Maternal age and body mass index as risk factors for rectovaginal colonization with group B streptococci.

OBJECTIVE: To examine the effect of including maternal age and body mass index (BMI; calculated as weight in kilograms divided by the square of height in meters) as additional risk factors in the traditional risk-based strategy at term pregnancies consisting of previous early-onset group B streptococcus (GBS) disease, GBS bacteriuria during pregnancy, maternal temperature of 38.0°C or more intrapartum, and rupture of membranes of 18 h or longer. METHODS: A secondary analysis of a Danish cohort including 902 pregnant women. Exposures were maternal age and pre-pregnancy BMI. Outcome was rectovaginal GBS colonization at the time of labor. The logistic regression analysis adjusted for parity, gestational age, vaginal delivery, and smoking. RESULTS: The GBS prevalence was 17% in the entire population, 35% among participants older than 40 years, and 23% among those with a BMI of 25 or greater. Including maternal "age > 40" as an additional risk factor increased the sensitivity of the risk-based strategy from 21% to 26% and decreased the specificity from 90% to 87%. Inclusion of "BMI ≥ 25" increased the sensitivity from 21% to 57% and decreased the specificity from 90% to 59%. CONCLUSIONS: Maternal age and BMI might be included as additional risk factors in risk-based programs for identification of GBS-positive laboring women to receive intrapartum antibiotics prophylaxis.

Polymerase chain reaction for Group B Streptococci (GBS) at labor highly correlates with vaginal GBS load.

OBJECTIVE: To explore factors associated with a high vaginal GBS load during labor considering (1) the recto-vaginal GBS load at 35-37 weeks' gestation determined by culture and (2) the vaginal GBS colonization determined by a polymerase chain reaction (PCR) assay during labor. METHODS: From an unselected cohort of 902 pregnant women, we obtained (1) recto-vaginal swabs for culture of GBS at 35-37 weeks' gestation (GBSrectovag-36), (2) vaginal swabs for GBS PCR detection at labor (PCRvag-labor), and (3) vaginal swabs for culture of GBS at labor (GBSvag-labor). The GBS load was classified semi quantitatively according to a culture protocol without prior broth enrichment of the swab samples: none (0), few (+), some (++), or many (+++) GBS colonies. RESULTS: Among 902 unselected pregnant women, 859 (95%) had a vaginal swab culture taken at labor, which was classified semi quantitatively. High load GBSvag-labor (+++) were found in 31 participants. GBSrectovag-36 showed a sensitivity of 90% (28/31) and a PPV of 23% (28/121), whereas PCRvag-labor had a sensitivity of 98% (30/31, non-significant difference) and a PPV of 42% (30/71, p < .01). PCR at labor had a lower sensitivity (78%) for detection of vaginal colonization with GBS at labor (any load) compared to recto/vaginal colonization with GBS at 36 weeks (92%). Vaginal colonization with GBS at 36 weeks seemed to have a lower sensitivity for detecting GBS in vagina at labor for high load (48%) and for any load (39%). CONCLUSION: PCR at labor has higher detection rate (non-significant) and PPV in identification of laboring women with a high load of vaginal GBS compared with recto-vaginal culture at 36 weeks' gestation.

Improvement of selection of pregnant women for intrapartum polymerase chain reaction screening for vaginal Group B Streptococci (GBS) colonization by adding GBS urine screening at 35-37 weeks of pregnancy.

OBJECTIVE: To evaluate whether systematic antepartum screening for Group B Streptococci (GBS) by urine culture improves the risk factor-based selection of pregnant women for intrapartum GBS screening with a rapid polymerase chain reaction (PCR) assay. METHODS: A prospective observational study was conducted between April 2013 and June 2014. GBS colonization judged by urine culture at 35-37 weeks of gestation was compared with the result of a vaginal GBS PCR test at labor as outcome. The results of urine culture were used as exposure variable. The PCR test was performed on intrapartum vaginal samples. RESULTS: Screening for urine GBS in 902 unselected pregnant Danish women at 35-37 weeks of gestation predicted intrapartum PCR GBS status with a sensitivity of 33.6%. A positive predictive value of 41.2% was seen among women with low GBS counts (<104 CFU/mL) and 83.3% among women with high GBS counts (≥104 CFU/mL). Systematic GBS screening of urine at 35-37 weeks of gestation added 30.9% extra women for intrapartum GBS PCR screening in the study group. CONCLUSION: Systematic antepartum GBS screening of urine should be implemented in order to improve risk stratification for early onset GBS by offering laboring women an intrapartum GBS PCR test.

Urine dipstick for predicting intrapartum recto-vaginal colonisation by group B streptococci.

INTRODUCTION: In pregnant women, bacteriuria with group B streptococci (GBS) may be associated with a high degree of recto-vaginal GBS colonisation and therefore an increased risk of early-onset GBS disease. The aim of this study was to assess the performance of routine use of dipstick urine analysis during pregnancy for prediction of recto-vaginal GBS colonisation at the time of labour. METHODS: Among 902 unselected Danish pregnant women, we obtained results from 1) dipstick urine analysis, 2) urine culture carried out during pregnancy, if indicated, and 3) recto-vaginal culture at labour. The inclusion criteria were age > 18 years and gestational age ≥ 37 weeks. RESULTS: Intrapartum recto-vaginal GBS colonisation was predicted by a positive urine dipstick with 5% sensitivity only. CONCLUSION: Dipstick urine analysis had a low sensitivity for predicting intrapartum recto-vaginal colonisation with GBS. FUNDING: none. TRIAL REGISTRATION: not relevant.

Group B streptococci cultured in urine during pregnancy associated with preterm delivery: a selection problem?

Objective: To investigate an association between Group B streptococci (GBS) in urine culture during pregnancy and preterm delivery. Methods: A population-based cohort consisted of all the pregnant women (n = 36,097) from the catchment area of Lillebaelt Hospital, Denmark, during the period January 2002 -December 2012. The cohort of 34,285 singleton pregnancies used in this study was divided into three groups. Group I (N = 249) included women whose urine culture was positive for GBS; group II (N = 5765) included women whose urine culture was negative for GBS; and group III (N = 28 271) included women whose urine had not been cultured during pregnancy. Primary outcome was preterm delivery before 37 weeks' gestation (PTD). Results: We did not find an association between PTD and GBS bacteriuria in the cultured groups (odds ratios (OR) = 0.89; 95% CI: 0.5-1.4) ( Table 1 ). After controlling for potential confounders, the PTD remained not associated with GBS bacteriuria (adjusted OR = 0.99; 95% CI: 0.6-1.6). Combined, the cultured groups (I and II) were associated with a statistically significant higher risk for PTD, when compared with the group with no urine specimens taken for culture (OR = 1.96; 95% CI: 1.8-2.2 and adjusted or 1.80; 95% CI 1.6-2.0). The cultured group of women differed considerably from the group of women with no urine specimens taken for culture on the vast majority of variables examined. Conclusions: No association between asymptomatic GBS bacteriuria and preterm delivery among women with singleton pregnancy and urine specimens cultured during pregnancy was found. Previous suggestions of such association may have been compromised by a selection problem for testing due to a high-risk profile of pregnancy complications in pregnant women selected for urine culture.

Using virtual-reality simulation to ensure basic competence in hysteroscopy.

BACKGROUND: Hysteroscopy is a technically challenging procedure. Specialty curricula of obstetrics and gynaecology appraise hysteroscopy for trainees but there is no present evidence-based training program that certifies the fundamental technical skills before performance on patients. The objectives of this study were to develop and gather validity evidence for a simulation-based test that can ensure basic competence in hysteroscopy. METHODS: We used the virtual-reality simulator HystMentor™. Six experts evaluated the feasibility and clinical relevance of the simulator modules. Six modules were selected for the test and a pilot study was carried out. Subsequently, medical students, residents, and experienced gynaecologists were enrolled for testing. Outcomes were based on generated simulator metrics. Validity evidence was explored for all five sources of evidence (content, response process, internal structure, relations to other variables, consequences of testing). RESULTS: Inter-case reliability was high for four out of five metrics (Cronbach's alpha ≥ 0.80). Significant differences were identified when comparing the three groups' performances (p values < 0.05). Participants' clinical experience was significantly correlated to their simulator test score (Pearson's r = 0.49, p < 0.001). A single medical student managed to achieve the established pass/fail score (6.7% false positive) and three experienced gynaecologists failed the test (27.3% false negative). CONCLUSIONS: We developed a virtual-reality simulation-based test in hysteroscopy with supporting validity evidence. The test is intended to ensure competency in a mastery learning program where trainees practise on the simulator until they are able to pass before they proceed to supervised training on patients.

Risk-based approach versus culture-based screening for identification of group B streptococci among women in labor.

OBJECTIVE: To compare a risk-based and culture-based screening approach for identification of group B streptococci (GBS) vaginal colonization using an intrapartum rectovaginal culture as the reference standard. METHODS: Pregnant women attending the prenatal clinic at Lillebaelt Hospital, Kolding, Denmark, between April 1, 2013, and June 30, 2014, were invited to participate in a prospective observational study. For prepartum culture-based screening, vaginal and rectal culture samples were obtained and, for reference, standard, paired vaginal and rectal culture samples were collected during labor. Risk factors for risk-based screening were previous early-onset GBS, GBS bacteriuria during pregnancy, maternal temperature ≥38.0°C intrapartum, and rupture of membranes for more than 18 hours. RESULTS: The intrapartum rectovaginal GBS colonization rate was 30% (32/108) among participants with risk factors and 15% (123/794) among participants without risk factors. Culture-based screening demonstrated a sensitivity, specificity, positive predictive value, negative predictive value, and positive likelihood ratio in predicting intrapartum GBS carriage of 78% (95% confidence interval [CI] 71-84), 95% (94-97), 78% (70-84), 95% (94-97), and 17 (12-23), respectively; for risk-based screening, these values were 21% (15-28), 90% (87-92), 30% (22-38), 85% (83-86), and 2 (1-3), respectively. CONCLUSIONS: Culture-based screening performed considerably better than a risk-based approach in identifying intrapartum GBS colonization.

Number of colony forming units in urine at 35-37 weeks' gestation as predictor of the vaginal load of Group B Streptococci at birth.

OBJECTIVE: To evaluate GBS colony numbers in the urine at 35-37 weeks' gestation to predict the load of GBS-colonization of the vagina at birth. STUDY DESIGN: In this prospective observational study, we included 902 unselected pregnant women. Exposure was GBS colony forming units (CFU) per mL urine at 35-37 weeks' gestation. Outcome was vaginal GBS colonization at birth as assessed by a semi-quantitative culture of a vaginal swab sample (negative, +1, +2, +3). RESULTS: Bacteriuria with GBS at 35-37 weeks' gestation performed with a sensitivity of 30% concerning any degree of vaginal GBS colonization at birth (31 of 104 cases); 19% for light (+1), 17% for medium (+2), and 52% for high load (+3) vaginal GBS colonization. The colony count in case of GBS bacteriuria at 35-37 weeks' gestation performed with positive predictive values of 35% for <104 CFU/mL, 70% for 104 CFU/mL, and 67% for >104 CFU/mL. CONCLUSION: Even though the urinary GBS CFU at 35-37 weeks' gestation is strongly associated with a high load of vaginal GBS colonization intrapartum, it may not perform satisfactorily as a standalone-screening marker for risk of early-onset GBS disease.

Intrapartum PCR assay versus antepartum culture for assessment of vaginal carriage of group B streptococci in a Danish cohort at birth.

The aim of this study was to compare the performances of two strategies for predicting intrapartum vaginal carriage of group B streptococci (GBS). One strategy was based on an antepartum culture and the other on an intrapartum polymerase chain reaction (PCR). We conducted a prospective observational study enrolling 902 pregnant women offered GBS screening before delivery by two strategies. The Culture-strategy was based on vaginal and rectal cultures at 35-37 weeks' gestation, whereas the PCR-strategy was based on PCR assay on intrapartum vaginal swab samples. An intrapartum vaginal culture for GBS was used as the reference standard from which the performances of the 2 strategies were evaluated. The reference standard showed a GBS-prevalence of 12%. The culture-strategy performed with a sensitivity of 82%, specificity of 91%, positive predictive value (PPV) of 55%, negative predictive value (NPV) of 98%, and Likelihood ratio (LH+) of 9.2. The PCR-strategy showed corresponding values as sensitivity of 83%, specificity of 97%, PPV of 78%, NPV of 98%, and LH+ of 27.5. We conclude that in a Danish population with a low rate of early-onset neonatal infection with GBS, the intrapartum PCR assay performs better than the antepartum culture for identification of GBS vaginal carriers during labor.

Risk-based screening combined with a PCR-based test for group B streptococci diminishes the use of antibiotics in laboring women.

OBJECTIVE: To assess the performance of a polymerase chain reaction - group B streptococci test (PCR-GBS test) - in deciding antibiotic prophylaxis in term laboring women. STUDY DESIGN: In this observational study, we enrolled 902 unselected Danish term pregnant women. During labor, midwives obtained vaginal swabs that were used for both GBS cultures (reference standard) and for the PCR-GBS test. Furthermore, we recorded the presence of risk factors for EOGBS (Early Onset Group B Streptococcal disease): (1) Bacteriuria during current pregnancy, (2) Prior infant with EOGBS (3) Temperature above 38.0°C during labor, and (4) Rupture of membranes ≥18h. RESULTS: The prevalence of GBS carriers was 12% (104 of 902), the sensitivity of the PCR-GBS test 83% (86 of 104), and the specificity 97% (774 of 798). Among the 108 with one or more EOGBS-risk factors, GBS was present in 23% (25 of 108), the sensitivity 92% (23 of 25), and the specificity 89% (74 of 83). CONCLUSION: In programs that aim to treat all laboring women with vaginal GBS-colonization (12% in the present study) with penicillin, the PCR-GBS will perform well (sensitivity 83% and specificity 97%). In programs aiming to treat only GBS-carriers among those with risk factors of EOGBS, a reduction of penicillin usage by two-thirds from 12% to 4% may be possible.

Low to Moderate Average Alcohol Consumption and Binge Drinking in Early Pregnancy: Effects on Choice Reaction Time and Information Processing Time in Five-Year-Old Children.

BACKGROUND: Deficits in information processing may be a core deficit after fetal alcohol exposure. This study was designed to investigate the possible effects of weekly low to moderate maternal alcohol consumption and binge drinking episodes in early pregnancy on choice reaction time (CRT) and information processing time (IPT) in young children. METHOD: Participants were sampled based on maternal alcohol consumption during pregnancy. At the age of 60-64 months, 1,333 children were administered a modified version of the Sternberg paradigm to assess CRT and IPT. In addition, a test of general intelligence (WPPSI-R) was administered. RESULTS: Adjusted for a wide range of potential confounders, this study showed no significant effects of average weekly maternal alcohol consumption during pregnancy on CRT or IPT. There was, however, an indication of slower CRT associated with binge drinking episodes in gestational weeks 1-4. CONCLUSION: This study observed no significant effects of average weekly maternal alcohol consumption during pregnancy on CRT or IPT as assessed by the Sternberg paradigm. However, there were some indications of CRT being associated with binge drinking during very early pregnancy. Further large-scale studies are needed to investigate effects of different patterns of maternal alcohol consumption on basic cognitive processes in offspring.

Cytokines associated with necrotizing enterocolitis in extremely-low-birth-weight infants.

BACKGROUND: The goal was to identify cytokines associated with necrotizing enterocolitis (NEC). Based on our earlier reports of decreased tissue expression of transforming growth factor (TGF)-ß, we hypothesized that infants with NEC also have low blood TGF-ß levels. We further hypothesized that because fetal inflammation increases the risk of NEC, infants who develop NEC have elevated blood cytokine levels in early neonatal period. METHODS: Data on 104 extremely-low-birth-weight infants with NEC and 893 without NEC from 17 centers were analyzed. Clinical information was correlated with blood cytokine levels on postnatal day 1 (D1), D3, D7, D14, and D21. RESULTS: Male gender, non-Caucasian/non-African American ethnicity, sepsis, lower blood TGF-ß and interleukin (IL)-2 levels, and higher IL-8 levels were associated with NEC. The NEC group had lower TGF-ß levels than controls since D1. The diagnosis of NEC was associated with elevated IL-1ß, IL-6, IL-8, IL-10, monocyte chemoattractant protein-1/CC-motif ligand-2, macrophage inflammatory protein-1ß/CC-motif ligand-3, and C-reactive protein. CONCLUSION: Clinical characteristics, such as gender and ethnicity, and low blood TGF-ß levels are associated with higher risk of NEC. Infants who developed NEC did not start with high blood levels of inflammatory cytokines, but these rose mainly after the onset of NEC.

Separable sustained and selective attention factors are apparent in 5-year-old children.

In adults and older children, evidence consistent with relative separation between selective and sustained attention, superimposed upon generally positive inter-test correlations, has been reported. Here we examine whether this pattern is detectable in 5-year-old children from the healthy population. A new test battery (TEA-Ch(J)) was adapted from measures previously used with adults and older children and administered to 172 5-year-olds. Test-retest reliability was assessed in 60 children. Ninety-eight percent of the children managed to complete all measures. Discrimination of visual and auditory stimuli were good. In a factor analysis, the two TEA-Ch(J) selective attention tasks (one visual, one auditory) loaded onto a common factor and diverged from the two sustained attention tasks (one auditory, one motor), which shared a common loading on the second factor. This pattern, which suggests that the tests are indeed sensitive to underlying attentional capacities, was supported by the relationships between the TEA-Ch(J) factors and Test of Everyday Attention for Children subtests in the older children in the sample. It is possible to gain convincing performance-based estimates of attention at the age of 5 with the results reflecting a similar factor structure to that obtained in older children and adults. The results are discussed in light of contemporary models of attention function. Given the potential advantages of early intervention for attention difficulties, the findings are of clinical as well as theoretical interest.

Early signs of autism in toddlers: a follow-up study in the Danish National Birth Cohort.

To identify possible early signs of autism spectrum disorder (ASD) within the Danish National Birth Cohort, we studied prospectively collected interviews from 76,441 mothers about their children's development and behaviour at 6 and 18 months. In Danish national registries, 720 children with ASD and 231 children with intellectual disability (ID) were identified. At 6 months, associations between early signs and ASD or ID were found only in few areas. At 18 months social, language, and motor skills were delayed, and suspicion of vision and hearing problems were increased for both groups. Signs distinguishing ASD from ID were unclear, and the positive predictive values regarding ASD were below 10 % for individual predictors and aggregated risk scores.

Cervical ultrasound elastography may hold potential to predict risk of preterm birth.

INTRODUCTION: Freehand ultrasound real-time elastography (RTE) is a simple technique allowing direct visualization of the elastogramme superimposed on the B-mode -image. The objective of RTE is to investigate stiffness and related parameters such as local tissue strain with a view to adding new information related to tissue morphology and architecture. MATERIAL AND METHODS: This was a pilot study in 12 healthy pregnant women who underwent transvaginal ultrasound. The RTE (Hitachi) information was colour-coded and superimposed on the B-mode scan. Elastography images were analyzed by means of a software tool to identify thresholds for the colours red (soft), green (medium hard) and blue (hard). The cervical strain rate was measured in three different parts. Additional information obtained included age of gestation, number of pregnancies and deliveries, previous preterm births and gestational age at delivery in current pregnancy. RESULTS: The softness of cervix increases towards portio. Within the colour spectrum, green was predominant. Strain ratio can be used as a comparative index among different subjects rather than as an absolute strain measurement. CONCLUSION: The elastographic image allowed for easy correlation between colour distribution and the anatomical structures as it is superimposed on the B-mode image. The elasticity of the cervix increases towards portio. FUNDING: Not relevant. TRIAL REGISTRATION: Not relevant.

Association between blood spot transforming growth factor-ß and patent ductus arteriosus in extremely low-birth weight infants.

Permanent ductal closure involves anatomic remodeling, in which transforming growth factor (TGF)-ß appears to play a role. Our objective was to evaluate the relationship, if any, between blood spot TGF-ß on day 3 and day 7 of life and patent ductus arteriosus (PDA) in extremely low birth weight (ELBW) infants. Prospective observational study involving ELBW infants (n = 968) in the National Institute of Child Health and Human Development Neonatal Research Network who had TGF-ß measured on filter paper spot blood samples using a Luminex assay. Infants with a PDA (n = 493) were significantly more immature, had lower birth weights, and had higher rates of respiratory distress syndrome than those without PDA (n = 475). TGF-ß on days 3 and 7 of life, respectively, were significantly lower among neonates with PDA (median 1,177 pg/ml [range 642-1,896]; median 1,386 pg/ml [range 868-1,913]) compared with others without PDA (median 1,334 pg/ml [range 760-2,064]; median 1,712 pg/ml [range 1,014-2,518 pg/ml]). The significant difference persisted when death or PDA was considered a composite outcome. TGF-ß levels were not significantly different among subgroups of infants with PDA who were not treated (n = 51) versus those who were treated medically (n = 283) or by surgical ligation (n = 159). TGF-ß was not a significant predictor of death or PDA (day 3 odds ratio [OR] 0.99, 95 % confidence interval [CI] 0.83-1.17; day 7 OR 0.88, 95 % CI 0.74-1.04) on adjusted analyses. Our results suggest that blood spot TGF-ß alone is unlikely to be a reliable biomarker of a clinically significant PDA or its responsiveness to treatment.

Amniotic fluid inflammatory cytokines: potential markers of immunologic dysfunction in autism spectrum disorders.

OBJECTIVES: The aim of the study was to analyze cytokine profiles in amniotic fluid (AF) samples of children developing autism spectrum disorders (ASD) and controls, adjusting for maternal autoimmune disorders and maternal infections during pregnancy. METHODS: AF samples of 331 ASD cases and 698 controls were analyzed for inflammatory cytokines using Luminex xMAP technology utilizing a historic birth cohort. Clinical data were retrieved from nationwide registers, and case-control differences in AF cytokine levels were assessed using chi-square tests, logistic and tobit regression models. RESULTS: Overall, individuals with ASD had significantly elevated AF levels of TNF-α and TNF-ß compared to controls. Analyzing individuals diagnosed only with ICD-10 codes yielded significantly elevated levels of IL-4, IL-10, TNF-α and TNF-ß in ASD patients. Restricting analysis to infantile autism cases showed significantly elevated levels of IL-4, TNF-α and TNF-ß compared to controls with no psychiatric comorbidities. Elevated levels of IL-6 and IL-5 were found in individuals with other childhood psychiatric disorders (OCPD) when compared to controls with no psychiatric comorbidities. CONCLUSIONS: AF samples of individuals with ASD or OCPD showed differential cytokine profiles compared to frequency-matched controls. Further studies to examine the specificity of the reported cytokine profiles in ASD and OCPD are required.

Joint analysis of SNPs and proteins identifies regulatory IL18 gene variations decreasing the chance of spastic cerebral palsy.

Cerebral palsy (CP) is a permanent disorder, affecting 2-3 per 1,000 live born children, disturbing movement and posture. Spastic limbs affects about 70-80% of the CP children, and this group is the target of our study. CP is considered a multifactorial condition believed to be provoked by, for example, preterm birth, infection during pregnancy, neural disorders, and genetics, to mention some. Interestingly, the cytokine network is believed to be involved in many of these disorders. In this study, including 203 spastic CP cases and 167 controls, we measured the levels of 25 cytokine proteins, and genotyped 159 SNPs in their gene loci. Using logistic regression, we estimated the genetic association of SNP genotypes to spastic CP. In addition, fitting a Tobit regression model for each protein and each SNP in the respective gene loci, we estimated three regression coefficients corresponding three different effects of the genetic variation on the protein level. Intriguingly, two IL18 loci SNPs (rs549908:A>C and rs1290349:C>A) showed a protective effect against spastic CP, and interestingly both were associated to a decreased epidemiological expression of IL-18 protein. By joining protein data to genetic information, we have provided new data suggesting IL18's involvement in the pathogenesis of spastic CP.

C-reactive protein and preterm delivery: clues from placental findings and maternal weight.

To study the association between maternal C-reactive protein (CRP) and preterm delivery (PTD) pathways, CRP was measured in maternal plasma collected at mid-pregnancy (n = 1310). PTD was subdivided into spontaneous (sPTD) or medically indicated (MI-PTD). Histologic chorioamnionitis (HCA) was determined by placental histopathology (n = 1076). Adjusted CRP levels were elevated for sPTD (5.5 µg/mL) versus term deliveries (4.8 µg/mL) and higher in sPTD with HCA (6.3 µg/mL). After removing HCA, an interaction between body mass index (BMI) and sPTD in relation to CRP was noted. In BMI-stratified models, an association between CRP and sPTD among women with prepregnancy BMI >25 (8.9 µg/mL for sPTD; 7.2 µg/mL for term) was absent among women with lower BMI. We propose that this remaining association in overweight/obese women suggests that CRP may mark an obesity/inflammation PTD pathway that is distinct from the pathway indicated by HCA.