scholarly journals Comparison of Two Commercially Available qPCR Kits for the Detection of Candida auris

2021 ◽  
Vol 7 (2) ◽  
pp. 154
Author(s):  
Janko Sattler ◽  
Janina Noster ◽  
Anne Brunke ◽  
Georg Plum ◽  
Pia Wiegel ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Positive ◽  
Limit Of Detection ◽  
Detection Methods ◽  
Limited Data ◽  
Candida Auris ◽  
Human Blood Samples ◽  
Newcastle Upon Tyne ◽  
Positive Results

Candida auris is an emerging pathogen with resistance to many commonly used antifungal agents. Infections with C. auris require rapid and reliable detection methods to initiate successful medical treatment and contain hospital outbreaks. Conventional identification methods are prone to errors and can lead to misidentifications. PCR-based assays, in turn, can provide reliable results with low turnaround times. However, only limited data are available on the performance of commercially available assays for C. auris detection. In the present study, the two commercially available PCR assays AurisID (OLM, Newcastle Upon Tyne, UK) and Fungiplex Candida Auris RUO Real-Time PCR (Bruker, Bremen, Germany) were challenged with 29 C. auris isolates from all five clades and eight other Candida species as controls. AurisID reliably detected C. auris with a limit of detection (LoD) of 1 genome copies/reaction. However, false positive results were obtained with high DNA amounts of the closely related species C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. The Fungiplex Candida Auris RUO Real-Time PCR kit detected C. auris with an LoD of 9 copies/reaction. No false positive results were obtained with this assay. In addition, C. auris could also be detected in human blood samples spiked with pure fungal cultures by both kits. In summary, both kits could detect C. auris-DNA at low DNA concentrations but differed slightly in their limits of detection and specificity.

scholarly journals Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia

2020 ◽  
Author(s):  
Nor Afizah Nuin ◽  
Angelica Fiona Tan ◽  
Yao Long Lew ◽  
Kim A Piera ◽  
Timothy William ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Plasmodium Knowlesi ◽  
Plasmodium Species ◽  
Detection Methods ◽  
Clinical Samples ◽  
Public Health Issue ◽  
Gene Target

Abstract Background : The monkey parasite Plasmodium knowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesi is now the dominant cause of human malaria. Molecular detection methods for P. knowlesi are essential for accurate diagnosis and in monitoring progress towards malaria elimination of other Plasmodium species. However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples. Methods : Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi 18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed P. knowlesi gene target. Diagnostic accuracy was evaluated using 52 P. knowlesi, 25 P. vivax , 21 P. falciparum , and 10 P. malariae clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated. Results : The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesi was comparable at 98.1% (95%CI 89.7-100) and 100% (95%CI 93.2-100) respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesi was high at 98.8% (95%CI 93.4-100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting P. vivax , P. falciparum , and P. malariae was comparable to P. knowlesi . The abTES™ assay demonstrated a lower LOD for P. knowlesi of ≤0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. Hospital microscopy demonstrated a sensitivity of 78.8% (95%CI 65.3-88.9) and specificity of 80.4% (95%CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi . Conclusion : The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of P. knowlesi infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.

scholarly journals Implications of Limits of Detection of Various Methods for Bacillus anthracis in Computing Risks to Human Health

2009 ◽  
Vol 75 (19) ◽  
pp. 6331-6339 ◽  
Author(s):  
Amanda B. Herzog ◽  
S. Devin McLennan ◽  
Alok K. Pandey ◽  
Charles P. Gerba ◽  
Charles N. Haas ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Bacillus Anthracis ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Risk Assessment ◽  
Detection Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Environmental Matrices ◽  
Limits Of Detection

ABSTRACT Used for decades for biological warfare, Bacillus anthracis (category A agent) has proven to be highly stable and lethal. Quantitative risk assessment modeling requires descriptive statistics of the limit of detection to assist in defining the exposure. Furthermore, the sensitivities of various detection methods in environmental matrices are vital information for first responders. A literature review of peer-reviewed journal articles related to methods for detection of B. anthracis was undertaken. Articles focused on the development or evaluation of various detection approaches, such as PCR, real-time PCR, immunoassay, etc. Real-time PCR and PCR were the most sensitive methods for the detection of B. anthracis, with median instrument limits of detection of 430 and 440 cells/ml, respectively. There were very few peer-reviewed articles on the detection methods for B. anthracis in the environment. The most sensitive limits of detection for the environmental samples were 0.1 CFU/g for soil using PCR-enzyme-linked immunosorbent assay (ELISA), 17 CFU/liter for air using an ELISA-biochip system, 1 CFU/liter for water using cultivation, and 1 CFU/cm2 for stainless steel fomites using cultivation. An exponential dose-response model for the inhalation of B. anthracis estimates of risk at concentrations equal to the environmental limit of detection determined the probability of death if untreated to be as high as 0.520. Though more data on the environmental limit of detection would improve the assumptions made for the risk assessment, this study's quantification of the risk posed by current limitations in the knowledge of detection methods should be considered when employing those methods in environmental monitoring and cleanup strategies.

scholarly journals Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

2007 ◽  
Vol 53 (12) ◽  
pp. 2042-2050 ◽  
Author(s):  
Brent C Satterfield ◽  
David A Kulesh ◽  
David A Norwood ◽  
Leonard P Wasieloski ◽  
Michael R Caplan ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
False Positive ◽  
Yersinia Pseudotuberculosis ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Specific Cell ◽  
Single Nucleotide ◽  
Positive Results

AbstractBackground: False-positive results are a common problem in real-time PCR identification of DNA sequences that differ from near neighbors by a single-nucleotide polymorphism (SNP) or deletion. Because of a lack of sufficient probe specificity, post-PCR analysis, such as a melting curve, is often required for mutation differentiation.Methods: Tentacle Probes™, cooperative reagents with both a capture and a detection probe based on specific cell-targeting principles, were developed as a replacement for 2 chromosomal TaqMan–minor groove binder (MGB) assays previously developed for Yersinia pestis and Bacillus anthracis detection. We compared TaqMan-MGB probes to Tentacle Probes for SNP and deletion detection based on the presence or absence of a growth curve.Results: With the TaqMan-MGB Y. pestis yp48 assays, false-positive results for Yersinia pseudotuberculosis occurred at every concentration tested, and with the TaqMan-MGB B. anthracis gyrA assays, false-positive results occurred in 21 of 29 boil preps of environmental samples of near neighbors. With Tentacle Probes no false-positive results occurred.Conclusions: The high specificity exhibited by Tentacle Probes may eliminate melting curve analysis for SNP and deletion mutation detection, allowing the diagnostic use of previously difficult targets.

scholarly journals Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
L. Leach ◽  
Y. Zhu ◽  
S. Chaturvedi
Keyword(s):  
Health Care ◽  
Real Time ◽  
Real Time Pcr ◽  
Multidrug Resistant ◽  
Effective Control ◽  
Pcr Assay ◽  
Candida Auris ◽  
Content Type ◽  
The Real ◽  
Positive Results

ABSTRACT Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 (ITS2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris. The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities.

scholarly journals 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

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.

scholarly journals Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia

2020 ◽  
Author(s):  
Nor Afizah Nuin ◽  
Angelica Fiona Tan ◽  
Yao Long Lew ◽  
Kim A Piera ◽  
Timothy William ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Plasmodium Knowlesi ◽  
Plasmodium Species ◽  
Detection Methods ◽  
Clinical Samples ◽  
Public Health Issue ◽  
Gene Target

Abstract Introduction: The monkey parasite Plasmodium knowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesi is now the dominant cause of human malaria. Molecular detection methods for P. knowlesi are essential for accurate diagnosis and in monitoring progress towards malaria elimination of other Plasmodium species. However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples. Methods: Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi 18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed P. knowlesi gene target. Diagnostic accuracy was evaluated using 52 P. knowlesi, 25 P. vivax, 21 P. falciparum, and 10 P. malariae clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated. Results: The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesi was comparable at 98.1% (95%CI 89.7-100) and 100% (95%CI 93.2-100) respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesi was high at 98.8% (95%CI 93.4-100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting P. vivax, P. falciparum, and P. malariae was comparable to P. knowlesi. The abTES™ assay demonstrated a lower LOD for P. knowlesi of ≤0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. Hospital microscopy demonstrated a sensitivity of 78.8% (95%CI 65.3-88.9) and specificity of 80.4% (95%CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi. Conclusion: The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of P. knowlesi infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.

scholarly journals Comparative Evaluation of Two Commercial Real-time Pcr Kits (Quantifast™ and Abtes™) for the Detection of Plasmodium Knowlesi and Other Plasmodium Species in Sabah, Malaysia

2020 ◽  
Author(s):  
Nor Afizah Nuin ◽  
Angelica Fiona Tan ◽  
Yao Long Lew ◽  
Kim A Piera ◽  
Timothy William ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Plasmodium Knowlesi ◽  
Plasmodium Species ◽  
Detection Methods ◽  
Clinical Samples ◽  
Public Health Issue ◽  
Gene Target ◽  
Negative Controls

Abstract Introduction: The monkey parasite Plasmodiumknowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesiis now the dominant cause of human malaria. Molecular detection methods for P. knowlesiare essential for accurate diagnosis and in monitoring progress towards malaria elimination of other Plasmodium species.However,recent commercially available PCR malaria kits have unpublished P. knowlesigene targets or have not been evaluated against clinical samples.Methods:Two real-time PCR methods currently used in Sabahfor confirmatorymalaria diagnosis and surveillance reportingwere evaluated: theQuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi18S SSU rRNA;and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosedP. knowlesi gene target. Diagnostic accuracywas evaluatedusing 52P. knowlesi, 25P. vivax, 21 P. falciparum, and 10 P. malariaeclinical isolates, and 26 malaria negative controls, and compared against a validated referencenested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated. Results:The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesiwas comparable at 98.1% (95%CI 89.7-100) and 100%(95%CI 93.2-100) respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesiwas high at 98.8% (95%CI 93.4-100) for both assays.The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits fordetecting P. vivax, P. falciparum, and P. malariae was comparable to P. knowlesi.The abTES™ assay demonstrated a lower LOD for P. knowlesiof ≤0.125 parasites/µL compared to QuantiFast™ witha LOD of20 parasites/µL.Hospital microscopy demonstrated a sensitivity of 78.8% (95%CI 65.3-88.9) and specificity of 80.4% (95%CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi.Conclusion:The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of P. knowlesi infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supportingits use as a second-line referral-laboratory diagnostic toolin Sabah, Malaysia.

scholarly journals A Rapid and Automated Sample-to-Result Candida auris Real-Time PCR Assay for High-Throughput Testing of Surveillance Samples with the BD Max Open System

2019 ◽  
Vol 57 (10) ◽  
Author(s):  
L. Leach ◽  
A. Russell ◽  
Y. Zhu ◽  
S. Chaturvedi ◽  
V. Chaturvedi
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Open System ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Pcr Assay ◽  
Candida Auris ◽  
Content Type ◽  
Clinical Sensitivity

ABSTRACT The 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 (L. Leach, Y. Zhu, and S. Chaturvedi, J Clin Microbiol 56:e01223-17, 2018, https://doi.org/10.1128/JCM.01223-17). The assay played a crucial role in the ongoing investigations of the 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 the 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 quadruplicate yielded assay precision. BD Max optimum assay conditions were as follows: DNA extraction at 75°C for 20 min and the use of PerfeCTa multiplex quantitative PCR (qPCR) ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR. 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 results of the manual assay. The BD Max assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-h workday.

scholarly journals Isıl İşlem Görmüş Sütlerde Salmonella Typhimurium Canlı Hücrelerinin PMA/Real-Time PCR ile Belirlenmesi

2017 ◽  
Vol 5 (5) ◽  
pp. 518
Author(s):  
Zülal Kesmen ◽  
Hakiye Aslan
Keyword(s):  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
False Positive ◽  
Live Cells ◽  
Dead Cell ◽  
Milk Samples ◽  
Heat Treated ◽  
Pcr Method ◽  
Positive Results

Applying different technological processes during the production of food has a lethal effect on the bacteria but DNA of these bacterial strains may cause false positive results when detected by real time PCR technique because they preserve their existence for a certain period of time. To overcome this shortcoming of the real time PCR technique, a new method has been developed in recent years, based on the removal of dead cell DNA from the medium by treatment with Propodium Monoazide (PMA) before DNA extraction. In this study, real-time PCR method was combined with PMA application for the detection of live cells of Salmonella Typhimurium in heat treated milk samples. For this purpose, milk samples inoculated with S. Tyhimurium were heat treated at different temperatures (60, 65, 70 and 75°C) and times (15, 60, 300, 900 sec) and number of live bacteria was determined comparatively by direct real-time PCR, PMA/real-time PCR and conventional cultural method. As a result, unlike the direct real time PCR technique, PMA/real-time PCR method prevents to a certain extent of false positive results from dead cells at all tested temperatures and times but higher results were obtained from PMA/real-time PCR method when compared to conventional cultural results. Therefore, further studies should be carried out to optimize the conditions of the PMA application in order to eliminate the high positive results detected by the PMA / real-time PCR method

Isolation and identification of Marek’s disease virus (MDV) from feather follicle epithelium and internal organs of diseased chickens in Dakahlia Governorate, Egypt

2019 ◽  
Vol 20 (2) ◽  
pp. 6-11
Author(s):  
Aly El-Kenawy ◽  
Mohamed El-Tholoth ◽  
Emad A
Keyword(s):  
Real Time ◽  
Disease Virus ◽  
Real Time Pcr ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Field Samples ◽  
Feather Follicle ◽  
Positive Results

In the present study, a total of 16 samples including feather follicle epithelium, ovary, spleen and kidney (4 samples for each organ) were collected from diseased chicken flocks suspected to be infected with Marek’s disease virus (MDV) at Dakahlia Governorate, Egypt during the period from October 2016 to October 2017. Each sample was pooled randomly from three to five birds (90 to 360 days old). The isolation of the suspected virus from the collected samples was carried out via chorioallantoic membranes (CAMs) of 12 days old embryonated chicken eggs (ECEs). Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard MDV. MDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Molecular identification of virus was carried out by conventional polymerase chain reaction (PCR) and real- time PCR in four selected samples. The results revealed that 14 samples (87.5%) including 4 (100%) samples from feather follicle epithelium, ovary and kidney and 2 (50%) samples from spleen, showed positive results in virus isolation after 3rd passage. The positive results percentage by AGPT for field samples were 50% (8 out of 16 samples), while after the 3rd passage in ECEs were 37.5% (6 out of 16 samples) and the positive results percentage by IFAT for field samples were 62.5% (10 out of 16 samples), while after the 3rd passage in ECEs were 81.25 % (13 out of 16 samples). Viral nucleic acid was detected in all selected samples by conventional and real- time PCR. The results indicate that feather follicle epithelium is the best organ for MDV detection. IFAT is superior over AGPT in virus detection. Conventional and real - time PCR could be efficiently used for molecular detection of the virus.

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