Indirect Screening Research Articles

Commentary on Ondersma et al. (2012): Beyond the quest for the perfect test—drug use screening in pregnancy

Unchanged since its articulation in 1968 by the World Health Organization, the object of screening remains ‘to discover among the apparently well who are in fact suffering from disease’ 1. Guidelines for a good screening test remain the following: the availability of treatment, latent disease state, patient acceptability and a balance of benefits to harms on the population level. Ondersma et al. 2 propose a novel screening instrument for drug use in the prenatal period. The authors are to be commended in approaching the subject area with an understanding of the shame and stigma that often accompanies drug use during pregnancy. To overcome what they term ‘hesitancy in disclosure’, the authors propose and validate an indirect screening method (Wayne Indirect Drug Use Screener: WIDUS). Using both urine and hair toxicology as the gold standard, the indirect screening instrument performed similarly to other published screening instruments and better than a direct screener, the Drug Abuse Screening Test (DAST), in their sample. As an obstetrician gynecologist who provides care to pregnant women with drug problems, I understand the importance of this topic. However, I am uncertain as to the benefit of a new screening instrument in general, and have reservations about the clinical utility of indirect methods in particular. From the 4Ps Plus to the Zuckerman–Kuhlman Personality Questionnaire (ZKPQ), the Substance Use Screening and Assessment Instruments Database managed by the Alcohol and Drug Abuse Institute at the University of Washington currently catalogues 904 unique instruments, of which 15 have been developed specifically for or validated in pregnant women 3. To my knowledge, there is a single systematic review of substance screening during pregnancy. Bradley et al. investigated seven alcohol screening instruments and failed to demonstrate any large differences in test characteristics between them 4. To date, no single method is recommended by professional societies in the United States 5, 6 or Canada 7. The overall absence of any evidence privileging one screening method over another begs the question as to whether additional instruments need to be developed. One of the problems with screening for drug use during pregnancy is the inconsistent uptake of screening among providers. Although universal screening is recommended 8, it is far from achieved 9. More common in the United States is selective screening based on race, poverty and poor pregnancy outcomes 10. This selective screening is highly discretionary 11, and betrays a disturbing bias among providers of women's health care towards the legal coercion of pregnant women with drug problems 12. A new screening instrument is unlikely to change these realities. Unlike direct screening methods which rely on self-report, indirect methods attempt to capture use without using a tool that refers to drug use. The concept and appeal of indirect screening has existed for several decades 13. However, the evidence to support its superiority is lacking. For example, a recent review of one of the most used indirect screeners (Substance Abuse Subtle Screening Inventory: SASSI) showed no benefit in detecting substance use correctly compared with direct methods 14. Screening only makes public health sense if linked to evidence-based interventions. Clinically, a positive screen invites the opportunity to discuss drug problems and provide brief interventions based upon the individual's readiness to change. Ondersma et al. note that the primary motivation for indirect screening is that under-reporting leads to missed opportunities for interventions. However, non-disclosure reflects a lack of readiness to change (a context in which brief interventions are not indicated) or a mistrust of the provider/clinical encounter (a context in which brief interventions may be harmful), or both. Therefore, it is difficult to see how indirect screening would lead to brief interventions without confrontation, an approach that Ondersma et al. describe correctly as ‘inaccurate, unethical and counterproductive’ 2. In this regard, the clinical utility of indirect screening appears more similar to toxicology testing than to direct screening and therefore raises similar ethical questions 15, specifically the potential harms of false positive results. Using the data from Ondersma et al.'s Table 3, I calculate a false positive rate of at least 7% (cut score 4) to as high as 34% (cut score 2) in the WIDUS screen, which is higher than the DAST-10 in their sample (range 2–10%). This is concerning, as one of the consequences of a positive screen may be a report to Child Protective Services. In conclusion, I believe that we need to move beyond the quest for the perfect test and focus instead on educating providers and empowering mothers such that the complex issues of drug use in pregnancy can be addressed safely and with compassion. None.

Genomic regions for canopy temperature and their genetic association with stomatal conductance and grain yield in wheat.

Stomata are the site of CO2 exchange for water in a leaf. Variation in stomatal control offers promise in genetic improvement of transpiration and photosynthetic rates to improve wheat performance. However, techniques for estimating stomatal conductance (SC) are slow, limiting potential for efficient measurement and genetic modification of this trait. Genotypic variation in canopy temperature (CT) and leaf porosity (LP), as surrogates for SC, were assessed in three wheat mapping populations grown under well-watered conditions. The range and resulting genetic variance were large but not always repeatable across days and years for CT and LP alike. Leaf-to-leaf variation was large for LP, reducing heritability to near zero on a single-leaf basis. Replication across dates and years increased line-mean heritability to ~75% for both CT and LP. Across sampling dates and populations, CT showed a large, additive genetic correlation with LP (rg=-0.67 to -0.83) as expected. Genetic increases in pre-flowering CT were associated with reduced final plant height and both increased harvest index and grain yield but were uncorrelated with aerial biomass. In contrast, post-flowering, cooler canopies were associated with greater aerial biomass and increased grain number and yield. A multi-environment QTL analysis identified up to 16 and 15 genomic regions for CT and LP, respectively, across all three populations. Several of the LP and CT QTL co-located with known QTL for plant height and phenological development and intervals for many of the CT and LP quantitative trait loci (QTL) overlapped, supporting a common genetic basis for the two traits. Notably, both Rht-B1b and Rht-D1b dwarfing alleles were paradoxically positive for LP and CT (i.e. semi-dwarfs had higher stomatal conductance but warmer canopies) highlighting the issue of translation from leaf to canopy in screening for greater transpiration. The strong requirement for repeated assessment of SC suggests the more rapid CT assessment may be of greater value for indirect screening of high or low SC among large numbers of early-generation breeding lines. However, account must be taken of variation in development and canopy architecture when interpreting performance and selecting breeding lines on the basis of CT.

G.P.111 Exudative retinopathy in facioscapulohumeral muscular dystrophy: When to screen? When to monitor? When to treat?

Abstract Visual loss caused by exudative retinal detachment is a rare but preventable association of FSHD. Underlying peripheral retinal vascular telangiectasis is however very common. Focal retinal hypovascularity may be a developmental abnormality parallel to aberrations in myogenesis. Since asymptomatic telangiectasis appears likely to remain static for many years, the question arises as to proper practice in relation to indirect ophthalmoscopic screening of FSHD patients, particularly children, and if and when to treat visualized exudates, which may track from the retinal periphery to the posterior pole. Accumulated international experience has demonstrated the efficacy of unusually aggressive and frequent treatment, typically with Laser photocoagulation directed at leaking vessels, even when vision is significantly impaired. However, once macular damage is established, visual loss may be irreversible. In advanced stages, painful glaucoma may lead to enucleation. Family follow-up now underlines the particular importance of reviewing patients from FSHD families with a history of overt retinal vascular disease. Two members of one family lost vision unilaterally in mid-life; only one seen early enough to treat successfully. Retinal abnormalities are (like the myopathy) asymmetrical; bilateral visual loss is rare but real. It is concluded that (i) severe and early-onset FSHD patients with clinical deafness are more likely to have treatable retinal exudates, and should be monitored closely, especially in early childhood. (ii) Retinal disease may present before muscle disease. (iii) Atypical cases of Coats Disease (e.g. when bilateral, in women, or in older patients) may justify genetic testing, with caution, for FSHD. (iv) All FSHD patients should have one indirect ophthalmoscopy examination and report unexplained visual symptoms immediately. (v) Peripheral exudates should be monitored closely, but exudates threatening the macula will mandate urgent treatment.

The sensitivity, specificity and predictive values of raised plasma metal ion levels in the diagnosis of adverse reaction to metal debris in symptomatic patients with a metal-on-metal arthroplasty of the hip

Plasma levels of cobalt and chromium ions and Metal Artefact Reduction Sequence (MARS)-MRI scans were performed on patients with 209 consecutive, unilateral, symptomatic metal-on-metal (MoM) hip arthroplasties. There was wide variation in plasma cobalt and chromium levels, and MARS-MRI scans were positive for adverse reaction to metal debris (ARMD) in 84 hips (40%). There was a significant difference in the median plasma cobalt and chromium levels between those with positive and negative MARS-MRI scans (p < 0.001). Compared with MARS-MRI as the potential reference standard for the diagnosis of ARMD, the sensitivity of metal ion analysis for cobalt or chromium with a cut-off of > 7 µg/l was 57%. The specificity was 65%, positive predictive value was 52% and the negative predictive value was 69% in symptomatic patients. A lowered threshold of > 3.5 µg/l for cobalt and chromium ion levels improved the sensitivity and negative predictive value to 86% and 74% but at the expense of specificity (27%) and positive predictive value (44%). Metal ion analysis is not recommended as a sole indirect screening test in the surveillance of symptomatic patients with a MoM arthroplasty. The investigating clinicians should have a low threshold for obtaining cross-sectional imaging in these patients, even in the presence of low plasma metal ion levels.

The rapid and direct determination of ATPase activity by ion exchange chromatography and the application to the activity of heat shock protein-90

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2mm×6mm i.d.), under a three-solvent gradient elution mode and UV detection at 256nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples.

Predictive Utility of Brief Alcohol Use Disorders Identification Test (AUDIT) for Human Immunodeficiency Virus Antiretroviral Medication Nonadherence

ABSTRACT Alcohol use negatively affects adherence to antiretroviral therapy (ART), thus human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) care providers need accurate, efficient assessments of alcohol use. Using existing data from an efficacy trial of 2 cognitive-behavioral ART adherence interventions, the authors sought to determine if results on 2 common alcohol screening tests (Alcohol Use Disorders Identification Test—Consumption [AUDIT-C] and its binge-related question [AUDIT-3]) predict ART nonadherence. Twenty-seven percent of the sample (n = 308) were positive on the AUDIT-C and 34% were positive on the AUDIT-3. In multivariate analyses, AUDIT-C–positive status predicted ART nonadherence after controlling for race, age, conscientiousness, and self-efficacy (P = .036). Although AUDIT-3–positive status was associated with ART nonadherence in unadjusted analyses, this relationship was not maintained in the final multivariate model. The AUDIT-C shows potential as an indirect screening tool for both at-risk drinking and ART nonadherence, underscoring the relationship between alcohol and chronic disease management.

Progesterone receptor up-regulation: a diagnostic tool for the illicit use of oestrogens in adult beef cattle

The monitoring of gene regulation via mRNA levels to detect anabolic sex steroid administration in cattle is a novel approach to detecting the illicit treatment of livestock in meat production. A previous study revealed that progesterone receptor (PR) gene expression levels were increased in the bulbourethral glands and prostates of 17β-oestradiol-treated prepubertal calves, suggesting that the PR can be used as a specific molecular biomarker for oestrogen treatment. The aim of this study was to verify the specificity and applicability of the PR to detect the illegal use of 17β-oestradiol in sexually mature beef cattle. Accessory sex glands were sampled from 42 male beef cattle that were divided into six experimental groups, including two control groups, K1 and K2. Group A cattle were treated with 17β-oestradiol (five weekly intramuscular doses of 20 mg), and group B cattle were treated with dexamethasone (40 daily doses of 0.7 mg per os). Group C cattle received an implant of Revalor-200 (200 mg of trenbolone acetate and 20 mg of 17β-oestradiol), and group D cattle received Revalor-200 plus dexamethasone (0.7 mg daily per os). 17β-Oestradiol, either alone or in combination with other steroids, up-regulated the PR gene and protein expression, even in the absence of detectable histological changes in the accessory sex glands, confirming the high sensitivity of PR gene expression as an indirect diagnostic screening tool to detect illicit oestrogen treatment in sexually mature male bovine.

EBV‐associated hemophagocytic syndrome

General aspect of the marrow: macrophages with prominent phagocytosis of red cells, platelets, and erythroblasts. The marrow is overall hypocellular (MGG, original magnification ×400). High-power view of two characteristic hemophagocytic macrophages. (A) A macrophage with ingested red cells, platelets, erythroblasts, neutrophil, and assorted debris. (B) A macrophage which has engulfed a large number of erythroblasts (MGG, original magnification ×1,000). Hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome characterized by fever, pancytopenia, organomegaly, multiorgan dysfunction, and hemophagocytosis by a large number of macrophages in bone marrow and other tissues [1]. Central to the pathophysiology is the impaired activity of NK and cytotoxic T lymphocytes resulting in uncontrolled immune activation, hypercytokinemia, and proliferation of macrophages. The diagnosis of HLH relies upon well-established criteria [2]. The syndrome has been described in familial conditions or as a secondary phenomenon to infections, malignancies, and collagen vascular diseases. HLH in the context of infection is best described in association with Epstein-Barr virus (EBV). EBV-associated HLH can occur either in primary infection or as a consequence of viral reactivation. EBV can also be the trigger in both familial and secondary cases. Although uncommon, hemophagocytic syndrome is a serious complication of EBV infection and can be fatal in many cases. Early recognition and prompt initiation of treatment are of great importance to reduce the high morbidity and mortality associated with this condition. The history and representative images from a case of EBV-associated hemophagocytic syndrome are described. The patient's consent has been obtained. A 25-year-old woman was admitted with fever, pancytopenia, and hepatic dysfunction. Five weeks previously, she was diagnosed with infectious mononucleosis on the basis of pharyngitis, fever, and positive EBV antibodies. In the weeks after diagnosis, she continued to complain of intermittent fevers and sweating. Past medical and family history were unremarkable. Physical examination revealed temperature 38°C, icteric sclera, and hepatosplenomegaly. Laboratory values included hemoglobin 8.1 g/dL, leucocytes 2,100 μL−1, neutrophils 720 μL−1, platelets 40,000 μL−1, bilirubin 7.4 mg/dL, ALT 135 U/L, LDH 1,080 U/L, triglycerides 459 mg/dL, and ferritin 6,070 ng/mL. Peripheral blood film morphology was unremarkable. The direct and indirect antiglobulin test and screening for cold autoagglutinins gave negative results. The reticulocyte automated count and absolute value were 1.11% and 31,900 μL−1, respectively, indicating impaired production of red cells. Bone marrow aspiration showed numerous macrophages with striking phagocytosis of blood cells and marrow precursors (Images 1 and 2). EBV serologies indicated primary infection, and the EBV-PCR result was 18,000 copies/mL. Genetic testing for familial HLH was negative. The patient was treated with methylprednisolone and intravenous immunoglobulins and eventually recovered completely.

Review of the Use of Digital Imaging in Retinopathy of Prematurity Screening

Retinopathy of prematurity (ROP) is a vision-threatening vasoproliferative condition of premature infants worldwide. As survival rates of younger and smaller infants improve, more babies are at risk for the development of ROP and blindness. Meanwhile, fewer ophthalmologists are available for bedside indirect ophthalmoscopy screening examinations. Remote digital imaging is a promising method with which to identify those infants with treatment-requiring or referral-warranted ROP quickly and accurately, and may help circumvent issues regarding the limited availability of ROP screening providers. The Retcam imaging system is the most common system for fundus photography, with which high-quality photographs can be obtained by trained non-physician personnel and evaluated by a remote expert. It has been shown to have high reliability and accuracy in detecting referral-warranted ROP, particularly at later postmenstrual ages. Additionally, the method is generally well received by parents and is highly cost-effective.

Towards an indirect screening technique facilitating detection of cellular populations bearing specific cell surface markers

We describe and demonstrate a technique for detection of cell surface antigens, with potential use in tissue antigen research. Briefly, a small volume of wetted chromatographic beads (specifically, 100 μL of Nickel-nitriloacetic acid (NTA) agarose) was bound to small quantities (specifically, ∼0.1-0.2 μg) of a single-chain Fv antibody recognizing the Class I MHC heavy chain antigen. The beads were then used to capture and detect cells bearing this antigen, through SDS polyacrylamide gel electrophoresis of boiled bead-cell complexes. A key feature of this method is its use of "signal amplification." Although the antibody-mediated binding and immobilization of intact cells involves relatively small numbers of antibodies, and cells, what is being detected is simply the presence of cells. Each bound cell contains a number of abundant proteins that are present in millions of copies per cell, and the abundance of these proteins ensures that they are detectable on SDS-PAGE, signaling cell-binding. As few as 10(10) -10(12) scFv antibodies immobilized on 100 μL of Ni-NTA beads are thus enough for the trapping of enough cells to allow visualization of their abundant proteins. Conceptually, this method could be easily developed and applied to detection of cells bearing any other cell specific antigen.

Screening for recombinant human erythropoietin using [Hb], reticulocytes, the OFFhr score, OFF z score and Hb z score: status of the Blood Passport

Haemoglobin concentration ([Hb]), reticulocyte percentage (retic%) and OFF(hr score) are well-implemented screening tools to determine potential recombinant human erythropoietin (rHuEpo) abuse in athletes. Recently, the International Cycling Union implemented the OFF(z score) and the Hb(z score) in their anti-doping testing programme. The aim of this study is to evaluate the sensitivity of these indirect screening methods. Twenty-four human subjects divided into three groups with eight subjects each (G1; G2 and G3) were injected with rHuEpo. G1 and G2 received rHuEpo for a 4-week period with 2 weeks of "boosting" followed by 2 weeks of "maintenance" and a wash-out period of 3 weeks. G3 received rHuEpo for a 10-week period (boost = 3 weeks; maintenance = 7 weeks; wash out = 1 week). Three, seven and eight of the 24 volunteers exceeded the cut-off limits for OFF(hr score), [Hb] and retic%, respectively. One subject from G1, nobody from G2, and seven subjects from G3 exceeded the cut-off limit for Hb(z score.) In total, ten subjects exceeded the cut-off limit for the OFF(z score); two subjects from G1, two subjects from G2 and six subjects from G3. In total, indirect screening methods were able to indicate rHuEpo injections in 58% of subjects. However, 42% of our rHuEpo-injected subjects were not detected. It should be emphasised that the test frequency in real world anti-doping is far less than the present study, and hence the detection rate will be lower.

Limited value of testing for intrinsic factor antibodies with negative gastric parietal cell antibodies in pernicious anaemia

Background:The appropriate testing strategy for diagnosing pernicious anaemia using gastric parietal cell (GPC) and/or intrinsic factor antibodies (IFA) is controversial. Intrinsic factor antibodies are found in only about 70% of...

Hand-Held Indirect Calorimeter Offers Advantages Compared with Prediction Equations, in a Group of Overweight Women, to Determine Resting Energy Expenditures and Estimated Total Energy Expenditures during Research Screening

ObjectiveTo compare standardized prediction equations to a hand-held indirect calorimeter in estimating resting energy and total energy requirements in overweight women. DesignResting energy expenditure (REE) was measured by hand-held indirect calorimeter and calculated by prediction equations Harris-Benedict, Mifflin-St Jeor, World Health Organization/Food and Agriculture Organization/United Nations University (WHO), and Dietary Reference Intakes (DRI). Physical activity level, assessed by questionnaire, was used to estimate total energy expenditure (TEE). SubjectsSubjects (n=39) were female nonsmokers older than 25 years of age with body mass index more than 25. Statistical analysesRepeated measures analysis of variance, Bland-Altman plot, and fitted regression line of difference. A difference within ±10% of two methods indicated agreement. ResultsSignificant proportional bias was present between hand-held indirect calorimeter and prediction equations for REE and TEE (P<0.01); prediction equations overestimated at lower values and underestimated at higher values. Mean differences (±standard error) for REE and TEE between hand-held indirect calorimeter and Harris-Benedict were −5.98±46.7 kcal/day (P=0.90) and 21.40±75.7 kcal/day (P=0.78); between hand-held indirect calorimeter and Mifflin-St Jeor were 69.93±46.7 kcal/day (P=0.14) and 116.44±75.9 kcal/day (P=0.13); between hand-held indirect calorimeter and WHO were −22.03±48.4 kcal/day (P=0.65) and −15.8±77.9 kcal/day (P=0.84); and between hand-held indirect calorimeter and DRI were 39.65±47.4 kcal/day (P=0.41) and 56.36±85.5 kcal/day (P=0.51). Less than 50% of predictive equation values were within ±10% of hand-held indirect calorimeter values, indicating poor agreement. ConclusionsA significant discrepancy between predicted and measured energy expenditure was observed. Further evaluation of hand-held indirect calorimeter research screening is needed.

Assessing the Reliability of Scores Produced by the Substance Abuse Subtle Screening Inventory

The Substance Abuse Subtle Screening Inventory (SASSI) is a 10 scale indirect screening instrument used to detect substance use disorders. The current meta-analytic study described reliability reporting practices across 48 studies involving the SASSI. Reliability generalization methods were then employed to evaluate typical score reliability for the screening measure. Results showed approximately 73% of studies did not report reliability estimates. Analysis of data from the remaining studies revealed adequate reliability for the total scale (α = .87) and face valid scales (FVA α = .88 and FVOD α = .92), but substantially lower reliability estimates for the indirect scales (range of α = .23–.65). The study's findings underscore the need for improved reliability reporting for the SASSI and suggest cautious use of the measure, especially its indirect scales, as an indicator of problematic substance use/abuse in clinical settings.

Human Metapneumovirus Reinfection among Children in Thailand Determined by ELISA Using Purified Soluble Fusion Protein

Human metapneumovirus (hMPV) is a newly discovered paramyxovirus that causes acute respiratory illness. Despite apparent near-universal exposure during early childhood, immunity is transient. An indirect screening enzyme-linked immunosorbent assay using a recombinant soluble fusion (F) glycoprotein derived from hMPV was used to test for anti-F IgG in 1,380 pairs of acute- and convalescent-stage serum samples collected from children in Kamphaeng Phet, Thailand. Of the 1,380 serum sample pairs tested, 1,376 (99.7%) showed evidence of prior infection with hMPV. Sixty-six paired specimens demonstrated a >or=4-fold rise in titer, for an overall reinfection rate of 4.9%. Two children demonstrated evidence of an initial infection. Forty-eight of the 68 new infections or reinfections occurred in 2000, accounting for 13.2% of all nonflaviviral febrile illnesses in the study population in that year. Of 68 positive cases, 85.3% complained of cough and 66.2% complained of rhinorrhea, compared with 61.4% and 49.0% of negative cases, respectively (P < .01). All positive samples were also tested for an increase in titer of antibodies to respiratory syncytial virus F, and 27% exhibited a >or=4-fold rise. These results demonstrate that hMPV reinfections cause illness at a rate equal to that seen for initial infections. hMPV may have a more significant impact in older children than previously realized and may be the cause of significant outbreaks in this population.

Update on the Genetics of Stroke and Cerebrovascular Disease 2007

The genetic story-of-the-year for 2007 is undoubtedly the dramatic and surprising success of large-scale genome-wide association (GWA) studies in finding consistent and replicable genetic markers of several complex diseases of adulthood. While GWA studies of stroke are on the verge of being reported, much can be learned already from reports of other complex diseases with robust genetic associations identified since the spring of 2007. These have included coronary artery disease (CAD),1–6 type 2 diabetes,3,7–9 rheumatoid arthritis,3,10 bipolar disorder,3 Crohn disease,3 multiple sclerosis11 and amyotrophic lateral sclerosis.12 Cumulatively, these findings have begun to transform our understanding of the genetics of complex diseases. Also, these studies might help discover new biological mechanisms, diagnostic methods and treatments for complex adult diseases.13–15 Genetic association studies, in their most common incarnation, compare cases and matched controls at the genomic level. If resources and computing power permitted, the most thorough comparison would involve direct sequence analysis of the individual 3.2 billion nucleotide bases per human genome and detection of patterns that differ statistically between the genomes of cases and controls. Although we are still a few years away from such studies, in September 2007 the first complete genomic sequence of a single human being was reported.16 This tour-de-force provided proof-of-concept that complete genomic sequencing is possible, albeit at a cost of tens of millions of dollars and hundreds of human-years of effort. However, the cost and time are expected to fall markedly and quickly. In the absence of complete directly determined individual sequence data, current comparative cases and control studies must use indirect screening sets of DNA markers. The most frequently used markers are derived from commonly occurring single letter changes in the DNA sequence, called single nucleotide polymorphisms (SNPs). SNPs can be chosen from within candidate …

Prebreeding in Common Bean and Use of Genetic Diversity from Wild Germplasm

ABSTRACTCommon bean (Phaseolus vulgaris L.) is the most widely consumed grain legume in the world. This cultigen was domesticated from wild P. vulgaris, an indeterminate viny plant, distributed from Mexico to Argentina in midaltitude neotropical and subtropical regions. To colonize such diverse ecological niches, the species possesses many adaptation traits and a wealth of genetic diversity. However, breeding programs are limited by the under‐utilization of the available genetic diversity because of the necessity of prebreeding exotic material. Due to partial reproductive isolation between the domesticated Andean and Mesoamerican gene pools, hybridizations between wild and domesticated types of P. vulgaris from the same gene pool offer greater potential to enhance the variation in the crop. Evaluations of wild P. vulgaris accessions have shown resistance to insects and diseases and higher N, Fe, and Ca content in seeds, which will ultimately contribute to improvements in nutritional quality and yield. Recurrent and inbred backcross methods are being used for the transfer of both qualitative and quantitative traits from wild into domesticated forms of P. vulgaris; specific data on yield and 100‐seed weight are presented. The prebreeding efforts will be enhanced by (i) information on gene pool origins, domestication syndrome traits, molecular diversity, and mapping data of the wild forms; (ii) indirect screening for biotic and abiotic stresses; and (iii) marker‐assisted selection.

Comparison of hybridoma screening methods for the efficient detection of high-affinity hapten-specific monoclonal antibodies

This study compares diverse microplate-based hybridoma screening methods for the generation of hapten-(aflatoxin-) specific monoclonal antibodies (MAbs). Standard indirect enzyme-linked immunosorbent assay (ELISA) screenings (with immobilization of hapten–protein conjugate and use of enzyme-labeled anti-mouse IgG as tracer) were compared with direct ELISAs (with antibody immobilization and use of a hapten-enzyme conjugate as tracer). Although direct ELISA is rarely used for routine hybridoma screenings, it showed considerable advantages compared to the indirect assays. Standard indirect ELISA screening can lead to a considerable number of false positives (up to about 50% false positives of all 373 supernatants tested) if the antibody concentrations in the supernatants are too high. Direct ELISAs gave useful screening results for the different supernatant dilutions chosen. At most 3 false positives were detected out of 373 supernatants. However, the sensitivity of the direct ELISA screening is generally lower compared to indirect ELISA, and individual high-affinity MAbs might be classified as false negative. Therefore, a modified indirect ELISA screening was also developed. It includes pre-incubation of the supernatants in anti-mouse IgG-coated microplates which are then transferred into the (indirect) hapten conjugate-coated microplates. This screening method leads to excellent results with good overall selectivity and sensitivity. It can also be conveniently combined with the direct ELISA screening. Using these improved screening methods, aflatoxin-specific MAbs could be generated with IC50 values down to 3 ng/l (aflatoxin concentration).

Mass spectrometry-based biochemical assays for enzyme-inhibitor screening

Screening for inhibitors of pharmacologically-relevant enzymes is in many cases an important starting point in drug discovery. While fluorescence-based detection techniques play an important role in high-throughput screening, mass spectrometry (MS)-based assays have gained in importance in recent years. The current review focuses on the different assay formats and applications of MS-based enzyme-inhibitor screening. MS-based assays can be categorized into direct and indirect screening methods. Direct screening methods isolate and detect the enzyme-inhibitor complex or the inhibitor to prove activity against the drug target. Active compounds that form enzyme-inhibitor complexes can be analyzed directly by MS as intact complex (direct infusion), or after isolation from the unbound (non-active) compounds by capillary isoelectric focusing (CIEF), frontal affinity chromatography (FAC), size-exclusion chromatography (SEC), immobilized enzymes or ultrafiltration (UF), and dissociation. Indirect screening methods rely on detection of reporter molecules to measure enzyme-inhibitory activity of analytes. When using native substrates as reporter molecules, the assay provides a functional response rather than relying on detection of inhibitor binding alone. Indirect screening for activity is performed by continuous-flow systems (CFSs), flow-injection analysis (FIA), high-performance liquid chromatography (HPLC) or matrix-assisted laser desorption/ionization (MALDI), coupled to MS. MS-based screening methods can screen various kinds of samples (e.g., combinatorial libraries and natural-product extracts), screen large libraries, provide activity information, mass information, structural information and affinity data, and achieve high throughputs.

Contribution of genetic analysis in screening for MEN1 among patients with sporadic disease and one or more typical manifestation

Multiple Endocrine Neoplasia type 1 (MEN1) is an autosomal dominant hereditary syndrome (OMIM 131100) due to MEN1 gene mutations, predisposing to the development of hyperplasic and tumoral lesions of neuroendocrine tissues. Since the identification of the gene in 1997, more than 400 different mutations of MEN1 have been registered. Genotypic analysis of MEN1 remains fastidious and must be reserved to targeted situations. If the lesions appear in a familial assessed context, there is a strong argument to search for MEN1 mutation. This is not the case in a sporadic context. With experience acquired in our laboratory, we evaluated the frequency of MEN1 mutations in patients with sporadic presentations. Our aim was to better define criteria for MEN1 genotypic analysis. One hundred and twenty four blood samples from unrelated patients, who gave their written informed consent, were analyzed. These patients exhibited 1 to 4 manifestations of MEN1 without any familial context. After DNA extraction, the analysis was undertaken by PCR-sequencing of all the MEN1 coding exons and exon/intron boundaries or by PCR of the pre-screened fragments alone, a technique made possible by indirect screening mutation methods. Mutations were identified by comparing the sequences to the reference MEN1 sequence available from GENBANK (U93237.1). Mutations were identified in 19 patients, with variable prevalence according to clinical manifestations: 100% for patients with 4 manifestations, 45.5% for patients with 3 manifestations, 19% for patients with 2 manifestations and 2% for patients with only one manifestation. Mutations were: 11 point variations (58%), including 2 splicing sites and 8 frameshift mutations (42%) including 5 deletions, 2 insertions and 1 insertion/deletion; one mutation was identified twice. We showed a relationship between clinical presentation and MEN1 mutation identification, especially with the number of clinical manifestations but also with the type of manifestation. Pancreatic manifestations were significantly linked with probability of mutation. In a sporadic context with at least two established manifestations of MEN1, the overall probability of identifying a mutation was 26%, warranting MEN1 genotypic analysis.