Diagnosis of Invasive Mold Infection by Real-Time Quantitative PCR

Pham, Audrey S.; Tarrand, Jeffrey J.; May, Gregory S.; Lee, Ming S.; Kontoyiannis, Dimitrios P.; Han, Xiang Y.

doi:10.1309/rq05pp9neg6dadxr

Cited by 67 publications

(28 citation statements)

References 18 publications

(22 reference statements)

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“…Most protocols (21/24) attaining the threshold could also reproducibly detect the sample with 5 genomes/ml, although at this concentration protocols 14, 17, and 19 generated positivity rates of 50% and 33.3%, respectively. Eleven protocols (1,3,4,6,7,10,12,13,19, 21, and 25) were able to detect the 1-genome/ml sample. At this concentration, not every 1-ml aliquot will contain DNA (Table 1), and logistical regression analysis generated a probability of 63.21% that an individual 1-ml aliquot of the 1-genome/ml specimen would actually contain target, and this would be accentuated if volumes less than 1 ml were used for the initial DNA extraction.…”

Section: Vol 49 2011mentioning

confidence: 99%

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Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

White¹,

et al. 2011

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A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (>100 l) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

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Section: Vol 49 2011mentioning

confidence: 99%

“…Aspergillus PCR assays have been used to successfully detect DNA in serum. Although the methodology varies, the pooled sensitivity and specificity of serum PCR were 72% and 96.5%, respectively (2,3,10,12,14).…”

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confidence: 99%

Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

White¹,

et al. 2011

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“…In such cases, PCR facilitates species identification, which cannot be achieved through microscopy but can serve an important role in guiding antifungal therapy. Multiplex PCR has also been tested as a method to detect fungal species in whole-blood (236,238,(243)(244)(245), serum (246), or BAL fluid (247) samples from patients at high risk for IFIs. The results are variable, but most studies report superior sensitivities and specificities of Ͼ80% (236,238,243,248).…”

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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“…However, a range of chemistry types are currently used (SYBR green, hybridization probes, and hydrolysis probes), and another highly debated area is the choice of the target DNA. Several multicopy genes, such as ribosomal genes-5.8S (24), 28S (2,5), and, most frequently, 18S (4,15,16,18), and the mitochondrial gene (3,7,25)-have been investigated for the assessment of real-time PCR assay in the diagnosis of IA. However, the designs of these studies are very diverse, making it impossible to compare the diagnostic performance with each type of DNA target.…”

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confidence: 99%

Ribosomal and Mitochondrial DNA Target for Real-Time PCR Diagnosis of Invasive Aspergillosis

et al. 2011

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The aim of the present study was to assess the diagnostic efficacy of a combination of two quantitative Aspergillus PCR assays, targeting a mitochondrial and a ribosomal target, in patients with risk factors for invasive aspergillosis (IA) and positive galactomannan (GM) antigen. Forty-four patients with hematological malignancies and risk factors for IA according to revised European Organization for Research on Treatment of Cancer and the Mycoses Study Group criteria (EORTC/MSG) criteria and presenting at least two sequential GM-positive sera were included in the study. Mitochondrial PCR was carried out prospectively on all GMpositive serum samples. Ribosomal PCR was carried out retrospectively on frozen stored sera. The sensitivities of mitochondrial and ribosomal PCRs were 58% and 50%, respectively. The diagnostic test performance was improved by using a combination of both PCR assays and by considering a patient PCR positive when at least two positive results were obtained. The sensitivity, specificity, and positive and negative likelihood ratios were 65%, 94%, and 11.8 and 0.37, respectively. A significant association between fatal outcome at 90 days and positive results of ribosomal PCR assays was observed (adjusted hazard ratio ‫؍‬ 8.2; 95% confidence interval [CI] ‫؍‬ 1.0 to 65.8; P ‫؍‬ 0.048). Our results showed that the combination of two PCR assays targeting mitochondrial and ribosomal Aspergillus DNA improves the sensitivity of PCR in the diagnosis of IA in hematological patients with risk factors and positive GM results. This study also confirms that a positive PCR result is associated with a poor prognosis in these patients and should lead to specific antifungal therapy being introduced immediately.

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Diagnosis of Invasive Mold Infection by Real-Time Quantitative PCR

Cited by 67 publications

References 18 publications

Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

Ribosomal and Mitochondrial DNA Target for Real-Time PCR Diagnosis of Invasive Aspergillosis

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