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Increased peripheral leukemia blasts leading to false-positive blood culture
Blood Cells, Molecules and Diseases 64 (2017) 8–9
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Letter to the Editor Increased peripheral leukemia blasts leading to false-positive blood culture Keywords: Acute myeloid leukemia Peripheral blood blasts Bacteremia
To the Editor, Fever is often the first and only sign of infection. Whenever a bloodstream infection is suspected, blood cultures are obtained to confirm the diagnosis, allow for susceptibility testing, and insure the patient is treated with an appropriate antimicrobial. Inaccuracies from blood work can lead to under diagnosis of a potentially serious pathology, or conversely, unnecessary administration of antibiotics. Continuous-monitoring blood culture systems (CMBSC) are widely used in clinical laboratories and they significantly decrease time to pathogen detection. The blood sample is inoculated directly into a broth culture that contains a fluorescent-sensing mechanism to detect CO2 production, or a colorimetric sensor that responds to shifts in pH due to changes in CO2 concentration. When a bottle signals positive, it typically indicates the presence of metabolically active microorganism producing CO2 in the culture [1]. However CMBSC are not 100% accurate. False-negative rates vary from 0.2% to 1%, while false-positive rates are between 1.4% and 6.2% [2]. A false-positive reading means that increased CO2 production was detected, but an organism cannot be visualized on microscopic evaluation nor isolated in subculture. Causes of falsepositive culture readings may include overfilling the culture bottle with blood, host cell lysis, and leukocytosis. We present the case of an AML patient with ~ 33 × 10− 9 blasts/L who had a false-positive blood culture, and provide a brief discussion of blast leukocytosis causing false-positive blood cultures. A 47-year-old man presented with fever, fatigue, and mucositis. He had a history of relapsed/refractory AML, diagnosed three years previously and twice underwent stem cell transplantation. At the time of presentation, he was on protocol therapy with azacitidine and nivolumab for relapsed disease. On admission he was hemodynamically stable with a maximum temperature of 38.6 °C. White blood cell (WBC) count was 37 × 109/L with 87% blasts. Chest computerized tomography scan showed no evidence of infection. A blood culture was obtained on presentation followed by empirical antimicrobial therapy with meropenem, daptomycin, caspofungin and acyclovir. At our institution a blood culture is collected using one culture bottle (BD BACTEC Plus Aerobic/F medium; Becton Dickinson, Inc.) and one lysis-centrifugation tube (ISOLATOR™, Alere, Inc.). The culture bottle and tube are inoculated at the bedside and transported to the laboratory. The culture bottle is placed into the
http://dx.doi.org/10.1016/j.bcmd.2017.02.005 1079-9796/© 2017 Elsevier Inc. All rights reserved.
BACTEC FX instrument (Becton Dickinson, Inc.), a CMBCS. The lysis-centrifugation tubes are processed per the manufacturer's recommendation and cultured to four agar plates: one 5% sheep blood, two chocolate, and one buffered charcoal, yeast extract. The plates are incubated overnight and examined for microbial growth once a day for four days. The patient's blood culture bottle flagged positive following 14 h of incubation, a finding typically associated with bacteremia. The gram stain smear was negative for any organism and subculture showed no growth after 72 h of incubation. The blood culture plates from the lysis-centrifugation tube were also negative following four days of incubation. The blood culture bottle was deemed a false-positive result, most likely due to the increased WBC count. High WBC counts may contribute to false-positive culture results as the metabolism of leukocytes also generate CO2, referred to as ‘background increases in CO2’ [1]. To our knowledge, leukocytosis has been reported to give erroneous results in two other AML patients [3,4], and all cases are presented in Table 1. Qian et al. [5] investigated the identification of blood cultures through amplification and sequencing of 16S rRNA. While successful for positive cultures, they were unable to amplify any bacterial nucleic acid in any of the 70 false-positive samples. Elevated leukocyte counts (N10.5 × 109/L) were more frequently associated with false-positives versus true-positives to a statistically significant degree. Daxboeck et al., [6] also investigated false-positive results and were also unable to amplify any bacterial rRNA, however most of the patients in their study were neutropenic. They concluded that leukocytes were not a significant factor in their study. It should be noted that the sample size of the former was much larger (n = 76) than that of the latter (n = 13). Neither of the studies accounted for underlying hematological comorbidities. The disparity in these results demonstrates the need for further research with standardized parameters to conclusively assess the impact of leukocytes on CMBSC. An interesting finding from the study by Qian et al. was the time to positivity was significantly shorter for false-positive (majority b 24 h) than for true-positive (all N24 h) cultures. Although the percentage of false-positive results in CMBSC is low, it is important to consider whether leukocytosis with blasts may skew results in leukemia patients. Patients with active acute leukemia may be at higher risk for false-positive results from increased peripheral blast counts. The generation of CO2 by metabolically active leukemic blasts may be interpreted incorrectly by CMBSC as the presence of microorganisms. A hypoxic microenvironment and CO2 production are both previously described features of leukemic proliferation, and targeting metabolic vulnerabilities is a viable anti-leukemia therapeutic approach [7,8]. CMBSC CO 2 plots from false-positive blood cultures have not been previously studied in detail. The CO 2 plot from our case is shown in Fig. 1A alongside a CO2 plots from a true-positive culture (Fig. 1B). As has been noted previously in our laboratory, a true-positive CO 2 plot shows an
Letter to the Editor
9
Table 1 Comparison of 3 AML patients with leukocytosis with blood cultures flagged positive and no growth on subculture. Reference
Patient
Automated system
WBC (×109/L)
Blast %
[3] [4] Case
67-yr-old man with fever and asthenia 56-yr-old woman with fever and malaise 47-yr-old man with fever and throat pain
BACTEC NR 730/BacT/Alert BACTEC 9240 BACTEC FX
150 446 37
95 100 87
exponential rise in fluorescence indicative of microbial growth, whereas a false-positive due to leukocytosis and blasts shows a steady linear increase in CO2 production. Thus, examination of CO2 plots may suggest whether a blood culture is falsely positive due to increased blasts. Delay in treatment of immunocompromised acute leukemia patients with bloodstream infections could have rapidly fatal consequences. Therefore appropriate antibiotic administration for each clinical situation remains essential, regardless of any suspicion of false-positive test results. The differential diagnosis for a leukemia patient with elevated peripheral blast count and a rapidly positive blood culture can include false-positivity due to blast CO 2
production, while antibiotics are administered and comprehensive evaluation underway. Conflicts of interest None. References [1] CLSI, Principles and Procedures for Blood Cultures; Approved Guideline, 2007 27. [2] R. Ziegler, I. Johnscher, P. Martus, et al., Controlled clinical laboratory comparison of two supplemented aerobic and anaerobic media used in automated blood culture systems to detect bloodstream infections, J. Clin. Microbiol. 36 (1998) 657–661. [3] R.M.M.R. Martinez, Y. Partal, J. Casas, J. Llosa, M. Almagro, An infrequent cause of false-positive blood cultures, Clin. Microbiol. Newsl. 15 (1993) 7–8. [4] Nico E.I. Meessen, E. CMvP, Jan A. Jacobs, False-positive blood culture in a patient with acute myeloid leukemia, Clin. Microbiol. Infect. 5 (1999) 769–770. [5] Q. Qian, Y.W. Tang, C.P. Kolbert, et al., Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results, J. Clin. Microbiol. 39 (2001) 3578–3582. [6] F. Daxboeck, H.J. Dornbusch, R. Krause, et al., Verification of false-positive blood culture results generated by the BACTEC 9000 series by eubacterial 16S rDNA and panfungal 18S rDNA directed polymerase chain reaction (PCR), Diagn. Microbiol. Infect. Dis. 48 (2004) 1. [7] J. Benito, Y. Shi, B. Szymanska, et al., Pronounced hypoxia in models of murine and human leukemia: high efficacy of hypoxia-activated prodrug PR-104, PLoS One 6 (8) (2011), e23108. [8] E.D. Lagadinou, A. Sach, K. Callahan, et al., BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cell, Cell Stem Cell 12 (3) (2013) 329–341.
Maliha Khan Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Rabbia Siddiqi Department of Internal Medicine, Dow University of Health Sciences, Karachi, Pakistan Marina Konopleva Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Micah M. Bhatti Department of Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Christopher B. Benton Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Corresponding author at: Department of Leukemia, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 428, Houston, TX 77030, United States. E-mail address: [email protected]
Fig. 1. Blood culture bottle CO2 plot summary. Each plot has a dual x-axis with fluorescence units on the left axis and positivity on the right axis. ‘Fluorescent units’ are directly related to CO2 production and ‘Positivity’ is when the bottle signals positive by the instrument (Positive = 1). The y-axis is incubation time of the culture bottle in days and hours. A) CO2 plot summary of the false-positive blood culture described in the case. B) CO2 plot summary of a true-positive blood culture that signaled positive on the same day as the false-positive culture.
23 February 2017 Available online 28 February 2017
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Letter to the Editor Increased peripheral leukemia blasts leading to false-positive blood culture Keywords: Acute myeloid leukemia Peripheral blood blasts Bacteremia
To the Editor, Fever is often the first and only sign of infection. Whenever a bloodstream infection is suspected, blood cultures are obtained to confirm the diagnosis, allow for susceptibility testing, and insure the patient is treated with an appropriate antimicrobial. Inaccuracies from blood work can lead to under diagnosis of a potentially serious pathology, or conversely, unnecessary administration of antibiotics. Continuous-monitoring blood culture systems (CMBSC) are widely used in clinical laboratories and they significantly decrease time to pathogen detection. The blood sample is inoculated directly into a broth culture that contains a fluorescent-sensing mechanism to detect CO2 production, or a colorimetric sensor that responds to shifts in pH due to changes in CO2 concentration. When a bottle signals positive, it typically indicates the presence of metabolically active microorganism producing CO2 in the culture [1]. However CMBSC are not 100% accurate. False-negative rates vary from 0.2% to 1%, while false-positive rates are between 1.4% and 6.2% [2]. A false-positive reading means that increased CO2 production was detected, but an organism cannot be visualized on microscopic evaluation nor isolated in subculture. Causes of falsepositive culture readings may include overfilling the culture bottle with blood, host cell lysis, and leukocytosis. We present the case of an AML patient with ~ 33 × 10− 9 blasts/L who had a false-positive blood culture, and provide a brief discussion of blast leukocytosis causing false-positive blood cultures. A 47-year-old man presented with fever, fatigue, and mucositis. He had a history of relapsed/refractory AML, diagnosed three years previously and twice underwent stem cell transplantation. At the time of presentation, he was on protocol therapy with azacitidine and nivolumab for relapsed disease. On admission he was hemodynamically stable with a maximum temperature of 38.6 °C. White blood cell (WBC) count was 37 × 109/L with 87% blasts. Chest computerized tomography scan showed no evidence of infection. A blood culture was obtained on presentation followed by empirical antimicrobial therapy with meropenem, daptomycin, caspofungin and acyclovir. At our institution a blood culture is collected using one culture bottle (BD BACTEC Plus Aerobic/F medium; Becton Dickinson, Inc.) and one lysis-centrifugation tube (ISOLATOR™, Alere, Inc.). The culture bottle and tube are inoculated at the bedside and transported to the laboratory. The culture bottle is placed into the
http://dx.doi.org/10.1016/j.bcmd.2017.02.005 1079-9796/© 2017 Elsevier Inc. All rights reserved.
BACTEC FX instrument (Becton Dickinson, Inc.), a CMBCS. The lysis-centrifugation tubes are processed per the manufacturer's recommendation and cultured to four agar plates: one 5% sheep blood, two chocolate, and one buffered charcoal, yeast extract. The plates are incubated overnight and examined for microbial growth once a day for four days. The patient's blood culture bottle flagged positive following 14 h of incubation, a finding typically associated with bacteremia. The gram stain smear was negative for any organism and subculture showed no growth after 72 h of incubation. The blood culture plates from the lysis-centrifugation tube were also negative following four days of incubation. The blood culture bottle was deemed a false-positive result, most likely due to the increased WBC count. High WBC counts may contribute to false-positive culture results as the metabolism of leukocytes also generate CO2, referred to as ‘background increases in CO2’ [1]. To our knowledge, leukocytosis has been reported to give erroneous results in two other AML patients [3,4], and all cases are presented in Table 1. Qian et al. [5] investigated the identification of blood cultures through amplification and sequencing of 16S rRNA. While successful for positive cultures, they were unable to amplify any bacterial nucleic acid in any of the 70 false-positive samples. Elevated leukocyte counts (N10.5 × 109/L) were more frequently associated with false-positives versus true-positives to a statistically significant degree. Daxboeck et al., [6] also investigated false-positive results and were also unable to amplify any bacterial rRNA, however most of the patients in their study were neutropenic. They concluded that leukocytes were not a significant factor in their study. It should be noted that the sample size of the former was much larger (n = 76) than that of the latter (n = 13). Neither of the studies accounted for underlying hematological comorbidities. The disparity in these results demonstrates the need for further research with standardized parameters to conclusively assess the impact of leukocytes on CMBSC. An interesting finding from the study by Qian et al. was the time to positivity was significantly shorter for false-positive (majority b 24 h) than for true-positive (all N24 h) cultures. Although the percentage of false-positive results in CMBSC is low, it is important to consider whether leukocytosis with blasts may skew results in leukemia patients. Patients with active acute leukemia may be at higher risk for false-positive results from increased peripheral blast counts. The generation of CO2 by metabolically active leukemic blasts may be interpreted incorrectly by CMBSC as the presence of microorganisms. A hypoxic microenvironment and CO2 production are both previously described features of leukemic proliferation, and targeting metabolic vulnerabilities is a viable anti-leukemia therapeutic approach [7,8]. CMBSC CO 2 plots from false-positive blood cultures have not been previously studied in detail. The CO 2 plot from our case is shown in Fig. 1A alongside a CO2 plots from a true-positive culture (Fig. 1B). As has been noted previously in our laboratory, a true-positive CO 2 plot shows an
Letter to the Editor
9
Table 1 Comparison of 3 AML patients with leukocytosis with blood cultures flagged positive and no growth on subculture. Reference
Patient
Automated system
WBC (×109/L)
Blast %
[3] [4] Case
67-yr-old man with fever and asthenia 56-yr-old woman with fever and malaise 47-yr-old man with fever and throat pain
BACTEC NR 730/BacT/Alert BACTEC 9240 BACTEC FX
150 446 37
95 100 87
exponential rise in fluorescence indicative of microbial growth, whereas a false-positive due to leukocytosis and blasts shows a steady linear increase in CO2 production. Thus, examination of CO2 plots may suggest whether a blood culture is falsely positive due to increased blasts. Delay in treatment of immunocompromised acute leukemia patients with bloodstream infections could have rapidly fatal consequences. Therefore appropriate antibiotic administration for each clinical situation remains essential, regardless of any suspicion of false-positive test results. The differential diagnosis for a leukemia patient with elevated peripheral blast count and a rapidly positive blood culture can include false-positivity due to blast CO 2
production, while antibiotics are administered and comprehensive evaluation underway. Conflicts of interest None. References [1] CLSI, Principles and Procedures for Blood Cultures; Approved Guideline, 2007 27. [2] R. Ziegler, I. Johnscher, P. Martus, et al., Controlled clinical laboratory comparison of two supplemented aerobic and anaerobic media used in automated blood culture systems to detect bloodstream infections, J. Clin. Microbiol. 36 (1998) 657–661. [3] R.M.M.R. Martinez, Y. Partal, J. Casas, J. Llosa, M. Almagro, An infrequent cause of false-positive blood cultures, Clin. Microbiol. Newsl. 15 (1993) 7–8. [4] Nico E.I. Meessen, E. CMvP, Jan A. Jacobs, False-positive blood culture in a patient with acute myeloid leukemia, Clin. Microbiol. Infect. 5 (1999) 769–770. [5] Q. Qian, Y.W. Tang, C.P. Kolbert, et al., Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results, J. Clin. Microbiol. 39 (2001) 3578–3582. [6] F. Daxboeck, H.J. Dornbusch, R. Krause, et al., Verification of false-positive blood culture results generated by the BACTEC 9000 series by eubacterial 16S rDNA and panfungal 18S rDNA directed polymerase chain reaction (PCR), Diagn. Microbiol. Infect. Dis. 48 (2004) 1. [7] J. Benito, Y. Shi, B. Szymanska, et al., Pronounced hypoxia in models of murine and human leukemia: high efficacy of hypoxia-activated prodrug PR-104, PLoS One 6 (8) (2011), e23108. [8] E.D. Lagadinou, A. Sach, K. Callahan, et al., BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cell, Cell Stem Cell 12 (3) (2013) 329–341.
Maliha Khan Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Rabbia Siddiqi Department of Internal Medicine, Dow University of Health Sciences, Karachi, Pakistan Marina Konopleva Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Micah M. Bhatti Department of Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Christopher B. Benton Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Corresponding author at: Department of Leukemia, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 428, Houston, TX 77030, United States. E-mail address: [email protected]
Fig. 1. Blood culture bottle CO2 plot summary. Each plot has a dual x-axis with fluorescence units on the left axis and positivity on the right axis. ‘Fluorescent units’ are directly related to CO2 production and ‘Positivity’ is when the bottle signals positive by the instrument (Positive = 1). The y-axis is incubation time of the culture bottle in days and hours. A) CO2 plot summary of the false-positive blood culture described in the case. B) CO2 plot summary of a true-positive blood culture that signaled positive on the same day as the false-positive culture.
23 February 2017 Available online 28 February 2017