Direct Detection of Legionella Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture

Control organisms.All experiments to optimize PCR conditions, as well as dilution studies to evaluate sensitivity and plasmid construction, were performed using L. pneumophilaserogroup 1 (ATCC 33152). Other strains of Legionella, used for validation of the assay, are listed in Table1, and included L. pneumophilaserogroups 1 to 6, as well as several other strains ofLegionella, representing the most commonly isolated non-L. pneumophila species. The specificity of the assay was assessed using a panel of control strains of bacteria (Table2), representing both commonly isolated respiratory pathogens and nonpathogens that might be detected in respiratory specimens.

Clinical specimens.A retrospective review of positiveLegionella culture results at the Mayo Clinic, from 1979 to 1999, revealed nine BAL specimens for which frozen, archived material was available. In seven of these cases, frozen cell suspensions were available; in two cases, only supernatant was available for analysis. In addition, cell suspensions from 10 BAL specimens which were culture-negative for Legionella, were randomly selected from similarly archived material. These cell suspensions were originally prepared from BAL specimens using a cytospin method. Only cell suspensions having >2 × 10⁶ cells counted by microscopy on a 4-mm² grid were cultured forLegionella spp. and archived. Cultures of BAL or lung biopsy specimens were performed at the time the specimens were collected using standard techniques (43); cultures were not repeated on archived portions of these specimens during the present study. In all cases, archived results of DFA assays for Legionella, performed on fresh specimens, were also available. A retrospective review, also from 1979 to 1999, revealed nine open lung biopsy specimens culture positive for Legionella species, for which a total of 16 formalin-fixed, paraffin-embedded tissue blocks were available for evaluation. Eight open lung biopsy specimens, from the same time period, all showing nonspecific histologic findings of pneumonia or pneumonitis and all culture-negative forLegionella species, were also selected for evaluation. A single tissue block was used from each of these culture-negative cases.

BAL processing and culture methodology.Prior to culture, BAL specimens were centrifuged for 15 min at 1,200 × gand 2,500 rpm, and the top 7.5 ml of the resulting suspension was removed. The remaining cell concentrate was mixed and used for culture. Fresh tissue from open lung biopsy specimens was homogenized in enriched brain heart infusion broth (Difco formulation; Becton Dickinson, Sparks, Md.) prior to plating. Culture forLegionella species was performed on BCYEα and BCYEα with polymyxin B, anisomycin, and vancomycin (Becton Dickinson), and plates were incubated at 35°C, in room air, for up to 14 days. Organisms from characteristic colonies were Gram stained and identified to the species level using a commercially available panel of fluorescein isothiacyanate (FITC)-labeled antibodies (SciMedX, Denville, N.J.).

Histopathologic examination.Tissues stained with hematoxylin and eosin and WS stain were cut and stained concurrent with and consecutive to sections taken for ISH, DFA assay, and PCR. All tissue sections were cut at 4 μm. Hematoxylin and eosin staining and WS staining were carried out using standard histologic laboratory methods (39).

The WS-stained slides were evaluated in a blinded manner by one of the authors (M.C.A.) Slides positive for Legionella-like organisms showed dark-brown-staining bacillary structures (Fig.1).

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Fig. 1.

Legionella bacilli, demonstrated by WS staining (left) and by ISH (right).

DFA detection.Prior to direct examination, fresh BAL specimen was centrifuged for 5 min, at 2,500 rpm, and the resultant supernatant was removed (in some cases this supernatant was later used in the PCR assay; see below, under “Real-time PCR”). The cell pellet was resuspended in normal saline, with lysis agent and/or mucolytic agents added as necessary. This cell suspension was used both for DFA assay and PCR (see below). For each smear to be examined by DFA, 200 μl was cytocentrifuged at 700 rpm, for 7 min, onto a clean glass slide and allowed to air dry. Histologic sections were deparaffinized through xylene and graded ethanol dilutions and allowed to air dry prior to examination. DFA assay was performed per the manufacturer's instructions (SciMedX). Two polyvalent FITC-labeled rabbit anti-Legionella conjugate pools were applied to a separate replicate smear or tissue section. FITC-labeled negative rabbit globulin was applied to a third replicate of the specimen, to serve as a negative control. Antibody pools were also applied to a positive control slide prepared from a knownLegionella-positive lung tissue specimen in formalin. Positives were interpreted based on the presence of fluorescent bacillary structures in a given smear or section.

ISH.ISH was performed by a procedure, as previously described with some modifications (34).

(i) Oligonucleotide probes.Two oligonucleotide probes (Table 3), both directed against the 16S rRNA sequence of L. pneumophila, were used. One probe was previously described (19). The other probe was designed based on the analysis of sequence matches and mismatches (GenBank). The specificity of probes was checked against the sequences of other bacteria, fungi, parasites, and animals, using Genetics Computer Group software (Madison, Wis.). Probes were 3′ tailed with digoxigenin-11-dUTP (Enzo Diagnostic, Inc.) and then diluted to a final concentration of 2.0 ng/μl in hybridization buffer.

(ii) Pretreatment of sections for ISH.Paraffin sections, after deparaffinizing and rehydration, were rinsed twice in diethyl pyrocarbonate-treated H₂O for 2 min each. Endogenous alkaline phosphatase activity was quenched with 0.2 M HCl for 20 min at room temperature, and slides were microwaved for 10 min in 10 mM citric acid, pH 6.0, and cooled to room temperature. Sections were then digested with proteinase K (25 μg/ml; Sigma, St. Louis, Mo.) in 10 mM phosphate-buffered saline, pH 7.2, for 10 min at room temperature, followed by acetylation for 15 min with freshly prepared 0.6% acetic anhydride in 0.1 M triethanolamine (pH 8.0). Prehybridization was performed for 30 min, at room temperature, with a mixture containing 50% deionized formamide (Sigma), 10% dextran sulfate (Sigma), 1× Denhardt's solution (Sigma), 3× standard saline citrate (SSC), salmon sperm DNA (100 μg/ml, Sigma), yeast tRNA (125 μg/ml), polyadenylic-cytidylic acid (10 μg/ml), Tris (0.05 M), EDTA (5 mM), 600 mM NaCl, and 0.1% sodium pyrophosphate (inorganic).

(iii) Hybridization and post hybridization washes.Following prehybridization, residual prehybridization buffer was thoroughly removed from around the tissue section. An oligonucleotide probe cocktail specific for L. pneumophila (2 ng/μl in prehybridization buffer) was applied to sections. A Sigmacote (Sigma) coverglass was placed on each slide, and the slides were heat treated at 95°C for 5 min and hybridized in a humid environment for 3 h at 50°C. Sections were rinsed twice in 2× SSC for 10 min at room temperature, washed in 0.5× SSC at 37°C for 20 min (to remove excess probe), and rinsed twice in buffer A (1% normal sheep serum in 0.3% Triton X-100) for 2 min at room temperature.

(iv) Immunochemical detection.After posthybridization washing, digoxigenin-labeled probes were detected according to the manufacturer's instructions (digoxigenin detection kit; Boehringer Mannheim). Briefly, after preincubation of sections for 30 min in blocking buffer A (1% normal swine serum and 0.3% Triton X-100), the sections were incubated in a 1:200 dilution of alkaline phosphatase-conjugated antidigoxigenin Fab fragment in blocking buffer A for 1 h at room temperature. Rinsing with buffer A and buffer C (Tris-HCl and MgCl, pH 9.5) was performed, and sections were subsequently reacted with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyphosphate (BCIP), forming an insoluble blue precipitate at the site of reaction. Sections were then rinsed in buffer C, counterstained with 0.1% nuclear fast red, rinsed again in buffer C, dehydrated in graded ethanols, and cleared in xylene, and a coverslip was placed on each section with a xylene-based synthetic mounting medium. Positive interpretation of a slide was based on the presence of blue-staining bacillary structures, against a pink-red background (Fig. 1).

(v) ISH negative controls and probe specificity tests.Negative controls used for ISH consisted of (i) omission of the probes from the hybridization reaction; (ii) slides hybridized with nonlabeled probe; (iii) cross-reactivity testing for target specificity, using an ISH probe for albumin, hybridized to Legionella-positive tissue sections; and (iv) cross-reactivity testing for probe specificity, using five additional specimens with tissue involvement by other gram-positive and gram-negative bacteria.

Real-time PCR.PCR and product detection were performed simultaneously on LightCycler instrumentation (Roche Molecular Biochemicals). The LightCycler is a combined thermocycler and fluorimeter that offers rapid PCR thermocycling (20 to 40 min). The temperature is controlled with circulated heated and ambient air. Samples and PCR master mix are contained in 30-μl glass cuvettes (58). Sample detection is based on the principle of fluorescence resonance energy transfer (57, 58), with adjacent hybridization probes directed against the intended PCR product. With fluorescein serving as the donor fluorophore and LC-Red 640 (Roche Molecular Biochemicals) serving as the acceptor fluorophore, the presence of PCR amplicons can be assessed by detection of LC-Red 640 fluorescence. Samples can be assayed for the presence of this signal during each PCR cycle, and the cycle number at which the signal is first detected can be correlated to the original concentration of target. The specificity of amplification can be confirmed by melting curve analysis. Single melting peaks can be generated by depicting the negative derivative of fluorescence versus temperature (−dF/dT) over the course of a gradual PCR product melt (see below, “PCR cycling and melting curve conditions”).

(i) Extraction of control bacterial strains.Pure culture isolates, used as control organisms, were extracted by two different methods. (i) In the case of gram-negative isolates, includingLegionella species, bacterial colonies were suspended in sterile H₂O, to a turbidity of approximately 1 McFarland standard, lysed in a 100°C heat block for 10 min, and centrifuged for 1 min at 20,000 × g . The resulting supernatant was used for analysis. (ii) In the case of gram-positive isolates, bacterial colonies were suspended in 1.0 ml of 1 N NaOH, to a turbidity of approximately 1 McFarland standard and incubated at room temperature for 5 min. The cells were pelleted, washed in an equal volume of 0.5 M Tris-HCl (pH 8.0), pelleted again, and resuspended in 100 μl of H₂O. After heating in a 100°C heat block for 10 min, the suspension was centrifuged for 1 min at 20,000 × g , and the supernatant was used for analysis. Control extracts were used at a final dilution of 1:100 in sterile H₂O. The presence of amplifiable DNA in specificity controls was verified by utilizing broad-range 16S ribosomal (rDNA) amplification by standard methods (23). Specific PCR assays were used to verify the presence of nucleic acid in extracts of mycobacteria and chlamydiae.

(ii) Extraction of BAL specimens.BAL specimens were extracted using Chelex 100 (InstaGene Matrix; Bio-Rad Laboratories, Hercules, Calif.). Briefly, the specimen was mixed thoroughly, and 20 μl was added to 200 μl of InstaGene matrix. The resulting solution was mixed and placed in a 100°C heat block for 10 min. After mixing again, the sample was centrifuged for 2 min at 12,000 rpm. The resulting supernatant was used for analysis.

(iii) Extraction of tissue specimens.Tissue sections (25 μm thick) were cut consecutively with other sections that were used for DFA and ISH assays. The sections were each cut with a clean blade and placed in a sterile glass tube. Sections were deparaffinized in xylene, washed twice in absolute ethanol, subjected to proteinase K digestion overnight at 55°C, and then placed in a 100°C heat block for 15 min. Extractions were carried out using QIAmp DNA spin columns (Qiagen Inc., Valencia, Calif.), following a standard protocol. DNA was eluted from the spin columns twice, each using 50 μl of elution buffer AE (from the QIAmp DNA mini kit). Elution buffer was incubated in the column for 1 min at room temperature, prior to the first elution, and for 5 min at RT prior to the second elution. The first and second eluants were analyzed separately by PCR.

(iv) Primers and probes.All nucleic acid targets for primers and probes used in the study are listed in Table 3. Amplicons were kept to a minimum length (105 bp for the 5S gene and 124 bp formip) in order to enhance the utility of these assays in formalin-fixed tissue. Probes were constructed in order to juxtapose donor (fluorescein) and acceptor (LC-Red 640) fluorophore dyes, when probes were annealed to amplicon. The mip primer-probe set was constructed for specific detection of L. pneumophila(Legionella pneumophila species-specific LC-PCR). The 5S rRNA primer-probe set was constructed to allow detection of all commonLegionella species (Legionella genus LC-PCR). Primers for the latter assay were slightly truncated versions of the 20-mer sequences, L5SL9 and L5SR93, previously reported by Mahbubani et al. (35). Due to constraints imposed by the size and sequence of the 5S amplicon, only a single probe, a 23-bp portion of the 50-mer probe, also reported by Mahbubani et al. (35) was used. This probe was labeled at its 5′ end with LC-Red 640, while the fluorescein label was placed near the 3′ end of the reverse primer, for fluorescence resonance energy transfer signal production.

(v) PCR master mix (5S).A 5-μl aliquot of sample (5 μl of H₂O was used as a negative control for each run) was added to 15 μl of PCR mix in each sample cuvette. Optimized PCR master mix consisted of 50 mM KCl–20 mM Tris-HCl (pH 8.4) with a 0.1 mM concentration of each of the deoxyribonucleoside triphosphates, 6 mM MgCl₂, a 0.5 μM concentration of both 5S primers, a 0.1 μM concentration of the single 5S probe, 0.05% IGEPAL CA-630 (Sigma), 0.025% bovine serum albumin, and 0.025 U of PLATINUM Taq DNA Polymerase (Life Technologies, Rockville, Md.) per μl.

(vi) PCR Master Mix(mip).A 5-μl aliquot of sample (5 μl of H₂O was used as a negative control for each run) was added to 15 μl of PCR mix in each sample cuvette. Optimized master mix consisted of 50 mM KCl–20 mM Tris-HCl (pH 8.4) with a 0.2 mM concentration of each of the deoxyribonucleoside triphosphates, 6 mM MgCl₂, a 0.5 μM concentration of both mipprimers, a 0.2 μM concentration of the fluorescein mipprobe, a 0.4 μM concentration of the LC-Red 640 mip probe, 0.05% IGEPAL CA-630 (Sigma), 0.025% bovine serum albumin, and 0.025 U of PLATINUM Taq DNA Polymerase (Life Technologies) per μl.

(vii) PCR cycling and melting curve conditions.PCR reagents and specimen extracts were sealed in glass capillary cuvettes with plastic plugs, centrifuged to allow mixing and to drive the mix into the distal end of each tube, and then placed on the LightCycler instrument. The cycling protocol was identical for both themip and the 5S amplification reactions: one cycle of 95°C for 2 min followed by 50 cycles of denaturation at 95°C, annealing for 10 s at 57°C, and extension for 5 s at 72°C. Melting curves were generated as follows: starting at 55°C, the thermal chamber temperature was slowly raised to 85°C, during which time fluorescence was measured at frequent intervals. Analysis of PCR amplification and melting curves was carried out using LightCycler software.

(viii) Sensitivity and inhibition studies.The sensitivities of both PCR assays were assessed by testing serial dilutions of a known number of CFU of L. pneumophila. PCR inhibition was assessed by spiking all culture- and PCR-negative eluants of both BAL and tissue specimens with low concentrations of L. pneumophila. Concentrations of organism used were within 1 log unit of each assay's limit of sensitivity. Inhibition was demonstrated by loss of amplification signal and/or by appearance of signal at a later cycle number than that seen in similar concentrations of organisms, diluted in sterile water as a control.

Analysis of results.For direct (WS, DFA, ISH) assays, positives were defined by the presence of five or more identifiable bacilli, with the proper staining characteristics for the given assay (as defined above). For PCR, positives were defined by a fluorescent signal from the reporter dye (either during PCR amplification or during melting curve analysis) of three times the baseline level of fluorescence (in turn defined by the signal from the negative control cuvette for each run). The results of culture were considered to be the gold standard against which all other assays were compared. Several tissue isolates were originally designated “Legionnaires' disease bacillus” (LDB), as they were recovered before methods were in use for species determination. These were all retrospectively classified asL. pneumophila, based on the results of ISH, for the current study. Analysis of results was based on the number of specimens, rather than on the number of cases or patients tested. For BAL fluid samples, cells and supernatants from a single lavage procedure were classified as a single specimen. A breakdown of results, by case, as well as by specimen, is given in Tables 4 and5.