Towards precision quantification of contamination in metagenomic sequencing experiments.

Metagenomic next-generation sequencing (mNGS) experiments involving small amounts of nucleic acid input are highly susceptible to erroneous conclusions resulting from unintentional sequencing of occult contaminants, especially those derived from molecular biology reagents. Recent work suggests that, for any given microbe detected by mNGS, an inverse linear relationship between microbial sequencing reads and sample mass implicates that microbe as a contaminant. By associating sequencing read output with the mass of a spike-in control, we demonstrate that contaminant nucleic acid can be quantified in order to identify the mass contributions of each constituent. In an experiment using a high-resolution (n = 96) dilution series of HeLa RNA spanning 3-logs of RNA mass input, we identified a complex set of contaminants totaling 9.1 ± 2.0 attograms. Given the competition between contamination and the true microbiome in ultra-low biomass samples such as respiratory fluid, quantification of the contamination within a given batch of biological samples can be used to determine a minimum mass input below which sequencing results may be distorted. Rather than completely censoring contaminant taxa from downstream analyses, we propose here a statistical approach that allows separation of the true microbial components from the actual contribution due to contamination. We demonstrate this approach using a batch of n = 97 human serum samples and note that despite E. coli contamination throughout the dataset, we are able to identify a patient sample with significantly more E. coli than expected from contamination alone. Importantly, our method assumes no prior understanding of possible contaminants, does not rely on any prior collection of environmental or reagent-only sequencing samples, and does not censor potentially clinically relevant taxa, thus making it a generalized approach to any kind of metagenomic sequencing, for any purpose, clinical or otherwise.

Dynamic outcome prediction in a socio-demographically diverse population-based cohort of extremely preterm neonates.

OBJECTIVE: Accurate outcome prediction is crucial for counseling parents and providing individualized treatment to extremely premature infants. We sought to improve upon existing prediction model by using a diverse population-based cohort of extremely premature live births (⩽28 weeks' gestation) for survival and survival without severe neonatal morbidity at different times throughout the first week of life and to evaluate potential differences by race/ethnicity and maternal education. STUDY DESIGN: Retrospective cohort study of all California live births from 2007 through 2011 with linked birth, death and hospital discharge records. RESULTS: A total of 6009 infants were included. In the validation data set at time of delivery, the area under the receiver-operating characteristic curve for the model containing all predictors was 0.863 for survival and 0.789 for survival without severe morbidity. The marginal probability of survival without severe neonatal morbidity of an Asian infant born to a mother with <12 years of education compared with the reference (Caucasian infant, mother with ⩾12 years of education) was -0.23 (95% confidence interval (CI) -0.31 to -0.15) for all infants at time of birth and -0.28 (95% CI -0.39 to -0.18) for infants with attempted resuscitation. Notably, no other differences by racial/ethnic category and maternal education emerged. CONCLUSIONS: Probabilities of survival and survival without major morbidity change rapidly throughout the first week of life. Extremely premature infants born to Asian mothers with less than a high school education appear to have a lower probability to survive without significant morbidity compared with their Caucasian peers.

Risk of preterm birth among women using drugs during pregnancy with elevated α-fetoprotein.

OBJECTIVE: Examine the risk of preterm birth (PTB) among women who use drugs during pregnancy and have elevated α-fetoprotein (AFP). STUDY DESIGN: The sample included California singleton live births in 2005 to 2010 contained within a hospital discharge database linked to the Prenatal Screening Program. A selection of mothers who did not use drugs was selected at a ratio of 4:1. Risk of PTB was calculated using adjusted odds ratios and 95% confidence intervals (CIs) for women who did or did not use drugs by their AFP percentile. RESULTS: We identified 7190 women who used drugs and selected 28 760 women who did not. Of women using cocaine with AFP ⩾95th percentile, 43.8% delivered prematurely. Women using drugs with AFP ⩾95th percentile were 11 to 35 times as likely to deliver <32 weeks. CONCLUSION: The combination of drug use and elevated AFP results in high rates of PTB. This combination results in an additive risk.

Population-based risks of mortality and preterm morbidity by gestational age and birth weight.

OBJECTIVE: The objective of this study is to examine the effect of small or large for gestational age (SGA/LGA) status on mortality and morbidity by gestational age. STUDY DESIGN: Logistic binomial regression was used to calculate relative risks (RRs) and 95% confidence intervals for infant mortality and preterm morbidities for SGA or LGA compared with appropriately grown (AGA) deliveries stratified by gestational age group. RESULTS: Compared with AGA infants of similar gestational age, SGA infants were at increased risk for infant mortality. Mortality risk was decreased for LGA infants born between 25 and 27 weeks (RR: 0.6) but increased for LGA infants born between 28 and 31 weeks (RR: 1.9). Risk of preterm morbidity was increased for SGA infants born between 28 and 38 weeks, but decreased for LGA infants born before 37 weeks. CONCLUSION: This study demonstrates the importance of considering birth weight for gestational age when evaluating morbidity and mortality risks.

Outcomes of pregnancies with more than one positive prenatal screening result in the first or second trimester.

OBJECTIVE: To describe adverse outcomes and fetal abnormalities in women with a positive prenatal screening result for more than one disorder. STUDY DESIGN: Study participants were drawn from a population of 452 901 women pregnant with singletons entering the California Prenatal Screening Program in their first-trimester. Risk assessment was provided for trisomy 21 and trisomy 18 in the first-trimester and trisomy 21, trisomy 18, neural tube defects, and Smith-Lemli-Opitz syndrome in the second-trimester. Inclusion in this study required positive screening for more than one of the screened conditions and a completed outcome of pregnancy survey. RESULTS: A total of 874 women met our study inclusion criteria. Over 25% of these pregnancies had a fetus with a chromosomal abnormality. Of the euploid pregnancies, 6.9% had a fetus with a major birth defect. Of the pregnancies with a fetus with neither a chromosomal abnormality nor a major birth defect, 9.3% ended in fetal demise. Overall, more than 50% of women with multiple positive screening results had either a fetus with a birth defect or a poor pregnancy outcome. CONCLUSION: Although it is rare to screen positive for more than one condition, such results indicate a very high risk for chromosomal abnormality, fetal demise, or structural abnormality.

Maternal characteristics and mid-pregnancy serum biomarkers as risk factors for subtypes of preterm birth.

OBJECTIVE: To examine the relationship between maternal characteristics, serum biomarkers and preterm birth (PTB) by spontaneous and medically indicated subtypes. DESIGN: Population-based cohort. SETTING: California, United States of America. POPULATION: From a total population of 1 004 039 live singleton births in 2009 and 2010, 841 665 pregnancies with linked birth certificate and hospital discharge records were included. METHODS: Characteristics were compared for term and preterm deliveries by PTB subtype using logistic regression and odds ratios adjusted for maternal characteristics and obstetric factors present in final stepwise models and 95% confidence intervals. First-trimester and second-trimester serum marker levels were analysed in a subset of 125 202 pregnancies with available first-trimester and second-trimester serum biomarker results. MAIN OUTCOME MEASURE: PTB by subtype. RESULTS: In fully adjusted models, ten characteristics and three serum biomarkers were associated with increased risk in each PTB subtype (Black race/ethnicity, pre-existing hypertension with and without pre-eclampsia, gestational hypertension with pre-eclampsia, pre-existing diabetes, anaemia, previous PTB, one or two or more previous caesarean section(s), interpregnancy interval ≥ 60 months, low first-trimester pregnancy-associated plasma protein A, high second-trimester α-fetoprotein, and high second-trimester dimeric inhibin A). These risks occurred in 51.6-86.2% of all pregnancies ending in PTB depending on subtype. The highest risk observed was for medically indicated PTB <32 weeks in women with pre-existing hypertension and pre-eclampsia (adjusted odds ratio 89.7, 95% CI 27.3-111.2). CONCLUSIONS: Our findings suggest a shared aetiology across PTB subtypes. These commonalities point to targets for further study and exploration of risk reduction strategies. TWEETABLE ABSTRACT: Findings suggest a shared aetiology across preterm birth subtypes. Patterns may inform risk reduction efforts.

Maternal serum markers, characteristics and morbidly adherent placenta in women with previa.

OBJECTIVE: To examine associations with morbidly adherent placenta (MAP) among women with placenta previa. STUDY DESIGN: Women with MAP (cases) and previa alone (controls) were identified from a cohort of 236,714 singleton pregnancies with both first and second trimester prenatal screening, and live birth and hospital discharge records; pregnancies with aneuploidies and neural tube or abdominal wall defects were excluded. Logistic binomial regression was used to compare cases with controls. RESULT: In all, 37 cases with MAP and 699 controls with previa alone were included. Risk for MAP was increased among multiparous women with pregnancy-associated plasma protein-A (PAPP-A) ⩾95th percentile (⩾2.63 multiple of the median (MoM); adjusted OR (aOR) 8.7, 95% confidence interval (CI) 2.8 to 27.4), maternal-serum alpha fetoprotein (MS-AFP) ⩾95th percentile (⩾1.79 MoM; aOR 2.8, 95% CI 1.0 to 8.0), and 1 and ⩾2 prior cesarean deliveries (CDs; aORs 4.4, 95% CI 1.5 to 13.6 and 18.4, 95% CI 5.9 to 57.5, respectively). CONCLUSION: Elevated PAPP-A, elevated MS-AFP and prior CDs are associated with MAP among women with previa.

Effect of magnitude and timing of maternal pregnancy blood lead (Pb) levels on birth outcomes.

OBJECTIVE: Associations between magnitude and timing of maternal pregnancy blood lead (Pb) levels (BLLs), birth weight, and total days of gestation were examined, as well as associations with related clinical diagnoses of low birth weight (LBW), preterm, and small-for-gestational-age (SGA) birth. STUDY DESIGN: Among a sample of 262 mother-infant pairs studied retrospectively, one-way analysis of variance and regression statistics were used to measure the relationship between level of maternal pregnancy BLLs and birth outcomes while controlling for key maternal and newborn factors. RESULTS: Women with maximum pregnancy BLLs (max-PBLLs) > or =10 microg/dl tended to give birth earlier and their babies were at substantially increased risk for preterm and SGA birth. By holding other explanatory factors constant, each unit increase in max-PBLL above 10 mug/dl was found to be associated with a decrease of -0.3 in total days of gestation. Compared to women with lower levels, women with max-PBLLs > or =10 microg/dl were at a threefold increased risk for preterm birth (adjusted OR=3.2, 95% CI 1.2-7.4) and more than a fourfold increased risk for having an SGA infant (adjusted OR=4.2, 1.3-13.9). Second trimester maximum BLLs > or =10 microg/dl were associated with a steep decrease in total days of gestation (a decrease of -1.0 days per each unit increase above 10 microg/dl). CONCLUSIONS: These data provide evidence of the adverse effects of maternal pregnancy BLLs, particularly when levels are > or =10 microg/dl. Prenatal Pb exposure at these levels was associated with significant decreases in total days of gestation and an increased risk of preterm and SGA birth.