Impact of long-term storage of plasma and cell-free DNA on measured DNA quantity and fetal fraction.

BACKGROUND AND OBJECTIVE: Optimal sample storage conditions are essential for non-invasive prenatal testing of cell-free fetal and total DNA. We investigated the effect of long-term storage of plasma samples and extracted cfDNA using qPCR. MATERIALS AND METHODS: Fetal and total cfDNA yield and fetal fraction were calculated before and after storage of plasma for 0-6 years at -25°C. Dilution experiments were performed to investigate PCR inhibition. Extraction with or without proteinase K was used to examine protein dissociation. Storage of extracted cfDNA was investigated by testing aliquots immediately, and after 18 months and 3 years of storage at -25°C. RESULTS: We observed a marked increase in the levels of amplifiable fetal and total DNA in plasma stored for 2-3 years, and fetal fraction was slightly decreased after 3 years of storage. cfDNA detection was independent of proteinase K during DNA extraction in plasma samples stored >2 years, indicating a loss of proteins from DNA over time, which was likely to account for the observed increase in DNA yields. Measured fetal and total DNA quantities, as well as fetal fraction, increased in stored, extracted cfDNA. CONCLUSION: Fetal and total cell-free DNA is readily detectable in plasma after long-term storage at -25°C. However, substantial variation in measured DNA quantities and fetal fraction means caution may be required when using stored plasma and extracted cfDNA for test development or validation purposes.

Noninvasive fetal RHD genotyping to guide targeted anti-D prophylaxis-an external quality assessment workshop.

BACKGROUND AND OBJECTIVES: Fetal RHD genotyping of cell-free fetal DNA from RhD-negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 28 laboratories. MATERIALS AND METHODS: Aliquots of pooled maternal plasma were sent to each laboratory. One sample was positive, and the second sample was negative for fetal RHD, verified by pre-workshop testing using quantitative real-time PCR (qPCR) analysis of RHD exons 4, 5, 7 and 10. Plasma samples were shipped at room temperature. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. Different methodological approaches were used, all employing qPCR with a total of eight different combinations of RHD exon targets. The samples were tested blindly. RESULTS: Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD-positive sample, and four inconclusive results were reported for the RHD-negative sample. All clinical conclusions were satisfactory. CONCLUSION: This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting.

Biology of human primitive erythroblasts for application in noninvasive prenatal diagnosis.

OBJECTIVE: Human primitive erythroblasts produced during early embryogenesis have been found in maternal circulation at early gestation and are considered good target cells for noninvasive prenatal diagnosis. We aimed to gain a better understanding of the biology of primitive erythroblasts and maximize their potential utility for noninvasive prenatal diagnosis. METHODS: Cells were obtained from first trimester human placental tissues. Biological properties including surface antigen composition, differentiation, proliferation, enucleation, and degeneration were studied as gestation progressed. A microdroplet culture system was developed to observe the behavior of these cells in vitro. RESULTS: Histology showed that primitive erythroblasts undergo maturation from polychromatic to orthochromatic erythroblasts and can differentiate spontaneously in vitro. Cell surface markers and nuclear gene expression suggest that the cells do not possess stemness properties, despite being primitive in nature. They have limited proliferative activity and highly deacetylated chromatin, but a microdroplet culture system can prolong their viability under normoxic conditions. No apoptosis was seen by 11 weeks' gestation, and there was no enucleation in vitro. CONCLUSION: These properties confirm that viable cells with intact nuclei can be obtained at very early gestation for genetic analysis.

Challenges in non-invasive prenatal screening for sub-chromosomal copy number variations using cell-free DNA.

Non-invasive prenatal screening (NIPS) has revolutionized the approach to prenatal fetal aneuploidy screening. Many commercial providers now offer analyses for sub-chromosomal copy number variations (CNVs). Here, we review the use of NIPS in the context of screening for microdeletions and microduplications, issues surrounding the choice of disorders tested for, and the advantages and disadvantages associated with the inclusion of microdeletions to current NIPS. Several studies have claimed benefits; however, we suggest that microdeletions have not demonstrated a low enough false positive rate to be deemed practical or ethically acceptable, especially considering their low positive predictive values. Because a positive NIPS result should be confirmed using diagnostic techniques, and false positive rates are as high as 90% for some microdeletions, diagnostic testing seems preferable when the goal is to maximize the detection of microdeletion or microduplication syndromes.

Has noninvasive prenatal testing impacted termination of pregnancy and live birth rates of infants with Down syndrome?

BACKGROUND: Implementation of noninvasive prenatal testing (NIPT) as a highly accurate aneuploidy screening test has raised questions around whether the high uptake may result in more terminations of pregnancies and fewer births of children with Down syndrome (DS). AIM: The aim of the study was to investigate the impact of NIPT on termination and live birth rates for DS. METHODS: Literature reporting pregnancy outcomes following NIPT was reviewed. Termination rates were calculated for women with a high-risk NIPT result for DS. Two audits of pregnancy outcomes where NIPT indicated DS were conducted in the United Kingdom and Singapore. RESULTS: Fourteen studies from the United States, Asia, Europe, and the United Kingdom were included in the review. Live births of children with DS were reported in 8 studies. Termination rates following NIPT were unchanged or decreased when compared to termination rates prior to the introduction of NIPT. Audits found 15 of 43 women in the United Kingdom and 2 of 6 in Singapore continued pregnancies following a high-risk NIPT result. CONCLUSIONS: Termination rates following the detection of DS by NIPT are unchanged or decreased compared to historical termination rates. Impact on live birth rates may be minimal in settings where termination rates fall. Population-based studies are required to determine the true impact.

Synthetic combinations of missense polymorphic genetic changes underlying Down syndrome susceptibility.

Single nucleotide polymorphisms (SNPs) are important biomolecular markers in health and disease. Down syndrome, or Trisomy 21, is the most frequently occurring chromosomal abnormality in live-born children. Here, we highlight associations between SNPs in several important enzymes involved in the one-carbon folate metabolic pathway and the elevated maternal risk of having a child with Down syndrome. Our survey highlights that the combination of SNPs may be a more reliable predictor of the Down syndrome phenotype than single SNPs alone. We also describe recent links between SNPs in p53 and its related pathway proteins and Down syndrome, as well as highlight several proteins that help to associate apoptosis and p53 signaling with the Down syndrome phenotype. In addition to a comprehensive review of the literature, we also demonstrate that several SNPs reside within the same regions as these Down syndrome-linked SNPs, and propose that these closely located nucleotide changes may provide new candidates for future exploration.

A crucial role for DOK1 in PDGF-BB-stimulated glioma cell invasion through p130Cas and Rap1 signalling.

DOK1 regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility. Mechanisms regulating tumour cell motility are essential for invasion and metastasis. We report here that PDGF-BB-mediated glioma cell invasion and migration are dependent on the adaptor protein downstream of kinase 1 (DOK1). DOK1 is expressed in several glioma cell lines and in tumour biopsies from high-grade gliomas. DOK1 becomes tyrosine phosphorylated upon PDGF-BB stimulation of human glioma cells. Knockdown of DOK1 or expression of a DOK1 mutant (DOK1FF) containing Phe in place of Tyr at residues 362 and 398, resulted in inhibition of both the PDGF-BB-induced tyrosine phosphorylation of p130Cas (also known as BCAR1) and the activation of Rap1. DOK1 colocalises with tyrosine phosphorylated p130Cas at the cell membrane of PDGF-BB-treated cells. Expression of a non-tyrosine-phosphorylatable substrate domain mutant of p130Cas (p130Cas15F) inhibited PDGF-BB-mediated Rap1 activation. Knockdown of DOK1 and Rap1 inhibited PDGF-BB-induced chemotactic cell migration, and knockdown of DOK1 and Rap1 and expression of DOK1FF inhibited PDGF-mediated three-dimensional (3D) spheroid invasion. These data show a crucial role for DOK1 in the regulation of PDGF-BB-mediated tumour cell motility through a p130Cas-Rap1 signalling pathway. [Corrected]

Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

BACKGROUND: Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. METHODS: Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. RESULTS: Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. CONCLUSIONS: We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

Non-invasive prenatal diagnosis of achondroplasia and thanatophoric dysplasia: next-generation sequencing allows for a safer, more accurate, and comprehensive approach.

OBJECTIVE: Accurate prenatal diagnosis of genetic conditions can be challenging and usually requires invasive testing. Here, we demonstrate the potential of next-generation sequencing (NGS) for the analysis of cell-free DNA in maternal blood to transform prenatal diagnosis of monogenic disorders. METHODS: Analysis of cell-free DNA using a PCR and restriction enzyme digest (PCR-RED) was compared with a novel NGS assay in pregnancies at risk of achondroplasia and thanatophoric dysplasia. RESULTS: PCR-RED was performed in 72 cases and was correct in 88.6%, inconclusive in 7% with one false negative. NGS was performed in 47 cases and was accurate in 96.2% with no inconclusives. Both approaches were used in 27 cases, with NGS giving the correct result in the two cases inconclusive with PCR-RED. CONCLUSION: NGS provides an accurate, flexible approach to non-invasive prenatal diagnosis of de novo and paternally inherited mutations. It is more sensitive than PCR-RED and is ideal when screening a gene with multiple potential pathogenic mutations. These findings highlight the value of NGS in the development of non-invasive prenatal diagnosis for other monogenic disorders.

Non-invasive prenatal diagnostic testing for ß-thalassaemia using cell-free fetal DNA and next generation sequencing.

OBJECTIVE: To develop an accurate non-invasive prenatal test using next generation sequencing (NGS) for HbE and the four most common ß-thalassaemia mutations found in South East Asia (namely -28A > G, CD17A > T, CD41/42(-TTCT) and IVS-II-654C > T). METHODS: Cell-free DNA was extracted from maternal plasma from 83 families where both parents were carriers of the HbE mutation or one of four common ß-thalassaemia mutations. Overlapping PCR amplicons covering each mutation were generated, pooled and sequenced using the Illumina MiSeq. Fastq files were analysed to detect inheritance of the paternal mutation. RESULTS: In two cases where the fathers were compound heterozygotes for HbE and -28A > G, the fetus was correctly diagnosed as having inherited one of the paternal mutations. In 35/85 cases, the paternal mutation was not detected, and in 50/85 cases, it was classified as inherited. Overall sensitivity for detection of paternal mutations was 100% (95% CI: 92.4-100%), and specificity was 92.1% (95% CI: 79.2-97.3%). CONCLUSION: We demonstrated that detection of paternal mutations using NGS can be readily achieved with high sensitivity and specificity, removing the need for an invasive test in 50% of pregnancies at risk of a thalassaemia in cases where the father and mother carry a different mutation. © 2014 John Wiley & Sons, Ltd.

Detection of aneuploidy from single fetal nucleated red blood cells using whole genome sequencing.

OBJECTIVE: The objective of the study was to detect aneuploidy in single fetal nucleated red blood cells (FNRBCs) from placental villi using whole genome amplification (WGA) and next generation sequencing. METHODS: Three single FNRBCs per sample were manually picked from villi collected from ten women undergoing elective first-trimester termination of pregnancy, and one or two cells were picked from each of four aneuploid chorionic villus samples. Following WGA and addition of adaptor and index sequences, samples were sequenced on the Illumina MiSeq. Leading and trailing 15 bases were trimmed, and reads were aligned to the human reference genome. Z-scores were calculated to determine deviation of the mean of the test from reference samples, with a score of 3 used as the threshold for classification of a particular chromosome as trisomic. RESULTS: We successfully made correct diagnoses from ten single cells isolated from villi from two cases of trisomy 21 (one case from a single cell and one from two cells), two cases of trisomy 18 (two cells each), and a case of trisomy 15 (three cells). CONCLUSION: With their faithful representation of fetal genome, diagnosis using single FNRBCs provides a definitive result compared with non-invasive prenatal testing using cell-free fetal DNA, and is a safer alternative to invasive amniocentesis.

Factors impacting influenza vaccination of urban low-income Latino children under nine years requiring two doses in the 2010-2011 season.

The Advisory Committee on Immunization Practices (ACIP) recommends that certain children under 9 years of age receive two influenza vaccine doses in a season for optimal protection. Recent data indicate that many of these children fail to receive one or both of these needed doses. Contributing factors to under-vaccination of this population remain unclear. Caregivers of children aged 6 months-8 years requiring two influenza vaccine doses in the 2010-2011 season were identified from households enrolled in four urban Head Start programs. Recruitment and survey administration were conducted between March and June 2011. The impact of caregiver, provider, and practice-based factors on influenza vaccine receipt was assessed using bivariate and multivariable logistic regression analyses. Caregivers (n = 128) were predominantly mothers, Latina, Spanish-speaking, and non-U.S. born. Few children received one (31 %) or both (7 %) influenza vaccine doses. Caregivers who discussed influenza vaccination with providers were more likely to know their child needed two doses (55 vs. 35 %, p < 0.05) and have a fully vaccinated child (11 vs. 0 %, p < 0.05). Among caregivers whose child received the first dose, those who reported being told when to return for the second dose were also more likely to have a fully vaccinated child (35 vs. 0 %, p = 0.05). Belief in influenza vaccine effectiveness was positively associated with vaccination (p < 0.001), while safety concerns were negatively associated (p < 0.05). This study highlights the importance of provider-family communication about the two-dose regimen as well as influenza vaccine effectiveness and safety.

Stability of cell-free DNA from maternal plasma isolated following a single centrifugation step.

OBJECTIVE: Cell-free fetal DNA can be used for prenatal testing with no procedure-related risk to the fetus. However, yield of fetal DNA is low compared with maternal cell-free DNA fragments, resulting in technical challenges for some downstream applications. To maximize the fetal fraction, careful blood processing procedures are essential. We demonstrate that fetal fraction can be preserved using a single centrifugation step followed by postage of plasma to the laboratory for further processing. METHODS: Digital PCR was used to quantify copies of total, maternal, and fetal DNA present in single-spun plasma at time points over a two-week period, compared with immediately processed double-spun plasma, with storage at room temperature, 4°C, and -80°C representing different postage scenarios. RESULTS: There was no significant change in total, maternal, or fetal DNA copy numbers when single-spun plasma samples were stored for up to 1 week at room temperature and 2 weeks at -80°C compared with plasma processed within 4 h. Following storage at 4°C no change in composition of cell-free DNA was observed. CONCLUSIONS: Single-spun plasma can be transported at room temperature if the journey is expected to take one week or less; shipping on dry ice is preferable for longer journeys.

Safe, accurate, prenatal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA.

OBJECTIVE: To improve the prenatal diagnosis of thanatophoric dysplasia by defining the change in fetal size across gestation and the frequency of sonographic features, and developing non-invasive molecular genetic diagnosis based on cell-free fetal DNA (cffDNA) in maternal plasma. METHODS: Fetuses with a confirmed diagnosis of thanatophoric dysplasia were ascertained, records reviewed, sonographic features and measurements determined. Charts of fetal size were then constructed using the LMS (lambda-mu-sigma) method and compared with charts used in normal pregnancies and those complicated by achondroplasia. Cases in this cohort referred to our Regional Genetics Laboratory for molecular diagnosis using cffDNA were identified and results reviewed. RESULTS: Forty-two cases were scanned in our units. Commonly reported sonographic features were very short and sometimes bowed femora, frontal bossing, cloverleaf skull, short fingers, a small chest and polyhydramnios. Limb shortening was obvious from as early as 13 weeks' gestation, with minimal growth after 20 weeks. Analysis of cffDNA in three of these pregnancies confirmed the presence of the c.742C>CT (p.Arg248Cys) or the c.1948A>AG (p.Lys650Glu) mutation in the fibroblast growth factor receptor 3 gene. CONCLUSION: These data should improve the accuracy of the sonographic diagnosis of thanatophoric dysplasia and have implications for reliable and safe targeted molecular confirmation using cffDNA.

The clinical implementation of non-invasive prenatal diagnosis for single-gene disorders: challenges and progress made.

Recently, we have witnessed the rapid translation into clinical practice of non-invasive prenatal testing for the common aneuploidies, most notably within the United States and China. This represents a lucrative market with testing being driven by companies developing and offering their services. These tests are currently aimed at women with high/medium-risk pregnancies identified by serum screening and/or ultrasound scanning. Uptake has been impressive, albeit limited to the commercial sector. However, non-invasive prenatal diagnosis (NIPD) for single-gene disorders has attracted less interest, no doubt because this represents a much smaller market opportunity and in the majority of cases has to be provided on a bespoke, patient or disease-specific basis. The methods and workflows are labour-intensive and not readily scalable. Nonetheless, there exists a significant need for NIPD of single-gene disorders, and the continuing advances in technology and data analysis should facilitate the expansion of the NIPD test repertoire. Here, we review the progress that has been made to date, the different methods and platform technologies, the technical challenges, and assess how new developments may be applied to extend testing to a wider range of genetic disorders.

Clinical impact of healthcare-associated respiratory syncytial virus in hospitalized adults.

OBJECTIVE: To describe the clinical impact of healthcare-associated (HA) respiratory syncytial virus (RSV) in hospitalized adults. DESIGN: Retrospective cohort study within a prospective, population-based, surveillance study of RSV-infected hospitalized adults during 3 respiratory seasons: October 2017-April 2018, October 2018-April 2019, and October 2019-March 2020. SETTING: The study was conducted in 2 academically affiliated medical centers. PATIENTS: Each HA-RSV patient (in whom RSV was detected by PCR test ≥4 days after hospital admission) was matched (age, sex, season) with 2 community-onset (CO) RSV patients (in whom RSV was detected ≤3 days of admission). METHODS: Risk factors and outcomes were compared among HA-RSV versus CO-RSV patients using conditional logistic regression. Escalation of respiratory support associated with RSV detection (day 0) from day -2 to day +4 was explored among HA-RSV patients. RESULTS: In total, 84 HA-RSV patients were matched to 160 CO-RSV patients. In HA-RSV patients, chronic kidney disease was more common, while chronic respiratory conditions and obesity were less common. HA-RSV patients were not more likely to be admitted to an ICU or require mechanical ventilation, but they more often required a higher level of care at discharge compared with CO-RSV patients (44% vs 14%, respectively). Also, 29% of evaluable HA-RSV patients required respiratory support escalation; these patients were older and more likely to have respiratory comorbidities, to have been admitted to intensive care, and to die during hospitalization. CONCLUSIONS: HA-RSV in adults may be associated with escalation in respiratory support and an increased level of support in living situation at discharge. Infection prevention and control strategies and RSV vaccination of high-risk adults could mitigate the risk of HA-RSV.

Digital PCR analysis of maternal plasma for noninvasive detection of sickle cell anemia.

BACKGROUND: Cell-free fetal DNA (cffDNA) constitutes approximately 10% of the cell-free DNA in maternal plasma and is a suitable source of fetal genetic material for noninvasive prenatal diagnosis (NIPD). The objective of this study was to determine the feasibility of using digital PCR for NIPD in pregnancies at risk of sickle cell anemia. METHODS: Minor-groove binder (MGB) TaqMan probes were designed to discriminate between wild-type hemoglobin A and mutant (hemoglobin S) alleles encoded by the HBB (hemoglobin, beta) gene in cffDNA isolated from maternal plasma samples obtained from pregnancies at risk of sickle cell anemia. The fractional fetal DNA concentration was assessed in male-bearing pregnancies with a digital PCR assay for the Y chromosome-specific marker DYS14. In pregnancies with a female fetus, a panel of biallelic insertion/deletion polymorphism (indel) markers was developed for the quantification of the fetal DNA fraction. We used digital real-time PCR to analyze the dosage of the variant encoding hemoglobin S relative to that encoding wild-type hemoglobin A. RESULTS: The sickle cell genotype was correctly determined in 82% (37 of 45) of male fetuses and 75% (15 of 20) of female fetuses. Mutation status was determined correctly in 100% of the cases (25 samples) with fractional fetal DNA concentrations >7%. The panel of indels was informative in 65% of the female-bearing pregnancies. CONCLUSIONS: Digital PCR can be used to determine the genotype of fetuses at risk for sickle cell anemia. Optimization of the fractional fetal DNA concentration is essential. More-informative indel markers are needed for this assay's comprehensive use in cases of a female fetus.

Change in functional status associated with respiratory syncytial virus infection in hospitalized older adults.

BACKGROUND: Respiratory syncytial virus (RSV) causes severe respiratory illnesses in infants and older adults. Older adults are frequently hospitalized with RSV illness and may experience loss of function. This study evaluated longitudinal changes in function associated with RSV hospitalization in older adults. METHODS: Adults ≥60 years hospitalized with laboratory-confirmed RSV were enrolled (N = 302). Demographics and comorbidities were collected. Functional status was assessed 2 weeks pre-hospitalization by recall, at enrollment, hospital discharge and 2, 4, and 6 months post-discharge using the Lawton-Brody Instrumental Activities of Daily Living (IADL) (scale 0-8) and Barthel ADL Index (scale 0-100). RESULTS: RSV-associated hospitalization resulted in acute functional loss. Median IADL (5 vs. 3, p < 0.0001) and ADL (90 vs. 70, p < 0.0001) scores decreased significantly from pre-hospitalization to admission and remained decreased at discharge. There were no statistically significant differences between pre-hospitalization and 2-, 4-, or 6-month scores. However, 33% and 32% of subjects experienced decreased 6-month IADL and ADL scores, respectively. Additionally, 14% required a higher level of care at discharge. When stratified by pre-hospitalization living situation, 6-month IADL scores declined significantly for those admitted from a skilled nursing facility (3 vs. 1, p = 0.001). In multivariate analysis, male sex and diabetes were associated with a 6-month decline in ADL score of ≥10. CONCLUSIONS: Older adults hospitalized with RSV demonstrate acute functional decline that may become prolonged. Pre-hospitalization living situation may predict patient outcomes. Further study is needed with hospitalized age-matched controls and refined measurement tools to better define the specific impact of RSV on function.

Whole-Chromosome Karyotyping of Fetal Nucleated Red Blood Cells Using the Ion Proton Sequencing Platform.

The current gold standard for the definitive diagnosis of fetal aneuploidy uses either chorionic villus sampling (CVS) or amniocentesis, both of which are which are invasive procedures carrying a procedure-related risk of miscarriage of up to 0.1-0.2%. Non-invasive prenatal diagnosis using fetal nucleated red blood cells (FNRBCs) isolated from maternal peripheral venous blood would remove this risk of miscarriage since these cells can be isolated from the mother's blood. We aimed to detect whole-chromosome aneuploidies from single nucleated fetal red blood cells using whole-genome amplification followed by massively parallel sequencing performed on a semiconductor sequencing platform. Twenty-six single cells were picked from the placental villi of twelve patients thought to have a normal fetal genotype and who were undergoing elective first-trimester surgical termination of pregnancy. Following karyotyping, it was subsequently found that two of these cases were also abnormal (one trisomy 15 and one mosaic genotype). One single cell from chorionic villus samples for two patients carrying a fetus with trisomy 21 and two single cells from women carrying fetuses with T18 were also picked. Pooled libraries were sequenced on the Ion Proton and data were analysed using Ion Reporter software. We correctly classified fetal genotype in all 24 normal cells, as well as the 2 T21 cells, the 2 T18 cells, and the two T15 cells. The two cells picked from the fetus with a mosaic result by CVS were classified as unaffected, suggesting that this was a case of confined placental mosaicism. Fetal sex was correctly assigned in all cases. We demonstrated that semiconductor sequencing using commercially available software for data analysis can be achieved for the non-invasive prenatal diagnosis of whole-chromosome aneuploidy with 100% accuracy.

Severe Clinical Outcomes Among Adults Hospitalized With Respiratory Syncytial Virus Infections, New York City, 2017-2019.

OBJECTIVES: Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in older adults. We assessed severe clinical outcomes among hospitalized adults that were associated with RSV infections. METHODS: We performed a nested retrospective study in 3 New York City hospitals during 2 respiratory viral seasons, October 2017-April 2018 and October 2018-April 2019, to determine the proportion of patients with laboratory-confirmed RSV infection who experienced severe outcomes defined as intensive care unit (ICU) admission, mechanical ventilation, and/or death. We assessed factors associated with these severe outcomes and explored the effect of RSV-associated hospitalizations on changes in the living situations of surviving patients. RESULTS: Of the 403 patients studied (median age, 69 years), 119 (29.5%) were aged ≥80. Severe outcomes occurred in 19.1% of patients, including ICU admissions (16.4%), mechanical ventilation (12.4%), and/or death (6.7%). Patients admitted from residential living facilities had a 4.43 times higher likelihood of severe RSV infection compared with patients who were living in the community with or without assistance from family or home health aides. At discharge, 56 (15.1%) patients required a higher level of care than at admission. CONCLUSIONS: RSV infection was associated with severe outcomes in adults. Living in a residential facility at admission was a risk factor for severe outcomes and could be a proxy for frailty rather than an independent risk factor. Our data support the development of prevention strategies for RSV infection in older populations, especially older adults living in residential living facilities.