LETTER TO THE EDITOR
Detection of Multiple Fungal Species in Blood Samples by Real-Time PCR: an Interpretative Challenge Catriona L. Halliday,a,b Tania C. Sorrell,b,c,d Sharon C.-A. Chena,b,c Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, Westmead Hospital, Sydney, Australiaa; Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, Westmead, Australiab; Sydney Medical School, University of Sydney, Westmead, Australiac; Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, New South Wales, Australiad
he speed and high sensitivity of broad-range panfungal PCR assays over culture, using clinical specimens, are appealing, particularly when the causative agent is unknown (1, 2). The sensitivity, specificity, and accuracy (3) of modern assays is high for tissue and sterile-site specimens (1, 4) but not blood, where the fungal burden is low (⬍10 CFU/ml). Indeed, ⬍1 CFU/ml is found in 25 to 50% of patients with candidemia (5–7). Thus, we read with interest that Zhao et al., using an Exserohilum rostratum-specific and a panfungal, real-time PCR assay (8), detected DNA of 11 fungal species (1 E. rostratum and 10 nonExserohilum species) in the blood of patients who had received contaminated methylprednisolone from the batch causing the E. rostratum meningitis outbreak in the United States (9, 10). The single diagnosis of E. rostratum meningitis is plausible given the medical history. By panfungal PCR, 12/24 (50%) blood samples yielded one or more of the following: Aspergillus fumigatus (n ⫽ 5 samples), Cryptococcus neoformans (n ⫽ 1), 4 different dematiaceous fungi (n ⫽ 4), and Pichia sp., Capnodium sp., Phlebia sp., and Filobasidium uniguttulatum (n ⫽ 1 each) (9). This high PCR positivity rate and breadth of pathogens detected are unexpected; they warrant consideration of other causes of PCR-positive signals. The authors were meticulous in minimizing airborne spores and carryover contamination, yet even in real-time systems, contamination by environmental fungi and/or fungal DNA introduced in commercial reagents can occur. Up to 13% (28/182) of blood samples from healthy volunteers tested Aspergillus PCR positive in one study (11–13). Administration of intravenous antibiotics may also introduce fungal DNA contamination (7). Most fungal species detected by Zhao et al. were environmental saprophytes or plant pathogens (9). Although any fungus can cause infection, such positive PCR signals in blood require careful interpretation, especially if they are based on a single test. To avoid false-positive results, the authors of a meta-analysis of Aspergillus PCR testing of blood from hematology patients recommended that two PCR-positive tests be required for a positive result (14). Establishing a threshold cycle (CT) cutoff in real-time systems to distinguish between positive and negative PCR signals is not always straightforward and is influenced by gene targets, patient population, and clinical context. Application of a CT cutoff of 35 in the panfungal PCR of Zhao et al. yielded an average cutoff for healthy volunteers (35.6; range, 34.9 to 37.0) that was only 2 cycles above that for infected patients (33.4; upper CT, 34.8). It is uncertain how this cutoff was determined. The identities of all PCR products that generated a CT as well as those with a CT of ⬍35 would be of interest. Curiously, the PCR signal from a synovial fluid specimen with a CT of 35.1 was deemed a negative result and the amplified product was not sequenced. For blood Aspergillus
September 2014 Volume 52 Number 9
PCR testing, Millon et al. consistently found CT values of ⬎36 and recommended a positive result at a CT of ⬍43 (7). That a mixture of fungi was observed in three blood samples due to differences in internal transcribed spacer 1 (ITS1) and ITS2 sequences is also intriguing. In mixed infection, one expects to see sequence chromatograms with more than one peak for each of these targets. This renders a 100% sequence match with database sequences for fungal identity (as used by the authors) highly unlikely. Species identity based on polymorphisms within the ITS1 and ITS2 loci are typically concordant. Finally, the clinical setting in which the real-time panfungal PCR assay is performed influences its utility and accuracy. If it is used to screen patients for early infection, validation of its ability to distinguish positive from negative results is required, as are studies outside the setting of patients at high risk (14–16). Further evaluation of broad-range assays to screen for fungal infection is needed. REFERENCES 1. Lau A, Chen S, Sorrell T, Carter D, Malik R, Martin P, Halliday C. 2007. Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens. J. Clin. Microbiol. 45:380 – 385. http://dx.doi.org/10.1128/JCM.01862-06. 2. Perfect JR. 2013. Fungal diagnostics: how do we do it and can we do better? Curr. Med. Res. 29(Suppl 4):S3–S11. http://dx.doi.org/10.1185 /03007995.2013.780861. 3. Burd EM. 2010. Validation of laboratory-developed molecular assays for infectious diseases. Clin. Microbiol. Rev. 23:550 –576. http://dx.doi.org /10.1128/CMR.00074-09. 4. Willinger BA, Obradovic A, Selitsch B, Beck-Mannagetta J, Buzina W, Braun H, Apfalter P, Hirschl AM, Makristathis A, Rotter M. 2003. Detection and identification of fungi from fungus balls of the maxillary sinus by molecular techniques. J. Clin. Microbiol. 41:581–585. http://dx .doi.org/10.1128/JCM.41.2.581-585.2003. 5. White PL, Shetty A, Barnes RA. 2003. Detection of seven Candida species using the Light-Cycler system. J. Med. Microbiol. 52:229 –238. http://dx .doi.org/10.1099/jmm.0.05049-0. 6. Pfeiffer CD, Samsa GP, Schell WA, Reller LB, Perfect JR, Alexander BD. 2011. Quantitation of Candida CFU in initial blood positive blood cultures. J. Clin. Microbiol. 49:2879 –2883. http://dx.doi.org/10.1128/JCM .00609-11. 7. Millon L, Piarroux R, Deconinck E, Bulabois CE, Grenouillet F, Rohrlich P, Costa JM, Bretagne S. 2005. Use of real-time PCR to process the first galactomannan-positive serum sample in diagnosing invasive
Editor: G. V. Doern Address correspondence to Sharon C.-A. Chen, [email protected]. For the author reply, see doi:10.1128/JCM.01711-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.01685-14
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tion. J. Mol. Recognit. 18(2):119 –138. http://dx.doi.org/10.1002/jmr.687 .PMID15565717. Palmer J, Francesconi A, Kasai M, Walsh TJ, Orle KA. 2001. Sources of false positive Aspergillus DNA by PCR from normal human blood, abstract J-844. In Final Prog. 41st Intersci. Conf. Antimicrob. Agents Chemother., Chicago, IL. American Society for Microbiology, Washington, DC. Mengoli C, Cruciani M, Barnes R, Loeffler J, Donnelly JP. 2009. Use of PCR for diagnosis of invasive aspergillosis: systematic review and metaanalysis.LancetInfect.9:89 –96.http://dx.doi.org/10.1016/S1473-3099(09) 70019-2. Nguyen MH, Wissel MC, Shields RK, Salomoni MA, Hao B, Press EG, Shields RM, Cheng S, Mitsani D, Vadnerkar A, Silveria FP, Kleiboeker SB, Clancy CJ. 2012. Performance of Candida real-time polymerase chain reaction, -D-glucan assay, and blood cultures in the diagnosis of invasive candidiasis. Clin. Infect. Dis. 54:1240 –1248. http://dx.doi.org/10.1093 /cid/cis200. Lau A, Halliday C, Chen SC, Playford EG, Stanley K, Sorrell TC. 2010. Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR. J. Clin. Microbiol. 48:811– 816. http://dx.doi.org/10.1128/JCM.01650-09.
Journal of Clinical Microbiology
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aspergillosis. J. Clin. Microbiol. 43:5097–5101. http://dx.doi.org/10.1128 /JCM.43.10.5097-5101.2005. Zhao Y, Park S, Kreiswirth BN, Ginocchio CC, Veyret R, Laayoun A, Troesch A, Perlin DS. 2009. Rapid real-time nucleic acid sequence-based amplification-molecular beacon platform to detect fungal and bacterial bloodstream infections. J. Clin. Microbiol. 47:2067–2078. http://dx.doi .org/10.1128/JCM.02230-08. Zhao Y, Armeanu E, DiVerniero R, Lewis TA, Dobson RC, Kontoiannis DP, Roilides E, Walsh TJ, Perlin DS. 2014. Fungal DNA detected in blood samples of patients who received contaminated methylprednisolone injections reveals increased complexity of causative agents. J. Clin. Microbiol. 52:2212–2215. http://dx.doi.org/10.1128/JCM.00854-14. Kauffman CA, Pappas PG, Patterson TF. 2013. Fungal infections associated with contaminated methylprednisolone injections. N. Engl. J. Med. 368:2495–2500. http://dx.doi.org/10.1056/NEJMra1212617. Fredricks DN, Smith C, Meier A. 2005. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J. Clin. Microbiol. 43:5122–5128. http://dx.doi.org/10.1128/JCM.43.10 .5122-5128.2005. Daly R, Hearn MT. 2005. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and produc-
Detection of Multiple Fungal Species in Blood Samples by Real-Time PCR: an Interpretative Challenge Catriona L. Halliday,a,b Tania C. Sorrell,b,c,d Sharon C.-A. Chena,b,c Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, Westmead Hospital, Sydney, Australiaa; Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, Westmead, Australiab; Sydney Medical School, University of Sydney, Westmead, Australiac; Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, New South Wales, Australiad
he speed and high sensitivity of broad-range panfungal PCR assays over culture, using clinical specimens, are appealing, particularly when the causative agent is unknown (1, 2). The sensitivity, specificity, and accuracy (3) of modern assays is high for tissue and sterile-site specimens (1, 4) but not blood, where the fungal burden is low (⬍10 CFU/ml). Indeed, ⬍1 CFU/ml is found in 25 to 50% of patients with candidemia (5–7). Thus, we read with interest that Zhao et al., using an Exserohilum rostratum-specific and a panfungal, real-time PCR assay (8), detected DNA of 11 fungal species (1 E. rostratum and 10 nonExserohilum species) in the blood of patients who had received contaminated methylprednisolone from the batch causing the E. rostratum meningitis outbreak in the United States (9, 10). The single diagnosis of E. rostratum meningitis is plausible given the medical history. By panfungal PCR, 12/24 (50%) blood samples yielded one or more of the following: Aspergillus fumigatus (n ⫽ 5 samples), Cryptococcus neoformans (n ⫽ 1), 4 different dematiaceous fungi (n ⫽ 4), and Pichia sp., Capnodium sp., Phlebia sp., and Filobasidium uniguttulatum (n ⫽ 1 each) (9). This high PCR positivity rate and breadth of pathogens detected are unexpected; they warrant consideration of other causes of PCR-positive signals. The authors were meticulous in minimizing airborne spores and carryover contamination, yet even in real-time systems, contamination by environmental fungi and/or fungal DNA introduced in commercial reagents can occur. Up to 13% (28/182) of blood samples from healthy volunteers tested Aspergillus PCR positive in one study (11–13). Administration of intravenous antibiotics may also introduce fungal DNA contamination (7). Most fungal species detected by Zhao et al. were environmental saprophytes or plant pathogens (9). Although any fungus can cause infection, such positive PCR signals in blood require careful interpretation, especially if they are based on a single test. To avoid false-positive results, the authors of a meta-analysis of Aspergillus PCR testing of blood from hematology patients recommended that two PCR-positive tests be required for a positive result (14). Establishing a threshold cycle (CT) cutoff in real-time systems to distinguish between positive and negative PCR signals is not always straightforward and is influenced by gene targets, patient population, and clinical context. Application of a CT cutoff of 35 in the panfungal PCR of Zhao et al. yielded an average cutoff for healthy volunteers (35.6; range, 34.9 to 37.0) that was only 2 cycles above that for infected patients (33.4; upper CT, 34.8). It is uncertain how this cutoff was determined. The identities of all PCR products that generated a CT as well as those with a CT of ⬍35 would be of interest. Curiously, the PCR signal from a synovial fluid specimen with a CT of 35.1 was deemed a negative result and the amplified product was not sequenced. For blood Aspergillus
September 2014 Volume 52 Number 9
PCR testing, Millon et al. consistently found CT values of ⬎36 and recommended a positive result at a CT of ⬍43 (7). That a mixture of fungi was observed in three blood samples due to differences in internal transcribed spacer 1 (ITS1) and ITS2 sequences is also intriguing. In mixed infection, one expects to see sequence chromatograms with more than one peak for each of these targets. This renders a 100% sequence match with database sequences for fungal identity (as used by the authors) highly unlikely. Species identity based on polymorphisms within the ITS1 and ITS2 loci are typically concordant. Finally, the clinical setting in which the real-time panfungal PCR assay is performed influences its utility and accuracy. If it is used to screen patients for early infection, validation of its ability to distinguish positive from negative results is required, as are studies outside the setting of patients at high risk (14–16). Further evaluation of broad-range assays to screen for fungal infection is needed. REFERENCES 1. Lau A, Chen S, Sorrell T, Carter D, Malik R, Martin P, Halliday C. 2007. Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens. J. Clin. Microbiol. 45:380 – 385. http://dx.doi.org/10.1128/JCM.01862-06. 2. Perfect JR. 2013. Fungal diagnostics: how do we do it and can we do better? Curr. Med. Res. 29(Suppl 4):S3–S11. http://dx.doi.org/10.1185 /03007995.2013.780861. 3. Burd EM. 2010. Validation of laboratory-developed molecular assays for infectious diseases. Clin. Microbiol. Rev. 23:550 –576. http://dx.doi.org /10.1128/CMR.00074-09. 4. Willinger BA, Obradovic A, Selitsch B, Beck-Mannagetta J, Buzina W, Braun H, Apfalter P, Hirschl AM, Makristathis A, Rotter M. 2003. Detection and identification of fungi from fungus balls of the maxillary sinus by molecular techniques. J. Clin. Microbiol. 41:581–585. http://dx .doi.org/10.1128/JCM.41.2.581-585.2003. 5. White PL, Shetty A, Barnes RA. 2003. Detection of seven Candida species using the Light-Cycler system. J. Med. Microbiol. 52:229 –238. http://dx .doi.org/10.1099/jmm.0.05049-0. 6. Pfeiffer CD, Samsa GP, Schell WA, Reller LB, Perfect JR, Alexander BD. 2011. Quantitation of Candida CFU in initial blood positive blood cultures. J. Clin. Microbiol. 49:2879 –2883. http://dx.doi.org/10.1128/JCM .00609-11. 7. Millon L, Piarroux R, Deconinck E, Bulabois CE, Grenouillet F, Rohrlich P, Costa JM, Bretagne S. 2005. Use of real-time PCR to process the first galactomannan-positive serum sample in diagnosing invasive
Editor: G. V. Doern Address correspondence to Sharon C.-A. Chen, [email protected]. For the author reply, see doi:10.1128/JCM.01711-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.01685-14
Journal of Clinical Microbiology
p. 3515–3516
jcm.asm.org
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Downloaded from http://jcm.asm.org/ on May 21, 2015 by UNIVERSITY OF LEEDS
T
Letter to the Editor
8.
9.
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tion. J. Mol. Recognit. 18(2):119 –138. http://dx.doi.org/10.1002/jmr.687 .PMID15565717. Palmer J, Francesconi A, Kasai M, Walsh TJ, Orle KA. 2001. Sources of false positive Aspergillus DNA by PCR from normal human blood, abstract J-844. In Final Prog. 41st Intersci. Conf. Antimicrob. Agents Chemother., Chicago, IL. American Society for Microbiology, Washington, DC. Mengoli C, Cruciani M, Barnes R, Loeffler J, Donnelly JP. 2009. Use of PCR for diagnosis of invasive aspergillosis: systematic review and metaanalysis.LancetInfect.9:89 –96.http://dx.doi.org/10.1016/S1473-3099(09) 70019-2. Nguyen MH, Wissel MC, Shields RK, Salomoni MA, Hao B, Press EG, Shields RM, Cheng S, Mitsani D, Vadnerkar A, Silveria FP, Kleiboeker SB, Clancy CJ. 2012. Performance of Candida real-time polymerase chain reaction, -D-glucan assay, and blood cultures in the diagnosis of invasive candidiasis. Clin. Infect. Dis. 54:1240 –1248. http://dx.doi.org/10.1093 /cid/cis200. Lau A, Halliday C, Chen SC, Playford EG, Stanley K, Sorrell TC. 2010. Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR. J. Clin. Microbiol. 48:811– 816. http://dx.doi.org/10.1128/JCM.01650-09.
Journal of Clinical Microbiology
Downloaded from http://jcm.asm.org/ on May 21, 2015 by UNIVERSITY OF LEEDS
11.
aspergillosis. J. Clin. Microbiol. 43:5097–5101. http://dx.doi.org/10.1128 /JCM.43.10.5097-5101.2005. Zhao Y, Park S, Kreiswirth BN, Ginocchio CC, Veyret R, Laayoun A, Troesch A, Perlin DS. 2009. Rapid real-time nucleic acid sequence-based amplification-molecular beacon platform to detect fungal and bacterial bloodstream infections. J. Clin. Microbiol. 47:2067–2078. http://dx.doi .org/10.1128/JCM.02230-08. Zhao Y, Armeanu E, DiVerniero R, Lewis TA, Dobson RC, Kontoiannis DP, Roilides E, Walsh TJ, Perlin DS. 2014. Fungal DNA detected in blood samples of patients who received contaminated methylprednisolone injections reveals increased complexity of causative agents. J. Clin. Microbiol. 52:2212–2215. http://dx.doi.org/10.1128/JCM.00854-14. Kauffman CA, Pappas PG, Patterson TF. 2013. Fungal infections associated with contaminated methylprednisolone injections. N. Engl. J. Med. 368:2495–2500. http://dx.doi.org/10.1056/NEJMra1212617. Fredricks DN, Smith C, Meier A. 2005. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J. Clin. Microbiol. 43:5122–5128. http://dx.doi.org/10.1128/JCM.43.10 .5122-5128.2005. Daly R, Hearn MT. 2005. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and produc-
