Stand-alone Assays Research Articles

Benefits of simultaneous screening and quantitation of drugs and their metabolites in post-mortem samples by liquid chromatography-high resolution mass spectrometry

Compare a novel method for the simultaneous screening and quantitation of drugs and their metabolites in post-mortem blood and urine with an established two-step process involving a screen followed by quantitation using stand-alone assays. Determine if adoption of the novel method provides any benefits. Introduction: toxicological analysis of post-mortem samples may be required as part of the investigation into the death of an individual. Post-mortem blood and urine are usually screened to identify any compounds present. This is followed by separate quantitative assays to determine the concentrations of toxicologically significant compounds in the blood. This process is labour intensive, time-consuming, requires a broad range of analytical instrumentation and often requires a large sample volume. The consolidation of screening and quantitation into a single methodology has the potential to improve the efficiency of analysis, reduce the sample volume required and decrease turnaround times. Recent publications have demonstrated the suitability of liquid chromatography-high resolution mass spectrometry (LC-HRMS) for the combined screening and quantitation approach (Bidney, J Anal Toxicol., 2017, 41, 181–195; Partridge, J Anal Toxicol., 2018, 42, 220–231). However, there are no published studies which compare a combined approach by LC-HRMS with existing two-step workflows to determine if there are benefits to adopting the combined approach. Post-mortem blood ( n = 200) and urine ( n = 103) samples were screened using an established gas chromatography-mass spectrometry (GCMS) screen. Screening results were compared to those obtained using a novel LC-HRMS methodology using Q-ExactiveTM technology. Linear regression analysis was performed to compare quantitative blood results generated using LC-HRMS for 42 toxicologically significant compounds, with results generated using established stand-alone quantitative assays by GCMS and high performance-liquid chromatography (HPLC). Turnaround time data for quantitative results pre and post the introduction of LC-HRMS was compared to determine the difference in turnaround times. RLC-HRMS screening detected key compounds in 125% more instances than GCMS. LC-HRMS also showed a 60% increase in the number of compounds detected in comparison to GCMS. Quantitative values generated using the LC-HRMS assay were within ±10% of values obtained using the established methods by GCMS or HPLC. Retrospective analysis of turnaround times pre and post the adoption of LC-HRMS showed an average decrease in turnaround time of 32% (range 6-69%). Combining screening and quantitation reduced staffing requirements by 2 days for opiate quantitation and 1 day for most other analytes, and significantly reduced sample volume requirements. Recent increases in substance misuse have resulted in an increase in the number of post-mortem samples submitted for toxicological analysis (Niles, Popul Health Manag., 2020, 24, 43–51). Timely analysis of post-mortem samples is a requirement of the Chief Coroner in England (Chief Coroner, 2016). A comparison of results generated by LC-HRMS and an existing two-step workflow has shown that the LC-HRMS methodology is well placed to allow the laboratory to meet these challenges by improving the quality of screening results, reducing the sample volume required, and decreasing turnaround time and staffing requirements. LC-HRMS delivers significant decreases in turnaround time and staffing and sample volume requirements. It also increases the number of compounds detected. This demonstrates that adopting LC-HRMS has the potential to improve analytical quality and efficiency, allowing the toxicology laboratory to meet the challenges of increasing workload, a wider range of substance misuse, and decreasing turnaround times.

Diagnostic Stewardship Approaches to Clostridioides difficile Infection in the Era of Two-Step Testing: a Shifting Landscape

To discuss the current strategies and impact of diagnostic stewardship for Clostridioides difficile infection. The diagnosis of C. difficile infection is challenging due to complex epidemiology and the limitations of a single assay that is adequate for diagnosis. Overdiagnosis with sensitive molecular assays is common due to the prevalence of colonization with C. difficile. To overcome these challenges, multiple diagnostic stewardship strategies have been successfully deployed to optimize C. difficile testing. Diagnostic stewardship strategies should be implemented at every stage of C. difficile testing in order to limit testing to patients with a high pre-test probability, minimize the limitations of stand-alone assays, and guide clinicians to appropriate management through clear result reporting and interpretation.

Advanced Diagnostic Strategies for Clostridium difficile Infection (CDI).

Clostridium difficile infection (CDI) is a leading cause of nosocomial intestinal diseases and produces considerable morbidity and mortality among inpatients globally. The increasing incidence of CDI, especially of its severe cases, creates a growing financial burden internationally. The pathogen responsible for CDI, C. difficile, is an anaerobic, spore-bearing Gram-positive bacillus that colonizes gastrointestinal tract in humans and animals. The bacterium can transfer from one host to another via the oral-fecal route and is consequently a public health concern in the community as well as hospitals. CDI also has implications for the personal hygiene of patients and medical workers. Classical risk factors for CDI include old age, long inpatient stay, and use of antibacterial agents. Clinical manifestations and a positive laboratory assay are both needed for an accurate diagnosis of CDI. Laboratory testing for C. difficile should distinguish between toxigenic and non-toxigenic strains, as well as asymptomatic colonization and the clinical manifestations of infection. Most stand-alone assays do not attain the desired sensitivity and specificity, and thus multistep algorithms and automated assays have emerged in recent years. Hospitals have adopted various assays, combining their conjunct advantages to enable rapid results, accurate diagnosis, and decreased expenditure. This strategy ultimately prevents misdiagnosis and over-diagnosis simultaneously. In this review, we describe common strategies for diagnosis of CDI and compare commercially available multistep and automatic assays.

CON4EI: Evaluation of QSAR models for hazard identification and labelling of eye irritating chemicals

Assessment of ocular irritation is a regulatory requirement in safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as stand-alone assays. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed to assess the reliability of eight in vitro test methods and computational models as well as establishing an optimal tiered-testing strategy.For three computational models (Toxtree, and Case Ultra EYE_DRAIZE and EYE_IRR) performance parameters were calculated. Coverage ranged from 15 to 58%. Coverage was 2 to 3.4 times higher for liquids than for solids. The lowest number of false positives (5%) was reached with EYE_IRR; this model however also gave a high number of false negatives (46%). The lowest number of false negatives (25%) was seen with Toxtree; for liquids Toxtree predicted the lowest number of false negatives (11%), for solids EYE_DRAIZE did (17%). It can be concluded that the training sets should be enlarged with high quality data. The tested models are not yet sufficiently powerful for stand-alone evaluations, but that they can surely become of value in an integrated weight-of-evidence approach in hazard assessment.

Reference control data obtained from an in vivo comet-micronucleus combination assay using Sprague Dawley rats

According to the International Conference on Harmonization Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (ICH S2(R1)), a positive response in any in vitro assay necessitates additional in vivo test(s) (other tissue/endpoint) in addition to the erythrocyte micronucleus test when Option 1 of the test battery is selected. When Option 2 of the test battery is selected, a bacterial gene mutation test and two in vivo tests with different tissues/endpoint are required. The in vivo alkaline comet assay is recommended as the second in vivo test because it can detect a broad spectrum of DNA damage in any tissue and can be combined with the erythrocyte micronucleus test. Considering animal welfare, a combination assay is preferable to an individual assay. Thus, we validated the protocol for the in vivo comet-micronucleus combination assay in rats with three daily administrations and determined the dose of the positive control (ethyl methanesulfonate; EMS, 200mg/kg/day). We also collected the negative control (vehicle) and positive control (EMS) data from the comet (liver, stomach, and kidney) and micronucleus (bone marrow) combination assay using male Sprague Dawley (SD) rats. The negative control data were comparable to our historical control data obtained from stand-alone assays. The positive control data showed clear and consistent positive responses in both endpoints.

Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays.

ELISPOT, ELISA and flow cytometry techniques are often used to study the function of immune system cells. It is tempting to speculate that these assays can be used interchangeably, providing similar information about the cytokine secreting activity of cells: the higher the number of cytokine-positive cells measured by flow cytometry, the higher the number of cytokine-secreting cells expected to be detected by ELISPOT and the larger the amount of secreted cytokine expected to be measured by ELISA. We have analyzed the expression level and secretion capacity of IFNγ from peripheral blood mononuclear cells isolated from five healthy donors and stimulated by calcium ionomycin mixed with phorbol 12-myristate 13-acetate in a non-specific manner in side-by-side testing using ELISPOT, ELISA and flow cytometry assays. In our study, we observed a general correlation in donors’ ranking between ELISPOT and flow cytometry; ELISA values did not correlate with either ELISPOT or flow cytometry. However, a detailed donor-to-donor comparison between ELISPOT and flow cytometry revealed significant discrepancies: donors who have similar numbers of IFNγ-positive cells measured by flow cytometry show 2–3-fold differences in the number of spot-forming cells (SFCs) measured by ELISPOT; and donors who have the same number of SFCs measured by ELISPOT show 30% differences in the number of IFNγ-positive cells measured by flow cytometry. Significant discrepancies between donors were also found when comparing ELISA and ELISPOT techniques: donors who secreted the same amount of IFNγ measured by ELISA show six-fold differences in the number of SFCs measured by ELISPOT; and donors who have 5–7-times less secreted IFNγ measured by ELISA show a two-fold increase in the number of SFCs measured by ELISPOT compared to donors who show a more profound secretion of IFNγ measured by ELISA. The results of our study suggest that there can be a lack of correlation between IFNγ values measured by ELISPOT, ELISA and flow cytometry. The higher number of cytokine-positive cells determined by flow cytometry is not necessarily indicative of a higher number of cytokine-secreting cells when they are analyzed by either ELISPOT or ELISA. Our ELISPOT vs. ELISA comparison demonstrates that the higher number of SFCs observed in ELISPOT does not guarantee that these cells secrete larger amounts of cytokines compared to donors with lower SFC numbers. In addition, our data indicate that ELISPOT, ELISA and flow cytometry should be performed as complementary, rather than stand-alone assays: running these assays in parallel on samples from the same donors may help to better understand the mechanisms underlying the physiology of cytokine-secreting cells.

Decorin binding proteins A and B in the serodiagnosis of Lyme disease in North America.

The laboratory diagnosis of Lyme disease is based upon the detection of antibodies generated against Borrelia burgdorferi using a two-tier assay, typically consisting of an enzyme-linked immunosorbent assay (ELISA), followed by a Western blot. This system, put into place to address the nonspecificity associated with standalone first-tier assays, is insensitive for diagnosing early infection, when most people seek care. The use of bacterial lysates or whole-protein antigens as first-tier assay targets contributes to nonspecificity due, in part, to the presence of cross-reactive epitopes that are also found in other bacteria. This precludes their use as sensitive standalone assays. The use of peptides containing linear epitopes that are highly specific for B. burgdorferi offers a method for reducing this cross-reactivity. In the present study, we mapped the linear epitopes of the prominently expressed Borrelia adhesins decorin binding protein A (DbpA) and DbpB. We identified several epitopes in each protein that were highly conserved among North American strains of B. burgdorferi, and we screened peptides containing specific epitopes using serum panels from early and late Lyme disease patients. The individual peptides primarily detected IgM but not IgG, while the proteins efficiently detected both IgM and IgG. While no individual peptide demonstrated better utility for antibody detection than its respective whole protein, an assay containing a combination of a DbpA and a DbpB peptide adequately detected both IgM and IgG, accurately identifying 87.5% (84/96) of the early Lyme disease patients and 80.0% (16/20) of the late Lyme disease patients.

Development of allele-specific multiplex PCR to determine the length of poly-T in intron 8 of CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis has been implemented for Cystic Fibrosis (CF) carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD). Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele results, unless a patient is R117H positive, or to have the poly-T analysis as a standalone reflex test using the same commercial platform. There are other standalone assays developed to detect poly-T alleles, such as heteroduplex analysis, High Resolution Melting (HRM) curve analysis, allele-specific PCR (AS-PCR) and Sanger sequencing. In this report, we developed a simple and easy-to-implement multiplex AS-PCR assay using unlabeled standard length primers, which can be used as a reflex or standalone test for CFTR poly-T track analysis. Out of 115 human gDNA samples tested, results from our new AS-PCR matched to the previous known poly-T results or results from Sanger sequencing.

Efficacy of multipurpose contact lens solutions against ESBL-positive Escherichia coli, MRSA, and Candida albicans clinical isolates.

The antimicrobial effects of multipurpose contact lens solutions (MPSs) have been evaluated according to ISO 14729 standards. The aim of this study was to assess the efficacy of commercially available MPSs against extended-spectrum beta lactamases (ESBL)-producing Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans clinical isolates. Three commercially available contact lens solutions (Opti-Free Expresss, ReNu MultiPlus, and Solo Care Aqua) were tested against 18 ESBL-positive E. coli clinical strains, 20 MRSA clinical strains, and 20 C. albicans clinical strains. The stand-alone assays for bacteria and fungi were performed according to ISO 14729 criteria, and all samples were evaluated after 2, 4, and 24 hours of incubation. The numbers of viable microorganisms were evaluated by the plate-counting method. All MPSs demonstrated at least 3 log reduction in colony-forming units (CFU) for all bacterial isolates and 1 log reduction in CFU for all yeast isolates, which meets ISO 14729 standards. Although no statistically significant differences were obtained among MPSs for bacterial isolates, variable responses were observed against clinical isolates: 5% povidone-iodine was more effective compared with Solo Care Aqua for C. albicans clinical strains (P<0.05); and all solutions were effective after an incubation time of only 2 hrs. The MPSs showed good activity against S. aureus, E. coli, and C. albicans. Although effective log reductions were provided with all MPSs, the reduction was variable depending on the strains tested. Multipurpose contact lens solutions should be tested under ISO 14729 standards for both standard and clinical strains.

Population genetics of 30 insertion-deletion polymorphisms in two Chinese populations using Qiagen Investigator® DIPplex kit.

Insertion-deletion polymorphisms (INDELs) are short length diallelic polymorphisms caused by the insertion or deletion of several bases. INDEL markers can serve as useful supplementary or stand-alone assays for human identification. The Qiagen Investigator(®) DIPplex kit multiplexes 30 autosomal INDELs plus amelogenin for forensic use. The objective of this study was to estimate genetic diversity of 30 INDEL markers in the Han (the largest ethnic group of China, n=565) and She population (almost the smallest ethnic group of China, n=119), and to evaluate their usefulness in forensic genetics. In the Han and She, the mean observed heterozygosity values were 0.4133 and 0.3896, and the combined matching probability values were 1.80×10(-11) and 3.17×10(-11), respectively. Furthermore, the allele frequencies for each locus were compared with those in other reported Chinese subpopulations, and the forensic efficacy was compared between this kit and in-house developed INDEL assay. This study demonstrates that the Investigator(®) DIPplex kit can be used as a supplementary tool for human identity testing in China.

Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection

SummaryBackgroundDiagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection.MethodsIn this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12 420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative.FindingsClinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12–2·31). Multistage algorithms performed better than did standalone assays.InterpretationWe noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection.FundingDepartment of Health and Health Protection Agency, UK.

Combination of Culture, Antigen and Toxin Detection, and Cytotoxin Neutralization Assay for Optimal Clostridium difficile Diagnostic Testing

There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings. To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile. One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples. Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing. The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenic C difficile.

A validation study of the Qiagen Investigator DIPplex® kit; an INDEL-based assay for human identification

Marker sets that are based on small insertion/deletion (INDEL) alleles can serve as useful supplementary or stand-alone assays for human identification. A validation study has been performed on a human identification assay based on a panel of 30 INDELs and amelogenin using the Investigator DIPplex® kit (Qiagen). The assay was able to type DNA from a number of forensically relevant sample types and obtain full profiles with 62pg of template DNA and partial profiles with as little as 16pg of template DNA. The assay is reproducible, precise, and non-overlapping alleles from minor contributors were detectable in mixture analysis ranging from 6:1 to 19:1 mixtures. Population studies were performed on the 30 indels, and there were no significant departures from Hardy-Weinberg equilibrium or significant linkage disequilibrium between the markers (after correction for sampling). In all populations, the random match probability was 1.43 × 10(-11) or less, and the power of exclusion was greater than .999999999. We also discovered several microvariant alleles in our population samples. The data support that the Investigator DIPplex® kit provides a powerful supplement or stand-alone capability for human identity testing.

New combination test for hepatitis C virus genotype and viral load determination using Amplicor GT HCV MONITOR test v2.0

To develop a new sensitive and inexpensive hepatitis C virus (HCV) combination test (HCV Guideline test) that enables the determination of HCV genotypes 1, 2 and 3, and simultaneous determination of HCV viral load using commercial Amplicor GT HCV MONITOR test v2.0 (microwell version). The HCV Guideline test used the PCR product generated in commercial Amplicor GT HCV Monitor test v2.0 for viral load measurement using microwell plate version of Amplicor HCV Monitor and also captured on separate plates containing capture probes and competitive oligonucleotide probes specific for HCV genotypes 1, 2 and 3, The HCV genotype was subsequently determined using the biotin-labeled PCR product and five biotin-labeled HCV-specific probes. The sensitivity of the HCV Guideline test was 0.5 KIU/mL. Specificity of the HCV Guideline test was confirmed by direct sequencing of HCV core region and molecular evolutionary analyses based on a panel of 31 samples. The comparison of the HCV Guideline test and an in-house HCV core genotyping assay using 252 samples from chronic hepatitis C patients indicated concordant results for 97.2% of samples (59.5% genotype 1, 33.7% genotype 2, 6.0% genotype 3, and 0.8% mixed genotypes). Similarly, the HCV Guideline test showed concordance with a serological test, and the serological test failed to assign any serotype in 12.7% of the samples, indicating a better sensitivity of the HCV Guideline test. Clinically, both viral load and genotypes (1, 2 and 3) have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C based on guidelines and they are, in normal circumstances, performed as separate stand-alone assays. The HCV Guideline test is a useful method for screening large cohorts in a routine clinical setting for determining the treatment regimen and for predicting the outcome of antiviral therapy of chronic hepatitis C.