The Pan Genera Detection Immunoassay: a Novel Point-of-Issue Method for Detection of Bacterial Contamination in Platelet Concentrates

Bacterial strains and culture conditions.The two strains Escherichia coli ATCC 35218 and K. pneumoniae ATCC 13882 were purchased from the American Type Culture Collection (ATCC) (LGC Promochem GmbH, Wesel, Germany). Strains K. pneumoniae PEI-B-08-08 and Staphylococcus aureus PEI-B-23-04 were obtained from the Paul-Ehrlich-Institute (PEI) (Langen, Germany). E. coli strain L01207081 and K. pneumoniae strain L01204084 were provided by Verax Biomedical Inc. (Worcester, MA). Bacterial spore suspensions with defined titers of Bacillus subtilis ATCC 35031 (SGM Biotech Inc., Bozeman, MT) were cultured in Trypticase soy broth (bioMérieux, Nürtingen, Germany) under aerobic conditions at 37°C for 24 to 48 h. All other K. pneumoniae and E. coli strains were previously isolated from patient specimens at our hospital and were characterized in our microbiological laboratory by standard methods.

PC collection.Apheresis-derived single-donor PCs were obtained from the transfusion service Uni.Blutspendedienst OWL (Bad Oeynhausen, Germany). PCs were prepared using the Haemonetics MCS+ (Haemonetics GmbH, Munich, Germany) from healthy blood donors and stored in gas-permeable containers (LN994CF-CPP; Haemonetics GmbH) at 20 to 24°C with agitation. Predonation sampling was performed after donor arm disinfection using a single-swab method with 70% isopropyl alcohol. The final PC volume was approximately 235 ml.

PGD testing.The PGD test (Verax Biomedical Inc.) was performed according to the manufacturer's instructions. Briefly, 500 μl of PC sample was mixed with 8 drops of reagent 1 (water, methanol, surfactants, and preservative [ProClin300]) by inverting the tube three times, followed by centrifugation at 11,000 × g for 5 min. Subsequently, the supernatant was removed and 8 drops of reagent 2 (water, sodium hydroxide, surfactants, and preservative [sodium azide]) were added to the cell pellet. Sample preparations were not vortexed before the addition of reagent 3 (Tricine buffer with surfactants, anticoagulants, protein stabilizers [bovine, mouse, and rabbit], and preservatives [ProClin300 and sodium azide]). In a change from the manufacturer's instructions, the pellet was loosened carefully from the bottom of the tube with a disposable pipette but was not divided into three or four fragments. The maximum residence time of reagent 2 did not exceed 2 min. Afterwards, 4 drops of reagent 3 were added, the pellet was completely resuspended by vortexing, and the total sample volume was transferred to the test device. Test performance and interpretation of results were implemented as described by the manufacturer.

Detection of bacterial proliferation in PCs.Before bacterial inoculation, all PCs used for spiking experiments were sampled to ensure baseline sterility of the original apheresis bags. Five milliliters of sample was inoculated into both the aerobic (BacT/Alert SA; bioMérieux, Nürtingen, Germany) and anaerobic (BacT/Alert SN; bioMérieux) culture bottles and incubated for up to 7 days. The time periods until detection of bacterial contamination using the PGD test were compared during PC storage at 20 to 24°C after inoculation with <1 CFU/ml of K. pneumoniae PEI-B-08-08, E. coli ATCC 35218, S. aureus PEI-B-23-04, and B. subtilis ATCC 35031. Bacterial titers of <1 CFU/ml were achieved by 10-fold serial dilution of stationary-grown overnight cultures in phosphate-buffered saline (PBS), followed by inoculation of 1 ml of the respective dilution (K. pneumoniae, 10⁻⁷; E. coli, 10⁻⁸; S. aureus, 10⁻⁷; and B. subtilis, 10⁻⁵). To control bacterial inoculation of a PC, samples were taken immediately after inoculation (0 h), as well as after 24 h, and analyzed with the BacT/Alert 3D continuous-monitoring system as described above. Sampling of appropriate sample volumes was performed by sterile welding of satellite bags at 0, 6, 12, 24, 30, 48, 54, and 72 h after inoculation directly prior to analysis. Subsequently, 500 μl of PC was analyzed using the PGD test as described above.

To monitor bacterial growth kinetics, 100-μl aliquots of 10-fold serial dilutions of PC samples were plated in triplicate onto tryptone soy agar (TS) and incubated at 37°C for at least 48 h. After incubation, the number of colonies was counted and the initial bacterial concentration was calculated.

Detection limit for Gram-negative strains.To analyze the detection limit of the PGD test for different Gram-negative strains, PCs were spiked with eight different E. coli and five different K. pneumoniae strains and incubated at 22°C with agitation for 48 h. Samples were diluted in a 10-fold dilution series with sterile PCs, three negative controls were added randomly, and all samples were analyzed by PGD. Test devices were read by five independent experimenters in a blinded trial. Each individual result was scored (positive, 1 points; arguable, 0.5 point; negative, 0 points), and total results were evaluated as follows: (i) 0 to 1, negative; (ii) 1.5 to 2.5, arguable, and (iii) 3 to 5, positive.

RAPD PCR analysis and serotyping.Bacteria were harvested from cultures grown overnight, and bacterial DNA was extracted using the QIAamp DNA blood kit (protocol D; Qiagen, Hilden, Germany) according to the manufacturer's instructions. Nucleic acids were eluted with 200 μl elution buffer (Qiagen). Randomly amplified polymorphic DNA (RAPD) PCR analysis was performed using arbitrary primers (ERIC-1, 5′-ATGTAAGCTCCTGGGGATTCAC-3′; ERIC-2, 5′-AAGTAAGTGACTGGGGTGAGCG-3′) (36). DNA amplification was carried out in 0.2-ml tubes containing 45 μl reaction mix and 5 μl DNA extract. The reaction mixture consisted of 1× AmpliTaq buffer, including 1.5 mM MgSO₄ (Applied Biosystems, Foster City, CA), 200 μM each deoxynucleoside triphosphate, 2000 nM each primer, and 5 U of AmpliTaq DNA polymerase (Applied Biosystems). DNA amplification was carried out with the following thermal cycling profile: preliminary denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 30 s, annealing at 35°C for 60 s, and extension at 72°C for 120 s. DNA fragments were separated by gel electrophoresis in a 1.5% agarose gel (Carl Roth, Karlsruhe, Germany) in 1× UltraPure Tris-borate-ETA (TBE) buffer (pH 8.0) (Gibco Technologies, Paisley, Scotland) containing 500 ng/ml ethidium bromide. After addition of 2 μl of 6× loading dye (Fermentas, St. Leon-Rot, Germany), 10 μl of PCR product was loaded onto the gel. The pUC mix 8 DNA ladder (Fermentas) was used as a molecular size marker. Electrophoresis was carried out at room temperature and at a constant voltage of 6 V/cm in 0.5× TBE buffer.

Serotyping of O and H antigens of E. coli strains was performed by the Robert Koch Institute (RKI) (Wernigerode, Germany).

Performance and implementation.The PGD test is very easy to perform, with a short hands-on time. Altogether, the processing of one sample at a time took 8 min. Reading of the test device took a maximum of an additional 6 min (1 min per result interpretation, six different times in case of negative results). The PGD test procedure took 77 min before negatively tested PCs were accessible for transfusion release, whereas the earliest positive results could be obtained after 37 min. Increasing processing to up to six samples at a time did not considerably influence the hands-on time or time to result.

Detection of bacterial proliferation in PCs.PCs were analyzed after inoculation with 0.06 CFU/ml K. pneumoniae PEI-B-08-08, 0.19 CFU/ml E. coli ATCC 35218, 0.07 CFU/ml B. subtilis ATCC 35031, or 0.13 CFU/ml S. aureus PEI-B-23-04 at eight different times during storage (Fig. 1), under conditions which might be encountered in practice. The BacT/Alert automated culture system was used to confirm successful contamination. For each inoculated PC, bacteria were detected in a minimum of one BacT/Alert culture bottle at the time of inoculation, whereas bacterial contamination was detected for all four strains under both aerobic and anaerobic conditions after 24 h (data not shown). Subsequently, the applicability of the PGD test was analyzed with regard to sensitivity and the time period until detection of bacterial contamination. Contamination with S. aureus was initially detected after 48 h, with a corresponding titer of 4.67 × 10⁶ CFU/ml (Fig. 1A). The bacterial titer of 8.4 × 10³ CFU/ml at the previous sampling point was very close to the specified detection limit of 8.2 × 10³ CFU/ml, resulting in a statistical contingency to obtain a positive or negative result. The PGD test revealed an initially positive result for B. subtilis after 72 h, with a corresponding titer of 4.63 × 10⁴ CFU/ml (Fig. 1B). At the previous sampling point, the bacterial titer of 667 CFU/ml was too low to obtain a positive result. In summary, the results obtained for the Gram-positive strains correlated well with the analytical sensitivities reported by the manufacturer (S. aureus, 8.2 × 10³ CFU/ml; B. cereus [representative for B. subtilis], 1.2 × 10⁴ CFU/ml).

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FIG. 1.

Detection of bacterial proliferation by the PGD immunoassay. One single apheresis-derived PC unit was spiked with <1 CFU/ml of the indicated bacterium and stored at 22°C with agitation. Samples were taken immediately (0 h) and at 6, 12, 24, 48, 54, and 72 h after inoculation. The PGD test was performed as described in Materials and Methods. Bacteria were enumerated by colony-forming assay. The dotted horizontal line represents the lower detection limit of the PGD assay for the respective strain.

In contrast, PGD testing of PCs inoculated with Gram-negative bacteria demonstrated analytical sensitivities that were considerably divergent from the specified lower detection limits, followed by a delayed detection of bacteria. The earliest positive result for the PC unit inoculated with E. coli was obtained after 54 h, with a bacterial titer of 9.20 × 10⁶ CFU/ml (Fig. 1C), whereas the detection limit reported by the manufacturer was 2.8 × 10⁴ CFU/ml. For the PC inoculated with K. pneumoniae, the PGD test revealed an arguably positive result, with very faint bands after 30 h of incubation, with a corresponding bacterial titer of 2.13 × 10⁷ CFU/ml (Fig. 1D). The lower detection limit was indicated by the manufacturer to be 2.0 × 10⁴ CFU/ml; therefore, the PGD test should already provide a positive result after 24 h, as well as at this sampling point. Definite positivity was not achieved until sampling after 48 h, with a corresponding bacterial titer of 8.97 × 10⁸ CFU/ml. Additionally, we observed difficulties in obtaining valid procedural control windows when analyzing high titers of bacteria, especially for K. pneumoniae. According to our consultation with the manufacturer, valid procedural control windows are not necessary in the case of a clearly positive result (personal communication).

Detection limits for different E. coli and K. pneumoniae strains.To analyze the detection limits of the PGD test for K. pneumoniae and E. coli in detail, PCs were spiked with eight different E. coli and five different K. pneumoniae strains and incubated at 22°C with agitation for 48 h. The two strains E. coli L01207081 and K. pneumoniae L01204084, which were used for the FDA validation studies, were used as reference strains. Samples were diluted in a 10-fold dilution series with sterile PCs and analyzed by PGD. Test devices were read by five independent experimenters in a blinded trial (Table 1). To allow for the subjectivity of reports by each individual experimenter and to obtain an objective qualitative result, we scored each individual result and calculated the total results as described in Materials and Methods. In general, very faint bands complicated the result interpretation, which is clearly comprehensible on the basis of the test device interpretation presented in Table 1. The reference strain E. coli L01207081, as well as E. coli isolate A001S, were detected in the given range of 2.8 × 10⁴ CFU/ml with a maximum score of 5.0 (positive, concordant results by all experimenters). In contrast, E. coli isolates MS82 and MS88 were detected (score, 5.0) only with bacterial titers of 10⁸ CFU/ml and 10⁷ CFU/ml, respectively. E. coli isolates 079 and 008 were definitely detected (score, 5.0) with a bacterial titer of 10⁸ CFU/ml, whereas arguable results were obtained for titers of 10⁷ CFU/ml (score, 2.0 or 2.5). Only E. coli strain ATCC 35218 (arguable; score, 2.0) and isolate 835 (positive; score, 5.0) were detectable with bacterial titers of 10⁶ CFU/ml. Accordingly, the K. pneumoniae reference strain L01204084 was detected (score, 5.0) with a bacterial titer of 7.95 × 10³ CFU/ml, which is closely below the given range of 2.0 × 10⁴ CFU/ml. However, similarly to the detection of E. coli, K. pneumoniae strains PEI-B-08-08 and ATCC 13882 and isolate 13734 were positive only at a concentration of 10⁸ CFU/ml (score, 5.0). Only isolate 01662 (score, 4.0) was also detectable with a bacterial titer of 10⁷ CFU/ml.

RAPD-PCR analysis and serotyping.To study the molecular relatedness of K. pneumoniae and E. coli strains and to infer a potential connection to the differences in detection limit, RAPD-PCR analysis was performed using two single-oligonucleotide primers with arbitrary sequences. To determine whether the composition of E. coli LPS influences the immunologic detection, strains were serotyped regarding their O and H side chains (Table 1). Primer ERIC-1 yielded six different banding patterns for the different K. pneumoniae strains and seven different patterns for E. coli, whereas primer ERIC-2 yielded five different patterns for K. pneumoniae and six different banding patterns for E. coli (data not shown). Regarding both primers, a molecular relatedness was detectable only for the two E. coli strains A001S and L01204081 (cluster EC-VI) (Table 1). Both strains AS001 and L01204081 had no H antigen, whereas the O antigens are not typeable so far. Interestingly, these two E. coli strains were detected by the PGD assay at as low as 10⁴ CFU/ml, in accordance with the detection limit reported by the manufacturer. All other strains demonstrated no molecular relatedness based on RAPD-PCR profile and serotyping results (Table 1).