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Evaluation of Bloodstream Infections During Chemotherapy-Induced Febrile Neutropenia in Patients with Malignant Hematological Diseases: Single Center Experience

From year to year, it is important to get an overview of the occurrence of causative agents in febrile neutropenic patients to determine the empiric treatment. Thus our aims were to evaluate a four-year period regarding the prevalence of bloodstream infections and the most important causative agents. During this period, 1,361 patients were treated in our hematology ward because of various hematological disorders. 812 febrile episodes were recorded in 469 patients. At that time, 3,714 blood culture (BC) bottles were sent for microbiological investigations, 759 of them gave positive signal. From the majority of positive blood culture bottles (67.1%), Gram-positive bacteria, mainly coagulase-negative staphylococci (CNS), were grown. Gram-negative bacteria were isolated from 32.9% of the positive blood culture bottles, in these cases the leading pathogen was Escherichia coli. The high prevalence of CNS was attributed to mainly contamination, while lower positivity rate for Gram-negative bacteria was associated with the use of broad-spectrum empiric antibiotic treatment.


Introduction
Infectious complications, especially bloodstream infections (BSIs), are major causes of morbidity and mortality among patients who suffered from malignant hematological diseases and treated with intensive chemotherapeutic regimens [1,2]. In these clinical settings, bacterial cultures from blood are of great diagnostic value and the gold standard to detect bloodstream infections; in addition to this, the results of blood cultures provide epidemiological data which are useful to determine empiric antibiotic therapy. However, the diagnosis of BSIs is still challenging in this patient group, because about half of all BSI cases are culture negative mainly because of the frequently used prophylactic antibiotics [3]. To overcome the inhibitory effect of antibiotics, special blood culture bottles containing resin have been developed; thus, modest increase in the sensitivity of culture has been achieved [3]. In the early 1970s, introducing empiric treatment protocols and antibiotic prophylaxis, increasing use of certain chemotherapeutic drugs associated with frequent oral mucositis, and frequent use of central venous catheters have changed the spectrum of pathogens in febrile neutropenic patients shifting it from Gram-negative to Gram-positive bacteria, especially viridans group streptococci and coagulase-negative staphylococci [3][4][5][6].
The aim of this study was to evaluate occurrence of bacterial species causing bloodstream infections due to febrile neutropenic episodes in the hematology ward of the University Hospital in Szeged, Hungary, between 2005 and 2008.

Patients
Between 2005 and 2008, 469 patients with febrile neutropenia (230 females and 239 males, median age 60 years) were observed in our department with various hematological diseases. Collected data from patients' documentations included demographics of patients, diagnosis, febrile episodes, source of fever and source of infection, neutrophil count, and clinical signifi cance of the isolated organism. Infectious complications were categorized into three groups: 1 -fever of unknown origin (FUO), 2 -microbiologically documented infection (MDI), and 3 -clinically documented infection (CDI).

Definitions
Febrile neutropenia was defi ned if a single oral temperature was measured higher than 38.3 °C, or temperature was 38.0 °C or higher for 1 h. Neutropenia was defi ned as absolute neutrophil count (ANC) less than 0.5 × 10 9 /l or less than 1.0 × 10 9 /l and rapidly declined below 0.5 × 10 9 /l [7]. A single positive blood culture (BC) was considered signifi cant if the isolated strain was clinically relevant cause of the infection. Common skin contaminants (CNS, propionibacteria) were considered signifi cant only if they could be found in two consecutive BC samples or if there were concurrent skin, soft tissue, or catheter-related infections. BSI was defi ned as polymicrobial if more than 1 bacteria grew from BC on the same day. Microbiological results were retrieved from the Clinical Microbiological Laboratory information system. Medical database of patients was used to collect information on the hematologic diseases, presence of febrile neutropenic episode, duration of neutropenia, and source of infection.

Analysis of blood cultures
BC samples were taken at the onset of fever. In patients having central venous catheters, BCs were taken from both central and peripheral veins. For collection of blood culture, the blood culture system (BD Bactec, Beckton Dickinson, USA) including aerobic, anaerobic bottles, and bottles for fungi was used. After taking blood, bottles were immediately placed in an incubator, where these were incubated for 5-14 days depending on the type of the putative pathogens. In the case of positive signal produced by the instrument on the basis of bacterial or fungal growth, microscopic examinations (phase contrast microscopy and examination of Gram-stained preparations) and culture were performed. For aerobic culture, Columbia blood agar supplemented with 5% sheep blood (BioMérieux, Marcy l'Etoile, France), chocolate agar supplemented with Poly-ViteX (BioMérieux, Marcy l'Etoile, France), eosin methylene blue (Lab M, UK) and Sabouraud Chloramphenicol (Bio-Rad, France) agars, and, for anaerobic culture, Schaedler agar supplemented with 5% sheep blood (Bio-Mérieux, Marcy l'Etoile, France) were inoculated with one drop of blood. Plates were incubated at 37 °C for 24 h in a 5% CO 2 incubator or 37 °C for 24 h under normal atmosphere or at 37 °C for 48 h in an anaerobic cabinet (Concept 400; Ruskinn Technology Ltd., Bridgend, UK) under a gas composition of 85% N 2 , 10% H 2 , and 5% CO 2 . From pure culture, antibiotic susceptibility tests were per-formed on the basis of Clinical Laboratory Standard Institute recommendations [8][9][10][11].

Antibiotic protocol
At the onset of fever, after taking BC, broad spectrum antibiotics were started empirically (piperacillin-tazobactam, cefepime, and imipenem or meropenem). Antibiotic dosage was modifi ed according to the patient's renal function. Patients were examined once daily by their physician to detect potential source of infection. After 48-72 h observations, the patient's condition was reevaluated. Changes in empiric antibiotic therapy depended on BC results and clinical response. In afebrile and culture-negative patients with stable clinical state, empiric antibiotic treatment was continued until ANC reached 500/μl. Vancomycin was used in patients with central venous devices, persistent fever, and hypotension [12]. On day 4-5, in patients with persistent fever suggesting fungal infection on the basis of clinical signs and computed tomography (CT) scans, amphotericin B was applied.
Among Gram-positive isolates, coagulase-negative staphylococci (CNS) were identifi ed in 331 cases. These blood culture samples were collected from 161 febrile episodes of 149 patients. In 50 febrile neutropenic episodes, CNS were relevant as a causative agent of fever because of the coexistence of skin, soft tissue, and central venous catheter-related infections. In the case of the remaining 111 cases, contamination could be the source of CNS.
Among rarely isolated pathogens, Listeria sp. was identifi ed from one patient with acute myeloid leukemia (AML) due to second relapse in 2006. The patient had clinically documented pneumonia and was treated with ampicillin. In 2006 and 2008, Burkholderia cepacea was identifi ed from two patients. One of them was observed with AML and did not receive any chemotherapeutic treatment. Blood culture sample was taken from peripheral vein. In this case, the patient was treated empirically with levofl oxacin. The other patient suffered from relapsed Hodgkin lymphoma and was treated according to ESHAP chemotherapeutic regimen (combination of high-dose cytosine-arabinosid, methylprednisolone, cisplatin, and etoposide) through the central venous line. Two pairs of blood culture samples were taken due to febrile episode from catheter, and B. cepacea was grown from each samples. N. farcinica was isolated from a patient with large granular lymphocytic (LGL) leukemia. He underwent six cycles of combined chemotherapeutic treatment (cyclo- phosphamide, vincristine, and prednisolone) and later had long term steroid therapy due to Coombs positive hemolytic anemia and active hemolytic events. On hospital admission, CT scan showed multiple lesions with perifocal oedema, but stereotactic core biopsy from lesions could not be performed because of the patient's severe clinical status. From one pair of blood culture, N. farcinica was isolated, but the patient died before adequate therapy could start. During the examined period, four cases of bacteremia caused by Fusobacterium nucleatum could be observed in this patient group. Two patients had acute myeloid leukemia, one patient had acute myelomonocytic leukemia, and one patient suffered from pre-B-cell lymphoblastic leukemia. Two patients received chemotherapy before positive blood culture; one of them had oral mucositis associated with the applied chemotherapy.

Discussion
Febrile neutropenia is the most important complication of chemotherapy in patients with hematologic malignancy. This may have infl uence on the applied chemotherapy; dose reduction or treatment delays can be observed frequently when febrile neutropenia is present, and this also has unfavorable long-term outcome in otherwise curable malignancy [13]. Bloodstream infections are among the most important bacterial infections, despite the development in the fi eld of microbiological diagnosis and antimicrobial therapy; these infections are responsible for the large proportion of nosocomial infections worldwide. In the early 1960s, the importance of bloodstream infection in neutropenic patients had been recognized; thus, empirical treatment protocols were established for mainly Gramnegative bacteria [14]. Later, the spectrum of pathogens associated with BSI shifted from Gram-negative bacteria to Gram-positive bacteria due to the increased use of antibiotic prophylaxis and indwelling catheters allowing colonizations and infections with the skin fl ora. Nowadays, the most common pathogens isolated from blood are coagulase-negative staphylococci and various antibiotic-resistant bacteria. In the majority of cases, the source of these infections is unknown in spite of various efforts to fi nd them. Recognition of changes in the epidemiology of BSIs is very important to modify the antibiotic policy because, on the basis of these fi ndings, we can reduce the infectionrelated morbidity and mortality [15,16]. During the fouryear study period, the incidence of bacteremia was 20.4%. Similar fi ndings were reported in the literature; Klastersky et al. showed that the incidence of bacteremia was 23% when they examined over two thousand patients with febrile neutropenia in cancer patients, while Viscoli et al. found that bacteremia occurred in 29% of patients with febrile neutropenia [17,18]. Our fi ndings correlated with the abovementioned literature data because, from the majority of blood culture bottles (13.7%), Gram-positive bacteria were isolated. Sixty-fi ve percent of Gram-positive bacteria belonged to coagulase-negative staphylococci. However, in a study by Winston et al. in North America, Gramnegative bacteria (55.6%) [19] were responsible for the majority of bacteremia in febrile neutropenic patients. At the same time, other authors from Italy or France showed that the most important isolates in neutropenic patients are Gram-positive bacteria, including coagulase-negative staphylococci or streptococci, while Gram-negative organisms including E. coli or Klebsiella spp. and P. aeruginosa constitute smaller portion of the isolates [20,21]. In our case, the most frequently used empirical treatment in this patient group is piperacillin-tazobactam or, if the patient has colitis or the possibility of abdominal infection is arisen, imipenem or meropenem is the frequently used antibiotic. Thus, the increased incidence of Gram-positive bacteria can be explained by the applied empirical antibiotic treatment, while the presence of coagulase-negative staphylococci could be attributed to the frequently used central venous catheters. The incidence of bacterial species in blood cultures can be infl uenced by the applied chemotherapy. In our case, 30% of patients with acute leukemia received high-dose Ara-C chemotherapy, and 15% and 12% of patients were treated with fl udarabin and Ara-C plus idarubicin, respectively. On the basis of literature data, increasing prevalence of Gram-positive cocci in febrile neutropenic patients could be observed after highdose cytarabine chemotherapy; this was confi rmed by our results [21]. Cordonnier et al. showed that the prevalence of staphylococci is higher than the prevalence of streptococci and enterococci in febrile neutropenic patients [21]. Similarly, our results confi rmed this because, among Gram-positive bacteria, the majority of the isolated strains were coagulase-negative staphylococci; 6.7% and only 2.4% of Gram-positive bacteria belonged to Enterococcus spp. and β-hemolytic streptococci, respectively. A total of 331 blood culture samples proved to be positive for coagulase-negative staphylococci and were collected from 161 febrile episodes of 149 patients. In 50 febrile neutropenic episodes, coagulase-negative staphylococci were relevant pathogen of fever because of the coexistence of skin, soft tissue, and central venous catheter-related infection. The remaining 111 cases were supposed to be contamination. From 32.9% of positive blood cultures bottles, Gramnegative bacteria were cultured; the majority of these proved to be positive for E. coli (52%). The second most common isolate was P. aeruginosa (14%), while the third one was Klebsiella spp. (9.6%). Similar fi ndings by Ramphal could be found [16]. In this review, the results of four articles were analyzed, and, among Gram-negative organisms, the most important pathogens were also E. coli, Klebsiella spp., and P. aeruginosa [16].
Among rarely isolated bacteria, Achromobacter xylosoxidans and Burkholderia cepacia are usually associated with catheter-related sepsis, while S. maltophilia can cause mainly nosocomial bacteremia [22] and the possible source of Haemophilus infl uenzae, Neisseria spp., and Gemella spp. is the damaged oral mucosa. Because of the possible presence of unusual pathogens, such as anaerobic bacteria or fastidious microorganisms, the use of various blood culture bottles including anaerobic bottles should be considered.
Because of its rapid progression of infection in febrile neutropenic patients and diffi culties in distinguishing infection from noninfected patients on the basis of clinical presentation in this patient group, the use of empirical antibiotic treatment is essential and may provide the possibility to reach better outcome.