scholarly journals Ultrasensitive Detection of Clostridium difficile Toxins Reveals Suboptimal Accuracy of Toxin Gene Cycle Thresholds for Toxin Predictions

2019 ◽  
Vol 57 (6) ◽  
Author(s):  
Johanna Sandlund ◽  
Mark H. Wilcox
Keyword(s):  
Clinical Practice ◽  
Nucleic Acid ◽  
Clostridium Difficile ◽  
Predictive Accuracy ◽  
High Sensitivity ◽  
Nucleic Acid Amplification ◽  
Toxin Gene ◽  
Ultrasensitive Detection ◽  
Surrogate Markers ◽  
Recent Interest

ABSTRACT The use of nucleic acid amplification tests (NAATs) for the diagnosis of Clostridium (Clostridioides) difficile infection (CDI) leads to overdiagnosis. To improve the clinical specificity of NAATs, there has been a recent interest in using toxin gene cycle thresholds (CTs) to predict the presence and absence of toxins. Although there is an association between CT values and fecal toxin concentrations, the predictive accuracy of the former is suboptimal for use in clinical practice. Ultrasensitive toxin immunoassays to quantify free toxins in stool offer a novel option for high-sensitivity fecal toxin detection rather than using surrogate markers for prediction.

scholarly journals Nucleic Acid Amplification Test Quantitation as Predictor of Toxin Presence in Clostridium difficile Infection

2017 ◽  
Vol 56 (3) ◽  
Author(s):  
M. J. T. Crobach ◽  
N. Duszenko ◽  
E. M. Terveer ◽  
C. M. Verduin ◽  
E. J. Kuijper
Keyword(s):  
Nucleic Acid ◽  
Clostridium Difficile ◽  
Characteristic Curve ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
Quantitative Results ◽  
Testing Algorithm

ABSTRACT Multistep algorithmic testing in which a sensitive nucleic acid amplification test (NAAT) is followed by a specific toxin A and toxin B enzyme immunoassay (EIA) is among the most accurate methods for Clostridium difficile infection (CDI) diagnosis. The obvious shortcoming of this approach is that multiple tests must be performed to establish a CDI diagnosis, which may delay treatment. Therefore, we sought to determine whether a preliminary diagnosis could be made on the basis of the quantitative results of the first test in algorithmic testing, which provide a measure of organism burden. To do so, we retrospectively analyzed two large collections of samples ( n = 2,669 and n = 1,718) that were submitted to the laboratories of two Dutch hospitals for CDI testing. Both hospitals apply a two-step testing algorithm in which a NAAT is followed by a toxin A/B EIA. Of all samples, 208 and 113 samples, respectively, tested positive by NAAT. Among these NAAT-positive samples, significantly lower mean quantification cycle ( C q ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean C q values of 24.4 versus 30.4 and 26.8 versus 32.2; P < 0.001 for both cohorts). Receiver operating characteristic curve analysis was performed to investigate the ability of C q values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal C q cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, C q values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

scholarly journals Mass screening of asymptomatic persons for SARS-CoV-2 using saliva

2020 ◽  
Author(s):  
Isao Yokota ◽  
Peter Y Shane ◽  
Kazufumi Okada ◽  
Yoko Unoki ◽  
Yichi Yang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Mass Screening ◽  
Disease Outbreaks ◽  
High Sensitivity ◽  
Nucleic Acid Amplification ◽  
Contact Tracing ◽  
Global Pandemic ◽  
Screening Study ◽  
Highly Correlated ◽  
Few Data

Abstract Background COVID-19 has rapidly evolved to become a global pandemic due largely to the transmission of its causative virus through asymptomatic carriers. Detection of SARS-CoV-2 in asymptomatic people is an urgent priority for the prevention and containment of disease outbreaks in communities. However, few data are available in asymptomatic persons regarding the accuracy of PCR testing. Additionally, although self-collected saliva has significant logistical advantages in mass screening, its utility as an alternative specimen in asymptomatic persons is yet to be determined. Methods We conducted a mass-screening study to compare the utility of nucleic acid amplification, such as reverse transcriptase polymerase chain reaction (RT-PCR) testing, using nasopharyngeal swabs (NPS) and saliva samples from each individual in two cohorts of asymptomatic persons: the contact tracing cohort and the airport quarantine cohort. Results In this mass-screening study including 1,924 individuals, the sensitivity of nucleic acid amplification testing with nasopharyngeal and saliva specimens were 86% (90%CI:77-93%) and 92% (90%CI:83-97%), respectively, with specificities greater than 99.9%. The true concordance probability between the nasopharyngeal and saliva tests was estimated at 0.998 (90%CI:0.996-0.999) on the estimated airport prevalence at 0.3%. In positive individuals, viral load was highly correlated between NPS and saliva. Conclusion Both nasopharyngeal and saliva specimens had high sensitivity and specificity. Self-collected saliva is a valuable specimen to detect SARS-CoV-2 in mass screening of asymptomatic persons.

Amplification-Free DNA Detection Using a Microtip-Sensor Decorated With LNA Probes for Rapid TB Screening

2011 ◽  
Author(s):  
Shinnosuke Inoue ◽  
Woon-Hong Yeo ◽  
Jong-Hoon Kim ◽  
Jae-Hyun Chung ◽  
Kyong-Hoon Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Staphylococcus Epidermidis ◽  
Genomic Dna ◽  
Bacterial Culture ◽  
Low Cost ◽  
Surrogate Marker ◽  
High Sensitivity ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Highly Sensitive

Tuberculosis (TB) is an epidemic affecting one-third of the world’s population, mostly in developing and low-resource settings. People having active pulmonary TB are considered highly infectious; therefore, it is critical to identify and treat these patients rapidly before spreading to others. However, the most reliable TB diagnostic methods of bacterial culture or nucleic acid amplification are time-consuming and expensive. The challenge of TB diagnosis lies in highly sensitive and specific screening with low cost. Here, we present an LNA-modified microtip-sensor, which is capable of selectively detecting low-abundance DNA from bacteria. When genomic DNA of Bacillus Calmette-Gue´rin (BCG, a surrogate marker of Mycobacterium bovis), and genomic DNA of Staphylococcus epidermidis (S. epi) are used, the microtip-sensor yields the detection limit of 1,000 copies/mL within 20 minutes. The high sensitivity and specificity approaching nucleic acid amplification methods can potentially overcome the current challenges for rapid TB screening.

scholarly journals Clostridium difficile Testing Algorithm: Is There a Difference in Patients Who Test Positive by Enzyme Immunoassay vs. Those Who Only Test Positive by Nucleic Acid Amplification Methodology?

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S394-S394
Author(s):  
Jonathan Polak ◽  
Ogheneruona Odili ◽  
Mary Ashleigh Craver ◽  
Anthony Mayen ◽  
Kyle Purrman ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Clostridium Difficile ◽  
Length Of Stay ◽  
Enzyme Immunoassay ◽  
Serum Lactate ◽  
Nucleic Acid Amplification ◽  
Categorical Variables ◽  
Grant Recipient ◽  
Stool Specimens ◽  
Chi Square Analysis

Abstract Background Testing for Clostridium difficile infection (CDI) commonly involves checking for the presence of toxins A and B by enzyme immunoassay (EIA) or nucleic acid amplification (NAA). The former is very specific, but not very sensitive. The latter is very sensitive. Beginning in 2011, our hospital incorporated an algorithm that involved testing liquid stool specimens for glutamate dehydrogenase (GDH) and toxin by EIA. For discrepant results, the stool specimen was tested for the presence of toxin by NAA. We sought to determine whether there was a difference in the baseline characteristics or outcomes between the two groups. Methods We performed a chart review of all subjects who tested positive for CDI by either method between 2011 and 2016 at Vidant Medical Center, a 909 bed, tertiary care teaching hospital. Testing was only performed on liquid stool specimens. Subjects less than 18 years of age were excluded. Repeat positive specimens were excluded. We collected demographic data including age, gender, baseline temperature, white blood cell count, and serum lactate and albumin. Length of stay and in-hospital mortality were also determined for both groups. Comparison of the two groups was done using t-test for continuous and chi-square analysis for categorical variables. Results Over the 6 year period, there were 535 positive test results. 243 specimens tested positive by EIA/GDH (EIA +); 292 specimens tested positive by GDH/NAA (NAA +). Compared with the EIA + group, the NAA + group was younger (61.8 years vs. 65.1 years, P = 0.01). There were no statistical differences in the presence of abdominal tenderness, temperature &gt;38oC, serum albumin, serum lactate, length of stay, or mortality between the two groups. The EIA + group was statistically more likely to have leukocytosis (WBC &gt;20,000 cells/mm3) at the time of the CDI testing compared with the NAA + group (P = 0.0002). Conclusion There do appear to be some clinical differences in the presentation of subjects who test positive for CDI by EIA/GDH compared with those who test positive only by GDH/NAA. These differences do not appear to affect length of stay or mortality. Disclosures P. P. Cook, Gilead: Grant Investigator, Grant recipient; Merck: Grant Investigator, Grant recipient; Pfizer: Grant Investigator and Shareholder, Grant recipient

scholarly journals Ultrasensitive CRISPR-based diagnostic for field-applicable detection ofPlasmodiumspecies in symptomatic and asymptomatic malaria

2020 ◽  
Vol 117 (41) ◽  
pp. 25722-25731 ◽  
Author(s):  
Rose A. Lee ◽  
Helena De Puig ◽  
Peter Q. Nguyen ◽  
Nicolaas M. Angenent-Mari ◽  
Nina M. Donghia ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sensitivity And Specificity ◽  
Parasite Density ◽  
Point Of Care ◽  
High Sensitivity ◽  
World Health ◽  
Ultrasensitive Detection ◽  
Asymptomatic Malaria ◽  
Radical Cure ◽  
Asymptomatic Carriers

Asymptomatic carriers ofPlasmodiumparasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation ofPlasmodium falciparum,Plasmodium vivax,Plasmodium ovale, andPlasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min forPlasmodiumspecies-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. OurP. falciparumandP. vivaxassays exhibited 100% sensitivity and specificity on clinical samples (5P. falciparumand 10P. vivaxsamples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite densityP. falciparuminfections.

A novel fluorescent assay for the ultrasensitive detection of miRNA-21 with the use of G-quadruplex structures as an immobilization material for a signal indicator

2019 ◽  
Vol 55 (45) ◽  
pp. 6453-6456 ◽  
Author(s):  
Ze-Zhou Yang ◽  
Zhi-Bin Wen ◽  
Xin Peng ◽  
Ya-Qin Chai ◽  
Wen-Bin Liang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Three Dimensional ◽  
Nucleic Acid Amplification ◽  
Ultrasensitive Detection ◽  
Fluorescent Assay ◽  
Target Recycling ◽  
Dna Walker ◽  
G Quadruplex

A fluorescent assay for the ultrasensitive detection of miRNA-21 is based on immobilization of PPIX as signal indicators in massive G-quadruplex structures obtained by target recycling, three-dimensional DNA walker and RCA coupled cascade nucleic acid amplification.

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