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Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Torquetenovirus (TTV) is present in biological fluids from healthy individuals and measurement of its titer is used to assess immune status in individuals with chronic infections and after transplants. We assessed if the titer of TTV in saliva varied with the presence of SARS-CoV-2 in the nasopharynx and could be a marker of COVID-19 status. Saliva from 91 individuals positive for SARS-CoV-2 in nasal-oropharyngeal samples, and from 126 individuals who were SARS-CoV-2-negative, all with mild respiratory symptoms, were analyzed. Both groups were similar in age, gender, symptom duration and time after symptom initiation when saliva was collected. Titers of TTV and SARS-CoV-2 were assessed by gene amplification. Loss of smell (p = 0.0001) and fever (p = 0.0186) were more prevalent in SARS-CoV-2-positive individuals, while sore throat (p = 0.0001), fatigue (p = 0.0037) and diarrhea (p = 0.0475) were more frequent in the SARS-CoV-2 negative group. The saliva TTV and nasal-oropharyngeal SARS-CoV-2 titers were correlated (p = 0.0085). The TTV level decreased as symptoms resolved in the SARS-CoV-2 infected group (p = 0.0285) but remained unchanged in the SARS-CoV-2 negative controls. In SARS-CoV-2 positive subjects who provided 2-4 saliva samples and in which TTV was initially present, the TTV titer always decreased over time as symptoms resolved. We propose that sequential TTV measurement in saliva is potentially useful to assess the likelihood of symptom resolution in SARS-CoV-2-positive individuals and to predict prognosis.

Knowledge of the role of Neotropical montane landscapes in shaping genetic connectivity and local adaptation is essential for understanding the evolutionary processes that have shaped the extraordinary species diversity in these regions. In the present study, we examined the landscape genetics, estimated genetic diversity, and explored genetic relationships with morphological variability and reproductive strategies in seven natural populations of Cattleya liliputana (Orchidaceae). Nuclear microsatellite markers were used for genetic analyses. Spatial Bayesian clustering and population-based analyses revealed significant genetic structuring and high genetic diversity (He = 0.733 ± 0.03). Strong differentiation was found between populations over short spatial scales (FST = 0.138, p < 0.001), reflecting the landscape discontinuity and isolation. Monmonier´s maximum difference algorithm, Bayesian analysis on STRUCTURE and principal component analysis identified one major genetic discontinuity between populations. Divergent genetic groups showed phenotypic divergence in flower traits and reproductive strategies. Increased sexual reproductive effort was associated with rock outcrop type and may be a response to adverse conditions for growth and vegetative reproduction. Here we discuss the effect of restricted gene flow, local adaptation and phenotypic plasticity as drivers of population differentiation in Neotropical montane rock outcrops.

The bacterial community and genes involved in geobiocycling of arsenic (As) from sediment impacted by long-term gold mining were characterized through culture-based analysis of As-transforming bacteria and metagenomic studies of the arsC, arrA, and aioA genes. Sediment was collected from the historically gold mining impacted Mina stream, located in one of the world's largest mining regions known as the "Iron Quadrangle". A total of 123 As-resistant bacteria were recovered from the enrichment cultures, which were phenotypically and genotypically characterized for As-transformation. A diverse As-resistant bacteria community was found through phylogenetic analyses of the 16S rRNA gene. Bacterial isolates were affiliated with Proteobacteria, Firmicutes, and Actinobacteria and were represented by 20 genera. Most were AsV-reducing (72%), whereas AsIII-oxidizing accounted for 20%. Bacteria harboring the arsC gene predominated (85%), followed by aioA (20%) and arrA (7%). Additionally, we identified two novel As-transforming genera, Thermomonas and Pannonibacter. Metagenomic analysis of arsC, aioA, and arrA sequences confirmed the presence of these genes, with arrA sequences being more closely related to uncultured organisms. Evolutionary analyses revealed high genetic similarity between some arsC and aioA sequences obtained from isolates and clone libraries, suggesting that those isolates may represent environmentally important bacteria acting in As speciation. In addition, our findings show that the diversity of arrA genes is wider than earlier described, once none arrA-OTUs were affiliated with known reference strains. Therefore, the molecular diversity of arrA genes is far from being fully explored deserving further attention.

Southeast Brazil is a neotropical region composed of a mosaic of different tropical habitats and mountain chains, which allowed for the formation of bird-rich communities with distinct ecological niches. Although this region has the potential to harbor a remarkable variety of avian parasites, there is a lack of information about the diversity of malarial parasites. We used molecular approaches to characterize the lineage diversity of Plasmodium and Haemoproteus in bird communities from three different habitats in southeast Brazil based on the prevalence, richness and composition of lineages. We observed an overall prevalence of 35.3%, with a local prevalence ranging from 17.2% to 54.8%. Moreover, no significant association between prevalence and habitat type could be verified (p>0.05). We identified 89 Plasmodium and 22 Haemoproteus lineages, with 86% of them described for the first time here, including an unusual infection of a non-columbiform host by a Haemoproteus (Haemoproteus) parasite. The composition analyses of the parasite communities showed that the lineage composition from Brazilian savannah and tropical dry forest was similar, but it was different from the lineage composition of Atlantic rainforest, reflecting the greater likeness of the former habitats with respect to seasonality and forest density. No significant effects of habitat type on lineage richness were observed based on GLM analyses. We also found that sites whose samples had a greater diversity of bird species showed a greater diversity of parasite lineages, providing evidence that areas with high bird richness also have high parasite richness. Our findings point to the importance of the neotropical region (southeast Brazil) as a major reservoir of new haemosporidian lineages.

Background Redondovirus (ReDoV) is a DNA virus present in the respiratory tract of many healthy individuals. Since SARS-CoV-2, the virus responsible for COVID-19, also primarily infects the same site, we evaluated whether ReDoV was present at increased frequency in patients with COVID-19 and influenced infection parameters. Methods Saliva samples were collected weekly from 59 individuals with COVID-19 and from 132 controls. ReDoV was detected by polymerase chain reaction and the genotypes were identified by metagenomics. Torque Teno Virus (TTV) in these samples were previously reported. Results ReDoV was detected in saliva more frequently from COVID-19 patients (72.9%) than from controls (50.0%) (p = 0.0015). There were no associations between ReDoV detection and either continuous or intermittent SARS-CoV-2 shedding, the duration of SARS-CoV-2 detection in saliva, patients’ sex or if infection was by the B1 or Gamma strain. The two ReDoV strains, Brisavirus and Vientovirus, were present in equivalent frequencies in ReDoV-positive COVID-19 patients and controls. Phylogenetic analysis suggested that the two ReDoV strains in Brazil were similar to strains previously detected on other continents. Conclusion ReDoV expression in saliva is increased in males and females in Brazil with mild COVID-19 but its presence does not appear to influence properties of the SARS-CoV-2 infection.

The accurate diagnosis and seroprevalence investigations of Zika virus (ZKV) infections remain complex due to cross reactivity with other flaviviruses. Two assay formats, both using labelled Zika virus NS1 antigen as a revealing agent (a double antigen binding assay, DABA, and an immunoglobulin Ig capture assay, G capture) were initially developed and compared with the indirect EuroimmunZ assay for the detection of anti-Zika antibody. Of 147 pre-Zika period serum samples, 39 (27%) were reactive in the EuroimmunZ or the DABA assays, 28 sera concordantly so. Such false reactivity was influenced by the serotype of Dengue virus (DV) to which individuals had been exposed to. Thus, of sera from patients undergoing secondary Dengue virus infection of known serotype, 91%, 45% and 28% of Dengue virus serotype 2, 3 and 4 respectively were reactive in one or more of the three assays. A novel method of quenching false sero-reactivity was therefore developed for the DABA and G capture assays. Initial addition of a single homologous Dengue virus serotype 3 NS1Ag quench significantly ablated false reactivities in the pre-Zika period sera. An equipotent quadrivalent quench comprising homologous Dengue virus serotypes 1 to 4 NS1Ag was shown to be optimum yet retained sensitivity for the detection of specific anti-Zika antibody. Comparing DABA and G capture assays using quenched and unquenched conjugates in comparison with EuroimmunZ early in the course of PCR-confirmed infection indicated that a significant component of the apparent early anti-ZIKA antibody response is likely to be due to a Zika virus-driven anamnestic anti-Dengue virus response. The increased specificity provided by homologous antigen quenching is likely to provide a significant improvement in sero-diagnostics and to be of clinical value.

Cryptococcal infections are mainly caused by members of the Cryptococcus neoformans species complex (molecular types VNI, VNII, VNB, VNIV and the AD hybrid VNIII). PCR of the mating type loci and MLST typing using the ISHAM-MLST consensus scheme were used to evaluate the genetic relationship of 102 (63 clinical and 39 environmental) C. neoformans isolates from Uberaba, Brazil and to correlate the obtained genotypes with clinical, antifungal susceptibility and virulence factor data. All isolates were mating type alpha. MLST identified 12 known and five new sequence types (ST). Fourteen STs were identified within the VNI isolates, with ST93 (57/102, 56%) and ST77 (19/102, 19%) being the most prevalent. From the nine VNII isolates previously identify by URA5-RFLP only four (ST40) were confirmed by MLST. The remaining five grouped within the VNB clade in the phylogenetic analysis corresponding to the sequence type ST504. Other two environmental isolates also grouped within VNB clade with the new sequence type ST527. The four VNII/ST40 isolates were isolated from CSF. The two VNIV sequence types (ST11 and ST160) were isolated from blood cultures. Two of six patients evaluated with more than one isolates had mixed infections. Amongst the VNI isolates 4 populations were identified, which showed differences in their susceptibility profiles, clinical outcome and virulence factors. These results reinforce that ST93 is the most prevalent ST in HIV-infected patients in the Southeastern region of Brazil. The finding of the VNB molecular type amongst environmental Brazilian isolates highlights that this genotype is not restricted to the African continent.

Objective: Socio-emotional skills, including social competence and social cognition, form the basis for robust relationships and wellbeing. Despite their importance, these skills are poorly defined and measured, particularly in children with developmental vulnerabilities. As a consequence, targets for effective management and treatment remain unclear. We aimed to i) phenotype social competence and social cognition in typically developing children (TDC) and in children with neurodevelopmental or mental health disorders (clinical groups) and ii) establish the relationships between these child-direct measures and parent ratings of social competence and behavior., Method: Using a multi-site, cross-sectional study design, we recruited 513 TDC and 136 children with neurodevelopmental (autism spectrum disorder [ASD], attention deficit hyperactivity disorder [ADHD]) or mental health (Anxiety Disorder [ANX]) diagnoses (age range 5-15 years). We administered the Paediatric Evaluation of Emotions, Relationships and Socialisation (PEERS) to children, and parents completed standardised questionnaires rating children's socio-emotional function., Results: Standardised parent questionnaires revealed a global pattern of everyday socio-emotional impairment that was common to all clinical groups, while PEERS identified disorder-specific socio-cognitive profiles for children with ASD, ADHD and ANX. Compared to TDCs, children with ASD demonstrated global socio-cognitive impairment. Children with ADHD were impulsive, demonstrating difficulties managing speed accuracy trade-offs. Children with ANX exhibited slowed social decision-making, but otherwise intact skills., Conclusions: Standardized parent questionnaires of child socio-emotional function reveal differences between children with typical and atypical development, but do not yield disorder-specific, socio-emotional profiles. In contrast, findings from the PEERS objective assessment suggest that that ASD, ADHD and ANX are associated with distinct socio-cognitive phenotypes, to more accurately guide and target management and treatment of impaired social competence., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Anderson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In interpreting attention-deficit/hyperactivity disorder (ADHD) symptoms, categorical and dimensional approaches are commonly used. Both employ binary symptom counts which give equal weighting, with little attention to the combinations and relative contributions of individual symptoms. Alternatively, symptoms can be viewed as an interacting network, revealing the complex relationship between symptoms. Using a novel network modelling approach, this study explores the relationships between the 18 symptoms in the Diagnostic Statistical Manual (DSM-5) criteria and whether network measures are useful in predicting outcomes. Participants were from a community cohort, the Children's Attention Project. DSM ADHD symptoms were recorded in a face-to-face structured parent interview for 146 medication naïve children with ADHD and 209 controls (aged 6-8 years). Analyses indicated that not all symptoms are equal. Frequencies of endorsement and configurations of symptoms varied, with certain symptoms playing a more important role within the ADHD symptom network. In total, 116,220 combinations of symptoms within a diagnosis of ADHD were identified, with 92% demonstrating a unique symptom configuration. Symptom association networks highlighted the relative importance of hyperactive/impulsive symptoms in the symptom network. In particular, the 'motoric'-type symptoms as well as interrupts as a marker of impulsivity in the hyperactive domain, as well as loses things and does not follow instructions in the inattentive domain, had high measures of centrality. Centrality-measure weighted symptom counts showed significant association with clinical but not cognitive outcomes, however the relationships were not significantly stronger than symptom count alone. The finding may help to explain heterogeneity in the ADHD phenotype., Competing Interests: PH’s institution has received payment from Shire for advisory boards; Eli Lilly, Janssen, and Shire for speaker’s bureau. This does not alter our adherence to PLOS ONE policies on sharing data and materials. TS, CM, RB, DE, VA, BJ, JN and ES have no conflicts to declare.

Fathers are underrepresented in interventions focussing on child well-being, yet research suggests their involvement may be critical to enhancing intervention effectiveness. This study aimed to provide the first Australian benchmark of rates of father attendance across several child mental health services. Retrospective casefile reviews were conducted to obtain data on father and mother attendance at 10 Australian child mental health services. A total of 2128 casefile records were retrospectively examined to extract family-level data. The main outcome measures were rates of father and mother attendance at sessions involving parents, and rates of father- and mother-instigated referral to services. Across services, fathers attended on average 48.2% (range 39.7% to 72.0%) of total parent sessions, with an average of 68.4% (range 53.1% to 88.1%) of fathers attending at least one session. Mothers attended sessions at significantly higher rates; an average of 92.8% of total parent sessions and 96.9% attendance for at least one session. For self-referred families, on average 12.6% of referrals were from fathers, and 87.4% were from mothers. These results indicate that rates of father attendance at Australian child mental health services vary, but are significantly lower than attendance rates for mothers. This may compromise the quality and outcomes of child mental health services in Australia. Routine monitoring of rates of father attendance is needed, as are strategies to enhance father engagement., Competing Interests: The authors have declared that no competing interests exist.

Verbal working memory (WM) comprises different processes (encoding, maintenance, retrieval) that are often compromised in brain diseases, but their neural correlates have not yet been examined in childhood and adolescence. To probe WM processes and associated neural correlates in developmental samples, and obtain comparable effects across different ages and populations, we designed an adapted Brown-Peterson task (verbal encoding and retrieval combined with verbal and visual concurrent tasks during maintenance) to implement during functional magnetic resonance imaging (fMRI). In a sample of typically developing children and adolescents (n = 16), aged 8 to 16 years, our paradigm successfully identified distinct patterns of activation for encoding, maintenance, and retrieval. While encoding activated perceptual systems in posterior and ventral visual regions, retrieval activated fronto-parietal regions associated with executive control and attention. We found a different impact of verbal versus visual concurrent processing during WM maintenance: at retrieval, the former condition evoked greater activations in visual cortex, as opposed to selective involvement of language-related areas in left temporal cortex in the latter condition. These results are in accord with WM models, suggesting greater competition for processing resources when retrieval follows within-domain compared with cross-domain interference. This pattern was found regardless of age. Our study provides a novel paradigm to investigate distinct WM brain systems with reliable results across a wide age range in developmental populations, and suitable for participants with different WM capacities.

This study aimed to determine the long term effects of resolution of SDB in preschool children, either following treatment or spontaneous recovery, on cognition and behavior. Children diagnosed with SDB at 3-5y (N = 35) and non-snoring controls (N = 25), underwent repeat polysomnography (PSG) and cognitive and behavioral assessment 3 years following a baseline study. At follow-up, children with SDB were grouped into Resolved and Unresolved. Resolution was defined as: obstructive apnea hypopnea index (OAHI) ≤1 event/h; no snoring detected on PSG; and no parental report of habitual snoring. 57% (20/35) of children with SDB received treatment, with SDB resolving in 60% (12/20). 43% (15/35) were untreated, of whom 40% (6/15) had spontaneous resolution of SDB. Cognitive reduced between baseline and follow-up, however this was not related to persistent disease, with no difference in cognitive outcomes between Resolved, Unresolved or Control groups. Behavioral functioning remained significantly worse in children originally diagnosed with SDB compared to control children, regardless of resolution. Change in OAHI did not predict cognitive or behavioral outcomes, however a reduction in nocturnal arousals, irrespective of full resolution, was associated with improvement in attention and aggressive behavior. These results suggest that resolution of SDB in preschool children has little effect on cognitive or behavioral outcomes over the long term. The association between sleep fragmentation and behavior appears independent of SDB, however may be moderated by concomitant SDB. This challenges the assumption that treatment of SDB will ameliorate associated cognitive and behavioural deficits and supports the possibility of a SDB phenotype.

Background: While schools can provide a comprehensive sampling frame for community-based studies of children and their families, recruitment is challenging. Multi-level approaches which engage multiple school stakeholders have been recommended but few studies have documented their effects. This paper compares the impact of a standard versus enhanced engagement approach on multiple indicators of recruitment: parent response rates, response times, reminders required and sample characteristics., Methods: Parents and teachers were distributed a brief screening questionnaire as a first step for recruitment to a longitudinal study, with two cohorts recruited in consecutive years (cohort 1 2011, cohort 2 2012). For cohort 2, additional engagement strategies included the use of pre-notification postcards, improved study materials, and recruitment progress graphs provided to school staff. Chi-square and t-tests were used to examine cohort differences., Results: Compared to cohort 1, a higher proportion of cohort 2 parents responded to the survey (76% versus 69%; p < 0.001), consented to participate (71% versus 56%; p < 0.001), agreed to teacher participation (90% versus 82%; p < 0.001) and agreed to follow-up contact (91% versus 80%; p < 0.001). Fewer cohort 2 parents required reminders (52% versus 63%; p < 0.001), and cohort 2 parents responded more promptly than cohort 1 parents (mean difference: 19.4 days, 95% CI: 18.0 to 20.9, p < 0.001)., Conclusion: These results illustrate the value of investing in a relatively simple multi-level strategy to maximise parent response rates, and potentially reduce recruitment time and costs.

Folate and other methyl-donor pathway components are widely supplemented due to their ability to prevent prenatal neural tube defects. Several lines of evidence suggest that these supplements act through epigenetic mechanisms (e.g. altering DNA methylation). Primary among these are the experiments on the mouse viable yellow allele of the agouti locus (A(vy)). In the Avy allele, an Intracisternal A-particle retroelement has inserted into the genome adjacent to the agouti gene and is preferentially methylated. To further test these effects, we tested the same diet used in the Avy studies on wild-derived Peromyscus maniculatus, a native North American rodent. We collected tissues from neonatal offspring whose parents were fed the high-methyl donor diet as well as controls. In addition, we assayed coat-color of a natural variant (wide-band agouti = A(Nb)) that overexpresses agouti as a phenotypic biomarker. Our data indicate that these dietary components affected agouti protein production, despite the lack of a retroelement at this locus. Surprisingly, the methyl-donor diet was associated with defects (e.g. ovarian cysts, cataracts) and increased mortality. We also assessed the effects of the diet on behavior: We scored animals in open field and social interaction tests. We observed significant increases in female repetitive behaviors. Thus these data add to a growing number of studies that suggest that these ubiquitously added nutrients may be a human health concern.

Unlabelled: Traumatic brain injury is (TBI) a leading cause of morbidity and mortality in youth. Adult survivors of a severe pediatric TBI are vulnerable to global impairments, including greater employment difficulties, poor quality of life (HRQoL) and increased risk of mental health problems. When estimating the health related quality of life in adolescents, the presence of anxiety and depression and the quality of social relationships are important considerations, because adolescents are entrenched in social development during this phase of maturation. The influence of anxiety, depression and loneliness on health related quality of life in adolescent survivors of TBI has not been documented. This pilot study aimed to identify and measure the relationship between anxiety, depression and loneliness and perceived health related quality of life in adolescent survivors of a TBI., Method: mixed method/cohort pilot study (11 adolescents, mild to severe TBI; 9 parents), using self-report and proxy-report measures of anxiety, depression, health related quality of life, loneliness and clinical psychiatric interviews (adolescent only)., Results: Self-reported depression was significantly correlated with self-reported HRQoL (rs [11] = -0.88, p<0.001). Age at injury was significantly correlated with self-reported HRQoL (rs [11] = -0.68, p = 0.02). Self-reported depression predicted self-reported HRQoL (R2 = 0.79, F [1, 10] = 33.48, p<0.001), but age at injury did not (R2 = 0.19, F [1, 10] = 2.09, p = 0.18)., Conclusions: Our results suggest that depression is a predictor of health related quality of life in youth post-TBI. The possibility of using targeted assessment and therapy for depression post-TBI to improve health related quality of life should be explored.