Abstract
This study aimed to assess the clinical utility of PCR for the analysis of bacteria and fungi from blood for the management of febrile neutropenic patients with hematologic malignancies. Using a PCR system able to detect a broad range of bacteria and fungi, we conducted a prospective pilot study of periodic analyses of blood from patients following intensive chemotherapy. When fever occurred, it was treated with empirical antibiotic therapy, basically without knowledge of the PCR results. In 23 febrile episodes during the neutropenic period, bacteria were detected by PCR in 11 cases, while the same species were identified by blood culture in 3 cases. In 10 out of 11 PCR-positive cases, fever could be managed by empirical therapy. In the empirical-therapy-resistant case, the identification of Stenotrophomonas maltophilia by PCR led to improvement of fever. No fungi were detected by PCR in febrile cases, while Aspergillus fumigatus was detected in one afebrile patient, several days before a clinical diagnosis was made. In subsequent sporadic PCR analyses in 15 cases of febrile neutropenia, bacteria were detected by both PCR and blood culture in 7 cases and by PCR alone in 6. Fungi were not detected. While fever was improved by empirical therapy in 12 out of the 13 PCR-positive cases, the identification of Pseudomonas aeruginosa by PCR in one therapy-resistant case contributed to the successful treatment of persistent fever. Our results indicate that PCR analysis of bacteria from blood provides essential information for managing empirical-therapy-resistant febrile neutropenia.
Management of febrile neutropenia in hematological patients undergoing intensive chemotherapy is important, because bacterial or fungal infections during prolonged neutropenia are major causes of morbidity and mortality in these patients (3, 14, 17, 26, 28, 32). These infections can rapidly become life-threatening if not treated appropriately and promptly (5, 23, 32). Therapeutic decisions should ideally be made based on microbial isolation. However, the sensitivity of microbial culture tests remains low. Despite clinicians' best efforts, it has been shown that specific pathogens were identified in only about 20 to 30% of febrile neutropenia cases (19, 20, 28, 33, 39). PCR-based molecular detection of fungal DNA from blood may be a promising tool for the early diagnosis of invasive fungal diseases; however, standardization and clinical validation are tasks that remain (1, 2, 9, 13, 22, 29). PCR analysis of bacteria from blood is reported to be more sensitive than blood culture (16, 19, 37-40). Nevertheless, prompt empirical therapy with broad-spectrum antimicrobial agents, sometimes modified by the results of blood cultures, has led to a dramatic reduction in the mortality rate in febrile neutropenia (14, 17, 23, 26, 28, 30, 32, 34). Thus, even though bacterial PCR analysis is a sensitive tool for diagnosing bacterial infections, it is more costly than blood culture, and its general utility during empirical antibiotic therapy in febrile neutropenia patients remains unclear.
To address this issue, we initially performed a pilot study to prospectively detect bacterial or fungal infections in peripheral blood after chemotherapy, using a PCR analysis system able to detect a broad range of species of bacteria (24, 36) and fungi. In this pilot study, the PCR results were not disclosed to the physicians unless more than two physicians agreed that disclosure of the PCR results was required for treatment. Based on the results of this study, we performed bacterial PCR analysis in sporadic febrile neutropenic cases but disclosed the PCR results as soon as possible. We observed that, although bacteria were detected by PCR analysis more often than by blood cultures, most febrile neutropenia cases could be managed with empirical antibacterial therapy, without access to PCR data. However, PCR analysis was useful in the diagnosis and treatment of empirical-therapy-resistant febrile neutropenia.
MATERIALS AND METHODS
Ethics.
This study was approved by the Human Research Ethics Committee of Mie University. Written informed consent was obtained from all patients recruited.
Blood collection, culture, and serodiagnostic tests.
For febrile neutropenia, we undertook blood cultures at least twice to increase the sensitivity. We routinely collected 2.5 ml of blood into a pediatric aerobic bottle and 5 ml into an anaerobic bottle; this is the usual procedure in Japan.
EDTA-anticoagulated peripheral blood (1 ml) was collected after the venipuncture site was wiped with 70% alcohol followed by two cycles of 2 min of disinfection with povidone-iodine. Samples for blood culture were cultured in an automated system (BacT/Alert 3D; bioMérieux, France). (1-3)-β-d-Glucan (BDG) and galactomannan (GM) were measured in each sample using the Beta-glucan test (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and Platelia Aspergillus enzyme immunoassay (EIA) (Bio-Rad, Marnes-la-Coquette, France).
Collection and handling of blood specimens for PCR analysis.
Extraction and purification of DNA from bacteria and fungi were performed by modification of previously described methods (24, 36). To prevent contamination, all procedures were performed inside a laminar flow clean bench and no more than two tubes were opened simultaneously. We used hydrophobic filter barrier pipette tips. Pipettes were disinfected with 5% sodium hypochlorite and exposed to UV light after the experiments. EDTA-anticoagulated peripheral blood (1 ml) was collected and centrifuged at 1,000 × g for 10 min. Based on the sensitivity test (data not shown), we used the supernatant and buffy coat for detection of bacteria and fungus, respectively. Thus, the supernatant was centrifuged at 13,000 × g for 10 min, and the pellet was washed with phosphate-buffered saline. Bacterial DNA was extracted and purified from the pellet, using Mora extract (Kyokuto Seiyaku, Co., Ltd., Tokyo, Japan), in accordance with the manufacturer's instructions. For extraction and purification of fungal DNA, the buffy coat from whole blood was washed twice with phosphate-buffered saline (PBS) and centrifuged at 3,000 × g for 10 min. The supernatant was decanted off, and the pellet was reacted with 50 μl of lysis buffer (COBAS Amplicor S.E.T.S II kit; Roche Diagnostics, Meylan, France) at room temperature for 2 min and then centrifuged at 1,000 × g for 1 min. The pellet was again incubated with lysis buffer at 90°C for 20 min and then centrifuged at 13,000 × g for 10 min. Fungal DNA was extracted and purified from the pellet, using Mora extract (Kyokuto Seiyaku).
Amplification and detection of bacterial DNA.
Amplification and detection of bacterial DNA was performed as previously reported (24, 36). Briefly, the complete 16S rRNA gene was amplified by PCR using two oligonucleotide primers: UN-F (5′-CAGCAGCCGCGCTAATAC-3′) and UN-R (5′-CCGTCAATTCCTTTGAGTTT-3′). PCRs were carried out in a DNA thermal cycler (GeneAmp PCR system 9600; Applied Biosystems, Foster City, CA) with preliminary denaturation at 95°C for 10 min, followed by 45 cycles of amplification consisting of denaturation at 94°C, primer annealing at 62°C, and elongation at 72°C, each lasting for 1 min. For amplification of DNA, we used AmpliTaq Gold (Applied Biosystems), based on our observation that identical results could be obtained with AmpliTaq Gold LD (Applied Biosystems), which rigorously excludes the contamination of endogenous E. coli DNA to a level as low as possible (data not shown). For species identification, positive PCR products (402 bp) were sequenced using the ABI PRISM BigDye terminator cycle sequencing ready reaction kit and ABI PRISM 377 genetic analyzer (Applied Biosystems). For phylogenetic identification, sequences were compared with those of known bacteria listed in official databases using the BLAST program available at the National Center for Biotechnology Information (http://ncbi.nlm.nih.gov).
Amplification and detection of fungal DNA.
The complete 18S rRNA gene was amplified by the first-round PCR using two oligonucleotide primers: Fung-F (5′-TTCGATGGTAGGATAGTGGCC-3) and B4R (5′-TGATCGTCTTCGATCCCCTA-3′). PCRs were carried out in a DNA thermal cycler with preliminary denaturation at 94°C for 5 min, followed by 40 cycles of amplification consisting of 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min. After amplification, all PCR products (683 bp) were precipitated by the addition of ethanol and amplified by nested PCR. The primers, except those for Zygomycota, were designed to separate PCR products into groups based on their susceptibilities to antifungal agents. We used reacting systems for the group resistant to fluconazole (FLCZ) (Fung-F and n-Asp/Pen R [5′-AGCCAGTGAAGGCCATG-3′]; 410 bp), the group moderately resistant to FLCZ (Fung-F and n-C.glab-R [5′-CCAACGGACAAGGACTTGG-3′]; 405 bp), the group susceptible to FLCZ (n-Candida-F [5′-TTTGATGCGTACTGGACCCA-3′] and B4R; 337 bp), and a wide-range fungal group (n-Fung-F [5′-GAATAAGGGTTCGATTCCGG-3′] and n-Fung-R [5′-CCCCGACCGTCCCTATTAAT-3′]; 410 bp). We planned for wide-range fungal PCR to be simultaneously performed in the second-round PCR to detect the small amounts of fungal DNA that could not be detected by first-round PCR and the sequence identified. We also aimed to validate that the gene products amplified by the susceptibility primers were indeed derived from fungal DNA and to obtain the same level of sensitivity to detect fungal species other than Candida sp., Aspergillus sp., and Penicillium sp. that were detectable by nested PCR with these susceptibility primers. The temperature conditions and number of cycles were the same as in the first-round PCR. Species were identified in any positive PCR products using the same methods as for bacteria. We performed the specificity test for the following bacteria and fungus and obtained the expected results.
Positive and negative controls and validation of bacterial and fungal PCR.
In each PCR assay, we had positive and negative controls. The positive controls for bacterial PCR were 100 CFU/ml of Staphylococcus aureus ATCC 29213, 500 CFU/ml of Candida albicans JCM1542, 500 CFU/ml of C. glabrata JCM1539, and 500 CFU/ml of Aspergillus fumigatus JCM1617. The negative control was nuclease-free water. When these positive and negative controls did not work as expected, we considered the assay as inappropriate. We validated bacterial and fungal PCR using 18 strains of nine bacteria and 26 strains of 13 fungi (data not shown). The sensitivity to detect fungus was 100 CFU/ml, while that to detect bacteria was 50 CFU/ml. Furthermore, in healthy donors, bacteria were detected in 2 (2.3%) of 87 samples. The detected bacteria, methicillin-resistant S. aureus (MRSA) and S. epidermidis, were both bacteria indigenous to the skin (M. Masuya, personal communication, December 2009).
Prospective study population, setting, and design.
From April 2007 to May 2008, 7 patients receiving intensive chemotherapy for acute leukemia or myelodysplastic syndrome (MDS) were enrolled in this study. Their characteristics are listed in Table 1. After intensified chemotherapy, EDTA-anticoagulated whole-blood specimens (1 ml) were collected for PCR analysis once a week during the neutropenic period (neutrophil counts of ≤500/μl) or during febrile episodes during the neutropenic period. Blood samples for culture and BDG and Aspergillus antigen analysis were collected simultaneously. The PCR results were not known to the physicians and were thus not utilized in their clinical management of the patients. However, the results were disclosed if the patient's fever continued despite empirical therapies and if more than two physicians requested the PCR results. Levofloxacin (300 mg/day) and fosfluconazole (200 mg/day) or itraconazole (ITC) oral solution (200 mg/day) were administered prophylactically in all patients. However, if patients developed a fever (≧38.0°C) during the neutropenic period, antibiotic therapy was initiated, based on the guideline of the Infectious Diseases Society of America (14, 30). Body temperature was measured at an axillary fossa after perspiration was wiped off, according to routine practice in Japan.
TABLE 1.
Patient characteristics in prospective studya
| Patient | Age (yr) | Sex | Primary disease | Disease status | Chemotherapy regimen |
|---|---|---|---|---|---|
| 1 | 24 | Male | AML (M5a) | 1st relapse | Reinduction therapy (FLAG-M), UR-BMT (TBI and CY) |
| 2 | 43 | Male | T-LBL | Refractory | CBT (CA, CY, and TBI) |
| 3 | 44 | Male | MDS (RAEB) | 1st CR | CBT (G-CSF, CA, CY, and TBI) |
| 4 | 57 | Male | MDS, AML | Just after diagnosis | Induction therapy (DNR and Ara-C) |
| 5 | 62 | Male | Ph-negative ALL | Just after diagnosis, 2nd CR | Induction therapy (Ara-C, ETP, and Dex) RICBT (Flu, Mel, and 4 Gy TBI) |
| 6 | 64 | Female | AML (M2) | 2nd CR | 4th consolidation (A triple V) |
| 7 | 78 | Male | MDS, AML | Just after diagnosis | Induction therapy (DNR and BHAC) |
AML, acute myeloid leukemia; T-LBL, T-cell lymphoblastic lymphoma; MDS, myelodysplastic syndrome; RAEB, refractory anemia with excess of blasts; Ph, Philadelphia chromosome; ALL, acute lymphoblastic leukemia; CR, complete remission; FLAG-M, fludarabine, cytosine arabinoside (Ara-C), granulocyte colony-stimulating factor (G-CSF), novantrone; UR-BMT, unrelated bone marrow transplantation; CBT, cord blood stem cell transplantation; CA, cytarabine; CY, cyclophosphamide; TBI, total body irradiation; DNR, daunorubicin; ETP, etoposide; Dex, dexamethasone; RICBT, reduced intensity cord blood transplantation; Mel, melphalan; A triple V, cytosine arabinoside, etoposide, vincristine, and vindesine; BHAC, behenoyl cytarabine.
Sporadic analysis of bacteria by PCR in febrile neutropenia.
In the sporadic study, PCR analysis of bacteria and fungi was performed in patients with febrile neutropenia. The febrile neutropenia was defined as the patient having a single body temperature of 38°C or greater and having a neutrophil count of ≤500 neutrophils/μl. Fever was treated empirically, as in the pilot study, but physicians were informed of the PCR results as soon as possible.
RESULTS
PCR and blood culture results from the prospective pilot study.
To elucidate the clinical usefulness of PCR analysis in the treatment of febrile neutropenia, we first performed a prospective pilot study in neutropenic patients who received intensive chemotherapy. As described in Materials and Methods, peripheral blood was collected once a week or during febrile episodes during the neutropenic period after chemotherapy and was subjected to PCR analysis, blood culture, and serological examinations. In this study, PCR results were not routinely disclosed to the physicians.
A total of 7 patients were registered in this pilot study (Table 1), all of whom had more than one febrile episode during the neutropenic period. Bacteria were detected by PCR in 11 out of 23 episodes of febrile episodes, while they were detected by blood culture in 3 episodes. No fungi were detected either by PCR or blood culture during these episodes (Table 2). In these afebrile cases, bacteria were detected by PCR alone in 3 cases, and by blood culture alone in 3 different cases. Fungus was detected by PCR in one afebrile case (Table 2).
TABLE 2.
Prospective pilot study PCR and blood culture results for bacteria and fungi
| PCR result | No. of cases with blood culture result of: |
Total no. of cases | |
|---|---|---|---|
| Positive | Negative | ||
| Cases with fever | |||
| Positive | 3 | 8 | 11 |
| Negative | 0 | 12 | 12 |
| Total | 3 | 20 | 23 |
| Cases without fever | |||
| Positive | 0 | 4a | 4 |
| Negative | 3 | NAb | 3 |
| Total | 3 | 4 | 7 |
One fungal infection is included.
NA, not applicable.
Characteristics of 3 febrile cases where bacteria were detected by PCR analysis and blood culture.
As mentioned above, the same bacterial species were identified by blood culture and PCR in 3 febrile cases in the prospective study (Table 3). In these cases, the same bacterial species were repeatedly detected on more than one occasion from the first day of fever and then became undetectable after empirical antibiotic therapy. Notably, blood samples were positive for bacteria by PCR 2 and 9 days before positive blood cultures were detected in cases 1 and 3, respectively. These data indicate a high specificity and sensitivity of PCR for the analysis of bacteremia. Case 3 (Table 3; Fig. 1) was the only case in the prospective study where the PCR result was disclosed by request of the physicians, because of resistance to empirical therapy. This patient was a 64-year-old woman with acute myeloid leukemia (AML) (M2), at the nadir of consolidation chemotherapy (Table 1 [patient 6]). Her fever failed to subside despite empirical therapy with cefepime (FEP) or meropenem (MEM). Disclosure of PCR and sequencing results at this point revealed that Stenotrophomonas maltophilia had been detectable for 9 days, from the first day of fever. The trimethoprim-sulfamethoxazole combination could not be administered because of adverse events, and minocycline (MIN) was therefore added to the antibiotics, and her fever subsided. S. maltophilia subsequently became undetectable by PCR, in accordance with her clinical improvement. Although S. maltophilia was detected once by blood culture, it only became detectable after 7 days in culture, when MIN had already been started. This suggests that PCR analysis of blood would be a useful tool for the detection of pathogens in the cases where bacteria grow poorly or slowly in blood culture.
TABLE 3.
Characteristics of bacterial PCR- and blood culture-positive cases
| Casea | Duration of PCR positivity (days) | Isolateb |
Clinical course | |
|---|---|---|---|---|
| PCR | Blood culture | |||
| 1 | 2 | E. faecalis | E. faecalis | Improved with empirical therapy |
| 2 | 6 | S. maltophilia | S. maltophilia | Improved with empirical therapy |
| 3 | 14 | S. maltophilia | S. maltophilia | Resistance to empirical therapy (disclosure of PCR results) |
Fever was present in each case.
Results are shown for Enterococcus faecalis and Stenotrophomonas maltophilia.
FIG. 1.
A case of empirical therapy-resistant PCR-positive febrile neutropenia. A 64-year-old female with acute myeloid leukemia (M2) had an episode of fever during the nadir period after consolidation chemotherapy. Empirical therapy with cefepime (FEP) and meropenem (MEM) was ineffective. Because PCR results disclosed that S. maltophilia had been detectable from the first day of fever, minocycline (MIN) was added to the therapy. Thereafter, her fever subsided and S. maltophilia became undetectable. BT, body temperature; CRP, C-reactive protein; N.A., not applicable.
Characteristics of 8 febrile and 3 afebrile cases where bacteria were detected by PCR alone.
As described, bacteria were only detected by PCR in 8 febrile cases (Table 4 [cases 1 to 8]) and 3 afebrile cases (Table 4 [cases 9 to 11]). Notably, in 3 febrile cases (cases 1, 4, and 6), the same bacteria were repeatedly detected over periods of 11, 8, and 16 days, respectively. In one case (patient 6), P. aeruginosa as identified by PCR of blood, was also detected from a culture of sputum. From the clinical perspective, all PCR-positive febrile cases improved after empirical antibiotic therapy. A representative case is shown in Fig. 2 (Table 4 [case 1]); Klebsiella oxytoca was detected in blood by bacterial PCR, but not blood culture, from the first day of fever during the neutropenic period after chemotherapy. C-reactive protein was also elevated. Fever was reduced after empirical antibiotic therapy with FEP, and K. oxytoca became undetectable. Similar findings were seen in the other PCR-positive cases. These results suggested that the highly sensitive PCR method allowed the detection of pathogenic bacteria that were not detectable by blood cultures, but that these bacterial infections were generally manageable with empirical antibiotic therapy.
TABLE 4.
Characteristics of bacterial PCR-only positive cases
| Case | Duration of PCR positivity (days) | PCR isolatesa | Blood culture isolates | Fever | Clinical course |
|---|---|---|---|---|---|
| 1 | 11 | K. oxytoca | − | + | Improved with empirical therapy |
| 2 | 1 | S. bovis | − | + | Improved with empirical therapy |
| 3 | 1 | F. nucleatum | − | + | Improved with empirical therapy |
| 4 | 8 | Impossible to analyze (double peak) | − | + | Improved with empirical therapy |
| 5 | 1 | MRCNS | − | + | Improved with empirical therapy |
| 6 | 16 | P. aeruginosa + B. fragilis | − | + | Improved with empirical therapy |
| 7 | 1 | More than 1 bacterium, including Enterococcus spp. | − | + | Improved with empirical therapy |
| 8 | 1 | S. intermedius | − | + | Improved with empirical therapy |
| 9 | 1 | S. epidermidis | − | − | Observation |
| 10 | 1 | S. bovis | − | − | Observation |
| 11 | 1 | MSSA | − | − | Observation |
Results are shown for the following organisms: Klebsiella oxytoca, Streptococcus bovis, Fusobacterium nucleatum, methicillin-resistant coagulase-negative staphylococci (MRCNS), Pseudomonas aeruginosa, Bacteroides fragilis, Streptococcus intermedius, Klebsiella pneumonia, Staphylococcus epidermidis, and methicillin-susceptible Staphylococcus aureus (MSSA).
FIG. 2.
A case of empirical-therapy-effective PCR-positive febrile neutropenia. A 24-year-old male with acute myelomonocytic leukemia (M5a) had an episode of fever during the nadir period of reinduction chemotherapy. Empirical therapy with cefepime (FEP) was effective. K. oxytoca had been PCR positive for more than 10 days from the first day of fever and became undetectable after therapy.
Several bacteria, however, were detected only once by PCR in 3 afebrile cases (Table 4 [cases 9 to 11]). Because the identified bacteria, such as Staphylococcus epidermidis and methicillin-susceptible Staphylococcus aureus (MSSA) were indigenous bacteria normally found on skin, and because the patients had no signs of infection, detection of these bacteria was thought to be due to contamination during the process of blood collection. Streptococcus bovis, however, might have played a pathogenic role, because the patient sometimes had low-grade fever.
Characteristics of 3 afebrile cases where bacteria were detected only by blood culture.
In 3 afebrile cases, several bacteria were detected only once by blood culture. These were Corynebacterium (n = 1) and methicillin-resistant coagulase-negative staphylococcus (n = 2), which are normally found on the skin. These patients had no signs of fever, and detection of these bacteria was thought to be due to contamination by normal skin bacteria during blood collection. To rule out the possibility of PCR inhibition, we added 102 CFU/ml of S. aureus (ATCC 29213) to DNA obtained from 3 samples that were blood culture positive and PCR negative and 17 samples that were blood culture negative and PCR negative. We then performed broad-range PCR. PCR products for S. aureus were detected in all samples from both groups. Furthermore, no PCR products of S. aureus were detected by the addition of DNA-free distilled water (dH2O).
One afebrile case where fungus was detected by PCR analysis.
No fungi were detected in patients with febrile neutropenia in the prospective study. At the time blood was collected for the prospective studies, the values of β-d-glucan and GM were not elevated. However, Aspergillus fumigatus was detected by PCR in one afebrile case (Table 2). This was an AML patient in relapse who received reinduction therapy (Table 1 [case 1]). Despite administration of oral ITC solution, regular screening with chest computed tomography (CT) revealed multiple small nodules with cavities in the lung field. Although both BDG and GM results had been negative, a high GM level (0.7) was detected in his bronchial alveolar lavage fluid. This patient was therefore diagnosed with probable invasive aspergillosis, according to European Organization of Research and Treatment of Cancer/Mycosis Study Group (EORTC/MSG) criteria (8). PCR analysis revealed that A. fumigatus had been continuously detectable for 13 days before suspected Aspergillus infection was detected by chest CT. A. fumigatus became undetectable after antifungal therapy with voriconazole.
PCR analysis in sporadic analyses.
The prospective pilot study showed that bacteria were detected more often by PCR than by blood culture in patients with febrile neutropenia. The study also showed that nondisclosure of PCR results could lead to late diagnosis, with possible disadvantages to the patients. We therefore performed sporadic PCR analysis of bacteria and fungi in febrile neutropenic patients and assessed its utility by disclosing the PCR results to the physicians as soon as possible. Table 5 shows details of the 15 cases of febrile neutropenia. In 13 out of 15 cases, bacteria were detected by PCR, and the same species were also identified by blood cultures in 7 of these cases. In all but one of the PCR-positive cases, fever was successfully treated by empirical antibiotic therapy. In the resistant case (Table 5 [case 11]), fever continued despite 3 series of empirical antibiotic therapies, including teicoplanin plus FEP, FEP plus MEM, and arbekacin plus pazufloxacin. Blood culture was negative, but Pseudomonas aeruginosa was identified by PCR analysis. The patient's fever subsided after substitution of arbekacin with amikacin. Thus, the PCR results provided essential information for the successful management of this patient with empirical therapy-resistant febrile neutropenia. In the cases of febrile neutropenia for sporadic analyses, no fungus was detected by blood culture or PCR. At the time blood was collected, the values of β-d-glucan and GM were not elevated.
TABLE 5.
Sporadic PCR analysis in febrile neutropeniaa
| Case | Age in yr (sex) | Diagnosis | PCR isolates | Blood culture isolates | Clinical course |
|---|---|---|---|---|---|
| 1 | 58 (female) | NHL | MRCNS | + | Improved with empirical therapy |
| 2 | 62 (male) | PTCL | E. faecium | + | Improved with empirical therapy |
| 3 | 53 (male) | NHL | E. coli | + | Improved with empirical therapy |
| 4 | 57 (male) | NHL | S. maltophilia | + | Improved with empirical therapy |
| 5 | 54 (female) | AML | P. aeruginosa | + | Improved with empirical therapy |
| 6 | 57 (male) | MM | C. freundii | + | Improved with empirical therapy |
| 7 | 62 (male) | MM | C. jeikeium | + | Improved with empirical therapy |
| 8 | 46 (female) | ALL | S. salivarius | − | Improved with empirical therapy |
| 9 | 59 (female) | ALL | Enterococcus sp. | − | Improved with empirical therapy |
| 10 | 59 (female) | ALL | P. aeruginosa | − | Improved with empirical therapy |
| 11 | 39 (female) | APL | P. aeruginosa | − | Resistant to empirical therapy |
| 12 | 65 (male) | AML | MSSA | − | Improved with empirical therapy |
| 13 | 79 (female) | NHL | S. maltophilia | − | Improved with empirical therapy |
| 14 | 62 (male) | MM | − | − | Improved with empirical therapy |
| 15 | 18 (female) | ALL | − | − | Improved with empirical therapy |
NHL, non-Hodgkin's lymphoma; PTCL, peripheral T-cell lymphoma; MM, multiple myeloma; APL, acute promyelocytic leukemia. Results are shown for the following organisms: methicillin-resistant coagulase-negative staphylococci (MRCNS), Enterococcus faecium,Escherichia coli, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Citrobacter freundii, Corynebacterium jeikeium, and methicillin-susceptible Staphylococcus aureus (MSSA).
DISCUSSION
Patients with hematologic malignancies who develop febrile neutropenia after intensified chemotherapy are promptly treated with empirical antibiotic therapy. Although increasing attention has been paid to the sensitivity and specificity of PCR for the diagnosis of bacterial and fungal infections (16, 19, 37-40), its clinical utility in general practice has not been well studied. In this study, we set up a PCR system able to detect a broad range of bacterial and fungal pathogens, except for zygomycetes and Fusarium. This was a wider range than with a commercial-based PCR detection system (SeptiFast; Roche, Basel, Switzerland). We assessed the clinical utility of this system for planning treatment for febrile neutropenia. PCR analysis proved more sensitive than blood cultures for the detection of bacteria in both prospective and sporadic studies, but most cases of febrile neutropenia were still manageable with empirical therapy, without recourse to PCR results. However, both studies showed that bacterial PCR analysis was useful in the treatment of empirical antibiotic therapy-resistant febrile neutropenia.
In common with other reports (17, 20, 37-40), the pilot and sporadic study showed that bacterial PCR analysis was more sensitive than blood culture results. The sensitivity of blood culture might be low because the volume of blood used for the culture was half that used in North America and Europe. Nonetheless, the pilot prospective PCR analysis suggested the following: (i) PCR results were mostly negative during the afebrile period after chemotherapy; (ii) when fever occurred, bacteria were detected by PCR from the first day of fever in most cases; (iii) bacteria were detectable during antimicrobial therapy; and (iv) bacteria became undetectable somewhat in accordance with clinical improvements. Contamination by indigenous bacteria was distinguishable in most cases by the identity of the bacterial species, frequency of detection, and clinical evaluation. Our findings therefore suggested that bacteria detected by PCR in febrile neutropenia patients were the cause of the fever, and PCR positivity correlated with clinical course. Because most of the bacteria that were considered to be contaminants were detected only once with PCR, while pathogenic bacteria were often repeatedly detected by PCR, analysis of 2 blood samples at different time points or from different locations could increase the specificity of PCR analysis for bacteria, as observed for PCR analysis of fungi (7, 12, 22).
In both our prospective and sporadic studies, most cases of febrile neutropenia in which bacteria were detected by PCR could be managed by empirical antibiotic therapy, regardless of PCR data. However, identification of bacteria by PCR in the 2 empirical therapy-resistant cases helped us to recognize and treat S. maltophilia and P. aeruginosa infections. These bacteria might not be detected because antibiotic agents would have been already initiated at the time of blood culture. S. maltophilia, especially, is an important opportunistic pathogen in cancer patients because of its intrinsic resistance to broad-spectrum antibiotics, making cotrimoxazole the main therapeutic option (11, 25, 31). Moreover, S. maltophilia and P. aeruginosa often acquire resistance to different antimicrobial agents (6, 18). Notably, in our study, S. maltophilia had been detected from the first day of fever, several days before empirical therapy was found to be ineffective. Our findings imply that bacterial PCR analysis would allow the early diagnosis of these empirical therapy-resistant bacteria.
In our prospective and sporadic study, no fungi were detected in febrile neutropenia patients by PCR and blood culture. The utility of PCR for the diagnosis of fungal infections is becoming more widely recognized than for bacteria (1, 4, 9, 22, 27), and several studies have shown PCR-based assays to be useful for the early diagnosis of fungal infections (7, 10, 12, 13, 15, 21, 35). The low incidence of fungal infection in our study might have been due to antifungal prophylaxis with fosfluconazole or ITC in all patients following intensive chemotherapy. In particular, after ITC began to be frequently used for prophylaxis, Candida species became less frequently detected. Nonetheless, in the prospective study, Aspergillus was repeatedly detected in one afebrile patient several days before the diagnosis of probable invasive aspergillosis was made, based on EORTC/MSG criteria. This result was consistent with recent reports suggesting that PCR analysis could be useful for the early diagnosis of invasive Aspergillus infection (7, 12, 13, 15). However, further studies are required to assess the utility of our PCR system for the diagnosis of invasive fungal infections.
In conclusion, although this study addressed a limited group of febrile neutropenia patients mostly due to bacteria, our results demonstrate that PCR-based analysis of bacteria in blood could be a valuable method for the diagnosis and treatment of febrile neutropenia, especially in patients with empirical-therapy-resistant neutropenic fever.
Acknowledgments
We thank all of the clinicians in the Department of Hematology and Oncology, Mie University Hospital, who assisted with the provision of data for this project.
Footnotes
Published ahead of print on 14 April 2010.
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