Development of a Nested Qualitative Real-Time PCR Assay To Detect
            <i>Aspergillus</i>
            Species DNA in Clinical Specimens

Halliday, Catriona; Wu, Qi; James, Gregory; Sorrell, Tania C.

doi:10.1128/jcm.43.10.5366-5368.2005

Cited by 39 publications

(19 citation statements)

References 15 publications

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“…Such a diagnostics-driven approach can therefore reduce unnecessary antifungal treatment, costs, and toxic side effects (2). Halliday et al included single and intermittent (within 14 days) positive PCR results for patient management (5). Another diagnostic approach used three markers instead of the usual two (6).…”

Section: Discussionmentioning

confidence: 99%

A Novel Extraction Method Combining Plasma with a Whole-Blood Fraction Shows Excellent Sensitivity and Reproducibility for Patients at High Risk for Invasive Aspergillosis

et al. 2012

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bDiagnosis of invasive aspergillosis (IA) is still a major problem in routine clinical practice. Early diagnosis is essential for a good patient prognosis. PCR is a highly sensitive method for the detection of nucleic acids and could play an important role in improving the diagnosis of fungal infections. Therefore, a novel DNA extraction method, ultraclean production (UCP), was developed allowing purification of both cellular and cell-free circulating fungal DNA. In this prospective study we evaluated the commercially available UCP extraction system and compared it to an in-house system. Sixty-three patients at high risk for IA were screened twice weekly, and DNA extracted by both methods was cross-analyzed, in triplicate, by two different real-time PCR assays. The negative predictive values were high for all methods (94.3 to 100%), qualifying them as screening methods, but the sensitivity and diagnostic odds ratios were higher using the UCP extraction method. Sensitivity ranged from 33.3 to 66.7% using the in-house extracts to 100% using the UCP extraction method. Most of the unclassified patients showed no positive PCR results; however, single-positive PCR replicates were observed in some cases. These can bear clinical relevance but should be interpreted with additional clinical and laboratory data. The PCR assays from the UCP extracts showed greater reproducibility than the inhouse method for probable IA patients. The standardized UCP extraction method yielded superior results, with regard to sensitivity and reproducibility, than the in-house method. This was independent of the PCR assay used to detect fungal DNA in the sample extracts.

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Section: Discussionmentioning

confidence: 99%

A Novel Extraction Method Combining Plasma with a Whole-Blood Fraction Shows Excellent Sensitivity and Reproducibility for Patients at High Risk for Invasive Aspergillosis

et al. 2012

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“…The shortcomings of this method may include increasing the likelihood of PCR bias by increasing the number of amplifications. However, nested realtime PCR has successfully been used previously to quantitatively detect Mycobacterium tuberculosis DNA (49, 50) and qualitatively detect Aspergillus DNA (19) in clinical samples, as well as Helminthosporium solani ITS DNA from soil (12).…”

Section: Discussionmentioning

confidence: 99%

Quantification of Nitrogen Reductase and Nitrite Reductase Genes in Soil of Thinned and Clear-Cut Douglas-Fir Stands by Using Real-Time PCR

Levy‐Booth

Winder

2010

Appl Environ Microbiol

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The abundance of nifH, nirS, and nirK gene fragments involved in nitrogen (N) fixation and denitrification in thinned second-growth Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest soil was investigated by using quantitative real-time PCR. Prokaryotic N cycling is an important aspect of N availability in forest soil. The abundance of universal nifH, Azotobacter sp.-specific nifH (nifH-g1), nirS, and nirK gene fragments in unthinned control and 30, 90, and 100% thinning treatments were compared at two long-term research sites on Vancouver Island, Canada. The soil was analyzed for organic matter (OM), total carbon (C), total N, NH 4 -N, NO 3 -N, and phosphorus (P). The soil horizon accounted for the greatest variation in nutrient status, followed by the site location. The 30% thinning treatment was associated with significantly greater nifH-g1 abundance than the control treatment in one site; at the same site, nirS in the mineral soil horizon was significantly reduced by thinning. The abundance of nirS genes significantly correlated with the abundance of nirK genes. In addition, significant correlations were observed between nifH-g1 abundance and C and N in the organic horizon and between nirS and nirK and N in the mineral horizon. Overall, no clear influence of tree thinning on nifH, nirS, and nirK was observed. However, soil OM, C, and N were found to significantly influence N-cycling gene abundance.

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“…The MagNA Pure LC total nucleic acid isolation kit (Roche Diagnostics) was used for extraction of blood, BAL, and CSF specimens with some modifications. Blood samples (500 l) were lysed with erythrocyte lysis buffer and incubated with sorbitol buffer and lyticase as outlined previously (11). BAL (600-l) and CSF (100-to 200-l) samples were centrifuged at 16,100 ϫ g for 10 min, and the pellet was resuspended in 200 l of sorbitol buffer (1 M sorbitol, 100 mM EDTA, and 0.1% 2-mercaptoethanol) (38) and 200 U lyticase (Sigma-Aldrich, Castle Hill, Australia).…”

Section: Fungal Isolates and Clinical Specimensmentioning

confidence: 99%

Reverse Line Blot Hybridization Assay for Identification of Medically Important Fungi from Culture and Clinical Specimens

et al. 2007

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We evaluated a combined panfungal PCR-reverse line blot (RLB) hybridization assay based on internal transcribed spacer 1 (ITS1) and ITS2 region polymorphisms to identify 159 Candida, Cryptococcus neoformans, and Aspergillus isolates (22 species). Its utility to identify fungal pathogens directly from 27 clinical specimens was also determined. ITS sequence analysis was performed to resolve discrepant identifications or where no RLB result was obtained. Species-specific ITS2-and ITS1-based probes correctly identified 155 of 159 isolates (98%) and 149 (93.7%) isolates, respectively. All strains were unambiguously differentiated with the exception of cross-reactivity between the Candida norvegensis probe and Candida haemulonii DNA product. Species identification of the pathogen was made for all 21 specimens (sensitivity of 100%) where species-specific probes were included in the RLB; however, there was no ITS2 probe-based hybridization signal for two specimens. Results were concordant with the culture results for 18 (85.7%) specimens. The assay was able to provide species identification in the absence of a culture result (two specimens) and to detect mixed infection (one specimen). The results indicate that the RLB assay is capable of reliably detecting yeasts and Aspergillus spp. in clinical specimens and that the incorporation of both ITS1-and ITS2-targeted probes is required for optimal sensitivity. The test has potential utility in the early diagnosis of invasive fungal infection, since "fungal" DNA was detected in all 27 specimens. Prior to incorporation of probes to detect other fungal species, ITS sequencing may be performed to achieve species identification.Invasive fungal infections (IFIs) are an important cause of morbidity and mortality in severely ill and immunocompromised patients. Recent epidemiological trends indicate a significant shift towards species of Candida and Aspergillus other than Candida albicans and Aspergillus fumigatus and a diverse range of less common fungal opportunists (27,29,35). Given the reduced susceptibility of many of these pathogens to standard antifungal agents (30,35,36), timely and accurate identification to the species level is essential in guiding clinical management. Conventional culture-based phenotypic identification methods, however, are slow and prone to misidentification, particularly with less common or unusual species (20, 34). In addition, the databases of commercial yeast identification systems do not contain all potential pathogens (34).Molecular approaches using PCR-based methods have been developed to provide rapid and accurate detection of fungi. In particular, the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the fungal ribosomal DNA gene complex, have shown promise as targets for species identification in a variety of formats, including DNA sequencing and DNA probe hybridization (9, 13, 22). Both length and sequence polymorphisms within the ITS region have permitted accurate identification of pathogenic yeasts and molds (5-7, 12, 14, 22, 25,...

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Development of a Nested Qualitative Real-Time PCR Assay To Detect Aspergillus Species DNA in Clinical Specimens

Cited by 39 publications

References 15 publications

A Novel Extraction Method Combining Plasma with a Whole-Blood Fraction Shows Excellent Sensitivity and Reproducibility for Patients at High Risk for Invasive Aspergillosis

A Novel Extraction Method Combining Plasma with a Whole-Blood Fraction Shows Excellent Sensitivity and Reproducibility for Patients at High Risk for Invasive Aspergillosis

Quantification of Nitrogen Reductase and Nitrite Reductase Genes in Soil of Thinned and Clear-Cut Douglas-Fir Stands by Using Real-Time PCR

Reverse Line Blot Hybridization Assay for Identification of Medically Important Fungi from Culture and Clinical Specimens

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