Study population and design
Patients with hematological malignancies or undergoing hematopoietic cell transplantation at the Seattle Cancer Care Alliance who developed pneumonia or pulmonary nodules underwent bronchoscopy with BAL. BAL fluid remaining after conventional microbiological and cytologic evaluations was processed as noted in the next sub-section. This was a retrospective study analyzing BAL fluid samples obtained from April 2002 to July 2003, and was approved by the Institutional Review Board at the Fred Hutchinson Cancer Research Center. This study involved 81 patients, 94 episodes of pneumonia, and 144 BAL samples. Note that multiple lobes were lavaged at the time of bronchoscopy in most subjects, resulting in an average of more than one BAL sample per episode. Analysis was done on an episode basis, with an episode defined as a single radiographically and temporally related pneumonia. If a subject had resolution of pulmonary infiltrates with appearance of a new infiltrate at a later time, this was considered a separate episode. Figure 1 depicts the algorithm used for the diagnosis of IPA using qPCR. Patients with proven or probable IPA were diagnosed using European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria [23]. Designation of clinical status was performed by an investigator who was blinded to qPCR results, with host factors, clinical criteria, and microbiological criteria abstracted from the medical record and entered into a relational database.
Processing of BAL fluid
The starting volume of BAL fluid was in the range of 2 to 5 ml. BAL fluid was centrifuged at 3200 rcf for 15 min at 4°C. The pellet was resuspended in a small volume of supernatant, with the final pellet fraction having a volume of 100 to 400 μl, depending on the degree of cellularity. The pellet and the remaining supernatant fraction were frozen in separate tubes at -80°C until DNA extraction.
DNA extraction from BAL fractions
DNA extraction of clinical samples and PCR set up was performed in a laminar flow hood within a laboratory that was exclusively used for pre-PCR processing. An optimized version of the MasterPure™ Yeast DNA Purification Kit (Epicentre® Biotechnologies, Madison, WI) was used for BAL DNA extraction. The 100% isopropanol, 70% ethanol and DNA grade water used for extraction were filtered in an Amicon Ultra-15 centrifugal filter unit with a molecular weight cut-off (MWCO) of 30 kDa (Millipore Corporation, Billerica, MA). Yeast Cell Lysis™ solution and MPC Protein Precipitation Reagent™ were UV irradiated at 240 mJ/cm2 with samples approximately 15 centimetres from the bulbs (Spectrolinker™, Westbury, NY). The silicon carbide sharps were washed 10 times in DNA free water and baked at 180°C for 48 h. DNA-free microcentrifuge tubes were used with DNA extraction (Eppendorf Biopur tubes, Eppendorf AG, Hamburg, Germany). Sham digest controls consisting of DNA free water were processed with every extraction run serving as negative controls to monitor for fungal contamination.
DNA was independently extracted from the pellet and supernatant fractions of the BAL; no whole BAL was processed. In the case of the supernatant fraction, extraction started with 0.5 ml of the supernatant from the protein precipitation step onwards. For the pellet fraction, an additional bead beating step was included. Two milliliter sterile screw-cap tubes were loaded with silicon carbide sharps of sizes 0.1 mm and 1 mm (BioSpec Products, Inc., Bartlesville, OK) at a 1:1 ratio up to a volume equivalent to 250 μl. Yeast Cell Lysis™ solution at a volume of 550 μl and BAL pellet at 100 – 400 μl, or 200 μl of water as digest control, were added to the tube. The contents of the tube were homogenized in a FastPrep®-24 System (MP Biomedicals, Solon, OH) at 5 m/s for 60 s. Each tube was incubated at 65°C for 45 min then kept on ice for 5 min. MPC Protein Precipitation Reagent™ was added at a volume of 325 μl for pellet and 450 μl for supernatant processing. The tubes were vortexed for 10 s and centrifuged at 11,000 rcf for 10 min. The resulting supernatant was transferred to a new micro-centrifuge tube containing an equal volume of 100% isopropanol pre-cooled to -20°C. The contents of the tube were mixed thoroughly by inversion and incubated at -20°C for 1 hour. Precipitated DNA was pelleted by centrifugation at 11,000 rcf for 10 min. This supernatant was removed and discarded. The pellet containing DNA was resuspended in 0.5 ml of pre-cooled (-20°C) 70% ethanol and vortexed. The tube was then centrifuged at 11,000 rcf for 5 min. This supernatant was removed to a level just short of disturbing the pellet. The remaining volume of ethanol was allowed to evaporate by air drying for 5 min within the laminar flow hood. The pellet was resuspended in 100 μl of 0.1% Triton-X pre-warmed to 65°C then incubated at room temperature for one minute with periodic gentle vortexing. The DNA was either used immediately for qPCR, stored at -20°C overnight or at -80°C for longer periods. If PCR inhibition was detected in the extracted samples, they were reprocessed from the protein precipitation step onwards (see Figure 1).
Preparation of fungal genomic DNA
Genomic DNA from fungi was extracted with an optimized MasterPure™ Yeast DNA Purification Kit (Epicentre® Biotechnologies, Madison, WI) in order to assess assay analytical sensitivity and specificity. Fungi were transferred into micro-centrifuge tubes from liquid media and centrifuged. Cell pellets were washed with 1 ml 1× PBS and centrifuged at 10,000 rcf for 3 min. The supernatant was discarded and cells resuspended in 500 μl Yeast Cell Lysis™ solution. The tube was vortexed at top speed for 10 s. The tube was incubated at 65°C for 1 h and then kept on ice for 5 min. For filamentous fungi, the pellet was ground with a micropestle at the start and during the 65°C incubation. Protein Precipitation Reagent™ was added at a volume of 400 μl to the tube and vortexed for 10 s. The tube was centrifuged to pellet cellular debris at 11,000 rcf for 10 min. The supernatant was transferred to a new micro-centrifuge tube containing an equal volume of 100% isopropanol pre-cooled to -20°C. The contents of the tube were thoroughly mixed by inversion and incubated at -20°C for 1 hr. Precipitated DNA was pelleted by centrifugation at 11,000 rcf for 10 min. The supernatant was removed and discarded. The pellet containing DNA was resuspended in pre-cooled (-20°C) 1 ml of 70% ethanol and vortexed at maximum speed for 10 s. The tube was then centrifuged at 11,000 rcf for 5 min. This supernatant was removed to a level just short of disturbing the pellet. The remaining volume was allowed to evaporate by air drying for 5 min. The pellet was resuspended in 100 μl of 0.1% Triton-X pre-warmed to 65°C and incubated at room temperature for 1 min with periodic gentle vortexing. The total nucleic acid in the extract was quantified using a UV spectrophotometer. For every 149 μg of total nucleic acid in the extract, 10 U of RiboShredder™ RNase Blend (Epicentre® Biotechnologies, Madison, WI) was used to remove RNA. RNA removal was confirmed by visualizing the pre- and post-treatment extract on a 1.5% agarose gel. DNA was quantified using a Qubit™ instrument and Quant-iT™ dsDNA HS Assay Kit (Invitrogen Corporation, Carlsbad, CA).
Quantitative PCR assays
Quantitative PCR assays in this study were based on Taqman™ chemistry and an Applied Biosystems 7500™ real-time instrument was used for detection. To prevent contamination, each PCR master mix without additional water component was filtered through a Microcon YM-100 centrifugal filter unit with a MWCO of 100 kDa (Millipore Corporation, Billerica, MA) at 650 rcf for 25 min and 1500 rcf for an additional 5 min before use. The additional water was independently filtered with an Amicon Ultra-15 centrifugal filter unit with a MWCO of 30 kDa using. DNA-free microcentrifuge tubes were used with the PCR set up (Eppendorf Biopur tubes, Eppendorf AG, Hamburg, Germany). No-template controls were run with each qPCR assay to monitor contamination. Each extracted BAL sample was run in duplicate reactions. Samples were interpreted as positive if both duplicates showed an increase in normalized relative florescence above the background and the multicomponent view demonstrated an increase in absolute florescence (as estimated by the 7500 System SDS software, Applied Biosystems).
(i) Internal amplification control (IAC) qPCR
The IAC qPCR was developed based on a 105 base template derived from the jellyfish aequorin gene which has a sequence of 5'- GCCTGGTGCAAAAATTGCTTATCAAATTGAACGGTCAATTGGAAGTGGCGGAAGAACAGCTATTGCAAACGC
CATCGCACAATACCATAAACACACTTGTCTTAG-3' [24]. The amplicon was detected with a forward primer 5'-GCC TGG TGC AAA AAT TGC TTA TC-3', reverse primer 5'- CTA AGA CAA GTG TGT TTA TGG TAT TG -3' and probe labelled with fluorescein (Quasar670) and quenched with BHQ2: 5'-Quasar670 CTT CCG CCA CTT CCA ATT GAC CGT TCA BHQ2-3' (Biosearch Technologies, Novato, CA). The IAC was multiplexed with the Aspergillus targeted 18S qPCR and the human targeted 18S extraction control qPCR to monitor inhibition in every qPCR reaction. If inhibition as assessed by > 2 cycle delay in the IAC threshold cycle was detected, DNA was re-purified and assayed again.
(ii) Extraction control qPCR
Successful DNA extraction was confirmed in all samples with a qPCR targeting the human 18S rRNA gene with forward primer 5'- CTC TTA GCT GAG TGT CCC GC -3', reverse primer 5'- CTT AAT CAT GGC CTC AGT TCC GA -3', and probe labelled with fluorescein (FAM) and quenched with TAMRA: 5'-FAM CCG AGC CGC CTG GAT ACC GCA GCT A TAMRA-3'. Each 50-μl PCR mixture contained 1× TaqMan® Buffer A, 6 mM of MgCl2, 1 mM of GeneAmp® dNTP Blend (12.5 mM with dUTP), 2.2 U of AmpliTaq Gold® DNA Polymerase, 0.05 U AmpErase® Uracil N-glycosylase (all from Applied Biosystems, Foster City, CA), 0.8 μM each of forward and reverse human targeted primers, 180 nM of human targeted probe, 0.24 μM each of forward and reverse of IAC primers, 180 nM of IAC probe, 0.002% of Triton-X 100, 105 copies of IAC template and 5 μl of DNA. The PCR cycling conditions consisted of an Uracil N-glycosylase activation at 50°C for 2 min, pre-melt at 95°C for 10 min and then 38 cycles of 95°C for 15 s (melt) and 65°C for 65 s (annealing and extension). A standard curve for quantifying human DNA was generated using human genomic DNA (Roche Applied Sciences, Indianapolis, IN) with dilutions ranging from 10,000 to 1 pg.
(iii) Aspergillustargeted 18S qPCR
The Aspergillus targeted qPCR amplified a 114 bp segment of the Aspergillus 18S rRNA gene with forward primer 5'- GAT AAC GAA CGA GAC CTC GG -3', reverse primer 5'- AGA CCT GTT ATT GCC GCG C -3' and probe 5'-FAM CTT AAA TAG CCC GGT CCG C BHQ-3' with minor groove binding modification. Each 50-μl PCR mixture contained 1× TaqMan® Buffer A, 6 mM of MgCl2, 1 mM of GeneAmp® dNTP Blend (12.5 mM with dUTP), 2.2 U of AmpliTaq Gold® DNA Polymerase, 0.05 U AmpErase® Uracil N-glycosylase (all from Applied Biosystems, Foster City, CA), 0.8 μM each of forward and reverse Aspergillus targeted primers, 200 nM of Aspergillus targeted probe, 0.4 μM each of forward and reverse of IAC primers, 190 nM of IAC probe, 0.002% of Triton-X 100, 105 copies of IAC template and 5 μl of DNA. The PCR cycling conditions consisted of an Uracil N-glycosylase activation at 50°C for 2 min, pre-melt at 95°C for 10 min and then 45 cycles of 95°C for 15 s (melt) and 65°C for 65 s (annealing and extension). A standard curve for quantifying Aspergillus DNA was generated using Aspergillus fumigatus genomic DNA (ATCC # MYA-1163) dilutions ranging from 1000 pg to 30 fg. All positive Aspergillus qPCRs for the first 48 episodes were subjected to sequencing using Big Dye terminators and an Applied Biosystems capillary sequencer to confirm identity with the expected target.
Analytical specificity testing
The analytical specificity of the Aspergillus qPCR was assessed by testing 1000 pg of genomic DNA from 29 different fungal species spanning 15 genera grown in culture. The following clinically or phylogenetically relevant fungal pathogens were chosen: Aspergillus fumigatus (ATCC # MYA-1163), Aspergillus oryzae (ATCC # 20719), Aspergillus ustus (ATCC # 20063), Aspergillus candidus (ATCC # 20022), Aspergillus terreus (ATCC # 10070), Aspergillus flavus (ATCC # MYA-3631), Candida albicans (ATCC # 90028), Candida glabrata (ATCC # 90876), Candida kefyr (ATCC # 28838), Candida guilliermondii (ATCC # 90877), Candida lusitaniae (ATCC # 42720), Candida dubliniensis (ATCC # MYA-580), Scedosporium apiospermum (ATCC # 28206), Scedosporium prolificans (ATCC # 90468), Paecilomyces variotti (ATCC # 10865), Penicillium chrysogenum (ATCC # 10108), Rhizopus oryzae (ATCC # 10260), Rhodotorula glutinis (ATCC # 16726), Absidia corymbifera (ATCC # 14058), Fusarium solani (ATCC # 56480), Mucor racemosus (ATCC # 42647), Rhizomucor miehei (ATCC # 46345), Cunninghamella bertholletiae (ATCC # 42155), Trichosporon cutaneum (ATCC # 38300), Candida parapsilosis (clinical isolate), Candida tropicalis (clinical isolate), Candida krusei (clinical isolate), Saccharomyces cerevisiae (Novagen, Madison, WI), and Cryptococcus neoformans (ATCC # 28958D-5). Cross-reactivity with 1 μg of human genomic DNA was also assessed.
Data analysis
Quantitative PCR results were compared with clinical diagnoses based on the EORTC/MSG criteria. Sensitivity, specificity and positive and negative likelihood ratios with their associated 95% confidence intervals were calculated. The negative and positive predictive values (NPV and PPV) were also calculated for these sequentially obtained samples. These diagnostic parameters were also calculated for culture, histology and both culture and histology combined. A receiver-operating characteristic (ROC) analysis was done using a computer program written with MathWorks MATLAB® software to assess how changing qPCR detection threshold affects sensitivity and 1-specificity.