Multiplex Blood PCR in Combination with Blood Cultures for

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Multiplex Blood PCR in Combination with Blood Cultures for

     JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 35103516 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00147-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

Multiplex Blood PCR in Combination with Blood Cultures for

    Improvement of Microbiological Documentation of

    Infection in Febrile Neutropenia

    1221211F. Lamoth,K. Jaton,G. Prodhom,L. Senn,J. Bille,T. Calandra,and O. Marchetti*

    12Infectious Diseases Service, Department of Medicine,and Institute of Microbiology,Centre Hospitalier Universitaire Vaudois and

    University of Lausanne, Lausanne, Switzerland

    Received 22 January 2010/Returned for modication 15 March 2010/Accepted 10 August 2010

The frequent lack of microbiological documentation of infection by blood cultures (BC) has a major impact

    on clinical management of febrile neutropenic patients, especially in cases of unexplained persistent fever. We assessed the diagnostic utility of the LightCycler SeptiFast test (SF), a multiplex blood PCR, in febrile

    neutropenia. Blood for BC and SF was drawn at the onset of fever and every 3 days of persistent fever. SF

    results were compared with those of BC, clinical documentation of infection, and standard clinical, radiolog- ical, and microbiological criteria for invasive fungal infections (IFI). A total of 141 febrile neutropenic episodes

    in 86 hematological patients were studied: 44 (31%) microbiologically and 49 (35%) clinically documented

    infections and 48 (34%) unexplained fevers. At the onset of fever, BC detected 44 microorganisms in 35/141 (25%) episodes. Together, BC and SF identied 78 microorganisms in 61/141 (43%) episodes (P 0.002 versus

    BC or SF alone): 12 were detected by BC and SF, 32 by BC only, and 34 by SF only. In 19/52 (37%) episodes

    of persistent fever, SF detected 28 new microorganisms (7 Gram-positive bacterial species, 15 Gram-negative bacterial species, and 6 fungal species [89% with a clinically documented site of infection]) whereas BC

    detected only 4 pathogens (8%) (P 0.001). While BC did not detect fungi, SF identied 5 Candida spp. and

    1 Aspergillus sp. in 5/7 probable or possible cases of IFI. Using SeptiFast PCR combined with blood cultures improves microbiological documentation in febrile neutropenia, especially when fever persists and invasive

    fungal infection is suspected. Technical adjustments may enhance the efciency of this new molecular tool in

    this specic setting.

     Febrile neutropenia is a frequent life-threatening complica- therapy (26, 28, 42). Homemade multiplex or broad-range PCR assays for the detection of bloodstream pathogens in tion in patients with hematological malignancies. Blood cul- cases of sepsis or febrile neutropenia have provided variable tures (BC) identify a pathogen in only 20 to 30% of febrile sensitivity and specicity compared with blood cultures (5, 8, 9, episodes, and the rate of microbiological documentation drops 28, 31, 42, 44). Their use is limited by the lack of standardized to less than 10 to 15% for those receiving antibiotics at time of technical procedures and commercially available systems. The sampling (6, 39). The majority of episodes are thus uniquely LightCycler SeptiFast test (SF) (Roche Diagnostics GmbH, managed based on the presence of fever and/or of a clinical site of infection. In addition, the low sensitivity of cultures for the Mannheim, Germany) is a real-time multiplex PCR amplica- detection of fungi is a major concern in these patients at high tion assay designed to detect a broad spectrum of bacteria and risk of invasive mycoses (13, 33). The diagnosis of invasive fungi in human blood from nonneutropenic patients with sep- fungal infections (IFI) based on clinical, radiological, and mi- sis (21, 30). The internal transcript spacer (ITS) region of the crobiological criteria according to the European Organization bacterial and fungal genome is the target selected for identi- for Research and Treatment of Cancer-Mycoses Study Group cation of 25 bloodstream pathogens. The aim of this study (EORTC-MSG) classication is often only presumptive and was to assess the appropriateness and utility of SF for the

    delayed (11). The persistence of fever despite broad-spectrum microbiological diagnosis of infection in febrile neutropenic

    antibacterial therapy is observed in one-third of cases, and the cancer patients.

     lack of identication of the causal pathogen results in empir-

     ical modication of antibacterial therapy and adjunction of MATERIALS AND METHODS antifungal therapy (10, 18, 29). Additional tools are thus Patients. This prospective, observational study was conducted in the isolation needed for the diagnosis of infection. ward of the Infectious Diseases Service at the University Hospital of Lausanne Molecular methods are able to rapidly detect microorgan- (Switzerland) between September 2006 and November 2007. Consecutive adult isms without incubation and despite ongoing antimicrobial hematological patients undergoing induction or consolidation chemotherapy for acute leukemia or autologous hematopoietic stem cell transplantation were en- rolled after written informed consent. The study protocol was approved by the Institutional Ethical Committee. * Corresponding author. Mailing address: Infectious Diseases Ser- Clinical management. Patients were hospitalized in positive-pressure high- vice, Department of Medicine, Centre Hospitalier Universitaire Vau- efciency particulate air-ltered isolation rooms. During the neutropenic period, dois and University of Lausanne, Rue du Bugnon 46, CH-1011 Lau- no antibacterial prophylaxis was used and patients with mucositis and oral and/or sanne, Switzerland. Phone: 41 21 314 10 26 or 10. Fax: 41 21 314 10 18. gastro-intestinal tract Candida colonization received uconazole prophylaxis. E-mail: Diagnostic workup of fever and empirical antibacterial therapy were conducted Published ahead of print on 18 August 2010. according to guidelines of the Infectious Diseases Society of America (IDSA)



TABLE 1. Analytical spectrum of the LightCycler SeptiFast test as true pathogens according to the recommendations for management of febrile neutropenia (2, 18, 34). Gram-positive Gram-negative In BC-negative episodes, microorganisms identied by SF were interpreted as bacterial species bacterial species Fungal species true pathogens in the presence of at least one of the following criteria: (i) the same microorganism was recovered at the same time by cultures from a clinically Staphylococcus aureus Escherichia coli Candida albicans aStaphylococcus epidermidisKlebsiella pneumoniae Candida tropicalis relevant site other than blood (e.g., central venous catheter, urine, sputum, or aStaphylococcus haemolyticusKlebsiella oxytoca Candida parapsilosis bronchoalveolar lavage [BAL] uid or from another normally sterile site) or (ii) Streptococcus pneumoniae Serratia marcescents Candida krusei a site of infection was documented clinically and/or radiologically in concomi- aStreptococcus pyogenesEnterobacter cloacae, Candida glabrata tance with the positive SF nding and the microorganism identied by SF was Enterobacter aerogenes aconsistent with a potential pathogen according to the site of infection (28). In the Aspergillus fumigatus Streptococcus agalactiaeProteus mirabilis aabsence of the criteria listed above, the signicance of a positive SF result Streptococcus mitisPseudomonas aeruginosa Enterococcus faecium Acinetobacter baumannii remained indeterminate. Positive SF ndings for fungi were interpreted accord- Enterococcus faecalis Stenotrophomonas ing to the EORTC-MSG diagnostic classication of fungal infections (11). maltophilia Fishers exact test and the nonparametric Mann-Whitney rank-sum test were used for the analysis of proportions and continuous variables, respectively. A a For coagulase-negative staphylococci and streptococci, a semiquantitative two-sided P value 0.05 was considered statistically signicant. analytical cutoff value has been set by the manufacturer for distinguishing be- tween true pathogens and contaminants from the skin ora. RESULTS 141 febrile neutropenic episodes occurred in 86 consecutive (18). Modication of antibacterial therapy and addition of antifungal therapy patients, with a median of 1 per patient (range, 1 to 6). A total were based on the clinical course (persistent fever for 72 h, clinical deteriora- of 237 sets of blood samples for cultures (BC) and SeptiFast tion, and/or a new or progressing focus of infection) and microbiological reas- sessment (18). Whereas clinical management was routinely based on BC results, PCR (SF) were obtained, with a median of 2 sets per patient SF results were not available in real time for therapeutic decisions. (range, 1 to 8); of those sets of samples, 144 (61%) were drawn Denitions. Standard denitions of neutropenia (neutrophil count 500/ while broad-spectrum antibiotic therapy was ongoing. The 3mm) and fever (measured once at 38.3?C or twice at 38.0?C for a 12-h characteristics of patients and febrile episodes are shown in period) were used (18). A new febrile episode was dened as a new onset of fever Table 2. after a 72-h period of apyrexia. The etiology of febrile episodes was classied according to denitions of the International Immunocompromised Host Society Febrile episodes were classied according to the ICHS def- (ICHS): microbiologically documented infections with bacteremia (MDI-B) or initions as follows: 35 (25%) were MDI-B, 9 (6%) were MDI- without bacteremia (MDI-NB), clinically documented infections (CDI), or fever NB, 49 (35%) were CDI, and 48 (34%) were FUO (Table 2). of unexplained origin (FUO) (1). Standard denitions (CDC and WHO) were Diagnostic performance of blood cultures and SeptiFast used for CDI classications (15). Mucositis with a WHO score of 2 and diarrhea with a frequency of 8 episodes/day were considered to represent CDI blood PCR at the onset of fever (day 0). At the onset of fever, (3). Invasive fungal infections (IFI) were classied according to the denitions of 44 microorganisms (21 Gram-negative and 23 Gram-positive the EORTC-MSG (11). bacteria; no fungi) were detected by BC in 35/141 (25%) fe- Blood sampling. Sets of blood samples for cultures (BC) and for testing with brile episodes. SF identied 46 species of microorganisms (29 the LightCycler SeptiFast test (Roche Diagnostics GmbH, Mannheim, Ger- Gram-negative and 13 Gram-positive bacteria and 4 fungi) in many) (SF) were drawn at the onset of fever and every 3 days in cases of persistent fever. The same procedure was applied for each new febrile neutro- 35/141 (25%) episodes. Together, BC and SF allowed the iden- penic episode. Blood (10 ml) was collected in each culture bottle (Bactec Plus tication of 78 species of microorganisms in 61/141 (43%) aerobic/F and Lytic anaerobic/F; Becton Dickinson, Sparks, MD). Each set of episodes (P 0.002 compared with BC or SF alone): 12 were samplings consisted of 4 pairs (i.e., 4 aerobic and 4 anaerobic bottles [80 ml]) of detected by both BC and SF, 32 by BC only, and 34 by SF only blood culture samples drawn simultaneously from the central venous catheter (2 pairs of samples consisting of 2 aerobic and 2 anaerobic bottles [40 ml]) and by (Table 3). peripheral venipuncture (2 pairs of samples consisting of 2 aerobic and 2 anaer- Among the 32 species of microorganisms detected by BC obic bottles [40 ml]) according to the IDSA recommendations (18). For SF only, 13 (41% [10 Gram-positive and 3 Gram-negative bacte- assays, 3-ml samples of blood were collected in EDTA tubes (Monovette K- ria]) were not included in the SF analytical spectrum. Among EDTA; Sarstedt, Numbrecht, Germany). Each set of samplings consisted of 4 the remaining 19 isolates, 7 were Streptococcus spp., 3 were tubes (i.e., 12 ml) drawn immediately after blood cultures from the central venous catheter (2 tubes [6 ml]) and by peripheral venipuncture (2 tubes [6 ml]). coagulase-negative staphylococci (2 of them were recovered in SF samples were sent within 1 h to the laboratory and stored at 2 to 8?C. only 1 of 4 pairs of bottles), and 6 were Escherichia coli. A Microbiological analyses. A Bactec 9240 automated blood culture system clinically or radiologically documented focus of infection was (Becton Dickinson, Sparks, MD) was used. The vials were incubated at 35?C for present in 18 (56%) cases (13 cases of gastrointestinal mucosi- 5 days. For SF assays, DNA was extracted from the EDTA whole-blood tubes within tis, 2 catheter-related infections, 2 pneumonias, 1 urinary tract 48 to 72 h after sampling. Specimen preparation and DNA amplication and infection), while the 14 (44%) remaining pathogens were as- detection were performed in a dedicated laboratory according to the manufac- sociated with a primary bacteremia without a documented turers recommendations (21, 30). Each specimen included an inhibition control, source. These 32 species of microorganisms were all consid- and each run included positive, negative, and extraction controls. The SF ana- ered true pathogens according to the denitions described in lytical spectrum includes the most common Gram-positive and Gram-negative bacteria, as well as pathogenic fungi (Table 1). For coagulase-negative staphy- Materials and Methods. lococci and streptococci, a semiquantitative analytical cutoff value has been set Conversely, SF detected 34 species of microorganisms (11 by the manufacturer in order to avoid false-positive results due to contamination Gram-positive and 19 Gram-negative bacteria and 4 fungi) by colonizing skin ora (21, 30). which were not recovered by BC in 4/35 (11%) cases of Data and statistical analyses. Positive results for SF and BC were compared for neutropenic patients at the onset of fever (day 0) and during persistent fever MDI-B, 2/9 (22%) cases of MDI-NB, 12/49 (24%) cases of lasting for 3 days or more (days 3, 6, 9, etc.). Discordant results were interpreted CDI, and 8/48 (17%) cases of FUO. Of the 34 species of mi- according to clinical assessment and radiological plus microbiological ndings. croorganisms detected, 22 (65%) were interpreted as true Microorganisms recovered by BC (including coagulase-negative staphylococci) pathogens, i.e., the presence of the same microorganism re- in at least one of the four pairs (at least one of the four aerobic and four covered by culture at a site other than blood (n 1 [urine]) or anaerobic bottles) of the sets of samples drawn at the same time were classied


    TABLE 2. Characteristics of patients and febrile episodes TABLE 3. Microorganisms detected at the onset of fever (day 0) by ablood culture and/or the LightCycler SeptiFast test (SF) Febrile neutropenic patient aValue characteristic (n 86) No. of species detected by: Male/female.................................................................... 53 (62)/33 (38) Species Median age in yr (range) ............................................. 54 (1771) Blood Blood SF culture culture only Hematological malignancies only and SF Acute myeloid leukemia/acute Gram-negative bacteria 11 10 19 lymphoblastic leukemia............................................. 37 (43)/9 (10) 6 2 Escherichia coli 5 Lymphoma...................................................................... 12 (14) 1 2 4 Klebsiella pneumoniae/K. oxytoca Multiple myeloma.......................................................... 22 (26) 1 Enterobacter cloacae/E. aerogenes Other ............................................................................... 6 (7) 1 11 Pseudomonas aeruginosa 1 Acinetobacter baumannii Chemotherapy 1 2 Stenotrophomonas maltophilia Induction/consolidation for acute leukemia............... 45 (52) bb Capnocytophaga gingivalis1 Autologous HSCT....................................................... 37 (43) bSphingomonas paucimobilis 1 Other ............................................................................... 4 (5) bLeptotrichia spp. 1 Median no. of days of neutropenia (range)................... 11 (1140) Gram-positive bacteria 21 2 11 Staphylococcus aureus 7 Febrile episodes (n 141) Staphylococcus epidermidis/S. haemolyticus 3 1 3 First febrile episodes..................................................... 85 (60) Streptococcus mitis/S. agalactiae 7 Recurrent febrile episodes ........................................... 56 (40) bStreptococcus salivarius1 Ongoing antimicrobial therapy at 1 Enterococcus faecium 1 onset of fever ............................................................. 51 (36) 1 Enterococcus faecalis bEnterococcus spp. (other),c 3 Microbiologically documented infections bGemella spp.2 (MDI).......................................................................... 44 (31) bBacillus cereus2 Bacteremic...................................................................... 35 (25) b Bacillus spp. (other)1 Single Gram-positive bacterial species ................... 13 (37) b Corynebacterium spp.1 Single Gram-negative bacterial species .................. 15 (43) Polymicrobial.............................................................. 7 (20) Fungi 0 c4 0 Nonbacteremic.............................................................. 9 (6) Aspergillus fumigatus 1 Candida albicans 1 Clinically documented infection (CDI) .......................... 49 (35) Candida tropicalis 2 Upper/lower gastrointestinal tract............................... 36 (73) Upper/lower airways ..................................................... 6 (12) Total 32 12 34 Catheter/skin/soft tissues .............................................. 7 (14)

     a Microorganisms included in the SF analytical spectrum that cannot be dis- Fever of unexplained origin (FUO)................................ 48 (34) tinguished at the species level have been coupled (e.g., K. pneumoniae/K. oxytoca, Episodes with persistent fever for 3 days ................... 52 (36) E. cloacae/E. aerogenes, S. epidermidis/S. haemolyticus, S. mitis/S. agalactiae). b Pathogens not included in the SF analytical spectrum. Invasive fungal infections c Enterococcus gallinarum, Enterococcus durans, and other Enterococcus spp.

     (EORTC-MSG criteria) ........................................... 7 (5) Probable.......................................................................... 2 Possible ........................................................................... 5

    a Values represent number (percent) except where otherwise indicated. and in cases of CDI compared with FUO (a median of 1 and b HSCT, hematopoietic stem cell transplantation. a range of 1 to 4 versus a median of 1 and a range of 1 to 2; P c Four cases of enterocolitis (Clostridium dif;cile infection), one of pneumonia 0.03). (Pneumocystis jiroveci), and four of urinary tract infections (two Escherichia coli, one Enterobacter cloacae, and one Klebsiella pneumoniae). Clostridium dif;cile Episodes with persistent fever for >3 days. Persistent fever and Pneumocystis jiroveci are not in the SF analytical spectrum. In one of the four for 3 days despite ongoing antimicrobial therapy was ob- urinary tract infections, SF detected the same microorganism (K. pneumoniae/K. served in 52/146 (36%) febrile episodes (19 MDI, 23 CDI, and oxytoca) in blood. 10 FUO). The median duration of fever in these episodes was

    7 days (range, 3 to 25).

    Additional species of microorganisms were detected by BC of microorganisms consistent with a clinically or radiologically

    on or beyond day 3 of persistent fever in 4 (8%) episodes. In documented site of infection (n 22 [2 cases of oral and 10 of

    contrast, the yield of SF was signicantly higher, with 28 new gastrointestinal mucositis and 3 cases of respiratory tract, 2 of

    bacterial or fungal species identied in 19 (37%) episodes of urinary tract, and 5 of catheter-related or skin or soft tissue

    persistent febrile neutropenia (P 0.001). The four species infections). The pathogenic role of the 12/34 (35%) remaining

    detected by BC (one Stenotrophomonas maltophilia, two S. species of microorganisms was undetermined in the absence of

    a consistent site of infection. Pseudomonas aeruginosa and epidermidis, and one S. haemolyticus), two of which were also

    Staphylococcus aureus accounted for 4/12 and 3/12 of those detected by SF, were resistant to ongoing antibacterial therapy. remaining microorganisms. The 28 additional species of microorganisms detected by SF

    and not by BC were 15 Gram-negative bacteria, 7 Gram-pos- The number of positive SF tubes per sampling (i.e., 4 tubes)

    was signicantly higher when the same microorganism was also itive bacteria, and 6 fungi. Twenty-ve (89%) of them were

    identied by BC (median, 3 [range, 1 to 4]) compared with identied in the presence of a consistent clinical site of infec- BC-negative episodes (median, 1 [range, 1 to 4; P 0.0001]) tion: 2 oral and 19 gastrointestinal cases of mucositis, 3 respi-


    TABLE 4. Microorganisms identied by the LightCycler SeptiFast test (SF) only in samples from patients with blood culture-negative

    neutropenic episodes and with persistent fever on day 3 or beyond 3 days after onset of fever according to the clinically and/or radiologically documented site of infection

    Species identied on indicated day of fever (no. of episodes) Site of infection 3 3 P. aeruginosa, S. aureus Upper gastrointestinal tract aaE. coli (3), Klebsiella spp.,Enterobacter spp.,, P. aeruginosa, Lower gastrointestinal tract P. aeruginosa (4), S. aureus, E. faecium, A. fumigatus, C. parapsilosis E. faecium (2), C. albicans (2), C. tropicalis Lung P. aeruginosa, S. pneumoniae C. albicans Catheter/skin S. aureus P. aeruginosa No site P. aeruginosa, A. baumannii

     a Klebsiella spp. and Enterobacter spp. cannot be distinguished at the species level by SF. ratory tract infections, and 1 catheter-related or soft tissue DISCUSSION

    infection (Table 4). The performance of the LightCycler SeptiFast test (SF) in Invasive fungal infections (IFI). IFI was diagnosed accord- combination with blood culture (BC) was assessed with a large ing to the EORTC-MSG criteria (11) in 7 (5%) febrile epi- sample of febrile neutropenic patients. When used together, sodes: 2 probable and 1 possible cases of aspergillosis, 3 pos- SF and BC signicantly increased the rate of microbiological sible cases of candidiasis (2 of which were associated with documentation at the onset of fever compared with the use of seroconversion of antimannan antibodies), and 1 possible IFI. BC or SF alone. While the yields of BC alone and SF alone While no fungemia was detected by BC, SF detected fungi in were similar at the initial presentation of fever, SF provided a 5/7 IFI cases (4 candidiasis and 1 aspergillosis) (Table 5). high number of positive results in cases of BC-negative persis- Fungi were detected by SF at a median of 9 days (range, 6 to tent neutropenic fever by identifying new bacterial and fungal 15) after the onset of fever, while IFI was diagnosed by microorganisms consistent with a clinically documented site of EORTC-MSG criteria after a median of 23 days (range, 8 to infection. 106) and empirical antifungal therapy had been started after a Studies performed with nonneutropenic patients with sepsis median of 7 days (range, 6 to 8). SF diagnosis preceded showed that SF may be used in addition to BC to improve the EORTC-MSG diagnosis in 3 cases. SF was negative in two

    timing and performance of the etiological documentation of cases of probable pulmonary aspergillosis, with positive bron- 23, 27, 35, 36, 38, 41, 43). Few data infection (4, 7, 12, 14, 21choalveolar lavage (BAL) galactomannan and negative blood are available on the utility of SF in febrile neutropenia, al- galactomannan test results. In addition, SF was positive for

    though recent studies have suggested that it may be a helpful fungi in 4 febrile episodes that were not classied as IFI ac-

    cording to EORTC-MSG criteria (Table 5). Enterocolitis supplementary tool for the detection of bacteria or fungi (24,

    37, 40). BC typically fail to detect pathogens when antimicro- (CDI) and gastrointestinal tract colonization with Candida

    bial therapy is ongoing. Our study identied BC-negative ep- spp. were documented in 2 cases (positive SF results for C.

    albicans and C. tropicalis). In the remaining 2 cases, no clinical isodesin particular, those occurring in patients with persis- symptoms or signs of infection were found. tent neutropenic feveras the clinical subset in which this

    TABLE 5. Characteristics of febrile episodes with invasive fungal infections (IFI) and/or positive LightCycler aSeptiFast test (SF) results for fungi

    Gastrointestinal IFI CT nding Ongoing Persistent tract mucositis Serological marker SF result b(EORTC-MSG fever consistent antifungal and Candida (GM, Mn, anti-Mn)ccriteria) with IFI ( 3 days) therapy colonization dPos GMn in BAL Probable aspergillosis (lungs) d(lungs) Probable aspergillosis Pos GMn in BAL Possible aspergillosis A. fumigatus Neg (lungs) dPossible candidiasis C. albicans/C. parapsilosis Pos anti-Mn (liver, spleen) d(empirical) Possible candidiasis C. albicans Pos anti-Mn (liver, lungs) Possible candidiasis C. tropicalis Neg (spleen) (empirical) Possible IFI C. albicans Neg (lungs) (empirical) e None A. fumigatus Neg None C. albicans Neg (prophylactic) None C. tropicalis Neg e None C. tropicalis Neg

    a , condition present; , condition absent. b GMn, galactomannan; Mn, mannan; anti-Mn, anti-mannan antibodies; BAL, bronchoalveolar lavage uid; Pos, positive; Neg, negative. c Values represent those seen at the time of a positive SF result or of an IFI diagnosis (EORTC-MSG criteria) when the SF result was negative. d Positive galactomannan, index 0.5; positive anti-mannan, 10 arbitrary units/ml. e Computed tomography (CT) not done.


    molecular diagnostic tool may best complement cultures for internal control results were negative in our cases, low-level

    DNA contamination cannot be ruled out. Blood sampling from the etiological diagnosis of bacterial and fungal infections.

    indwelling vascular catheters or by venipuncture may represent These data suggest that SF is an efcient tool for the early

    another source of contamination by cutaneous ora. On the diagnosis of IFI when combined with clinical, microbiological,

    and radiological criteria. As the time window of SF positivity other hand, the transient presence of viable bacteria or de-

    graded bacterial DNA may be detected in bloodstream sam- overlapped with the start of empirical antifungal therapy, SF

    ples due to the breakdown of the protective barriers of skin may have an impact on therapeutic decisions. Moreover, the

    detection of circulating microbial DNA after prolonged appro- and gastrointestinal tract mucosal membranes and/or due to priate anti-infective therapy in a patient with persistent signs of the effect of ongoing efcacious antimicrobial therapy.

    infection suggests a sustained release from the infectious focus. Although SF looks promising for the early diagnosis of IFI, This nding and those reported from recent studies on molec- the small number of probable or possible IFI and the absence ular diagnosis performed in critically ill patients may provide a of microbiologically or histopathologically proven cases are new insight into the natural history of infection in these high- limitations of this data set that do not allow the estimation of risk settings (8, 22, 42, 43). Although SF does not provide the tests diagnostic performance. The failure of SF to detect information on the susceptibility of microorganisms, the spe- two cases of probable pulmonary aspergillosis with negative cies identication may be useful for the reassessment of the galactomannan antigenemia might suggest that its utility for appropriateness of ongoing antimicrobial therapy when infec- the diagnosis of localized infections is limited. These results tion is not responding to treatment. are consistent with the limited sensitivity and specicity of

    The occurrence of about one-third false-negative SF results blood PCR methods for the diagnosis of aspergillosis reported has been reported in studies of nonneutropenic patients (22, in previous studies (25). SF detected Candida DNA in four

    43). While SF failed to detect frequent bloodstream pathogens cases of probable or possible invasive candidiasis, while BC such as E. coli, coagulase-negative staphylococci, and strepto- results remained negative. Minor differences among BC sys- cocci, about 40% of the false-negative SF results in our analysis tems with respect to detection of Candida spp. do not explain

    occurred with microorganisms not included in the SF analytical the discrepancy discussed above, which is consistent with the spectrum. The smaller blood volumes used for SF compared identication by PCR of the circulation of unviable fungal with BC may have contributed to the failure to detect some components or of very low viable fungal loads (17, 32). How- pathogens. The low sensitivity of SF for coagulase-negative ever, not having used BC bottles containing a specic fungal

    staphylococci and streptococci is possibly associated with the medium may have potentially limited the detection of yeasts by semiquantitative analytical cutoff value specically set in order cultures.

    to distinguish contamination from infection in nonneutropenic The SF has been designed for the detection of pathogens in patients with sepsis (21). Finally, the presence of high bacterial septic nonneutropenic patients, and adjustments of the micro- loads might have resulted in a paradoxical inhibition of the bial spectrum and the analytical technique may improve its molecular detection. efciency in febrile neutropenic patients (21). In particular, The absence of a reliable diagnostic gold standard is a com- reassessment of the semiquantitative analytical cutoff thresh- mon limitation for the assessment of new molecular tech- old for coagulase-negative staphylococci set by the manufac- niques: in particular, negative BC results due to ongoing anti- turer in order to distinguish true pathogens from contaminants microbial therapy or small circulating microbial loads and for intensive care unit (ICU) or emergency department pa- positive BC results due to contaminations represent a major tients might be appropriate for febrile neutropenic cancer pa- limitation for the interpretation of positive or negative SF tients, for whom a central line is very frequently used; the results (28). In this context, the calculation of sensitivity, spec- interpretations and implications for management of the recov- icity, positive and negative predictive values, and likelihood ery of coagulase-negative staphylococci from blood may sub- ratios as well as of the efciency of the SF may be inappropri- stantially differ from those in other clinical settings, and the ate. Experts have recommended the interpretation of positive absence of neutrophils in the analyzed blood may inuence the

    PCR results for BC-negative febrile episodes according to the detection of microorganisms. In addition, the technique in its clinical context (26, 28). In the present analysis, the majority of current version is work-intensive: automating the test protocols the microorganisms detected by SF could be considered to would facilitate its cost-effective use on a routine basis. represent the etiological cause in the presence of a clinically or In conclusion, the LightCycler SeptiFast test gives new in- radiologically documented site of infection. These results may sights into the natural history of infection during neutropenia. be particularly signicant for patients with persistent or pro- These data suggest that this multiplex PCR technique, when gressing signs of infection, who may benet from a modica- combined with BC, provides clinically relevant information for tion of empirical antimicrobial therapy (4, 26, 35). Our data the diagnosis of infection in cases of BC-negative febrile neu- also suggest that the number of positive SF tubes may contrib- tropenia, particularly in persistent fever despite antibacterial ute in discriminating true-positive from false-positive results. therapy, when a nonresponding bacterial infection or an inva- Although the detection of P. aeruginosa and S. aureus in blood sive fungal infection is suspected.

     cultures usually reects true infection, the unusually large pro-

     portion of these bacteria in a single SF tube in the absence of ACKNOWLEDGMENTS a documented focus may suggest a potential contamination.

    Despite the use of MagNA Pure water, contamination of PCR We thank Cyril Andre?, Rene? Brouillet, Monika Ochsner, Annie assays by waterborne bacteria such as Pseudomonas spp. is Savoie, and the staff of the Isolation Ward of the Infectious Diseases frequently reported (16, 19, 20). Although all the assay-specic Service and of the Laboratory of Microbiology and Molecular Diag-


nostics of the Institute of Microbiology for outstanding assistance in sted (ed.), APIC infection control and applied epidemiology: principles and practice. Mosby, St. Louis, MO. collection and management of clinical data and blood samples. 16. Grahn, N., M. Olofsson, K. Ellnebo-Svedlund, H. J. Monstein, and J. Financial support for this work was provided in the form of an Jonasson. 2003. Identication of mixed bacterial DNA contamination in unrestricted research grant from Roche Diagnostics GmbH, Rotkreuz, broad-range PCR amplication of 16S rDNA V1 and V3 variable regions by Switzerland, and Penzberg, Germany. pyrosequencing of cloned amplicons. FEMS Microbiol. Lett. 219:8791. Potential conict of interest: Jacques Bille, Thierry Calandra, and 17. Horvath, L. L., B. J. George, C. K. Murray, L. S. Harrison, and D. R. Oscar Marchetti attended advisory board meetings and/or gave lec- Hospenthal. 2004. 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