Virome Analysis and Association of Positive Coxsackievirus B Serology during Pregnancy with Congenital Heart Disease.

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.

The plasma virome in longitudinal samples from pregnant patients.

Introduction: Nucleic acid from viruses is common in peripheral blood, even in asymptomatic individuals. How physiologic changes of pregnancy impact host-virus dynamics for acute, chronic, and latent viral infections is not well described. Previously we found higher viral diversity in the vagina during pregnancy associated with preterm birth (PTB) and Black race. We hypothesized that higher diversity and viral copy numbers in the plasma would show similar trends. Methods: To test this hypothesis, we evaluated longitudinally collected plasma samples from 23 pregnant patients (11 term and 12 preterm) using metagenomic sequencing with ViroCap enrichment to enhance virus detection. Sequence data were analyzed with the ViroMatch pipeline. Results: We detected nucleic acid from at least 1 virus in at least 1 sample from 87% (20/23) of the maternal subjects. The viruses represented 5 families: Herpesviridae, Poxviridae, Papillomaviridae, Anelloviridae, and Flaviviridae. We analyzed cord plasma from 18 of the babies from those patients and found nucleic acid from viruses in 33% of the samples (6/18) from 3 families: Herpesviridae, Papillomaviridae, and Anelloviridae. Some viral genomes were found in both maternal plasma and cord plasma from maternal-fetal pairs (e.g. cytomegalovirus, anellovirus). We found that Black race associated with higher viral richness (number of different viruses detected) in the maternal blood samples (P=0.003), consistent with our previous observations in vaginal samples. We did not detect associations between viral richness and PTB or the trimester of sampling. We then examined anelloviruses, a group of viruses that is ubiquitous and whose viral copy numbers fluctuate with immunological state. We tested anellovirus copy numbers in plasma from 63 pregnant patients sampled longitudinally using qPCR. Black race associated with higher anellovirus positivity (P<0.001) but not copy numbers (P=0.1). Anellovirus positivity and copy numbers were higher in the PTB group compared to the term group (P<0.01, P=0.003, respectively). Interestingly, these features did not occur at the time of delivery but appeared earlier in pregnancy, suggesting that although anelloviruses were biomarkers for PTB they were not triggering parturition. Discussion: These results emphasize the importance of longitudinal sampling and diverse cohorts in studies of virome dynamics during pregnancy.

Multiple subtypes of coxsackievirus group B can cause congenital heart disease.

BACKGROUND: Different serotypes of coxsackievirus B (CVB), which is the most common cause of viral myocarditis, target cardiomyocytes through Coxsackie and Adenovirus Receptor and Decay-Accelerating Factor. Both receptors are expressed in the fetal heart. We hypothesized that infection with different serotypes of CVB during early pregnancy plays a role in pathogenesis of congenital heart defect (CHD). METHODS: In this study, we use a murine model to infect with CVB1, CVB4, and combination of CVB3 + CVB4 during a critical period in gestation. We examined offspring of pregnant mice for fetal death and heart defects following viral infection. RESULT: Fetuses from uninfected control dams showed normal heart development, while maternal CVB infection precipitates CHD: majorly ventricular septal defects (VSD) and non-compaction of ventricular myocardium (NC), with some infrequent cases of double outlet right ventricle, left ventricle wall rupture, right ventricle hypertrophy, and thickened/dysplastic semilunar valves. Infection of pregnant dams with CVB1 leads to 44% VSD and 41.2% NC cases, while with CVB4 leads to 31.7% VSD and 13.3% NC cases. Co-infection with CVB3 + CVB4 increases fetal pathology to 51.3% VSD and 41% NC cases. Infection can also result in fetal death, with higher incidences with CVB3 + CVB4 with 46.2% cases, compared to 33.3% by CVB1 and 21.7% by CVB4. Male fetuses were more susceptible to all phenotypes. CONCLUSION: Our report shows that prenatal CVB infections can lead to pathogenesis of certain heart defects in mouse model, particularly exacerbated with co-infections. This data confirms a link between prenatal CVB infection and CHD development and highlights it is not unique to just one serotype of CVB.

A Case of Persistent Human Pegivirus Infection in Two Separate Pregnancies of a Woman.

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.

Possible Association of Pulmonary Atresia with In-Utero Coxsackievirus B Exposure.

Gestational viral infection has been associated with congenital heart disease (CHD). Few studies, however, have studied the potential role of gestational Coxsackievirus B (CVB) exposure in the pathogenesis of CHD. We prospectively enrolled women with pregnancies affected by CHD to explore possible associations with in utero CVB exposure. Serum samples were obtained from 122 women referred for fetal echocardiography between 2006 and 2018. We quantified CVB IgG and IgM levels, with titers ≥ 15.0 U/mL considered positive and measured neutralizing antibodies for three CVB serotypes: CVB1, CVB3, and CVB4. Using data from the national enterovirus surveillance system, we compared the annual exposure rates for each serotype in our cohort to infections reported across the United States. 98 pregnancies with no genetic defects were included. Overall, 29.6% (29/98) had positive IgG and 4.1% (4/98) of women had positive CVB IgM titers. To explore first-trimester CVB exposure, we focused exclusively on the 26 women with positive IgG and negative IgM titers. 61.5% (16/26) had neutralizing antibodies against a single serotype and 38.5% (10/26) against multiple CVB serotypes. CVB4 neutralizing antibodies were the most common (65.4%, 17/26), followed by CVB3 (53.9%, 14/26) and CVB1 (30.8%, 8/26). Among these, 30.8% of babies presented pulmonary valve anomalies: 19.2% (5/26) pulmonary atresia, and 11.5% (3/26) pulmonary stenosis. 23.1% (6/26) of babies had coronary sinusoids. CVB exposure in our cohort mirrored that of reported infections in the United States. Our results suggest a possible association between gestational CVB exposure and specific CHD, particularly pulmonary valve anomalies and coronary sinusoids.

Coxsackievirus B3 Infection Early in Pregnancy Induces Congenital Heart Defects Through Suppression of Fetal Cardiomyocyte Proliferation.

Background Coxsackievirus B (CVB) is the most common cause of viral myocarditis. It targets cardiomyocytes through coxsackie and adenovirus receptor, which is highly expressed in the fetal heart. We hypothesized CVB3 can precipitate congenital heart defects when fetal infection occurs during critical window of gestation. Methods and Results We infected C57Bl/6 pregnant mice with CVB3 during time points in early gestation (embryonic day [E] 5, E7, E9, and E11). We used different viral titers to examine possible dose-response relationship and assessed viral loads in various fetal organs. Provided viral exposure occurred between E7 and E9, we observed characteristic features of ventricular septal defect (33.6%), abnormal myocardial architecture resembling noncompaction (23.5%), and double-outlet right ventricle (4.4%) among 209 viable fetuses examined. We observed a direct relationship between viral titers and severity of congenital heart defects, 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 congenital heart defects. We examined signaling pathways in CVB3-exposed hearts using RNA sequencing, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and immunohistochemistry. Signaling proteins of the Hippo, tight junction, transforming growth factor-ß1, and extracellular matrix proteins were the most highly enriched in CVB3-infected fetuses with ventricular septal defects. Moreover, cardiomyocyte proliferation was 50% lower in fetuses with ventricular septal defects compared with uninfected controls. Conclusions We conclude prenatal CVB3 infection induces congenital heart defects. Alterations in myocardial proliferate capacity and consequent changes in cardiac architecture and trabeculation appear to account for most of observed phenotypes.

Impact of pregnancy on autograft dilatation and aortic valve function following the Ross procedure.

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.

Familial Screening for Left-Sided Congenital Heart Disease: What Is the Evidence? What Is the Cost?

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.

Application of the aviation black box principle in pediatric cardiac surgery: tracking all failures in the pediatric cardiac operating room.

BACKGROUND: 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. STUDY DESIGN: 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. RESULTS: 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). CONCLUSIONS: 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.

Maternal ß-hemolytic streptococcal pharyngeal exposure and colonization in pregnancy.

OBJECTIVES: To report the pharyngeal colonization rate of ß-hemolytic streptococci and changes in the value of antistreptolysin O (ASO) and anti-DNase B serology titers during pregnancy. METHODS: Healthy pregnant women were recruited and blood was drawn in each trimester. The upper limit of normal (ULN) values for ASO and anti-DNase B was calculated for each trimester. Throat swabs were collected for culture and positive cultures were further assessed for the identification of serogroup of the isolated ß-hemolytic streptococcus. RESULTS: Out of a total of 126 pregnant women, 34.1% had positive throat cultures. Group C and group G strains were isolated in 18.2% of throat cultures while group F was detected in 13.5% of cases. The rate of colonization with GAS was 1.6%. There was an overall drop in ASO titer during pregnancy while anti-DNase B titers remained relatively unchanged. ULN values of 164(IU), 157(IU), and 156(IU) were calculated for ASO at the first, second, and third trimesters, respectively. Based on the ULN values, 28.6% of patients had recent streptococcal exposure. CONCLUSIONS: These results show that pregnant women act as a reservoir for spreading potentially immunogenic (groups C and G) and disease producing (group F) virulent strains of streptococci.

Congenital heart disease linked to maternal autoimmunity against cardiac myosin.

Structural congenital heart disease (CHD) has not previously been linked to autoimmunity. In our study, we developed an autoimmune model of structural CHD that resembles hypoplastic left heart syndrome (HLHS), a life-threatening CHD primarily affecting the left ventricle. Because cardiac myosin (CM) is a dominant autoantigen in autoimmune heart disease, we hypothesized that immunization with CM might lead to transplacental passage of maternal autoantibodies and a prenatal HLHS phenotype in exposed fetuses. Elevated anti-CM autoantibodies in maternal and fetal sera, as well as IgG reactivity in fetal myocardium, were correlated with structural CHD that included diminished left ventricular cavity dimensions in the affected progeny. Further, fetuses that developed a marked HLHS phenotype had elevated serum titers of anti-ß-adrenergic receptor Abs, as well as increased protein kinase A activity, suggesting a potential mechanism for the observed pathological changes. Our maternal-fetal model presents a new concept linking autoimmunity against CM and cardiomyocyte proliferation with cardinal features of HLHS. To our knowledge, this report shows the first evidence in support of a novel immune-mediated mechanism for pathogenesis of structural CHD that may have implications in its future diagnosis and treatment.

Defining the best practice patterns for the neonatal systemic-to-pulmonary artery shunt procedure.

OBJECTIVES: To assess variation in outcome measures and their associations with shunt thrombosis prophylaxis regimens after systemic-to-pulmonary artery shunt surgery across centers in the United States participating in the Pediatric Health Information System database. METHODS: We reviewed data on neonates who underwent an off-pump shunt procedure at 39 centers between 2000 and 2011. The overall variation in rates of discharge mortality and shunt-related complications were assessed by funnel plots. Complications were defined as revision/repeat of the shunt procedure during the same admission, institution of extracorporeal membrane oxygenation after surgery, and catheter interventions after shunt surgery. Bayesian hierarchical modeling was used to identify outliers. Shunt thrombosis prophylaxis regimens including the time of initiation of aspirin were compared between high and low outliers. RESULTS: A total of 2058 index operations were identified. Funnel plots highlighting the outcomes from various centers allowed discrimination of discharge mortality and complication rates around an aggregate of 6.7% and 12.3%, respectively. Bayesian modeling showed the presence of substantial variation in complication rates between centers; 20% of them were identified as outliers. Aspirin was initiated significantly earlier during the hospital course in centers with a lower composite rate of complications than those with higher rates (median initiation day of 2 [interquartile range (IQR), 1-3] in low outliers vs 4 [IQR, 3-6] in high outliers; P < .001). CONCLUSIONS: A substantial variation was found between hospitals in the rate of shunt-related complications. Centers with best outcomes implement aspirin earlier in their postoperative shunt thrombosis prophylaxis regimen.

Prioritizing quality improvement in pediatric cardiac surgery.

BACKGROUND: Several efforts are under way to conduct quality-improvement initiatives in pediatric cardiology and cardiac surgery. Our goal was to develop an objective prioritization scheme for such initiatives based on encounter frequency and relative contribution of quality measures of morbidity (and associated variances), particularly in the setting of low mortality. METHODS: We identified patients in the Pediatric Health Information System in Risk Adjustment for Congenital Heart Surgery 1 category 1 to 6 for 32 pediatric cardiac surgical procedures conducted between 2003 and 2011 (n = 67,550). These were examined for their overall contribution to mortality, intensive care unit and hospital lengths of stay (coefficient of variation and excess days), adverse events, and readmission rates. A ranking scheme was created on the basis of the outcome measures. Then we ordered the procedures across metrics to develop a prioritization scheme. RESULTS: Observed mortality rates were consistent with published rates. A few procedures accounted for significant variation in hospital and intensive care length of stay across the hospitals. Likewise, a few procedures accounted for most excess days of stay and readmission rates. Up to 60% of the hospital stay was accounted for by intensive care unit stay. Although there was a linear relationship between adverse event rates and Risk Adjustment for Congenital Heart Surgery 1 categories, a few procedures once again accounted for disproportionate event rates within and across their respective Risk Adjustment for Congenital Heart Surgery 1 categories. CONCLUSIONS: A small number of procedures account for a substantial burden of morbidity, even among low mortality risk groups. Consideration should be given to using such objective prioritization schemes to develop targeted quality-improvement measures.

Seasonality of hypoplastic left heart syndrome in the United States: a 10-year time-series analysis.

OBJECTIVE: Hypoplastic left heart syndrome is a major congenital heart defect and is associated with significant morbidity and mortality. Its etiology remains unknown although genetic studies imply complex inheritance. Anecdotal reports of cluster presentations suggest the possible involvement of an environmental component, although previous epidemiologic studies have been of limited scope. The objective of this study was to examine seasonal and temporal patterns of hypoplastic left heart syndrome births compared with other left-sided heart defects in the United States. METHODS: We conducted a retrospective analysis of the Pediatric Health Information System inpatient database from pediatric hospitals across the country from 1996 to 2006. Population and index case patterns were analyzed for each diagnostic category. An epidemiologic survey was performed through time-series analyses using Fisher's Kappa test and the Bartlett Kolmogorov-Smirnov test. The existence and strength of seasonality for the left-sided heart defects was quantified by the autoregression R2. RESULTS: A seasonal occurrence was found in hypoplastic left heart syndrome but not other left-sided heart diseases. Significant seasonal differences occurred each year, with peaks in summer months and troughs in winter months. The seasonality inversely correlated with the incidence of chromosomal and extracardiac anomalies; such anomalies were highest in interrupted aortic arch, which had a random pattern of presentation. CONCLUSIONS: There is a significant seasonal pattern in the presentation of hypoplastic left heart syndrome, with preponderance in summer months, in contrast to the random pattern in other left-sided heart diseases. Further studies are warranted to identify the influence of potential environmental factor(s) in hypoplastic left heart syndrome, as seen in diseases with seasonal patterns.

Transcription factor ETS1 is critical for human uterine decidualization.

The aim of this study was to examine whether the transcription factor ETS1 plays a critical role in the regulation of human decidualization. Decidual fibroblast cells were decidualized in vitro by treatment with medroxyprogesterone, estradiol (E(2)) and dibutyryl cyclic AMP or prostaglandin E(2) in the absence or presence of an ETS1 antisense oligonucleotide (oligo) that blocks the translation of ETS1 mRNA. Control experiments were performed using a control oligo that did not affect ETS1 expression and the induction of specific marker genes for decidualization. The ETS1 antisense oligo markedly inhibited ETS1 protein expression and significantly inhibited downstream targets of ETS1 action. On day 6 of culture, the decidualized fibroblast cells that had been exposed to the ETS1 antisense oligo contained 40-90% less mRNAs for prolactin, insulin growth factor binding protein 1 (IGFBP-1) and other decidualization-specific markers (laminin, tissue inhibitor of metalloproteinase-3 [TIMP3], endometrial bleeding associated factor [EBAF] and decorin) than those of control cells that had not been exposed to the ETS1 antisense oligo. GAPDH mRNA levels, which do not change during decidualization, were unaffected by either the ETS1 antisense or the control oligo. The cells decidualized in the presence of the ETS1 antisense oligo also released significantly less prolactin, EBAF and IGFBP-1 protein, determined by western blot analyses, than the control cells. Taken together, these findings strongly suggest that ETS1 plays a critical role in the induction of human decidualization.

Cannabinoid receptor I activation markedly inhibits human decidualization.

The role of cannabinoid receptor I (CBR-1) in the induction of decidualization was examined using decidual fibroblasts and human endometrial stromal cells as model systems. Decidual fibroblasts decidualized in vitro for 3 and 6 days in the presence of the CBR-1 agonist R(+)-WIN 55,212-2 mesylate (WIN, 0.1-10 microM) expressed less of the decidualization-specific markers prolactin, CBR-1, forkhead (FKHR), TIMP-3, laminin, endometrial bleeding associated factor (EBAF), decorin and insulin-like growth factor binding protein-1 (IGFBP-1) mRNA levels compared to control cells. The maximal decrease for each transcript was in the range of 50-99%. In contrast, cells exposed to the CBR-1 inhibitor AM-251 (1 microM) expressed about two-fold higher levels of the decidualization-specific marker gene mRNAs. The WIN-exposed cells showed a marked decrease in intracellular cAMP levels and a progressive, concentration-dependent increase in DNA fragmentation (TUNEL assay) and caspase 3 levels during decidualization compared to control cells. These studies strongly suggest that activation of CBR-1 inhibits human decidualization and stimulates apoptosis by a cAMP-dependent mechanism.