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-