Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

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Diagnostic Microbiology and Infectious Disease

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Use of multiplex PCR in diagnosis of bloodstream infections inkidney patients

Natalia Fernández-Romero a,1, Inmaculada Quiles a,1, Carlos Jiménez b, María Ovidea Lopez Oliva b,Begoña Rivas b, Jesús Mingorance a, María Pilar Romero-Gómez a,⁎a Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Paseo de la Castellana, 261, 28046, Madrid, Spainb Servicio de Nefrología, Hospital Universitario La Paz, IdiPAZ, Paseo de la Castellana, 261, 28046, Madrid, Spain

a b s t r a c ta r t i c l e i n f o

⁎ Corresponding author. Tel.: +34-912071842; fax: +E-mail address: mromerogomez@salud.madrid.org (

1 These 2 authors contributed equally.

http://dx.doi.org/10.1016/j.diagmicrobio.2014.07.0010732-8893/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Fernández-RomeMicrobiol Infect Dis (2014), http://dx.doi.o

Article history:Received 26 January 2014Received in revised form 5 June 2014Accepted 2 July 2014Available online xxxx

Keywords:Multiplex PCR for diagnostic of sepsis inkidney patients

The LightCycler® SeptiFast Test (Roche Diagnostics GmbH, Mannheim, Germany) was prospectivelycompared with the standard blood culture technique in a series of 86 kidney patients. The sensitivity ofthe PCR compared with the culture was 71%, and the specificity was 88%. All the species identified by culturein these patients were in the SeptiFast panel. The median time to results was 1 day for the PCR, 3 days forpositive cultures, and 5 days for negative cultures.

34-917277372.M.P. Romero-Gómez).

ro N, et al, Use of multiplex PCR in diagnosisrg/10.1016/j.diagmicrobio.2014.07.001

© 2014 Elsevier Inc. All rights reserved.

Bloodstream infections (BSIs) are an important cause of morbidityand mortality in kidney patients (Abbott et al., 2001), septic shockbeing the most severe complication and increasing overall mortality(Harbarth et al., 2002). The case fatality rate from BSI causing organdysfunction ranges from 25% to 50% (Palmer et al., 2000).

BSI is frequently a consequence of urinary tract infection, with ahigh prevalence of gram-negative bacilli (Chuang et al., 2005;Merçon et al., 2010). Microbiological and epidemiological dataindicate that a limited number of bacteria and fungi are responsiblefor the majority of all BSIs in kidney transplant recipients andhemodialysis patients (Al-Hasan et al., 2009, 2011; Li and Chow,2012; Silva et al., 2010). Early detection and adequate treatment ofcausative pathogens within the first 6–12 h is critical for a favorableoutcome in patients with BSI (Garnacho-Montero et al., 2003;Morrell et al., 2005), but the microbiological “gold standard” for thedetection and identification of bacterial and fungal BSIs is the bloodculture (BC), which is limited by high volume requirements andprolonged incubation times. In an effort to address some of theselimitations, many advances have been developed to improve thesensitivity and reduce the time to identification of pathogens inblood cultures (Romero-Gómez, 2011; Romero-Gómez et al., 2012).Molecular amplification techniques have been developed to detectthe pathogens directly on blood (Haag et al., 2013; Loonen et al.,2014; Samuel et al., 2013; Schreiber et al., 2013; Wojewoda et al.,

2013), replacing the incubation step in blood culture, but their use islimited by some important technical drawbacks: 1) detection andidentification can be achieved in one step by amplification ofuniversal targets (usually 16S, 23S rRNA genes or their associatedITS regions) and multiplexing, but it is difficult to span the broadrange of pathogens that can be found in blood cultures and maintainaccuracy in species identification without adding lengthy sequenc-ing or hybridization steps; 2) bacterial loads in blood may be as lowas a few colony-forming units per milliliter, so the methods must bevery sensitive, but then environmental and reagent contaminationwith bacterial DNA becomes a serious concern. The LightCycler®SeptiFast Test (Roche Diagnostics GmbH, Mannheim, Germany) is acommercial multiplex real-time PCR assay that uses DNA-freereagents (M Grade) to detect directly in whole blood any of apanel of 25 of the most frequent pathogens (bacteria and fungi)found in blood cultures (Table S1) (Lehmann et al., 2008). The assayhas been tested in blood samples from diverse patient groups,including intensive care patients (Lodes et al., 2012; Regueiro et al.,2010; Yanagihara et al., 2010), transplant recipients (Rath et al.,2012), pediatric patients (Torres-Martos et al., 2012), and even insamples different from blood (Fernández et al., 2010; Mencacciet al., 2011).

The microorganisms detected by SeptiFast include the mostcommon pathogens found in positive blood cultures from kidneypatients suggesting that the technique might be especially well suitedfor this patient group. On this ground, the objective of this study wasto evaluate the utility of the LightCycler® SeptiFast multiplex real-time PCR assay compared to those of conventional blood culture forthe diagnosis of BSIs in renal patients.

of bloodstream infections in kidney patients, Diagn

Table 1Comparison of BC and SeptiFast results.

BC positive BC negative Total

SeptiFast positive 15 9 24SeptiFast negative 6 66 72Total 21 75 96

The table shows the number of positive and negative blood cultures and the number ofpositive and negative SeptiFast assays. The sensitivity of the PCR compared with the BCwas 71%; the specificity was 88% (PPV 63%, NPV 92%).

Table 2Microorganisms identified.

Microorganisms Total Positive withboth methods

Positive onlywith SeptiFast

Positive onlywith BC

S. aureus 7 4 2 1K. pneumoniae 7 1 4 2E. coli 6 4 1 1E. faecalis 5 3 1 1Streptococcus pneumoniae 2 1 1 0Enterobacter aerogenes/Enterobacter cloacae

2 0 2 0

Pseudomonas aeruginosa 1 0 1 0Proteus mirabilis 1 0 0 1S. epidermidis 1 0 0 1

2 N. Fernández-Romero et al. / Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

1. Materials and methods

1.1. Study site, patients, and tests

Subjects were recruited in Hospital Universitario La Paz in Madrid,Spain, between September 2009 and June 2012. Ninety-sevenSeptiFast tests were performed on blood samples from 86 subjects,aged 20–96 years (median age 63 years), as an adjunct diagnostic toolfor the diagnosis of sepsis. Ward submitting samples was thenephrology department, and all the patients were registered asnephrology patients.

1.2. Inclusion criteria

Patients were considered for inclusion in the study if they had aknown or suspected infection on the basis of clinical data at the timeof screening and if they exhibited 2 or more signs of the systemicinflammatory response syndrome (SIRS) within a 24-h period (Boneet al., 2009). Only subjects admitted to the hospital and for whomblood culture results were available were included in this analysis.

1.3. Sample processing

Blood was collected for culture by the use of sterile technique.During a febrile episode, a single venipuncture was used to drawsamples for 2 or 3 pairs of aerobic/anaerobic BC bottles of Bact/Alert(bioMérieux, Marcy l’Etoile, France) or BACTEC 9240 (BectonDickinson, Franklin Lakes, NJ, USA). Blood samples from patientsthat were receiving antibiotic therapy were inoculated into Bact/Alertbottles with activated charcoal (BacT/ALERT FA FAN® Aerobic andBacT/ALERT FN FAN Anaerobic) while samples from patients withoutantibiotic therapy were inoculated into BACTEC 9240 bottles(BACTEC™ Standard Aerobic/F Medium and BACTEC™ StandardAnerobic/F Medium). Ten milliliters of whole blood was used foreach of a pair of aerobic/anaerobic BC bottles, and immediately afterthe blood for the culture was drawn, 1–3 mL of whole blood wascollected in sterile BD Vacutainer® Plus Plastic K2EDTA Tubes forthe PCR assay. The blood culture system and the local laboratorysoftware automatically registered time to positive BC. In case ofpositivity, Gram staining and culture on solid medium wereperformed; definitive organism identification and antibiotic sus-ceptibility were determined with MALDI-TOF-MS (Bruker DaltonikGmbH, Bremen, Germany) and Vitek 2 system (bioMérieux,Durham, NC, USA) respectively.

Total nucleic acid extraction was done from 0.4-mL whole bloodspecimens collected in sterile BD Vacutainer® Plus Plastic K2EDTAtubes for the PCR assay. Samples were subjected to mechanical lysiswith ceramic beads in a MagNA Lyser® instrument (Roche Diagnos-tics GmbH) for 70 s at 7000 rpm. After lysis, total DNA was extractedusing a MagNA Pure Compact system and the MagNA Pure CompactNucleic Acid Isolation Kit I (Roche Diagnostics GmbH) (Regueiro et al.,2010). Detection and identification of microorganisms were done bythe SeptiFast multiplex real-time PCR in a LightCycler® 2.0 Instru-ment and analysis of melting curves according to the manufacturer'sinstructions (Roche Diagnostics GmbH). The SeptiFast assay isperformed in 3 independent capillary tubes, 1 for gram negatives,1 for gram positives, and 1 for fungi, and the probes are detected in 5different channels, so melting peaks are analysis is distributedamong different tubes and different fluorescence channels, allowingwide (3–5 °C) melting peak differences in the same tube andchannel (Lehmann et al., 2008).

Blood samples for SeptiFast were collected from the samevenipuncture stick that the blood cultures were collected, and testingwas performed once daily from Monday to Friday, for samplescollected in the previous 24 h.

Please cite this article as: Fernández-Romero N, et al, Use of multiplex PMicrobiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.20

Blood culture was used as the reference assay. BSI was defined as apositive blood culture result. Interpretation of blood culture findingswas performed according to established clinical and microbiologicalstandards. The significance of microorganisms identified by PCR wasevaluated retrospectively by taking into account the identity of themicroorganism detected and by comparing PCR results with corre-spondingBCfindings and/or fromprevious patient samples (respiratorysamples, intra-abdominal fluid, urine, catheter, surgical wound, etc.)obtained within 4 days before or after the onset of the septic episode.

2. Results

A total of 96 samples from 81 patients from the nephrologydepartment were evaluated by both conventional blood culturetechniques and the SeptiFast PCR; 37 were women (45%). The patientshad a mean age of 58 years (median 63, range: 21–96). Twenty-sixpatients were dialysis patients, 46 were renal transplant patients, and 6were chronic renal failure patients and suffered acute renal failure.Forty-four patients had received previous antibiotic treatment. Themost common infections concurrent with blood culture requests werethe urinary tract (13.4%), followed by the respiratory tract (10.4%),central venous catheter–related infections (8.9%), intra-abdominalinfections (8.8%), skin and soft tissues infections (8.5%), gastrointestinalinfections (7.5%), osteoarticular infections (5.3%), and surgical woundinfection (4.4%). Twenty-twopatients (32.8%) had anunknownprimarysource of BSI.

Blood cultures were positive in 21 samples and negative in 75samples, while PCR was positive in 24 samples and negative in 72(Table 1). There were 6 SeptiFast-negative and culture-positivesamples. These might be due to the small sample volume used(0.4 mL), which is 10–20 times lower than the blood volume used toinoculate the bottles. Fifteen samples were positive by both, cultureand SeptiFast; in 13 of them, the samemicroorganismwas detected bythe 2 methods, in 1, the PCR detected Escherichia coli and Enterococcusfaecalis, while the culture detected only E. coli. In 2 samples, theresults were discordant, in 1, SeptiFast detected Klebsiella pneumoniae,while Staphylococcus epidermidis was obtained in the blood culture,and in the other, SeptiFast detected Staphylococcus aureus, while agram-positive Bacillus was observed by microscopy in the culturebottles but could not be isolated in plates and identified (Table 2).

CR in diagnosis of bloodstream infections in kidney patients, Diagn14.07.001

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Except for this, all the species identified by culture were in theSeptiFast panel (Lehmann et al., 2008). In terms of detection, thesensitivity of the PCR compared with the BC was 71%; the specificitywas 88% (positive predictive value [PPV] 63%, negative predictivevalue [NPV] 92%).

The turnaround time for final microbial identification of pathogensof blood culture isolates was compared with the time for microbialDNA detection by SeptiFast. In this study, SeptiFast tests wereperformed once daily from Monday to Friday for samples collectedin the previous 24 h; the samples for PCR were stored at 8 °C, if notwere testing immediately, and therefore, the turnaround time rangesfrom 0.25 to 2 days (average 1 day). For culture positive samples, arange from 0.5 to 4 days (median 3 days) was obtained formicrobiological identification, calculated from the arrival of theblood culture bottle, while for negative samples, the mean and themedian turnaround times were 5 days.

3. Discussion

Blood culture is the most widely utilized technique for thediagnosis of BSIs. However, despite its high specificity, its sensitivityremains low, and the mean time for obtaining results varies from 3 to4 days for positive results to 5 days for negative ones. This studyaimed to evaluate a real-time multiplex PCR assay as an adjunct toblood culture for the rapid identification of BSIs.

The performance of SeptiFast in our study in terms of sensitivityand specificity is similar to that found in the meta-analysis performedby Chang et al. (2013) and the study of Skvarc et al. (2013), but theturnaround time is shorter. As expected, all the microorganismsdetected by blood cultures were in the SeptiFast panel, so no false-negative results of PCR were due to the presence of microorganismsnot included in the panel. There was a good concordance between theresults of blood culture and PCR, with the 2 methods having similaroperating characteristics; the accuracy was 84.38% with NPV and PPVof 91.67% and 62.50%, respectively (95% confidence intervals, 82.12–96.57 and 40.76–80.45%). There were 15 discordant cases, represent-ed by 6 blood culture-positive and PCR-negative results and 9 bloodculture-negative and PCR-positive results (4 samples SeptiFastmethod detected polymicrobial infections). In 4 of these, the bloodsamples were obtained after the initiation of antimicrobial treatment,and in 2 cases, the patients had not received antibiotic treatment.Theremight bedifferent reasons for thediscordant results. PCRmight benegative in blood culture positive samples because PCR is limited by itstest panel (whichwasnot the case in thiswork) and by the small samplevolume used: 0.4 mL of blood is used for a single PCR assay, while 2 or 3bottles, each one inoculatedwith 10 mLof blood, are used for culture, sothe volume of blood used for culture is 10–20 times higher than thatused for PCR. On the other hand, PCRmight be positive in blood culture-negative samples because thePCRassaymaydetect DNA fromdead cellsor from viable cells unable to growth in culture bottles due to thepresence of antibiotics carried in the blood samples.

SeptiFast assays were performed once daily fromMonday to Fridayfor samples collected in the previous 24 h. This means that we havenot exploited the full potential of the method, and the response timemight be improved. In spite of this, the faster availability of resultsmight have an impact in reducing treatments or narrowing theirspectra. Moreover, given the good specificity and NPV and the shorterturnaround time of the negative results, SeptiFast data might becombined with C-reactive protein, procalcitonin, and the results ofprevious microbiological findings in other compartments to takedecisions (Lodes et al., 2012; Loonen et al., 2014). This will needspecifically designed studies (Fitting et al., 2012) and might be mostuseful in clinical contexts in which the SeptiFast panel of pathogens isrepresentative of the vast majority of findings, as is the case in kidneypatients (Li and Chow, 2012).

Please cite this article as: Fernández-Romero N, et al, Use of multiplex PMicrobiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.20

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.diagmicrobio.2014.07.001.

Conflict of interest

The authors received research funding from Roche DiagnosticsGmbH for this project.

References

Abbott KC, Duran M, Hypolite I, Ko CW, Jones CA, Agodoa LY. Hospitalizations forbacterial endocarditis after renal transplantation in the United States. J Nephrol2001;14(5):353–60.

Al-Hasan MN, Razonable RR, Eckel-Passow JE, Baddour LM. Incidence rate and outcomeof Gram-negative bloodstream infection in solid organ transplant recipients. Am JTransplant 2009;9(4):835–43.

Al-Hasan MN, Huskins WC, Lahr BD, Eckel-Passow JE, Baddour LM. Epidemiology andoutcome of Gram-negative bloodstream infection in children: a population-basedstudy. Epidemiol Infect 2011;139(5):791–6.

Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, KnausWA, et al. Definitions for sepsisand organ failure and guidelines for the use of innovative therapies in sepsis. TheACCP/SCCM Consensus Conference Committee. American College of ChestPhysicians/Society of Critical Care Medicine. 1992. Chest 2009;136(5 Suppl):1644–55.

Chang S-S, Hsieh W-H, Liu T-S, Lee S-H, Wang C-H, Chou H-C, et al. Multiplex PCRsystem for rapid detection of pathogens in patients with presumed sepsis - asystemic review and meta-analysis. PLoS One 2013;8(5):e62323.

Chuang P, Parikh CR, Langone A. Urinary tract infections after renal transplantation: aretrospective review at two US transplant centers. Clin Transplant 2005;19(2):230–5.

Fernández AL, Varela E, Martínez L, Martínez A, Sierra J, González-Juanatey JR, et al.Evaluation of a multiplex real-time PCR assay for detecting pathogens in cardiacvalve tissue in patients with endocarditis. Rev Esp Cardiol 2010;63(10):1205–8.

Fitting C, Parlato M, Adib-Conquy M, Memain N, Philippart F, Misset B, et al. DNAemiadetection by multiplex PCR and biomarkers for infection in systemic inflammatoryresponse syndrome patients. PLoS One 2012;7(6):e38916.

Garnacho-Montero J, Garcia-Garmendia JL, Barrero-Almodovar A, Jimenez-Jimenez FJ,Perez-Paredes C, Ortiz-Leyba C. Impact of adequate empirical antibiotic therapy onthe outcome of patients admitted to the intensive care unit with sepsis. Crit CareMed 2003;31(12):2742–51.

Haag H, Locher F, Nolte O. Molecular diagnosis of microbial aetiologies using SepsiTestTM in thedaily routine of a diagnostic laboratory. Diagn Microbiol Infect Dis 2013;76(4):413–8.

Harbarth S, Ferrière K, Hugonnet S, Ricou B, Suter P, Pittet D. Epidemiology andprognostic determinants of bloodstream infections in surgical intensive care. ArchSurg 2002;137(12):1353–9. [discussion 1359].

Lehmann LE, Hunfeld K-P, Emrich T, Haberhausen G, Wissing H, Hoeft A, et al. Amultiplex real-time PCR assay for rapid detection and differentiation of 25 bacterialand fungal pathogens from whole blood samples. Med Microbiol Immunol 2008;197(3):313–24.

Li PK-T, Chow KM. Infectious complications in dialysis—epidemiology and outcomes.Nat Rev Nephrol 2012;8(2):77–88.

Lodes U, Bohmeier B, Lippert H, König B, Meyer F. PCR-based rapid sepsis diagnosiseffectively guides clinical treatment in patients with new onset of SIRS.Langenbecks Arch Surg 2012;397(3):447–55.

Loonen AJM, de Jager CPC, Tosserams J, Kusters R, Hilbink M, Wever PC, et al.Biomarkers andmolecular analysis to improve bloodstream infection diagnostics inan emergency care unit. PLoS One 2014;9(1):e87315.

Mencacci A, Leli C, Cardaccia A, Montagna P, Moretti A, Bietolini C, et al. Comparison ofconventional culture with SeptiFast real-time PCR for microbial pathogen detectionin clinical specimens other than blood. J Med Microbiol 2011;60(Pt. 12):1774–8.

Merçon M, Regua-Mangia AH, Teixeira LM, Irino K, Tuboi SH, Goncalves RT, et al.Urinary tract infections in renal transplant recipients: virulence traits ofuropathogenic Escherichia coli. Transplant Proc 2010;42(2):483–5.

Morrell M, Fraser VJ, Kollef MH. Delaying the empiric treatment of candida bloodstreaminfection until positive blood culture results are obtained: a potential risk factor forhospital mortality. Antimicrob Agents Chemother 2005;49(9):3640–5.

Palmer SM, Alexander BD, Sanders LL, Edwards LJ, Reller LB, Davis RD, et al. Significanceof blood stream infection after lung transplantation: analysis in 176 consecutivepatients. Transplantation 2000;69(11):2360–6.

Rath P-M, Saner F, Paul A, Lehmann N, Steinmann E, Buer J, et al. Multiplex PCR for rapidand improved diagnosis of bloodstream infections in liver transplant recipients.J Clin Microbiol 2012;50(6):2069–71.

RegueiroBJ, Varela-Ledo E,Martinez-Lamas L, Rodríguez-Calvin J, SantosA, Gomez-Tato A,et al. Automated extraction improves multiplex molecular detection of infection inseptic patients. PLoS One 2010;5(10):e13387.

Romero-Gómez MPMJ. The effect of the blood culture bottle type in the rate of directidentification from positive cultures by matrix-assisted laser desorption/ionisationtime-of-flight (MALDI-TOF) mass spectrometry. J Infect 2011;62(3):251–3.

Romero-Gómez M-P, Gómez-Gil R, Paño-Pardo JR, Mingorance J. Identification andsusceptibility testing of microorganism by direct inoculation from positive bloodculture bottles by combining MALDI-TOF and Vitek-2 Compact is rapid andeffective. J Infect 2012;65(6):513–20.

Samuel LP, Tibbetts RJ, Agotesku A, Fey M, Hensley R, Meier FA. Evaluation of amicroarray-based assay for rapid identification of Gram-positive organisms andresistance markers in positive blood cultures. J Clin Microbiol 2013;51(4):1188–92.

CR in diagnosis of bloodstream infections in kidney patients, Diagn14.07.001

4 N. Fernández-Romero et al. / Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

Schreiber J, Nierhaus A, Braune SA, de Heer G, Kluge S. Comparison of three differentcommercial PCR assays for the detection of pathogens in critically ill sepsis patients.Med Klin Intensivmed Notfallmed 2013;108(4):311–8.

SilvaM,Marra AR, Pereira CAP, Medina-Pestana JO, Camargo LFA. Bloodstream infectionafter kidney transplantation: epidemiology, microbiology, associated risk factors,and outcome. Transplantation 2010;90(5):581–7.

Skvarc M, Stubljar D, Rogina P, Kaasch AJ. Non-culture-based methods todiagnose bloodstream infection: does it work? Eur J Microbiol Immunol (Bp)2013;3(2):97–104.

Please cite this article as: Fernández-Romero N, et al, Use of multiplex PMicrobiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.20

Torres-Martos E, Pérez-Ruiz M, Pedrosa-Corral I, Peña-Caballero M, Jiménez-Valera MM,Pérez-Ramírez MD, et al. Evaluation of the LightCycler(®) SeptiFast test in newbornsand infants with clinical suspicion of sepsis. Enferm Infecc Microbiol Clin 2012:12–6.

Wojewoda CM, Sercia L, Navas M, Tuohy M, Wilson D, Hall GS, et al. Evaluation of theVerigene Gram-positive blood culture nucleic acid test for rapid detection ofbacteria and resistance determinants. J Clin Microbiol 2013;51(7):2072–6.

Yanagihara K, Kitagawa Y, Tomonaga M, Tsukasaki K, Kohno S, Seki M, et al. Evaluationof pathogen detection from clinical samples by real-time polymerase chain reactionusing a sepsis pathogen DNA detection kit. Crit Care 2010:1–7.

CR in diagnosis of bloodstream infections in kidney patients, Diagn14.07.001