Probability Of False Negative Results Research Articles

Considerations about Reducing False-Negative PCR Test for COVID-19.

Polymerase chain reaction (PCR) for the detection of nucleic acids is the gold standard test for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is the probability of false-negative results with this test, which poses a threat to public health. Here, we highlight some important factors that should be considered for reducing the false-negative results of the SARS-CoV-2 PCR test.

Optimal group testing strategy for the mass screening of SARS-CoV-2

We analyze the group testing strategy that maximizes the efficiency of the SARS-CoV-2 screening test while ensuring its effectiveness, where the effectiveness of group testing guarantees that negative results from pooled samples can be considered presumptive negative. Two aspects of test efficiency are considered, one concerning the maximization of the welfare throughput and the other concerning the maximization of the identification rate (namely, identifying as many infected individuals as possible). We show that compared with individual testing, group testing leads to a higher probability of false negative results but a lower probability of false positive results. To ensure the test effectiveness, both the group size and the prevalence of SARS-CoV-2 must be below certain respective thresholds. To achieve test efficiency that concerns either the welfare throughput maximization or the identification rate maximization, the optimal group size is jointly determined by the test accuracy parameters, the infection prevalence rate, and the relative importance of identifying infected subjects. We also show that the optimal group size that maximizes the welfare throughput is weakly smaller than the one that maximizes the identification rate.

Occupancy modeling and resampling overcomes low test sensitivity to produce accurate SARS-CoV-2 prevalence estimates

BackgroundWe evaluated whether occupancy modeling, an approach developed for detecting rare wildlife species, could overcome inherent accuracy limitations associated with rapid disease tests to generate fast, accurate, and affordable SARS-CoV-2 prevalence estimates. Occupancy modeling uses repeated sampling to estimate probability of false negative results, like those linked to rapid tests, for generating unbiased prevalence estimates.MethodsWe developed a simulation study to estimate SARS-CoV-2 prevalence using rapid, low-sensitivity, low-cost tests and slower, high-sensitivity, higher cost tests across a range of disease prevalence and sampling strategies.ResultsOccupancy modeling overcame the low sensitivity of rapid tests to generate prevalence estimates comparable to more accurate, slower tests. Moreover, minimal repeated sampling was required to offset low test sensitivity at low disease prevalence (0.1%), when rapid testing is most critical for informing disease management.ConclusionsOccupancy modeling enables the use of rapid tests to provide accurate, affordable, real-time estimates of the prevalence of emerging infectious diseases like SARS-CoV-2.

SARC-F as a case-finding tool for sarcopenia according to the EWGSOP2. National validation and comparison with other diagnostic standards

BackgroundSarcopenia is a potentially reversible condition, which requires proper screening and diagnosis.AimsTo validate a Polish version of sarcopenia screening questionnaire (SARC-F), and assess its clinical performance.MethodsCross-sectional validation study in community-dwelling subjects ≥ 65 years of age. Diagnosis of sarcopenia was based on the 2018 2nd European Working Group on Sarcopenia in Older People (EWGSOP2) consensus. Hand grip and 4-m gait speed were measured, and the Polish version of SARC-F was administered.ResultsThe mean (SD) age of 73 participants (21.9% men) was 77.8 (7.3) years. Seventeen participants (23.3%) fulfilled the EWGSOP2 criteria of sarcopenia, and 9 (12.3%) criteria for severe sarcopenia. Fourteen (19.2%) participants fulfilled the SARC-F criteria for clinical suspicion of sarcopenia. The Cronbach’s alpha coefficient for internal was 0.84. With EWGSOP2 sarcopenia as a gold standard, the sensitivity of SARC-F was 35.3% (95% CI 14.2–61.7, p = 0.33), specificity was 85.7% (95% CI 73.8–93.6, p < 0.0001). The corresponding positive and negative predictive values were 42.9% (p = 0.79) and 81.4% (p < 0.0001), respectively. The probability of false-positive result was 14.3% (95% CI 6.4–26.2, p < 0.0001) and the probability of false-negative result was 64.7% (95% CI 38.3–85.8, p = 0.33). Overall the predictive power of SARC-F was low (c-statistic 0.64).DiscussionSARC-F is currently recommended for sarcopenia case finding in general population of older adults. However, its sensitivity is low, despite high specificity.ConclusionsAt present SARC-F is better suited to rule out sarcopenia then to case-finding. Further refinement of screening for sarcopenia with the use of SARC-F seems needed.

Real-life validation of the Panbio™ COVID-19 antigen rapid test (Abbott) in community-dwelling subjects with symptoms of potential SARS-CoV-2 infection.

BackgroundRT-qPCR is the reference test for identification of active SARS-CoV-2 infection, but is associated with diagnostic delay. Antigen detection assays can generate results within 20 min and outside of laboratory settings. Yet, their diagnostic test performance in real life settings has not been determined.MethodsThe diagnostic value of the Panbio™ COVID-19 Ag Rapid Test (Abbott), was determined in comparison to RT-qPCR (Seegene Allplex) in community-dwelling mildly symptomatic subjects in a medium (Utrecht, the Netherlands) and high endemic area (Aruba), using two concurrently obtained nasopharyngeal swabs.Findings: 1367 and 208 subjects were enrolled in Utrecht and Aruba, respectively. SARS-CoV-2 prevalence, based on RT-qPCR, was 10.2% (n = 139) and 30.3% (n = 63) in Utrecht and Aruba respectively. Specificity of the Panbio™ COVID-19 Ag Rapid Test was 100% (95%CI: 99.7–100%) in both settings. Test sensitivity was 72.6% (95%CI: 64.5–79.9%) in the Netherlands and 81.0% (95% CI: 69.0–89.8%) in Aruba. Probability of false negative results was associated with RT-qPCR Ct-values, but not with duration of symptoms. Restricting RT-qPCR test positivity to Ct-values <32 yielded test sensitivities of 95.2% (95%CI: 89.3–98.5%) in Utrecht and 98.0% (95%CI: 89.2–99.95%) in Aruba.InterpretationIn community-dwelling subjects with mild respiratory symptoms the Panbio™ COVID-19 Ag Rapid Test had 100% specificity, and a sensitivity above 95% for nasopharyngeal samples when using Ct-values <32 cycles as cut-off for RT-qPCR test positivity. Considering short turnaround times, user friendliness, low costs and opportunities for decentralized testing, this test can improve our efforts to control transmission of SARS-CoV-2.

COVID-19 Testing.

ObjectivesTo illustrate how patient risk and clinical costs are driven by false-positive and false-negative results.MethodsMolecular, antigen, and antibody testing are the mainstay to identify infected patients and fight the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To evaluate the test methods, sensitivity (percent positive agreement [PPA]) and specificity (percent negative agreement [PNA]) are the most common metrics utilized, followed by the positive and negative predictive value—the probability that a positive or negative test result represents a true positive or negative patient. The number, probability, and cost of false results are driven by combinations of prevalence, PPA, and PNA of the individual test selected by the laboratory.ResultsMolecular and antigen tests that detect the presence of the virus are relevant in the acute phase only. Serologic assays detect antibodies to SARS-CoV-2 in the recovering and recovered phase. Each testing methodology has its advantages and disadvantages.ConclusionsWe demonstrate the value of reporting probability of false-positive results, probability of false-negative results, and costs to patients and health care. These risk metrics can be calculated from the risk drivers of PPA and PNA combined with estimates of prevalence, cost, and Reff number (people infected by 1 positive SARS-CoV-2 carrier).

Probability of false-negative results in microscopical detection of cutaneous leishmaniasis: more accurate screening by kDNA-PCR during epidemiological survey.

Leishmaniasis has an important impact on global public health, and the common form of thedisease is cutaneous form as well in Iran. Different species of Leishmania parasite make variable clinical manifestations, so prompt diagnosis and recognition at thespecies level are important due to their impact on the treatment and outcome of the disease.We aimed to examine the potential existence ofthe Leishmania parasite genome in the exudate materials derived from lesions of the cutaneous leishmaniasis suspected patients referred to Varamin Health Center Laboratory, that were reported negative microscopically. Regarding the object of the study, kDNA-Nested-PCR was used.A 570bp band equal to what expected for Leishmania major was amplified in 18 out of 29 tested samples (62%). Findings indicate the effectivness of kDNA as a high copy number gene to avoid false-negative results.

The role of stimulated thyroglobulin in the prognosis of persistent papillary thyroid carcinoma

Objective —to study the incidence of regional metastases after radioiodine therapy in the absence of routine prophylactic neck dissection in patients with papillary thyroid carcinoma (PTC), depending on the levels of stimulated thyroglobulin (TG) and its use for predicting disease persistence. Material and methods. Treatment outcomes were investigated for 738 patients (78.2 % female, 21.8 % male; mean age 49.1 ± 11.9 years) with PTC who underwent thyroidectomy with neck dissection of varying volume in medical institutions of Ukraine with further radioiodine therapy. Risk groups are formed according to ATA 2015 recommendations. The stimulated TG testing was conducted in different laboratories in Ukraine and by different methods, but with comparable reference ranges. Statistical calculationswere done woth MSExcel, AtteStat and StatPlus 7 (AnalystSoftInc.), ROCanalysis, Z-test and Fisher-Freeman-Halton exact test.Particle confidence intervals were determined using Wilson method and VassarStats online calculator. Results and discussion. 738 patients were examined, of whom 22.6 %, 47.3 % and 30.1 % were in the low, medium, and high-risk groups. After the 131I therapy and SPECT (single-photon emission computed tomography) incidence of metastases was 19.1 % overall (2.6 % iodine negative), and 10.8 %, 16.3 %, and 29.7 %, respectively, depending on the TG level. Choosing a cut-off threshold for TG = 0.5 ng/ml (without taking into account the risk group) provides a minimum probability of missing metastases (4.96 %, 95 % CI: 2.42—9.89). In total, 148 (20.1 %) patients were identified in the total of 738 subjects with TG stimulated levels < 0.5 ng/ml, of which 7 (0.9 %) had metastasized after 131I. In 590 (79.9 %) patients with TG ≥ 0.5 ng/ml, 134 (18.2 %) had metastases, while in the low-risk group, 18 (10.8 %) had metastases, including 4 (2.3 %/22.2 %) were found to be iodine-negative. In 12 patients in the TG subgroup < 0.5 ng/ml, 4 (0.54 %/2.4 %) cases with metastases were identified, whereas in the TG subgroup ≥ 0.5 ng/ml, 14 (1,90 %/8.38 %). In the intermediate-risk group, 57 (16.33 %) patients had metastases and all of them were in the subgroup with TG ≥ 0.5 ng/ml. Patients with iodine-positive metastases had higher TG values than patients with iodine-negative ones, but the difference was not statistically significant. The highest incidence of metastases was recorded in the high-risk group, in which 66 (8.94 %/29.73 %) were detected. In all risk groups at TG values < 0.5 ng/ml, the detectability of metastases was low and practically independent of risk level (p > 0.05).At TG values ≥ 0.5 ng / ml, the difference in the detection of metastases between subgroups withmetastaticpositive and metastaticnegative were statistically significant: p < 0.05 in the low-risk group and p < 0.001 in the medium- and high-risk groups. Соnclusions. After thyroidectomy without routine neck dissection, the incidence of metastases in whole body 131I scintigraphy and SPECT was relatively high in all stratification risk groups (low — 10.8 %, medium — 16.3 %, high — 29.7 %). An elevated level of stimulated TG can be considered as a predictor of metastasis, which can be used to individualize further tactics for the treatment of patients with PTC. When the cut-off threshold TG = 0.5 ng/ml is selected, the highest sensitivity(0.95) and the predictive value of the negative result (-PV = 0.953) are provided, specificity and overall diagnostic efficiency are 0.236 and 37.26 %, respectively. Almost the same sensitivity and specificity (0.794 and 0.784, respectively) are reached at the cut-off threshold TG = 7.5 ng/ml (DE = 78.59 %). Regardless of the stratification risk groups in patients with stimulated TG up to 0.5 ng/ml, the probability of a false-negative result is 4.96 %, the detection of metastasis is quite low (0.54 %/0 %/0.41 %) and the need for radioiodine therapy is questionable. In patients with low and intermediate risk, postoperative values from 1.0 to 10.0 ng/ml are optimistic, but they do not completely exclude the detection of metastases (the probability of false-negative result is 6.4—24.2 %). With postoperative values of stimulated TG ≥ 10 ng/ml, the likelihood of persistence increases (probability of false-negative result > 26.2 %), which requires additional therapy.

Risk of false compliance/non-compliance decisions for sterility test due to false-negative and false-positive test results

The sterility conditions of pharmaceutical products should consider the manufacturing conditions and the result obtained from a sterility test. The statistical and analytical limitations of the sterility test may lead to false-negative or false-positive results. Experimental determination of the probability of false-negative and false-positive results requires a very large number of assays, which may be impracticable. Thus, the aim of this work was to estimate the risk of false decisions related to sterility test due to the false-negative or false-positive test results, using a theoretical approach. The probability of false-negative results was estimated by considering the expected level of contamination in the sample, the tested volume for each item, the total volume of each item, and the number of tested items. The probability of false-positive results due to environmental contamination was estimated by considering the microbial load per plate, the sampled air volume, the total volume of the safety cabinet, the number of air changes per hour, and the volume of the test tube used in the sterility test. A MS-Excel spreadsheet provided in supplementary material can be used to estimate the probability of false-negative or false-positive test results. The probabilities of false-negative and false-positive results considering the experimental conditions were found to be 0.0045% and 0.070%, respectively. The probabilities of false-negative or false-positive test results may be combined with previous knowledge about the probability of a non-sterile lot (Sterility Assurance Level – SAL) using the Bayesian approach. The Bayesian approach allows the risk of false compliance/non-compliance decisions to be estimated. Risks to consumer and producer are significantly affected by prior knowledge of the sterility assurance level.

Tuberculin skin test and predictive host factors for false-negative results in patients with pulmonary and extrapulmonary tuberculosis.

Tuberculin skin test (TST) has been the standard test for screening for Mycobacterium tuberculosis infection for decades. Identifying persons with latent tuberculosis infection (LTBI) is crucial, as they constitute a reservoir that sustains the global tuberculosis (TB) epidemic. However, different factors, such as HIV infection, can lower the sensitivity of the test. The aim of this study was to determine the TST sensitivity in active TB patients and to ascertain risk factors that could be associated with false-negative results. Retrospective cohort study of all active TB notifications with a TST result (n=8833), from 2008 to 2015. TST results were interpreted using a 5mm and 10mm cutoff. Bivariate and multivariate logistic regression analysis were used to evaluate the association of sociodemographic and clinical factors with false-negative TST results and to develop predictive risk models. TST presented an overall sensitivity of 63.8% (5mm) and 56.1% (10mm). HIV infection was the risk factor with the strongest association with false-negative results (aOR 4.65-5mm; aOR 5.05-10mm). Other factors such as chronic renal failure (CRF) (aOR 1.55-5mm; aOR 1.73-10mm), alcohol abuse (aOR 1.52-5mm; aOR 1.31-10mm), drug abuse (aOR 1.90-5mm; aOR 1.76-10mm) or age ≥65years (OR 1.69-5mm and 10mm) were also associated with a probability of false-negative results. These results highlight the importance of knowing which factors influence TST results, such as HIV status, substance abuse or age, thus improving its usefulness as a screening method for LTBI.

Study design, errors and sample size calculation in medical research

The choice of an appropriate study design is one of the crucial steps in the research process after framing a research question. A single research question may fit into different study designs. Each design has its own merits and drawbacks; diligence in implementing the methodology and data collection reflects good study design. Sample size justification and power analysis are foundations of a study design. They should ideally be settled when framing a research question and creating the study design. An adequate sample size minimises random error or chance occurrence. 'A just large enough' sample supports the researcher to estimate expected cost, time and feasibility. The sample 'size' is a tug-of-war between reality and scientific effectiveness and is highly influenced by study designs. Null hypothesis (H0) is the assumption that there is no difference in the treatment groups, whereas an assumption that there is a difference is called alternate hypothesis (Ha). Type I error (α) finds difference in the absence of one (false-positive conclusion), whereas Type II error (β) indicates probability of false-negative results. If the calculated P value is smaller than α, the researcher rejects the null hypothesis (H0) and welcomes the alternative hypothesis (Ha). There are several validated software available for sample size calculation. Sample size tends to be smaller for means than percentages. As the sample size increases, the P value tends to become small. Finally, a statistically significant result might not always be clinically relevant.

Diagnostic Accuracy of Age-Adjusted D-Dimer Cutoff in Suspected Cases of Pulmonary Embolism

IntroductionCurrent guidelines recommend use of clinical pretest probability scores, d-dimer assay, and imaging studies in the diagnosis of pulmonary embolism (PE). Studies have shown that normal d-dimer levels with low-intermediate probability in Wells score can safely exclude PE in most cases. Usually, d-dimer level of less than 500 µg/L is used to decide that no further imaging studies such as computed tomography pulmonary angiography (CTPA) or ventilation perfusion (V/Q) scan) are required. However, the conventional cutoff of 500 µg/L may not be as specific in all patients as d-dimer levels increase with age. Thus, use of age-adjusted d-dimer cutoff has been advocated which is defined as (age × 10) µg/L in patients 50 years or older.ObjectiveTo study if age-adjusted d-dimer is helpful in decreasing imaging studies without missing the diagnosis of PE in suspected cases.MethodsWe did an observational retrospective study of all consecutive adult patients who presented to the emergency department from January 2014 to December 2016. The patients who were clinically suspected with PE and underwent subsequent workup along with d-dimer assays were included for analysis. The number of imaging studies performed for PE diagnosis were recorded.ResultsWe included 3,197 patients in the study. Median age was 55 years, and 67% were females (Table 1). Total 2,937 (92%) patients had elevated d-dimer levels based on conventional d-dimer cutoff compared to 2,526 (79%) patients based on age-adjusted d-dimer cutoff. The age-adjusted d-dimer cutoff was more specific (21% vs 8%, p-value<0.0001) but less sensitive (100% vs 95%, p-value= 0.15) than conventional cutoff (Table 2). The use of age-adjusted d-dimer cutoff would have avoided 409 (13% of total) imaging studies (Table 3). However, 2 cases (0.7% of total) with PE, which would have underwent imaging study with conventional cutoff, would have been missed with age-adjusted d-dimer cutoff. The Wells score was 3, intermediate pretest probability, for both cases of PE.ConclusionAge-adjusted d-dimer cutoff can significantly decrease the number of imaging studies performed in cases of suspected PE. However, as shown in our study, there is a chance of missing cases with actual PE. Thus, clinicians should always be aware of a rare probability of false negative result with age-adjusted d-dimer levels. Further stratification of the probability risk or the prompt use of imaging studies in cases with higher clinical suspicion can help in accurate diagnosis of PE. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vigilancia tecnológica e inteligencia competitiva de un desarrollo tecnológico para la detección deGiardia, una innovación en salud

RESUMENLa giardiasis es una enfermedad causada por Giardia duodenalis, protozoario enteropatógeno que también es responsable de la enfermedad diarreica aguda y la gastroenteritis en humanos. Dadas las características de excreción intermitente propias de este parásito, la probabilidad de obtener resultados falsos negativos durante el proceso de diagnóstico es alta. En el Grupo de Parasitología del Instituto Nacional de Salud de Colombia se identificaron proteínas inmunogénicas presentes en quistes y trofozoítos del parásito en aislamientos colombianos de Giardia con potencial comercial y posibilidad de ser patentadas. Se realizó un estudio exploratorio descriptivo a partir de las características de la técnica en estudio, su estado de desarrollo y la viabilidad comercial de la invención. Se encontraron 390 patentes y se seleccionaron cinco invenciones similares que no representaban ningún riesgo frente a una eventual solicitud de patente. De los 36 estuches para el diagnóstico de Giardia disponibles en el mercado y las 21 licencias sanitarias otorgadas hasta diciembre del 2017, sólo cuatro y tres, respectivamente, proponen técnicas similares a la de la estudiada, pero ninguna de ellas cuenta con un registro de patente relacionado. La técnica en desarrollo es innovadora y puede aplicarse en el campo de la salud pública en la Región de las Américas con beneficios previsibles.

Prospective Comparison of F-18 Choline PET/CT Scan Versus Axial MRI for Detecting Bone Metastasis in Biochemically Relapsed Prostate Cancer Patients.

We compared fluor-18 choline positron emission tomography/computed tomography (PET/CT) and axial skeleton magnetic resonance imaging (MRI) prospectively obtained for the detection of bone metastases in non-castrated patients with biochemically recurrent prostate cancer following primary treatment. PET/CT was performed 45 min post-injection of 3–4 MBq/kg F-18 methyl choline. MRI included T1- and fluid sensitive T2-weighted images of the spine and pelvis. Readers were initially blinded from other results and all scans underwent independent double reading. The best valuable comparator (BVC) defined the metastatic status. On the basis of the BVC, 15 out of 64 patients presented with 24 bone metastases. On a patient level, the sensitivity and specificity of MRI and PET were not significantly different. On a lesion level, the sensitivity of MRI was significantly better compared to PET, and the specificity did not differ significantly. In conclusion, axial MRI is an interesting screening tool for the detection of bone metastases because of its low probability of false negative results. However, F-18 choline PET is a valuable addition as it can overrule false positive MRI results and detect non-axial metastases.

Validation of the accuracy of needle placement as used in diagnostic local analgesia of the maxillary nerve for investigation of trigeminally mediated headshaking in horses

Diagnostic local anaesthesia of the maxillary nerve is a valuable aid in the diagnosis of trigeminally mediated headshaking in horses. Our objective is to validate the accuracy of needle placement...

Performance criteria for rapid screening methods to detect mycotoxins

The paper describes the validation of screening methods that are used for official control to classify samples into negative and suspect positive samples. The concept is based on the principle that negative samples are considered as compliant, whereas suspect positive samples need to be re-analysed with confirmatory methods. An important performance criterion often used is a maximum value of 5% for the probability of false negative results obtained on samples that contain the analyte at the legal limit. Since the result of analysis is a binary decision, specific validation schemes need to be applied. The paper places emphasis on practical aspects of the calculation of the method performance characteristics, which are required to check whether the methods fulfil the performance criterion. The paper shows that screening methods based on a visual inspection, e.g. a dipstick, require special data treatment. In contrast there are many methods where the classification into negative and suspect positive samples is based on the comparison of a measured response against a cut-off value. This type of methods can be validated with quantitative statistics. The paper also elaborates on the calculation of the rate of false positive results of compliant samples. In addition the impact on the economical aspect of the use of the screening method is estimated, taking into account external factors such as the cost ratio between the screening and the confirmatory method and the occurrence of non-compliant samples in the entire population of the samples.

The Majority of MicroRNAs Detectable in Serum and Saliva Is Concentrated in Exosomes

There is an increasing interest in using microRNAs (miRNA) as biomarkers in autoimmune diseases. They are easily accessible in many body fluids but it is controversial if they are circulating freely or are encapsulated in microvesicles, particularly exosomes. We investigated if the majority of miRNas in serum and saliva are free-circulating or concentrated in exosomes. Exosomes were isolated by ultracentrifugation from fresh and frozen human serum and saliva. The amount of selected miRNAs extracted from the exosomal pellet and the exosome-depleted serum and saliva was compared by quantitative RT-PCR. Some miRNAs tested are ubiquitously expressed, others were previously reported as biomarkers. We included miRNAs previously reported to be free circulating and some thought to be exosome specific. The purity of exosome fraction was confirmed by electronmicroscopy and western blot. The concentration of miRNAs was consistently higher in the exosome pellet compared to the exosome-depleted supernatant. We obtained the same results using an equal volume or equal amount of total RNA as input of the RT-qPCR. The concentration of miRNA in whole, unfractionated serum, was between the exosomal pellet and the exosome-depleted supernatant. Selected miRNAs, which were detectable in exosomes, were undetectable in whole serum and the exosome-depleted supernantant. Exosome isolation improves the sensitivity of miRNA amplification from human biologic fluids. Exosomal miRNA should be the starting point for early biomarker studies to reduce the probability of false negative results involving low abundance miRNAs that may be missed by using unfractionated serum or saliva.

Radioligand binding assays in the drug discovery process: potential pitfalls of high throughput screenings.

Radioligand binding assays evaluating directly the ability of a drug to interact with a defined molecular target is part of the drug discovery process. The need for a high throughput rate in screening drugs is actually leading to simplified experimental schemes that increase the probability of false negative results. Special concern involves voltage-gated ion channel drug discovery where a great care is required in designing assays because of frequent multiplicity of (interacting) binding sites. To clearly illustrate this situation, three different assays used in the academic drug discovery program of the authors were selected because they are rich of intrinsic artifacts: (I) (20 mmol/l caffeine almost duplicated [3H]ryanodine binding (89% higher than control) to rat heart microsomes at 0.3 mumol/l free calcium but did not exert any effect when using a high (107 mumol/l) free calcium, as mostly used in ryanodine binding assays; (II) An agonist for the ionotropic glutamate receptor of the kainate type can distinctly affect [3H]kainate binding to chicken cerebellum membranes depending on its concentration: unlabelled kainic acid per se either stimulated about 30% (at 50-100 nmol/l), had no effect (at 200 nmol/l) or even progressively decreased (at 0.3-2 mumol/l) the binding of 5 nmol/l [3H]kainate, emphasizing the risk of using a single concentration for screening a drug; (III) in a classical [3H]flunitrazepam binding assay, the stimulatory effect of a GABA (gamma-aminobutyric acid) agonist was only observed when using extensively washed rat brain synaptosomes (10 mumol/l GABA increased flunitrazepam binding by 90%). On the other hand, the inhibitory effect of a GABA antagonist was only observed when using crude synaptosomes (10 mumol/l bicuculine reduced [3H]flunitrazepam binding by 40%). It can be concluded that carefully designed radioligand assays which can be performed in an academic laboratory are appropriate for screening a small number of drugs, especially if these are potential hits because of their rational design. Therefore, the low throughput rate could be partially balanced by a higher performance when compared to what is done in a robotic high throughput screening where simplification of assay conditions can lead to false negative results.

Herd-level prevalence of Johne's disease in Utah and adjacent areas of the intermountain west as detected by a bulk-tank milk surveillance project

The objectives of this study were to estimate the dairy herd-level prevalence of Johne's disease (JD) in Utah and nearby areas of the intermountain west and to estimate the sensitivity of a single bulk-tank milk test for JD detection. Two milk samples from all bulk tanks on the study farms were collected 1 mo apart. Samples were frozen and shipped to a laboratory for JD testing. An ELISA to measure total IgG antibody specific against Mycobacterium avium ssp. paratuberculosis, the etiological agent that causes JD, and a quantitative real-time PCR to detect M. avium ssp. paratuberculosis DNA were used; both tests were designed for bulk milk. Of the dairy farms in the study area, 170/246 (69%) participated. Positive JD results were found in bulk milk from 67/170 (39%) of dairy farms in Utah and adjacent areas. There were 138 JD-positive bulk-tank results from 241 bulk-tank samples from the 67 positive herds. The sensitivity of the bulk milk testing for detection of JD was 138/241(57%). From the 103 JD-negative farms, 235 bulk-tank samples tested negative for JD. The probability of false-negative results on a single bulk-milk sample was (1 – 0.57)=0.43. For farms with 1 bulk tank, 2 samples collected 1 mo apart, with both samples testing negative (by both ELISA and quantitative real-time PCR) for JD, the true-negative probability was [1 – (0.43)2]=(1 – 0.18)=82%. For farms with at least 2 bulk tanks, at least 4 samples tested, with all results negative for JD, the true-negative probability was at least 97%. Results support other estimates that prevalence of JD has increased over the last 15 to 20 yr. However, the prevalence detected was 3 times that from a recent report where 13% of dairy herds in the western US were positive. The increase in JD suggests that current control programs, at least as applied, are not effective. Bulk milk testing is a practical way to screen dairy herds for presence of JD. Studies are needed regarding the use of individual cow milk tests for accuracy, practicality, and effectiveness in reducing the prevalence of JD in dairy herds.

Study of binding affinity and selectivity of perylene and coronene derivatives towards duplex and quadruplex DNA by ESI‐MS

In this paper, we report an extensive electrospray ionization mass spectrometry (ESI-MS) study of the noncovalent interactions between different intermolecular and intramolecular G-quadruplex structures and several perylene and coronene ligands. The selectivity of these compounds toward quadruplex structures with respect to duplex DNA, a fundamental topic for the biological evaluation and the pharmacological application of these ligands as potential chemotherapeutic agents, has also been investigated. After exploring this topic according to the classical approach based on the very simple duplex model of an autocomplementary dodecamer, we extended our analysis reporting for the first time a competition ESI-MS experiment in the presence of genomic DNA fragments. Whereas those ligands showing a high level of selectivity between quadruplex and duplex oligonucleotides, in terms of binding constants and percentage of bound DNA, confirmed their selectivity in the competition experiment, the contrary was not always true: some ligands showing poor selectivity with the autocomplementary dodecamer resulted selective in the presence of genomic DNA fragments. This result suggests that physiologically nonrelevant interactions are possible with a short duplex oligonucleotide. This means that the dodecamer can fail in representing a biologically significant structural model, or, better, that it can be used to quickly screen potentially selective molecules, but bearing in mind the high probability of false negative results.