study clinicians. Statistical analyses were performed using Stata 14.1 (Stata Corp., College Station, TX). Study variables were counted and summarized in frequency tables. Univariate and multivariate analyses were performed to identify risk factors for CMV, B19V and EV neonatal infection, separately. Firth logistic regression was used in order Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 5 / 17 to address issues of separability, small sample sizes and bias of the parameter estimates. Variables that were found to be significantly associated with CMV, B19V and EV acquisition in the univariate analysis together with those related at a significance level of p<0.10 were entered into a multivariate model. Agreement between the results of DUC RT-PCR assays and those of NPA RT-PCR was assessed through Kappa statistic. Ethical considerations This protocol and all supporting documentation (Informed consent documents, Study questionnaires) were approved by the local bioethics committee of CISM (Comite ´Institucional de Bioe ´tica para Sau ´de) and by the National Bioethics Committee of Maputo in Mozambique, and by the Ethics Committee of the Hospital Clı ´nic in Barcelona, Spain. Participants were asked to express their willingness to participate in the study by signing (or thumb-printing in case they were illiterate) a consent form. Participation in this study was voluntary, and studyrelated procedures did not interfere with the pregnant women’s or children’s standard clinical care. Results Maternal characteristics During the study period, 118 pregnant women were recruited upon delivery at MDH (Fig 1). Table 1 summarizes the socio-demographic and clinical characteristics of participant mothers. Median age of recruited women was 22 years (Interquartile range, IQR 19–29), with >75% being younger than 30 years of age. Nearly a third of women (31.4%, 37/118) were confirmed HIV positive and only one of 118 had a positive syphilis test. Malaria test results were registered as negative in 5/118 women and no information about this disease was available for the rest of mothers. CMV IgG AB were detected in all (100%) women. Distal villous hypoplasia was observed in 27/117 (23.0%) of placental tissue samples evaluated. Neither microorganisms nor other significant findings were detected after evaluating tissue sections with standard tissue staining or in the immunohistochemical studies (Fig 2). No maternal risk factors associated to neonatal cCMV were found in the univariate analysis and thus, multivariate analysis was not performed (Table 1). Neonatal outcomes and infant follow-up One hundred and eighteen delivery outcomes from the 118 pregnant women were recorded (100%) at MDH. Characteristics of neonates born of mothers participating in the study are shown in Table 2. Neonatal outcomes included 110 live term babies, 7 preterm and 1 case of stillbirth born at 41 weeks of GA, whose mother refused permission to sample the foetus. Twenty-one (17.8%) newborns had low-birth-weight and three of them (2.5%) presented microcephaly at birth[48]. At least one specimen was collected from 117 neonates. A hundred and fifteen of the 117 DUC samples obtained were valid for viral determination. All of them were negative for B19V and EV. Three of the 115 (2.6%) valid DUC samples were positive for CMV. This virus was also detected in 6/96 (6.3%) valid NPA samples obtained. A total of 7/117 (6.0%) newborns tested had at least one positive sample for CMV. Prevalence of cCMV infection measured by DUC among HIV-exposed neonates was 2.7% (1/37) and 8.1% (3/37) when CMV was detected through NPA, although no significant difference was found compared to the prevalence among HIV-unexposed in both cases (Table 1). One of the NPA-CMV positive cases was born Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 6 / 17 preterm and with low birth weight while all neonates DUC-CMV positive were healthy at birth. CMV infection was associated to a higher risk of microcephaly at birth when CMV was determined by NPA (OR 9.65, 95% CI 1.07–87.12, p = 0.04) in the univariate analysis (Table 2). Through HMSS, 7/117 infants born to mothers participating in the study were detected as admissions at MDH at least once during their first 6 months of life (Table 2). Two of them admitted within the first 24h after birth died due to clinical sepsis. Other causes of admission included perinatal asphyxia, bronchiolitis, diarrhoea and malaria. Among neonates were CMV was detected, only the preterm baby with a positive NPA for CMV was admitted at birth. In all of these cases, children were discharged fully recovered. No additional follow-up investigations were conducted. Comparison of dried umbilical cord and nasopharyngeal aspirate RT-PCR Assays for CMV determination. A total of 94 pairs of samples (DUC and NPA) from 94 neonates were Fig 1. Study profile. ANC: antenatal clinics; IC: informed consent; One stillbirth whose mother refused to take samples. RT-PCR: real-time polymerase chain reaction, DUC: dried umbilical cord, NPA: nasopharyngeal aspirate. https://doi.org/10.1371/journal.pone.0194186.g001 Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 7 / 17 available for testing CMV by RT-PCR assay and comparing results. Of these, 2/94 neonates (2.1%) were positive for CMV by any test. A newborn with a positive RT-PCR assay through DUC had a negative NPA result, and the NPA RT-PCR assay also identified four additional neonates as infected although their DUC RT-PCR were negative (Fig 1). The overall concordance of the RT-PCR assay through DUC with their correspondent through NPA was moderate (Kappa = 0.42 and p<0.001). Table 1. Socio-demographic and clinical characteristics of mothers participating in the study and univariate analysis of maternal risk factors associated to congenital CMV infection measured by different specimens. Total mothers recruited, n = 118 Mothers of neonates CMV positive by DUC,n = 3ᶤ Crude OR (95%CI)ᵟ pvalueᵟ Mothers of neonates CMV positive by NPA,n = 6ᶽ Crude OR (95%CI)ᵟ pvalueᵟ Socio-demographic characteristics n (%) n (%) n (%) Age in years 0.61 0.61 <21 51 (43.2) 2 (66.7) 1.00 4 (66.7) 1.00 22 to 29 40 (33.9) 0 (0) 0.23 (0.01– 4.92) 1 (16.7) 0.23 (0.01– 4.92) 30 27 (22.9) 1 (33.3) 1.05 (0.13– 8.42) 1 (16.7) 1.05 (0.13– 8.42) Seconday or tertiary education 30 (25.4) 0 (0.0) 0.40 (0.02– 8.06) 0.55 3 (50.0) 3.23 (0.68– 15.36) 0.14 Employment 6 (5.1) 0 (0.0) 2.34 (0.1150.28 0.59 0 (0.0) 1.92 (0.89– 41.36) 0.68 Obstetric History n (%) n (%) n (%) Age of first pregnancy (median±IQR)) 18.0 (17–20) 18.0 (17–18) 0.91 (0.27– 3.11) 0.89 18.0 (18–18) 1.03 (0.76– 1.41) 0.84 Gravidity (mean±SD) 2.7 (±0.2) 3.7 (±2.2) 1.27 (0.81– 2.01) 0.30 1.5 (±0.2) 0.63 (0.31– 1.29) 0.21 Previous abortion n(%) 11 (9.3) 0 (0.0) 1.26 (0.61– 25.97) 0.88 0 (0.0) 0.75 (0.89– 14.43) 0.85 History of current pregnancy n (%) n (%) n (%) At least 3 antenatal visits during the pregnancy 69 (58.5) 2 (66.7) 0.78 (0.98– 6.15) 0.81 5 (83.3) 1.22 (0.19– 8.01) 0.83 Gestational hypertension 7 (6.1) 0 (0.0) 2.73 (0.12– 62.25) 0.53 1 (16.7) 3.38 (0.47– 24.33) 0.23 Vaginal discharge 2 (1.7) 0 (0.0) 6.31 (0.25– 157.63) 0.26 0 (0.0) 2.72 (0.12– 62.86) 0.53 Investigations n (%) n (%) n (%) Syphilis positive 1 (0.5) 0 (0.0) 9.95 (0.34– 290.3) 0.32 0 (0.0) 5.12 (0.19– 140.92) 0.33 HIV positive 37 (31.4) 1 (2.7) 1.26 (0.16– 9.89) 0.83 3 (50.0) 2.19 (0.46– 10.30) 0.32 HIV positive in HAART 33 (89.2) 1 (100.0) 1.25 (0.11– 4.43) 0.86 3 (100.0) 2.60 (0.48– 14.04) 0.25 Anemia (<11g/dL) 55 (72.4) 0 (0.0) 0.13 (0.01– 3.26) 0.21 2 (66.7) 0.59 (0.07– 4.88) 0.63 CMV IgG serum antibodies 118 (100) 3 (100) 0.03 (0.00– 1.81) 0.09 6 (100) 0.07 (0.06– 3.92) 0.20 IQR (interquartile range). SD: standard deviation. HAART: highly active antiretroviral therapy. CMV: cytomegalovirus. DUC: Dried umbilical cord. ᶤ Results based on 115 valid DUC samples. NPA: Nasopharingeal aspirate. ᶽ Results based on 96 valid NPA samples OR: odds ratio. CI; confindence intervals. ᵟ OR and P-value derived from Firth logistic regression. https://doi.org/10.1371/journal.pone.0194186.t001 Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 8 / 17 Discussion This study is a first attempt at proactively investigating vertical transmission of CMV, B19V and EV in Mozambique. The study was an opportunistic attempt to explore congenital transmission of these viruses, and was not specifically designed to evaluate the performance of the RT-PCR method of identification of cCMV in neonates as compared with the “gold-standard” for the detection of CMV (isolation of the virus in rapid cultures or PCR assay) since that has already been demonstrated[49–51]. The study shows a prevalence of cCMV infection assessed through dried umbilical cord samples of 2.6% and through NPA of 6.3%. Although specimens utilized in this study have not been validated for their use in cCMV diagnosis, they highlight a high burden of vertical CMV transmission. Contrarily, B19V and EV congenital transmission in the newborns was not found in this cohort although only DUC samples were analyzed. The prevalence of cCMV in this study detected through DUC was likely underestimated, as it has been demonstrated that real-time dried-blood-spot PCR assay has a lower sensitivity compared with the standard saliva rapid culture[26]. The dried umbilical cord samples have previously been used for retrospective studies to diagnose cCMV[29,30] and although this sample type has never been compared to other accepted specimens for cCMV diagnosis, it would however be reasonable to assume that a positive DUC sample is likely a true positive. On the other hand, NPA has never been previously assessed as a specimen valid for cCMV diagnosis. A significant limitation of this study includes the fact that up to 30% of CMV-seropositive women will secrete CMV in their vaginal fluid, something that could possibly contaminate with CMV Fig 2. Placental histology from an infant with cytomegalovirus detected in dried umbilical cord blood. Distal villous hypoplasia: many terminal villi are extremely small, with reduced stroma and capillaries number. No inmunohistochemical evidence of CMV could be found. https://doi.org/10.1371/journal.pone.0194186.g002 Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth PLOS ONE | https://doi.org/10.1371/journal.pone.0194186 March 14, 2018 9 / 17 41. Cutland CL, Schrag SJ, Zell ER, Kuwanda L, Buchmann E, Velaphi SC, et al. Maternal HIV infection and vertical transmission of pathogenic bacteria. Pediatrics. 2012; 130(3):e581–90. https://doi.org/10. 1542/peds.2011-1548 PMID: 22869824. 42. Sacoor C, Nhacolo A, Nhalungo D, Aponte JJ, Bassat Q, Augusto O, et al. Profile: Manhica Health Research Centre (Manhica HDSS). Int J Epidemiol. 2013; 42(5):1309–18. Epub 2013/10/26. https://doi. org/10.1093/ije/dyt148 PMID: 24159076. 43. WHO. Use of Antiretroviral Drugs for Treating Pregnant Women and Preventing HIV Infection in Infants. 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