Multicenter Clinical Validation of a Cartridge-Based Real-Time PCR System for Detection of Coccidioides spp. in Lower Respiratory Specimens

ABSTRACT Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for Coccidioides spp. directly in lower respiratory secretions and compared them to today's “gold standard,” fungal culture. The GeneSTAT Coccidioides assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at −70°C. These samples were tested by the GeneSTAT Coccidioides assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of Coccidioides, except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT Coccidioides assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.

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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 the BD Max Open System

Journal of Clinical Microbiology ◽

10.1128/jcm.00630-19 ◽

2019 ◽

Vol 57 (10) ◽

Cited By ~ 6

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.

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Comparison and Recommendations for Use of Dientamoeba fragilis Real-Time PCR Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.01466-18 ◽

2019 ◽

Vol 57 (5) ◽

Cited By ~ 5

Author(s):

Rory Gough ◽

John Ellis ◽

Damien Stark

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Content Type ◽

Prevalence Studies ◽

Dientamoeba Fragilis ◽

Stool Samples ◽

Pcr Method ◽

Genetic Signatures

ABSTRACT Dientamoeba fragilis is a gastrointestinal trichomonad parasite whose pathogenicity is yet to be determined. The difficulty involved in microscopically diagnosing D. fragilis in feces led to the development of real-time PCR methodologies for the detection of D. fragilis in stool samples. Prevalence studies in Europe show much higher levels of infection where a laboratory-developed real-time assay is the predominant assay for the detection of Dientamoeba fragilis than in regions that use the EasyScreen assay for detection of gastrointestinal pathogens. The aim of this study was to compare a commercially available Dientamoeba fragilis assay (Genetic Signatures EasyScreen assay) to a widely used laboratory-developed real-time PCR method. Two hundred fifty fecal samples were screened using the laboratory-developed real-time assay on four real-time PCR platforms producing a number of discrepant results. Limit-of-detection studies were undertaken to attempt to resolve sensitivity for each platform tested. The presence or absence of Dientamoeba fragilis DNA in discrepant samples was shown using PCR amplicon next-generation sequencing. Eukaryotic 18S diversity profiling was conducted on discrepant samples to identify the presence or absence of additional protozoan species in samples that may be responsible for cross-reactivity seen in these samples. The results revealed the potential for multiple false-positive results when using the laboratory-developed real-time assay across multiple real-time platforms using manufacturer default settings. This report provides recommendations to resolve these issues where possible and suggestions for future prevalence studies, and it emphasizes the EasyScreen assay as the molecular method of choice as well as the need for standardization of detection assays across all nations screening for D. fragilis.

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Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR

Foods ◽

10.3390/foods9030332 ◽

2020 ◽

Vol 9 (3) ◽

pp. 332

Author(s):

Jasmin Wrage ◽

Oxana Kleyner ◽

Sascha Rohn ◽

Jürgen Kuballa

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Detection Method ◽

Immunoglobulin E ◽

Limit Of Detection ◽

Cocos Nucifera ◽

Cross Reactivity ◽

Pcr Assay ◽

Probe System ◽

Ige Mediated

So far, only a few cases of immunoglobulin E (IgE)-mediated coconut allergies have been described in the literature. Due to a growing consumption of coconut-containing foods in occidental countries, the number of coconut allergies may also increase. As there is no causative immunotherapy in clinical routine, appropriate food labelling is particularly important, also with regard to cross-contamination, to prevent serious health consequences. The purpose of this study was to develop a DNA-based detection method for coconut (Cocos nucifera). Initially, three sets of coconut-specific primers were designed and tested. A TaqMan™ probe was then developed to identify and quantify coconut by real-time PCR assay. With 27 other plant and animal species, the specificity of the primer/probe system was tested and cross reactivity was excluded. In a dilution series, a limit of detection of 1 pg/µL was determined. Thus, the developed real-time PCR assay is a suitable method to detect coconut in food.

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Real-Time PCR Detection of Parapoxvirus DNA,

Clinical Chemistry ◽

10.1373/clinchem.2005.060335 ◽

2006 ◽

Vol 52 (2) ◽

pp. 316-319 ◽

Cited By ~ 53

Author(s):

Andreas Nitsche ◽

Mathias Büttner ◽

Sonja Wilhelm ◽

Georg Pauli ◽

Hermann Meyer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Probit Analysis ◽

Animal Origin ◽

Pcr Assay ◽

Medical Practitioners ◽

B2l Gene ◽

Zoonotic Infections

Abstract Background: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported. Methods: A minor groove binder–based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms. Results: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed. Conclusion: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.

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Development of an Immunochromatographic Assay Kit Using Fluorescent Silica Nanoparticles for Rapid Diagnosis of Acanthamoeba Keratitis

Journal of Clinical Microbiology ◽

10.1128/jcm.02595-14 ◽

2014 ◽

Vol 53 (1) ◽

pp. 273-277 ◽

Cited By ~ 10

Author(s):

Koji Toriyama ◽

Takashi Suzuki ◽

Tomoyuki Inoue ◽

Hiroshi Eguchi ◽

Saichi Hoshi ◽

...

Keyword(s):

Silica Nanoparticles ◽

Real Time ◽

Real Time Pcr ◽

High Sensitivity ◽

Acanthamoeba Keratitis ◽

Cross Reactivity ◽

Immunochromatographic Assay ◽

Content Type ◽

Fluorescent Silica Nanoparticles ◽

Positive Results

We developed an immunochromatographic assay kit that uses fluorescent silica nanoparticles bound to anti-Acanthamoebaantibodies (fluorescent immunochromatographic assay [FICGA]) and evaluated its efficacy for the detection ofAcanthamoebaand diagnosis ofAcanthamoebakeratitis (AK). The sensitivity of the FICGA kit was evaluated using samples ofAcanthamoebatrophozoites and cysts diluted to various concentrations. A conventional immunochromatographic assay kit with latex labels (LICGA) was also evaluated to determine its sensitivity in detectingAcanthamoebatrophozoites. To check for cross-reactivity, the FICGA was performed by using samples of other common causative pathogens of infectious keratitis, such asPseudomonas aeruginosa,Staphylococcus aureus,Staphylococcus epidermidis, andCandida albicans. Corneal scrapings from patients with suspected AK were tested with the FICGA kit to detect the presence ofAcanthamoeba, and the results were compared with those of real-time PCR. The FICGA kit detected organisms at concentrations as low as 5 trophozoites or 40 cysts per sample. There were no cross-reactivities with other pathogens. The FICGA was approximately 20 times more sensitive than the LICGA for the detection ofAcanthamoebatrophozoites. The FICGA kit yielded positive results for all 10 patients, which corresponded well with the real-time PCR results. The FICGA kit demonstrated high sensitivity for the detection ofAcanthamoebaand may be useful for the diagnosis of AK.

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An Optimized Real-Time qPCR Method for the Effective Detection of Human Malaria Infections

Diagnostics ◽

10.3390/diagnostics11050736 ◽

2021 ◽

Vol 11 (5) ◽

pp. 736

Author(s):

Saiful Arefeen Sazed ◽

Mohammad Golam Kibria ◽

Mohammad Shafiul Alam

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Plasmodium Species ◽

Cost Effective ◽

Plasmodium Malariae ◽

Cross Reactivity ◽

Diagnostic Pcr ◽

Pcr Method ◽

Human Plasmodium

Polymerase chain reaction, although an expensive method for the detection of human Plasmodium spp., is still considered the finest for the diagnosis of malaria. The conventional diagnostic PCR is an inexpensive process but consumes a lot of time, reagents and lacks sensitivity. On the other hand, real-time PCR assays currently being used are mostly probe-based expensive methods and sometimes not feasible to detect all the species in a single amplification reaction condition. Here we have established a real-time PCR method that is time and cost effective with a single protocol to detect and distinguish five human Plasmodium species using the existing primers efficiently. The primers used here are being used in the conventional method and the sensitivity as well as specificity of this method has also been immensely improved (100%). The lower limit of detection for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae are 0.064 parasites/µL, 1.6 parasites/µL, and 0.32 parasites/µL respectively and no cross reactivity was observed. Besides, we have analyzed melt curves that can be used for further species confirmation and validation purposes using multiplex systems. This method, therefore, can be considered as an alternative to the existing lineup for molecular diagnosis of malaria in endemic countries.

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Use of Quantitative Real-Time PCR for Direct Detection of Serratia marcescens in Marine and Other Aquatic Environments

Applied and Environmental Microbiology ◽

10.1128/aem.02755-13 ◽

2013 ◽

Vol 80 (5) ◽

pp. 1679-1683 ◽

Cited By ~ 18

Author(s):

Jessica Joyner ◽

David Wanless ◽

Christopher D. Sinigalliano ◽

Erin K. Lipp

Keyword(s):

Serratia Marcescens ◽

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Florida Keys ◽

Sponge Tissue ◽

Acropora Palmata ◽

Quantitative Real Time Pcr ◽

Coral Mucus ◽

Content Type

ABSTRACTSerratia marcescensis the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coralAcropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detectS. marcescensdirectly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted theluxSgene and was able to distinguishS. marcescensfrom otherSerratiaspecies with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence ofS. marcescensfor as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmentalS. marcescensin complex sewage influent samples at up to 761 CE ml−1and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml−1. This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

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Development of a Real-Time PCR Assay for Identification of Coccidioides immitis by Use of the BD Max System: TABLE 1

Journal of Clinical Microbiology ◽

10.1128/jcm.02731-14 ◽

2015 ◽

Vol 53 (3) ◽

pp. 926-929 ◽

Cited By ~ 13

Author(s):

Marilyn Mitchell ◽

Dominic Dizon ◽

Robert Libke ◽

Michael Peterson ◽

David Slater ◽

...

Keyword(s):

Sample Preparation ◽

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Hospital Setting ◽

Internal Standard ◽

Specimen Preparation ◽

Coccidioides Immitis ◽

Rt Pcr ◽

Content Type

Rapid real-time PCR (RT-PCR) can be performed in a community hospital setting to identifyCoccidioidesspecies using the new Becton Dickinson molecular instrument BD Max. Following sample preparation, DNA extraction and PCR were performed on the BD Max using the BD Max extraction kit ExK-DNA-1 test strip and a master mix prepared by BioGX (Birmingham, AL). Sample preparation took 2 h, and testing on the BD Max took an additional 2 h. Method sensitivity and specificity were evaluated along with the limits of detection to confirm that this convenient method would provide medically useful information. Using serial dilutions, the lower limit of detection was determined to be 1 CFU/μl. Testing with this method was validated using samples from various body sites, including bronchial alveolar lavage (BAL) fluid; sputum and lung tissue samples; and pleural and spinal fluids. Safety protocols were established, and specimen preparation processes were developed for the various types of specimens. The range for the cycle threshold (CT) indicating adequate fluorescent signal to signify a positive result was established along with the acceptable range for the internal standard. Positive controls run with each batch were prepared by spiking a pooled BAL fluid specimen with a known dilution ofCoccidioides immitisorganism. Our experience with testing >330 patient samples shows that clinically relevant information can be available within 4 h using an RT-PCR method on the BD Max to identifyCoccidioidesspp., with sensitivity equivalent to culture.

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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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Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens

10.1101/2021.04.08.21254780 ◽

2021 ◽

Author(s):

Sudha Chaturvedi ◽

Tanya R Victor ◽

Anuradha Marathe ◽

Ketevan Sidamonidze ◽

Kelly L Crucillo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Coccidioides Immitis ◽

Coccidioides Posadasii ◽

Pcr Assay ◽

Valley Fever ◽

Clinical Specimens ◽

Wide Range

Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 129 isolates and three primary specimens as C. posadasii and 23 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the surveillance of coccidioidomycosis.

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