Therapeutic impact and diagnostic performance of multiplex PCR in patients with malignancies and suspected sepsis

Journal of Infection (2010) 61, 335e342

www.elsevierhealth.com/journals/jinf

Therapeutic impact and diagnostic performance of multiplex PCR in patients with malignancies and suspected sepsis `le Maubon a,*, Rebecca Hamidfar-Roy b, Ste ´phane Courby c, Danie ´lien Vesin d, Max Maurin e, Patricia Pavese f, Nadia Ravanel e, Aure ´ Pelloux a, Claude-Eric Bulabois c, Jean-Paul Brion f, Herve b,d Jean-Franc ¸ois Timsit a

Infectious Agent Department, Parasitology-Mycology Laboratory, Albert Michallon Teaching Hospital, 38043 Grenoble Cedex 9, France b Medical ICU, Albert Michallon Teaching Hospital, 38043 Grenoble Cedex 9, France c Hematology Department, Albert Michallon Teaching Hospital, 38043 Grenoble Cedex 9, France d Inserm U823, University Grenoble 1 e Albert Bonniot Institute 38 000 Grenoble, France e Infectious Agent Department, Bacteriology Laboratory, Albert Michallon Teaching Hospital, 38043 Grenoble, France f Infectious Diseases Department, Albert Michallon Teaching Hospital, 38043 Grenoble Cedex 9, France Accepted 8 July 2010 Available online 15 July 2010

KEYWORDS Sepsis diagnosis; Cancer; Hematological malignancies; Multiplex PCR; Therapeutic impact

Summary Objectives: New molecular methods allow rapid pathogen detection in patients with sepsis, but their impact on treatment decisions remains to be established. We evaluated the therapeutic usefulness of multiplex PCR testing in patients with cancer and sepsis. Methods: 110 patients with cancer and sepsis were included prospectively and underwent LightCycler
SeptiFast (LC-SF) multiplex PCR testing in addition to standard tests. Two independent panels of experts assessed the diagnosis in each patient based on medical record data; only one panel had the LC-SF results. The final diagnosis established by a third panel was the reference standard. Results: The final diagnosis was documented sepsis in 50 patients (55 microorganisms), undocumented sepsis in 54, and non-infectious disease in 6. LC-SF detected 17/32 pathogens recovered from blood cultures (BC) and 11/23 pathogens not recovered from BC; 12 microorganisms were detected neither by BC nor by LC-SF. LC-SF produced false-positive results in 10 cases. The LC-SF results would have significantly improved treatment in 11 (10%) patients and prompted immediate antimicrobial therapy not given initially in 3 patients.

* Corresponding author. Tel.: þ33 476 76 54 90; fax: þ33 476 76 56 60. E-mail address: [email protected] (D. Maubon). 0163-4453/$36 ª 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2010.07.004

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D. Maubon et al. Conclusions: In cancer patients with suspected sepsis, LC-SF detected 11/55 (20%) true pathogens not recovered from BCs and would have improved the initial management in 11/110 (10%) patients. ª 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction Sepsis is a major health problem worldwide.1 Prompt administration of appropriate antimicrobials is crucial to decrease the high morbidity and mortality rates of sepsis, particularly as recent years have seen an increase in the proportion of sepsis patients who have immune deficiencies and a high risk of progression to severe sepsis.2 The diagnosis of sepsis relies heavily on blood cultures (BCs) done to detect circulating living bacteria or fungi and to determine their species and susceptibility to antimicrobials. However, the sensitivity of BCs is limited and the culturing, identification, and drug-susceptibility testing process takes time.3 Therefore, probabilistic antimicrobial therapy is given immediately after sample collection. To improve the early detection of pathogens causing sepsis, molecular tools have been developed.3,4 Polymerase chain reaction (PCR) tests for detecting the DNA of bacteria, viruses, parasites, and fungi in body fluids have undergone considerable development over the last 10 years. The main advantages of PCR tests are high sensitivity, high specificity, and availability of the results within a few hours. However, the presence of DNA in a specimen does not necessarily reflect the presence of live microorganisms. In blood samples, for instance, a positive PCR test may reflect the presence of isolated DNA or transient bacteremia.5 Thus, the clinical relevance of a positive PCR test in a patient with negative BCs remains unclear. Several multiplex PCR tests for detecting microbial DNA in patients with sepsis were recently introduced on the market.3 Among them, LightCycler SeptiFast (LC-SF, Roche Diagnostics, GmbH, Penzberg, Germany) allows, in a 6 h time, the qualitative detection of 25 pathogen DNAs and simultaneous identification at the species level of 14 bacteria and 6 fungi, in whole blood samples. The pathogens detected by LC-SF account for 90% of sepsis cases in immunocompromised patients.6,7 LC-SF was the focus of several studies published in 2008 and 2009.8e16 Most of these studies compared the yields of LC-SF and BCs. PCR testing consistently produced higher yields and, overall, the studies suggested a possible role for PCR as an adjunct to BCs and other standard tests. A positive PCR result in a patient with negative BCs may reflect either a false-positive PCR result or a false-negative BC result. Differentiating these two situations relies on a careful clinical assessment of all the data from the patient. Therefore, the diagnosis made by a panel of experts based on the full range of information available for each patient is a suitable reference standard for diagnostic evaluations of PCR. The uncertainties surrounding the clinical usefulness and therapeutic impact of multiplex PCR testing in patients with suspected sepsis prompted us to evaluate the LC-SF kit in patients with solid or hematological malignancies. Our objective was to evaluate the diagnostic accuracy of LC-SF for detecting causative pathogens. We also assessed

whether the LC-SF results suggested new diagnostic hypotheses and whether they influenced treatment decisions. To achieve our objectives, we conducted a prospective study involving a review of patient records by three independent panels of experts.

Patients and methods We conducted a prospective multidisciplinary study from October 2007 to September 2008 at the Grenoble Teaching Hospital in France. This study was approved by the regional and national ethics committees and was registered at the AFSSAPS (#2007-A01053-50) and ClinicalTrials (#NCT00561639). All patients or their relatives gave written informed consent prior to study inclusion.

Patients During the study period, 110 patients with solid or hematological malignancies were admitted for suspected infection with at least one sign of sepsis, with or without organ dysfunction, as defined by the Systemic Inflammatory Response Syndrome (SIRS) criteria.17 All patients were managed in accordance with standard practice and French recommendations.18,19 The following tests were thus performed: leukocyte count, C-reactive protein and procalcitonin measurement, two sets of bacterial and fungal BCs, urine culture, chest radiograph and, when appropriate, specific viral and fungal tests (Cytomegalovirus PCR and detection of galactomannan by the Platelia Aspergillus test -Bio-Rad, Marnes-la-Coquette, France-). For each included patient, a single 5-ml blood sample was collected in a sterile EDTA tube after the first set of BCs using the same sampling material. Two more sets of BC were collected secondarily. Only one sample of blood was collected for the LC-SF test. Bacteraemia and candidemia were defined when a bloodstream infection pathogen was identified from one of the blood culture specimen. Considering facultative pathogenic skin bacteria at least two positive separate BC from different sites were required. Proven, probable or possible aspergillosis was defined using the European Organization for Research and Treatment of Cancer (EORTC) criteria.20 LC-SF was performed for the study but the results were not available to the clinicians managing the patients. Multiple episodes in the same patients were to be included only if separated by at least 1 month. Laboratory results, imaging study findings, and clinical events were collected prospectively by clinical investigators with the help of a clinical research assistant.

Multiplex PCR procedure LC-SF can detect 14 bacterial species and 6 fungal species (including Aspergillus fumigatus). For each included patient,

Impact of multiplex PCR in cancer patients with sepsis a single 5-ml blood sample was collected in a sterile EDTA tube after the first set of BCs using the same sampling material. Only one sample of blood was collected for the LC-SF test. The sample was stored in the laboratory at þ4  C for no longer than 3 days, which is the maximum delay to avoid a loss of sensitivity of the test according to the manufacturer’s instructions. Twice a week, multiplex PCR testing was performed in the laboratory using the LightCycler
2.0 (Roche Diagnostics, GmbH, Penzberg, Germany), as recommended by the manufacturer. The remaining blood (3.5 ml) was frozen at 20  C. MGrade
reagents and plasticware from Roche Diagnostics were used for each step of the procedure to avoid bacterial or fungal DNA contamination. DNA was extracted under a laminar flow cabinet with a UV germicidal lamp, and a stringent decontamination procedure was used for each step of the PCR. A control reagent, a negative control, and an internal control were included in each PCR run or sample. The entire PCR procedure lasted about 6.5 h. The melting curves were marked manually by a biologist, and the SeptiFast Software Set v2.0 was used to determine the corresponding melting temperature (Tm). Coagulase-negative staphylococci (CoNS) or Streptococcus spp., which had cutoff values greater than 20 cycles, were interpreted as contaminants by the SeptiFast Identification Software but were recorded in the patient’s chart. Samples containing PCR inhibitors (negative internal control) were subjected again to the entire PCR procedure.

Figure 1

337 LC-SF targets the Internal Transcribed Spacer sequences located between the bacterial 16S and 23S ribosomal DNA genes and the fungal 18S and 5.6S ribosomal DNA genes. Information on primer and probe sequences is the property of Roche Diagnostics (GmbH, Penzberg, Germany). Microorganism DNA was detected based on the presence of a specific melting curve. Quantification of the DNA load was generally not available, except for the 610 Gramþ and 640 Gramþ detection channels, for CoNS and Streptococcus spp., respectively, which were classified as possible contaminants.

Study design The general outline of the study is shown in Fig. 1. The study had no influence on patient management. Two multidisciplinary panels of experts met once a month to perform an assessment of the potential contribution of LC-SF to the management of the study patients. Since expert panel met no more than once a month, the sepsis episodes leading to patient’s inclusion were over at the time of the meeting, though some patients were still hospitalized due to their underlying pathologies. However, when the patient’s outcome was not obvious, medical files were analyzed during the next meeting and conclusions were drawn at that time. Each panel included an infectiologist, an intensivist, an oncologist, and a microbiologist (over 10 years of experience).

General study design: LC-SF: LightCycler
SeptiFast.

338 The panels worked independently from each other. Expert panel reviewed the whole patient’s story: all relevant medical events and biological results even before the LC-SF sample collection and before the sepsis event were available for the expert panel. For each episode of suspected sepsis, one panel was assigned at random to reviewing the patient data with, and the other without, the LC-SF results. All other data were available to both panels, whose members were free to ask questions of the attending physicians. Treatment was determined by the expert group based on the available guidelines and the patients’ clinical data. In case of discrepancies between the two groups of experts, the final consensus was given by the expert group C. This broader panel composed of three or more infectiologists, two intensivists, and three or more microbiologists (over 10 years of experience) assessed the differences between the conclusions of the panels that did and did not have access to the LC-SF results. Finally, the broader panel classified each episode as follows: documented sepsis with or without bacteremia; undocumented sepsis; or non-infectious disease mimicking sepsis. This classification served as the reference standard. Contribution of LC-SF to the treatment recommendations was evaluated using this reference standard.

Data analysis Numbers were expressed as frequencies and percentages for categorical variables, mean (SD) for normally distributed quantitative variables, and median [interquartile range] for nonnormally distributed quantitative variables. Cohen’s kappa was computed to evaluate concordance between blood culture results and LC-SF results. This method takes concordance due to chance into account, provides confidence intervals for the kappa coefficient, and tests for absence of agreement. A kappa value of 1 indicates perfect agreement and a kappa value of 0 no agreement. P values smaller than 0.05 were considered statistically significant. Statistical analyses were done using SAS 9.13 (Cary, NC).

Results Patients We included 110 consecutive patients with malignancies and suspected sepsis admitted over the 1 year study period. There were 67 women and 43 men with a mean age of 56.3 (13.7) years. Of the 110 patients, 56 (51%) had neutropenia (neutrophils <1G/L), 64 (58%) had severe sepsis, and 27 (25%) had septic shock. Most patients (97/110) received antimicrobials prior to inclusion. Table 1 shows the main features of the patients and episodes.

Microbiological results The broader panel classified 50 patients (55 recovered pathogens) as having documented sepsis, 54 as having undocumented sepsis, and 6 as having SIRS due to noninfectious causes. Of the 55 pathogens recovered in the 50 patients with documented sepsis, 32 were recovered by BCs after a mean of 43 (35) h. 28 microorganisms were

D. Maubon et al.

Table 1 Clinical and laboratory characteristics of the 110 included patients. Patient characteristics

Values

Female Age, mean  SD Direct admission McCabe score (n Z 86) Death expected within 1 year Death expected within 5 years Death not expected

43 (39.1%) 56.3  13.7 47 (43%) 18 (22%) 46 (42%) 22 (20%)

Malignancy Acute leukemia Lymphoma Myeloma Other hematological malignancy Solid tumor

48 (43.6%) 28 (25.5%) 10 (9.1%) 9 (8.2%) 15 (13.6%)

Prior treatment Chemotherapy Antimicrobials

81 (74%) 97 (88.2%)

Sepsis Characteristics Days from hospital admission to sepsis, median [IQR] Sepsis diagnosed within 24 h after admission SIRS criteria Fever or hypothermia Pulse rate >120 min SBP < 120 mm Hg or 50 mm Hg decrease Respiratory rate >30/min Neurological impairment Leukocytosis (>12.109/liter) or leukopenia (<4.109/L) Lowest temperature, median [IQR] Highest temperature, median [IQR]

5 [2e16] 27 (25%)

72 (65%) 30 (27%) 86 (78%) 30 (27%) 35 (32%) 90 (82%) 37 [36.6e37.3] 38.5 [37.9e39.3]

Severity Severe sepsis Septic shock Invasive mechanical ventilation Noninvasive mechanical ventilation Hospital death Neutropenia (<1  109/L)

64 27 29 12 41 56

Biomarkers C-reactive protein >40 mg/L Procalcitonin >0.5 mg/L (n Z 79)

80 (73%) 40 (36%)

Final Diagnosis Documented Sepsis

50 (45%)

Origin for documented Sepsis Lung Catheter Digestive tract Others

16 (32%) 9 (18%) 14 (28%) 11 (22%)

(58%) (25%) (26%) (11%) (37%) (51%)

SD: Standard deviation; IQR: Interquartile range; SIRS: Systemic Inflammatory Response Syndrome; SBP: systolic blood pressure

Impact of multiplex PCR in cancer patients with sepsis detected by LC-SF.BC and LC-SF results were in agreement in 17 patients. Fig. 2 shows the distributions of pathogens detected by BCs and LC-SF. The kappa coefficient for agreement between the two methods in the 110 patients was 0.48 [0.30; 0.66]; P < .0001. In 6 cases, the first set of the BCs sampled at inclusion was negative. To compare operating value of BC with the one of LC-SF, we deleted this 6 BC to evaluate sensitivity. Consequently, accuracy characteristics of both blood culture and LC-SF test for diagnosing infection were done only at time of inclusion. Sensitivity of BCs (26/55) was 47% (34%; 60%), specificity (57/60) was 95% (89.5%; 100%), positive predictive value (PPV) was 90% (79%; 100%), and negative predictive value (NPV) was 66% (56%; 76%). With LC-SF, sensitivity (28/55) was 51% (38%; 64%), specificity (50/60) was 83% (74%; 92%), positive predictive value was 74% (60%; 88%), and negative predictive value was 69% (59%; 79%). The performance of LC-SF was not different between patients with and without neutropenia (data not shown). Interestingly, LC-SF identified sepsis-causing pathogens in 11 patients with negative BCs (Fig. 2). The expert panel concluded that these 11 pathogens were responsible for the sepsis episode in regard of the patients’ files and also considering the outcome under antimicrobial therapy. Within these 11 patients, 9 had a favourable outcome under antimicrobial therapy targeting the pathogen detected by the LC-SF. Nevertheless 2 patients died under specific treatment, but the expert panel concluded that the pathogens detected by LC-SF were the cause of the sepsis in these patients. One LC-SF positive for Enterobacter cloacae/aerogenes was associated with a pleural fluid culture growing with E. cloacae in this patient. In another patient, P. aeruginosa was isolated at a concentration greater than 105 CFU/ml from the BAL with positive LC-SF to Pseudomonas aeruginosa. Of the two patients with LC-SF positive for E. coli, one had a cellulitis of the pelvis with isolated E. coli and one had a colorectal cancer. Another patient who had

Figure 2

339 positive LC-SF to Enterococcus faecalis presented an anal fissure, as the one with the LC-SF positive for Klebsiella Two of these positive LC-SFs patients were positive for A. fumigatus and were indeed probable invasive aspergillosis based on the criteria of the EORTC20 showing multiple positive galactomannan antigens (Platelia Aspergillus test,Bio-Rad, Marnes-la-Coquette, France) in sera as the main biological criteria. Last three patients (2 E. coli and 1 S. aureus isolated ) received antibiotics treatment when they were included and experts judged considering radio-clinical data and outcome that the LC-SF result was pertinent. Twelve infections with negative BC and negative LC-SF were diagnosed using other tests: 3 pathogens were isolated from quantitative culture of bronchoalveolar-lavage (2 E. coli, 1 S. aureus), 3 from per-operative abdominal sampling (2 Candida, 1 S. maltophilia). One patient had acute CMV infection. Galactomannan antigen was positive in one patient, who had also A. fumigatus in his sputum (probable aspergillosis). Four more cases of aspergillosis were classified as “possible aspergillosis” on clinical and radiological data based on the criteria of the EORTC.20 In 10 cases, LC-SF results were considered as clinical false-positive because patients recovered with antimicrobials not adapted to the LC-SF result. However, experts could not rule out the possible spontaneous patients’ recovery. The LC-SF results were not relevant at a clinical viewpoint and might be either due to false positivity of the test or to circulating DNA or transient bacteraemia. The different microorganisms detected in the 110 included patients with suspected sepsis are detailed in Table 2. Of the 55 pathogens, 2 were not detectable by LCSF, namely, Candida norvegiensis and Citrobacter braakii. In a single patient, the BC and LC-SF results were discordant regarding the pathogen species: BC was positive for E. cloacae and LC-SF for Klebsiella oxytoca/pneumoniae. This finding can be ascribed to a sequence similarity frequently described between Klebsiella spp. and

Distribution of pathogens identified by blood culture and LightCycler
SeptiFast.

340

D. Maubon et al.

Table 2 Microorganisms detected in our 110 cancer patients with suspected sepsis. BC, detected by blood culture only; BCþLC-SF, detected by both blood culture and LC-SF; LC-SF, detected by LC-SF only; Other, detected neither by BC nor LC-SF but detected by other samples culture, Cytomegalovirus PCR, and/or galactomannan detection and radio-clinical data for Aspergillus. BC Gram-negative E. coli Klebsiella spp. E. cloacae/aerogenes Citrobacter braakia P. aeruginosa Acinetobacter spp. S. maltophilia Gram-positive S. aureus S. epidermidis Other CNS S. oralis S. pneumoniae E. faecium E. faecalis Fungi Aspergillus fumigatus Candida norvegiensisa Candida spp. Virus Total a

4 1 1 1 1 1

BC þ LC-SF 5 2 1

Other 2

LC-SF 4 1 1 1

1

1

1

1

1

1 2 1 1

3 1 2

1

1

15

17

1

Total 15 4 3 1 2 1 2 3 1 2 1 4 1 3

5 1 1 1

2

7 1 3 1

12

11

55

Not detectable by LC-SF (LightCycler SeptiFast).

Enterobacter spp.,21 and is also reported in the LC-SF package insert for the ITS region (4% of the tested Enterobacter strains were identical to Klebsiella). The SeptiFast Identification Software version 2.0 made two interpretation errors. In 1 of these 2 patients, BCs were positive for Klebsiella pneumoniae, E. faecalis, and Streptococcus pneumoniae and LC-SF was positive for Klebsiella pneumoniae/oxytoca, P. aeruginosa, and S. aureus. The melting peak for S. pneumoniae was present (channel 670 Gramþ Tm value, 56  C) but was not reported by the software. This problem also occurred in a previous study.15 The other patient had two sets of blood cultures and a catheter tip culture positive for Streptococcus haemolyticus with a favorable outcome under vancomycin. LC-SF melting peak corresponding to CoNS was present but reported as a contaminant by the SeptiFast Identification software because the peak was lower than the proposed cutoff.

Evaluations of treatment by the panels of experts The two panels that reviewed the patient data with and without the LC-SF results, respectively, agreed about the best empirical antimicrobials for 70 (64.2%) of the 110 patients. Of the 32 patients with positive BCs, 4 would have been treated earlier had the LC-SF results been taken into account (E. cloacae 2, n Z 2; methicillin-resistant CoNS, n Z 1; and S. pneumoniae, n Z 1), 4 would have received adequate treatment (S. maltophilia, n Z 1; Enteroccoci

spp., n Z 2; and E. coli with an extended broad-spectrum beta-lactamase, n Z 1), and 1 would have received treatment for an adequate duration (fever of unknown origin and LC-SF positive for E. coli treated for only 3 days without catheter removal, followed by catheter-related E. coli bloodstream infection). Of the 69 patients with negative BCs, 1 would have received earlier treatment for probable invasive aspergillosis had the LC-SF results been taken into account, 1 patient would have received narrower-spectrum antimicrobial therapy, 4 patients with febrile neutropenia would have received the correct diagnosis (E. coli 1, n Z 1; E. cloacae, n Z 2; and S. aureus, n Z 1), and 1 patient would have received antimicrobial therapy for shock (LC-SF positive for E. coli) mistakenly ascribed to cardiac tamponade. The experts concluded that LC-SF would have improved the initial treatment prescribed by the clinicians in 11 (10%) patients and would have led to the immediate prescription of antimicrobials by the experts in 3 (2.8%) patients. The LC-SF result would also have established the cause of fever in 4 neutropenic patients, who were however treated with appropriate antimicrobials.

Discussion Identifying the cause of sepsis in patients with malignancies is challenging, as the BCs are often negative.22,23 Identification of the causative pathogen increases the chances of selecting appropriate early antimicrobials and, therefore,

Impact of multiplex PCR in cancer patients with sepsis improves patient outcomes.24 Thus, of our 110 patients with suspected sepsis, 97 received antimicrobials before study inclusion and only 32 had positive BCs. LC-SF identified 11 pathogens not detected by BCs in 11 patients who had sepsis according to our reference standard (in regard of the patients’ files and also considering the outcome under antimicrobial therapy). However, in 10 other patients, LC-SF yielded false-positive results according to our reference standard, although the experts could not definitively conclude that these apparently false-positive results were devoid of clinical significance. The absence of bacteriological/mycological origin of the disease was accepted by the expert panel in 6 cases. We could not be sure that in some cases the experts could have missed an undiagnosed bacteriological or mycological disease. The LC-SF results would have prompted immediate antimicrobial therapy in 3 patients who did not receive this treatment and major changes in the antimicrobial regimen in 11 patients. The performance of LC-SF in detecting fungi deserves special attention. All patients with positive LC-SF results for fungi had clinical or laboratory evidence of candidemia or invasive aspergillosis. This result is encouraging, as none of the other available PCR methods reliably diagnoses fungemia in sepsis episodes.25 Common difficulties include contamination with Aspergillus spp., which leads to a high false-positive rate, and lack of standardization of commercial kits.26 Although the EORTC does not include a positive molecular test among the criteria for invasive aspergillosis, studies suggest that LC-SF may assist in the diagnosis of invasive aspergillosis.13,27 Another interesting point is that no false-positive results for Candida spp. occurred in our study, even in patients heavily colonized by these fungi. A larger study focusing on this aspect is needed to further investigate the performance of LC-SF in diagnosing fungal causes of sepsis. Overall agreement between BCs and LC-SF in our study was 70%, in keeping with earlier studies showing agreement rates in the 71%e88% range. The main objective of these studies was to retrospectively compare LC-SF results to BC results in specific populations with sepsis (e.g., immunocompromised patients10,13,14 or patients with endocarditis16) or in unselected patients with sepsis.8,9 The three studies of patients with neutropenia and/or malignancies10,13,14 found that LC-SF that LC-SF was a useful adjunct to BCs for identifying the cause of sepsis. In addition to comparing the diagnostic performance of LC-SF to that of BCs, we evaluated the potential of LC-SF for improving the management of patients with malignancies and sepsis in everyday clinical practice (by evaluating the proportion of patients who would have a therapeutic benefit secondarily to the LC-SF result). Other studies also pointed out the importance of evaluating others aspects than the biological efficiency of the LC-SF test.12,14,15,28,29 A study of 436 patients with sepsis suggested that multiplex PCR might decrease the number of days on inadequate empirical antimicrobial therapy by providing the diagnosis faster than the BCs.28 However, although LC-SF results are obtained within 6 h, the test requires a level of technical expertise that is not, up to now, usually available around the clock. In a retrospective review by two infectiologists of the potential clinical impact of LC-SF in 72 patients with suspected sepsis, it was estimated that the LC-SF

341 results would have improved the treatment in 8% of cases.15 Similarly, using a more appropriate methodology (blind assessment by two independent multidisciplinary panels of experts), we found a therapeutic impact in 10% of cases. A preliminary study of the clinical impact of LC-SF12 in 72 patients whose physicians received the LC-SF results in real-time and could use them for their treatment decisions showed that, based on clinical relevance, sensitivity was 78% and specificity 99%, which was considerably better than in our study.12 This study12 does not allow an evaluation of outcomes without the LC-SF results. Interestingly, the recent study from Bloos et al. on 142 severe sepsis shows a correlation between positive PCR based pathogen detection and disease severity even in negative BC.29 Our study involved an evaluation of clinical decisions with no direct impact on the patient. Prospective patient inclusion followed by a review of the medical records was used to investigate both the diagnostic value and the potential clinical and therapeutical impact of the test. The experts assessed the contribution of LC-SF whenever 10 additional cases were reviewed, and a clear benefit of LC-SF was to lead to premature study discontinuation with routine use of LC-SF in all patients. This stopping criterion was not met in our study. The design of the present study only explored the potential impact of the result of the PCR but might not assess the impact of the procedure in the real practice. Another trial using a randomized controlled trial is needed to confirm these results. Furthermore, we assessed neither the cost of LC-SF testing nor the potential savings made when LC-SF results led to improved treatment decisions. Our results indicate that LC-SF has both advantages and shortcomings. Our study assessed a potential therapeutic impact for 10% of patients with sepsis and malignancies. These results are encouraging but the accuracy of the test can be improved: of special concern is the high rate of clinically irrelevant positive results but also failure of the LCSF to detect a significant number of species detected by blood culture. These issues show the limits encountered with this test and more generally with molecular diagnostic. Multiplex PCR for sepsis have only been recently developed and future will probably bring up a lot of other new interesting diagnostic tools but many key problems have already been highlighted in regard of the existing.30,31 These critical questions need to be answered to make these tools as reliable as possible for clinicians and the patients’ care.

Conflict of interest The authors have no conflicts of interest to declare.

Acknowledgments We are grateful to Jessica Catinella, Gerard Bargue `s, and Stephanie Herwegh for technical support; Caroline Tournegros, who was the clinical research assistant; to Professor Patrice Morand, Professor Rene ´e Grillot and Dr Bernadette Lebeau for helpful discussions and to Dr Agne `s Bonadona, Dr Carole Schwebel, Dr Yohann Dubois, Dr Cle ´mence Minet, Dr Olivier Epaulard and Dr Virginie Hincky-Vitrat for their respective participation.

342 All material needed for multiplex PCR testing was donated by Roche Diagnostics. This study was supported by academic grants from the De´le´gation a` la Recherche Clinique et a` l’Innovation (DRCI 2007-AO-01-05350) of the publicly funded Grenoble University Hospital and by research grants from Roche Diagnostics, Brahms Diagnostics, Gilead Sciences, Merck, and Pfizer.

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