Clinical exome sequencing uncovers genetic disorders in neonates with suspected hypoxic-ischemic encephalopathy: A retrospective analysis.

Hypoxic-ischemic encephalopathy (HIE) occurs in up to 7 out of 1000 births and accounts for almost a quarter of neonatal deaths worldwide. Despite the name, many newborns with HIE have little evidence of perinatal hypoxia. We hypothesized that some infants with HIE have genetic disorders that resemble encephalopathy. We reviewed genetic results for newborns with HIE undergoing exome or genome sequencing at a clinical laboratory (2014-2022). Neonates were included if they had a diagnosis of HIE and were delivered ≥35 weeks. Neonates were excluded for cardiopulmonary pathology resulting in hypoxemia or if neuroimaging suggested postnatal hypoxic-ischemic injury. Of 24 patients meeting inclusion criteria, six (25%) were diagnosed with a genetic condition. Four neonates had variants at loci linked to conditions with phenotypic features resembling HIE, including KIF1A, GBE1, ACTA1, and a 15q13.3 deletion. Two additional neonates had variants in genes not previously associated with encephalopathy, including DUOX2 and PTPN11. Of the six neonates with a molecular diagnosis, two had isolated HIE without apparent comorbidities to suggest a genetic disorder. Genetic diagnoses were identified among neonates with and without sentinel labor events, abnormal umbilical cord gasses, and low Apgar scores. These results suggest that genetic evaluation is clinically relevant for patients with perinatal HIE.

Hypnozoite depletion in successive Plasmodium vivax relapses.

Genotyping Plasmodium vivax relapses can provide insights into hypnozoite biology. We performed targeted amplicon sequencing of 127 relapses occurring in Indonesian soldiers returning to malaria-free Java after yearlong deployment in malarious Eastern Indonesia. Hepatic carriage of multiple hypnozoite clones was evident in three-quarters of soldiers with two successive relapses, yet the majority of relapse episodes only displayed one clonal population. The number of clones detected in relapse episodes decreased over time and through successive relapses, especially in individuals who received hypnozoiticidal therapy. Interrogating the multiplicity of infection in this P. vivax relapse cohort reveals evidence of independent activation and slow depletion of hypnozoites over many months by multiple possible mechanisms, including parasite senescence and host immunity.

Video education about genetic privacy and patient perspectives about sharing prenatal genetic data: a randomized trial.

BACKGROUND: Laboratories offering cell-free DNA often reserve the right to share prenatal genetic data for research or even commercial purposes, and obtain this permission on the patient consent form. Although it is known that nonpregnant patients are often reluctant to share their genetic data for research, pregnant patients' knowledge of, and opinions about, genetic data privacy are unknown. OBJECTIVE: We investigated whether pregnant patients who had already undergone cell-free DNA screening were aware that genetic data derived from cell-free DNA may be shared for research. Furthermore, we examined whether pregnant patients exposed to video education about the Genetic Information Nondiscrimination Act-a federal law that mandates workplace and health insurance protections against genetic discrimination-were more willing to share cell-free DNA-related genetic data for research than pregnant patients who were unexposed. STUDY DESIGN: In this randomized controlled trial (ClinicalTrials.gov Identifier: NCT04420858), English-speaking patients with singleton pregnancies who underwent cell-free DNA and subsequently presented at 17 0/7 to 23 6/7 weeks of gestation for a detailed anatomy scan were randomized 1:1 to a control or intervention group. Both groups viewed an infographic about cell-free DNA. In addition, the intervention group viewed an educational video about the Genetic Information Nondiscrimination Act. The primary outcomes were knowledge about, and willingness to share, prenatal genetic data from cell-free DNA by commercial laboratories for nonclinical purposes, such as research. The secondary outcomes included knowledge about existing genetic privacy laws, knowledge about the potential for reidentification of anonymized genetic data, and acceptability of various use and sharing scenarios for prenatal genetic data. Eighty-one participants per group were required for 80% power to detect an increase in willingness to share data from 60% to 80% (α=0.05). RESULTS: A total of 747 pregnant patients were screened, and 213 patients were deemed eligible and approached for potential study participation. Of these patients, 163 (76.5%) consented and were randomized; one participant discontinued the intervention, and two participants were excluded from analysis after the intervention when it was discovered that they did not fulfill all eligibility criteria. Overall, 160 (75.1%) of those approached were included in the final analysis. Most patients in the control group (72 [90.0%]) and intervention (76 [97.4%]) group were either unsure about or incorrectly thought that cell-free DNA companies could not share prenatal genetic data for research. Participants in the intervention group were more likely to incorrectly believe that their prenatal genetic data would not be shared for nonclinical purposes than participants in the control group (28.8% in the control group vs 46.2% in the intervention; P=.03). However, video education did not increase participant willingness to share genetic data in multiple scenarios. Non-White participants were less willing than White participants to allow sharing of genetic data specifically for academic research (P<.001). CONCLUSION: Most participants were unaware that their prenatal genetic data may be used for nonclinical purposes. Pregnant patients who were educated about the Genetic Information Nondiscrimination Act were not more willing to share genetic data than those who did not receive this education. Surprisingly, video education about the Genetic Information Nondiscrimination Act led patients to falsely believe that their data would not be shared for research, and participants who identified as racial minorities were less willing to share genetic data. New strategies are needed to improve pregnant patients' understanding of genetic privacy.

Deep Sequencing to Detect Diversity of Trypanosoma cruzi Infection in Patients Coinfected With Human Immunodeficiency Virus and Chagas Disease.

Chagas disease, caused by Trypanosoma cruzi, can reactivate and cause severe acute disease in immunocompromised patients such as those infected with human immunodeficiency virus (HIV). We conducted amplicon deep sequencing of a 327-bp fragment of the tcscd5 gene using an Ion Torrent PGM directly from clinical samples from HIV patients with high parasitemia. We describe the within-host diversity, both characterizing the discrete typing unit of the infections and confirming the presence of multistrain infections, directly from clinical samples. This method can rapidly provide information on the genetic diversity of T. cruzi infection, which can have direct impacts on clinical disease.

Privacy practices using genetic data from cell-free DNA aneuploidy screening.

PURPOSE: Cell-free fetal DNA (cfDNA) analyzes maternal and fetoplacental DNA, generating highly personal genetic information for both mother and fetus. This study aimed to determine how laboratories retain, use, and share genetic information from cfDNA. Other outcomes included laboratories' adherence to American Society of Human Genetics (ASHG) privacy principles, and the readability of privacy policies. METHODS: Laboratories offering cfDNA aneuploidy screening were identified from online searches, curated databases, and a genomics news website. Of 124 laboratories identified, 13 were commercial laboratories offering cfDNA aneuploidy screening in the United States, and were included. Genetic privacy policies from eligible laboratories were identified by reviewing requisition and consent forms, which were obtained online or by direct contact. RESULTS: Most laboratories use prenatal genetic information for research (n = 10, 77%), and more than half (n = 7, 54%) shared genetic information with others. Overall, laboratories inadequately disclosed privacy risks. In a readability analysis, 9 of 11 (82%) laboratories' genetic privacy policies were written at or above a 12th grade reading level. CONCLUSION: Most laboratories allowed for prolonged use and sharing of cfDNA data, demonstrated incomplete adherence to ASHG privacy recommendations, and provided consents written in college-level language. Laboratories should revise their consent forms, and providers should help patients understand these forms.

Privacy Risks in Prenatal Aneuploidy and Carrier Screening: What Obstetricians and Their Patients Need to Know.

Modern prenatal genetic screening techniques such as cell-free fetal DNA and expanded carrier screening genotype substantial amounts of maternal and fetoplacental DNA. Although DNA can be deidentified by stripping protected health information from genetic data, anonymized DNA can be reidentified using genetic databases, raising long-term genetic privacy concerns for both mother and fetus. In this commentary, we explore the evolution of prenatal genetic screening and how modern screening techniques may pose unanticipated privacy risks. We highlight knowledge gaps and outline steps to improve patient awareness of and control over their genetic privacy, including specific recommendations for laboratories and prenatal care practitioners who offer screening. We also encourage our colleagues who provide prenatal care to be well informed about the privacy implications of the genetic tests we order and to be vocal advocates for our patients' genetic privacy, both with the laboratories that perform these tests and in the public sphere.

Efficient Transmission of Mixed Plasmodium falciparum/vivax Infections From Humans to Mosquitoes.

BACKGROUND: In Southeast Asia, people are often coinfected with different species of malaria (Plasmodium falciparum [Pf] and Plasmodium vivax [Pv]) as well as with multiple clones of the same species. Whether particular species or clones within mixed infections are more readily transmitted to mosquitoes remains unknown. METHODS: Laboratory-reared Anopheles dirus were fed on blood from 119 Pf-infected Cambodian adults, with 5950 dissected to evaluate for transmitted infection. Among 12 persons who infected mosquitoes, polymerase chain reaction and amplicon deep sequencing were used to track species and clone-specific transmission to mosquitoes. RESULTS: Seven of 12 persons that infected mosquitoes harbored mixed Pf/Pv infection. Among these 7 persons, all transmitted Pv with 2 transmitting both Pf and Pv, leading to Pf/Pv coinfection in 21% of infected mosquitoes. Up to 4 clones of each species were detected within persons. Shifts in clone frequency were detected during transmission. However, in general, all parasite clones in humans were transmitted to mosquitoes, with individual mosquitoes frequently carrying multiple transmitted clones. CONCLUSIONS: Malaria diversity in human hosts was maintained in the parasite populations recovered from mosquitoes fed on their blood. However, in persons with mixed Pf/Pv malaria, Pv appears to be transmitted more readily, in association with more prevalent patent gametocytemia.

Plasmodium vivax chloroquine resistance links to pvcrt transcription in a genetic cross.

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.

Matched Placental and Circulating Plasmodium falciparum Parasites are Genetically Homologous at the var2csa ID1-DBL2X Locus by Deep Sequencing.

In pregnancy-associated malaria, infected erythrocytes accumulate in the placenta. It is unclear if in polyclonal infections this results in distinct peripheral and placental parasite populations. We used long amplicon deep sequencing of Plasmodium falciparum var2csa ID1-DBL2X from 15 matched peripheral and placental samples collected at delivery from a high transmission area to determine genetic homology. Despite substantial sequence variation and detecting 23 haplotypes, the matched pairs mostly contained the same genetic variants, with 11 pairs sharing 100% of their variants, whereas others showed some heterogeneity. Thus, at delivery, peripheral and placental parasites appear to intermix and placental genotypes can be inferred through peripheral sampling.

SeekDeep: single-base resolution de novo clustering for amplicon deep sequencing.

PCR amplicon deep sequencing continues to transform the investigation of genetic diversity in viral, bacterial, and eukaryotic populations. In eukaryotic populations such as Plasmodium falciparum infections, it is important to discriminate sequences differing by a single nucleotide polymorphism. In bacterial populations, single-base resolution can provide improved resolution towards species and strains. Here, we introduce the SeekDeep suite built around the qluster algorithm, which is capable of accurately building de novo clusters representing true, biological local haplotypes differing by just a single base. It outperforms current software, particularly at low frequencies and at low input read depths, whether resolving single-base differences or traditional OTUs. SeekDeep is open source and works with all major sequencing technologies, making it broadly useful in a wide variety of applications of amplicon deep sequencing to extract accurate and maximal biologic information.

Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity.

Background: Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine. Methods: Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset. Results: Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity. Conclusions: Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.

Partner-Drug Resistance and Population Substructuring of Artemisinin-Resistant Plasmodium falciparum in Cambodia.

Plasmodium falciparum in western Cambodia has developed resistance to artemisinin and its partner drugs, causing frequent treatment failure. Understanding this evolution can inform the deployment of new therapies. We investigated the genetic architecture of 78 falciparum isolates using whole-genome sequencing, correlating results to in vivo and ex vivo drug resistance and exploring the relationship between population structure, demographic history, and partner drug resistance. Principle component analysis, network analysis and demographic inference identified a diverse central population with three clusters of clonally expanding parasite populations, each associated with specific K13 artemisinin resistance alleles and partner drug resistance profiles which were consistent with the sequential deployment of artemisinin combination therapies in the region. One cluster displayed ex vivo piperaquine resistance and mefloquine sensitivity with a high rate of in vivo failure of dihydroartemisinin-piperaquine. Another cluster displayed ex vivo mefloquine resistance and piperaquine sensitivity with high in vivo efficacy of dihydroartemisinin-piperaquine. The final cluster was clonal and displayed intermediate sensitivity to both drugs. Variations in recently described piperaquine resistance markers did not explain the difference in mean IC90 or clinical failures between the high and intermediate piperaquine resistance groups, suggesting additional loci may be involved in resistance. The results highlight an important role for partner drug resistance in shaping the P. falciparum genetic landscape in Southeast Asia and suggest that further work is needed to evaluate for other mutations that drive piperaquine resistance.

Increased risk of low birth weight in women with placental malaria associated with P. falciparum VAR2CSA clade.

Pregnancy associated malaria (PAM) causes adverse pregnancy and birth outcomes owing to Plasmodium falciparum accumulation in the placenta. Placental accumulation is mediated by P. falciparum protein VAR2CSA, a leading PAM-specific vaccine target. The extent of its antigen diversity and impact on clinical outcomes remain poorly understood. Through amplicon deep-sequencing placental malaria samples from women in Malawi and Benin, we assessed sequence diversity of VAR2CSA's ID1-DBL2x region, containing putative vaccine targets and estimated associations of specific clades with adverse birth outcomes. Overall, var2csa diversity was high and haplotypes subdivided into five clades, the largest two defined by homology to parasites strains, 3D7 or FCR3. Across both cohorts, compared to women infected with only FCR3-like variants, women infected with only 3D7-like variants delivered infants with lower birthweight (difference: -267.99 g; 95% Confidence Interval [CI]: -466.43 g,-69.55 g) and higher odds of low birthweight (<2500 g) (Odds Ratio [OR] 5.41; 95% CI:0.99,29.52) and small-for-gestational-age (OR: 3.65; 95% CI: 1.01,13.38). In two distinct malaria-endemic African settings, parasites harboring 3D7-like variants of VAR2CSA were associated with worse birth outcomes, supporting differential effects of infection with specific parasite strains. The immense diversity coupled with differential clinical effects of this diversity suggest that an effective VAR2CSA-based vaccine may require multivalent activity.

Genomic Analysis of Multidrug-Resistant Escherichia coli from North Carolina Community Hospitals: Ongoing Circulation of CTX-M-Producing ST131-H30Rx and ST131-H30R1 Strains.

Escherichia coli sequence type 131 (ST131) predominates globally among multidrug-resistant (MDR) E. coli strains. We used whole-genome sequencing (WGS) to investigate 63 MDR E. coli isolates from 7 North Carolina community hospitals (2010 to 2015). Of these, 39 (62%) represented ST131, including 37 (95%) from the ST131-H30R subclone: 10 (27%) from its H30R1 subset and 27 (69%) from its H30Rx subset. ST131 core genomes differed by a median of 15 (range, 0 to 490) single-nucleotide variants (SNVs) overall versus only 7 within H30R1 (range, 3 to 12 SNVs) and 11 within H30Rx (range, 0 to 21). The four isolates with identical core genomes were all H30Rx. Epidemiological and clinical characteristics did not vary significantly by strain type, but many patients with MDR E. coli or H30Rx infection were critically ill and had poor outcomes. H30Rx isolates characteristically exhibited fluoroquinolone resistance and CTX-M-15 production, had a high prevalence of trimethoprim-sulfamethoxazole resistance (89%), sul1 (89%), and dfrA17 (85%), and were enriched for specific virulence traits, and all qualified as extraintestinal pathogenic E. coli The high overall prevalence of CTX-M-15 appeared to be possibly attributable to its association with the ST131-H30Rx subclone and IncF[F2:A1:B-] plasmids. Some phylogenetically clustered non-ST131 MDR E. coli isolates also had distinctive serotypes/fimH types, fluoroquinolone mutations, CTX-M variants, and IncF types. Thus, WGS analysis of our community hospital source MDR E. coli isolates suggested ongoing circulation and differentiation of E. coli ST131 subclones, with clonal segregation of CTX-M variants, other resistance genes, Inc-type plasmids, and virulence genes.

skelesim: an extensible, general framework for population genetic simulation in R.

Simulations are a key tool in molecular ecology for inference and forecasting, as well as for evaluating new methods. Due to growing computational power and a diversity of software with different capabilities, simulations are becoming increasingly powerful and useful. However, the widespread use of simulations by geneticists and ecologists is hindered by difficulties in understanding these softwares' complex capabilities, composing code and input files, a daunting bioinformatics barrier and a steep conceptual learning curve. skelesim (an R package) guides users in choosing appropriate simulations, setting parameters, calculating genetic summary statistics and organizing data output, in a reproducible pipeline within the R environment. skelesim is designed to be an extensible framework that can 'wrap' around any simulation software (inside or outside the R environment) and be extended to calculate and graph any genetic summary statistics. Currently, skelesim implements coalescent and forward-time models available in the fastsimcoal2 and rmetasim simulation engines to produce null distributions for multiple population genetic statistics and marker types, under a variety of demographic conditions. skelesim is intended to make simulations easier while still allowing full model complexity to ensure that simulations play a fundamental role in molecular ecology investigations. skelesim can also serve as a teaching tool: demonstrating the outcomes of stochastic population genetic processes; teaching general concepts of simulations; and providing an introduction to the R environment with a user-friendly graphical user interface (using shiny).

A Prolonged Outbreak of KPC-3-Producing Enterobacter cloacae and Klebsiella pneumoniae Driven by Multiple Mechanisms of Resistance Transmission at a Large Academic Burn Center.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter cloacae has been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in health care settings, including carbapenem-resistant Enterobacteriaceae (CRE). We experienced a prolonged outbreak of CRE E. cloacae and K. pneumoniae over a 3-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates by using WGS. Twenty-two clinical isolates of CRE, including E. cloacae (n = 15) and K. pneumoniae (n = 7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacae and K. pneumoniae This outbreak was primarily maintained by a clonal expansion of E. cloacae sequence type 114 (ST114) with distribution of multiple resistance determinants. Plasmid and transposon analyses suggested that the majority of blaKPC-3 was transmitted via an identical Tn4401b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of the strain and/or plasmid in association with a transposon, highlighting the versatility of KPC-producing Enterobacteriaceae in their ability to utilize multiple modes to resistance gene propagation.

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia.

Cambodia, in which both Plasmodium vivax and Plasmodium falciparum are endemic, has been the focus of numerous malaria-control interventions, resulting in a marked decline in overall malaria incidence. Despite this decline, the number of P vivax cases has actually increased. To understand better the factors underlying this resilience, we compared the genetic responses of the two species to recent selective pressures. We sequenced and studied the genomes of 70 P vivax and 80 P falciparum isolates collected between 2009 and 2013. We found that although P falciparum has undergone population fracturing, the coendemic P vivax population has grown undisrupted, resulting in a larger effective population size, no discernable population structure, and frequent multiclonal infections. Signatures of selection suggest recent, species-specific evolutionary differences. Particularly, in contrast to P falciparum, P vivax transcription factors, chromatin modifiers, and histone deacetylases have undergone strong directional selection, including a particularly strong selective sweep at an AP2 transcription factor. Together, our findings point to different population-level adaptive mechanisms used by P vivax and P falciparum parasites. Although population substructuring in P falciparum has resulted in clonal outgrowths of resistant parasites, P vivax may use a nuanced transcriptional regulatory approach to population maintenance, enabling it to preserve a larger, more diverse population better suited to facing selective threats. We conclude that transcriptional control may underlie P vivax's resilience to malaria control measures. Novel strategies to target such processes are likely required to eradicate P vivax and achieve malaria elimination.

Longitudinal Pooled Deep Sequencing of the Plasmodium vivax K12 Kelch Gene in Cambodia Reveals a Lack of Selection by Artemisinin.

The emergence of artemisinin resistance among Plasmodium falciparum in the Greater Mekong subregion threatens malaria control interventions and is associated with multiple unique mutations in K13 (PF3D7_1343700). The aim of this study was to survey Cambodian Plasmodium vivax for mutations in the K13 ortholog (K12, PVX_083080) that might similarly confer artemisinin resistance. Extracted DNA from Cambodian isolates collected between 2009 and 2012 was pooled by province and year and submitted for next-generation sequencing. Single-nucleotide polymorphisms (SNPs) were identified using a pile-up approach that detected minority SNPs. Among the 14 pools, we found six unique SNPs, including three nonsynonymous SNPs, across six codons in K12 However, none of the SNPs were orthologous to artemisinin resistance-conferring mutations in PF3D7_1343700, and nonsynonymous changes did not persist through time within populations. These results suggest a lack of selection in the P. vivax population in Cambodia due to artemisinin drug pressure.