Conventional Screening Procedures Research Articles

Advancements in biosensors for cancer detection: revolutionizing diagnostics.

Cancer stands as the reigning champion of life-threatening diseases, casting a shadow with the highest global mortality rate. Unleashing the power of early cancer treatment is a vital weapon in the battle for efficient and positive outcomes. Yet, conventional screening procedures wield limitations of exorbitant costs, time-consuming endeavors, and impracticality for repeated testing. Enter bio-marker-based cancer diagnostics, which emerge as a formidable force in the realm of early detection, disease progression assessment, and ultimate cancer therapy. These remarkable devices boast a reputation for their exceptional sensitivity, streamlined setup requirements, and lightning fast response times. In this study, we embark on a captivating exploration of the most recent advancements and enhancements in the field of electrochemical marvels, targeting the detection of numerous cancer biomarkers. With each breakthrough, we inch closer to a future where cancer's grip on humanity weakens, guided by the promise of personalized treatment and improved patient outcomes. Together, we unravel the mysteries that cancer conceals and illuminate a path toward triumph against this daunting adversary. This study celebrates the relentless pursuit of progress, where electrochemical innovations take center stage in the quest for a world free from the clutches of carcinoma.

Prospects for New Antibiotics Discovered through Genome Analysis

Abstract: Antibiotic-resistant illnesses are on the rise worldwide, and the pipeline for developing new antibiotics is drying up. As a result, researchers need to create novel compounds with antimicrobial action. Recent decades have seen a dearth of novel antibiotics because of the reliance on conventional empirical screening procedures using both natural and synthetic chemicals to find them. There is hope that the massive amount of bacterial genome sequence data that has become accessible since the sequencing of the first bacterial genome more than 20 years ago might help lead to the development of new antibiotic drugs. Genes with significant levels of conservation both within and between bacterial species can be found using comparative genomic techniques; these genes may be involved in essential bacterial functions. Bioactive chemicals found in natural products have been successfully used in treating everything from infectious diseases to cancer, but over the past 20-30 years, the effectiveness of screening methods based on fermentation has decreased. Researchers urgently need answers to the unmet demand for bacterial infection resistance. Now more than ever, with the advent of cheap, high-throughput genomic sequencing technology, natural product discovery can be revitalized. Using bioinformatics, investigators may foretell whether or not a certain microbial strain would generate compounds with novel chemical structures, which may have novel modes of action in inhibiting bacterial growth. This manuscript describes how this potential might be utilised, with a particular emphasis on manipulating the expression of dormant biosynthetic gene clusters that are hypothesised to encode new antibiotics. Additionally, it consolidates the work of the past and the present to utilise bacterial genomic data in the identification and development of new antibiotics.

Microfluidics for 3D Cell and Tissue Cultures: Microfabricative and Ethical Aspects Updates.

The necessity to improve in vitro cell screening assays is becoming ever more important. Pharmaceutical companies, research laboratories and hospitals require technologies that help to speed up conventional screening and therapeutic procedures to produce more data in a short time in a realistic and reliable manner. The design of new solutions for test biomaterials and active molecules is one of the urgent problems of preclinical screening and the limited correlation between in vitro and in vivo data remains one of the major issues. The establishment of the most suitable in vitro model provides reduction in times, costs and, last but not least, in the number of animal experiments as recommended by the 3Rs (replace, reduce, refine) ethical guiding principles for testing involving animals. Although two-dimensional (2D) traditional cell screening assays are generally cheap and practical to manage, they have strong limitations, as cells, within the transition from the three-dimensional (3D) in vivo to the 2D in vitro growth conditions, do not properly mimic the real morphologies and physiology of their native tissues. In the study of human pathologies, especially, animal experiments provide data closer to what happens in the target organ or apparatus, but they imply slow and costly procedures and they generally do not fully accomplish the 3Rs recommendations, i.e., the amount of laboratory animals and the stress that they undergo must be minimized. Microfluidic devices seem to offer different advantages in relation to the mentioned issues. This review aims to describe the critical issues connected with the conventional cells culture and screening procedures, showing what happens in the in vivo physiological micro and nano environment also from a physical point of view. During the discussion, some microfluidic tools and their components are described to explain how these devices can circumvent the actual limitations described in the introduction.

In Vitro Assessment of Bio-Functional Properties from Lactiplantibacillus plantarum Strains.

In recent years, alongside the conventional screening procedures for the evaluation of probiotics for human usage, the pharmaceutical and food industries have encouraged scientific research towards the selection of new probiotic bacterial strains with particular functional features. Therefore, this study intended to explore novel functional properties of five Lactiplantibacillus plantarum strains isolated from bee bread. Specifically, antioxidant, antimicrobial and β-glucosidase activities, exopolysaccharides (EPS) production and the ability to synthesize γ-aminobutyric acid (GABA) were evaluated. The results demonstrated that the investigated L. plantarum strains were effective in inhibiting the growth of some human opportunistic pathogens in vitro (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterococcus faecalis and Staphylococcus aureus). Moreover, the evaluation of antioxidant and β-glucosidase activity and of EPS and GABA production, revealed a different behavior among the strains, testifying how these properties are strongly strain-dependent. This suggests that a careful selection within a given species is important in order to identify appropriate strains for specific biotechnological applications. The results highlighted that the five strains of L. plantarum are promising candidates for application as dietary supplements in the human diet and as microbial cultures in specific food productions.

DNA Marker Screening for High-Risk Non-syndromic Hearing Loss Associated to Gene Mutations

Background:Congenital hearing loss is a debilitating disease affecting 1–3 out of 1,000 live births. According to WHO’s associated figures, in both ears, 278 million people globally suffer from moderate to extreme hearing loss. Most hearing-loss individuals live in developing countries. Many deafness and hearing problems cases were documented in our region. Those cases’ exact cause is still unknown, so we performed this study and aimed to screen DNA in high-risk Non-syndromic hearing loss patients in Arbil city. Methods: This researchscreened 132 blood samples from (80 newborns and 52 individuals) at (Hiwa Institute for deaf and mutes); their ages patients from 14 to 22 years old.MTRNR1genes were performed for molecular detection of mutant genes. The mutation gene wasamplified by multiplex tetra primer PCR. Result:G- mito-1555-F1, mito-1555-R1 (O), mito-1555-F2 (I), and mito-1555-R2 (I) hearing loss mutations were not observed in 132 blood samples from both classes and genotyped in MTRNR1. For mtDNA 12S rRNA mt.1555A>G, no mutant alleles were detected in all of the tests, and no false-positives were identified.Using all primers, fifty-two samples were easily separated on 2% agarose gel; two were outer primers, and others are inner primers. Two separate bands were observed with 52 molecular samples (wild type at 254bp and control at 341bp).Of 80 samples, 28 have control bands at 341 bp. We did not find any mutation in our 80 samples. Conclusion: MTRNR1 mutation genes were not present in collected samples in deafness-related mutation. Genetic tests for the deafness gene can better diagnose infant congenital NSHL cases than conventional screening procedures.

Circulating exosomal miRNAs in cardiovascular disease pathogenesis: New emerging hopes.

Cardiovascular diseases (CVDs) are one of the leading causes of morbidity and mortality. Standard therapies have failed to significantly increase patients' survival. Moreover, the majority of conventional screening procedures are ineffective for the diagnosis of CVDs at early stages. Accumulating evidence suggests that numerous cell types release a class of nano-sized vesicles named exosomes into the extracellular space. Exosomes are widely distributed in various body fluids and contain a number of diverse biomolecules such as proteins, lipids, and both mRNA and noncoding RNAs which reflect host-cell molecular architecture. MicroRNAs (miRNAs), which can be found in exosomes, could be taken up by both neighboring and distal cells. Not only has recent evidence indicated the regulatory role of exosomal miRNAs in the pathogenesis of CVD, but it has also been shown that differential expression of exosomal miRNAs in CVDs has made them promising biomarkers for early detection of CVDs. Owing to these remarkable features, exosomal miRNAs have emerged as hot spots in research. This review summarizes the role of exosomal miRNAs in the pathogenesis of CVDs and discusses their potential application in the clinical setting as both therapeutic and diagnostic tools.

Relative Gene Expression Analysis from Urine Samples of Patients with Prostate Disease

Conventional screening and diagnostic procedures in prostate complaints rely on PSA (Prostate Specific Antigen) concentration which is not specific for prostate cancer and frequently leads to unnecessary invasive procedures in order to exclude malignant disease. It is estimated that approximately 50% of persons who underwent tissue biopsy did so based on false positive PSA value. Therefore a proper and timely differential diagnosis of malignant disease using non-invasive techniques remains one of the biggest challenges in medicine. Urine is the invaluable source of biological information contained in small molecules i.e. RNA that is easily accessible and detectable using molecular genetics techniques. We describe economical and fast method for relative expression analysis applicable to any target gene using urine as a sample. Efficient non-invasive method for identification of malignant or high risk cases prove useful in reduction of patient distress during the diagnostic procedure and significantly reduce healthcare costs.

Generalizability theory reliability of written expression curriculum-based measurement in universal screening.

The purpose of this study was to examine the reliability of written expression curriculum-based measurement (WE-CBM) in the context of universal screening from a generalizability theory framework. Students in second through fifth grade (n = 145) participated in the study. The sample included 54% female students, 49% White students, 23% African American students, 17% Hispanic students, 8% Asian students, and 3% of students identified as 2 or more races. Of the sample, 8% were English Language Learners and 6% were students receiving special education. Three WE-CBM probes were administered for 7 min each at 3 time points across 1 year. Writing samples were scored for commonly used WE-CBM metrics (e.g., correct minus incorrect word sequences; CIWS). Results suggest that nearly half the variance in WE-CBM is related to unsystematic error and that conventional screening procedures (i.e., the use of one 3-min sample) do not yield scores with adequate reliability for relative or absolute decisions about student performance. In most grades, three 3-min writing samples (or 2 longer duration samples) were required for adequate reliability for relative decisions, and three 7-min writing samples would not yield adequate reliability for relative decisions about within-year student growth. Implications and recommendations are discussed. (PsycINFO Database Record

Expeditious screening of candidate proteins for microbial vaccines

Advancements in high-throughput “omics” technologies have revolutionized the way vaccine candidates are identified. Now every surface expressed protein that an organism produces can be identified in silico and possibly made available for the rapid development of recombinant/subunit vaccines. However, evaluating the antigenicity of a large number of candidate proteins is an immense challenge, typically requiring cloning of several hundred candidates followed by immunogenicity screening. Here we report the development of a rapid, high-throughput method for screening candidate proteins for vaccines. This method involves utilizing a coupled, cell-free transcription–translation system to screen tagged proteins that are captured at the C-termini using appropriate ligand coated wells in 96 well ELISA plates. The template DNA for the cell-free expression is generated by two sequential PCRs and includes gene coding sequences, promoter, terminator, other necessary cis-acting elements and appropriate tag sequences. The process generates expressible candidate proteins containing two different peptide tags at the N- and the C-termini of the protein molecules. Proteins are screened in parallel for their quantity and immunoreactivity with N-terminal tag antibodies and antisera raised against the pathogen of interest, respectively. Normalization against the total detectable bound protein in the control wells allows for the identification of highly immunoreactive candidates. For this study we selected 30 representatives of >300 potential candidate proteins from Mannheimia haemolytica, a bacterial agent of pneumonia in feedlot cattle for expression with N-terminal Strep-II and C-terminal His(x6)-tag and evaluated their relative immunoreactivities using Strep-tactin-HRP and rabbit antisera generated against M. haemolytica. Using this system we were able to swiftly and quantitatively analyze and rank the suitability of proteins to identify potentially viable vaccine candidates, with the majority of the high ranking candidates being associated with virulence and pathogenicity. The system is adaptable to any bacterial target and presents an alternative to conventional laborious cloning, expression and screening procedures.

MicroRNAs in Barrett's esophagus: future prospects.

MicroRNAs in Barrett's esophagus: future prospects.

Genetic Diversity Analysis of Some Barley Genotypes for Salt Tolerance Using SSR Markers

The aim of the present work was to evaluate the performance of 20 barley genotypes and find out the genetic diversity of these genotypes for salt tolerance using simple sequence repeats during two consecutive seasons; 2009/2010 and 2010/2011. Twenty barley genotypes differed in their tolerance potentiality against salinity were planted in two screening field experiments at two locations; Sakha, North Egypt (as a control) and El-Serw (as saline site) to detect their tolerance to salt stress. They were planted in a randomized complete block design with three replicates. Results revealed that the Egyptian barley cultivars Giza 123, California Mariout and genotype no.12 (line 12 from Cyprus) were salt tolerant besides genotype no.9 (Saiko) giving a moderate salt tolerance response and they all exhibited the highest mean values for some traits such as heading date and plant height under saline condition. Out of ten primers used, only six primers (Bmac0209, Bmac0316, Scssr03907, Bmag770, HVM67 and HVHOTRI) generated clear patterns with high polymorphism. This six discriminatory primer pairs were used to evaluate the genetic diversity of salt tolerance in the 20 barley genotypes. Based on phylogenic trees the data from the dendrogram constructed with SSR markers showed four clusters. All the salt tolerant genotypes and some moderately salt tolerant genotypes were found in two closely related clusters, while all the sensitive genotypes and moderate ones were closely related in the other two clusters. It was concluded that those barley genotypes which showed salt tolerance could serve as potentially novel germplasm that could be exploited for the development of new breeding lines with high level of salinity tolerance and to accelerate genetic advancement in barley and better cost efficient compared to conventional and tedious screening procedures under saline field conditions.

Strangles: Taking steps towards eradication

Strangles, caused by the host adapted Lancefield group C bacterium Streptococcus equi sub-species equi (S. equi), is one of the oldest recognised infectious diseases of horses and continues to cause significant welfare and economic cost throughout the world. The ability of S. equi to establish sub-clinical persistent infections primarily in the guttural pouches of convalescent horses has been instrumental to its success. However, the implementation of simple control measures that permit the identification and treatment of persistently infected carriers can prevent further outbreaks of disease at a local level.This review summarises some of the molecular mechanisms exploited by S. equi to cause disease. New qPCR and iELISA diagnostic tests replace culture methodologies as the gold standard for the detection of infected animals. A strategy to maximise the effective application of these tests to direct management methods for the eradication of S. equi infection is presented and the role of preventative vaccines is discussed.In contrast to current understanding, emerging data illustrates the dynamism of the global S. equi population and potential consequences for the effectiveness of currently available vaccines. The ability to use modern vaccines alongside conventional biosecurity and screening procedures will be critical to the large-scale prevention and even eradication of strangles, providing an opportunity to finally break the stranglehold that this disease has on the world's equine industry.

Production of Staphylococcal Enterotoxin R byStaphylococcus aureusStrains

Staphylococcal enterotoxin D and R (SED, SER) production was determined in 24 S. aureus strains harboring sed gene. Seven of them were not able to produce SED as evidenced by enzyme-linked immunosorbent assay and Western blotting. Sequencing revealed that all these strains harbor a variant of sed gene. Expression of SER was detectable in 22 out of 24 isolates, with variance in productivity ranging from ∼40 to 450 ng/mL. Out of the seven isolates not able to produce SED, three produced high amounts of SER (249-396 ng/mL), two produced less than 200 ng/mL of SER, and two were found to express no detectable amount of SER. Three of those were assigned to spa type t1677 with two being of agr type III and one of agr type I. One strain was t084, agr type II, one t603, agr type II, one 2920, agr type III, one t2920, agr type III, and one t5160, agr type I. Because conventional screening procedures involve only the detection of classical enterotoxins in food, the isolates not able to produce SED presented in this study could pose a threat to human health due to SER production.

Initial PET/CT Staging for Choroidal Melanoma: AJCC Correlation and Second Nonocular Primaries in 333 Patients

To report on whole body positron emission tomography/computed tomography (PET/CT) screening for metastasis at diagnosis of primary uveal melanoma. Since August 2003, 333 consecutive patients were diagnosed with uveal melanoma and underwent whole body screening for metastatic disease with PET/CT along with liver function tests and physical examination. Abnormal findings prompted further biopsies, blood tests, imaging, or clinical evaluations for confirmation. The presence of metastatic disease and second cancers were evaluated. Using the American Joint Committee on Cancer (AJCC) tumor, node, metastasis (TNM) 7th edition criteria, 104 tumors were classified T1 (31%), 162 T2 (49%), 37 T3 (11%), and 30 T4 (9%). Seven of 333 (2.1%; 95% confidence interval [CI] 0.8-4.3) patients had metastatic melanoma. One tumor was a T3 and 6 were T4. Thus, 3% of T3 and 20% of T4 melanomas were found to have metastases at the time of initial diagnosis. Ten patients (3.3%; 95% CI 0.9-5.5) had synchronous second cancers and 28 (8.4%) concurrent benign lesions. The most common metastatic sites were liver (7/7) and bone (2/7). This study suggests that PET/CT improves the yield of detecting both extrahepatic metastases, especially from tumors defined as AJCC-T4, and synchronous primary cancers, irrespective of the size of the uveal melanoma. With respect to liver metastases, PET/CT demonstrated high sensitivity and positive predictive values, indicating an overall better performance than conventional screening procedures.

Should breast cancer survivors be excluded from, or invited to, organised mammography screening programmes?

BackgroundThe prevalence of breast cancer in developed countries has steadily risen over recent decades. Immediate and long-term health needs of patients, including preventive care and screening services, are receiving increasing attention. A question still unresolved is whether breast cancer survivors should receive mammographic surveillance in the clinical or screening setting and, thus, whether they should be excluded from, or invited to, organised mammography screening programmes. The objective of this article is to discuss the many contradictory aspects of this matter.DiscussionProblems with mammographic surveillance of breast cancer survivors include: weak evidence of a reduction in mortality; lack of evidence in favour of one setting or the other; lack of evidence-based guidelines for the frequency and duration of surveillance; disproportionate emphasis placed on the first few years post-treatment, probably dictated by surgical and oncological priorities; a variety of screening policies, as these women are permanently or temporarily or partially excluded from many - but not all - organised screening programmes worldwide; an even greater disparity in follow-up protocols used in the clinical setting; a paucity of data on compliance to mammographic surveillance in both settings; and a difficulty in coordinating the roles of health care providers. In the future, the use of mammography in breast cancer survivors will be influenced by the inclusion of women aged > 69 years in organised screening programmes and the implementation of multidisciplinary breast units, and will probably be investigated by research activities on individual risk assessment and risk-tailored screening. In the interim, current problems can be partially alleviated with some technical solutions in screening data recording, patient flows, and care coordination.SummaryMammographic surveillance of breast cancer survivors is situated at the crossroads of numerous different specialist areas of breast cancer control and management. The solutions for current problems probably lie in some important modifications in the conventional screening procedure that are underway or under study. These developments appear to be directed towards a partial modification of the screening rationale, with an adaptation to meet the diversified breast care needs of women. The complexity of the matter constitutes a call to action for several entities to eliminate the barriers to effective research in this field.

Characterization of two major urinary metabolites of the PPARδ-agonist GW1516 and implementation of the drug in routine doping controls

Since January 2009, the list of prohibited substances and methods of doping as established by the World Anti-Doping Agency includes new therapeutics such as the peroxisome-proliferator-activated receptor (PPAR)-delta agonist GW1516, which is categorized as a gene doping substance. GW1516 has completed phase II and IV clinical trials regarding dyslipidemia and the regulation of the lipoprotein transport in metabolic syndrome conditions; however, its potential to also improve athletic performance due to the upregulation of genes associated with oxidative metabolism and a modified substrate preference that shifted from carbohydrate to lipid consumption has led to a ban of this compound in elite sport. In a recent report, two presumably mono-oxygenated and bisoxygenated urinary metabolites of GW1516 were presented, which could serve as target analytes for doping control purposes after full characterization. Hence, in the present study, phase I metabolism was simulated by in vitro assays employing human liver microsomal fractions yielding the same oxygenation products, followed by chemical synthesis of the assumed structures of the two abundant metabolic reaction products. These allowed the identification and characterization of mono-oxygenated and bisoxygenated metabolites (sulfoxide and sulfone, respectively) as supported by high-resolution/high-accuracy mass spectrometry with higher-energy collision-induced dissociation, tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Since urine samples have been the preferred matrix for doping control purposes, a method to detect the new target GW1516 in sports drug testing samples was developed in accordance to conventional screening procedures based on enzymatic hydrolysis and liquid-liquid extraction followed by liquid chromatography, electrospray ionization, and tandem mass spectrometry. Validation was performed for specificity, limit of detection (0.1 ng/ml), recovery (72%), intraday and interday precisions (7.7-15.1%), and ion suppression/enhancement effects (<10%).

Screening of TERC gene amplification as an additional genetic diagnostic test in detection of cervical preneoplastic lesions

TERC gene amplification was investigated as a possible diagnostic marker for use in routine cytological screening to improve the accuracy of conventional screening procedures in detection of cervical preneoplastic lesions. Cervical smears were screened and classified as low-grade or high-grade squamous intraepithelial lesions (LSIL or HSIL). A fluorescence in situ hybridization procedure using a TERC-specific DNA probe was performed on the same specimens and TERC gene copy number was evaluated. More than two signals per cell were defined as TERC positive. In cervical smears graded after conization as cervical intraepithelial neoplasia grade 1 (CIN 1), no TERC-positive cases were found in either LSIL or HSIL, and no TERC amplification was found in LSIL cases with histological results CIN 1 and CIN 2. Amplifications of the TERC gene first appeared in HSIL cases with CIN 2 histology. In the CIN 3 group, TERC-positive cases were present in both LSIL and HSIL; in these, there were no statistically significant differences between TERC-positive and TERC-negative cases. Statistically significant differences in TERC-positive cases were found between LSIL and HSIL without regard to the CIN grade. From the results obtained, it can be concluded that TERC gene amplifications inevitably lead to a high risk of CIN 3 in both LSIL and HSIL after cytological smear examination. A high CIN grade is not necessarily correlated with TERC amplification, but a positive TERC result certainly demands a high CIN classification.

18-FDG PET scan in the staging of recurrent melanoma: additional value and therapeutic impact.

Staging of melanoma patients by means of whole body functional imaging in a single evaluation session using positron emission tomography (PET) with fluorine-18- labelled deoxy-d-glucose (FDG) as a metabolic tracer has created much interest over the last decade. After enthusiastic pilot studies, more attention has been paid to the false-negative and false-positive results of this technique than to its true therapeutic impact. This study aimed to evaluate (1) the sensitivity and specificity of this technique at a single lesion level compared with conventional screening procedures (CSP) - both of these accompanied by careful clinical examination; and (2) the additional value of the PET scan at the level of the individual patient and its therapeutic impact for different types of melanoma recurrence. A consecutive series of 100 PET scans performed on 84 melanoma patients with regional or distant recurrence according to CSP (89 PET scans) or suspicion of recurrence, i.e. inconclusive CSP (11 PET scans), were retrospectively analysed and compared with the CSP results. At the single lesion level, PET scan and CSP showed a sensitivity of 85 and 81%, a specificity of 90 and 87% and an accuracy of 88 and 84%, respectively. PET provided false-negative results for small skin metastases and brain involvement; false-positive results were associated with unrelated benign or malignant tumours and peripheral soft tissue and bone uptake. PET scan showed an additional value over and above CSP at the individual patient's level by true upstaging in 10 cases, true downstaging in 24 cases and depiction of more lesions within the same stage of disease in 15 cases. The overall therapeutic impact reached 26%: 17 out of 71 (24%) cases with regional recurrence, one out of 18 cases (5.5%) with distant metastasis and eight out of 11 cases (73%) with suspicion of recurrence where CSP remained doubtful. However, in 19 cases comparison between CSP and PET resulted in discordant findings, suggesting upstaging in one area and downstaging at another site within the same patient. In conclusion, PET scan has an additional value in the staging of recurrent melanoma, providing it is accompanied by careful clinical examination and specific brain imaging. However, in the absence of evidence of metastasis or unrelated conditions at the same site on CSP, PET spots may represent false-positive images, which would falsely upgrade a patient to an incurable state, or they may be early true-positive findings, which will become evident during close follow-up.

Discrimination of Prohibited Oral Use of Salbutamol from Authorized Inhaled Asthma Treatment

The administration of salbutamol is permitted only by inhalation by the International Olympic Committee (IOC) for the management of asthma and exercise-induced asthma in athletes. The establishment of criteria to distinguish between the (IOC) authorized use (inhaled) and the (IOC) prohibited use (oral) of salbutamol appeared possible using simultaneous evaluation of variables based on the concentration of nonconjugated enantiomers of salbutamol excreted in urine. Urine was collected from asthmatic and nonasthmatic swimmers who had received various preexercise doses of oral (five doses of 4 mg) or inhaled (two doses of 100 microgram) salbutamol. Urine was also obtained from subjects who had received the maximum dosage of inhaled salbutamol advisable for competing athletes to provide protection from exercise-induced asthma and treatment of asthma (1600 microgram in 24 h, 800 microgram being in the last 4 h). All samples were analyzed to determine the total amount of unchanged salbutamol excreted in urine and the ratio between the S: and R: enantiomers. The discriminant function D = -3.776 + 1.46 x 10(-3) ([S:(+)] + [R:(-)]) + 1.012 ([S:(+)]/[R(-)]) can be used to classify data into two groups, inhaled and oral. The confirmatory criterion suggested (cutoff at D = 1.06, 4 SD from the mean D value of the inhaled distribution) has been verified in different sets of samples showing suspicious concentrations by conventional screening procedures in doping control. An 11.8% false-negative (oral classified as inhaled) rate is assumed with the confirmatory criterion proposed, but virtually no false positives (inhaled classified as oral) are obtained (<1 in 33 000). The overall procedure recommended is to screen all samples and to apply the confirmation criterion proposed to samples showing free racemic salbutamol concentrations >500 microgram/L by gas chromatography-mass spectrometry or free + conjugated racemic salbutamol concentrations >1400 microgram/L by ELISA.

Distinction of inhaled and oral salbutamol by urine analysis using conventional screening procedures for doping control.

Salbutamol administration in athletes is permitted only by inhalation, for the management of asthma. The authors discuss different criteria for suspecting oral use of salbutamol, taking into account the data obtained by application of two conventional screening procedures for doping control: gas chromatography/mass spectrometry (GC/MS) and enzyme-linked immunosorbent assay (ELISA). Urine samples obtained after administration of oral and inhaled salbutamol to asthmatic and nonasthmatic swimmers were analyzed using both analytical approaches. As expected, concentrations obtained by the ELISA test (detection of total salbutamol) were higher than those obtained using the GC/MS procedure (detection of nonsulfated salbutamol). After oral administration, the ELISA test detected significantly higher salbutamol concentrations than those detected after inhalation, reflecting the greater doses administered orally. Urine samples with total salbutamol greater than 1400 ng/mL were obtained after oral doses, but no sample reached this value after inhaled doses. Higher concentrations of nonsulfated salbutamol have also been detected after oral intake, although there is an overlap between the distributions of concentrations after oral and inhaled doses. A cut-off concentration of 500 ng/mL can be used for nonsulfated salbutamol to select suspicious samples, giving 11.8% false negative results and 4.3% false positive results. An additional criterion evaluated was the androsterone-salbutamol peak height ratio, which was lower after oral doses because of the higher concentrations of salbutamol in urine. This ratio was lower than 2 for all the samples collected after oral administration, although 6.8% false positive samples resulted because of low concentrations of androsterone in female urine. Several possibilities for detecting suspicious samples from athletes who have taken prohibited oral salbutamol are available with conventional screening procedures in doping control.