Anoop K. Brar

Background: We have previously shown coxsackievirus B (CVB) to be a potent inducer of congenital heart disease (CHD) in mice. The clinical relevance of these findings in humans and the roles of other viruses in the pathogenesis of CHD remain unknown. Methods: We obtained plasma samples, collected at all trimesters, from 89 subjects (104 pregnancies), 73 healthy controls (88 pregnancies), and 16 with CHD–affected birth (16 pregnancies), from the Perinatal Family Tissue Bank (PFTB). We performed CVB IgG/IgM serological assays on plasma. We also used ViroCap sequencing and PCR to test for viral nucleic acid in plasma, circulating leukocytes from the buffy coat, and in the media of a co-culture system. Results: CVB IgG/IgM results indicated that prior exposure was 7.8 times more common in the CHD group (95% CI, 1.14–54.24, adj. p-value = 0.036). However, the CVB viral genome was not detected in plasma, buffy coat, or co-culture supernatant by molecular assays, although other viruses were detected. Conclusion: Detection of viral nucleic acid in plasma was infrequent and specifically no CVB genome was detected. However, serology demonstrated that prior CVB exposure is higher in CHD-affected pregnancies. Further studies are warranted to understand the magnitude of the contribution of the maternal blood virome to the pathogenesis of CHD.

Human pegivirus (HPgV) is best known for persistent, presumably non-pathogenic, infection and a propensity to co-infect with human immunodeficiency virus or hepatitis C virus. However, unique attributes, such as the increased risk of malignancy or immune modulation, have been recently recognized for HPgV. We have identified a unique case of a woman with high levels HPgV infection in two pregnancies, which occurred 4 years apart and without evidence of human immunodeficiency virus or hepatitis C virus infection. The second pregnancy was complicated by congenital heart disease. A high level of HPgV infection was detected in the maternal blood from different trimesters by RT-PCR and identified as HPgV type 1 genotype 2 in both pregnancies. In the second pregnancy, the decidua and intervillous tissue of the placenta were positive for HPgV by PCR but not the chorion or cord blood (from both pregnancies), suggesting no vertical transmission despite high levels of viremia. The HPgV genome sequence was remarkably conserved over the 4 years. Using VirScan, sera antibodies for HPgV were detected in the first trimester of both pregnancies. We observed the same anti-HPgV antibodies against the non-structural NS5 protein in both pregnancies, suggesting a similar non-E2 protein humoral immune response over time. To the best of our knowledge, this is the first report of persistent HPgV infection involving placental tissues with no clear indication of vertical transmission. Our results reveal a more elaborate viral-host interaction than previously reported, expand our knowledge about tropism, and opens avenues for exploring the replication sites of this virus.

Human pegivirus (HPgV) is best known for persistent, presumably non-pathogenic, infection and a propensity to co-infect with human immunodeficiency virus or hepatitis C virus. However, unique at-tributes, such as the increased risk of malignancy or immune modulation, have been recently recognized for HPgV. We have identified a unique case of a woman with high levels HPgV infection in two preg-nancies, which occurred 4 years apart, without evidence of human immunodeficiency virus or hepatitis C virus infection. The second pregnancy was complicated by congenital heart disease. A high level of HPgV infection was detected in maternal blood from different trimesters by RT-PCR and identified as HPgV type 1 genotype 2 in both pregnancies. In the second pregnancy, the decidua and intervillous tissue of the placenta were positive for HPgV by PCR but not the chorion or cord blood (from both pregnancies), suggesting no vertical transmission despite high levels of viremia. The HPgV genome sequence was remarkably conserved over the 4 years. Using VirScan, sera antibodies for HPgV were detected in the first trimester of both pregnancies. We observed the same anti-HPgV antibodies against the non-structural NS5 protein in both pregnancies, suggesting a similar non-E2 protein humoral immune response over time. To the best of our knowledge, this is the first report of persistent HPgV infection involving placental tissues with no evidence of vertical transmission. Our results reveal a more elaborate viral-host interaction than previously reported, expand our knowledge about tropism, and opens avenues for exploring the replication sites of this virus.

Aims: Coxsackievirus B (CVB), the most common cause of viral myocarditis, targets cardiomyocytes through Coxsackie and Adenovirus Receptor, which is highly expressed in the fetal heart. We hypothesized CVB3, a well-recognized culprit for viral myocarditis, can precipitate congenital heart defects (CHD), when fetal infection occurs during critical window of gestation. Methods & Results: We infected C57Bl/6 pregnant mice with CVB3 during serial time points in early gestation (E5, E7, E9 and E11). We used different viral titers to examine possible dose- response relationship and assessed viral loads in various fetal organs as well as kinetics of virus passage into the fetus during gestation. Provided viral exposure occurred between E7-E9, we observed characteristic features of ventricular septal defect (33.6%), abnormal myocardial architecture resembling non-compaction (23.5%), and double outlet right ventricle (4.4%) among 209 viable fetuses examined. We observed a direct relationship between viral titers, severity and incidence of CHD, with apparent predominance among female fetuses. Infected dams remained healthy; we did not observe any maternal heart or placental injury suggestive of direct viral effects on developing heart as likely cause of CHD. We examined signaling pathways in CVB3-exposed hearts using RNAseq, KEGG enrichment analysis and immunohistochemistry. Signaling proteins of the Hippo, tight junction, transforming growth factor β1 and extracellular matrix proteins were the most highly enriched in CVB3-infected fetuses with VSD (log fold change >1.9, P<0.02). Moreover, cardiomyocyte proliferation was 50% lower in fetuses with VSD compared with uninfected controls. Conclusion: Prenatal CVB3 infection can induce CHD, provided the infection occurs during a critical window. Alterations in myocardial proliferate capacity and consequent changes in cardiac architecture and trabeculation appear to account for the majority of observed phenotypes. GRAPHICAL

The research for this study was undertaken to examine association between perinatal infection with Coxsackievirus B (CVB) and congenital heart defects (CHD). Suggestive, although inconclusive, data exists of an association between CVB infection during pregnancy and CHD. We present data from a clinical study showing that CVB infection in early pregnancy induces heart defects. In a prospective study of 123 pregnant women with pregnancies affected with CHD we found circulating CVB IgG and IgM in the serum. A significant number of the women were positive for CVB antibodies; 30.9% for IgG and 6.5% for IgM. Our data demonstrates not only an association of CVB with CHD in pregnant women but this is the first report showing significant association between in-utero CVB infection and Pulmonary Atresia (PA) and HLHS (AA/MS variant). Elevated levels of IgG were seen in 55.5% of women carrying babies with PA and 60% of women carrying babies with HLHS (AA/MS), with p-values of 0.01 and 0.03 respectively. To determine specific CVB serotypes involved, neutralizing antibody titers to CVB1, CVB3, and CVB4 were analyzed on samples with positive IgG/IgM levels, and showed a possible association between CVB4 and CHD in the cohort. Our study has broad implications for the future understanding and potential management of CHD as well as introduces a new avenue of research for cardiac defects.

To the Editor—The role of the maternal microbiome during pregnancy on fetal and long-term health has increasingly come under scrutiny. Although a number of studies have analyzed the bacterial flora found in diabetic patients, surprisingly few have focused on the composition of the virome, whether the diversity is similar to nondiabetics, and any possible resulting interactions with health or disease.

Objective The effects of pregnancy on autograft dilatation and neoaortic valve function in patients with a Ross procedure have not been studied. We sought to evaluate the effect of pregnancy on autograft dilatation and valve function in these patients with the goal of determining whether pregnancy is safe after the Ross procedure. Design A retrospective chart review of female patients who underwent a Ross procedure was conducted. Patients Medical records for 51 patients were reviewed. Among the 33 patients who met inclusion criteria, 11 became pregnant after surgery and 22 did not. Outcome Measures Echocardiographic reports were used to record aortic root diameter and aortic insufficiency before, during, and after pregnancy. Patient's charts were reviewed for reinterventions and complications. Primary endpoints included reinterventions, aortic root dilation of ≥5 cm, aortic insufficiency degree ≥ moderate, and death. Results There were 18 pregnancies carried beyond 20 weeks in 11 patients. There was no significant difference in aortic root diameter between nulliparous patients and parous patients prior to their first pregnancy (3.53 ± 0.44 vs 3.57 ± 0.69 cm, P = .74). There was no significant change in aortic root diameter after first pregnancy (3.7 ± 0.4 cm, P = .056) although there was significant dilatation after the second (4.3 ± 0.7 cm, P = .009) and third (4.5 ± 0.7 cm, P = .009) pregnancies. Freedom from combined endpoints was significantly higher for patients in the pregnancy group than those in the nonpregnancy group (P = .002). Conclusions Pregnancy was not associated with significantly increased adverse events in patients following the Ross procedure. Special care should be taken after the first pregnancy, as multiparity may lead to increased neoaortic dilatation.

Since the American Heart Association’s recommendation for familial screening of adults with congenital heart disease for bicuspid aortic valve, similar recommendations for other left-sided heart defects, such as hypoplastic left heart syndrome (HLHS), have been proposed. However, defining at-risk populations for these heart defects based on genetics is less straightforward due to the wide variability of inheritance patterns and non-genetic influences such as environmental and lifestyle factors. We discuss whether there is sufficient evidence to standardize echocardiographic screening for first-degree relatives of children diagnosed with HLHS. Due to variations in the inclusion of cardiac anomalies linked to HLHS and the identification of asymptomatic individuals with cardiac malformations, published studies are open to interpretation. We conclude that familial aggregation of obstructive left-sided congenital heart lesions in families with history of HLHS is not supported and recommend that additional screening should adopt a more conservative definition of what truly constitutes this heart defect. More thorough consideration is needed before embracing familial screening recommendations of families of patients with HLHS, since this could inflict serious costs on healthcare infrastructure and further burden affected families both emotionally and financially.

Cardiac surgical procedures are complex and require the coordinated action of many. This creates the potential for small failures that could be the substrate for subsequent morbidity or mortality. High-reliability science suggests that preoccupation with small failures can lead to improved outcomes. Failures of all magnitudes (ie, events) were captured within the pediatric cardiac operating room starting with a single surgeon in April 2008. As the surgical team became more familiar with the process, failure recording was extended to all surgeons and all surgical procedures performed until the conclusion of the study in December 2010. New recording processes were developed and used on a rolling basis during this study. With systematic capture, event rates increased (from occurring within 20% to 50% of operative procedures). Although we identified 9 recurrent patterns, 2 categories (ie, Equipment and Patient Instability) accounted for almost half of the events (45%). The greatest number of events occurred during the prebypass period (40.2%), compared with bypass (20.1%) and postbypass (32.3%) periods. These events were mainly difficulties in access (31.8%), equipment (42.4%), and patient instability (33.3%) in each of the epochs, respectively. Of all events, 7.3% occurred during nonbypass cases, 30.6% of these were communication events. Implementation of this initiative led to recognition of major system-wide issues (eg, need for change in the blood-product acquisition process). Preoccupation with all failures in the operating room can reveal important information about the operating room and perioperative microenvironment that can prompt substantive process changes both locally and within the larger health system.