Real-Time Polymerase Chain Reaction in Transfusion Medicine: Applications for Detection of Bacterial Contamination in Blood Products Jens Dreier, Melanie Störmer, and Knut Kleesiek Bacterial contamination of blood components, particu- larly of platelet concentrates (PCs), represents the great- est infectious risk in blood transfusion. Although the incidence of platelet bacterial contamination is approxi- mately 1 per 2000 U, the urgent need for a method for the routine screening of PCs to improve safety for patients had not been considered for a long time. Besides the culturing systems, which will remain the criterion stan- dard, rapid methods for sterility screening will play a more important role in transfusion medicine in the future. In particular, nucleic acid amplification techniques (NATs) are powerful potential tools for bacterial screening assays. The combination of excellent sensitivity and specificity, reduced contamination risk, ease of perfor- mance, and speed has made real-time polymerase chain reaction (PCR) technology an appealing alternative to conventional culture-based testing methods. When using real-time PCR for the detection of bacterial contamina- tion, several points have to be considered. The main focus is the choice of the target gene; the assay format; the nucleic acid extraction method, depending on the sample type; and the evaluation of an ideal sampling strategy. However, several factors such as the availability of bacterial-derived nucleic acid amplification reagents, the impracticability, and the cost have limited the use of NATs until now. Attempts to reduce the presence of contam- inating nucleic acids from reagents in real-time PCR have been described, but none of these approaches have proven to be very effective or to lower the sensitivity of the assay. Recently, a number of broad-range NATassays targeting the 16S ribosomal DNA or 23S ribosomal RNA for the detection of bacteria based on real-time technol- ogy have been reported. This review will give a short survey of current approaches to and the limitations of the application of real-time PCR for bacterial detection in blood components, with emphasis on the bacterial contamination of PCs. C 2007 Elsevier Inc. All rights reserved. D espite considerable advances in the safety of blood components, transfusion-associated bacterial infection remains an unresolved problem, with significant transfusion-related morbidity and mortality rates. Platelet concentrate (PC) transfu- sion-associated sepsis is now recognized as the most frequent infectious complication in transfusion therapy, surpassing by up to 2 orders of magnitude the incidence of transfusion-associated viral trans- mission. 1,2 The bacterial contamination of PCs is a major problem because of the current requirement to store PCs at room temperature with agitation (aerobic conditions) to preserve platelet function. At 22°C to 24°C, bacteria grow more easily than under lower temperature conditions, so that small bacterial inocula can grow to very high numbers within a short period. Consequently, older units are most likely to have high bacterial inocula and are therefore more likely to cause sepsis in recipients. The bacterial contamination rate of whole blood held at room temperature for 2 to 20 hours is reported to be 0.34%. 3 Recent reports suggest that platelet-related bacteremia occurs at a frequency approximately 50 times greater than that for red blood cells. 4 Approximately 80% of red cell– associated sepsis involved psychrophile bacteria or microbes capable of growth at refrigerated tem- peratures, including Yersinia enterocolitica (46%), Pseudomonas sp (25%), and Serratia sp (11%). 5 Cell-free products such as plasma and cryoprecipi- tates are stored frozen and are rarely associated with bacterial contamination. 1 Therefore, in the scope of bacterial safety, highest attention is given to PCs because they represent a good growth medium and the storage conditions support bacterial growth. 6 The reported prevalence of bacterially contami- nated PCs varies from 0.08% to 0.7% in countries that perform prospective testing, depending on their technology, testing protocols, and additional inter- vention methods. 7,8 Different screening methods have been devel- oped for bacterial detection and can be divided into culture and rapid-detection methods 9-18 ; but to date, none of these methods is sufficient as a perfect From the Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Univer- sitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany. Address reprint requests to Jens Dreier, Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany. E-mail: [email protected]0887-7963/07/$ - see front matter n 2007 Elsevier Inc. All rights reserved. doi: 10.1016/j.tmrv.2007.03.006 Transfusion Medicine Reviews, Vol 21, No 3 (July), 2007: pp 237-254 237
Transcript
Real-Time Polymerase Chain Reaction in Transfusion Medicine:Applications for Detection of Bacterial Contamination in
Blood Products
Jens Dreier, Melanie Störmer, and Knut Kleesiek
Bacterial contamination of blood components, particu-larly of platelet concentrates (PCs), represents the great-est infectious risk in blood transfusion. Although theincidence of platelet bacterial contamination is approxi-mately 1 per 2000U, the urgent need for amethod for theroutine screening of PCs to improve safety for patientshad not been considered for a long time. Besides theculturing systems, which will remain the criterion stan-dard, rapidmethods for sterility screeningwill play amoreimportant role in transfusion medicine in the future. Inparticular, nucleic acid amplification techniques (NATs)are powerful potential tools for bacterial screeningassays. The combination of excellent sensitivity andspecificity, reduced contamination risk, ease of perfor-mance, and speed has made real-time polymerase chainreaction (PCR) technology an appealing alternative toconventional culture-based testing methods. When usingreal-time PCR for the detection of bacterial contamina-tion, several points have to be considered. Themain focus
Transfusion Medicine Reviews, Vol 21, No 3 (July), 2007: pp 237-25
is the choice of the target gene; the assay format; thenucleic acid extraction method, depending on the sampletype; and the evaluation of an ideal sampling strategy.However, several factors such as the availability ofbacterial-derived nucleic acid amplification reagents, theimpracticability, and the cost have limited the use of NATsuntil now. Attempts to reduce the presence of contam-inating nucleic acids from reagents in real-time PCR havebeen described, but none of these approaches haveproven to be very effective or to lower the sensitivity ofthe assay. Recently, a number of broad-range NATassaystargeting the 16S ribosomal DNA or 23S ribosomal RNAfor the detection of bacteria based on real-time technol-ogy have been reported. This review will give a shortsurvey of current approaches to and the limitations of theapplication of real-time PCR for bacterial detection inblood components, with emphasis on the bacterialcontamination of PCs.C 2007 Elsevier Inc. All rights reserved.
5
From the Institut für Laboratoriums und Transfusionsmedizin,Herz und Diabeteszentrum Nordrhein-Westfalen, Univer-sitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen,Germany.
0887-7963/07/$ - see front mattern 2007 Elsevier Inc. All rights reserved.doi: 10.1016/j.tmrv.2007.03.006
D espite considerable advances in the safety ofblood components, transfusion-associated
bacterial infection remains an unresolved problem,with significant transfusion-related morbidity andmortality rates. Platelet concentrate (PC) transfu-sion-associated sepsis is now recognized as the mostfrequent infectious complication in transfusiontherapy, surpassing by up to 2 orders of magnitudethe incidence of transfusion-associated viral trans-mission.1,2 The bacterial contamination of PCs is amajor problem because of the current requirement tostore PCs at room temperature with agitation(aerobic conditions) to preserve platelet function.At 22°C to 24°C, bacteria grow more easily thanunder lower temperature conditions, so that smallbacterial inocula can grow to very high numberswithin a short period. Consequently, older units aremost likely to have high bacterial inocula and aretherefore more likely to cause sepsis in recipients.
The bacterial contamination rate of whole bloodheld at room temperature for 2 to 20 hours isreported to be 0.34%.3 Recent reports suggest thatplatelet-related bacteremia occurs at a frequencyapproximately 50 times greater than that for redblood cells.4 Approximately 80% of red cell–associated sepsis involved psychrophile bacteria ormicrobes capable of growth at refrigerated tem-peratures, including Yersinia enterocolitica (46%),
Pseudomonas sp (25%), and Serratia sp (11%).Cell-free products such as plasma and cryoprecipi-tates are stored frozen and are rarely associated withbacterial contamination.1 Therefore, in the scope ofbacterial safety, highest attention is given to PCsbecause they represent a good growth medium andthe storage conditions support bacterial growth.6
The reported prevalence of bacterially contami-nated PCs varies from 0.08% to 0.7% in countriesthat perform prospective testing, depending on theirtechnology, testing protocols, and additional inter-vention methods.7,8
Different screening methods have been devel-oped for bacterial detection and can be divided intoculture and rapid-detection methods9-18; but to date,none of these methods is sufficient as a perfect
4 237
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preventative screening for the detection of con-taminated units. General diagnostic difficulties forthe detection of bacterial contamination in bloodproducts are the limited diagnostic window, theheterogeneity of the target organisms, and theadvanced demand on sensitivity to detect lowbacterial cell numbers present at the beginning ofthe storage of blood products (less than 100 colonyforming units [CFU] in 200 mL). These problemsespecially occur in sterility controls of PCs becauseonly 5 days of storage is allowed, and the low initialcontamination level of the product is below thedetection limit of all methods. Even the mostsensitive assay based on the cultivation of bacteria(eg, automatic blood culturing systems) particularlydetects 1 CFU per assay, but slow-growing bacteriasuch as Propionibacterium sp may be missed or aredetected too late (5-7 days after PC preparation),when blood products have already been transfused.In conclusion, the detection of bacterial contamina-tion of blood components generally requires timefor the organisms to proliferate.2,9,19 This is incontrast to viral contamination of blood products, inwhich the virus or the immune response to the virususually can be detected from a sample obtained atthe time of donation.In line with this diagnostic dilemma, several
rapid methods have been developed for sterilitytesting of blood products.5,17 Moreover, moleculargenetic techniques are theoretically appealing forthe detection of bacteria in blood componentsbecause of their high sensitivity and specificity andthe rapidity of obtaining results. At present, the highpotential of nucleic acid amplification techniques(NATs) for application in sterility testing has notbeen exhausted. Such assays, adaptable to transfu-sion medicine, are not commercially available sofar, although several working groups have devel-oped and validated home-brewed NAT assays. Thisreview will give a short survey of the presentsituation with real-time molecular diagnosticsregarding the sterility testing of blood products.
MOLECULAR TECHNOLOGY
Real-Time PCR
Real-time PCR has revolutionized the wayclinical microbiology laboratories diagnose humanpathogens. The technique is extremely sensitive,rapid, capable of high throughput, and relativelyeasy to perform.20-23 With its ability to measure
PCR products as they accumulate in “real time,” ithas become possible to determine the amount ofPCR product accumulated during the exponentialphase of bacterial growth, which can in turn be usedfor the quantitation of nucleic acids. This testingmethod combines PCR chemistry with fluorescentprobe detection of amplified products in the samereaction vessel. In general, both PCR and amplifiedproduct detection can be completed in 1 hour orless, which is considerably faster than conventionalPCR and detection methods.24 Real-time PCR hasseveral advantages over other PCR-based quantifi-cation approaches, including the elimination ofpostamplification handling, no cross-contaminationwith PCR products, easier automation, and proces-sing of large numbers of samples. Sensitive andspecific detection is possible by using novelfluorescent probe technology. Three types ofnucleic acid detection methods have been usedmost frequently with real-time PCR testing plat-forms in clinical microbiology: 5' nuclease (Taq-Man probes), molecular beacons, and hybridizationprobes. These detection methods all rely on thetransfer of light energy between two adjacent dyemolecules, a process referred to as fluorescenceresonance energy transfer.20,24,25 In real-time PCRassays to detect bacterial contamination in bloodproducts, TaqMan probes were preferred10,14
because of the limited target regions that can beused for primer and probe hybridization. Especiallythe use of the locked nucleic acid and minor groovebinder probe technology in combination with shortoligonucleotides permits a better efficiency of PCRbecause of lower interference during the amplifica-tion process. The shorter length gives the probesbetter sequence specificity and lower fluorescentbackground in comparison with conventionalprobes.26-28 Furthermore, the chemical modifica-tion of the probes can improve the hybridizationaffinity for complementary sequences, increase themelting temperature (Tm) by several degrees, andtherefore improve the specificity of the assay. Usingreal-time PCR for the bacterial detection, severalpoints have to be considered. The main focus refersto the choice of the target gene and the assay format.At present, there are two real-time NAT assayspublished that detect bacterial contamination inPCs.14,18 These two NAT assays will be comparedin the following sections, and several other differentapproaches for the detection of bacterial contam-ination will be presented.
239STERILITY TESTING OF BLOOD COMPONENTS
Choice of Target for Broad-Range Bacterial NAT
A general requirement for both the primer andthe probe sequences is their unique ability toidentify a specific organism or an organism group(eg, transfusion-relevant bacteria species). More-over, the PCR primer has to be able to identify thetarget sequences in the specimen of interest withhigh efficiency and specificity (eg, blood, plasma,PC). The target nucleic acid sequence should beconserved in bacteria. If sequence data of theintended target area show a significant frequencyof polymorphisms, a more conserved site shouldbe chosen.20
For screening methods, all potentially occurringbacteria species have to be detected with nearly thesame efficiency. Consequently, broad-range ampli-fication techniques are preferred for this purpose,rather than the specific detection of a limited groupof bacterial genera (eg, Enterobacteriaceae29) orspecies (eg, Y enterocolitica30,31). In blood pro-ducts, a relatively limited spectrum of bacterialspecies has been isolated, depending on the productitself.1 In PCs, skin-associated gram-positive bac-
Fig 1. Comparison of bacterial marker molecules used as targetsattributed to E coli. The number of variable and therefore informative poet al.39 The information bits are expressed as a logarithm (base 2) of theof (E coli) positions. Abbreviations: groEL, gene that encodes the 60-polymerase β-subunit; rrn, ribosomal RNA operon; tDNA, transfer DNAelongation factor EF-Tu.
teria (mainly Staphylococcus sp, Bacillus cereus,and Propionibacterium acnes) have been implicatedin most contamination cases (up to 71%), but gram-negative organisms (mainly members of the Enter-obacteriaceae) also account for most (82%) of thetransfusion fatalities.1,4,5 A number of broad-rangePCR methods for the identification and detection ofbacteria have been reported, targeting the 16Sribosomal RNA (rRNA),14,29,31-35 23S rRNA,10,18
These targets are present in all bacteria andaccumulate mutations at a slow, constant rate.Hence, these genes were used as molecular clocksin a phylogenetic analysis of bacteria.39 However,these housekeeping genes exhibit different degreesof conservation (Fig 1). Highly variable regions ofgenes contain unique signatures for each bacterium,as well as information about the relationshipsbetween different bacteria. These DNA sequencesare used for phylogenetic analysis, identification,and specific detection of bacteria. Conserved
in broad-range NAT. The molecule size and copy numbers aresitions for the bacteria domain is depicted as described by Ludwignumber of possible character states (4 n; 20 aa) times the numbekd heat shock protein GroEL; rpoB, gene that encodes the RNA; tRNA, transfer RNA; tufA, gene that encodes the translationa
r
l
240 DREIER ET AL
regions of sequence are found in all known bacteria;hence, these regions are used for broad-range PCRto amplify intervening, variable, or diagnosticregions.40 In screening assays, these conservedregions are targets for broad-range primers andprobes that permit the detection of bacteriaindependently of bacterial culture or knowledgeof phylogenetic origin. The 16S ribosomal DNA(rDNA)–based techniques have historically beenmost commonly used; hence, in comparison toother genes, for example, 23S rRNA, a high numberof complete sequences for the 16S rRNA arecurrently available. Although, the overall phyloge-netic information content of the 23S rRNAmolecule is greater than that of the 16S rRNAmolecule (Fig 1),39 the 16S rRNA broad-rangePCR remains the criterion standard. Recently, thedevelopment of broad-range PCR targeting the16S-23S rRNA intergenic transcribed spacer (ITS)or 23S rRNA gene has become popular.10,41 Thelimited DNA sequence data and the lower degree ofconservation of the other bacterial housekeepinggenes such as tuf, groEL, or rpoB have beenneglected, but could be used for other diagnosticquestions, for example, viability assays. Althoughthe degree of conservation of the target gene is themajor criterion in broad-range bacteria PCR, thereare several additional points to consider whendesigning a molecular genetic bacterial screeningassay. For bacterial screening tests, the sensitivityof the assay is crucial. Thus, the copy number of thetarget molecule is a main factor. Looking at theDNA level, multicopy genes such as rRNA genesexhibit up to 12 copies per bacterial cell,42-44
compared with single-copy genes such as groEL ortufA that reveal only one copy per bacterial cell.This ratio has evidently shifted when looking atthe RNA level of these genes. Depending onthe growth rate, microorganisms can synthesizebetween 1000 and 50000 copies of rRNA mole-cules per cell, stabilized in the ribosomes innucleoprotein complexes. The use of RNA mole-cules as a target in NAT achieves enhancedsensitivity. This was demonstrated in bacterialreverse transcriptase PCR (RT-PCR).10,18 The useof ITS regions for molecular genetic detection at theRNA level is disadvantageous because these targetsare lost during processing of the primary transcript.When using RNA as a target in NAT, the copynumber controlled by the gene expression and half-
life of RNA is crucial. Thus, the regulation of geneexpression and stability of the synthesized RNA isof importance. For example, the groEL gene,encoding the heat shock protein Hsp60, is inducedby stress; components of the translation machinerysuch as EF-Tu or rRNA are extensively expressedin growth phases. The elongation factor EF-Tu isone of the most abundant proteins in bacteria,present in about 100000 molecules per cell, whichis as much as 5% of the total cell protein. It isencoded by 2 different genes called tufA and tufB.The amounts of EF-Tu relative to ribosomes varyfrom 3-fold to 14-fold depending on the growthconditions (corresponding to the transcription of tufgenes).45
Molecular Viability Markers
Ribonucleic acid stability is an important factorin gene regulation. In contrast to DNA, messengerRNA (mRNA) is turned over rapidly in viable cells,whereas rRNA has a stability comparable to DNA.Most mRNA species have half-lives measured inseconds to minutes.46 For example, tufAmRNA hasa half-life of 3.0 minutes.47 A wide range ofstabilities was observed for individual mRNAs ofEscherichia coli, although approximately 80%of all mRNAs have half-lives between 3 and8 minutes,48 which is much shorter than thatdescribed for rRNA molecules.10,49 The rRNAmolecules 16S and 23S rRNA do not indicate theviability status of cells killed under in vitroconditions. Hence, detection of mRNA by RT-PCR as opposed to DNA-based methods isconsidered a better indicator of cell viability. Theviability question is of importance when positiveresults are obtained with DNA-based NAT. Hence,the detection of bacterial DNA always indicatessterility problems, especially in blood products.However, PCR-positive results can lead to mis-interpretation. This problem is of major importancewith clinical specimens from patients who undergoantibiotic therapy. When using NAT for bacterialdetection, for example, for diagnosis of sep-sis,33,50,51 endocarditis,50,52-54 or meningitis,55,56
it has to be considered whether the detectedbacterial species are viable. At present, moleculargenetic methods cannot determine bacterial viabi-lity without doubt; and therefore, cultural methodsare still the criterion standard. However, in someinstances, the latter strategy has failings, namely,
241STERILITY TESTING OF BLOOD COMPONENTS
when slow-growing or uncultivable bacteria areinvolved. First approaches give hope for the futurethat molecular genetic viability testing will supple-ment culture methods.10,49 It has been demon-strated that the detection of bacterial groEL mRNAwith RT-PCR is a good marker for viability, incontrast to DNA or rRNA, which can still bedetected days after the death of the bacteria.
QUALITY CONTROL
Nucleic acid amplification technique assayshave to be validated according to the general andlegal requirements referring to accuracy, precision,specificity, sensitivity (detection limit, quantitationlimit), linearity, range, and robustness.57-61 In theInternational Conference on Harmonisation guide-lines for the validation of analytical processes,various aspects of the validation of qualitative testsare indicated, which also apply partly to the NATmethods. These requirements are not all currentlyfulfilled for bacterial NAT. Both qualitative mole-cular genetic bacterial detection (sterility testing)and quantitative determination of bacterial load aretaking place.14,44 The main focus in sterility testingof PCs refers to qualitative NAT assays. Animportant criterion for the evaluation of ananalytical method (including qualitatively) is thedetermination of the lower detection limit, requiringdefined standards. However, standards are not yetavailable for microbiological diagnostics and arethus produced by each laboratory individually.Ascertaining the target values of these standards isextremely problematic because no metrologicallycorrect measuring system for these parametersexists. In addition, reference methods for bacterialquantification are not available. Target values forthe so-called standards are therefore attained usingeither the routine methods biologically characteriz-ing the standard or the same method used to screenthe sample material. The routine methods used inmicrobiology are less reliable (incorrect andimprecise). The current practice is quantificationvia either (1) bacterial titer expressed as CFU or (2)the nucleic acid molecule for detection expressed incopies or genome equivalents. These problems arediscussed in the following section.
Determination of the Lower Detection Limit
A comparison between the detection limits of thedifferent bacterial NAT assays is difficult becausestandardized methods and reference materials are
not yet available. Real-time NAT assays permit thedetermination of the initial template concentrationand, therefore, an accurate estimation of themolecule number or the lower detection limit ofthe assay. For viral approaches, there is a directcorrelation between viral load and copy numberdetermined by real-time NAT.24,28 This is restrictedfor quantitation of bacteria and can be applied onlywhen the result is expressed in copy numbers andnot in CFUs, CFU equivalents, or cell number.
For viral NAT assays, standardization wasachieved by using well-characterized referencematerial, that is, calibrated material using acommon standard unit (international units).62,63
For bacteria, there are several problems andquestions regarding the establishment of a standardpreparation. Bacteria are living organisms and incontrast to viruses are changeable regarding theirmetabolism. Thus, conservation of a representativestate of bacterial growth is difficult or impossible.Nevertheless, first steps were taken to generatebacterial standards that can be used for spikingexperiments of blood components.64 The bacterialstandards that can be purchased from the PaulEhrlich Institute (Langen, Germany) can be char-acterized by their growth kinetics using animpedance registering system and are frozen inhuman albumin during their logarithmic growthperiod. The CFU before and after freezing of thebacteria can thus be described as constant.16,65
To determine the analytical sensitivity testing ofbacterial NAT, CFU standards can thus be used.18
This represents a compromise because bacteriastored frozen have downregulated metabolisms. Inthis lag phase of growth, fewer RNA copies percell can be detected than in growing cultures.Hence, in RNA-based NAT, the analytical sensi-tivity of the method is underestimated because ofthe fact that this standard does not reflect the realcell number as occurring in growing cultures, forexample, bacterial propagation in PCs. However,the introduction of CFU standards will helpcompare different DNA-based methods regardingtheir sensitivity. Besides the molecular geneticmethods, these bacterial standards are very usefulfor spiking experiments, where defined inocula areneeded.16,18,65,66 However, the extrapolation ofresults from an assay in spiked studies using freshcultures of “healthy” inoculates or frozen bacterialstandards to routine analysis can justifiably bequestioned.57 When determining CFU, results
242 DREIER ET AL
depend on various parameters, including cultureconditions, differences in species and strain, overallcell count, living cell count, metabolic state, growthbehavior, etc. Huge amounts of noncultivable deadcells or free nucleic acids that can be amplificated byDNA-based NAT complicate the equalization ofbacterial titer and CFU. Moreover, bacterial quanti-tation by real-time PCR assays is influenced by thevariation in the number of gene copies in a givenbacterial species. The copy numbers in a singlechromosome of the main target in broad-range PCR,the rRNA operon, have varieties between 1(Mycoplasma sp67), 4 (Pseudomonas aeruginosa),7 (E coli42), and 9 (Staphylococcus aureus43). Inaddition, chromosomal replication can furtherincrease the numbers of a given rRNA operon.The number of replication forks is directly related tothe generation time, which in turn depends on themetabolic state of the bacteria at the time ofsampling.44 Ignorance of the exact number ofcopies of rRNA operons in any given species atthe time of sampling represents the main limitationto an absolute determination of bacterial numbers byreal-time PCR based on rDNA.44 One real-timePCR assay developed based upon 16S rDNAamplification describes an analytical sensitivity of1 CFU equivalent per PCR for the gram-negativebacterium E coli (corresponding to 50 CFU/mL) inPCs,14,68 but without any given definition of thegrowing conditions of the cultures. However, gram-positive bacteria occur more frequently in PCs,1,5
which make especially high demands on the nucleicacid extraction procedure. As a consequence ofthese major differences in efficiency of nucleic acidextraction and the variation of copy numberinterspecies and intraspecies, standard curves forthe quantification of bacterial load are barelyapplicable for this purpose. The absolute quantifica-tion of CFU equivalents or cell numbers withstandard curves is thus problematic. For instance, 1CFU of S aureus contains more cells than 1 CFU ofE coli. This refers to the well-known dilemmaregarding NAT vs culture-based methods, where weare actually comparing “apples” with “oranges.”Among other things, the measured CFU leveldepends on bacterial viability; thus, stationarilygrown cultures, such as the standards used in thisstudy, are not comparable with logarithmicallygrown cultures.Most commonly, quantification or determination
of analytical sensitivity is performed with DNA
standards (amplicons, plasmids) for PCR or RNAstandards for RT-PCR. These molecules representparts of the target organism's genome and are usedto determine the bacterial load expressed in genomeequivalents or copy numbers. This control formsthe basis of an external standard curve created fromthe data produced by the individual amplification ofa dilution series of exogenous control. Theconcentration of an unknown sample, which isamplified in the same reaction but in a separatevessel, can then be found from the standard curve.However, this quantitation strategy only images theamplification steps of nucleic acids but ignores thenucleic acid extraction step, the most critical withinthe NAT assay.
As discussed, the overall procedure for moleculargenetic detection of bacteria is thus difficult toimage. It would therefore be better to separatescreening of the NAT assay (referring to themolecule for detection) and sample preparation(nucleic acid extraction). The NAT assay can thenbe characterized by the nucleic acid molecule fordetection, whereas the lower detection limit,precision, and correctness of the method can allbe determined. Sample preparation, that is, nucleicacid extraction, should be evaluated using definedamounts of molecule spiked in the matrix forscreening (here PCs). To measure the efficiency andyield of the nucleic acid released during lysis of thebacteria, defined bacterial suspensions should beused. They should be produced under standardizedculture conditions and be characterized with regardto overall cell count (counted using flow cytometry)and bacterial titer (expressed as CFU). Theperformance of the detection method can then beevaluated more successfully using the correlationbetween the bacterial count and the molecule count.Overall, improved standardization, or at least anestablished validation program, is required forbacterial NAT in the future. A further problemwhen evaluating these approaches is the compar-ability of the methods.
Quality Control and Quality Assurance
Importance has to be attached to the internalquality assurance scheme and, finally, participationin external proficiency trials, also called externalquality assurance programs.57,58 Such trials forqualitative PCR assays are available for mostclinically relevant viral and bacterial pathogens.Such external quality assurance programs for
243STERILITY TESTING OF BLOOD COMPONENTS
bacterial screening NAT assays are not availabletoday, and comparisons of different methods orlaboratory findings are difficult. All PCR assaysshould include an internal amplification control (asdiscussed below), a reagent control, and theprocessing of both negative and positive controls(run control).20,57 Hence, screening of PCs forbacterial contamination with NAT assays should
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Fig 2. Amplification plots of routine platelet screening using real-time RT-PCR. Nucleic acids of culture-negative tested PCs werextracted from 2.4 mL PC using magnetic separation technology and analyzed on the Rotorgene platform.18 For positive control (solid), PCas spiked with S epidermidis (103 CFU/mL). For the 2 low positive run controls (A, B: dashed), PCs were spiked with the PEI standards Spidermidis (A, 50 CFU/mL) or K pneumoniae (B, 50 CFU/mL). Abbreviations: FAM, 6-carboxy-fluorescein; JOE, 2V, 7V-dimethoxy-4V,5V-ichloro-6-carboxyfluorescein; Norm. Fluoro. FAM 23S rRNA, normalized fluorescence of the amplification plots detecting the bacterial3S ribosomal RNA in the FAM channel; Norm. Fluoro. JOE β2 microglobulin, normalized fluorescence of the amplification plots detectinge IC β2 microglobulin messenger RNA in the JOE channel; negative control, H2O (dotted).
ewed2th
include controls that can be used to check the testsuitability to ensure the reliability of the analyticalprocedure whenever used. This run control shouldbe at a concentration near the lower detection limitof the assay to challenge the detection system, yet ata high enough level to provide consistent positiveresults. For viral NAT assays, this is realized byincluding a working reagent, for example, plasma
244 DREIER ET AL
spiked with a hepatitis C virus sample calibratedagainst the WHO Hepatitis C Virus InternationalStandard.62,63 For bacterial NAT assays, thechoice of control material is more difficultbecause of the lack of standardization, asdiscussed below. Recently, PCs spiked withbacterial standards64 were applied for processingpositive controls, whereby the bacterial titer wastwice the 95% detection limit of the assay.18 Totake account of the bacterial differences, controlsfor gram-positive (Staphylococcus epidermidis)and gram-negative bacteria (Klebsiella pneumo-niae) were used, as shown in Figure 2. Inaddition, a sterile PC aliquot should be used asa nonreactive processing control to demonstratethat nonspecific PCR amplification and detectionof amplified product are not occurring. Inaddition, a negative control is optionally used todemonstrate that the reagents are not contami-nated with target nucleic acid and can be used tocompensate for background signal generated bythe reagents. This is of extreme importance forbroad-range bacterial NAT assays, where contam-ination is the omnipresent problem, as discussedin “Limitations of Bacterial NAT Assays byNucleic Acid Contamination.”
Internal Control
In addition, consideration should be given tothe incorporation of an internal control (IC) inNAT assays. An inherent problem in diagnosticPCR is the presence of amplification inhibitorsthat may cause false-negative results. Therefore,the addition of an amplifiable nucleic acid in thePCR assay serves as an IC, an important qualitycontrol, and has already been described for earlyPCR experiments.24 The use of an IC ismandatory for blood screening NAT tests, asdecided by the German federal licensing agencyPaul Ehrlich Institute. An IC for diagnostic NATassays should be easy to produce and tostandardize, without affecting the efficiency ofbacterial target amplification.An exogenous IC is added before nucleic acid
isolation (extraction control) or amplification(amplification control), where coamplification isperformed within the same reaction. Ideally, theseICs hybridize to the same primers, have identicalamplification efficiencies, and contain discriminat-ing features, such as length or sequence variations,targeted by hybridization probes. However, these
competitive ICs can lower the amplificationefficiency, which results in a lower detectionlimit.57 Therefore, noncompetitive IC templatesare used, where the target and IC are amplified withdifferent primer sets. The detection of modelviruses spiked to clinical specimens is a well-established system for exogenous ICs to monitorthe efficiency of extraction and amplification.Especially bacteriophages such as lambda69 orMS270 are often used and provide process controlin many NAT assays. The advantage of such modelviruses is the stability of RNA and the control ofdecapsulation of the viral DNA and RNA duringthe extraction procedure. One disadvantage is thatamplification of the IC may not accurately reflectamplification of the target.
An endogenous IC is a template that occursnaturally within the specimen being analyzed. Ingene expression analysis and virus screenings,housekeeping genes are often used as ICs andreferences for transcript quantification.71 Thesame approach was chosen for NAT to detectbacterial contamination in blood components.14,18
Mohammadi and coworkers assessed the efficacyof DNA extraction by coamplification of thehuman HLA-DQA gene that was coextracted fromPCs. In addition, the potential inhibition of thebacterial amplification system was controlled byspiking PCRs with bacterial control DNA fromBordetella avium.14
Another approach was performed by Störmeret al.18 The coamplification of the human glycer-aldehyde-3-phosphate dehydrogenase mRNAserved as an IC. Because of better performance induplex RT-PCR, another transcript from the humanhousekeeping gene β2 microglobulin was chosen.18
REAL-TIME PCR FOR BACTERIA SCREENING OFBLOOD COMPONENTS
The high sensitivity, specificity, and rapidity ofresults of molecular biologic methods have madethem appealing for the detection of contaminatedblood products. Until today, culture methods,which require a long time to indicate the presenceof bacteria, have remained the criterion standard,although there have been various approaches todetecting bacteria in PCs. Polymerase chainreaction has opened up new detection possibilitiesfor slow-growing pathogens, intracellular bacteria,and viable but nonculturable pathogens.57 Thedetection of bacteria in blood by real-time PCR
Table 1. Molecular Genetic Methods for Bacterial Detection in Blood Components
Abbreviations: ail, a chromosomal gene of Y enterocolitica that encodes a 17-kd outer membrane protein; groEL, gene that encodes the 60-kd heat shock protein GroEL; NS, not specified; virF,virulence gene of Yersinia species.
245
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246 DREIER ET AL
has been described in only a few studies, asshown in Table 1. It is stated that the ideal testshould be simple to perform; rapid; not prohibi-tively expensive; capable of detecting all organ-isms at low levels; and applicable for erythrocytes,platelets, and plasma products. Depending on thesample type, the extraction of nucleic acidsrequires a different treatment. Problems associatedwith the PCR detection of bacteria in clinicalsamples include the extraction difficulty of nucleicacids from the sample and their subsequentpurification to remove PCR inhibiting substances.Samples of whole blood or blood components arenotoriously bad PCR targets because they containinhibitory factors such as hemoglobin. Therefore,displayed studies are often optimized for the useof a specific sample matrix, especially for PCs.The use of a nonamplified chemiluminescence-linked universal bacterial rRNA probe was one ofthe first approaches that was able to detect 2.1 ×
Table 2. Comparison of Two Platelet Bacteria S
23S rRNA RT-PCR
Sampling Sample Apheresis-derived PCSampling time 24 h after preparation
Nucleic acidextraction
Sample volume 2.4 mLKit Chemagic viral DNA/RNA kitInstrument Magnetic Separation Module I (CheTechnology Magnetic bead technologyTime 75 minNucleic acid RNA and DNA
Controls Extraction control Coamplification of β2 microglobulimRNA
PCR inhibitioncontrol
Coamplification of β2microglobulin mRNA
Run control Coamplification of unspiked andspiked sampleswith low bacterial titers (S epiderm
*Volume (V) equivalent of PC set in NAT [(Vtemplate / Veluate) * Vsam
105 CFU/mL PC in 60 to 90 minutes. This NATassay can detect a wide variety of bacteria and hasbeen recently improved by semiautomation.74,75
Subsequently, the amplification of bacterial rRNAsequences has been evaluated by several investi-gators. Feng et al31 investigated the use of PCR inthe detection of Y enterocolitica in whole bloodthat detected 5 × 103 CFU/mL. This assayrequires 6 hours but is capable of detecting only1 bacterial pathogen. Recently, Sen developed a 5′nuclease TaqMan probe PCR assay based on thenucleotide sequence of the 16S rRNA gene from Yenterocolitica and detected as little as 30 CFU/mLin 2 hours.29
Broad-range primers were used by Harris andHartley33 in conventional PCR that detects 1 to 103
CFU/mL. Notably, a number of broad-range PCRmethods targeting the 16S rDNA for identificationand detection of bacteria based on real-time PCRhave been reported.14,29,30,34,35,77
creening Strategies Using Real-Time NAT
18 16S rDNA PCR14
Buffy coat–derived PC24 h after separation200 μLMagNA Pure total nucleic acid isolation kit
magen) MagNA Pure LC Instrument (Roche)Magnetic bead technology25-40 minDNAReal-time PCRUltrafiltration of reagents usingPlasmid Maxiprep binding columns,Sau3AI digestion of the PCR mix, UNG16S rDNA0.02 mL
50 CFU/mL) ABI 7700 (Applied Biosystems)
62 min4 h
n Coamplification ofhuman HLA-DQA DNACoamplification of spiked B aviumDNA (50 CFU/mL)
idis, K pneumoniae)
Coamplification of spiked B aviumDNA (50 CFU/mL)
ple per NAT reaction].
247STERILITY TESTING OF BLOOD COMPONENTS
Nadkarni et al described the use of universalprimers and probes targeting a conserved regionof the 16S rDNA to estimate the total bacterialload in clinical samples.44 Several investigatorsused this primer-probe system and adapted it forsterility testing of blood components.14,35
Mohammadi et al14 developed a broad-rangePCR assay based on real-time PCR technologyto monitor bacterial contamination in PCs. Thisassay enables the detection of 50 CFU equivalentsper milliliter PC. To remove contaminating DNAfrom the reaction mix, different approaches usingenzyme digestion, UV irradiation, and ultrafiltra-tion were discussed.68
A different approach was the development ofRNA targeting real-time RT-PCR assays for plateletbacteria screening.10,18 Two real-time RT-PCRmethods targeting conserved regions of the eubac-terial 23S rRNA gene or the groEL gene (encodingthe 60-kd heat shock protein Hsp60) were devel-oped10 and optimized for use in the routinelaboratory with a detection limit of 22 to 29 CFU/mL PC.18 Only these two investigators have pre-sented their real-time approach for platelet bacteriascreening to date. As shown in Table 2, both appealfor their sampling 24 hours after donation (apher-esis-derived PC66) or preparation (pool PC81) at theearliest. The 16S rDNA targeting assay combines anautomated DNA extraction from a 200-μL sampleand real-time PCR in 4 hours. Decontamination ofthe reagents in the extraction kit is achieved byfiltration through plasmid-binding columns,whereas the PCR mix is decontaminated usingenzyme digestion. Amplification of the coextractedhuman HLA-DQA DNA provides an extractioncontrol, and that of spiked B avium DNA anamplification control.14
In contrast, the 23S rRNA targeting assaycombines an automated high-volume DNA andRNA extraction from a 2.4-mL sample and real-time RT-PCR in 4 hours without decontaminationtreatment.18 Coamplification of β2 microglobulinmRNA serves as extraction as well as an amplifica-tion control. Furthermore, two run controls areincluded in each run, which contain low bacterialtiters to control the sensitivity of the whole process,as shown in Figure 2.
Sampling Strategy for Bacteria Screening
The efficacy of bacterial screening methodsand their usefulness as routine bacterial detection
techniques are influenced by many factors, suchas bacterial growth kinetics, the sample volume,and the time of sampling.82 In contrast to viruses,bacteria are not static and proliferate duringstorage; thus, the choice of the sampling day forthe bacterial screening is crucial. If sampling istoo late, it will limit the availability of compo-nents; but if it is too early, it will trigger therelease of components that would be detected asbacterially contaminated if tested later.83 Theinitial levels of bacteria in the PC units areusually exceedingly low, below the detectionlimits of current screening systems and belowthe level considered clinically significant (105
CFU/mL).84 Therefore, sampling on the day ofcollection invariably misses bacterial contamina-tion. With longer times between sampling andtesting, there is a higher probability that allcontaminated units will be identified.
Bacterial growth is variable and depends on theinitial bacterial load. Three bacterial groups can bedistinguished: bacteria that have no capacity toproliferate, bacteria that survive and are able togrow, and bacteria that remain in the lag phase ofgrowth for up to 6 days.83,85,86 Culture studiesperformed both on packed red cell units andplatelets have shown that culture on the day ofcollection invariably misses bacterially contami-nated PCs.9,82 It has been shown that sampling forcultural detection on day 2 would detect most of thebacteria capable of causing bacterial sepsis despitea very low initial inoculum.87
Studies to determine the optimal time forsampling PCs for real-time PCR screeningshowed that the sampling time is obviously notthat critical when PCs are contaminated with fast-growing organisms, resulting in a sufficientbacterial load in the blood product being tested.The detection of slow-growing organisms remainsmore problematic because the most commonorganisms concerning platelet contamination,such as S epidermidis, exhibited a characteristi-cally extended lag phase before rapid prolifera-tion after 48 hours.66 Therefore, it was concludedthat sampling should not be carried out earlierthan 24 hours after preparation (apheresis-derivedPC) or 48 hours after blood donation (pooledPC) because this preenrichment should enablemost of the contaminated PCs to be detected, asshown in Figure 3. Aseptic sampling could beachieved by the connection of a sterile transfer
Fig 3. Determination of the optimal sampling time for bacterial screening using real-time RT-PCR on a slow-growing organism. Onesingle apheresis-derived PC unit was spiked with approximately 1 CFU/mL of S epidermidis and stored at 22°C. Samples were taken induplicate before inoculation (negative control at 0 hour) and at different times (4, 14, 17, 24, 38, 44, 60, 72, and 96 hours afterinoculation) and enumerated by plating culture (line including points indicates the bacterial growth representing the bacterial load at thetime of sampling). Nucleic acids were extracted using magnetic separation technology and analyzed on the Rotorgene18 (threshold cycle[CT] values of the real-time RT-PCR detection are displayed in bars). Staphylococcus epidermidis showed an initially long lag phase andrapid growth after 44 hours of storage. Samples taken before and at 4, 14, and 17 hours after inoculation were tested negative (CT> 45) inreal-time RT-PCR. Bacteria were detected for the first time when sampling was performed 24 hours after inoculation. Here theamplification of a PCR product is first detected. This is determined by identifying the cycle number at which the reporter dye emissionintensity rises above a background noise. That cycle number is referred to as the threshold cycle.
248 DREIER ET AL
pack container to the PC unit using a steriletubing welder.66,81
Another critical factor is the sample volume thatcould have a significant impact on the amount ofproduct left for transfusion, particularly for randomdonor units. Therefore, a larger sample volumeimproves the sensitivity but depletes the product.The required sample volume should be such that thegiven sample will contain at least 1 organism.82 Asshown in the nucleic acid extraction section,commercially available extraction methods areable to process only a sample volume of up to1 mL. The risk of sampling errors is thereforeenormously high and cannot comply with require-ments mentioned above when sampling is processedtoo early. For the first time, the use of a high-volumeextraction method was adapted for use in plateletbacteria screening to minimize sampling errors andto improve the sensitivity of the method used.18
A different approach is incubation of the sampleat a higher temperature6,16 that could shorten thedetection times for slow-growing organisms. Withregard to sampling errors, taking the sample at an
early stage for incubation at a different temperaturemeans that contaminated units could be missed.Moreover, further incubation in a synthetic growthmedium at 35°C after sampling and after 24-hourstorage of the PC is another sampling strategyapproach.88,89 Nevertheless, 24-hour storage of thePC unit seems to be indispensable. This screeningprocedure is a justifiable compromise between thesensitivity of NAT assays, the diagnostic windowperiod, and the delay of the PC supply.
Nucleic Acid Extraction From Blood Products
The prerequisite benefit from the advantages ofPCR is efficient protocols for the extraction ofnucleic acids. The overall sensitivity of the assay isdetermined by the nucleic acid yield, its purity, andthe amount of sample equivalents that can betransferred to the amplification reaction. Untiltoday, an array of commercially available nucleicacid isolation systems has been developed, offeringstandardized, quality-controlled reagents with opti-mized compositions for all steps of the process.However, some fail to detect a minor amount of
249STERILITY TESTING OF BLOOD COMPONENTS
bacterial DNA among an abundance of humanDNA. Therefore, an optimal sample preparationprocedure should efficiently break very resistantbacteria cell walls, without being too harsh for thenucleic acids released from the cells that are easilylysed, and should remove substances that mayinhibit amplification, such as heme.20,90 Moreover,the released nucleic acids should be maintained intoa small volume of aqueous solution to protect themfrom degradation. With regard to the sensitivity ofthe assay, a maximum amount of the sample has tobe set in the nucleic acid extraction procedure.Extraction of clinical specimens can be accom-plished either by manual or by automated methods.Conventional manual sample preparation methodsare labor-intensive and susceptible to contamina-tion, handling variations, or errors.91 Automationaspires to avoid human errors, to improve exactness,to reproduce results, and to permit analysis ofsamples in large numbers. For PCs, commerciallyavailable extraction methods have been successfullyused to detect bacterial contaminations.10,14,18,74,75
Manual Nucleic Acid Extraction
Classic phenol extraction was successfully usedto prepare DNA for amplification in the earlydiagnostic applications of PCR. However, the useof corrosive and toxic agents represents a safetyhazard in the clinical laboratory. Several manualextraction kits using noncorrosive agents have beendeveloped.90 Researchers are therefore able tochoose the technique most suited to their target,source, or starting material. Classic methods ofDNA and RNA isolation are based on column orprecipitation methods. These techniques requirecentrifugation or vacuum steps and often have longprocessing times, as well as volume limitations.Moreover, the use of ethanol to precipitate thenucleic acids can inhibit the PCR when it is notproperly removed.20 Manual extraction of bacterialnucleic acids from PCs was performed with glass-fiber fleece immobilized in a special plastic filtertube and subjected to centrifugation.10 For efficientlysis of bacteria, mechanical and enzymatic degra-dation of cell walls was carried out. This approachleaves room for the option of isolating total nucleicacid or, after DNase treatment, pure RNA.
Automated Nucleic Acid Extraction
Magnetic separation techniques have severaladvantages over standard separation procedures.
This process is usually very simple, with only a fewhandling steps. Automated systems are typicallyclosed systems to minimize the contamination riskand walkaway systems that do not require constantattention.20 Furthermore, the recovery of nucleicacids from automated instruments is consistent andreproducible.20 However, besides these benefits,potential drawbacks such as the cost of equipmentand space in the laboratory also have to beconsidered. Most recent developments use magneticbeads that bind the nucleic acids to their silicasurface and transfer them through the various stepsof the extraction process.92 This technology wassuccessfully evaluated for the extraction of viralnucleic acids for routine blood donation screen-ing92,93 and adapted for the extraction for bacterialnucleic acids from PCs for sterility testing byStörmer and colleagues.18 Using the Chemagic viralDNA/RNA kit in conjunction with the MagneticSeparation Module I (Chemagen, Baesweiler,Germany), high-volume simultaneous extractionof total DNA and RNA is performed. This methoduses magnetic beads, consisting of an iron oxidecore surrounded by a nucleic acid–binding matrix,for direct capture of total nucleic acids. After celllysis, nucleic acids bind to these magnetic beads andcan be separated using the Chemagic MagneticSeparation Module I. The advantages of extractionwith this technology are the absence of a precipita-tion step, which often causes problems with yieldand purity; the simultaneous extraction of bothspecies of nucleic acids; and the possibility of usinga high sample volume of 2.4 mL with regard tominimizing the sampling error.94 Nucleic acidisolation of up to 12 high-volume samples in onebatch on the Chemagic Separation Module I isaccomplished in approximately 75 minutes.92 Adifferent method is the coverage of magnetic beadswith nucleic acid–binding matrices.14 The MagNAPure system (Roche Diagnostics, Mannheim, Ger-many) consists of a fully automated bench-topinstrument and ready-to-use nucleic acid isolationkits with prefilled cartridges, permitting automatedisolation of nucleic acids from 1 to 8 samples within25 to 40 minutes.
Limitations of Bacterial NAT Assays by NucleicAcid Contamination
Several factors have limited the direct amplificationof bacterial nucleotide amplification in bloodproducts. The major practical problems associated
250 DREIER ET AL
with the use of broad-range rDNA PCR are thecontamination of the assay by exogenous bacterialDNA of the nucleotide amplification reagents,particularly of the bacterially derived enzymes,and the presence of bacterial DNA sequencescommonly found in human blood.95 This compli-cation has led to a number of diagnostic laboratoriesabandoning the idea of adopting broad-range rDNAPCR techniques as part of their diagnostic service.To exclude carryover contamination from pre-viously amplified DNA, the enzyme uracil-N-glycosylase (UNG), which cleaves the uracil basefrom the phosphodiester backbone of uracil-con-taining DNA, could be used. The enzyme has noeffect on thymine-containing DNA.96 Because ofthe use of recombinant enzymes that wereexpressed and purified from E coli, additionalcontamination of the reagents by any kind ofeubacterial DNA and RNA cannot be excluded byusing UNG; and this limitation still represents aserious problem.Taq DNA polymerase has often been found to
be contaminated with bacterial DNA97,98 or rRNAbecause of its high affinity for DNA, which iscopurified during enzyme production. Severalinvestigators have reported this problem inbroad-range bacterial PCR and have attempted toovercome it using a number of different proce-dures.33 Recently, Corless et al99 have describednumerous problems associated with use of theTaqMan system and 16S rDNA PCR. Attempts todecontaminate PCR materials have involved meth-ods to destroy DNA, including UV irradiation,8-methoxypsoralen treatment, DNase treatment,restriction enzymes, and combinations of thesemethods.10,33,68,99,100 Meier et al101 investigated amethod to eliminate contamination of DNA withpsoralen. Psoralens are known to intercalate intodouble-stranded nucleic acids and form a covalentinterstrand cross-link after photoactivation withUV light. Therefore, use of 8-methoxypsoralen toextinguish the template activity of contaminatingDNA has been suggested.102 Some investigatorssolved the problem of contaminated reagents bypassing PCR reagents through centrifugal filterdevices because these filters permit decontamina-tion of all PCR reagents, including UNG, Taqpolymerase, primers, and probes, which was notpossible using other methods such as DNasetreatment.68 Furthermore, an additional approach
to remove contaminating DNA from the isolationkit reagents was filtration using a nucleic acidbinding column.14 However, it was found thatmost decontamination methods decreased PCRsensitivity10,99; and thus, none of these methodshas been shown to be entirely effective orreproducible. As a result, some conclude thatreducing the number of PCR cycles is the mosteffective way of avoiding the amplification ofcontaminant DNA that gives false-positive results;but this would lower the sensitivity of the assay aswell.33,44 Nucleic acid free enzymes are needed,but they are still not available. The expression ofDNA polymerases in eukaryotic systems wouldsolve the purification problem, but patent claims orthe in vitro diagnostic medical devices directiverepresents insurmountable obstacles.
CONCLUSIONS AND FUTURE PERSPECTIVES
Real-time NAT is a powerful tool in clinicaldiagnostics and will possibly acquire enhancedsignificance for sterility testing in transfusionmedicine. Initial skepticism about moleculargenetic tests has been observed, as occurred whenNAT assays were introduced for the virus screeningof blood products. The application of broad-rangebacterial NAT is demonstrated in several stu-dies.10,14,18 These methods are highly specific,very sensitive, and rapid. The relatively shortturnaround time of NAT assays provides thepotential for them to be used before the release ofPCs for transfusion.
Nucleic acid amplification technique screeningdoes not prevent all transfusion-associated bacterialseptic reactions, as is the case for other diagnosticmethods. Not every bacterial contamination playsan important role because not all species or evenisolates of certain species are able to grow withinhuman plasma or PCs. For instance, Propionibac-terium species were detected more frequently inPCs with culture-based methods than with NAT.14
Inoculating blood culture systems and anaerobiccultivation detects these bacteria after approxi-mately 3 to 7 days under optimized growthconditions; here the relevance for transfusionmedicine has to be discussed. Anaerobic P acnesshow only slow or no growth under PC storageconditions. In addition to slow growth, the levels ofbacteria in blood components are thought to be toolow to result in sepsis upon immediate transfusion.Hence, their clinical significance may be low.
251STERILITY TESTING OF BLOOD COMPONENTS
Hitherto, systematic studies of the outcome ofpatients transfused with P acnes–contaminated PCsto demonstrate the transfusion relevance are miss-ing. Propionibacterium acnes is not usuallyvirulent, and only a few cases have been describedin transfusion-related sepsis.103,104 In all cases, Pacnes was not isolated from the patients; and acause-and-effect relation was not confirmed.Depending on the species level, differences ofgrowth in PCs are observed. Therefore, detection ofbacteria in PCs with culture-based methods mustmean that these bacteria neither will propagate inthis milieu nor cause transfusion-related sepsis.
Because of the low bacteria titers of about 10 to100 bacteria per donation initially, the sensitivity ofthe detection method plays a crucial role. Micro-biological detection requires a long incubation time,whereas NAT assays should be sensitive and fastenough for the routine contamination screening ofPCs. To overcome sampling errors, a minimumpreincubation of PCs has been proposed.18,81 Thispreenrichment should enable most of the contami-nated PCs to be detected using current NATmethods; but it is to be expected that rare, slow-growing bacteria might escape such a detectionscheme. An essential requirement is the standardi-
zation of NAT assays for the broad-range detectionof bacteria. The dilemma is the lack of compar-ability to NAT methods and to other screeningmethods used for sterility testing.
In the future, molecular genetic sterility testingmay also be requested for other blood components.Besides cellular therapeutic agents, for example,human stem cells, dendritic cells, or T cells,molecular genetic tests are of interest becausemethods prescribed in international pharmacopedia(European Pharmacopedia, Japanese Pharmacope-dia, United States Pharmacopedia) are not applicablein all cases. Because of methodological limitations ortime-consuming diagnostics, rapidmethodswill haveto be applied, particularly if the blood product has arestricted durability or is prematurely needed forpatient therapy. For such tissue cultures, the sterilityassay has to also detect Mycoplasma species andfungal pathogens. Therefore, NAT assays developedfor PC screening will have to be adapted andvalidated for this latter purpose.
In conclusion, the high potential of bacterial NATscreening of blood components has been demon-strated; but further studies are needed to prove itsapplicability for the routine contamination screen-ing by transfusion services.
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