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Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood Andrea Bacconi, a Gregory S. Richmond, a Michelle A. Baroldi, a Thomas G. Laffler, a Lawrence B. Blyn, a Heather E. Carolan, a Mark R. Frinder, a Donna M. Toleno, a David Metzgar, a Jose R. Gutierrez, a Christian Massire, a Megan Rounds, a Natalie J. Kennel, a Richard E. Rothman, b Stephen Peterson, b Karen C. Carroll, c Teresa Wakefield, c David J. Ecker, a Rangarajan Sampath a Ibis Biosciences, Inc., Carlsbad, California, USA a ; Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA b ; The Johns Hopkins Hospital Clinical Microbiology Laboratory, Baltimore, Maryland, USA c The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and ac- curate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole- blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitiv- ity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Repli- cate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organ- isms responsible for bloodstream infections. A lthough molecular tests have been available for decades, there is no widely used molecular method to rapidly identify the organ- isms causing bloodstream infections and bacteremia directly from the blood. In the most serious cases. those of sepsis and septic shock the risk of mortality increases by the hour if appropriate antimicro- bial therapy is delayed (1). The long delays associated with culture methods for the detection and identification of organisms in blood force physicians to empirically treat patients with multiple broad- spectrum antimicrobial agents rather than to wait for more specific microbiological data. This is not ideal because of the toxicities of broad-spectrum agents, the fact that the empirical antimicrobial therapy might not be optimal for the infection being treated, the high costs associated with increased hospitalization time, and the impact on antimicrobial stewardship (1–5). Direct molecular identification of infecting microbes in blood would mitigate these issues. PCR and mass spectrometry methods used on positive blood cultures to identify microbes have been reported to decrease the time to an answer, but these strategies are still far from ideal, as they depend on the cultures to grow. More importantly, blood cultures are negative in 50% of the cases for which true bacterial or candidal infections are believed to exist (3, 6, 7). Some of these apparent false negatives result from a lack of bacteria in any one sample; however, the bacteria present in such samples are often rendered nonviable (and hence unculturable) with concurrent antibiotic treatment (8). Molecular methods have an inherent advantage in detecting such cryptic infections, as they do not rely on viability. A rapid and sensitive molecular method that detects a broad range of microbes directly in blood specimens would have a sig- nificant impact on the management of patients with suspected infections. However, the molecular detection and identification of microbes that may be present in low quantities in a large volume of blood are challenging. First, a relatively large volume of blood must be analyzed to provide a reasonable sampling of the blood compartment, where the distribution of the microbe may not be homogeneous (8–10). Second, diverse microbes must be effi- ciently lysed in a dense complex matrix of cellular material. Third, high quantities of human genomic DNA from white blood cells must be either copurified with microbial DNA or separated with- out losing the microbial DNA. Finally, amplification of target DNA and signal capture (by sequencing, probe capture, mass spectrometry, or another method) must be robust. This can be very challenging if genomic DNA is copurified with targeted mi- crobial DNA (11–13). A number of solutions to this problem have been proposed (14–19) but, to date, none have achieved the de- sired sensitivity (20). It should be noted that many of these meth- ods do appear to capture a significantly higher number of total positives than culture, and the “extra” detections are often corre- lated with clinical indications of bloodstream infection; however, Received 26 March 2014 Returned for modification 23 April 2014 Accepted 6 June 2014 Published ahead of print 20 June 2014 Editor: P. H. Gilligan Address correspondence to Rangarajan Sampath, [email protected]. A.B. and G.S.R. contributed equally to this work. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.00801-14 3164 jcm.asm.org Journal of Clinical Microbiology p. 3164 –3174 September 2014 Volume 52 Number 9 on December 17, 2020 by guest http://jcm.asm.org/ Downloaded from

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Page 1: Improved Sensitivity for Molecular Detection of Bacterial ... · Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood Andrea Bacconi, a Gregory

Improved Sensitivity for Molecular Detection of Bacterial andCandida Infections in Blood

Andrea Bacconi,a Gregory S. Richmond,a Michelle A. Baroldi,a Thomas G. Laffler,a Lawrence B. Blyn,a Heather E. Carolan,a

Mark R. Frinder,a Donna M. Toleno,a David Metzgar,a Jose R. Gutierrez,a Christian Massire,a Megan Rounds,a Natalie J. Kennel,a

Richard E. Rothman,b Stephen Peterson,b Karen C. Carroll,c Teresa Wakefield,c David J. Ecker,a Rangarajan Sampatha

Ibis Biosciences, Inc., Carlsbad, California, USAa; Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USAb; TheJohns Hopkins Hospital Clinical Microbiology Laboratory, Baltimore, Maryland, USAc

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids indiagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capableof detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background ofgenomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and ac-curate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitiv-ity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 mlof whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysisof 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens(10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Repli-cate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initialfindings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were consideredtrue positives. The integrated solution described here has the potential to provide rapid detection and identification of organ-isms responsible for bloodstream infections.

Although molecular tests have been available for decades, there isno widely used molecular method to rapidly identify the organ-

isms causing bloodstream infections and bacteremia directly fromthe blood. In the most serious cases. those of sepsis and septic shockthe risk of mortality increases by the hour if appropriate antimicro-bial therapy is delayed (1). The long delays associated with culturemethods for the detection and identification of organisms in bloodforce physicians to empirically treat patients with multiple broad-spectrum antimicrobial agents rather than to wait for more specificmicrobiological data. This is not ideal because of the toxicities ofbroad-spectrum agents, the fact that the empirical antimicrobialtherapy might not be optimal for the infection being treated, the highcosts associated with increased hospitalization time, and the impacton antimicrobial stewardship (1–5).

Direct molecular identification of infecting microbes in bloodwould mitigate these issues. PCR and mass spectrometry methodsused on positive blood cultures to identify microbes have beenreported to decrease the time to an answer, but these strategies arestill far from ideal, as they depend on the cultures to grow. Moreimportantly, blood cultures are negative in �50% of the cases forwhich true bacterial or candidal infections are believed to exist (3,6, 7). Some of these apparent false negatives result from a lack ofbacteria in any one sample; however, the bacteria present in suchsamples are often rendered nonviable (and hence unculturable)with concurrent antibiotic treatment (8). Molecular methodshave an inherent advantage in detecting such cryptic infections, asthey do not rely on viability.

A rapid and sensitive molecular method that detects a broadrange of microbes directly in blood specimens would have a sig-nificant impact on the management of patients with suspectedinfections. However, the molecular detection and identification of

microbes that may be present in low quantities in a large volume ofblood are challenging. First, a relatively large volume of bloodmust be analyzed to provide a reasonable sampling of the bloodcompartment, where the distribution of the microbe may not behomogeneous (8–10). Second, diverse microbes must be effi-ciently lysed in a dense complex matrix of cellular material. Third,high quantities of human genomic DNA from white blood cellsmust be either copurified with microbial DNA or separated with-out losing the microbial DNA. Finally, amplification of targetDNA and signal capture (by sequencing, probe capture, massspectrometry, or another method) must be robust. This can bevery challenging if genomic DNA is copurified with targeted mi-crobial DNA (11–13). A number of solutions to this problem havebeen proposed (14–19) but, to date, none have achieved the de-sired sensitivity (20). It should be noted that many of these meth-ods do appear to capture a significantly higher number of totalpositives than culture, and the “extra” detections are often corre-lated with clinical indications of bloodstream infection; however,

Received 26 March 2014 Returned for modification 23 April 2014Accepted 6 June 2014

Published ahead of print 20 June 2014

Editor: P. H. Gilligan

Address correspondence to Rangarajan Sampath,[email protected].

A.B. and G.S.R. contributed equally to this work.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.00801-14

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an inability to capture all positives detected by culture (false neg-atives) has been an issue.

We previously described PCR followed by electrospray ioniza-tion mass spectrometry (ESI-MS) technology using a broad bac-terial and candida detection research assay, which is able to detectand identify �800 clinically relevant bacteria and Candida spp.associated with human infections, including unculturable organ-isms, directly from blood specimens (21–27). The assay can alsodetect three classes of antibiotic resistance markers associated withresistance to methicillin (mecA), vancomycin (vanA and vanB),and carbapenems (blaKPC). Two previous PCR/ESI-MS studiesusing direct blood specimens reported on the speed, breadth ofbacterial coverage, and accuracy of bacterial and Candida identi-fication (28, 29) of this research assay. In these studies, PCR/ESI-MS consistently identified more positive specimens than cul-ture due to the well-known phenomenon that only about half ofthe blood specimens are positive in cases for which true infectionsare believed to exist (3, 6, 7). When using blood culture as a directcomparator method, however, PCR/ESI-MS of 1-ml direct bloodspecimens identified only about 50% of the culture-positive spec-imens, leaving a significant number of culture-confirmed infec-tions undetected. Thus, our goal was to improve the sensitivity ofdirect detection from human blood specimens compared to thatof culture-positive specimens without introducing additionalsteps that would compromise laboratory workflow.

Under the assumption that the sensitivity of molecular meth-ods is limited in part by small sample volumes paired with lowtiters and/or uneven distributions of pathogens in the blood-stream, we developed a nucleic acid extraction system capable ofprocessing 5 ml of blood. Lysis of all the cellular components ofthe blood, including human, bacterial, and candidal cells, wasachieved by high-impact percussive beating with zirconium-yt-trium beads, followed by automated extraction and purification ofthe total nucleic acids. The extracts were tested using a small num-ber of conserved-site PCRs targeting all bacterial and candidalspecies, with amplicon detection and organism identificationachieved through ESI-MS. Interference by human DNA was lim-ited by two strategies. First, PCR primers and formulations wereoptimized to be robust to large amounts of human DNA. Second,we developed a post-PCR analytical method that selectively en-riches for PCR amplicons and debulks the specimen of humanDNA to enable detection of the amplicon by ESI-MS.

Here, we evaluated the performance of the new extraction sys-

tem with high-blood-volume specimen preparation technologycoupled to a PCR/ESI-MS detection platform (available for re-search applications only; Ibis Biosciences, Abbott, Carlsbad, CA)through analytical limit of detection and breadth-of-coveragestudies using culture-quantified microbes spiked in whole blood.Whole-blood specimens from patients suspected to have blood-stream infections were also evaluated using the new system. ThePCR/ESI-MS results were compared to the culture results to eval-uate sensitivity and specificity, and a subset of PCR/ESI-MS-pos-itive/culture-negative samples were subjected to both direct se-quencing and repeat PCR/ESI-MS analysis of duplicate bloodspecimens in an effort to confirm the presence of the initiallyidentified microorganism. The system is able to rapidly detect abroad range of microorganisms with a workflow that is suitablefor hospital laboratories. The integrated processes that collectivelyimprove sensitivity and workflow are summarized in Fig. 1.

MATERIALS AND METHODSExtraction and analysis of DNA from 5 ml of whole blood. In this re-search application, genomic DNA was isolated from 5 ml of EDTA-treated whole-blood clinical specimens or 5 ml healthy volunteer bloodsamples spiked with cultured microbes. Two tubes of blood, one fromeach arm, were drawn (usually within 30 min of each other) as part of thestandard of care per hospital protocol. One additional sample was drawnfrom one arm (using the same venipuncture used for one of the culturesamples) for PCR/ESI-MS testing, yielding between 5 and 15 ml of blood.This single blood sample was distributed into multiple aliquots of 5 mland stored. In cases for which �10 ml was collected, the first 5 ml was usedfor the primary PCR/ESI-MS analysis and the second 5 ml was used forrepeat testing as needed. Each patient was sampled only once during anyhospital visit or admission for PCR/ESI-MS testing. The specimens werekept at 4°C refrigeration within 30 min of collection, shipped at 4°C, andkept frozen until analysis.

The blood samples were lysed in the presence of 665 �l lysis buffer(Abbott Molecular, Des Plaines, IL) 100 mM Tris solution containingguanidinium thiocyanate and detergent, 145 �l of 10% bovine serumalbumin (BSA) containing a pumpkin DNA extraction control (24), and 3g of 0.2-mm yttria-stabilized zirconium oxide beads using a large-volumebead mill homogenizer (under development at Ibis Biosciences, Abbott,Carlsbad, CA, and Omni International, Kennesaw, GA) (speed, 6.6 m/s;three 90-s cycles with 20-s dwell time). The sample tubes were centrifugedat 3,220 � g for 5 min. The supernatant fractions were processed by anautomated DNA extraction and PCR setup instrument (under develop-ment at Ibis Biosciences, and Precision System Science, Co. Ltd., Mat-sudo, Japan). The extraction system uses prefilled disposable cartridges

FIG 1 Workflow and timing of the steps in sample preparation and PCR/ESI-MS analysis of a single whole-blood specimen, with results reported in �6 h. Abatch of up to 6 samples can be tested simultaneously and the results reported in 8 h.

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containing DNA-free reagents and silica-coated magnetic particles (Ab-bott Molecular).

The eluates were transferred into 16 wells (30 �l per well) of a customPCR assay strip prefilled (25 �l per well) with 18 unique primer pairs andconcentrated PCR master mix. The primers of the bacteria and Candidaassay for bloodstream infections (BAC BSI assay; available for researchapplications only) were designed to hybridize to conserved genomic se-quences and amplify species-specific genetic signatures from a broadspectrum of bacteria and Candida spp.; target-specific primers yield sig-natures indicative of antibiotic resistance elements. The gene targets,primer sequences, and configuration were previously described in detail(28). The general PCR formulations and thermocycling conditions havealso been described elsewhere (25). Due to the high loads of white bloodcells in 5-ml whole-blood specimens, the primer and polymerase concentra-tions (detailed in Results) were optimized to enable the BAC BSI assay towithstand potentially extensive interference from high levels of human DNA(up to �12 �g per reaction). The PCR/ESI-MS data analysis and results in-clude a report of the organism names, level, and Q score. The Q score is theoutput of a principal component analysis and represents a relative measure ofthe strength of the data supporting identification. It is a single figure of meritthat describes the overall quality of the result derived from primer-dependentparameters, such as the number of primer pairs producing amplificationproducts compared to the maximum number expected for the identifiedorganism, the closeness of the match of those products to reference signaturesin the database for that organism, and the consistency of signal amplitudesacross multiple primer pairs. For the assay described here, organisms arereported above a threshold score of �0.85. The level is a reflection of signalabundance relative to a set of competitive PCR standards of known inputquantity and thus serves as an indirect estimate of how much specific templatewas amplified. This is calculated with reference to an internal calibrant con-struct (the amplification control), as described previously (30), and providesa relative measure of the genome (or copy number) concentration of anydetected target.

Sequencing and data analysis. For identification by sequencing, por-tions of the 16S rRNA gene (for bacteria) or the 28S rRNA gene (forCandida spp.) were amplified with the primers specified in the CLSIguidelines for the identification of bacteria and fungi (31), including theprimer pairs MM18-A (4F-TTGGAGAGTTTGATCCTGGCTC) and108R (GGCGTGGACTACCAGGGTATCT), 28SF (GGACTACCCGCTGAACTTAAGCATATCAATA) and 28SR (GGTTTTACACCCAAACACTCGCATAGAC), and M13F (CCCAGTCACGACGTTGTAAAACG) andM13R (AGCGGATAACAATTTCACACAGG), using Platinum Taq highfidelity (Invitrogen). Platinum Taq buffer was used with 200 �M eachdeoxynucleoside triphosphate (dNTP), 2 mM MgSO4, and 250 nM eachprimer. The reactions were cycled with the following conditions: 95°C for2 min, 8 cycles of 95°C for 15 s, 52°C for 45 s (increasing 0.6°C per cycle),and 68°C for 90 s, 27 cycles of 95°C for 15 s, 64°C for 15 s, and 68°C for 60s, followed by 4 min at 68°C. SeqWright, Inc., (Houston, TX) performedall sequencing.

The sequences were analyzed with Phred and Phrap and aligned withBioLign (http://en.bio-soft.net/dna/BioLign.html). The primers weretrimmed from the alignments and the sequences searched against Gen-Bank using NCBI BLAST. Using the CLSI MM18-A guidelines (31), thebest-matched organisms were reported at the genus and species levels with�99% identity. In some cases for which there were no perfect matches,�95% identity was used to report the most closely related genus, per theMM18-A guidelines.

Clinical specimens. Blood samples were collected from prospectivelyconsenting adults from January to April 2012 at The Johns Hopkins Hos-pital. The samples were obtained from subjects whose physicians orderedblood cultures due to clinical suspicion of a bloodstream infection. Thesubjects were considered eligible if they were �18 years old, were havingblood cultures drawn as part of clinical care, and were able to provideinformed consent. Two tubes of blood, one from each arm, were drawn(usually within 30 min of each other) as part of the standard of care per

hospital protocol. One additional sample was drawn from one arm (usingthe same venipuncture used for one of the culture samples) for PCR/ESI-MS testing, yielding between 5 and 15 ml of blood. This single bloodsample was distributed into multiple aliquots of 5 ml and stored. In casesfor which �10 ml was collected, the first 5 ml was used for the primaryPCR/ESI-MS analysis, and the second 5 ml was used for repeat testing asneeded. Each patient was sampled only once during any hospital visit oradmission for PCR/ESI-MS testing.

Microbiological methods. The subjects suspected of sepsis had twosets of blood cultures obtained after appropriate skin decontamination. Aset consisted of one BD Bactec Plus aerobic/F bottle and one BD Bacteclytic anaerobic/F bottle (BD Diagnostics). All bottles were sent promptlyto the laboratory within 1 h and incubated on the Bactec FX (BD Diag-nostics) continuously monitored blood culture system. The bottles wereincubated and monitored for 5 days before being called negative. Positiveblood cultures were removed immediately from the instrument, and aGram stain was performed. The peptide nucleic acid fluorescence in situhybridization (PNA-FISH) Enterococcus faecalis-other Enterococcus dualprobe and the PNA-FISH Candida albicans-Candida glabrata dual probewere used to rapidly identify Gram-positive cocci and yeast, respectively.All other pathogens were subcultured to the appropriate medium de-pending upon the Gram stain results and the type of bottle from whichthey were recovered. The organisms were subsequently identified by avariety of phenotypic methods, including the Phoenix automated micro-biology system (BD Diagnostics), classical biochemical analyses, cell wallfatty acid analysis using gas liquid chromatography (Sherlock microbialidentification system), and, rarely, 16S rRNA gene sequencing.

RESULTSOptimization of PCR and desalting steps for sensitivity in thepresence of high levels of nontarget DNA. Due to very high num-bers of white blood cells in the 5-ml blood specimens, the BAC assaywas optimized in the presence of high levels of human DNA (up to�12 �g per PCR). The optimal PCR conditions were determinedusing a systematic matrix analysis that varied primer, Mg��, andpolymerase concentrations, annealing time, and temperature. Theoptimum concentrations for each component were chosen as thosethat gave the maximum amplicon yields as determined by capillaryelectrophoresis. The results showed that increasing primer and poly-merase concentrations simultaneously to 750 �M and 2.2 units perreaction, respectively, resulted in a PCR yield in a 12-�g DNA back-ground of 86% (range, 56% to 120%) of the yield when 1 �g ofhuman DNA was present (data not shown). Changing the other pa-rameters resulted in negligible improvements to the previously re-ported PCR formulations and thermocycling conditions (25).

Prior to mass spectrometry, the amplicons were desalted byanion-exchange chromatography using an automated platform(30). In addition to salts, which interfere with mass spectrometry,this procedure removes �98% of human DNA background insamples containing 12 �g human DNA per PCR (data notshown). We discovered that during primary amine anion ex-change on irregularly shaped porous microparticle clusters, shortPCR amplicons bound to submicron-sized pores on the surfaceand within the porous cluster. The dual anion-exchange and sizeexclusion properties of the particles prevented the larger-sizedhuman genomic DNA from binding, effectively enriching thesample for PCR amplicons and improving detection by ESI-MS.

Levels of human DNA in 5 ml of blood. In order to define thelimit of human DNA background tolerated, healthy volunteer bloodsamples were spiked at the limit of detection (LOD) with multiplemicroorganisms and analyzed in the presence of a range of humanDNA concentrations. The total amount of DNA extracted from 5 ml

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of blood has been capped to deliver no more than 12 �g per PCR andpost-PCR amplicon enrichment reaction. Human DNA in whole-blood samples comes primarily from white blood cells. There is ap-proximately 72 �g of total human DNA in approximately 12 � 106

cells. The white blood cell counts from the population intended foruse showed that 90% of patients fell into a range between 0 and 16 �106 cells/ml (Fig. 2). Most patients had between 6 � 106 and 12 � 106

white blood cells per ml; 15.1, 14.3, and 14.3% of the subjects had 6 �106, 8 � 106, and 12 � 106 white blood cells/ml, respectively; 90% ofthe patients had �16 � 106 white blood cells/ml. To understand thecapability of the assay to withstand higher levels of human DNA, 5-mlwhole-blood samples were spiked at the assay LOD of 16 CFU/ml ofEnterococcus faecium (a vancomycin-resistant Enterococcus [VRE]species) and 4 CFU/ml of C. albicans with 5 � 106, 7 � 106, 8 �106, 10 � 106, 12 � 106, 23 � 106, and 40 � 106 white bloodcells/ml. PCR/ESI-MS correctly identified the spiked organisms atall white blood cell levels (data not shown). These results suggestthat microbial DNA was detected and correctly identified in pa-tients with the highest white blood cell counts observed in thisstudy.

Limits of detection for relevant organisms. To determine theanalytical sensitivity of the PCR/ESI-MS system with the 5-mlblood preparation system, we determined the limits of detectionfor Klebsiella pneumoniae (blaKPC

�), E. faecium (vanA� vanB�),Staphylococcus aureus (mecA�), and C. albicans. Each of theprimer pairs in the assay targets one or more of these organisms.Aliquots of uninfected blood were spiked with a dilution series ofmicrobes in 2-fold steps. The LOD was taken as the last dilutionstep at which at least 19 of 20 replicates (95%) were positive. TheLODs for each organism (inclusive of their resistance markers)were 16 CFU/ml for S. aureus, 16 CFU/ml for K. pneumoniae, 16CFU/ml for E. faecium, and4 CFU/ml for C. albicans.

Clinical specimens. To demonstrate the accuracy of organismidentification in clinical specimens, we tested 331 prospectivelycollected deidentified blood specimens from consenting patientsfrom the Johns Hopkins Medical Center emergency departmentunder institutional review board (IRB) approval (JHU IRB no.NA_00013251). The PCR/ESI-MS results from an analysis of 5 mlof EDTA-blood using the BAC assay were compared with the re-sults from standard clinical microbiology cultures. An analysis of331 subjects with suspected bloodstream infections (Table 1)yielded 35 PCR/ESI-MS-positive specimens (10.6%) compared to18 positive specimens by culture (5.4%). There were 15 samplesthat were positive by both methods. One specimen, sample 1,070,was polymicrobial. Culture identified two organisms, while PCR/ESI-MS identified three. Overall, for the 16 cases for which culturereported an organism, PCR identified the same organism in 15instances, for an accuracy of identification of 94%. The one mis-match was a case (sample 1,346) for which culture failed to iden-tify the organism in the primary culture used for comparison andPCR/ESI-MS identified it as Escherichia coli. The culture of a sub-sequent blood draw yielded E. coli, but this was not included in theanalysis.

Using culture as the comparator method, PCR/ESI-MS was83% sensitive and 94% specific (Table 2). When PCR/ESI-MS-positive but culture-negative specimens were confirmed by repeatPCR/ESI-MS testing of additional replicate specimens (Table 3)and the confirmed detections were considered true positives, sen-sitivity increased to 91% and specificity to 99% (see section be-low). In one specimen (sample 1,070), two organisms were iden-tified by culture; both were correctly identified in direct testing byPCR/ESI-MS. PCR/ESI-MS also detected a high level of Candidaglabrata in the same sample that culture failed to identify.

Sequencing is not an appropriate comparator method forPCR/ESI-MS. To determine whether sequencing can be used as acomparator method for PCR/ESI-MS, we attempted to sequencethe extracted DNA that was used for PCR/ESI-MS analysis usingprimers and protocols specified by the CLSI guidance documentfor the identification of bacteria and candida. We examined thespecimens from the patient population with suspected blood-stream infections and also extracted a second set of samples fromorthopedic tissues suspected of being infected (kindly provided byGarth Ehrlich [32]). The extracted blood specimens had a higherconcentration of human DNA (270 ng/�l) than did the tissuespecimens (27 ng/�l) but a substantially smaller amount of infect-ing bacterial DNA by PCR/ESI-MS (Fig. 3). Only two of the 35PCR/ESI-MS-positive specimens (including 15 that were also cul-ture positive) from patients with suspected bloodstream infec-tions were confirmed by sequencing. Sequencing confirmed thatthe organisms identified by PCR/ESI-MS in 27 of 36 (75%) tissuespecimens were present. No specimens were positive by sequenc-ing and negative by PCR/ESI-MS.

An examination of the data in Fig. 3 suggests that the success ofsequencing is dependent on both the concentration of the targetbacterial DNA and level of human DNA. Overall, 84% of sampleswith �0.4 �g of background DNA per PCR sequencing reactionand �40 genomes per PCR well were positive by sequencing. Incontrast, only 8% of the PCR/ESI-MS-positive samples contain-ing �0.4 �g DNA (all but one of the blood samples tested) werepositive by sequencing. Thus, in the absence of a method to re-move or reduce human DNA, 16S Sanger sequencing of nucleicacid extracts from whole-blood specimens is not a suitable

FIG 2 Frequency distribution plot for white blood cell counts obtained frompatients. Each bin has a width of 2 � 106 white blood cells/ml. The y axisrepresents the frequency of each bin relative to the total number of samples,and the x axis shows the range of white blood cell counts. The bracket at the topindicates that 90% of samples fall into the range from 0 to 16 � 106 cells/ml.The black regions of the bars correspond to the specimens that were positive byPCR/ESI-MS.

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method for detecting and identifying infecting bacteria or fungi,and it cannot be used as a comparator method for PCR/ESI-MS ofblood specimens.

Repeated analytical testing by PCR/ESI-MS. Many studieshave shown that molecular methods will find bacterial and can-didal DNA in blood extracts for which cultures are negative (19,20, 28, 29). In the absence of another technology sharing both thesensitivity and the breadth of coverage of the BAC BSI assay, it ischallenging to prove that PCR/ESI-MS identifications are correctin the absence of a confirming culture result. To partially addressthis issue, we retrieved frozen remnant blood samples of the PCR/ESI-MS-positive/culture-negative patient specimens and retestedthem on a different instrument with a different operator. The resultsare shown in Table 3. In 18 of 21 cases for which there was sufficientvolume of blood to retest, the results were identical in replicate testing

TABLE 1 Positive detections by culture or PCR/ESI-MS

Sampleno. Culture result PCR/ESI-MS result Q score

Level(GE/ml)a

IBIS0838 Klebsiella pneumoniae K. pneumoniae 0.98 332IBIS0917 Staphylococcus species, coagulase negative Staphylococcus epidermidis, mecA� 0.97 40IBIS1088 Escherichia coli E. coli 0.99 220IBIS1090 E. coli E. coli 0.96 12IBIS1126 K. pneumoniae K. pneumoniae 0.99 124IBIS1168 Streptococcus group G Streptococcus dysgalactiae 0.98 60IBIS1170 E. coli E. coli 0.98 544

Vancomycin-resistant Enterococcus faecium E. faecium, vanA� 0.96 120Not detected Candida glabrata 0.97 204

IBIS1185 Staphylococcus species, coagulase negative S. epidermidis, mecA� 0.99 444IBIS1195 E. coli E. coli 0.96 40IBIS1230 Staphylococcus aureus S. aureus 0.98 172IBIS1296 S. aureus S. aureus 0.99 80IBIS1346 Reported as positive, but no organism listed E. coli 0.97 12IBIS1366 S. aureus S. aureus 0.97 36IBIS1407 S. aureus S. aureus 0.99 764IBIS1414 K. pneumoniae K. pneumoniae 0.98 16IBIS0834 Viridans Streptococcus group Negative Not detected NAIBIS1016 E. coli Negative Not detected NAIBIS1051 Streptococcus pneumoniae Negative Not detected NAIBIS0840 Negative Bacteroides fragilis 0.97 72IBIS0852 Negative Finegoldia magna 0.90 20IBIS0868 Negative f. magna 0.88 16IBIS0869 Negative Bartonella henselae 0.96 160IBIS0933 Negative Acinetobacter baumannii 0.98 148IBIS0885 Negative K. pneumoniae 0.98 72IBIS0965 Negative S. aureus 0.97 16IBIS1000 Negative E. coli 0.98 292IBIS1006 Negative E. coli 0.97 100IBIS1023 Negative E. coli 0.95 20IBIS1083 Negative Serratia marcescens 0.99 172IBIS1097 Negative E. coli 0.98 192

Negative Pseudomonas aeruginosa 0.98 560IBIS1093 Negative Enterobacter cloacae complex 0.98 76IBIS1109 Negative E. coli 0.99 32IBIS1181 Negative Viridans/Mitis group Streptococcus 0.96 104IBIS1170 Negative C. glabrata 0.97 204IBIS0800 Negative K. pneumoniae 0.96 32IBIS1121 Negative Enterococcus faecalis 0.98 28IBIS1365 Negative K. pneumoniae 0.99 36IBIS1381 Negative E. faecalis 0.98 16IBIS1416 Negative E. coli 0.98 36a GE, genome copy; NA, not applicable.

TABLE 2 Concordance of culture results with PCR/ESI-MS on a per-sample basisa

PCR/ESI-MS result

Culture resultCulture and repeatedPCR/ESI-MS

Positive Negative Total Positive Negative Total

Positive (no.) 15 20 35 32 3 35Negative (no.) 3 293 296 3 293 296Total (no.) 18 313 331 35 296 331Sensitivity (%) 83 91Specificity (%) 94 99a Culture results are from direct comparison of culture and PCR/ESI-MS, and theculture and repeated PCR/ESI-MS results are from comparison of culture plus PCR/ESI-MS when replicated as a comparator method.

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in terms of the organism identified and similar in terms of the level ofmicrobial DNA detected, providing supporting evidence that the or-ganism DNA reported by PCR/ESI-MS was indeed present in thesesamples and not the result of postcollection contamination. The threespecimens that were negative upon retesting had yielded relativelylow-level signals on the initial test. In order to be counted as a detec-tion by PCR/ESI-MS, a critical number of spectra with positive peaksfor specific organisms must be achieved. Representative spectra fromthe specimens that were PCR/ESI-MS positive in duplicate indepen-dent tests are shown in Fig. 4. The species identification with thismethod results from the completion of a succession of well-definedtasks that are themselves submitted to stepwise stringent quality con-trol requirements, and hence repeatable identifications strongly indi-cate that these specimens contain the initially reported microbialDNA.

Expected levels of bacterial DNA in blood from patients withbloodstream infections. In order to estimate the true concentra-tion of pathogen DNA available in whole blood for molecularanalysis from patients with bloodstream infections, we analyzedthe data available in the literature. Organism-specific quantitativePCR has been used to measure the bacterial DNA present inwhole-blood specimens from patients with sepsis, pneumonia, orsuspected bloodstream infections (13, 33–48). Each of these stud-ies analyzed whole-blood specimens from patients with suspectedor confirmed infections rather than spiked samples for which thegenome-to-viable cell ratio is expected to be close to 1:1. Of 16such publications, 15 used a calibrated real-time PCR methodfocused on single organisms, and one publication reported a

quantitative 16S broad-range method (37). The investigators cal-ibrated their PCRs by using either an analytically prepared DNAreference standard of a single-copy gene (reporting results as bac-terial genome copies/ml) or quantified spikes of cultured mi-crobes (reporting results as CFU equivalents/ml of blood). Al-though the results of these experiments revealed some variabilitybetween different microorganisms, specific PCR methods, anddifferent patient populations, the central values (median andmean) for bacteria were typically between 1 � 103 and 1 � 104

genome copies per ml (Fig. 5). Thus, in subjects with suspected orconfirmed bloodstream infections, the amount of bacterial nu-cleic acid available for detection by molecular methods is approx-imately 2 to 3 orders of magnitude higher than what one mightexpect from the literature on culture-based quantitative microbi-ology. This explains the apparent inconsistency between the re-ported analytical LODs of PCR/ESI-MS, which are approximately16 CFU/ml for freshly spiked blood, the reported concentrationsof viable microbial cells in septic blood (averaging between 1 and10 CFU/ml), and the approximate clinical sensitivity of PCR/ESI-MS of 83 to 91%. The results reported here are consistent withquantitative PCR analysis of patient specimens (Fig. 5).

FIG 3 Microbial and human DNA loads define the functional limits of 16Ssequence analysis of clinical specimens. Blood sample extracts were obtainedusing the 5-ml DNA isolation protocol from whole-blood specimens collectedfrom patients suspected to have bacteremia. For tissues, 25-mg specimens werecollected from patients suspected of sterile-site bacterial/candidal infection.All PCR/ESI-MS-positive samples were further analyzed by Sanger sequenc-ing. The samples are plotted with respect to their total DNA load (y axis) andbacterial DNA detection level by PCR/ESI-MS (x axis). The horizontal dashedline indicates a 0.4-�g DNA load threshold above which only one tissue sampleis present and below which only one blood sample is represented. The verticalsolid line indicates the detection level of approximately 40 bacterial genomesper PCR, the threshold above which sequencing was successful.

TABLE 3 Replicate testing of PCR/ESI-MS-positive samples that werenegative by culture

PCR/ESI-MSreplicate 1

Qscore

Level(GE/ml)

PCR/ESI-MSreplicate 2

Qscore

Level(GE/ml)

Acinetobacterbaumannii

0.98 148 A. baumannii 0.98 88

Bacteroides fragilis 0.97 72 B. fragilis 0.9 16Bartonella henselae 0.96 160 B. henselae 0.98 128Candida glabrata 0.97 204 C. glabrata 0.97 276Enterobacter cloacae

complex0.98 76 E. cloacae

complex0.98 88

Enterococcus faecalis 0.98 28 No result NA NAE. faecalis 0.98 16 No result NA NAEscherichia coli 0.98 292 E. coli 0.99 236E. coli 0.97 100 E. coli 0.99 108E. coli 0.95 20 E. coli 0.98 24E. coli 0.98 192 E. coli 0.98 172E. coli 0.99 32 E. coli 0.98 40E. coli 0.98 36 E. coli 0.96 32Finegoldia magna 0.90 20 F. magna 0.99 32F. magna 0.88 16 F. magna 0.98 24Klebsiella

pneumoniae0.98 72 K. pneumoniae 0.98 92

K. pneumoniae 0.96 32 No result NA NAPseudomonas

aeruginosa0.98 560 P. aeruginosa 0.99 656

Serratia marcescens 0.99 172 S. marcescens 0.98 164Staphylococcus

aureus0.97 16 S. aureus 0.99 32

Viridans/MitisgroupStreptococcus

0.96 104 Viridans/MitisgroupStreptococcus

0.97 80

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DISCUSSION

In order for molecular assays to be optimally useful for diagnosingpatients with suspected systemic infections, the assay must (i) ac-curately and sensitively identify bacterial and candidal speciespresent in blood, (ii) provide rapid results, (iii) detect the mostimportant genetic mediators of antimicrobial drug resistance, and(iv) be carried out with a workflow and throughput suitable for ahospital laboratory. To meet these objectives, we developed aPCR/ESI-MS hardware platform and research assay with im-proved clinical sensitivity that facilitates workflow in a clinicallaboratory environment. The semiautomated workflow describedin Fig. 1 allows the first sample to obtain a result in approximately6 h for the first batch of 6 specimens. The modular nature of theworkflow permits additional rounds of samples to begin process-ing through the front-end lysis and extraction steps while the latermodules are still finishing the previous set of samples. This allowsfor samples that enter the testing laboratory later in the day to beincorporated into the testing workflow throughout the day. Im-proved sensitivity was achieved by extracting nucleic acids from a5-ml volume of blood, developing an automated specimen prep-aration technology to accommodate this volume, and optimizingthe entire remaining system to be tolerant to the high levels ofhuman DNA arising from human white blood cells. Unlike pro-tocols that separate human white blood cells from the bacteria or

those that separate bacterial DNA from human DNA after lysis,the procedure we developed retains all potential compartments ofbacterial DNA, including cell-associated bacteria, free bacteriaand free bacterial DNA, and it avoids the introduction of steps thatwould complicate the workflow and increase costs.

This improved approach resulted in PCR/ESI-MS detectingtwice as many positive samples (10.6%) as culture (5.4%) in the331 samples analyzed from patients suspected of having a bloodinfection. This is consistent with the well-established observationthat approximately half of the truly infected patients are not pos-itive by culture (3, 6, 7), often because samples are taken afterpatients began antimicrobial drug treatment. The analytical sen-sitivity for uninfected blood spiked with bacteria was improvedabout 5-fold compared to that of the previously reported 1-mlsample preparation method (28, 29). More importantly, the high-er-volume-sample preparation method integrated with the othersensitivity improvements increased the detection rate for speci-mens that were blood culture positive from about 50% for thelow-blood-volume PCR-ESI-MS assay to 83% for the high-vol-ume research protocol.

Previously published quantitative microbiology studies haveshown that the number of recoverable CFU of bacteria in theblood of patients with clinically significant bacteremia is low, typ-ically in the range of �1 to 30 CFU/ml (49–51). However, the CFU

FIG 4 Quantitative bacterial loads in whole blood determined by various methods. Q, interquartile range; I, range; S, �1 standard deviation; , cutoff; diamond, median.The PCR/ESI-MS values are those reported in Table 1 corrected for the dilution factors in sample preparation and converted to genome copies per ml of blood to beconsistent with the reported values for quantitative PCR. The analytical LOD for PCR/ESI-MS is indicated by the vertical dashed line.

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measured by quantitative microbiology represents only separableviable organisms that survive the plating process and not deadcells, cells that cannot form colonies, multiple cells in irreducibleclumps, or free microbial DNA that may have been liberated from

lysed cells in the blood compartment. Thus, the true concentra-tions of pathogen DNA available in whole blood for molecularanalysis in patients with bloodstream infections cannot be in-ferred from viable cell count (quantitative culture) data. Here, we

FIG 5 Spectra from representative PCRs. The spectra for primer pairs 348 (left) and 349 (right) are reported for sample 1,083 (Serratia marcescens detected inreplicates 1 and 2, first and second rows) and for sample 933 (Acinetobacter baumannii detected in replicates 1 and 2, third and fourth rows). For each primer pair,the conserved horizontal scale emphasizes the reproducibility of the detections between replicates, as can be verified by the vertical alignment of the peakscorresponding to the internal positive control of the assay (calibrant, [gray]) and to the detected species (blue). The vertical scales are normalized to the highestpeak present in the corresponding well. The spectra shown are for only two primer pairs; the reported species detections are further supported by similardetections of the corresponding amplicons using four additional primer pairs.

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report that in subjects with suspected or confirmed bloodstreaminfections, the amount of bacterial nucleic acid available for de-tection by molecular methods is approximately 2 to 3 orders ofmagnitude higher than what one might expect from the culture-based quantitative microbiology literature. This estimate of thebacterial DNA load in patient specimens may have clinical utility.Several studies have demonstrated that bacterial load measured byPCR correlates with disease severity and that the bacterial load ispredictive of which patients are likely to become severely ill (35,37, 41, 42, 52, 53). Two studies independently identified a bacterialload of 1 � 103 genome copies per ml of blood as a critical thresh-old above which patients had an increased risk of developing sep-tic shock with E. coli (37), S. aureus (37), or Streptococcus pneu-moniae (42), suggesting that quantitative measurement of thebacterial DNA load in blood may have clinical value. The analyt-ical sensitivity of PCR/ESI-MS (16 CFU/ml) is more than suffi-cient to detect the concentrations of organisms present in patientswith these serious infections.

A highly sensitive molecular test that detects a broad range ofbacteria and fungi presents challenges for validation. AlthoughPCR/ESI-MS-positive results can be compared to positive cultureresults, there is no good way to corroborate a PCR/ESI-MS-posi-tive result when cultures are negative other than to show thatPCR/ESI-MS gives the same result when testing additional speci-mens from the same patient. When replicate testing was per-formed, 91% of the PCR/ESI-MS results were corroborated.Broad amplification followed by sequencing failed to provide asufficiently sensitive comparator method for bloodstream infec-tions because it is difficult to amplify and sequence the smallamount of targeted bacterial DNA against an overwhelming back-ground of human DNA. Mass spectrometry of unfragmented am-plicons is, in comparison to sequencing, relatively unaffected bybackground genomic DNA but still provides species-specific sig-natures that can be matched to sequence database-derived signa-tures for unambiguous identification.

There is a growing body of evidence that rapid and accurateidentification of the microbes causing bloodstream infectionsprovide significant clinical and economic value. The use of molec-ular methods to identify culture-isolated organisms decreases theoverall time to identification, resulting in improved patient out-comes and significantly decreasing hospital costs (54–57). This is astrong step forward but requires time for culture and is not usefulwhen cultures from truly infected patients are negative (58, 59).The direct analysis of patient specimens would both increase thenumber of patients who benefit from the information and furtherdecrease the time to result by avoiding the lag time associated withgrowing cultures.

ACKNOWLEDGMENTS

This work was funded in part by the Middle Atlantic RCE Program (NI-AID/NIH grant 2 U54 AI057168 to R.E.R).

N.J.K. is a independent consultant under contract with Ibis Biosci-ences; all other authors affiliated with Ibis Biosciences are full-time em-ployees.

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