Development and Evaluation of a Calibrator Material for Nucleic Acid-Based Assays for Diagnosing Aspergillosis

Lyon, G. Marshall; Abdul-Ali, Deborah; Loeffler, Juergen; White, P. Lewis; Wickes, Brian L.; Herrera, Mónica; Alexander, Barbara D.; Baden, Lindsey R.; Clancy, Cornelius J.; Denning, David W.; Nguyen, M.H.; Sugrue, Michele W.; Wheat, L. Joseph; Wingard, John R.; Donnelly, J. Peter; Barnes, Rosemary Ann; Patterson, Thomas F.; Caliendo, Angela M.

doi:10.1128/jcm.00744-13

Cited by 23 publications

(10 citation statements)

References 6 publications

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“…Finally, based on the findings of the aforementioned studies, the Aspergillus Technology Consortium created and validated an Aspergillus sp. DNA calibrator material that can be used to standardize nucleic acid-based diagnostic assays, thus improving interlaboratory comparisons of qualitative and quantitative results between different techniques (184). Although the repercussions of these endeavors have yet to be realized in ensuing clinical trials, it is anticipated that they will reduce the inconsistencies observed in previous studies and will pave the way to large-scale implementation of Aspergillus sp.…”

Section: Pcrmentioning

confidence: 99%

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

et al. 2014

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SUMMARY Invasive fungal infections constitute a serious threat to an ever-growing population of immunocompromised individuals and other individuals at risk. Traditional diagnostic methods, such as histopathology and culture, which are still considered the gold standards, have low sensitivity, which underscores the need for the development of new means of detecting fungal infectious agents. Indeed, novel serologic and molecular techniques have been developed and are currently under clinical evaluation. Tests like the galactomannan antigen test for aspergillosis and the β-glucan test for invasive Candida spp. and molds, as well as other antigen and antibody tests, for Cryptococcus spp., Pneumocystis spp., and dimorphic fungi, have already been established as important diagnostic approaches and are implemented in routine clinical practice. On the other hand, PCR and other molecular approaches, such as matrix-assisted laser desorption ionization (MALDI) and fluorescence in situ hybridization (FISH), have proved promising in clinical trials but still need to undergo standardization before their clinical use can become widespread. The purpose of this review is to highlight the different diagnostic approaches that are currently utilized or under development for invasive fungal infections and to identify their performance characteristics and the challenges associated with their use.

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Section: Pcrmentioning

confidence: 99%

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

et al. 2014

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“…In an attempt to address qPCR assay variability, the EAPCRI collaborated with the Aspergillus Technology Consortium (AsTeC) and Invasive Aspergillosis Animal Models (IAAM) group to create an A. fumigatus DNA calibrator (49). Twelve clinical and research laboratories in the United States and Europe independently determined the concentration of DNA in the calibrator by performing replicate testing of a dilution series.…”

Section: Molecular Methods For Direct Detection Of Aspergillus In CLImentioning

confidence: 99%

Molecular Diagnostic Testing for Aspergillus

Powers-Fletcher

Hanson

2016

J Clin Microbiol

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The direct detection of Aspergillus nucleic acid in clinical specimens has the potential to improve the diagnosis of aspergillosis by offering more rapid and sensitive identification of invasive infections than is possible with traditional techniques, such as culture or histopathology. Molecular tests for Aspergillus have been limited historically by lack of standardization and variable sensitivities and specificities. Recent efforts have been directed at addressing these limitations and optimizing assay performance using a variety of specimen types. This review provides a summary of standardization efforts and outlines the complexities of molecular testing for Aspergillus in clinical mycology.

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“…Specific to mycology, the European Aspergillus PCR initiative (EAPCRI) has offered recommendations to provide optimal methodology when testing whole-blood and serum samples by Aspergillus PCR (5,6). An international standard has been developed, providing reference material for assay comparison and validation (7).…”

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Evaluation of a Commercially Developed Semiautomated PCR–Surface-Enhanced Raman Scattering Assay for Diagnosis of Invasive Fungal Disease

et al. 2014

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c Nonculture-based tests are gaining popularity in the diagnosis of invasive fungal disease (IFD), but PCR is excluded from disease-defining criteria because of limited standardization and a lack of commercial assays. Commercial PCR assays may have a standardized methodology while providing quality assurance. The detection of PCR products by a surface-enhanced Raman scattering (SERS) assay potentially provides superior analytical sensitivity and multiplexing capacity compared to that of realtime PCR. Using this approach, the RenDx Fungiplex assay was developed to detect Candida and Aspergillus. Analytical and clinical evaluations of the assay were undertaken using extraction methods according to European Aspergillus PCR Initiative (EAPCRI) recommendations. A total of 195 previously extracted samples (133 plasma, 49 serum, and 13 whole blood) from 112 patients (29 with proven/probable IFD) were tested. The 95% limit of detection of Candida and Aspergillus was 200 copies per reaction, with an overall reproducibility of 92.1% for detecting 20 input copies per PCR, and 89.8% for the nucleic acid extraction-PCR-SERS process for detecting fungal burdens of <20 genome equivalents per sample. A clinical evaluation showed that assay positivity significantly correlated with IFD (P < 0.0001). The sensitivity of the assay was 82.8% and was similar for both Candida (80.0%) and Aspergillus (85.7%). The specificity was 87.5% and was increased (97.5%) by using a multiple (>2 samples) PCR-positive threshold. In summary, the RenDx Fungiplex assay is a PCR-SERS assay for diagnosing IFD and demonstrates promising clinical performance on a variety of samples. This was a retrospective clinical evaluation, and performance is likely to be enhanced through a prospective analysis of clinical validity and by determining clinical utility.

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Development and Evaluation of a Calibrator Material for Nucleic Acid-Based Assays for Diagnosing Aspergillosis

Cited by 23 publications

References 6 publications

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

Molecular Diagnostic Testing for Aspergillus

Evaluation of a Commercially Developed Semiautomated PCR–Surface-Enhanced Raman Scattering Assay for Diagnosis of Invasive Fungal Disease

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