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1 Department of Microbiology, Barts and The London NHS Trust, Royal London Hospital, London, UK
2 ,4 Centre for Haematology2 and Centre for Infectious Disease4 , Institute for Cell and Molecular Sciences, Barts and The London School of Medicine and Dentistry, London, UK
3 Department of Respiratory Medicine, Barts and The London NHS Trust, St Bartholomew's Hospital, London
Correspondence
Mark Wilks
m.wilks{at}qmul.ac.uk
Received 5 January 2006
Accepted 9 May 2006
Abbreviations: Ct, threshold cycle; IA, invasive aspergillosis.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Clinical signs and symptoms of invasive aspergillosis (IA) are non-specific. Those patients most at risk for fungal infections often show little or no evidence of any systemic infective processes (Tomee & van der Werf, 2001). Lung biopsy remains the ‘gold standard’ investigation in forming a firm diagnosis of IA, allowing demonstration of septate, branching hyphae in tissue. However, profound neutropenia and thrombocytopenia are often contraindications to biopsy in the at-risk population (Soubani & Chandrasekar, 2002). Culture of sputum and broncho-alveolar lavage yield positivity rates of only 30 and 30–50 %, respectively, in patients with confirmed IA (Denning, 1998; Soubani & Chandrasekar, 2002). The presence of fungi in a broncho-alveolar lavage sample is more highly predictive of infection than isolation from sputum. In patients with haematological malignancy the isolation of Aspergillus spp. from sputum has a positive predictive value for infection of 80–90 % (Denning, 1998; Soubani & Chandrasekar, 2002) and should therefore always be assumed to be pathogenic in this patient group until proven otherwise. Blood cultures are usually negative, even when techniques such as lysis centrifugation are carried out (Denning, 2000; Mandell et al., 2000; Collier et al., 1998); therefore, microbiological cultures have a limited role in forming a diagnosis of IA.
High-resolution computed tomography scanning is a useful clinical investigation that supports the diagnosis of IA. However, the halo sign, which is highly suggestive of IA, only appears in 33–60 % of patients with IA (Singh & Paterson, 2005). In order to be clinically useful, a computed tomography scan must be performed within 1 week of the first symptoms, as 75 % of halo signs disappear within 1 week (Singh & Paterson, 2005). The air crescent sign, which correlates with neutrophil recovery (Soubani & Chandrasekar, 2002), is also highly suggestive of IA. However, this is a late sign of the infection (Singh & Paterson, 2005). These diagnostic limitations result in frequent empirical use of systemic antifungal agents, with significant costs and potential toxic side effects. The inability of traditional diagnostics to give an efficient and conclusive diagnosis of IA has led investigators to look at newer emerging molecular technologies. PCR is one such approach. Detecting the presence of fungal DNA in the blood may improve the diagnosis of aspergillosis.
One of the major hurdles of using PCR to diagnose aspergillosis is extraction of the DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Several fungal DNA extraction protocols have been described in the literature (Al-Samarrai & Schmid, 2000; Einsele et al., 1997; Griffin et al., 2002; Müller et al., 1998; Velegraki et al., 1999; Williamson et al., 2000). However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique.
In order to extract DNA from fungal cells, it is necessary to disrupt the cell wall. This can be achieved in a number of ways. In previous studies, freeze–thawing of microbes that are resistant to standard cell lysis, using liquid nitrogen or dry ice, has been shown to be successful (Griffin et al., 2002; Johnson et al., 1995; Loeffler et al., 2001). Homogenization has also been used to extract fungal DNA incorporating the use of glass-bead beating (Müller et al., 1998; Smit et al., 1999). Enzyme extraction is another method. Overnight incubation with lyticase breaks down components of the fungal cell wall, releasing fungal DNA (Williamson et al., 2000). Whilst these protocols give good results, they are complex, time-consuming and require a high skill level. We have amended and simplified these previously published techniques (Griffin et al., 2002; Müller et al., 1998; Williamson et al., 2000), omitting steps and using alternative buffers to make them more suitable for use in the routine laboratory. DNA yield and suitability for downstream amplification was compared by real-time PCR using the primers and probe from a previously published method (Challier et al., 2004).
The aim of this study was to compare six different extraction methods using three different operators with different levels of technical expertise. The methods compared were: (i) a freeze–thawing method (Griffin et al., 2002); (ii) a freeze–boiling method (Griffin et al., 2002); (iii) an enzyme extraction method (Williamson et al., 2000); (iv) a bead-beating method (Müller et al., 1998); (v) a mixture of bead beating and enzyme extraction (Müller et al., 1998; Williamson et al., 2000); and (vi) a bead-beating method using a different lysis buffer (in-house method). Each lysis procedure was followed by DNA purification using the Qiagen DNA Mini kit, as recommended by Loffler et al. (1997).
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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DNA extracted from each dilution was compared using real-time PCR to ascertain which method gave the best-quality DNA suitable for PCR. PCR was carried out using primers and a method described previously (Challier et al., 2004). DNA was confirmed as belonging to A. fumigatus by amplification using a set of 18S broad-spectrum primers (Innis et al., 1990), followed by sequencing of the product.
Extraction methods
Each extraction method was carried out in triplicate by three different operators of differing technical experience, ranging from no previous DNA extraction experience to a highly skilled technician.
Each procedure was followed by the addition of 20 µl proteinase K at a concentration of 20 mg ml–1 (Qiagen) and incubation for 2 h at 55 °C. DNA purification using the QIAamp DNA Mini kit (Qiagen) was carried out following the tissue protocol with the amendment of using a 100 µl elution volume.
Method 1: freeze–thaw. Ten microlitres of each dilution of conidia was added to 600 µl sorbitol buffer. Samples were frozen to –70 °C for 10 min and then thawed at room temperature.
Method 2: freeze–boil. Ten microlitres of each dilution of conidia was added to 600 µl sorbitol buffer. Samples were frozen to –70 °C for 10 min and then boiled at 100 °C for 2 min.
Method 3: enzyme digestion. Ten microlitres of each dilution of conidia was added to 600 µl sorbitol buffer. Lyticase (200 U; Sigma) was added and samples were incubated at 37 °C for 30 min.
Method 4: bead beating. Ten microlitres of each dilution of conidia was added to 600 µl sorbitol buffer. Beads (0.5 g, 0.5 mm diameter; Biospec) were added and the mixture was shaken in a Mini-BeadBeater-8 (Biospec) for 3 min on the ‘homogenize’ setting.
Method 5: enzyme digestion and bead beating. Ten microlitres of each dilution of conidia was added to 600 µl sorbitol buffer. Lyticase (200 U; Sigma) was added and samples were incubated at 37 °C for 30 min. The mixture was centrifuged at 11 000 g for 10 min. The supernatant was discarded and the pellet was resuspended in 360 µl AL buffer and 20 µl proteinase K at a concentration of 20 mg ml–1 (both from Qiagen). Beads were added and shaken in a Mini-BeadBeater-8, as in Method 4.
Method 6: bead beating with a different lysis buffer. Ten microlitres of each dilution of conidia was added to 360 µl AL buffer and 20 µl proteinase K at a concentration of 20 mg ml–1 (both from Qiagen). Beads were then added and shaken in a Mini-BeadBeater-8, as in Method 4.
Automated extraction. In addition to the manual methods described above, two commercial automated extraction platforms, EZ1 (Qiagen) and MagNA Pure (Roche), were evaluated. Extractions were carried out following the manufacturer's instructions.
Extraction from whole blood. Whole blood was collected from a healthy volunteer in a 4 ml EDTA vacutainer (BD) containing 7.2 mg EDTA. A. fumigatus fungal saline suspensions were prepared as described for the previous extractions, giving starting concentrations of 3 log10 to 7 log10 conidia ml–1. Ten microlitres of each suspension was added to 600 µl EDTA/whole blood. This mixture was centrifuged at 11 000 g for 10 min. The supernatant was discarded, the pellet was resuspended in 360 µl AL buffer (Qiagen) and the DNA was extracted using Method 6.
PCR protocol. The 25 µl PCR mixture contained 1x Taqman universal PCR Master Mix (Applied Biosystems), 500 nM each primer, 100 nM probe and 7 µl DNA. DNA was amplified in an ABI Prism 7900HT sequence detector (Applied Biosystems) in optical 384-well plates. Cycling conditions were 95 °C for 10 min, followed by 40 amplification cycles of 15 s of denaturation at 95 °C and 1 min of hybridization and elongation at 60 °C. The primers and probe were from a previously published method (Challier et al., 2004). The threshold cycle (Ct) value, which is inversely proportional to the log of the amount of target DNA initially present, was calculated using SDS software version 2.0 (Applied Biosystems). All samples were run in triplicate with negative and positive controls. The median value of the triplicate results was recorded.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and the whole process was completed within 2.5 h.
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Traditionally, purification of DNA has involved extraction methods that use toxic chemicals such as phenol/chloroform and are technically difficult to execute. In recent years, commercial kits have been used more frequently and have been shown to be less technically demanding and to be safer and quicker without loss of sensitivity (Loffler et al., 1997). Loffler et al. (1997) compared a number of commercial kits and the Qiagen DNA Mini kit was reported to be the best available for the extraction and purification of DNA from fungi. Extra steps are still required initially to lyse the cell prior to purification, as fungal cell walls are extremely strong and difficult to lyse by traditional extraction techniques.
These difficulties in lysing the cell wall have led to time-consuming, expensive and complex lysis methods. These include overnight incubations with enzymes (Williamson et al., 2000) and the use of liquid nitrogen with many intermediate steps (Einsele et al., 1997). These methods are not practical in the context of a routine diagnostic laboratory where high-throughput, reproducible results are required. The high level of technical expertise required and the time-consuming nature greatly increase the labour costs associated with extraction. The use of enzymes and consumables such as liquid nitrogen also significantly increase the cost of the method. The increased number of steps associated with these methods increases the chance of contamination occurring. Here, we have described a comparison of six methods that are suitable for use in a routine laboratory.
In this comparison of various cell lysis techniques, the bead-beating methods (Methods 4 and 6) were shown to give the greatest yield of DNA, and were the most reproducible and the quickest and simplest to perform. This DNA extraction procedure took 3 h to perform, in contrast with the freeze–thaw method, which took over 4 h to complete. The bead-beating method showed the least variability among the three operators, indicating not only that it is highly reproducible but also that it is not reliant on technical expertise, as there was little difference between the results obtained from the operator with little or no experience and those from the most experienced operator. By using a detergent buffer (Method 6) instead of sorbitol buffer (Method 4), the DNA yield was further improved. The freeze–boil method proved to be the least reliable technique, resulting in little, if any, DNA.
The Mini-BeadBeater-8 is a cell disrupter that violently agitates up to eight standard microcentrifuge tubes at a time. It is aerosol-free, thus reducing the opportunity for cross-contamination. A high-throughput version of the Mini-BeadBeater-8 is available that can homogenize up to 192 samples. The capital cost of these machines may be an issue, as the Mini-BeadBeater-8 costs approximately £880 and the Mini-BeadBeater-96 costs approximately £2600. Most of the ongoing costs of extracting the DNA are due to operator time. Consumable costs for this method are low compared with alternative methods that utilize enzymes or liquid nitrogen. The high capital cost of the equipment would quickly be outweighed by savings in labour and consumables due to the simple and rapid method described here.
The other potential obstruction to the use of this machine is the noise that it generates, requiring it to be placed in a separate room or ear defenders to be provided when it is in use. However, once these problems are overcome, this machine disrupts fungal cells quickly and efficiently.
Extraction Method 6, when applied to the extraction of fungal DNA from spiked whole blood, had a limit of detection of 10 conidia extracted from blood. Einsele et al. (1997) reported that the extraction method they utilized was able to detect 10 conidia ml–1, which is in line with our own findings. However, our extraction method is far simpler. This is a good limit of detection. However, clinically IA may present with fungal loads of <1 conidia ml–1. In this study, we used a volume of 600 µl, which may result in infections being missed due to the use of a small volume. The method we describe here could be used on larger volumes of blood. This could be achieved using larger capacity spin columns such as the DNA Midi kit (Qiagen), which can accept blood volumes of up to 2 ml, or the DNA Maxi kit (Qiagen), which has a capacity of 4 ml blood.
In conclusion, we have reported here a rapid, reproducible and simple method for the extraction of DNA from fungi. This is a method that can be used in the routine laboratory.
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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