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ORIGINAL ARTICLE MYCOLOGY

Diagnosis of chronic disseminated candidosis from liver biopsies


by a novel PCR in patients with haematological malignancies

M. Fleischhacker1, S. Schulz1, K. Johrens2, M. von Lilienfeld-Toal3, T. Held4, E. Fietze5, C. Schewe2, I. Petersen6 and
M. Ruhnke1
1) Medizinische Klinik und Poliklinik II, Charite Campus Mitte der Humboldt-Universitat zu Berlin, Berlin, 2) Institut fur Pathologie, Charite Campus Mitte,
Universitatsmedizin Berlin, Berlin, 3) Medizinische Klinik und Poliklinik III, Uniklinikum Bonn, Bonn, 4) Klinik fur Hamatologie, Onkologie und Tumorimmuno-
logie HELIOS Klinikum Berlin-Buch Klinikum Buch Klinik fur Hamatologie Onkologie und Tumorimmonologie, Schwanebecker Chaussee, Berlin,
5) Gemeinschaftspraxis fur Pathologie am Bundeswehrkrankenhaus, Scharnhorststr, Berlin and 6) Institut fur Pathologie, Universitatsklinikum Jena,
Ziegelmuhlenweg, Jena, Germany

Abstract

Hepatic Candida infection (HCI; known as chronic disseminated candidosis or CDC) is a distinct form of disseminated Candida infection
with predominant involvement of the liver. Diagnosis of HCI is usually made on clinical suspicion together with multiple lesions in liver
on ultrasound (US), CT and/or MRI scan. Fungal elements may not always be visible in liver tissue and mycological culture is frequently
negative, making the evidence for proven fungal disease difcult. We studied a novel commercially available low-cost and density-array
(LCD) chip technique for a molecular diagnosis of HCI. This is a two-step procedure with PCR amplication after DNA extraction fol-
lowed by hybridization on a small chip provided by the manufacturer (Fungi 2.1, Chipron GmbH). The analysis of DNA from 45 fungal
control strains showed an excellent specicity and sensitivity. The DNA from 11 liver biopsies of patients with haematological malignan-
cies suffering from CDC was analysed on the LCD chip and overall 11 fungal pathogens could be detected in eight liver biopsies, sup-
porting the clinical diagnosis of HCI/CDC. Analysis of liver biopsies from controls was negative for fungal DNA in all samples studied.
In conclusion, the novel LCD chip technique examined in our study was able to detect fungal pathogens in liver biopsies from patients
with haematological malignancies and suspected HCI/CDC but was negative in control biopsies.

Keywords: Candida, candidosis, CDC, diagnosis, fungal, haematological malignancies, liver, molecular, PCR
Original Submission: 12 July 2011; Revised Submission: 25 October 2011; Accepted: 30 October 2011
Editor: E. Roilides
Article published online: 3 November 2011
Clin Microbiol Infect 2012; 18: 10101016
10.1111/j.1469-0691.2011.03713.x
nated Candida infection. When both liver and spleen are
Corresponding author: M. Ruhnke, Medizinische Klinik und
involved, the term hepatosplenic candidosis (HSC) is used. HCI
Poliklinik II, Charite Campus Mitte der Humboldt-Universitat zu
Berlin, Chariteplatz 1, 10117 Berlin, Germany occurring in immunocompromised patients is typically associ-
E-mail: markus.ruhnke@charite.de ated with disseminated infection involving multiple organs.
Data were presented in part at the 50th Interscience Conference on Since rst reports in the early 1960s, HCI is recognized
Antimicrobial Agents and Chemotherapy (ICAAC), Boston, 12-15 increasingly as a complication of patients with acute leukaemia
September 2010.
and other haematological malignancies treated with chemo-
therapy [1]. Although more than 40 years have passed since
the rst patient was described, the optimal diagnosis, as well
as management of this condition, is not well established. Diag-
Introduction
nosis of HCI is usually made on clinical suspicion (after recov-
ery from prolonged granulocytopenia with persistent fever,
Hepatic Candida infection (HCI), also known as chronic anorexia and elevation of alkaline phosphatase) together with
disseminated candidosis (CDC), is a distinct form of dissemi- multiple lesions (hypodensities) in liver on ultrasound, com-

2011 The Authors


Clinical Microbiology and Infection 2011 European Society of Clinical Microbiology and Infectious Diseases
CMI Fleischhacker et al. Molecular diagnosis of hepatic candidosis 1011

puter tomography (CT) scan and/or magnetic resonance imag- molecular diagnostics were stored at )80C until used. The
ing (MRI) [2]. Among the imaging modalities, MRI has the high- third part was xed in neutral buffered formalin and embed-
est diagnostic level as a non-invasive tool for the detection of ded in parafn for histological work-up. Histological features
CDC/HCI but will not lead to proven diagnosis [3]. Proven that are regarded as suggestive of hepatosplenic candidosis
HCI requires a positive histology plus cultural evidence for were analysed, including changes in hepatic architecture, pres-
fungi from a (sterile obtained) liver biopsy [4]. ence of granulomas, portal and parenchymal inammation,
However, a liver biopsy may not always establish the de- ductal proliferation, sinusoidal dilatation, necrosis, brosis and
nite diagnosis or some patients may be ineligible for the pro- fatty change [13,14]. Inammatory foci containing fungal
cedure. In addition, fungal elements may not always be organisms were documented. In addition, biopsies were analy-
visible in liver tissue and mycological culture is frequently sed microbiologically using standard culture techniques.
negative. This makes the evidence for proven fungal disease
difcult [57]. Fungi are usually microscopically visible in DNA isolation from parafn-embedded tissue and reference
organ biopsies, but it has been often observed that yeast strains
forms and pseudohyphae are seen only in the central area of Parafn was removed by extraction (twice) with xylene and
the abscess. The abscess may be even missed if the liver centrifugation. The tissue was rehydrated by incubation in etha-
biopsy is not taken targeted by laparoscopy [8]. nol (100%, 96%, 75%). The DNA was isolated with the Fast-
The role of other non-cultural diagnostic techniques (e.g. DNA Spin Kit (MP Biomedicals, Illkirch Cedex, France)
PCR) for detection of fungi in tissue samples has been stud- according to the instructions of the supplier with minor modi-
ied primarily for mould infection in the lungs but not for cations. The samples were homogenized in the FastPrep
yeast infection in the liver [912]. In this study, we used a instrument three times for 30 s at a speed setting of 55 with
novel commercially available low-cost and density-array cooling intervals of 1 min on ice. Finally, the DNA was eluted
(LCD) chip technique for studying the role of this molecular from the binding matrix with 100 lL DES (part of the Fast-
tool in diagnosing HCI. DNA Spin Kit) and stored at )20C until used.
From 45 fungal control strains, DNA was isolated using a
standard procedure as described earlier using zymolase diges-
Materials and Methods
tion following phenol-chloroform extractions and ethanol pre-
cipitation and the DNA was stored at )20C until used [15].
Retrospectively from 2001 to 2010, we identied 12 patients
from hospital charts who had a liver biopsy performed because PCR, gelelectrophoresis and chip hybridization
of suspected HCI. In 11 patients, liver biopsies were taken After DNA extraction, the DNA is amplied by PCR with
guided by ultrasound. One patient who died underwent an primers supplied with the kit and hybridized onto the LCD
autopsy. One patient (patient 11) nally was diagnosed as hav- chip. The DNA was amplied in a total volume of 25 lL
ing bacterial liver abscesses and served as a control patient. The using Taq polymerase (Platinum Taq, Invitrogen GmbH,
remaining 11 patients (seven female/four male) had a mean age Karlsruhe, Germany) according to the instructions of the
of 48 years (range 1881 years) and received chemotherapy manufacturer of the LCD chip (Fungi 2.1; Chipron GmbH,
for haematological malignancies (nine acute leukaemias (eight Berlin, Germany). The cycling conditions were set to 3 min
acute myeloid leukaemia (AML)/one acute lymphocytic leukae- at 95C (initial denaturation), 30 s at 94C, 45 s at 56C,
mia (ALL)), one chronic myeloic leukaemia (CML) and one 45 s at 72C (3545 repetitions for amplication), 3 min at
non-Hodgkin lymphoma (NHL). HCI was clinically suggested by 72C for nal extension, and cooling at 4C, and a MJ
the appearance of new multiple hypodense lesions in liver (and Research PTC-100 cycler was used. The hands-on time for
spleen) assessed by radiological examination (CT, ultrasound 16 samples is <2 h. An aliquot of 7 lL of the PCR products
and/or MRI) and clinical signs and symptoms suggestive of HCI/ was run on a 2% agarose gel. In a second step, DNA was
CDC. Further clinical information is given in Table 3. spotted manually on LCD chips and hybridization was per-
In addition, 18 liver biopsies from patients without signs formed according to the instruction manual. According to
and symptoms of HCI/CDC who underwent diagnostic liver the manufacturer, the array is able to discriminate between
biopsy (e.g. after liver transplantation) were analysed and 25 different fungal species or species clusters, such as
served as controls. Candida albicans, Candida glabrata, Candida tropicalis, Candida
Biopsies were cut into three parts. Two parts were trans- parapsilosis, Candida krusei, Candida dubliniensis, Candida guillier-
ferred, each in 2 mL isotonic sodium chloride solution, for mondii, Candida pelliculosa, Candida lusitaniae, Candida lambica,
microbiological culture and molecular diagnostics. Samples for Candida kefyr, Aspergillus niger complex, Aspergillus fumigatus,

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Clinical Microbiology and Infection 2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 10101016
1012 Clinical Microbiology and Infection, Volume 18 Number 10, October 2012 CMI

Aspergillus avus, Aspergillus terreus, Aspergillus nidulans, Mucor not be detected by the predened array format were nega-
spp., Rhizomucor pusillus, Rhizomucor oryzae/Rhizomucor ar- tive by chip hybridization (Table 2).
rhizus, Rhizomucor azygosporus/Rhizomucor microspores, Rhizo- All 12 patients suffered from active haematological malig-
mucor stolonifer, Cryptococcus neoformans, Paecilomyces variotii, nancies (nine AML/one ALL, one NHL, one allogeneic bone
Scedosporium prolicans and Lichtheimia (Absidia) corymbifera. marrow transplantation for CML) and had radiological signs
For scanning and nal analysis a combination of a transmis- highly suggestive of HCI showing multiple new lesions in the
sion light scanner and image analysis software supplied by liver by abdominal ultrasound, which was conrmed by
the manufacturer was used. biphasic spiral liver computed tomography CT (ten patients)
or MRI (two patients) (Table 3).
Hepatic lesions showed the well-known abscess-like pat-
Results
tern previously described [16]. Many patients had a history of
a previous fungal disease, mostly proven/probable invasive pul-
Identication of reference strains according to the prede- monary aspergillosis (IPA), in six individuals, and possible IPA,
ned array format was correct in all isolates tested in one patient. Hepatic lesions developed while these patients
(Table 1). In addition, various other fungal strains that should were treated for pulmonary aspergillosis with broad-spectrum

TABLE 1. Identication of refer-


Control strain PCR result Chip hybridization Identication
ence strains according to a prede-
Lichtheimia (Absidia) corymbifera (IP 1280.81) Strong band Abidia corymbifera Correct ned array format
Aspergillus avus (ATCC 9643) Strong band A. avus Correct
Aspergillus fumigatus (DSM 819) Strong band A. fumigatus Correct
Aspergillus fumigatus (DSM 819) Strong band A. fumigatus Correct
Aspergillus nidulans (RKI 491/06) Strong band A. nidulans Correct
Aspergillus niger (clinical isolate) Strong band A. niger Correct
Aspergillus terreus (RKI 83/06) Strong band A. terreus Correct
Aspergillus terreus (RKI 533/02) Strong band A. terreus Correct
Aspergillus versicolor (RKI 826/02) Strong band A. versicolor Correct
Candida albicans (ATCC 90028) Strong band C. albicans Correct
Candida dubliniensis (CBS 8500) Strong band C. dubliniensis Correct
Candida glabrata (ATCC 90030) Strong band C. glabrata Correct
Candida guilliermondii (ATCC 90877) Strong band Pichia guilliermondii Correct
Candida kefyr (DSM 11954) Strong band Kluyveromyces marxianus Correct
Candida krusei (DSM 11956) Very weak band Issatchenkia orientalis Correct
Candida parapsilosis (ATCC 22019) Strong band C. parapsilosis Correct
Candida orthopsilosis (GHP 3540) Strong band C. parapsilosis Correct
Candida metapsilosis (GHP 3541) Strong band C. parapsilosis Correct
Candida parapsilosis (DSM 11955) Strong band C. parapsilosis Correct
Candida tropicalis (ATCC 28707) Strong band C. tropicalis Correct
Candida tropicalis (ATCC 750) Strong band C. tropicalis Correct
Cryptococcus neoformans (ATCC 62066) Strong band Crytococcus neoformans Correct
Mucor rouxii (IP 1248.80) Strong band Mucor Correct
Paecilomyces variotii (RKI 498/00) Strong band Paecilomyces variotii Correct
Rhizopus microsporus var. rhizopodi for. (IP 676.72) Strong band Rhizopus Correct
Rhizopus microsporus (IP 1123.75) Strong band Rhizopus Correct
Rhizomucor pusillus (IP 1127.75) Weak band Rhizomucor Correct
Rhizopus oryzae (IP 1443.83) Strong band Rhizopus Correct

TABLE 2. Control strains not


Chip
Control strain PCR result hybridization Identication detected by chip hybridization

Aspergillus alliaceusa Strong band No signal Not depicted on chip


Aureobasidium pullulansa Strong band No signal Not depicted on chip
Arthrobotrys oligosporaa Strong band No signal Not depicted on chip
Candida allociferiia Strong band No signal Not depicted on chip
Candida lipolyticab Strong band No signal Not depicted on chip
Cuninghamella bertholletiae (IP 1886.89) Strong band No signal Not depicted on chip
Fusarium oxysporum (RKI 592/07) Strong band No signal Not depicted on chip
Geotrichum candidum (clinical isolate) Weak band No signal Not depicted on chip
Penicillium purpurogenuma Strong band No signal Not depicted on chip
Pseudallescheria boydii (clinical isolate) Weak band No signal Not depicted on chip
Saccharomyces cerevisiae (ATCC 9763) Strong band No signal Not depicted on chip
Scopulariopsis brevicaulisa Strong band No signal Not depicted on chip
Syncephalastrum racemosum (IP 1541.84) Strong band No signal Not depicted on chip
Trichosporon asahii (GHP 3537)b Strong band No signal Not depicted on chip
Tritiratium oryzaea Strong band No signal Not depicted on chip
Ulcocladiuma Strong band No signal Not depicted on chip
a
Courtesy of H. Hof.
b
Courtesy of G. Haase.

2011 The Authors


Clinical Microbiology and Infection 2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 10101016
CMI

TABLE 3. Clinical data from 12 liver biopsies obtained from patients with HCI (CDC) in correlation with PCR results

Antifungal
Age Fungus in Radiology therapy
(years)/ biopsy (PAS/ (CT/abdominal PCR from liver before
Patient sex Diagnosis Sample Histology Grocott stain) Fungal culture ultrasound) Clinical diagnosis tissue biopsy (days)

1 48/f AML Liver biopsy Granuloma, necrosis, scars Negative Negative Multiple lesions Proven IPA (lungs) Candida albicans 90
liver Possible HCI
2 81/m B-NHL Autopsy Multiple white lesions on n.d. at autopsy Candida albicans + Single hepatic lesions Proven IPA (lungs) Aspergillus fumigatus 6
liver surface; malignant Aspergillus spp. Possible HCI Candida albicans
lymphoma in liver
3 56/m CML/allo-BMT Liver biopsy Necrosis Negative Negative Multiple lesions Possible HCI Candida albicans 78
liver/spleen
4 19/m AML Liver biopsy Granuloma, cholostatic Negative Negative Multiple lesions Possible HCI Candida dubliniensis 420
inammation liver/spleen
5 28/f ALL Liver biopsy Granuloma, brosis Yeasts Negative Multiple lesions Proven HCI Candida albicans 21
liver
6 46/f AML Liver biopsy Fibrosis, scars, necrosis, Yeasts n.d. Multiple lesions Proven HCI Candida lipolytica* 60
fatty changes liver
7 57/f AML Liver biopsy Fibrosis, fatty changes Negative n.d. Multiple lesions liver Possible HCI Candida tropicalis, 92
Candida glabrata
8 44/f AML Liver biopsy Fibrosis Negative Negative Multiple lesions Probable IPA (lung) Candida albicans 35
Liver Possible HCI
Fleischhacker et al.

9 56/f AML Liver biopsy Fibrosis, necrosis, fatty Negative Negative Multiple lesions Probable IPA (lung) Negative 45
changes liver/spleen Possible HCI
10 26/f AML Liver biopsy Fibrosis, fatty changes Negative Negative Multiple lesions Probable IPA (lung) Candida lipolytica* 14
liver/spleen Possible HCI
11* 56/m AML Liver biopsy Granuloma, brosis, Negative Negative Multiple lesions Probable IPA (lung) Negative 21
purulent inammation (growth S. aureus) liver and suspected Possible HCI
Liver cirrhosis cirrhosis
12 71/m AML Liver biopsy Fibrosis, fatty changes Negative Negative Multiple lesions Possible IPA (lung) Candida albicans 90
liver/spleen Possible HCI

AML, acute myeloid leukaemia; ALL, acute lymphocytic leukaemia; B-NHL, B-cell non-Hodgkin lymphoma; CML, chronic myeloid leukaemia; allo-BMT, allogeneic bone marrow transplantation; HCI, hepatic Candida infection; IPA, invasive
pulmonary aspergillosis; *, control patient; n.d., not detected.
Molecular diagnosis of hepatic candidosis
1013

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Clinical Microbiology and Infection 2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 10101016
1014 Clinical Microbiology and Infection, Volume 18 Number 10, October 2012 CMI

antifungal therapy (conventional amphotericin B, liposomal Diagnosis is primarily established on clinical ndings in immu-
amphotericin or voriconazole). All patients but one under- nocompromised patients (predominantly with acute leukae-
went CT or ultrasound-guided liver biopsy. One individual mia) with persistent fever not responsive to conventional
who died had an autopsy performed. Detection of yeasts with antibiotics, together with clinical ndings, such as gastrointes-
pseudohyphae in liver tissue by periodic acid-Schiff reaction tinal symptoms with hepatomegaly and laboratory signs (e.g.
staining succeeded in two patients (patients 5 and 6). Fungal elevated levels of alkaline phosphatase). Fever typically pre-
culture was negative in all but one biopsy (patient 2). In one sents as recurrent fever that occurs after neutrophil recov-
patient (patient 2), CT was performed 8 weeks before death ery and HCI is diagnosed when typical lesions in liver (and
from autopsy-proven disseminated aspergillosis, showing sometimes spleen and/or other organs) can be seen on com-
hepatic lesions of unclear aetiology. This patient suffered from puted tomography, ultrasound and/or magnetic resonance
aggressive non-Hodgkin lymphoma and at autopsy clinically imaging [3,5,17,18]. In the very rst reports on disseminated
silent recurrence of malignant lymphoma in the liver was Candida infections, which were called moniliasis until the
detected without further manifestations at other body sites. 1960s, fungal infections were found predominantly in the
This patient died from fulminant sepsis-like syndrome. Micro- lungs, kidneys, heart and gastrointestinal tract but less fre-
biological culture of the liver biopsy revealed growth of yeasts quently in the liver [1]. Prevalence of hepatic involvement in
and lamentous fungi (6 days of treatment with amphotericin disseminated Candida infection varied depending on the publi-
B before death). All other patients had received either empiri- cation but a mean prevalence of 14% has been suggested in
cal or prolonged pre-emptive antifungal therapy before the earlier reports [19]. Soon, it became clear that focal hepatic
biopsy was performed (14420 days, mean 87 days). In one Candida infection is a distinct clinical variant of disseminated
patient (patient 11), Staphyloccocus aureus was cultured from Candida infection (chronic disseminated candidosis, CDC) in
the liver biopsy, but no fungi were detected and PCR was neg- immunocompromised patients, mostly patients who have
ative. This patient was regarded as having bacterial liver received intense cytotoxic chemotherapy for acute leukaemia
abscesses and not HCI (control patient). Histological examina- [2,3,57,1922].
tion in this patient revealed clinically unsuspected liver cirrho- Lesions associated with HCI were described as granulo-
sis together with granuloma, brosis and purulent mas, microabscesses, centrilobular congestion, haemorrhagic
inammation, suggestive of bacterial infection. necrosis, bile stasis, inammatory parenchymal aggregates,
With the LCD chip, overall 11 fungal pathogens could be free yeasts in sinusoids and/or fatty changes [13,19].
detected in eight liver biopsies, with six Candida albicans and With regard to the denite diagnosis of HCI, the rst
one Candida dubliniensis as single pathogens, and two mixed denition of the diagnosis of invasive fungal diseases (IFD) in
infections, one with Candida albicans plus Aspergillus fumigatus haematopoietic stem cell transplant recipients was estab-
and one with Candida glabrata plus Candida tropicalis, respec- lished for research purposes. Patients with typical lesions for
tively. In two liver samples, PCR revealed a positive ampli- HCI on CT or ultrasound were regarded as probable IFD
cation product specic for a fungal pathogen. However, using without any need for mycological support. In the revised def-
the LCD chip array a specic fungal pathogen could not be initions, such cases are now classied as possible IFD [4]. In
identied. After additional sequencing of the PCR product, earlier reports, when patients may or may not have received
Candida lipolytica was identied in two samples. This pathogen Amphotericin B for suspected HCI, it has been suggested
will not be detected by the array and would be missed in tis- that liver biopsies usually revealed yeast and/or hyphal forms,
sue samples where Candida lipolytica is the infecting pathogen. but mycological cultures were frequently negative [5,19].
We also analysed the DNA isolated from 18 liver biopsies However, identiable Candida organisms may not be found in
that had been taken from patients without evidence for HCI. all tissue specimens and only indirect signs of HCI may be
All 18 samples gave no signal after hybridization onto the seen at histopathological examination [13].
chip (data not shown) and were negative when examined The majority of cases in our study had a possible diagnosis
with a pan-fungal real-time assay. of HCI because fungal elements were not visible in the
biopsy but radiology was highly suggestive of fungal liver dis-
ease. Interestingly, many of the patients studied here had a
Discussion
previous history of proven/probable invasive fungal disease in
the lungs (possible/probable aspergillosis) and have received
Chronic disseminated candidosis is a potentially life-threaten- systemic antifungal therapy. Only in two patients, could
ing invasive fungal disease in patients with haematological yeasts be detected in the liver sample. However, hepatic
malignancies which requires systemic antifungal therapy. Candida infection could not be proven by a positive culture

2011 The Authors


Clinical Microbiology and Infection 2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 10101016
CMI Fleischhacker et al. Molecular diagnosis of hepatic candidosis 1015

of the fungal organism in any of the cases. As outlined above, patients with proven invasive mould infection [28]. Recently,
all patients had received a substantial amount of systemic improvement has been achieved by establishing DNA micro-
antifungal therapy with Candida-active antifungal agents arrays to detect and identify DNA from fungal pathogens,
before the liver biopsy was taken. This underlines the diag- mostly Candida and Aspergillus species [29,30]. In contrast to
nostic dilemma when a tissue sample is taken under ongoing the assay studied here, these microarray systems are not
antifungal therapy. It has been earlier discussed that laparo- commercially available but may detect up to 14 different fun-
scopic liver biopsies may have higher yield in the diagnosis of gal pathogens from various samples (blood, bronchoalveolar
HCI because the procedure allows better sampling of hepatic lavage and tissue) in high-risk patients [30].
focal lesions [6,23]. Laparoscopy is nowadays rarely per- In conclusion, the novel LCD chip technique examined in
formed in patients with haematological malignancies to estab- our study was able to detect fungal pathogens in the majority
lish the diagnosis of HCI because radiological diagnosis with of liver biopsies from patients with suspected hepatic fungal
ultrasound, CT and/or MRI has markedly improved infection but was negative in control biopsies. Together with
[1618,2427]. In our patients, it cannot be excluded that a the clinical information, detection of fungal DNA in liver
laparoscopy-guided liver biopsy might have had a higher diag- biopsies may help to improve establishing the diagnosis of
nostic yield. Anttila et al. reported that the diagnosis of HCI CDC even during antifungal therapy.
was established signicantly more often when the biopsy was
targeted at white nodules than when targeted randomly or
Acknowledgements
at scars [6]. However, in this study many liver biopsies tar-
geted at white nodules had negative results at histological
and/or cytological examination as well. We thank Dr Kathrin Tintelnot (Robert-Koch-Institute/RKI
Molecular techniques may improve diagnosis of invasive isolates, Berlin), Professor Herbert Hof (University Hospital
fungal disease. Molecular methods for the identication of Mannheim), Professor Gerhard Haase (University Hospital
Aspergillus spp and other pathogenic fungi in parafn wax Aachen/GHP isolates) and Professor Olivier Lortholary
embedded tissues have been established [9,12]. In general, in (Institute Pasteur/IP isolates, Paris, France) for providing
these assays DNA was amplied by PCR using pan-fungal strains. We thank Dr Volker Heiser (Chipron GmbH, Berlin,
probes, and detected by Southern blot hybridization with a Germany) and Clarissa Radecke for technical assistance.
probe specic for Aspergillus fumigatus and other fungi [9,12].
In our study, the specicity and sensitivity for detecting
fungal DNA from a large collection of control and reference
Transparency Declaration
strains was good. All fungal isolates that are supposed to be
detectable by the LCD chip could be correctly identied All authors have substantially contributed to the manuscript
using reference strains. Furthermore, DNA from a large col- and can take public responsibility for the content. M. Ruhnke
lection of additional fungal control strains that should not be has received research grants from Deutsche Krebshilfe/Ger-
identied could be amplied by PCR but not the nal hybrid- man Cancer Aid, Pzer and Merck/MSD, served as a consul-
ization step on the LCD chip. These additional fungal strains tant and at the speakers bureau of Astellas, Basilea, Essex/
may cause invasive fungal disease in immunocompromised Schering-Plough, Gilead Sciences, Janssen, Merck/MSD,
patients but would not be identied by the array, which lim- Novartis, Pzer, Pliva (all not related to the manuscript). M.
its this method. Of interest, we found fungal DNA in two von Lilienfeld-Toal has received research grants from Merck/
liver samples from patients with possible CDC that could MSD and served as a consultant and at the speakers bureau
not be further identied after nal chip hybridization. After of Merck/MSD and Wyeth. M. Fleischhacker, S. Schulz, K.
additional sequencing of the PCR product, Candida lipolytica Johrens, T. Held, E. Fietze, C. Schewe, I. Petersen have noth-
was identied. This rare Candida species (as well as, for ing to declare.
example, Trichosporon spp.) is not within the detection scope
of the array and may limit the test method. However, the
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Clinical Microbiology and Infection 2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 10101016
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2011 The Authors


Clinical Microbiology and Infection 2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 10101016