Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/µL of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection of 39 copies/mL. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infection rates in Massachusetts and nationwide.

Detection of EGFR mutations in non-small cell lung cancer by droplet digital PCR.

Activating mutations in EGFR predict benefit from tyrosine kinase inhibitor therapy for patients with advanced non-small cell lung cancer. Directing patients to appropriate therapy depends on accurate and timely EGFR assessment in the molecular pathology laboratory. This article describes the analytical design, performance characteristics, and clinical implementation of an assay for the rapid detection of EGFR L858R and exon 19 deletion mutations. A droplet digital polymerase chain reaction (ddPCR) assay was implemented with probe hydrolysis-dependent signal detection. A mutation-specific probe was used to detect EGFR L858R. A loss of signal design was used to detect EGFR exon 19 deletion mutations. Analytical sensitivity was dependent on DNA input and was as low as 0.01% variant allele fraction for the EGFR L858R assay and 0.1% variant allele fraction for the EGFR exon 19 deletion assay. Correlation of 20 clinical specimens tested by ddPCR and next generation sequencing showed 100% concordance. ddPCR showed 53% clinical sensitivity in the detection of EGFR mutations in plasma cell-free DNA from patients with lung cancer. The median clinical turnaround time was 5 days for ddPCR compared to 13 days for next generation sequencing. The findings show that ddPCR is an accurate and rapid method for detecting EGFR mutations in patients with non-small cell lung cancer.

Role of Risk Taking and Inhibitory Control in Alcohol Use Among Maltreated Adolescents and Nonmaltreated Adolescents.

Exposure to childhood maltreatment is a significant risk factor for the development and persistence of problematic alcohol use. The present study examined the role of risk taking and inhibitory control, key cognitive processes believed to govern behavioral regulation, as mechanisms that underlie the association between childhood maltreatment and the early stages of alcohol use. A sample of 129 maltreated adolescents and 102 socioeconomic status-matched, nonmaltreated adolescents and their parents completed three annual assessments, including computer-administered tasks and adolescent- and parent-report questionnaires, across ages 12-13 through 14-15 years. Childhood maltreatment was not directly associated with alcohol use in middle adolescence but was significantly associated with deficits in inhibitory control in early adolescence, which, in turn, led to significantly increased alcohol use in middle adolescence. Indeed, decreased inhibitory control was significant as a mediator, highlighting the salient role of this cognitive process in the early stages of alcohol use among maltreated adolescents.

Behavioral and electrophysiological indices of inhibitory control in maltreated adolescents and nonmaltreated adolescents.

Early adverse experiences are believed to have a profound effect on inhibitory control and the underlying neural regions. In the current study, behavioral and event-related potential (ERP) data were collected during a go/no-go task from adolescents who were involved with the child welfare system due to child maltreatment (n = 129) and low-income, nonmaltreated adolescents (n = 102). The nonmaltreated adolescents were more accurate than the maltreated adolescents on the go/no-go task, particularly on the no-go trials. Paralleling the results with typically developing populations, the nonmaltreated adolescents displayed a more pronounced amplitude of the N2 during the no-go trials than during the go trials. However, the maltreated adolescents demonstrated a more pronounced amplitude of the N2 during the go trials than during the no-go trials. Furthermore, while the groups did not differ during the go trials, the nonmaltreated adolescents displayed a more negative amplitude of the N2 than the maltreated adolescents during no-go trials. In contrast, there was not a significant group difference in amplitude of the P3. Taken together, these results provide evidence that the early adverse experiences encountered by maltreated populations impact inhibitory control and the underlying neural activity in early adolescence.

Effects of a school readiness intervention on electrophysiological indices of external response monitoring in children in foster care.

This study examined the impact of a school readiness intervention on external response monitoring in children in foster care. Behavioral and event-related potential (ERP) data were collected during a flanker task from children who received the Kids In Transition to School (KITS) Program (n = 26) and children who received services as usual (n = 19) before and after the intervention. While there were no significant group differences on the behavioral data, the ERP data for the two groups of children significantly differed. Specifically, in contrast to the children who received services as usual, the children who received the KITS Program displayed greater amplitude differences between positive and negative performance feedback over time for the N1, which reflects early attention processes, and feedback-related negativity, which reflects evaluation processes. In addition, although the two groups did not differ on amplitude differences between positive and negative performance feedback for these ERP components before the intervention, the children who received the KITS Program displayed greater amplitude differences than the children who received services as usual after the intervention. These results suggest that the KITS Program had an effect on responsivity to external performance feedback, which may be beneficial during the transition into kindergarten.

General Cognitive Ability as an Early Indicator of Problem Behavior Among Toddlers in Foster Care.

OBJECTIVE: Children who have experienced maltreatment and subsequent placement in foster care are at increased risk of problem behavior. Increased knowledge of the development of problem behavior in this population, particularly during toddlerhood, can greatly inform preventive intervention efforts. This study examined variability in problem behavior among toddlers entering new foster care placements and identified related child and parenting characteristics. METHODS: Ninety-one toddlers in foster care (mean = 2.26 years) and their caregivers completed an initial assessment and were reassessed 6 months later. A child's general cognitive ability was assessed via performance on a standardized developmental measure, and child problem behavior, parenting stress, frequency of family routines, and harsh discipline were assessed via caregiver report. RESULTS: Upon entering a new foster care placement and 6 months after placement, respectively, 38% and 25% of the toddlers were within the borderline clinical or clinical range in terms of problem behavior when assessed using the Child Behavior Checklist. There was not a significant difference in problem behavior over this 6-month period, suggesting that problem behavior was quite stable among the toddlers as a group. However, general cognitive ability was a significant predictor of individual differences in change in problem behavior, with toddlers with lower general cognitive ability displaying increased problem behavior over this period. CONCLUSION: An increased number of toddlers in foster care displayed clinically significant levels of problem behavior, further demonstrating that these children are an extremely high-risk group. The association between general cognitive ability and change in problem behavior highlights the importance of early developmental screenings, which may help identify children at greatest risk of problem behavior and most in need of preventive intervention efforts.

Exploring the Psychometric Properties of the Parent Daily Report - Toddler Version (PDR-T).

The present study examined the psychometric properties of a brief parent-report daily checklist of toddler behavior (Parent Daily Report - Toddler Version; PDR-T). Data were collected from three groups of 18-36 month-olds who were followed longitudinally for approximately 1 year: 1) internationally adopted children (n = 156), 2) children placed in foster care due to child maltreatment (n = 79), and 3) community comparison children raised by their biological families (n = 80). An exploratory factor analysis of this measure resulted in three factors, measuring aggressive/noncompliant, positive, and distress behaviors. While there were estimation issues with the positive and distress factors, the aggressive/noncompliant factor exhibited invariance across time and groups, and partial invariance between genders. Significant correlations were observed between this factor and measures of externalizing behavior and inhibitory control (r = .26-.56), but not shyness, fearfulness, or negative affect. This provides support for both convergent and discriminant validity. Reliability of this factor was adequate to good across time and group. Results provide preliminary support for the utility, reliability, and consistency of one factor of the PDR-T as an easy parent-report tool to assess daily patterns and changes in child aggressive/noncompliant behavior over time.

Effects of a school readiness intervention on hypothalamus-pituitary-adrenal axis functioning and school adjustment for children in foster care.

Maltreated children in foster care are at high risk for dysregulated hypothalamus-pituitary-adrenal (HPA) axis functioning and educational difficulties. The present study examined the effects of a short-term school readiness intervention on HPA axis functioning in response to the start of kindergarten, a critical transition marking entry to formal schooling, and whether altered HPA axis functioning influenced children's school adjustment. Compared to a foster care comparison group, children in the intervention group showed a steeper diurnal cortisol slope on the first day of school, a pattern previously observed among nonmaltreated children. A steeper first day of school diurnal cortisol slope predicted teacher ratings of better school adjustment (i.e., academic performance, appropriate classroom behaviors, and engagement in learning) in the fall of kindergarten. Furthermore, the children's HPA axis response to the start of school mediated the effect of the intervention on school adjustment. These findings support the potential for ameliorative effects of interventions targeting critical transitional periods, such as the transition of formal schooling. This school readiness intervention appears to influence stress neurobiology, which in turn facilitates positive engagement with the school environment and better school adjustment in children who have experienced significant early adversity.

Improving kindergarten readiness in children with developmental disabilities: Changes in neural correlates of response monitoring.

Among children diagnosed with developmental delays, difficulties in self-regulation are prominent and have been linked to school readiness problems. The current study sought to examine the impact of the Kids in Transition to School (KITS) school readiness intervention program on self-regulation, with a specific focus on response monitoring skills, among children with developmental delays. Children (n = 20 in the KITS group and n = 21 in a services as usual group) were administered a flanker task during which event-related potential data were collected to examine group differences in response monitoring. Findings indicated that children in the KITS group showed significant enhancement of a neural index of response monitoring post-intervention. Specifically, the KITS group showed a significant change in the magnitude of their feedback-related negativity in response to negative performance feedback from baseline to post-intervention, whereas children in the services as usual group did not. There were no significant differences between the groups for the error-related negativity or the error-related positivity on incorrect trials nor were there group differences in behavioral performance on the task at the post-intervention assessment. Overall, these findings provide support for the plasticity of response monitoring skills in young children and support the growing literature demonstrating improved self-regulation outcomes via intervention that enhances children's response monitoring.

Income, neural executive processes, and preschool children's executive control.

This study aimed to specify the neural mechanisms underlying the link between low household income and diminished executive control in the preschool period. Specifically, we examined whether individual differences in the neural processes associated with executive attention and inhibitory control accounted for income differences observed in performance on a neuropsychological battery of executive control tasks. The study utilized a sample of preschool-aged children (N = 118) whose families represented the full range of income, with 32% of families at/near poverty, 32% lower income, and 36% middle to upper income. Children completed a neuropsychological battery of executive control tasks and then completed two computerized executive control tasks while EEG data were collected. We predicted that differences in the event-related potential (ERP) correlates of executive attention and inhibitory control would account for income differences observed on the executive control battery. Income and ERP measures were related to performance on the executive control battery. However, income was unrelated to ERP measures. The findings suggest that income differences observed in executive control during the preschool period might relate to processes other than executive attention and inhibitory control.

Preliminary evidence of the impact of early childhood maltreatment and a preventive intervention on neural patterns of response inhibition in early adolescence.

Maltreated youths in foster care often experience negative developmental and psychological outcomes, which have been linked with poor response inhibition. Recent evidence suggests that childhood maltreatment is also associated with alterations in the neural circuitry underlying response inhibition. However, a burgeoning line of research has begun to explore the mitigating effects of preventive interventions on neural functioning. The current study used event-related functional magnetic resonance imaging to explore the impact of early childhood maltreatment and a preventive intervention on response inhibition in early adolescence. Thirty-six demographically similar adolescents (ages 9-14 years) completed a Go/NoGo task. The sample included nonmaltreated adolescents (n = 14) and maltreated adolescents who were in foster care as preschoolers and randomly assigned to receive services as usual (n = 11) or a preventive intervention, Multidimensional Treatment Foster Care for Preschoolers (n = 11). The groups demonstrated similar behavioral performance but significantly different neural patterns. The maltreated adolescents who received services as usual demonstrated subcortical hypoactivity during successful response inhibition and subcortical hyperactivity during unsuccessful response inhibition. In contrast, the nonmaltreated adolescents and maltreated adolescents who received the intervention exhibited strikingly similar neural patterns during successful response inhibition, but the maltreated adolescents who received the intervention demonstrated prefrontal hypoactivity during unsuccessful response inhibition. These findings offer preliminary evidence that early childhood maltreatment alters the neural patterns underlying response inhibition in early adolescence and that participating in a preventive intervention could mitigate maltreatment-related effects on these neural systems.

Detection of Mismatch Repair Deficiency and Microsatellite Instability in Colorectal Adenocarcinoma by Targeted Next-Generation Sequencing.

Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome-associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.

The Clinical Implications of Inconsistently Methylated Results from Glioblastoma MGMT Testing by Replicate Methylation-Specific PCR.

The methylation status of the promoter of the O6-methylguanine DNA methyltransferase gene (MGMT) is an established prognostic and predictive biomarker of glioblastoma (GBM). At the Center for Advanced Molecular Diagnostics, MGMT testing is performed by methylation-specific PCR with multiple replicates, leading to three types of reportable results: methylated, unmethylated, and inconsistently methylated. An inconsistently methylated result is reported when a methylated peak is seen in some but not all of the PCR replicates from a single DNA sample. To better understand the clinical implications of these results, we performed a retrospective review of all MGMT testing at our laboratory over a 5-year period, and correlated test results with outcome and specimen-quality data. This review yielded several novel findings. First, inconsistent MGMT methylation on replicate methylation-specific PCR is not uncommon, composes 12% (58/465) of our GBM results. Second, inconsistently methylated GBM cases are associated with relatively poor overall survival (more similar to unmethylated than to methylated cases). Third and interestingly, there appears to be a dose-response relationship between patient survival and the extent of methylation in inconsistently methylated GBMs. Finally, our analyses of specimen-quality data suggest that a combination of technical factors (eg, small samples) and tumor biology may explain inconsistent MGMT results on replicate methylation-specific PCR testing.

Impulsivity and the association between the feedback-related negativity and performance on an inhibitory control task in young at-risk children.

Identifying neurocognitive processes associated with effective inhibitory control is particularly relevant for individuals at high risk for disruptive behaviors, such as maltreated children. Performance feedback processing during a flanker task was investigated in maltreated preschool-aged children (N = 67) via an event-related potential component, the feedback-related negativity (FRN). The functionality of the FRN in children with high impulsivity was of interest, as impulsivity was associated with an exaggerated FRN in previous research. Results showed that high impulsivity was associated with an exaggerated FRN and greater post-error slowing. For children with high impulsivity, there was a correlation between the FRN and accuracy, which was not found in children with low impulsivity. This suggests that an exaggerated FRN is particularly important for children with high impulsivity to maintain effective inhibitory control.

HPA stability for children in foster care: mental health implications and moderation by early intervention.

Research on stress-sensitive biological systems has typically focused on activation at one time, yet recent theories emphasize dynamic, context-specific adaptation. This study tested hypothesized calibration of one such system by examining both mean levels and longitudinal stability of daily cortisol--reflecting hypothalamic-pituitary-adrenal axis activation--in children exposed to high-risk versus lower-risk caregiving contexts. Context-specific effects of longitudinal cortisol profiles were addressed via relations with child psychiatric symptoms. Children from regular foster care, foster children participating in a family-based intervention, and community comparison children (n = 96 total) collected saliva samples for cortisol assay at 29 timepoints across 6+ years. High-risk (regular foster care) children showed lower and more variable cortisol levels than their lower-risk (treatment foster care, community comparison) counterparts. For the high-risk children only, higher and more stable cortisol related to elevated anxiety symptoms. Implications for contextual calibration of stress systems and family intervention mechanisms are discussed.

Child diurnal cortisol rhythms, parenting quality, and externalizing behaviors in preadolescence.

This study examined a neurobiologically informed model of the emergence of child externalizing behaviors in an ethnically diverse community sample of 232 9-12 year old children. Replicating extensive prior research, our analyses revealed that parents' inconsistent discipline and poor quality monitoring were predictive of child externalizing behavior. In addition, poor parental monitoring, but not inconsistent discipline, was associated with children having a significantly flatter morning-to-evening cortisol slope, which was in turn, related to higher levels of externalizing behaviors. An indirect effect of parental monitoring on externalizing behaviors, through child diurnal cortisol rhythms, was also supported. These findings highlight the role of the hypothalamic-pituitary-adrenal (HPA) axis and its hormonal end product, cortisol, in the relationship between the caregiving environment and the development of externalizing behaviors.

Pulmonary large cell carcinoma lacking squamous differentiation is clinicopathologically indistinguishable from solid-subtype adenocarcinoma.

CONTEXT: Pulmonary large cell carcinoma (LCC) includes tumors not readily diagnosed as adenocarcinoma (ADC) or squamous cell carcinoma on morphologic grounds, without regard to immunophenotype, according to the World Health Organization (WHO). This ambiguous designation may cause confusion over selection of mutation testing and directed therapies. Several groups have proposed the use of immunohistochemistry (IHC) to recategorize LCC as ADC or squamous cell carcinoma; however, it remains unclear if strictly defined LCCs are a clinicopathologically distinct lung tumor subset. OBJECTIVE: To compare the pathologic, molecular, and clinical features of 2 morphologically similar tumors: solid-subtype ADC and LCC. DESIGN: Tumors were included on the basis of solid growth pattern; tumors with squamous or neuroendocrine differentiation were excluded. Solid ADC (n = 42) and LCC (n = 57) were diagnosed by using WHO criteria (5 intracellular mucin droplets in ≥2 high-power fields for solid ADC) and tested for KRAS, EGFR, and ALK alterations. RESULTS: Both solid ADC and LCC groups were dominated by tumors with "undifferentiated"-type morphology and both had a high frequency of thyroid transcription factor 1 expression. KRAS was mutated in 38% of solid ADCs versus 43% of LCCs (P = .62). One ALK-rearranged and 1 EGFR-mutated tumor were detected in the solid ADC and LCC groups, respectively. There were no significant differences in clinical features or outcomes; the prevalence of smoking in both groups was greater than 95%. CONCLUSIONS: Other than a paucity of intracellular mucin, LCC lacking squamous or neuroendocrine differentiation is indistinguishable from solid-subtype ADC. We propose the reclassification of these tumors as mucin-poor solid adenocarcinomas.

Patterns of brain activation in foster children and nonmaltreated children during an inhibitory control task.

Children in foster care have often encountered a range of adverse experiences, including neglectful and/or abusive care and multiple caregiver transitions. Prior research findings suggest that such experiences negatively affect inhibitory control and the underlying neural circuitry. In the current study, event-related functional magnetic resonance imaging was employed during a go/no go task that assesses inhibitory control to compare the behavioral performance and brain activation of foster children and nonmaltreated children. The sample included two groups of 9- to 12-year-old children: 11 maltreated foster children and 11 nonmaltreated children living with their biological parents. There were no significant group differences on behavioral performance on the task. In contrast, patterns of brain activation differed by group. The nonmaltreated children demonstrated stronger activation than did the foster children across several regions, including the right anterior cingulate cortex, the middle frontal gyrus, and the right lingual gyrus, during correct no go trials, whereas the foster children displayed stronger activation than the nonmaltreated children in the left inferior parietal lobule and the right superior occipital cortex, including the lingual gyrus and cuneus, during incorrect no go trials. These results provide preliminary evidence that the early adversity experienced by foster children impacts the neural substrates of inhibitory control.

Immediate Effects of a School Readiness Intervention for Children in Foster Care.

RESEARCH FINDINGS: School readiness is a strong predictor of adjustment in elementary school and beyond. Children in foster care are at particular risk for academic and social difficulties in school. Limitations in self-regulatory skills and caregiver involvement among these children might contribute to a lack of school readiness. This study presents the immediate effects on school readiness of a targeted, short-term intervention designed to improve children's early literacy, prosocial, and self-regulatory skills during the summer before kindergarten entry: Kids in Transition to School (KITS). Using a randomized controlled trial design, 192 children in foster care were assigned to either an intervention or services as usual comparison condition. Multimethod, multiagent assessments were conducted immediately prior to and following the completion of the intervention. The results from structural equation modeling indicated that the intervention had significant, positive effects on early literacy and self-regulatory skills. PRACTICE: An efficacious, short-term, readily scalable, theoretically-based intervention targeted at specific vulnerabilities for children in foster care may help to improve their school readiness and eventual school adjustment.

Early adverse care, stress neurobiology, and prevention science: lessons learned.

There is growing evidence that some of the difficulties observed among children who have experienced early adverse care (e.g., children internationally adopted from institutional care and maltreated children in foster care) involve experience-induced alterations in stress-responsive neurobiological systems, including the hypothalamic-pituitary-adrenocortical (HPA) system. Thus, incorporating stress neurobiology into prevention research could aid in identifying the children most in need of preventive intervention services, elucidating the mechanisms of change in effective interventions, and providing insight into the differential responses of children to effective interventions. However, integrating stress neurobiology and prevention research is challenging. In this paper, the results of studies examining HPA system activity in children who have experienced early adverse care are reviewed, the implications of these results for prevention research are discussed, and critical steps for successfully incorporating stress neurobiology into prevention research are identified.