Effect of different sample volumes on the DNA extraction of Aspergillus fumigatus from whole blood

Schulz, Bettina; Weber, Kai; Radecke, C.; Scheer, Carola; Ruhnke, Markus

doi:10.1111/j.1469-0691.2009.02797.x

Cited by 8 publications

(5 citation statements)

References 14 publications

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“…From 45 fungal control strains, DNA was isolated using a standard procedure as described earlier using zymolase digestion following phenol-chloroform extractions and ethanol precipitation and the DNA was stored at )20°C until used [15].…”

Section: Methodsmentioning

confidence: 99%

Diagnosis of chronic disseminated candidosis from liver biopsies by a novel PCR in patients with haematological malignancies

Fleischhacker

Schulz

Jöhrens

et al. 2012

Clinical Microbiology and Infection

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Hepatic Candida infection (HCI; known as chronic disseminated candidosis or CDC) is a distinct form of disseminated Candida infection with predominant involvement of the liver. Diagnosis of HCI is usually made on clinical suspicion together with multiple lesions in liver on ultrasound (US), CT and/or MRI scan. Fungal elements may not always be visible in liver tissue and mycological culture is frequently negative, making the evidence for proven fungal disease difficult. We studied a novel commercially available low-cost and density-array (LCD) chip technique for a molecular diagnosis of HCI. This is a two-step procedure with PCR amplification after DNA extraction followed by hybridization on a small chip provided by the manufacturer (Fungi 2.1, Chipron GmbH). The analysis of DNA from 45 fungal control strains showed an excellent specificity and sensitivity. The DNA from 11 liver biopsies of patients with haematological malignancies suffering from CDC was analysed on the LCD chip and overall 11 fungal pathogens could be detected in eight liver biopsies, supporting the clinical diagnosis of HCI/CDC. Analysis of liver biopsies from controls was negative for fungal DNA in all samples studied. In conclusion, the novel LCD chip technique examined in our study was able to detect fungal pathogens in liver biopsies from patients with haematological malignancies and suspected HCI/CDC but was negative in control biopsies.

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Section: Methodsmentioning

confidence: 99%

Diagnosis of chronic disseminated candidosis from liver biopsies by a novel PCR in patients with haematological malignancies

Fleischhacker

Schulz

Jöhrens

et al. 2012

Clinical Microbiology and Infection

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“…The use of larger sample volumes allows the detection of lower concentrations of fungal burden because both the overall amount of target and the opportunity for retrieval are increased (23). Paradoxically, too much extrinsic DNA may inhibit the PCR process, so it is essential that an internal control be used to prevent the reporting of false-negative results.…”

Section: Discussionmentioning

confidence: 99%

Aspergillus PCR: One Step Closer to Standardization

White¹,

et al. 2010

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PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as "compliant" or "noncompliant," according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 l showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (>3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 l.

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“…The IH‐PCR method was performed as described earlier by Schabereiter‐Gurtner et al . In short, after hypotonic lysis of erythrocytes and enzymatic lysis of leucocytes (erythrocytes‐white‐cell‐lysis‐blood, EWCLB), the EWCLB‐treated sample was transferred into a new tube containing approximately 60‐70 mg of glass beads (acid‐washed, 710‐1180 lm; Sigma, Steinheim, Germany) . DNA extraction was modified according to the EAPCRI recommendation and performed using the High Pure PCR template preparation kit (Roche Diagnostics, Mannheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

Prospective evaluation of the SeptiFAST multiplex real‐time PCR assay for surveillance and diagnosis of infections in haematological patients after allogeneic stem cell transplantation compared to routine microbiological assays and an in‐house real‐time PCR method

Elges

Arnold

Liesenfeld

et al. 2017

Mycoses

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We prospectively evaluated a multiplex real-time PCR assay (SeptiFast, SF) in a cohort of patients undergoing allo-BMT in comparison to an in-house PCR method (IH-PCR). Overall 847 blood samples (mean 8 samples/patient) from 104 patients with haematological malignancies were analysed. The majority of patients had acute leukaemia (62%) with a mean age of 52 years (54% female). Pathogens could be detected in 91 of 847 (11%) samples by SF compared to 38 of 205 (18.5%) samples by BC, and 57 of 847 (6.7%) samples by IH-PCR. Coagulase-negative staphylococci (n=41 in SF, n=29 in BC) were the most frequently detected bacteria followed by Escherichia coli (n=9 in SF, n=6 in BC). Candida albicans (n=17 in SF, n=0 in BC, n=24 in IH-PCR) was the most frequently detected fungal pathogen. SF gave positive results in 5% of samples during surveillance vs in 26% of samples during fever episodes. Overall, the majority of blood samples gave negative results in both PCR methods resulting in 93% overall agreement resulting in a negative predictive value of 0.96 (95% CI: 0.95-0.97), and a positive predictive value of 0.10 (95% CI: -0.01 to 0.21). SeptiFast appeared to be superior over BC and the IH-PCR method.

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Effect of different sample volumes on the DNA extraction of Aspergillus fumigatus from whole blood

Abstract: Five methods were compared, using conventional PCR, for the isolation of DNA from Aspergillus fumigatus conidia from 1-3-mL samples of whole blood. A lower detection threshold of Aspergillus conidia was achieved using 3-mL rather than 1-mL samples with three of five methods tested.

Cited by 8 publications

References 14 publications

Diagnosis of chronic disseminated candidosis from liver biopsies by a novel PCR in patients with haematological malignancies

Diagnosis of chronic disseminated candidosis from liver biopsies by a novel PCR in patients with haematological malignancies

Aspergillus PCR: One Step Closer to Standardization

Prospective evaluation of the SeptiFAST multiplex real‐time PCR assay for surveillance and diagnosis of infections in haematological patients after allogeneic stem cell transplantation compared to routine microbiological assays and an in‐house real‐time PCR method

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