JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3475–3481 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00542-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.
The Pan Genera Detection Immunoassay: a Novel Point-of-Issue
Method for Detection of Bacterial Contamination
in Platelet Concentrates
Tanja Vollmer, Dennis Hinse, Knut Kleesiek, and Jens Dreier*
Institut fu?r Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen,
Universita?tsklinik der Ruhr-Universita?t Bochum, Bad Oeynhausen, Germany
Received 12 March 2010/Returned for modi？cation 21 April 2010/Accepted 2 August 2010
Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk in transfusion-
transmitted sepsis. Recently the Pan Genera Detection (PGD) system was developed and FDA licensed for screening of bacterial contamination of PCs directly prior to transfusion. The test principle is based on the
immunological detection of lipopolysaccharide (for Gram-negative bacteria) or lipoteichoic acid (for Gram-
positive bacteria). In the present study we analyzed the applicability of this method with regard to detection limit, practicability, implementation, and performance. PCs were spiked with Staphylococcus aureus, Bacillus
subtilis, and ；ve different Klebsiella pneumoniae strains, as well as eight different Escherichia coli strains. The
presence of bacteria was assessed by the PGD immunoassay, and bacteria were enumerated by plating cultures. Application of the PGD immunoassay showed that it is a rapid test with a short hands-on time for sample
processing and no demand for special technical equipment and instrument operation. The lower detection
limits of the assay for Gram-positive bacteria showed a good agreement with the manufacturer’s speci；cations 34(8.2 10to 5.5 10CFU/ml). For some strains of K. pneumoniae and E. coli, the PGD test showed analytical 64sensitivities (>10CFU/ml) that were divergent from the designated values (K. pneumoniae, 2.0 10CFU/ml; 4E. coli, 2.8 10CFU/ml). Result interpretation is sometimes dif；cult due to very faint bands. In conclusion, our study demonstrates that the PGD immunoassay is an easy-to-perform bedside test for the detection of
bacterial contamination in PCs. However, to date there are some shortcomings in the interpretation of results
and in the detection limits for some strains of Gram-negative bacteria.
Bacterial contamination of platelet concentrates (PCs) still sampling strategy, carrying a high risk of sampling errors. The represents an ongoing risk in transfusion-transmitted sepsis. In initial levels of most skin-based bacteria in PC units are usually 2004, sterility testing of PCs was recommended by the Amer- remarkably low, and it has been demonstrated that 57% of ican Association of Blood Banks, and the detection of bacterial cultures are false negative at low contamination levels of 10 to contamination in PCs has been implemented in several blood 100 CFU per PC unit (22). Application of rapid detection centers and transfusion services as routine quality control test- methods combined with an early sampling strategy will also ing (11). However, transfusion-transmitted bacterial sepsis has invariably miss bacterial contamination as a result of the sam- still not been completely eliminated, with septic complications pling error. Therefore, a prolonged time frame between sam- observed particularly with older PCs (6, 8, 11, 12, 20, 26, 32, pling and testing will increase the probability that most con- 34). At present, the detection of microbiological contamina- taminated PCs will be identi？ed. With cultural approaches, tion in PCs can be divided into two major methodological results are not available in time to avoid transfusion of the concepts (19): (i) incubation or cultivation methods and (ii) contaminated PC. rapid detection methods, such as nucleic acid ampli？cation Hence, substantial interest focuses on rapid detection meth- techniques (NAT) (9, 16, 28), ，uorescence-activated cell sort- ods for bacterial screening combined with a late sampling strat- ing (FACS) (10, 13, 17, 30, 31), or immunological detection egy. In this context, a sensitive, speci？c, cost-effective, rapid, methods (Pan Genera Detection [PGD] system) (24, 25). and easy-to-perform point-of-issue bacterial detection test im- Incubation or cultivation methods are currently the most mediately before transfusion (24, 25), requiring only a small sensitive detection methods and are utilized predominantly for test sample volume, is considered optimal. Recently, the Pan sterility testing of PCs (7). Nevertheless, culture-based meth- Genera Detection (PGD) system was developed and FDA ods require 24 h prior to sampling and at least 18 h to 24 h of licensed [501(k) clearance] for the screening of bacterial con- incubation to obtain a positive result (5, 15, 23, 24). Therefore, tamination directly prior to transfusion. Experience regarding culture-based methods have to be combined with an early the implementation and performance of this technology has seldom been reported (24, 25). A ？rst study using the PGD test for screening of 7,733 whole-blood derived PCs has been pub- * Corresponding author. Mailing address: Institut fu?r Laboratori- lished (38). The test principle is based on the immunological ums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nord- detection of the conserved bacterial antigens lipopolysaccha- rhein-Westfalen, Universita?tsklinik der Ruhr-Universita?t Bochum, ride (LPS) (for Gram-negative bacteria) or lipoteichoic acid Georgstrasse 11, 32545 Bad Oeynhausen, Germany. Phone: 49-5731- (LTA) (for Gram-positive bacteria) by lateral-，ow immuno- 97-1391. Fax: 49-5731-97-2307. E-mail: firstname.lastname@example.org. precipitation. In a previous study, we developed a novel rapid Published ahead of print on 11 August 2010.
3476 VOLLMER ET AL. J. CLIN. MICROBIOL.
different E. coli and ？ve different K. pneumoniae strains and incubated at 22?C screening method based on ，ow cytometric detection (10), with agitation for 48 h. Samples were diluted in a 10-fold dilution series with which was compared with the PGD test, among others. Pre- sterile PCs, three negative controls were added randomly, and all samples were liminary results revealed that the PGD test detected Gram- analyzed by PGD. Test devices were read by ？ve independent experimenters in positive bacteria in the given range, but Gram-negative bacte- a blinded trial. Each individual result was scored (positive, 1 points; arguable, 0.5 rial species such as Klebsiella pneumoniae were detected with 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. considerably divergent detection limits, as speci？ed by the RAPD PCR analysis and serotyping. Bacteria were harvested from cultures manufacturer. Based on these data, we have evaluated this grown overnight, and bacterial DNA was extracted using the QIAamp DNA effect systematically and in detail in the present study. blood kit (protocol D; Qiagen, Hilden, Germany) according to the manufactur- er’s instructions. Nucleic acids were eluted with 200 l elution buffer (Qiagen). Randomly ampli？ed polymorphic DNA (RAPD) PCR analysis was performed MATERIALS AND METHODS using arbitrary primers (ERIC-1, 5 -ATGTAAGCTCCTGGGGATTCAC-3 ; ERIC-2, 5 -AAGTAAGTGACTGGGGTGAGCG-3 ) (36). DNA ampli？cation Bacterial strains and culture conditions. The two strains Escherichia coli was carried out in 0.2-ml tubes containing 45 l reaction mix and 5 l DNA ATCC 35218 and K. pneumoniae ATCC 13882 were purchased from the Amer- extract. The reaction mixture consisted of 1 AmpliTaq buffer, including 1.5 mM ican Type Culture Collection (ATCC) (LGC Promochem GmbH, Wesel, Ger- MgSO(Applied Biosystems, Foster City, CA), 200 M each deoxynucleoside 4many). Strains K. pneumoniae PEI-B-08-08 and Staphylococcus aureus PEI-B- triphosphate, 2000 nM each primer, and 5 U of AmpliTaq DNA polymerase 23-04 were obtained from the Paul-Ehrlich-Institute (PEI) (Langen, Germany). (Applied Biosystems). DNA ampli？cation was carried out with the following E. coli strain L01207081 and K. pneumoniae strain L01204084 were provided by thermal cycling pro？le: preliminary denaturation at 95?C for 5 min, followed by Verax Biomedical Inc. (Worcester, MA). Bacterial spore suspensions with de- 45 cycles of denaturation at 95?C for 30 s, annealing at 35?C for 60 s, and ？ned titers of Bacillus subtilis ATCC 35031 (SGM Biotech Inc., Bozeman, MT) extension at 72?C for 120 s. DNA fragments were separated by gel electrophore- were cultured in Trypticase soy broth (bioMe?rieux, Nu?rtingen, Germany) under sis in a 1.5% agarose gel (Carl Roth, Karlsruhe, Germany) in 1 UltraPure aerobic conditions at 37?C for 24 to 48 h. All other K. pneumoniae and E. coli Tris-borate-ETA (TBE) buffer (pH 8.0) (Gibco Technologies, Paisley, Scotland) strains were previously isolated from patient specimens at our hospital and were containing 500 ng/ml ethidium bromide. After addition of 2 l of 6 loading dye characterized in our microbiological laboratory by standard methods. (Fermentas, St. Leon-Rot, Germany), 10 l of PCR product was loaded onto the PC collection. Apheresis-derived single-donor PCs were obtained from the gel. The pUC mix 8 DNA ladder (Fermentas) was used as a molecular size transfusion service Uni.Blutspendedienst OWL (Bad Oeynhausen, Ger- marker. Electrophoresis was carried out at room temperature and at a constant many). PCs were prepared using the Haemonetics MCS (Haemonetics voltage of 6 V/cm in 0.5 TBE buffer. GmbH, Munich, Germany) from healthy blood donors and stored in gas- Serotyping of O and H antigens of E. coli strains was performed by the Robert permeable containers (LN994CF-CPP; Haemonetics GmbH) at 20 to 24?C Koch Institute (RKI) (Wernigerode, Germany). with agitation. Predonation sampling was performed after donor arm disin- fection using a single-swab method with 70% isopropyl alcohol. The ？nal PC volume was approximately 235 ml. PGD testing. The PGD test (Verax Biomedical Inc.) was performed accord- RESULTS ing to the manufacturer’s instructions. Brie，y, 500 l of PC sample was mixed with 8 drops of reagent 1 (water, methanol, surfactants, and preservative Performance and implementation. The PGD test is very [ProClin300]) by inverting the tube three times, followed by centrifugation at easy to perform, with a short hands-on time. Altogether, the 11,000 g for 5 min. Subsequently, the supernatant was removed and 8 drops of processing of one sample at a time took 8 min. Reading of the reagent 2 (water, sodium hydroxide, surfactants, and preservative [sodium test device took a maximum of an additional 6 min (1 min per azide]) were added to the cell pellet. Sample preparations were not vortexed before the addition of reagent 3 (Tricine buffer with surfactants, anticoagulants, result interpretation, six different times in case of negative protein stabilizers [bovine, mouse, and rabbit], and preservatives [ProClin300 results). The PGD test procedure took 77 min before nega- and sodium azide]). In a change from the manufacturer’s instructions, the pellet tively tested PCs were accessible for transfusion release, was loosened carefully from the bottom of the tube with a disposable pipette but whereas the earliest positive results could be obtained after 37 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 min. Increasing processing to up to six samples at a time did pellet was completely resuspended by vortexing, and the total sample volume was not considerably in，uence the hands-on time or time to result. transferred to the test device. Test performance and interpretation of results Detection of bacterial proliferation in PCs. PCs were ana- were implemented as described by the manufacturer. lyzed after inoculation with 0.06 CFU/ml K. pneumoniae PEI- Detection of bacterial proliferation in PCs. Before bacterial inoculation, all B-08-08, 0.19 CFU/ml E. coli ATCC 35218, 0.07 CFU/ml B. 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 subtilis ATCC 35031, or 0.13 CFU/ml S. aureus PEI-B-23-04 at aerobic (BacT/Alert SA; bioMe?rieux, Nu?rtingen, Germany) and anaerobic eight different times during storage (Fig. 1), under conditions (BacT/Alert SN; bioMe?rieux) culture bottles and incubated for up to 7 days. The which might be encountered in practice. The BacT/Alert au- time periods until detection of bacterial contamination using the PGD test were tomated culture system was used to con？rm successful con- 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. tamination. For each inoculated PC, bacteria were detected in subtilis ATCC 35031. Bacterial titers of 1 CFU/ml were achieved by 10-fold a minimum of one BacT/Alert culture bottle at the time of serial dilution of stationary-grown overnight cultures in phosphate-buffered sa- inoculation, whereas bacterial contamination was detected for line (PBS), followed by inoculation of 1 ml of the respective dilution (K. pneu- all four strains under both aerobic and anaerobic conditions 787