Concomitant Clinical Sensitivity (CCS) and Cross-Reactivity

BackgroundThere are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasises early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unreliable in inexperienced hands and complicated by infections that have similar presentations. This study was to develop and evaluate the use of recombinase polymerase amplification (RPA) assay for the detection of Mycobacterium ulcerans at the point of patient care.MethodsA specific fragment of IS2404 of M. ulcerans was amplified in 15 min at a constant temperature of 42°C, using the RPA assay and analysed on a portable fluorometre. Themethod was tested for sensitivity and specificity with molecular standard of IS2404 DNA fragment, various M.ulcerans strains, other mycobacteria and environmentally associated bacteria. Additionally, the assay performance as a diagnostic tool was tested with archived DNA from symptomatic patients. All results were compared with that of a highly sensitive IS2404 PCR.ResultsThe detection limit was 50 copies of IS2404 in 15 min using plasmid standard and 125 fg with genomic Mu DNA equivalent 25 genomic copies. The assay was highly specific in detecting all strains of M. ulcerans with no observed cross reactivity with other mycobacteria and common skin colonising bacteria. The clinical sensitivity and specificity of the BU-RPA assay using clinical samples was 86% and 100% respectively.ConclusionWe have developed a real-time isothermal RPA assay for the detection of M. ulcerans as a cheaper alternative to PCR. Combining this assay with a simple extraction protocol will maximise its use as point-of-care test for Buruli ulcer.

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Comparison of commercial RT-PCR diagnostic kits for COVID-19

10.1101/2020.04.22.056747 ◽

2020 ◽

Cited By ~ 3

Author(s):

Puck B. van Kasteren ◽

Bas van der Veer ◽

Sharon van den Brink ◽

Lisa Wijsman ◽

Jørgen de Jonge ◽

...

Keyword(s):

Limit Of Detection ◽

Cross Reactivity ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Specific Method ◽

Preliminary Evaluation ◽

Rt Pcr ◽

Accurate Identification ◽

Clinical Sensitivity ◽

Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

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Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

PLoS ONE ◽

10.1371/journal.pone.0245164 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245164

Author(s):

Yee Ling Lau ◽

Ilyiana binti Ismail ◽

Nur Izati binti Mustapa ◽

Meng Yee Lai ◽

Tuan Suhaila Tuan Soh ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Sensitivity And Specificity ◽

Reverse Transcription ◽

Cross Reactivity ◽

Recombinase Polymerase Amplification ◽

Analytical Sensitivity ◽

Clinical Sensitivity ◽

Primary Means ◽

Amplification Assay ◽

Recombinase Polymerase Amplification Assay

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

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Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™ - Antigen-detecting point-of-care device for SARS-CoV-2

10.1101/2021.03.02.21252430 ◽

2021 ◽

Author(s):

Lisa Johanna Krüger ◽

Julian A.F. Klein ◽

Frank Tobian ◽

Mary Gaeddert ◽

Federica Lainati ◽

...

Keyword(s):

Viral Load ◽

Limit Of Detection ◽

Point Of Care ◽

Scale Up ◽

Cross Reactivity ◽

Ease Of Use ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Clinical Sensitivity ◽

Lateral Flow Assays

Background: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and Methods: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results: Study conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

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Clinical evaluation of the Abbott Alinity SARS-CoV-2 spike-specific quantitative IgG and IgM assays among infected, recovered, and vaccinated groups

Journal of Clinical Microbiology ◽

10.1128/jcm.00388-21 ◽

2021 ◽

Author(s):

Madhusudhanan Narasimhan ◽

Lenin Mahimainathan ◽

Ellen Araj ◽

Andrew E. Clark ◽

John Markantonis ◽

...

Keyword(s):

Immune Responses ◽

Clinical Performance ◽

Antibody Test ◽

Cross Reactivity ◽

Antibody Responses ◽

Spike Protein ◽

Clinical Sensitivity ◽

Health Infrastructure ◽

Archived Samples ◽

Significant Burden

The COVID-19 pandemic continues to impose a significant burden on global health infrastructure. While identification and containment of new cases remains important, laboratories must now pivot and consider an assessment of SARS-CoV-2 immunity in the setting of the recent availability of multiple COVID-19 vaccines. Here we have utilized the latest Abbott Alinity semi-quantitative IgM and quantitative IgG spike protein (SP) serology assays (IgMSP and IgGSP) in combination with Abbott Alinity IgG nucleocapsid (NC) antibody test (IgGNC) to assess antibody responses in a cohort of 1236 unique participants comprised of naïve, SARS-CoV-2 infected, and vaccinated (including both naïve and recovered) individuals. The IgMSP and IgGSP assays were highly specific (100%) with no cross-reactivity to archived samples collected prior to the emergence of SARS-CoV-2, including those from individuals with seasonal coronavirus infections. Clinical sensitivity was 96% after 15 days for both IgMSP and IgGSP assays individually. When considered together, the sensitivity was 100%. A combination of NC- and SP-specific serologic assays clearly differentiated naïve, SARS-CoV-2-infected, and vaccine-related immune responses. Vaccination resulted in a significant increase in IgGSP and IgMSP values, with a major rise in IgGSP following the booster (second) dose in the naïve group. In contrast, SARS-CoV-2 recovered individuals had several fold higher IgGSP responses than naïve following the primary dose, with a comparatively dampened response following the booster. This work illustrates the strong clinical performance of these new serological assays and their utility in evaluating and distinguishing serological responses to infection and vaccination.

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MILESTONES MARKING THE KNOWLEDGE OF ADVERSE REACTIONS TO FOOD IN THE DECADE OF THE 1980s

PEDIATRICS ◽

10.1542/peds.96.2.380b ◽

1995 ◽

Vol 96 (2) ◽

pp. 380-381

Author(s):

Betty Miller

Keyword(s):

Food Allergy ◽

Adverse Reactions ◽

Food Additives ◽

Food Challenge ◽

Cross Reactivity ◽

Allergic Reactions ◽

Double Blind ◽

Specific Patient ◽

Clinical Sensitivity ◽

A Prospective Study

Purpose of the Study. This study outlines the significant advances made to our understanding of adverse reactions to foods and food additives in the last decade. The milestones are listed in order of overall importance and are discussed in depth in the review. 1. Establishing double-blind, placebo-controlled food challenge (DBPCFC) as the "gold-standard" for defining specific patient population to be used in scientific studies. (VanMetre, May, Bock) 2. Recognition of the key role food allergy plays in the pathogenesis of atopic dermatitis. (Sampson, May) 3. Identifying the group of foods most likely to be associated with true allergic reactions. Analysis of DBPCFC in children has shown that 93% of allergic reactions occurred to eight foods (in order of frequency): egg, peanut, milk, soy, tree nuts, crustacean-type shellfish, fish, and wheat. Corn and chocolate allergy was rarely found. (Bock, Sampson, Atkins) 4. Food allergy has a natural history. In a prospective study of 501 children, Bock found that, of 15 cases of allergy proven by DBPCFC in the first year of life, none of the 15 cases was reactive beyond 24 months of age. In contrast, long-term follow-up of patients who have experienced peanut anaphylaxis revealed that clinical sensitivity lasts for at least 14 years. (Bock, Atkins) 5. Food allergy cross-sensitivity (clinical reactivity) does not extend equally to all members of a biologic food family. Although immunologic cross-reactivity between peanut, soy, and other peas/beans could be regularly found in allergic patients, clinically important cross-reactions demonstrated by DBPCFC were rare. (Bernhisel -Broadbent, Sampson)

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HRP2 and HRP3 cross-reactivity and implications for HRP2-based RDT use in regions with Plasmodium falciparum hrp2 gene deletions

Malaria Journal ◽

10.1186/s12936-021-03739-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Amy Kong ◽

Scott A. Wilson ◽

Yong Ah ◽

Douglas Nace ◽

Eric Rogier ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Clinical Sample ◽

Cross Reactivity ◽

Sub Saharan Africa ◽

Gene Deletions ◽

Clinical Sensitivity ◽

Gene Coding ◽

Malaria Rapid Diagnostic Tests ◽

Sub Saharan ◽

Multiplex Bead

Abstract Background The Plasmodium falciparum antigen histidine rich protein 2 (HRP2) is a preferred target for malaria rapid diagnostic tests (RDTs) because of its abundant production by the parasite and thermal stability. As a result, a majority of RDTs procured globally target this antigen. However, previous reports from South America and recent reports from sub-Saharan Africa and Asia indicate that certain P. falciparum parasites have deletions of the gene coding for HRP2. The HRP2 antigen is paralogous to another P. falciparum antigen HRP3 and some antibodies to HRP2 cross-react with HRP3. Multiple parasites have been described with deletions of one or both hrp2 and hrp3 genes. It is unclear how the various combinations of hrp2 and hrp3 deletion genotypes affect clinical sensitivity of HRP2-based RDTs. Methods Cross-reactivity between HRP2 and HRP3 was tested on malaria RDTs using culture-adapted P. falciparum parasites with both hrp2 and hrp3 intact or with one or both genes deleted. Ten-fold serial dilutions of four culture-adapted P. falciparum parasites [3D7 (hrp2+/hrp3+), Dd2 (hrp2−/hrp3+), HB3 (hrp2+/hrp3−) and 3BD5 (hrp2−/hrp3−)] ranging from 100,000 to 0.01 parasites/µL were prepared. HRP2, Plasmodium lactate dehydrogenase (pLDH) and aldolase concentrations were determined for the diluted samples using a multiplex bead assay. The samples were subsequently tested on three RDT products designed to detect P. falciparum by HRP2 alone or in combination with pLDH. Results At parasite densities of approximately 1000 parasites/µL, parasites that expressed either hrp2 or hrp3 were detected by all three RDTs. Multiplex based antigen measurement using HRP2- conjugated beads demonstrated higher antigen concentration when both hrp2 and hrp3 genes were intact (3D7 parasites, 47.9 ng/ml) compared to HB3 (3.02 ng/mL) and Dd2 (0.20 ng/mL) strains that had one gene deleted. 3D7 at 10 parasites/µL (0.45 ng/mL) was reactive on all three RDT products whereas none of the other parasites were reactive at that density. Conclusions Above a certain antigen threshold, HRP3 cross-reactivity on HRP2-based RDTs is sufficient to mask the effects of deletions of hrp2 only. Studies of hrp2 deletion and its effects on HRP2-based RDTs must be studied alongside hrp3 deletions and include clinical sample reactivity on HRP2-based tests.

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A rapid and automated sample-to-result Candida auris real-time PCR assay for high-throughput testing of surveillance samples with BD MAX™ open system

10.1101/608190 ◽

2019 ◽

Author(s):

L. Leach ◽

A. Russell ◽

Y. Zhu ◽

S. Chaturvedi ◽

V. Chaturvedi

Keyword(s):

New York ◽

Real Time ◽

High Throughput ◽

Open System ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Pcr Assay ◽

Candida Auris ◽

Clinical Sensitivity

ABSTRACTThe multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (Leach et al. JCM. 2018: 56, e01223-17). The assay played a crucial role in the ongoing investigations of C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using BD MAX™ open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris, and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in the quadruplicate yielded assay precision. BD MAX™ optimum assay conditions were: DNA extraction at 75°C for 20 min, and the use of PerfeCTa Multiplex qPCR ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR reaction. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD MAX™ System with 96% clinical sensitivity and 94% accuracy compared to the manual assay. BD MAX™ assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-hour workday.

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Development of a PTHrP Chemiluminescent Immunoassay to Assess Humoral Hypercalcemia of Malignancy

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.2076 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A1015-A1015

Author(s):

Susan Louise Ashrafzadeh-Kian ◽

Joshua Bornhorst ◽

Alicia Algeciras-Schimnich

Keyword(s):

Limit Of Detection ◽

Reference Interval ◽

Cross Reactivity ◽

Humoral Hypercalcemia Of Malignancy ◽

Clinical Sensitivity ◽

Hypercalcemia Of Malignancy ◽

Humoral Hypercalcemia ◽

Hook Effect ◽

Limit Of Quantitation ◽

Acridinium Ester

Abstract Background: Measurement of parathyroid hormone related peptide (PTHrP) is helpful in the diagnosis and clinical management of patients suspected of humoral hypercalcemia of malignancy (HHM). In these patients uncontrolled release of PTHrP by tumor cells is responsible for the hypercalcemia and PTH concentrations are typically suppressed. Objective: Develop a sensitive and specific assay for quantitation of PTHrP in plasma. Method: Calibrators (PTHrP 1-86) and samples (50uL) were incubated with an anti-PTHrP goat polyclonal acridinium ester labeled antibody. Complexes were transferred and incubated in a microplate coated with an anti-PTHrP polyclonal rabbit antibody. After washing, the acridinium ester generated signal, which is directly proportional to the amount of PTHrP in sample, was quantified. Results: In this assay PTHrp was stable for 24 hours ambient, 3 days refrigerated, 34 days frozen and through 3 freeze/thaws. Intra and inter-assay imprecision in EDTA plasma (~0.16-35.0 pmol/L) ranged from 2.2-8.6% and 5-15%, respectively. The limit of detection was 0.04 pmol/L and the limit of quantitation was 0.16 pmol/L (15% CV). The analytical measuring range was 0.39-50.5 pmol/L (slope of 1.07 and r2 of 0.99). Average spike recovery was 98% (range 85-108%). The assay was not affected by hemoglobin of ≤500 mg/dL, triglycerides of ≤2000 mg/dL, or bilirubin of ≤50mg/dL. No hook effect was noted up to 500 pmol/L. PTH (1-84) did not cross-react in the assay. C-terminal PTHrP(107-139), and N-terminal PTHrP(1-36) had no significant cross-reactivity (≤1.1%). Mid-PTHrP(38-94) had 8.3% cross-reactivity. Comparison with an in-house PTHrP assay (n=267) showed an r2 of 0.96, and slope of 2.25 by Passing-Bablok regression fit. The 97.5% reference interval for PTHrP (n=114) was ≤0.7 pmol/L, however a higher concentration (≤4.2 pmol/L) was identified as a more specific clinical cut-off. A retrospective clinical validation study showed that using ≤4.2 pmol/L resulted in a 91% clinical sensitivity and a 98% clinical specificity. Conclusion: We have developed an analytically and clinically sensitive and specific PTHrP immunoassay. A cutoff of ≤4.2 pmol/L is clinically useful in the evaluation of patients suspected of hypercalcemia of malignancy.

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Performance characteristics of the VIDAS® SARS-COV-2 IgM and IgG serological assays

10.1101/2020.09.28.20196030 ◽

2020 ◽

Author(s):

Nathalie Renard ◽

Soizic Daniel ◽

Nadège Cayet ◽

Matthieu Pecquet ◽

Frédérique Raymond ◽

...

Keyword(s):

Clinical Performance ◽

Cross Reactivity ◽

Epidemiological Surveillance ◽

Test Results ◽

Rt Pcr ◽

Serological Tests ◽

Clinical Sensitivity ◽

Healthy Donors ◽

Coefficients Of Variation ◽

Respiratory Virus Infection

ABSTRACTThe COVID-19 pandemic, caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to spread worldwide. Serological testing for SARS-CoV-2-specific antibodies plays an important role in understanding and controlling the pandemics, notably through epidemiological surveillance. Well validated and highly specific SARS-CoV-2 serological assays are urgently needed. We describe here the analytical and clinical performance of VIDAS® SARS-CoV-2 IgM and VIDAS® SARS-CoV-2 IgG, two CE-marked, EUA-authorized, automated, qualitative assays for the detection of SARS-CoV-2-specific IgM and IgG, respectively. Both assays showed high within-run and within-laboratory precision (coefficients of variation < 11.0%) and very low cross-reactivity towards sera of patients with a past common coronavirus or respiratory virus infection. Clinical specificity determined on up to 989 pre-pandemic healthy donors was ≥ 99% with a narrow 95% confidence interval for both IgM and IgG assays. Clinical sensitivity was determined on up to 232 samples from 130 RT-PCR-confirmed SARS-CoV-2 patients. The positive percent agreement (PPA) with SARS-CoV-2 PCR reached 100% at ≥ 16 days (VIDAS® SARS-CoV-2 IgM) and ≥ 32 days (VIDAS® SARS-CoV-2 IgG) of symptom onset. Combined IgM/IgG test results improved the PPA compared to each test alone. SARS-CoV-2 IgG seroconversion followed closely that of SARS-CoV-2 IgM and remained stable over time, while SARS-CoV-2 IgM levels rapidly declined. Interestingly, SARS-CoV-2-specific IgM and IgG responses were significantly higher in COVID-19 hospitalized vs. non-hospitalized patients. Altogether, the VIDAS® SARS-CoV-2 IgM and IgG assays are highly specific and sensitive serological tests suitable for the reliable detection of past acute SARS-CoV-2 infections.

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