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Keywords:

  • Candida;
  • candidosis;
  • CDC;
  • diagnosis;
  • fungal;
  • haematological malignancies;
  • liver;
  • molecular;
  • PCR

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Clin Microbiol Infect 2012; 18: 1010–1016

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 difficult. 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 amplification after DNA extraction followed 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 specificity and sensitivity. The DNA from 11 liver biopsies of patients with haematological malignancies suffering from CDC was analysed on the LCD chip and overall 11 fungal pathogens could be detected in eight liver biopsies, supporting 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Hepatic Candida infection (HCI), also known as chronic disseminated candidosis (CDC), is a distinct form of disseminated Candida infection. When both liver and spleen are involved, the term hepatosplenic candidosis (HSC) is used. HCI occurring in immunocompromised patients is typically associated with disseminated infection involving multiple organs.

Since first reports in the early 1960s, HCI is recognized increasingly as a complication of patients with acute leukaemia and other haematological malignancies treated with chemotherapy [1]. Although more than 40 years have passed since the first patient was described, the optimal diagnosis, as well as management of this condition, is not well established. Diagnosis of HCI is usually made on clinical suspicion (after recovery from prolonged granulocytopenia with persistent fever, anorexia and elevation of alkaline phosphatase) together with multiple lesions (hypodensities) in liver on ultrasound, computer tomography (CT) scan and/or magnetic resonance imaging (MRI) [2]. Among the imaging modalities, MRI has the highest diagnostic level as a non-invasive tool for the detection of CDC/HCI but will not lead to proven diagnosis [3]. Proven HCI requires a positive histology plus cultural evidence for fungi from a (sterile obtained) liver biopsy [4].

However, a liver biopsy may not always establish the definite diagnosis or some patients may be ineligible for the procedure. In addition, fungal elements may not always be visible in liver tissue and mycological culture is frequently negative. This makes the evidence for proven fungal disease difficult [5–7]. Fungi are usually microscopically visible in organ biopsies, but it has been often observed that yeast forms and pseudohyphae are seen only in the central area of the abscess. The abscess may be even missed if the liver biopsy is not taken targeted by laparoscopy [8].

The role of other non-cultural diagnostic techniques (e.g. PCR) for detection of fungi in tissue samples has been studied primarily for mould infection in the lungs but not for yeast infection in the liver [9–12]. In this study, we used a novel commercially available low-cost and density-array (LCD) chip technique for studying the role of this molecular tool in diagnosing HCI.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Retrospectively from 2001 to 2010, we identified 12 patients from hospital charts who had a liver biopsy performed because of suspected HCI. In 11 patients, liver biopsies were taken guided by ultrasound. One patient who died underwent an autopsy. One patient (patient 11) finally was diagnosed as having bacterial liver abscesses and served as a control patient. The remaining 11 patients (seven female/four male) had a mean age of 48 years (range 18–81 years) and received chemotherapy for haematological malignancies (nine acute leukaemias (eight acute myeloid leukaemia (AML)/one acute lymphocytic leukaemia (ALL)), one chronic myeloic leukaemia (CML) and one non-Hodgkin lymphoma (NHL). HCI was clinically suggested by the appearance of new multiple hypodense lesions in liver (and spleen) assessed by radiological examination (CT, ultrasound and/or MRI) and clinical signs and symptoms suggestive of HCI/CDC. Further clinical information is given in Table 3.

In addition, 18 liver biopsies from patients without signs and symptoms of HCI/CDC who underwent diagnostic liver biopsy (e.g. after liver transplantation) were analysed and served as controls.

Biopsies were cut into three parts. Two parts were transferred, each in 2 mL isotonic sodium chloride solution, for microbiological culture and molecular diagnostics. Samples for molecular diagnostics were stored at −80°C until used. The third part was fixed in neutral buffered formalin and embedded in paraffin for histological work-up. Histological features that are regarded as suggestive of hepatosplenic candidosis were analysed, including changes in hepatic architecture, presence of granulomas, portal and parenchymal inflammation, ductal proliferation, sinusoidal dilatation, necrosis, fibrosis and fatty change [13,14]. Inflammatory foci containing fungal organisms were documented. In addition, biopsies were analysed microbiologically using standard culture techniques.

DNA isolation from paraffin-embedded tissue and reference strains

Paraffin was removed by extraction (twice) with xylene and centrifugation. The tissue was rehydrated by incubation in ethanol (100%, 96%, 75%). The DNA was isolated with the FastDNA® Spin Kit (MP Biomedicals, Illkirch Cedex, France) according to the instructions of the supplier with minor modifications. The samples were homogenized in the FastPrep® instrument three times for 30 s at a speed setting of 5·5 with cooling intervals of 1 min on ice. Finally, the DNA was eluted from the binding matrix with 100 μL DES (part of the FastDNA® Spin Kit) and stored at −20°C until used.

From 45 fungal control strains, DNA was isolated using a standard procedure as described earlier using zymolase digestion following phenol-chloroform extractions and ethanol precipitation and the DNA was stored at −20°C until used [15].

PCR, gelelectrophoresis and chip hybridization

After DNA extraction, the DNA is amplified by PCR with primers supplied with the kit and hybridized onto the LCD chip. The DNA was amplified in a total volume of 25 μL using Taq polymerase (Platinum Taq®, Invitrogen GmbH, Karlsruhe, Germany) according to the instructions of the manufacturer of the LCD chip (Fungi 2.1; Chipron GmbH, Berlin, Germany). The cycling conditions were set to 3 min at 95°C (initial denaturation), 30 s at 94°C, 45 s at 56°C, 45 s at 72°C (35–45 repetitions for amplification), 3 min at 72°C for final extension, and cooling at 4°C, and a MJ Research PTC-100 cycler was used. The hands-on time for 16 samples is <2 h. An aliquot of 7 μL of the PCR products was run on a 2% agarose gel. In a second step, DNA was spotted manually on LCD chips and hybridization was performed according to the instruction manual. According to the manufacturer, the array is able to discriminate between 25 different fungal species or species clusters, such as Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida dubliniensis, Candida guilliermondii, Candida pelliculosa, Candida lusitaniae, Candida lambica, Candida kefyr, Aspergillus niger complex, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, Mucor spp., Rhizomucor pusillus, Rhizomucor oryzae/Rhizomucor arrhizus, Rhizomucor azygosporus/Rhizomucor microspores, Rhizomucor stolonifer, Cryptococcus neoformans, Paecilomyces variotii, Scedosporium prolificans and Lichtheimia (Absidia) corymbifera. For scanning and final analysis a combination of a transmission light scanner and image analysis software supplied by the manufacturer was used.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Identification of reference strains according to the predefined array format was correct in all isolates tested (Table 1). In addition, various other fungal strains that should not be detected by the predefined array format were negative by chip hybridization (Table 2).

Table 1.   Identification of reference strains according to a predefined array format
Control strainPCR resultChip hybridizationIdentification
Lichtheimia (Absidia) corymbifera (IP 1280.81)Strong band Abidia corymbifera Correct
Aspergillus flavus (ATCC 9643)Strong band A. flavus 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 detected by chip hybridization
Control strainPCR resultChip hybridizationIdentification
  1. aCourtesy of H. Hof.

  2. bCourtesy of G. Haase.

Aspergillus alliaceus a Strong bandNo signalNot depicted on chip
Aureobasidium pullulans a Strong bandNo signalNot depicted on chip
Arthrobotrys oligospora a Strong bandNo signalNot depicted on chip
Candida allociferii a Strong bandNo signalNot depicted on chip
Candida lipolytica b Strong bandNo signalNot depicted on chip
Cuninghamella bertholletiae (IP 1886.89)Strong bandNo signalNot depicted on chip
Fusarium oxysporum (RKI 592/07)Strong bandNo signalNot depicted on chip
Geotrichum candidum (clinical isolate)Weak bandNo signalNot depicted on chip
Penicillium purpurogenum a Strong bandNo signalNot depicted on chip
Pseudallescheria boydii (clinical isolate)Weak bandNo signalNot depicted on chip
Saccharomyces cerevisiae (ATCC 9763)Strong bandNo signalNot depicted on chip
Scopulariopsis brevicaulis a Strong bandNo signalNot depicted on chip
Syncephalastrum racemosum (IP 1541.84)Strong bandNo signalNot depicted on chip
Trichosporon asahii (GHP 3537)bStrong bandNo signalNot depicted on chip
Tritiratium oryzae a Strong bandNo signalNot depicted on chip
Ulcocladium a Strong bandNo signalNot depicted on chip

All 12 patients suffered from active haematological malignancies (nine AML/one ALL, one NHL, one allogeneic bone marrow transplantation for CML) and had radiological signs highly suggestive of HCI showing multiple new lesions in the liver by abdominal ultrasound, which was confirmed by biphasic spiral liver computed tomography CT (ten patients) or MRI (two patients) (Table 3).

Table 3.   Clinical data from 12 liver biopsies obtained from patients with HCI (CDC) in correlation with PCR results
PatientAge (years)/sexDiagnosisSampleHistologyFungus in biopsy (PAS/Grocott stain)Fungal cultureRadiology (CT/abdominal ultrasound)Clinical diagnosisPCR from liver tissueAntifungal therapy before biopsy (days)
  1. 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.

 148/fAMLLiver biopsyGranuloma, necrosis, scarsNegativeNegativeMultiple lesions liverProven IPA (lungs) Possible HCI Candida albicans 90
 281/mB-NHLAutopsyMultiple white lesions on liver surface; malignant lymphoma in livern.d. at autopsyCandida albicans + Aspergillus spp.Single hepatic lesionsProven IPA (lungs) Possible HCI Aspergillus fumigatus Candida albicans 6
 356/mCML/allo-BMTLiver biopsyNecrosisNegativeNegativeMultiple lesions liver/spleenPossible HCI Candida albicans 78
 419/mAMLLiver biopsyGranuloma, cholostatic inflammationNegativeNegativeMultiple lesions liver/spleenPossible HCI Candida dubliniensis 420
 528/fALLLiver biopsyGranuloma, fibrosisYeastsNegativeMultiple lesions liverProven HCI Candida albicans 21
 646/fAMLLiver biopsyFibrosis, scars, necrosis, fatty changesYeastsn.d.Multiple lesions liverProven HCI Candida lipolytica* 60
 757/fAMLLiver biopsyFibrosis, fatty changesNegativen.d.Multiple lesions liverPossible HCI Candida tropicalis, Candida glabrata 92
 844/fAMLLiver biopsyFibrosisNegativeNegativeMultiple lesions LiverProbable IPA (lung) Possible HCI Candida albicans 35
 956/fAMLLiver biopsyFibrosis, necrosis, fatty changesNegativeNegativeMultiple lesions liver/spleenProbable IPA (lung) Possible HCINegative45
1026/fAMLLiver biopsyFibrosis, fatty changesNegativeNegativeMultiple lesions liver/spleenProbable IPA (lung) Possible HCI Candida lipolytica* 14
11*56/mAMLLiver biopsyGranuloma, fibrosis, purulent inflammation Liver cirrhosisNegativeNegative (growth S. aureus)Multiple lesions liver and suspected cirrhosisProbable IPA (lung) Possible HCINegative21
1271/mAMLLiver biopsyFibrosis, fatty changesNegativeNegativeMultiple lesions liver/spleenPossible IPA (lung) Possible HCI Candida albicans 90

Hepatic lesions showed the well-known abscess-like pattern previously described [16]. Many patients had a history of a previous fungal disease, mostly proven/probable invasive pulmonary aspergillosis (IPA), in six individuals, and possible IPA, in one patient. Hepatic lesions developed while these patients were treated for pulmonary aspergillosis with broad-spectrum antifungal therapy (conventional amphotericin B, liposomal amphotericin or voriconazole). All patients but one underwent CT or ultrasound-guided liver biopsy. One individual who died had an autopsy performed. Detection of yeasts with pseudohyphae in liver tissue by periodic acid-Schiff reaction staining succeeded in two patients (patients 5 and 6). Fungal culture was negative in all but one biopsy (patient 2). In one patient (patient 2), CT was performed 8 weeks before death from autopsy-proven disseminated aspergillosis, showing hepatic lesions of unclear aetiology. This patient suffered from aggressive non-Hodgkin lymphoma and at autopsy clinically silent recurrence of malignant lymphoma in the liver was detected without further manifestations at other body sites. This patient died from fulminant sepsis-like syndrome. Microbiological culture of the liver biopsy revealed growth of yeasts and filamentous fungi (6 days of treatment with amphotericin B before death). All other patients had received either empirical or prolonged pre-emptive antifungal therapy before the biopsy was performed (14–420 days, mean 87 days). In one patient (patient 11), Staphyloccocus aureus was cultured from the liver biopsy, but no fungi were detected and PCR was negative. This patient was regarded as having bacterial liver abscesses and not HCI (control patient). Histological examination in this patient revealed clinically unsuspected liver cirrhosis together with granuloma, fibrosis and purulent inflammation, suggestive of bacterial infection.

With the LCD chip, overall 11 fungal pathogens could be detected in eight liver biopsies, with six Candida albicans and one Candida dubliniensis as single pathogens, and two mixed infections, one with Candida albicans plus Aspergillus fumigatus and one with Candida glabrata plus Candida tropicalis, respectively. In two liver samples, PCR revealed a positive amplification product specific for a fungal pathogen. However, using the LCD chip array a specific fungal pathogen could not be identified. After additional sequencing of the PCR product, Candida lipolytica was identified in two samples. This pathogen will not be detected by the array and would be missed in tissue samples where Candida lipolytica is the infecting pathogen.

We also analysed the DNA isolated from 18 liver biopsies that had been taken from patients without evidence for HCI. All 18 samples gave no signal after hybridization onto the chip (data not shown) and were negative when examined with a pan-fungal real-time assay.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Chronic disseminated candidosis is a potentially life-threatening invasive fungal disease in patients with haematological malignancies which requires systemic antifungal therapy. Diagnosis is primarily established on clinical findings in immunocompromised patients (predominantly with acute leukaemia) with persistent fever not responsive to conventional antibiotics, together with clinical findings, such as gastrointestinal symptoms with hepatomegaly and laboratory signs (e.g. elevated levels of alkaline phosphatase). Fever typically presents as recurrent fever that occurs after neutrophil recovery and HCI is diagnosed when typical lesions in liver (and sometimes spleen and/or other organs) can be seen on computed tomography, ultrasound and/or magnetic resonance imaging [3,5,17,18]. In the very first reports on disseminated Candida infections, which were called moniliasis until the 1960s, fungal infections were found predominantly in the lungs, kidneys, heart and gastrointestinal tract but less frequently in the liver [1]. Prevalence of hepatic involvement in disseminated Candida infection varied depending on the publication but a mean prevalence of 14% has been suggested in earlier reports [19]. Soon, it became clear that focal hepatic Candida infection is a distinct clinical variant of disseminated Candida infection (chronic disseminated candidosis, CDC) in immunocompromised patients, mostly patients who have received intense cytotoxic chemotherapy for acute leukaemia [2,3,5–7,19–22].

Lesions associated with HCI were described as granulomas, microabscesses, centrilobular congestion, haemorrhagic necrosis, bile stasis, inflammatory parenchymal aggregates, free yeasts in sinusoids and/or fatty changes [13,19].

With regard to the definite diagnosis of HCI, the first definition of the diagnosis of invasive fungal diseases (IFD) in haematopoietic stem cell transplant recipients was established for research purposes. Patients with typical lesions for HCI on CT or ultrasound were regarded as probable IFD without any need for mycological support. In the revised definitions, such cases are now classified as possible IFD [4]. In earlier reports, when patients may or may not have received Amphotericin B for suspected HCI, it has been suggested that liver biopsies usually revealed yeast and/or hyphal forms, but mycological cultures were frequently negative [5,19]. However, identifiable Candida organisms may not be found in all tissue specimens and only indirect signs of HCI may be seen at histopathological examination [13].

The majority of cases in our study had a possible diagnosis of HCI because fungal elements were not visible in the biopsy but radiology was highly suggestive of fungal liver disease. Interestingly, many of the patients studied here had a previous history of proven/probable invasive fungal disease in the lungs (possible/probable aspergillosis) and have received systemic antifungal therapy. Only in two patients, could yeasts be detected in the liver sample. However, hepatic Candida infection could not be proven by a positive culture of the fungal organism in any of the cases. As outlined above, all patients had received a substantial amount of systemic antifungal therapy with Candida-active antifungal agents before the liver biopsy was taken. This underlines the diagnostic dilemma when a tissue sample is taken under ongoing antifungal therapy. It has been earlier discussed that laparoscopic liver biopsies may have higher yield in the diagnosis of HCI because the procedure allows better sampling of hepatic focal lesions [6,23]. Laparoscopy is nowadays rarely performed in patients with haematological malignancies to establish the diagnosis of HCI because radiological diagnosis with ultrasound, CT and/or MRI has markedly improved [16–18,24–27]. In our patients, it cannot be excluded that a laparoscopy-guided liver biopsy might have had a higher diagnostic yield. Anttila et al. reported that the diagnosis of HCI was established significantly more often when the biopsy was targeted at white nodules than when targeted randomly or at scars [6]. However, in this study many liver biopsies targeted at white nodules had negative results at histological and/or cytological examination as well.

Molecular techniques may improve diagnosis of invasive fungal disease. Molecular methods for the identification of Aspergillus spp and other pathogenic fungi in paraffin wax embedded tissues have been established [9,12]. In general, in these assays DNA was amplified by PCR using pan-fungal probes, and detected by Southern blot hybridization with a probe specific for Aspergillus fumigatus and other fungi [9,12].

In our study, the specificity and sensitivity for detecting fungal DNA from a large collection of control and reference strains was good. All fungal isolates that are supposed to be detectable by the LCD chip could be correctly identified using reference strains. Furthermore, DNA from a large collection of additional fungal control strains that should not be identified could be amplified by PCR but not the final hybridization step on the LCD chip. These additional fungal strains may cause invasive fungal disease in immunocompromised patients but would not be identified by the array, which limits this method. Of interest, we found fungal DNA in two liver samples from patients with possible CDC that could not be further identified after final chip hybridization. After additional sequencing of the PCR product, Candida lipolytica was identified. This rare Candida species (as well as, for example, Trichosporon spp.) is not within the detection scope of the array and may limit the test method. However, the most common Candida and Aspergillus spp. as well as some other pathogenic fungi could be identified correctly.

In a study on respiratory tract biopsies, two semi-nested PCR assays for Aspergillus spp. and Zygomycetes offered a reliable aetiological diagnosis that was superior to culture in patients with proven invasive mould infection [28]. Recently, improvement has been achieved by establishing DNA microarrays to detect and identify DNA from fungal pathogens, mostly Candida and Aspergillus species [29,30]. In contrast to the assay studied here, these microarray systems are not commercially available but may detect up to 14 different fungal pathogens from various samples (blood, bronchoalveolar lavage and tissue) in high-risk patients [30].

In conclusion, the novel LCD chip technique examined in our study was able to detect fungal pathogens in the majority of liver biopsies from patients with suspected hepatic fungal infection but was negative in control biopsies. Together with the clinical information, detection of fungal DNA in liver biopsies may help to improve establishing the diagnosis of CDC even during antifungal therapy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

We thank Dr Kathrin Tintelnot (Robert-Koch-Institute/RKI isolates, Berlin), Professor Herbert Hof (University Hospital Mannheim), Professor Gerhard Haase (University Hospital Aachen/GHP isolates) and Professor Olivier Lortholary (Institute Pasteur/IP isolates, Paris, France) for providing strains. We thank Dr Volker Heiser (Chipron GmbH, Berlin, Germany) and Clarissa Radecke for technical assistance.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

All authors have substantially contributed to the manuscript and can take public responsibility for the content. M. Ruhnke has received research grants from Deutsche Krebshilfe/German Cancer Aid, Pfizer and Merck/MSD, served as a consultant and at the speakers’ bureau of Astellas, Basilea, Essex/Schering-Plough, Gilead Sciences, Janssen, Merck/MSD, Novartis, Pfizer, Pliva (all not related to the manuscript). M. von Lilienfeld-Toal has received research grants from Merck/MSD and served as a consultant and at the speakers’ bureau of Merck/MSD and Wyeth. M. Fleischhacker, S. Schulz, K. Jöhrens, T. Held, E. Fietze, C. Schewe, I. Petersen have nothing to declare.

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References
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