Guidance for Studies Evaluating the Accuracy of Tuberculosis Triage Tests.

Approximately 3.6 million cases of active tuberculosis (TB) go potentially undiagnosed annually, partly due to limited access to confirmatory diagnostic tests, such as molecular assays or mycobacterial culture, in community and primary healthcare settings. This article provides guidance for TB triage test evaluations. A TB triage test is designed for use in people with TB symptoms and/or significant risk factors for TB. Triage tests are simple and low-cost tests aiming to improve ease of access and implementation (compared with confirmatory tests) and decrease the proportion of patients requiring more expensive confirmatory testing. Evaluation of triage tests should occur in settings of intended use, such as community and primary healthcare centers. Important considerations for triage test evaluation include study design, population, sample type, test throughput, use of thresholds, reference standard (ideally culture), and specimen flow. The impact of a triage test will depend heavily on issues beyond accuracy, primarily centered on implementation.

Improvements and cost-effective measures to the automated intermittent water renewal system for toxicity testing with sediments.

The push to make bioassays more sensitive has meant an increased duration of testing to look at more chronic endpoints. To conduct these longer bioassays through the use of traditional bioassay methods can be difficult, as many traditional bioassays have employed manual water changes, which take considerable time and effort. To that end, static-renewal systems were designed to provide researchers a technique to ease the manual water change burden. One of the most well-known static-renewal designs, the static intermittent renewal system (STIR) was produced by the United States Environmental Protection Agency in 1993. This system is still being used in laboratories across the globe today. However, these initial designs have become rather dated as new technologies and methods have been developed that make these systems easier to build and operate. The following information details changes to the initial design and a proof of concept experiment with the benthic invertebrate, Chironomus tepperi, to validate the modifications to the original system.

Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays.

BACKGROUND: Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system. METHODS: C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy. RESULTS: The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture. CONCLUSIONS: The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

An efficient design strategy for logistic regression using outcome- and covariate-dependent pooling of biospecimens prior to assay.

Potential reductions in laboratory assay costs afforded by pooling equal aliquots of biospecimens have long been recognized in disease surveillance and epidemiological research and, more recently, have motivated design and analytic developments in regression settings. For example, Weinberg and Umbach (1999, Biometrics 55, 718-726) provided methods for fitting set-based logistic regression models to case-control data when a continuous exposure variable (e.g., a biomarker) is assayed on pooled specimens. We focus on improving estimation efficiency by utilizing available subject-specific information at the pool allocation stage. We find that a strategy that we call "(y,c)-pooling," which forms pooling sets of individuals within strata defined jointly by the outcome and other covariates, provides more precise estimation of the risk parameters associated with those covariates than does pooling within strata defined only by the outcome. We review the approach to set-based analysis through offsets developed by Weinberg and Umbach in a recent correction to their original paper. We propose a method for variance estimation under this design and use simulations and a real-data example to illustrate the precision benefits of (y,c)-pooling relative to y-pooling. We also note and illustrate that set-based models permit estimation of covariate interactions with exposure.

A Mutagenic Primer Assay for Genotyping of the CRHR1 Gene Rare Variant rs1876828 (A/G) in Asians: A Cost-Effective SNP Typing.

BACKGROUND: Today, the genetic and genomic research entered in a new era of high-throughput genotyping technology. However, mutagenic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is still a choice of genotyping method in molecular epidemiological research. It has been extensively used for the detection of risk alleles, if the target SNP has no natural discriminating restriction site. We undertook this study to develop a mutagenic primer assay for a CRHR1 rare gene variant: rs1876828 (A/G) and to determine their allele frequency in north Indian children. METHODS: The mutagenic primers were designed and assay conditions were optimized to perform mutagenic PCR-RFLP in 550 subjects. The efficiency of assay and results were validated by sequencing. RESULTS: This study demonstrated that the mutagenic primer assay is feasible and applicable to discriminate CRHR1 gene rare variant rs1876828 (A/G) and the "frequency of allele "G" was 100% in north Indian asthmatics as well as normal subjects. CONCLUSION: This method can be used for both large- and small-scale study of complex genetic, where CRHR1 gene plays the pivotal roles.

Development of a Low-Cost Ecotoxicological Bioassay Based on the Feeding Behaviour of the Aquatic Snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca).

Conventional acute tests are not suited to assess the effects of toxicants, because they do not use the concentrations that are usually found in natural ecosystems. By contrast, nonlethal realistic concentrations may cause deleterious effects on animal fitness as a consequence of behaviour impairment. Behaviour is a good integrative variable of complex biochemical and physiological processes. Therefore, bioassays based on behaviour are a useful tool in ecotoxicology. In this study, two bioassays were conducted: (1) acute bioassay (48 h) of acetone on the aquatic snail Potamopyrgus antipodarum, and (2) video-recording behavioural bioassay with pulse exposures to acetone to assess its effects on feeding behaviour. In the latter, animals were exposed to three pulses of acetone (24 h each) with 6 days of postexposure after each pulse. This design allowed us to assess the degree of feeding behaviour recovery after exposure and the effects of repeated pulses. Our results show that postexposure periods have an important effect on the recovery of normal feeding behaviour and that this developed bioassay is an ecotoxicological tool with a relatively low-cost and a short-time consuming. The application of this new tool to different ecotoxicological requirements is discussed.

Systematic neonatal screening for severe combined immunodeficiency and severe T-cell lymphopenia: Analysis of cost-effectiveness based on French real field data.

BACKGROUND: The inclusion of severe combined immunodeficiency (SCID) in a Europe-wide screening program is currently debated. OBJECTIVE: In making a case for inclusion in the French newborn screening program, we explored the costs incurred and potentially saved by early management of SCID. METHODS: For test costs, a microcosting study documented the resources used in a laboratory piloting a newborn screening test on Guthrie cards using the T-cell receptor excision circle quantification method. For treatment costs, patients with SCID admitted to the national reference center for primary immunodeficiency in France between 2006 and 2010 were included. Costs of admission were estimated from actual national production costs. We estimated the costs for patients who underwent early versus delayed hematopoietic stem cell transplantation (HSCT; age, ≤3 vs. >3 months, respectively). RESULTS: The unit cost of the test varied between €4.69 and €6.79 for 33,800 samples per year, depending on equipment use and saturation. Of the 30 patients included, 27 underwent HSCT after age 3 months. At 1 year after HSCT, 10 of these had died, and all 3 patients undergoing early transplantation survived. The medical costs for HSCT after 3 months were €195,776 (interquartile range, €165,884-€257,160) versus €86,179 (range, €59,014-€272,577) when performed before 3 months of age. In patients undergoing late transplantation, active infection contributed to high cost and poor outcome. CONCLUSION: Early detection of SCID could reduce the cost of treatment by €50,000-100,000 per case. Assuming a €5 unit cost per test, the incidence required to break even is 1:20,000; however, if the survival advantage of HSCT before 3 months is confirmed, universal screening is likely to be cost-effective.

Rapid detection of HLA-B*51 by real-time polymerase chain reaction and high-resolution melting analysis.

HLA-B*51, a class I human leukocyte antigen (HLA) molecule, is the strongest known genetic risk factor for Behçet disease. However, there are only few articles reporting methods to determine the presence or absence of HLA-B51. For this reason, we designed and developed an easy, fast, and inexpensive real-time high-resolution melting (HRM) assay to detect HLA-B*51. We genotyped 61 samples by our HRM assay and by conventional polymerase chain reaction, and no discrepancies were found between results. Besides, a subgroup of 25 samples was also genotyped in a different laboratory, and another subgroup of 16 samples was obtained from the International Histocompatibility Working Group DNA Bank, and a full concordance of results was observed with those obtained by HRM. Regarding the identifying system evaluated, we obtained 100% of specificity, sensibility, and repeatability, and 0% of false positive and false negative rates. Therefore, this HRM analysis is easily applicable to the rapid detection of HLA-B*51, exhibits a high speed, and requires a very low budget.

Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985.

Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.

Paper-based device for rapid typing of secondary human blood groups.

We report the use of bioactive paper for typing of secondary human blood groups. Our recent work on using bioactive paper for human blood typing has led to the discovery of a new method for identifying haemagglutination of red blood cells. The primary human blood groups, i.e., ABO and RhD groups, have been successfully typed with this method. Clinically, however, many secondary blood groups can also cause fatal blood transfusion accidents, despite the fact that the haemagglutination reactions of secondary blood groups are generally weaker than those of the primary blood groups. We describe the design of a user-friendly sensor for rapid typing of secondary blood groups using bioactive paper. We also present mechanistic insights into interactions between secondary blood group antibodies and red blood cells obtained using confocal microscopy. Haemagglutination patterns under different conditions are revealed for optimization of the assay conditions.

The utility of yeast as a tool for cell-based, target-directed high-throughput screening.

Many Neglected Tropical Diseases (NTDs) have recently been subject of increased focus, particularly with relation to high-throughput screening (HTS) initiatives. These vital endeavours largely rely of two approaches, in vitro target-directed screening using biochemical assays or cell-based screening which takes no account of the target or targets being hit. Despite their successes both of these approaches have limitations; for example, the production of soluble protein and a lack of cellular context or the problems and expense of parasite cell culture. In addition, both can be challenging to miniaturize for ultra (u)HTS and expensive to utilize. Yeast-based systems offer a cost-effective approach to study and screen protein targets in a direct-directed manner within a eukaryotic cellular context. In this review, we examine the utility and limitations of yeast cell-based, target-directed screening. In particular we focus on the currently under-explored possibility of using such formats in uHTS screening campaigns for NTDs.

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters.

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample.

Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis.

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160 °C) value of 6.6 ± 0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

Mouse assay for determination of arsenic bioavailability in contaminated soils.

A mouse assay for measuring the relative bioavailability (RBA) of arsenic (As) in soil was developed. In this study, results are presented of RBA assays of 16 soils, including multiple assays of the same soils, which provide a quantitative assessment of reproducibility of mouse assay results, as well as a comparison of results from the mouse assay with results from a swine and monkey assay applied to the same test soils. The mouse assay is highly reproducible; three repeated assays on the same soils yielded RBA estimates that ranged from 1 to 3% of the group mean. The mouse, monkey, and swine models yielded similar results for some, but not all, test materials. RBA estimates for identical soils (nine test soils and three standard reference materials [SRM]) assayed in mice and swine were significantly correlated (r = 0.70). Swine RBA estimates for 6 of the 12 test materials were higher than those from the mouse assay. RBA estimates for three standard reference materials (SRM) were not statistically different (mouse/swine ratio ranged from 0.86-1). When four test soils from the same orchard were assessed in the mouse, monkey, and swine assays, the mean soil As RBA were not statistically different. Mouse and swine models predicted similar steady state urinary excretion fractions (UEF) for As of 62 and 74%, respectively, during repeated ingestion doses of sodium arsenate, the water-soluble As form used as the reference in the calculation of RBA. In the mouse assay, the UEF for water soluble As(V) (sodium arsenate) and As(III) (sodium [meta] arsenite) were 62% and 66%, respectively, suggesting similar absolute bioavailabilities for the two As species. The mouse assay can serve as a highly cost-effective alternative or supplement to monkey and swine assays for improving As risk assessments by providing site-specific assessments of RBA of As in soils.

A myo-inositol bioassay utilizing an auxotrophic strain of S. cerevisiae.

Myo-inositol is a six­carbon sugar that is essential for the growth of mammalian cells and must be obtained through either extracellular uptake or de novo biosynthesis. The physiological importance of myo-inositol stems from its incorporation into phosphoinositides and inositol phosphates, which serve a variety of signaling, regulatory, and structural roles in cells. To study myo-inositol metabolism and function, it is essential to have a reliable method for assaying myo-inositol levels. However, current approaches to assay myo-inositol levels are time-consuming, expensive, and often unreliable. This article describes a simple new myo-inositol bioassay that utilizes an auxotrophic strain of S. cerevisiae to measure myo-inositol concentration in solutions. The accuracy of this method was confirmed by comparing assay values to those obtained by tandem mass spectrometry (LC-MS/MS). It is easy to perform, inexpensive, does not require sophisticated equipment, and is specific for myo-inositol.

"Covalent biosensing" enables a one-step, reagent-less, low-cost and highly robust assay of SARS-CoV-2.

We have established a new protocol for detecting severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) using a peptidomimetic to covalently detect a viral marker protease.

Quantitative biomarker assay with microfluidic paper-based analytical devices.

This article describes the use of microfluidic paper-based analytical devices (muPADs) to perform quantitative chemical assays with internal standards. MicroPADs are well-suited for colorimetric biochemical assays; however, errors can be introduced from the background color of the paper due to batch difference and age, and from color measurement devices. To reduce errors from these sources, a series of standard analyte solutions and the sample solution are assayed on a single device with multiple detection zones simultaneously; an analyte concentration calibration curve can thus be established from the standards. Since the muPAD design allows the colorimetric measurements of the standards and the sample to be conducted simultaneously and under the same condition, errors from the above sources can be minimized. The analytical approach reported in this work shows that muPADs can perform quantitative chemical analysis at very low cost.

Requirements for screening and confirmatory methods for the detection and quantification of marine biotoxins in end-product and official control.

An overview is given of the biological origin of phycotoxins, as well as their chemical characteristics. Major poisoning types are described and examples of poisoning events are given to illustrate the importance of the phenomenon to both shellfish consumers and the shellfish producing industry. The characteristics of phycotoxins as natural products, the lack of predictability of their occurrence, economic drivers and the freshness of shellfish consumed in many countries result in a number of requirements for methods to be used in the efficient detection of these compounds. Subsequently, the performance of mouse bioassays and mass spectrometry as detection tools are compared for examples from Irish and French monitoring programmes to assess the usefulness of qualitative and quantitative tools in official control, and their fitness for purpose compared with the requirements. The final part of the paper critically reviews methods available for the end-product and official control of shellfish toxins and their use in screening and confirmatory approaches in monitoring. Recent expert consultations on the methodology for phycotoxins at European and global level are summarised and recommendations are made for future progress in this area.

Cost analysis of smear microscopy and the Xpert assay for tuberculosis diagnosis: average turnaround time.

INTRODUCTION: Rapid and accurate tuberculosis detection is critical for improving patient diagnosis and decreasing tuberculosis transmission. Molecular assays can significantly increase laboratory costs; therefore, the average time and economic impact should be evaluated before implementing a new technology. The aim of this study was to evaluate the cost and average turnaround time of smear microscopy and Xpert assay at a university hospital. METHODS: The turnaround time and cost of the laboratory diagnosis of tuberculosis were calculated based on the mean cost and activity based costing (ABC). RESULTS: The average turnaround time for smear microscopy was 16.6 hours while that for Xpert was 24.1 hours. The Xpert had a mean cost of USD 17.37 with an ABC of USD 10.86, while smear microscopy had a mean cost of USD 13.31 with an ABC of USD 6.01. The sensitivity of smear microscopy was 42.9% and its specificity was 99.1%, while the Xpert assay had a sensitivity of 100% and a specificity of 96.7%. CONCLUSIONS: The Xpert assay has high accuracy; however, the turnaround time and cost of smear microscopy were lower than those of Xpert.