Diagnostic Microbiology and Infectious Disease 78 (2014) 141–143
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Identification of fungal pathogens from clinical specimens using multi-locus PCR coupled with electrospray ionization mass spectrometry Chi-Jung Wu a, b,⁎, Ya-Ping Chen b, Hsuan-Chen Wang a, Ih-Jen Su a, Wen-Chien Ko b, Jiann-Shiuh Chen c, Chao-Neng Cheng c, Nan-Yao Lee b, H. Sunny Sun d, Chia-Yu Chi a, c, Tsai-Yun Chen b,⁎ a
National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan Department of Internal Medicine, National Cheng Kung University Hospital and Medical College, Tainan, Taiwan Department of Pediatrics, National Cheng Kung University Hospital and Medical College, Tainan, Taiwan d Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan b c
a r t i c l e
i n f o
Article history: Received 14 May 2013 Received in revised form 14 August 2013 Accepted 23 August 2013 Available online 20 November 2013
a b s t r a c t Polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was successfully used to identify a variety of fungi, including mixed fungal species, from 10 of 12 clinical specimens (10 culture-negative) of biopsy tissues or body fluids from patients with fungal infections. The application of PCR/ESI-MS for identifying fungal pathogens is discussed. © 2014 Elsevier Inc. All rights reserved.
Keywords: Electrospray ionization mass spectrometry Fungal infection PCR Paraffin
Rapid and accurate detection of the fungal pathogens that cause invasive fungal infections (IFIs) is critical for optimal patient care. Recently, the use of multi-locus polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) has been demonstrated to have the capacity for rapid and highthroughput genotypic characterization of a broad range of fungal organisms in bronchoalveolar lavage (BAL) (Shin et al., 2013). In this study, clinical specimens of biopsy tissues, body fluids, and BAL were used in PCR/ESI-MS analyses to evaluate the usefulness of this novel platform in IFI diagnoses. Non-consecutive patients with clinical suspicion of fungal infections that were culture-negative or difficult-to-treat were referred to the study by the infectious diseases specialists at National Cheng Kung University Hospital between June 2012 and March 2013. The study was approved by the institutional review board of the hospital, and patients were enrolled after informed consent. Patients’ clinical data were reviewed and characterized according to the definitions of IFIs from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group consensus group (De Pauw et al., 2008). The clinical information and types of specimens collected are listed in Table 1. Two formalin-fixed, ⁎ Corresponding authors. C.-J. Wu is to be contacted at Tel.: +886-6-7000123x65220; fax: +886-6-208-3466. T.-Y. Chen, Tel.: +886-6-2353535x4559; fax: +886-6-2752037. E-mail addresses: [email protected] (C.-J. Wu), [email protected] (T.-Y. Chen). 0732-8893/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.08.007
paraffin-embedded (FFPE) tissues and one fresh biopsy tissue were used as control samples. Clinical specimens distributed to culture and PCR/ESI-MS analyses were processed with aseptic techniques, whereas those that went to histopathological examination (and formalin fixation and paraffin embedding) were not. Fungal culturing and morphological or phenotypic identifications were performed on fresh specimens using standard procedures in clinical microbiology laboratories (ASM Press, 2010). For PCR/ESI-MS analyses, the specimens were lysed with a PLEX-ID bead beater, and the microbial DNA was extracted from the homogenates and purified using the UMD-Universal extraction kit (4 cases per serving) (Molzym, Bremen, Germany); DNA from FFPE tissues was extracted using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Extracted DNAs were automatically amplified and analyzed using PCR/ESI-MS (PLEX-ID) broad fungal assay (BFA) (Abbott Laboratories, Abbott Park, IL, USA), following the manufacturer’s instructions (Shin et al., 2013). To prevent contamination, the PCR setup, DNA extraction, PCR amplification, and PCR/ESI-MS analyses were performed in four separate rooms with dedicated consumables. Sample preparation and DNA extraction was performed in a biosafety cabinet illuminated with ultraviolet light when not in use. Organism identification was based on the total mass and base compositions of the PCR amplicons compared to those in the molecular signature database established by the PLEX-ID manufacturer (Shin et al., 2013). To determine the limit of detection by PCR/ESI-MS, two independent tenfold-dilution panels (10 1–10 3 cfu/mL) of Candida albicans ATCC 14053 and Aspergillus fumigatus BCRC 30099 were prepared by spiking colonies into 3 ml
142
C.-J. Wu et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 141–143
EDTA-anticoagulated peripheral blood from healthy volunteers and subjected to PCR/ESI-MS BFA analyses (Loeffler et al., 2000). The experiment was performed twice, and the limit of detection was 10 cfu/mL for both organisms. Using PCR/ESI-MS, a variety of fungi, including mixed fungal species, in 10 of the 12 clinical specimens (10 of which culturenegative) with suspected IFIs were identified (Table 1). The fungi detected included yeasts (Candida spp., Cryptococcus spp., and Malassezia spp.), filamentous fungi (Aspergillus spp., Acremonium spp., Cladosporium spp., and Coniosporium spp.), and Pneumocystis spp., although 2 results from FFPE tissues might be due to contaminants in the FFPE tissues. Except in patients 5 and 8, in which the PCR/ESI-MS could not differentiate between two closely related species (Aspergillus flavus and Aspergillus oryzae), the fungi detected were identified to a species level. This ability to identify multiple fungal species in a single specimen is an important advantage over pan-fungal PCR-based sequencing, another commonly used
technique, which often fails if two or more organisms co-exist in one specimen (Shin et al., 2013). Out of 4 culture-negative fresh biopsy tissue specimens, Candida albicans, Candida tropicalis and Pneumocystis jiroveci were identified in patients 1, 2, and 6, respectively, and the results were consistent with clinical features. A retrospective study found that 24 patients with hepatosplenic candidiasis all had negative culture results (De Castro et al., 2012). The PCR-based technique is more sensitive than microbiological culturing (89% vs. 56%) for the detection of IFIs (Buitrago et al., 2012). The high culture-negative rate in IFIs necessitates development of more sensitive detection methods, such as molecular identification. Our data suggest that the PCR/ESI-MS is capable of detecting and characterizing small amounts of fungal DNA in culture-negative specimens, and the identification result might be useful for selecting appropriate antifungal therapy. Of the 7 FFPE tissues tested, fungi were detected by PCR/ESI-MS in 4 of the 5 culture-negative clinical specimens with suspected IFIs and
Table 1 Summary of clinical information and laboratory results in the study. Case Age/Underlying no. diseases
Fresh tissues 1 22 y/leukemia
Infectious diseases
Specimens
HSC
Histology/GM indexa
2 3
5 y/neuroblastoma 52 y/ESRD
FN with skin rash PD peritonitis
Liver Hyphae biopsy Skin biopsy Hyphae/pseudohyphae PD fluid NP
4
23 y/nil
Meningitis
CSF
NP
5
43 y/leukemia
IMD, lung
Sputum
NP/4.46
6
37 y/AIDS
Pneumonia
BAL
NP
Lung biopsy
Cytomegalovirus inclusion body and granulomatous inflammation Interstitial inflammation
7
Lung biopsy Formalin-fixed, paraffin-embedded (FFPE) tissues Lung 8 50 y/leukemia IMD, lungd biopsy
9
17 y/leukemia
68 y/diabetes
10 39 y/leukemia
IMD, lung
IMD, sphenoid sinus Sphenoid sinus biopsy IMD, lung Lung biopsy
11 28 y/leukemia
HSC
Liver biopsy
12 54 y/leukemia
Hepatosplenic abscesses
Liver biopsy
Lung mass
FFPE tissue, lung FFPE tissue, oropharynx Fresh lung biopsy tissue
Control samples 13 74 y/lymphoma 14 65 y/nil 15 66 y/lymphoma
Oropharyngeal tuberculosis Lung mass
Right angle, broad pauciseptate hyphae, suggestive of mucormycosis or aspergillosis/0.91 Fungal hyphae, suggestive of aspergillosis/0.31
PCR/ESI-MS BFA identificationb
Fungal culture identification
EORTC/MSG criteria
Comparison of results of PCR/ESI-MS with conventional methods or clinical course
Candida albicans
No growth
Proven IFI
Compatible
Candida tropicalis Candida parapsilosis Cryptococcus neoformans C. albicans and Aspergillus flavus/ oryzae Pneumocystis jirovecic P. jirovecic
No growthc C. parapsilosis
Proven IFI Proven IFI
Compatible Compatible
C. neoformans
Proven IFI
Compatible
No growth
Probable IFI
Compatible
No growth
NA
Compatible
No growth
NA
Compatible
No detection
No growth
Possible IFI
NA
Aspergillus flavus/ oryzae
No growth
Proven IFI
Compatibled
Aspergillus fumigatus
No growth
Proven IFI
Compatible
No growth
Proven IFI
Most likely a contaminant
No growth
possible IFI
Most likely contaminants
No growth
possible IFI
NA
Coniosporium sp. Septated acute angle branching hyphae, suggestive of aspergillosis/0.15 Inflammation and fibrosis Acremonium persicinum and Cladosporium cladosporioides Suppurative granulomatous No detection inflammation. Lymphoma
Malassezia pachydermatis Granulomatous inflammation, A. persicinum and acid-fast bacilli C. cladosporioides Lymphoma No detection
No growth NP No growth
Most likely contaminants Most likely contaminants Compatible
AIDS = acquired immunodeficiency syndrome; CSF = cerebrospinal fluid; ESRD = end-stage renal disease; FN = febrile neutropenia; HSC = hepatosplenic candidiasis; IMD = invasive mold disease; NA = not applicable; NP = not performed; PD = peritoneal dialysis. a Serum galactomannan index (Platelia Aspergillus EIA, Bio-Rad Laboratories): A cut-off index of 0.5 is considered positive. b Seven FFPE tissues were analyzed independently using PCR/ESI-MS in 7 separate BFA plates at different times. c Cytomegalovirus was detected simultaneously using the PCR/ESI-MS Broad Viral Assay. The clinical condition improved with ganciclovir and trimethoprim/ sulfamethoxazole treatment. d Pneumonia resolved with voriconazole treatment.
C.-J. Wu et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 141–143
in 2 control samples. Formalin prevents the amplification of long fungal DNA fragments due to the degradation of DNA. The amplicon sizes obtained using PCR/ESI-MS BFA ranged from 72 to 154 base pairs, lengths that are more suitable for detecting fungal DNA in FFPE tissues than large amplicon sizes (Cabaret et al., 2011; Shin et al., 2013). A. flavus/oryzae and A. fumigatus were identified in patients 8 and 9, respectively, and the results were concordant with clinical and laboratory features of the patients. Nevertheless, the results of PCR/ESI-MS analyses should be interpreted cautiously for FFPE tissues. For example, C. tropicalis was the presumptive causative organism of hepatosplenic candidiasis in patient 11, who had experienced a prior episode of C. tropicalis fungemia and concurrent liver and spleen microabscesses revealed by a computed tomography during a neutropenic period. Acremonium persicinum and Cladosporium cladosporioides detected by PCR/ESI-MS from FFPE liver tissue from this patient are more likely to be contaminants in the paraffin section, an idea that is further supported by an identical result from another FFPE tissue from a lesion site on a patient with documented tuberculosis (patient 14). The 2 patients received biopsies for histopathology examination (and formalin fixation and paraffin embedding of tissues) 2 weeks apart, while their FFPE were retrieved, processed (using separate sets of QIAamp Tissue Kit), and examined by PCR/ESIMS 7 months apart. The long period between two independent PCR/ ESI-MS analyses and absence of both organisms in other samples precluded the possibility of contamination during sample processing or DNA extraction in PCR/ESI-MS analyses. Similarly improbable results also arose in the FFPE tissues from patients 10 and 13, in which Malassezia pachydermatis and Coniosporium spp. were detected. There have been no reports of pulmonary infection due to either organism. Our study has some limitations. The sensitivity of pathogen detection by PCR/ESI-MS compared to detection by cultures or microscopic examinations could not be determined due to small sample size. Furthermore, because cases of Mucorales infection did not occur in this or a previous study (Shin et al., 2013), the diagnostic performance of PCR/ESI-MS (with specific primers targeting mito-
143
chondrial cytB and the short rRNA subunit of Mucorales) for detecting Mucorales could not be evaluated. Additional studies that enroll more patients are needed to evaluate its diagnostic performance for Mucorales and other less common fungal species. In conclusion, PCR/ESI-MS is capable of rapid detection of a broad range of fungi, including mixed fungal species, directly from clinical specimens. Fresh samples are preferred to avoid misinterpretation due to contaminants existing in FFPE tissues.
Acknowledgments This study was supported by grants from the National Health Research Institutes (IV-101-SP-18). We thank Miss Ming-I Hsieh for the laboratory work.
References Buitrago MJ, Aguado JM, Ballen A, Bernal-Martinez L, Prieto M, Garcia-Reyne A, et al. Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease. Clin Microbiol Infect 2012;19:E271–7. Cabaret O, Toussain G, Abermil N, Alsamad IA, Botterel F, Costa JM, et al. Degradation of fungal DNA in formalin-fixed paraffin-embedded sinus fungal balls hampers reliable sequence-based identification of fungi. Med Mycol 2011;49:329–32. De Castro N, Mazoyer E, Porcher R, Raffoux E, Suarez F, Ribaud P, et al. Hepatosplenic candidiasis in the era of new antifungal drugs: a study in Paris 2000–2007. Clin Microbiol Infect 2012;18:E185–7. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et al. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis 2008;46:1813–21. Loeffler J, Henke N, Hebart H, Schmidt D, Hagmeyer L, Schumacher U, et al. Quantification of fungal DNA by using fluorescence resonance energy transfer and the Light Cycler system. J Clin Microbiol 2000;38:586–90. Manual of clinical microbiology10th ed. . Washington, DC: ASM Press; 2010. Shin JH, Ranken R, Sefers SE, Lovari R, Quinn CD, Meng S, et al. Detection, identification, and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus PCR coupled with electrospray ionization/mass spectrometry. J Clin Microbiol 2013;51:136–41.
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Identification of fungal pathogens from clinical specimens using multi-locus PCR coupled with electrospray ionization mass spectrometry Chi-Jung Wu a, b,⁎, Ya-Ping Chen b, Hsuan-Chen Wang a, Ih-Jen Su a, Wen-Chien Ko b, Jiann-Shiuh Chen c, Chao-Neng Cheng c, Nan-Yao Lee b, H. Sunny Sun d, Chia-Yu Chi a, c, Tsai-Yun Chen b,⁎ a
National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan Department of Internal Medicine, National Cheng Kung University Hospital and Medical College, Tainan, Taiwan Department of Pediatrics, National Cheng Kung University Hospital and Medical College, Tainan, Taiwan d Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan b c
a r t i c l e
i n f o
Article history: Received 14 May 2013 Received in revised form 14 August 2013 Accepted 23 August 2013 Available online 20 November 2013
a b s t r a c t Polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was successfully used to identify a variety of fungi, including mixed fungal species, from 10 of 12 clinical specimens (10 culture-negative) of biopsy tissues or body fluids from patients with fungal infections. The application of PCR/ESI-MS for identifying fungal pathogens is discussed. © 2014 Elsevier Inc. All rights reserved.
Keywords: Electrospray ionization mass spectrometry Fungal infection PCR Paraffin
Rapid and accurate detection of the fungal pathogens that cause invasive fungal infections (IFIs) is critical for optimal patient care. Recently, the use of multi-locus polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) has been demonstrated to have the capacity for rapid and highthroughput genotypic characterization of a broad range of fungal organisms in bronchoalveolar lavage (BAL) (Shin et al., 2013). In this study, clinical specimens of biopsy tissues, body fluids, and BAL were used in PCR/ESI-MS analyses to evaluate the usefulness of this novel platform in IFI diagnoses. Non-consecutive patients with clinical suspicion of fungal infections that were culture-negative or difficult-to-treat were referred to the study by the infectious diseases specialists at National Cheng Kung University Hospital between June 2012 and March 2013. The study was approved by the institutional review board of the hospital, and patients were enrolled after informed consent. Patients’ clinical data were reviewed and characterized according to the definitions of IFIs from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group consensus group (De Pauw et al., 2008). The clinical information and types of specimens collected are listed in Table 1. Two formalin-fixed, ⁎ Corresponding authors. C.-J. Wu is to be contacted at Tel.: +886-6-7000123x65220; fax: +886-6-208-3466. T.-Y. Chen, Tel.: +886-6-2353535x4559; fax: +886-6-2752037. E-mail addresses: [email protected] (C.-J. Wu), [email protected] (T.-Y. Chen). 0732-8893/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.08.007
paraffin-embedded (FFPE) tissues and one fresh biopsy tissue were used as control samples. Clinical specimens distributed to culture and PCR/ESI-MS analyses were processed with aseptic techniques, whereas those that went to histopathological examination (and formalin fixation and paraffin embedding) were not. Fungal culturing and morphological or phenotypic identifications were performed on fresh specimens using standard procedures in clinical microbiology laboratories (ASM Press, 2010). For PCR/ESI-MS analyses, the specimens were lysed with a PLEX-ID bead beater, and the microbial DNA was extracted from the homogenates and purified using the UMD-Universal extraction kit (4 cases per serving) (Molzym, Bremen, Germany); DNA from FFPE tissues was extracted using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Extracted DNAs were automatically amplified and analyzed using PCR/ESI-MS (PLEX-ID) broad fungal assay (BFA) (Abbott Laboratories, Abbott Park, IL, USA), following the manufacturer’s instructions (Shin et al., 2013). To prevent contamination, the PCR setup, DNA extraction, PCR amplification, and PCR/ESI-MS analyses were performed in four separate rooms with dedicated consumables. Sample preparation and DNA extraction was performed in a biosafety cabinet illuminated with ultraviolet light when not in use. Organism identification was based on the total mass and base compositions of the PCR amplicons compared to those in the molecular signature database established by the PLEX-ID manufacturer (Shin et al., 2013). To determine the limit of detection by PCR/ESI-MS, two independent tenfold-dilution panels (10 1–10 3 cfu/mL) of Candida albicans ATCC 14053 and Aspergillus fumigatus BCRC 30099 were prepared by spiking colonies into 3 ml
142
C.-J. Wu et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 141–143
EDTA-anticoagulated peripheral blood from healthy volunteers and subjected to PCR/ESI-MS BFA analyses (Loeffler et al., 2000). The experiment was performed twice, and the limit of detection was 10 cfu/mL for both organisms. Using PCR/ESI-MS, a variety of fungi, including mixed fungal species, in 10 of the 12 clinical specimens (10 of which culturenegative) with suspected IFIs were identified (Table 1). The fungi detected included yeasts (Candida spp., Cryptococcus spp., and Malassezia spp.), filamentous fungi (Aspergillus spp., Acremonium spp., Cladosporium spp., and Coniosporium spp.), and Pneumocystis spp., although 2 results from FFPE tissues might be due to contaminants in the FFPE tissues. Except in patients 5 and 8, in which the PCR/ESI-MS could not differentiate between two closely related species (Aspergillus flavus and Aspergillus oryzae), the fungi detected were identified to a species level. This ability to identify multiple fungal species in a single specimen is an important advantage over pan-fungal PCR-based sequencing, another commonly used
technique, which often fails if two or more organisms co-exist in one specimen (Shin et al., 2013). Out of 4 culture-negative fresh biopsy tissue specimens, Candida albicans, Candida tropicalis and Pneumocystis jiroveci were identified in patients 1, 2, and 6, respectively, and the results were consistent with clinical features. A retrospective study found that 24 patients with hepatosplenic candidiasis all had negative culture results (De Castro et al., 2012). The PCR-based technique is more sensitive than microbiological culturing (89% vs. 56%) for the detection of IFIs (Buitrago et al., 2012). The high culture-negative rate in IFIs necessitates development of more sensitive detection methods, such as molecular identification. Our data suggest that the PCR/ESI-MS is capable of detecting and characterizing small amounts of fungal DNA in culture-negative specimens, and the identification result might be useful for selecting appropriate antifungal therapy. Of the 7 FFPE tissues tested, fungi were detected by PCR/ESI-MS in 4 of the 5 culture-negative clinical specimens with suspected IFIs and
Table 1 Summary of clinical information and laboratory results in the study. Case Age/Underlying no. diseases
Fresh tissues 1 22 y/leukemia
Infectious diseases
Specimens
HSC
Histology/GM indexa
2 3
5 y/neuroblastoma 52 y/ESRD
FN with skin rash PD peritonitis
Liver Hyphae biopsy Skin biopsy Hyphae/pseudohyphae PD fluid NP
4
23 y/nil
Meningitis
CSF
NP
5
43 y/leukemia
IMD, lung
Sputum
NP/4.46
6
37 y/AIDS
Pneumonia
BAL
NP
Lung biopsy
Cytomegalovirus inclusion body and granulomatous inflammation Interstitial inflammation
7
Lung biopsy Formalin-fixed, paraffin-embedded (FFPE) tissues Lung 8 50 y/leukemia IMD, lungd biopsy
9
17 y/leukemia
68 y/diabetes
10 39 y/leukemia
IMD, lung
IMD, sphenoid sinus Sphenoid sinus biopsy IMD, lung Lung biopsy
11 28 y/leukemia
HSC
Liver biopsy
12 54 y/leukemia
Hepatosplenic abscesses
Liver biopsy
Lung mass
FFPE tissue, lung FFPE tissue, oropharynx Fresh lung biopsy tissue
Control samples 13 74 y/lymphoma 14 65 y/nil 15 66 y/lymphoma
Oropharyngeal tuberculosis Lung mass
Right angle, broad pauciseptate hyphae, suggestive of mucormycosis or aspergillosis/0.91 Fungal hyphae, suggestive of aspergillosis/0.31
PCR/ESI-MS BFA identificationb
Fungal culture identification
EORTC/MSG criteria
Comparison of results of PCR/ESI-MS with conventional methods or clinical course
Candida albicans
No growth
Proven IFI
Compatible
Candida tropicalis Candida parapsilosis Cryptococcus neoformans C. albicans and Aspergillus flavus/ oryzae Pneumocystis jirovecic P. jirovecic
No growthc C. parapsilosis
Proven IFI Proven IFI
Compatible Compatible
C. neoformans
Proven IFI
Compatible
No growth
Probable IFI
Compatible
No growth
NA
Compatible
No growth
NA
Compatible
No detection
No growth
Possible IFI
NA
Aspergillus flavus/ oryzae
No growth
Proven IFI
Compatibled
Aspergillus fumigatus
No growth
Proven IFI
Compatible
No growth
Proven IFI
Most likely a contaminant
No growth
possible IFI
Most likely contaminants
No growth
possible IFI
NA
Coniosporium sp. Septated acute angle branching hyphae, suggestive of aspergillosis/0.15 Inflammation and fibrosis Acremonium persicinum and Cladosporium cladosporioides Suppurative granulomatous No detection inflammation. Lymphoma
Malassezia pachydermatis Granulomatous inflammation, A. persicinum and acid-fast bacilli C. cladosporioides Lymphoma No detection
No growth NP No growth
Most likely contaminants Most likely contaminants Compatible
AIDS = acquired immunodeficiency syndrome; CSF = cerebrospinal fluid; ESRD = end-stage renal disease; FN = febrile neutropenia; HSC = hepatosplenic candidiasis; IMD = invasive mold disease; NA = not applicable; NP = not performed; PD = peritoneal dialysis. a Serum galactomannan index (Platelia Aspergillus EIA, Bio-Rad Laboratories): A cut-off index of 0.5 is considered positive. b Seven FFPE tissues were analyzed independently using PCR/ESI-MS in 7 separate BFA plates at different times. c Cytomegalovirus was detected simultaneously using the PCR/ESI-MS Broad Viral Assay. The clinical condition improved with ganciclovir and trimethoprim/ sulfamethoxazole treatment. d Pneumonia resolved with voriconazole treatment.
C.-J. Wu et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 141–143
in 2 control samples. Formalin prevents the amplification of long fungal DNA fragments due to the degradation of DNA. The amplicon sizes obtained using PCR/ESI-MS BFA ranged from 72 to 154 base pairs, lengths that are more suitable for detecting fungal DNA in FFPE tissues than large amplicon sizes (Cabaret et al., 2011; Shin et al., 2013). A. flavus/oryzae and A. fumigatus were identified in patients 8 and 9, respectively, and the results were concordant with clinical and laboratory features of the patients. Nevertheless, the results of PCR/ESI-MS analyses should be interpreted cautiously for FFPE tissues. For example, C. tropicalis was the presumptive causative organism of hepatosplenic candidiasis in patient 11, who had experienced a prior episode of C. tropicalis fungemia and concurrent liver and spleen microabscesses revealed by a computed tomography during a neutropenic period. Acremonium persicinum and Cladosporium cladosporioides detected by PCR/ESI-MS from FFPE liver tissue from this patient are more likely to be contaminants in the paraffin section, an idea that is further supported by an identical result from another FFPE tissue from a lesion site on a patient with documented tuberculosis (patient 14). The 2 patients received biopsies for histopathology examination (and formalin fixation and paraffin embedding of tissues) 2 weeks apart, while their FFPE were retrieved, processed (using separate sets of QIAamp Tissue Kit), and examined by PCR/ESIMS 7 months apart. The long period between two independent PCR/ ESI-MS analyses and absence of both organisms in other samples precluded the possibility of contamination during sample processing or DNA extraction in PCR/ESI-MS analyses. Similarly improbable results also arose in the FFPE tissues from patients 10 and 13, in which Malassezia pachydermatis and Coniosporium spp. were detected. There have been no reports of pulmonary infection due to either organism. Our study has some limitations. The sensitivity of pathogen detection by PCR/ESI-MS compared to detection by cultures or microscopic examinations could not be determined due to small sample size. Furthermore, because cases of Mucorales infection did not occur in this or a previous study (Shin et al., 2013), the diagnostic performance of PCR/ESI-MS (with specific primers targeting mito-
143
chondrial cytB and the short rRNA subunit of Mucorales) for detecting Mucorales could not be evaluated. Additional studies that enroll more patients are needed to evaluate its diagnostic performance for Mucorales and other less common fungal species. In conclusion, PCR/ESI-MS is capable of rapid detection of a broad range of fungi, including mixed fungal species, directly from clinical specimens. Fresh samples are preferred to avoid misinterpretation due to contaminants existing in FFPE tissues.
Acknowledgments This study was supported by grants from the National Health Research Institutes (IV-101-SP-18). We thank Miss Ming-I Hsieh for the laboratory work.
References Buitrago MJ, Aguado JM, Ballen A, Bernal-Martinez L, Prieto M, Garcia-Reyne A, et al. Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease. Clin Microbiol Infect 2012;19:E271–7. Cabaret O, Toussain G, Abermil N, Alsamad IA, Botterel F, Costa JM, et al. Degradation of fungal DNA in formalin-fixed paraffin-embedded sinus fungal balls hampers reliable sequence-based identification of fungi. Med Mycol 2011;49:329–32. De Castro N, Mazoyer E, Porcher R, Raffoux E, Suarez F, Ribaud P, et al. Hepatosplenic candidiasis in the era of new antifungal drugs: a study in Paris 2000–2007. Clin Microbiol Infect 2012;18:E185–7. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et al. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis 2008;46:1813–21. Loeffler J, Henke N, Hebart H, Schmidt D, Hagmeyer L, Schumacher U, et al. Quantification of fungal DNA by using fluorescence resonance energy transfer and the Light Cycler system. J Clin Microbiol 2000;38:586–90. Manual of clinical microbiology10th ed. . Washington, DC: ASM Press; 2010. Shin JH, Ranken R, Sefers SE, Lovari R, Quinn CD, Meng S, et al. Detection, identification, and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus PCR coupled with electrospray ionization/mass spectrometry. J Clin Microbiol 2013;51:136–41.
