Automated and Manual Methods of DNA Extraction for
            <i>Aspergillus fumigatus</i>
            and
            <i>Rhizopus oryzae</i>
            Analyzed by Quantitative Real-Time PCR

Francesconi, Andrea; Kasai, Miki; Harrington, Susan M.; Beveridge, Mara; Petraitienė, Rūta; Petraitis, Vidmantas; Schaufele, Robert L.; Walsh, Thomas J.

doi:10.1128/jcm.02246-07

Cited by 46 publications

(29 citation statements)

References 38 publications

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“…was developed by using the multicopy 28S rRNA target. The pan-Aspergillus NASBA assay demonstrated a high sensitivity, with the detection limit being 1 CFU of conidia or even lower (66% of the runs detected 0.1 CFU), which is comparable to the detection limit of the qPCR used in this and other studies (6,27,31). Good linearity was also observed by this assay when TNAs extracted from serially diluted conidial suspensions were used.…”

Section: Discussionsupporting

confidence: 51%

Detection of Aspergillus fumigatus in a Rat Model of Invasive Pulmonary Aspergillosis by Real-Time Nucleic Acid Sequence-Based Amplification

Zhao¹,

Park²,

Warn

et al. 2010

J Clin Microbiol

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Invasive pulmonary aspergillosis (IPA) is a major cause of morbidity and mortality in immunocompromised patients. Therapeutic success depends on an early diagnosis and the early initiation of antifungal therapy (5). The rapid and sensitive detection of Aspergillus from clinical samples may facilitate the early diagnosis of IPA. Molecular diagnostics have emerged as promising methods for the diagnosis of a variety of infectious diseases, including fungal infections (22). Given the advantages of detection sensitivity and speed, real-time quantitative PCR (qPCR) has been extensively studied and explored as a tool for use for the detection and identification of Aspergillus and other pathogenic fungi in clinical samples (9-12, 31). However, a lack of standardization often makes it difficult to compare results between laboratories, which is critical for the ultimate approval of a test as a standard diagnostic test for IPA (2).Nucleic acid sequence-based amplification (NASBA) is an isothermal amplification process that specifically amplifies RNA even in the presence of genomic DNA (30). The amplification is highly robust, yielding Ͼ10 12 amplicons in 30 min (4). The amplified single-stranded RNA can easily be detected in real time by the use of molecular beacon (MB) probes, which are self-reporting, hairpin-structured, single-stranded nucleic acid (NA) probes that brightly fluoresce when they are bound to their targets (33). NASBA-based assays for the detection of Aspergillus have been described by a few study groups (14, 36). However, to our knowledge, real-time NASBA has not been applied to the detection of Aspergillus fumigatus from an animal model of IPA.The object of the study described here was to evaluate a highly sensitive real-time NASBA assay for its ability to detect Aspergillus burdens in an inhalational rat model of IPA at selected time points in the course of infection and to compare the diagnostic yield with the yields obtained by culture and qPCR. MATERIALS AND METHODSAnimal model. Thirty male Sprague-Dawley rats (Charles River, Margate, United Kingdom) weighing 200 to 250 g were used in the experiment, after being allowed 7 days to acclimatize. The rats were housed in cages provided with HEPA filters and were allowed free access to food and water. The rats were immunosuppressed with cyclophosphamide (75 mg/kg of body weight intraperitoneally twice) plus prednisolone (Depo-medrone; 16 mg/kg intramuscularly once) 2 days before infection. The animals received eurofloxacin (Baytril; 10 mg/kg subcutaneously daily) from day 2 preinfection to prevent secondary bacterial infections. A frozen glycerol stock of A. fumigatus A1163 was cultured into Sabouraud dextrose agar (SAB) for 10 days. The spores were harvested by the use of phosphate-buffered saline (PBS) plus 0.05% Tween 80 and counted with a hemocytometer. The rats were infected with 4 ϫ 10 8 /ml spores in an inhalation acrylic chamber in a class II hood for an hour (28). The rats (n ϭ 5) were checked for symptoms such as body weight loss, dyspnea, and le...

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Section: Discussionsupporting

confidence: 51%

Detection of Aspergillus fumigatus in a Rat Model of Invasive Pulmonary Aspergillosis by Real-Time Nucleic Acid Sequence-Based Amplification

Zhao¹,

Park²,

Warn

et al. 2010

J Clin Microbiol

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“…Examples of lysis techniques utilized are enzymatic digestion processes that often rely on use of toxic chemicals, such as phenol-chloroform, mechanical disruption using glass beads, and sonication (120). In an effort to overcome this barrier, automated extraction methods have been developed that are able to decrease the time for sample processing and lessen the possibility of errors (121). However, it is still unclear whether these techniques alone adequately disrupt the fungal cell wall and significantly improve the sensitivity of fungal PCR assays (121,122).…”

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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“…Until now, only a limited number of mucormycete-specific PCR methods have been published. Moreover, only some of them allow the quantification of the fungal DNA load in samples (6)(7)(8)(9)(10), which can be important to differentiate between a real infection and contamination/colonization of the sample/patient.…”

mentioning

confidence: 99%

Rapid Detection and Identification of Mucormycetes in Bronchoalveolar Lavage Samples from Immunocompromised Patients with Pulmonary Infiltrates by Use of High-Resolution Melt Analysis

et al. 2014

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e Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by highresolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/ HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.

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Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR

Cited by 46 publications

References 38 publications

Detection of Aspergillus fumigatus in a Rat Model of Invasive Pulmonary Aspergillosis by Real-Time Nucleic Acid Sequence-Based Amplification

Detection of Aspergillus fumigatus in a Rat Model of Invasive Pulmonary Aspergillosis by Real-Time Nucleic Acid Sequence-Based Amplification

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

Rapid Detection and Identification of Mucormycetes in Bronchoalveolar Lavage Samples from Immunocompromised Patients with Pulmonary Infiltrates by Use of High-Resolution Melt Analysis

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