Proficiency testing for SARS-CoV-2 in assuring the quality and overall performance in viral RNA detection in clinical and public health laboratories.

Diagnostic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone significant changes over the duration of the pandemic. In early 2020, SARS-CoV-2 specific nucleic acid testing (NAT) protocols were predominantly in-house assays developed based on protocols published in peer reviewed journals. As the pandemic has progressed, there has been an increase in the choice of testing platforms. A proficiency testing program for the detection of SARS-CoV-2 by NAT was provided to assist laboratories in assessing and improving test capabilities in the early stages of the pandemic. This was vital in quality assuring initial in-house assays, later commercially produced assays, and informing the public health response. The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) offered three rounds of proficiency testing for SARS-CoV-2 to Australian and New Zealand public and private laboratories in March, May, and November 2020. Each round included a panel of five specimens, consisting of positive (low, medium or high viral loads), inconclusive (technical specimen of selected SARS-CoV-2 specific genes) and negative specimens. Results were received for round 1 from 16, round 2 from 97 and round 3 from 101 participating laboratories. Improvement in the accuracy over time was shown, with the concordance of results in round 1 being 75.0%, in round 2 above 95.0% for all samples except one, and for round 3 above 95.0%. Overall, participants demonstrated high capabilities in detecting SARS-CoV-2, even in samples of low viral load, indicating excellent testing accuracy and therefore providing confidence in Australian and New Zealand public and private laboratories test results.

Niclosamide-A promising treatment for COVID-19.

Vaccines have reduced the transmission and severity of COVID-19, but there remains a paucity of efficacious treatment for drug-resistant strains and more susceptible individuals, particularly those who mount a suboptimal vaccine response, either due to underlying health conditions or concomitant therapies. Repurposing existing drugs is a timely, safe and scientifically robust method for treating pandemics, such as COVID-19. Here, we review the pharmacology and scientific rationale for repurposing niclosamide, an anti-helminth already in human use as a treatment for COVID-19. In addition, its potent antiviral activity, niclosamide has shown pleiotropic anti-inflammatory, antibacterial, bronchodilatory and anticancer effects in numerous preclinical and early clinical studies. The advantages and rationale for nebulized and intranasal formulations of niclosamide, which target the site of the primary infection in COVID-19, are reviewed. Finally, we give an overview of ongoing clinical trials investigating niclosamide as a promising candidate against SARS-CoV-2.

Proficiency testing for bacterial whole genome sequencing in assuring the quality of microbiology diagnostics in clinical and public health laboratories.

The adoption of whole genome sequencing (WGS) data over the past decade for pathogen surveillance, and decision-making for infectious diseases has rapidly transformed the landscape of clinical microbiology and public health. However, for successful transition to routine use of these techniques, it is crucial to ensure the WGS data generated meet defined quality standards for pathogen identification, typing, antimicrobial resistance detection and surveillance. Further, the ongoing development of these standards will ensure that the bioinformatic processes are capable of accurately identifying and characterising organisms of interest, and thereby facilitate the integration of WGS into routine clinical and public health laboratory setting. A pilot proficiency testing (PT) program for WGS of infectious agents was developed to facilitate widely applicable standardisation and benchmarking standards for WGS across a range of laboratories. The PT participating laboratories were required to generate WGS data from two bacterial isolates, and submit the raw data for independent bioinformatics analysis, as well as analyse the data with their own processes and answer relevant questions about the data. Overall, laboratories used a diverse range of bioinformatics tools and could generate and analyse high-quality data, either meeting or exceeding the minimum requirements. This pilot has provided valuable insight into the current state of genomics in clinical microbiology and public health laboratories across Australia. It will provide a baseline guide for the standardisation of WGS and enable the development of a PT program that allows an ongoing performance benchmark for accreditation of WGS-based test processes.

Laboratory biosafety measures involving SARS-CoV-2 and the classification as a Risk Group 3 biological agent.

The current public health emergency surrounding the COVID-19 pandemic, that is the illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in thousands of cases in Australia since 25 January 2020 when the first case was diagnosed. This emerging virus presents particular hazards to researchers and laboratory staff in a clinical setting, highlighted by rapid and widespread global transmission. Based on the epidemiological and clinical data that have become available in mid-2020, we propose the interim classification of SARS-CoV-2 as a Risk Group 3 organism is reasonable, and discuss establishing Biosafety Level 3 (BSL-3) regulations accordingly. Despite its global spread, the reported mortality rate of SARS-CoV-2 ranging from 0.13% to 6.22% is considerably less than that of other Risk Group 4 agents including Ebola and Marburg viruses with fatality rates as high as 90%. In addition, studies have demonstrated that approximately 86% of patients presenting with severe courses of the disease are aged 70 years or above, with the presence of comorbid conditions such as cardiovascular and respiratory system diseases in the majority of all fatal cases. In contrary to recent discussions surrounding the protective and administrative measures needed in a laboratory, the emerging evidence surrounding mortality rate, distinct demographics of severe infections, and the presence of underlying diseases does not justify the categorisation of SARS-CoV-2 as a Risk Group 4 organism. This article summarises biosafety precautions, control measures and appropriate physical containment facilities required to minimise the risk of laboratory-acquired infections with SARS-CoV-2.

SARS-CoV-2 seroprevalence and asymptomatic viral carriage in healthcare workers: a cross-sectional study.

OBJECTIVE: To determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers. DESIGN: A cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020. SETTING: University Hospitals Birmingham NHS Foundation Trust (UHBFT), UK. PARTICIPANTS: 545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded. INTERVENTION: Participants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked. MAIN OUTCOME MEASURE: Proportion of participants demonstrating infection and positive SARS-CoV-2 serology. RESULTS: The point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ2=21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%), with lower rates observed in participants working in intensive care (14.8%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02). CONCLUSIONS AND RELEVANCE: We identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices.

A Randomized Crossover Study to Assess the Usability of Two New Vision Tests in Patients with Low Vision.

SIGNIFICANCE: Well-established charts such as Early Treatment Diabetic Retinopathy Study are able to quantify visual acuity (VA) with a low cutoff of 1.6 logMAR. Below this point, nonquantitative measures, such as count fingers, hand movements, and light perception, are used. There is a need for more reproducible, comparable, and reliable ways to measure VA changes in this patient cohort. PURPOSE: The purpose of this study was to examine and compare the ability of the Berkeley Rudimentary Vision Test (BRVT) and the Freiburg Acuity Test (FrACT) to quantify VA in low-vision patients who score nonnumerical VAs in standard charts. METHODS: Fifty adult participants with VA ≤1.0 logMAR in both eyes were recruited from the Oxford Eye Hospital, Oxford, United Kingdom. Correlation between FrACT and BRVT results and the correlation between VA and daily living activities were analyzed statistically. Potential predictors of differences were investigated. RESULTS: The BRVT was significantly faster to conduct (P = .002), but FrACT was able to quantify vision numerically in a greater proportion of eyes. The κ agreement between tests was 0.26. The difference increased systematically with the VA reduction (P < .0001). The Bland-Altman analysis showed a skew to measurement of lower logMAR VA indicating better vision measured on the FrACT. The only significant predictor of difference between the tests was binocular VA (coefficient, -0.445; P = .001). CONCLUSIONS: Both tests are suitable for a very low-vision population. The BRVT is a faster test to administer, but FrACT provides a numerical result in more eyes. The poor intertest repeatability indicates that they cannot be used interchangeably. The BRVT generally reported poorer vision than did the FrACT. The medium of presentation, such as a computer screen or externally lit print medium, is likely to be the biggest factor in these differences and warrants further investigation.

Increased central adiposity and decreased subcutaneous adipose tissue 11ß-hydroxysteroid dehydrogenase type 1 are associated with deterioration in glucose tolerance-A longitudinal cohort study.

OBJECTIVE AND CONTEXT: Increasing adiposity, ageing and tissue-specific regeneration of cortisol through the activity of 11ß-hydroxysteroid dehydrogenase type 1 have been associated with deterioration in glucose tolerance. We undertook a longitudinal, prospective clinical study to determine if alterations in local glucocorticoid metabolism track with changes in glucose tolerance. DESIGN, PATIENTS, AND MEASUREMENTS: Sixty-five overweight/obese individuals (mean age 50.3 ± 7.3 years) underwent oral glucose tolerance testing, body composition assessment, subcutaneous adipose tissue biopsy and urinary steroid metabolite analysis annually for up to 5 years. Participants were categorized into those in whom glucose tolerance deteriorated ("deteriorators") or improved ("improvers"). RESULTS: Deteriorating glucose tolerance was associated with increasing total and trunk fat mass and increased subcutaneous adipose tissue expression of lipogenic genes. Subcutaneous adipose tissue 11ß-HSD1 gene expression decreased in deteriorators, and at study completion, it was highest in the improvers. There was a significant negative correlation between change in area under the curve glucose and 11ß-HSD1 expression. Global 11ß-HSD1 activity did not change and was not different between deteriorators and improvers at baseline or follow-up. CONCLUSION: Longitudinal deterioration in metabolic phenotype is not associated with increased 11ß-HSD1 activity, but decreased subcutaneous adipose tissue gene expression. These changes may represent a compensatory mechanism to decrease local glucocorticoid exposure in the face of an adverse metabolic phenotype.

Liver homing of clinical grade Tregs after therapeutic infusion in patients with autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an immune-mediated disease with no curative treatment. Regulatory T cell (Treg) therapy is potentially curative in AIH given the critical role of Tregs in preventing autoimmunity. To work effectively, adoptively transferred Tregs must migrate to and survive within the inflamed liver. We conducted a proof-of-concept study aiming to assess the safety and liver-homing properties of good manufacturing practice (GMP)-grade autologous Tregs in patients with AIH. METHODS: Autologous polyclonal GMP-grade Tregs were isolated using leukapheresis and CliniMACS, labelled with indium tropolonate and re-infused intravenously to 4 patients with AIH. GMP-Treg homing to the liver was investigated with longitudinal gamma camera and SPECT-CT scanning. GMP-Treg immunophenotype, function and immunometabolic state were assessed during the study. RESULTS: We observed that the isolated Tregs were suppressive and expressed CXCR3, a chemokine receptor involved in recruitment into the inflamed liver, as well as Treg functional markers CD39, CTLA-4 and the transcription factor Foxp3. Serial gamma camera and SPECT-CT imaging demonstrated that 22-44% of infused Tregs homed to and were retained in the livers of patients with autoimmune hepatitis for up to 72 h. The infused cells did not localise to any off-target organs other than the spleen and bone marrow. GMP-Tregs were metabolically competent and there were no infusion reactions or high-grade adverse effects after Treg infusion. CONCLUSION: Our novel findings suggest that the liver is a good target organ for Treg cellular therapy, supporting the development of clinical trials to test efficacy in autoimmune hepatitis and other autoimmune liver diseases. LAY SUMMARY: Autoimmune liver diseases occur when the body's immune cells target their own liver cells. Regulatory T cells (Tregs) prevent autoimmunity, thus they are a potential therapy for autoimmune liver diseases. In patients with autoimmune hepatitis, Treg infusion is safe, with nearly a quarter of infused Tregs homing to the liver and suppressing tissue-damaging effector T cells. Thus, Tregs are a potentially curative immune cell therapy for early autoimmune liver diseases.

Optimisation of dark adaptation time required for mesopic microperimetry.

BACKGROUND: Macular Integrity Assessment (MAIA) microperimetry is increasingly used in clinical and research settings to assess point retinal sensitivity and fixation stability. Testing occurs under mesopic conditions, commonly after a period of dark adaptation. Our aim was to identify the minimum length of adaptation required to optimise microperimetry performance. METHODS: MAIA microperimetry using the 10-2 grid was performed on 40 right eyes of 40 healthy participants aged 18-73 with no ocular pathology and vision of at least 0.1 logMAR after ambient light exposure, with 0, 5, 10, 15, 20 and 30 min of adaptation in mesopic settings. Ten right eyes of 10 participants with choroideremia were also tested following 0 and 20 min of adaptation. We further tested 10 right eyes of 10 healthy participants after bright light exposure, with 0, 10 and 20 min of adaptation. We compared changes in threshold sensitivity and fixation stability across time points. RESULTS: Microperimetry performance did not improve with increasing adaptation time in healthy participants or patients with choroideremia after ambient light exposure. After bright light exposure, we found microperimetry thresholds improved after 10 min of adaptation, but did not improve further at 20 min. CONCLUSION: Mesopic adaptation is not required before MAIA microperimetry after exposure to ambient light. Ten minutes of adaptation is sufficient after exposure to a bright light stimulus, such as ophthalmoscopy or retinal imaging. The brief time of dark adaptation required corresponds to cone adaptation curves and provides further evidence for cone-mediated central retinal function under mesopic conditions.

Proficiency testing for the detection of Middle East respiratory syndrome coronavirus demonstrates global capacity to detect Middle East respiratory syndrome coronavirus.

The first reported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection was identified in Saudi Arabia in September 2012, since which time there have been over 2000 laboratory-confirmed cases, including 750 deaths in 27 countries. Nucleic acid testing (NAT) is the preferred method for the detection of MERS-CoV. A single round of a Proficiency Testing Program (PTP) was used to assess the capability of laboratories globally to accurately detect the presence of MERS-CoV using NAT. A panel of eleven lyophilized specimens containing different viral loads of MERS-CoV, common coronaviruses, and in vitro RNA transcripts was distributed to laboratories in all six World Health Organization regions. A total of 96 laboratories from 79 countries participating in the PTP, with 76 of 96 (79.2%) reporting correct MERS-CoV results for all nine scored specimens. A further 10 laboratories (10.4%) scored correctly in eight of nine specimens of the PTP. The majority of laboratories demonstrated satisfactory performance in detecting the presence of MERS-CoV using NAT. However, some laboratories require improved assay sensitivity, reduced cross contamination of samples, and improved speciation of coronavirus subtypes for potentially complex clinical specimens. Further PTP and enhanced links with expert laboratories globally may improve the laboratory performance.

Slowly progressive retinitis pigmentosa caused by two novel mutations in the MAK gene.

BACKGROUND: The growing number of clinical trials currently underway for inherited retinal diseases has highlighted the importance of achieving a molecular diagnosis for all new cases presenting to hospital eye services. The male germ cell-associated kinase (MAK) gene encodes a cilium-associated protein selectively expressed in the retina and testis, and has recently been implicated in autosomal recessive retinitis pigmentosa (RP). Whole exome sequencing has previously identified a homozygous Alu insertion in probands with recessive RP and nonsense and missense mutations have also been reported. MATERIALS AND METHODS: Here we describe two novel mutations in different alleles of the MAK gene in a 75-year-old British female, who had a clinical diagnosis of RP () with onset in the fourth decade and no relevant family history. The mutations were established through next generation sequencing of a panel of 111 genes associated with RP and RP-like phenotypes. RESULTS: Two novel null mutations were identified within the MAK gene. The first c.1195_1196delAC p.(Thr399fs), was a two base-pair deletion creating a frame-shift in exon 9 predicted to result in nonsense-mediated decay. The second, c.279-2A>G, involved the splice acceptor consensus site upstream of exon 4, predicted to lead to aberrant splicing. CONCLUSIONS: The natural history of this individual's RP is consistent with previously described MAK mutations, being significantly milder than that associated with other photoreceptor ciliopathies. We suggest inclusion of MAK as part of wider genetic testing in all individuals presenting with RP.

Ebola Preparedness: Diagnosis Improvement Using Rapid Approaches for Proficiency Testing.

The unprecedented 2015 Ebolavirus (EBOV) outbreak in West Africa was declared a public health emergency, making diagnosis and quality of testing a global issue. The accuracy of laboratory diagnostic capacity for EBOV was assessed in 2014 to 2016 using a proficiency testing (PT) strategy developed by the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) in Biosecurity. Following a literature search, EBOV-specific gene targets were ranked according to the frequency of their use in published methods. The most commonly used gene regions (nucleoprotein [NP], glycoprotein [GP], and RNA-dependent RNA polymerase [L]) were selected for the design of in vitro RNA transcripts to be included in the simulated EBOV specimens used for EBOV detection with PCR-based assays. Specimens were tested for stability and found to be stable on long-term storage (1 year) at -80°C and on shorter-term storage in lyophilized form (1 week at ambient temperature and a subsequent week at -80°C). These specimens were used in three EBOV PTs offered from April 2014 to March 2016. In the first and third PTs, all laboratories (3/3 and 9/9, respectively) correctly identified specimens containing EBOV RNA transcripts, while in the second PT, all but one laboratory (5/6) correctly confirmed the presence of EBOV. The EBOV PT panel was useful for ensuring the competency of laboratories in detecting EBOV in the absence of readily available clinical samples. The simulated EBOV specimen was safe, stable, and reliable and can be used in lyophilized form for future EBOV PT programs, allowing simplicity of transport.

Single centre outcomes from definitive chemo-radiotherapy and single modality radiotherapy for locally advanced oesophageal cancer.

BACKGROUND: Definitive chemo-radiotherapy (dCRT) has been advocated as an alternative to surgical resection for the treatment of locally advanced oesophageal cancer (OC). We have retrospectively reviewed 4 years' experience of patients (pts) who underwent contemporary staging and were treated with concurrent chemo-radiotherapy (dCRT) or single modality radical radiotherapy (RT) with curative intent. METHODS: Retrospective analysis permitted identification of consecutive patients who underwent contemporary staging prior to non-surgical treatment for locally advanced oesophageal carcinoma. The primary outcomes were overall survival (OS) and disease-free survival (DFS), adjusted for baseline differences in age, tumour staging and histological cell type. All patients were treated with either dCRT or single modality RT within a single centre between 2009 and 2012. RESULTS: We identified 235 patients in total [median age 69.8 years, male =130 pts, female =105 pts, adenocarcinoma (ACA) =85 pts, squamous =150 pts]. A total of 190 pts received dCRT and 45 patients were treated with RT. All patients were staged with CT of chest, abdomen and pelvis, 226 patients underwent endoscopic ultrasound (EUS), and 183 patients had PET-CT. Patients treated with dCRT demonstrated longer OS (27 vs. 25 months respectively, P=0.02) and DFS (31 vs. 16 months respectively, P=0.01) compared to those treated with RT. More advanced tumour stage (stage 3 vs. stage 1/2) at presentation conferred poorer OS (32 vs. 38.2 months, P=0.02) and DFS (11 vs. 28 months, P=0.013). We demonstrated an acceptable toxicity profile with only 77 patients (32.8%) suffering grade 3 toxicity and 9 patients (4.2%) experiencing grade 4 toxicity by CTC criteria. The NG/PEG feeding rates were 4% across all treated patients. CONCLUSIONS: This retrospective analysis is in keeping with current treatment paradigms emphasising the importance and safety of concurrent CRT in maximising curative potential for patients undergoing non-surgical treatment of OC. Although retrospective, in comparison to similar retrospective series from both our centre and historical literature, this data suggest improvements in OS and DFS, possibly due to improved patient selection through the use of more effective tumour staging.

Longitudinal changes in glucocorticoid metabolism are associated with later development of adverse metabolic phenotype.

OBJECTIVE: Dysregulation of enzymes that control local tissue steroid metabolism has been implicated in the pathogenesis of obesity and insulin resistance; however, longitudinal changes in glucocorticoid metabolism have not been investigated. This study was performed to evaluate the role of glucocorticoid metabolism in the development of insulin resistance and obesity and to identify biomarkers for future development of metabolic disease. DESIGN: This was a prospective longitudinal observation study conducted over 5 years. METHODS: A 24-h collection was used to serially analyze urinary glucocorticoid and mineralocorticoid metabolites in 57 obese and overweight patients with no prior diagnosis of diabetes mellitus, recruited from the community. RESULTS: Baseline higher 5α-reductase (5αR) activity, but not 11ß-hydroxysteroid dehydrogenase type 1 activity, was predictive of increased fasting insulin at final visit (11.4 compared with 7.4 mU/l in subjects with lower 5αR activity, P<0.05), area under the curve insulin response to oral glucose tolerance test (176.7 compared with 89.1 mU/l.h, P<0.01), and homeostasis model assessment (HOMA2-IR; 1.3 compared with 0.8, P<0.01). Higher total glucocorticoid production was associated with abnormal glucose tolerance and increased BMI. During this study, systolic blood pressure increased (equivalent to ∼1 mmHg/year), as did plasma sodium levels; this evidence of increased mineralocorticoid activity was associated with increased aldosterone metabolites and decreased 11ß-hydroxysteroid dehydrogenase type 2 activity. CONCLUSIONS: Increased 5αR activity and glucocorticoid secretion rate over time are linked with the development of metabolic disease, and may represent targets for therapeutic intervention, which merits further study.

OPCRIT+: an electronic system for psychiatric diagnosis and data collection in clinical and research settings.

BACKGROUND: The increasingly large sample size requirements of modern adult mental health research suggests the need for a data collection and diagnostic application that can be used across a broad range of clinical and research populations. Aims To develop a data collection and diagnostic application that can be used across a broad range of clinical and research settings. METHOD: We expanded and redeveloped the OPCRIT system into a broadly applicable diagnostic and data-collection package and carried out an interrater reliability study of this new tool. RESULTS: OPCRIT+ performed well in an interrater reliability study with relatively inexperienced clinicians, giving a combined, weighted kappa of 0.70 for diagnostic reliability. CONCLUSIONS: OPCRIT+ showed good overall interrater reliability scores for diagnoses. It is now incorporated in the electronic patient record of the Maudsley and associated hospitals. OPCRIT+ can be downloaded free of charge at http://sgdp.iop.kcl.ac.uk/opcritplus.

Changes in body weight during pharmacological treatment of depression.

The risk of weight gain is an important determinant of the acceptability and tolerability of antidepressant medication. To compare changes in body weight during treatment with different antidepressants, body weight and height were measured at baseline and after 6, 8, 12 and 26 wk treatment with escitalopram or nortriptyline in 630 adults with moderate-to-severe unipolar depression participating in GENDEP, a part-randomized open-label study. Weight increased significantly more during treatment with nortriptyline compared to escitalopram. The weight gain commenced during the first 6 wk of nortriptyline treatment, reached on average 1.2 kg at 12 wk (0.44-point BMI increase), and continued throughout the 6-month follow-up period. Participants who were underweight at baseline gained most weight. Participants who were obese at baseline did not gain more weight during treatment. Weight gain occurred irrespective of whether weight loss was a symptom of current depressive episode and was identified as an undesired effect of the antidepressant by most participants who gained weight. There was little weight change during treatment with escitalopram, with an average increase of 0.14 kg (0.05-point BMI increase) over 12 wk of treatment. In conclusion, treatment with the tricyclic antidepressant nortriptyline was associated with moderate weight gain, which cannot be explained as a reversal of symptomatic weight loss and is usually perceived as an undesired adverse effect. While treatment with nortriptyline may be recommended in underweight subjects with typical neurovegetative symptoms, escitalopram is a suitable alternative for subjects at risk of weight gain.

Utility of the pooling approach as applied to whole genome association scans with high-density Affymetrix microarrays.

BACKGROUND: We report an attempt to extend the previously successful approach of combining SNP (single nucleotide polymorphism) microarrays and DNA pooling (SNP-MaP) employing high-density microarrays. Whereas earlier studies employed a range of Affymetrix SNP microarrays comprising from 10 K to 500 K SNPs, this most recent investigation used the 6.0 chip which displays 906,600 SNP probes and 946,000 probes for the interrogation of CNVs (copy number variations). The genotyping assay using the Affymetrix SNP 6.0 array is highly demanding on sample quality due to the small feature size, low redundancy, and lack of mismatch probes. FINDINGS: In the first study published so far using this microarray on pooled DNA, we found that pooled cheek swab DNA could not accurately predict real allele frequencies of the samples that comprised the pools. In contrast, the allele frequency estimates using blood DNA pools were reasonable, although inferior compared to those obtained with previously employed Affymetrix microarrays. However, it might be possible to improve performance by developing improved analysis methods. CONCLUSIONS: Despite the decreasing costs of genome-wide individual genotyping, the pooling approach may have applications in very large-scale case-control association studies. In such cases, our study suggests that high-quality DNA preparations and lower density platforms should be preferred.

The Bipolar Association Case-Control Study (BACCS) and meta-analysis: No association with the 5,10-Methylenetetrahydrofolate reductase gene and bipolar disorder.

Bipolar disorder (BD) is a complex genetic disease for which the underlying pathophysiology has yet to be fully explained. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in folate-mediated one-carbon metabolism and folate deficiency can be associated with psychiatric symptoms. A single base variant in MTHFR gene (C677T) results in the production of a mildly dysfunctional thermolabile enzyme and has recently been implicated in BD. We conducted an association study of this polymorphism in 897 patients with bipolar I or bipolar II disorder, and 1,687 healthy control subjects. We found no evidence for genotypic or allelic association in this sample. We also performed a meta-analysis of our own, and all published data, and report no evidence for association. Our findings suggest that the MTHFR C677T polymorphism is not involved in the genetic etiology of clinically significant BD.

Suicidal ideation during treatment of depression with escitalopram and nortriptyline in genome-based therapeutic drugs for depression (GENDEP): a clinical trial.

BACKGROUND: Suicidal thoughts and behaviours during antidepressant treatment, especially during the first weeks of treatment, have prompted warnings by regulatory bodies. The aim of the present study is to investigate the course and predictors of emergence and worsening of suicidal ideation during tricyclic antidepressant and serotonin reuptake inhibitor treatment. METHODS: In a multicentre part-randomised open-label study, 811 adult patients with moderate to severe unipolar depression were allocated to flexible dosage of escitalopram or nortriptyline for 12 weeks. The suicidality items of three standard measures were integrated in a suicidal ideation score. Increases in this score were classified as treatment emergent suicidal ideation (TESI) or treatment worsening suicidal ideation (TWOSI) according to the absence or presence of suicidal ideation at baseline. RESULTS: Suicidal ideation decreased during antidepressant treatment. Rates of TESI and TWOSI peaked in the fifth week. Severity of depression predicted TESI and TWOSI. In men, nortriptyline was associated with a 9.8-fold and 2.4-fold increase in TESI and TWOSI compared to escitalopram, respectively. Retirement and history of suicide attempts predicted TWOSI. CONCLUSION: Increases in suicidal ideation were associated with depression severity and decreased during antidepressant treatment. In men, treatment with escitalopram is associated with lower risk of suicidal ideation compared to nortriptyline. Clinicians should remain alert to suicidal ideation beyond the initial weeks of antidepressant treatment. TRIAL REGISTRATION: EudraCT (No.2004-001723-38) and ISRCTN (No. 03693000).

Genetic predictors of increase in suicidal ideation during antidepressant treatment in the GENDEP project.

The aim of this study was to investigate genetic predictors of an increase in suicidal ideation during treatment with a selective serotonin reuptake inhibitor or a tricyclic antidepressant. A total of 796 adult patients with major depressive disorder who were treated with a flexible dosage of escitalopram or nortriptyline in Genome-based Therapeutic Drugs for Depression (GENDEP) were included in the sample and provided data on suicidal ideation. Nine candidate genes involved in neurotrophic, serotonergic, and noradrenergic pathways were selected based on previous association studies with suicidal ideation or behavior. Using a logistic regression model, 123 polymorphisms in these genes were compared between subjects with an increase in suicidal ideation and those without any increase in suicidal ideation. Polymorphisms in BDNF, the gene encoding the brain-derived neurotrophic factor, were significantly associated with an increase in suicidal ideation. The strongest association was observed for rs962369 in BDNF (p=0.0015). Moreover, a significant interaction was found between variants in BDNF and NTRK2, the gene encoding the BNDF receptor (p=0.0003). Among men taking nortriptyline, suicidality was also associated with rs11195419 SNP in the alpha(2A)-adrenergic receptor gene (ADRA2A) (p=0.007). The associations observed with polymorphisms in BDNF suggest the involvement of the neurotrophic system in vulnerability to suicidality. Epistasis between BDNF and NTRK2 suggests that genetic variations in the two genes are involved in the same causal mechanisms leading to suicidality during antidepressant treatment. Among men, genetic variation in noradrenergic signaling may interact with norepinephrine reuptake-inhibiting antidepressants, thereby contributing to suicidality.