Abstract
Purpose. Tissue samples from patients with suspicion of deep or subcutaneous fungal infections were analysed at the Portuguese Reference Mycology Laboratory according to a proposed diagnostic approach, which aims to constitute a rapid and accurate diagnosis for these fungal infections.
Abstract
Methodology. Forty-six tissue biopsy samples were analysed over a period of 26 months, using a diagnostic approach that includes culture, panfungal PCR and Aspergillus-directed PCR.
Abstract
Results/Key findings. Overall, 23 samples were reported as negative while the remaining 23 were reported as positive for fungi (PCR, culture and/or histology). PCR showed an estimated detection limit of 12 pg DNA µl -1 . From the 46 samples, 30 were negative for fungal DNA while 16 gave positive results. From these, 12 cases were detected by panfungal PCR and six cases by PCR directed toward Aspergillus. In 61 % of the cases, there was concordance between molecular and cultural methods. Aetiological agents identified were Candida albicans, C. glabrata, C. tropicalis, Trichosporon montevideense, Alternaria spp., Exophiala sp., Trichoderma sp., Histoplasma spp., Aspergillus fumigatus, Trichophyton rubrum and Paracoccidioides brasiliensis.
Abstract
Our results showed that the proposed polyphasic approach appears to be a useful strategy in the detection of fungi from tissue samples, allowing a better prognosis. In further studies, the inclusion of a higher number of samples and the implementation of more genus-specific PCRs will certainly contribute to an increase in the specificity and sensitivity of this method.
INTRODUCTION
Invasive fungal infections are increasing significantly due to advances in medical care in immunocompromised populations. These infections are difficult to diagnose and treat, and thus pose a challenge to laboratory technicians and clinicians [1] .
INTRODUCTION
According to the revised definitions of invasive fungal disease (EORTC/MSG) [2] , a proven invasive fungal infection is defined by the demonstration of tissue invasion by fungal structures or by positive culture from a normally sterile sample. Nevertheless, histopathological examination requires experience and sometimes cultures remain negative after weeks of incubation or show very slow growth rates, representing a major drawback in regard to achieving an early diagnosis. The lack of positive histology or culture from tissues makes it difficult to classify an invasive infection as proven and, in the majority of the cases, these infections may only be classified as possible or probable. These classifications are based on host factors and clinical and mycological criteria [2] .
INTRODUCTION
Detection of fungal DNA in tissue samples should be standardized in order to improve diagnosis, thereby leading to a better patient outcome.
INTRODUCTION
Aiming to contribute to future discussion and standardization of the diagnosis of deep fungal infections, this study presents the experience of the Portuguese Reference Laboratory in the diagnosis of invasive and subcutaneous fungal infections, using a polyphasic approach that includes cultural and molecular methodologies. The specific goals of this study were the performance evaluation of our panfungal/Aspergillus PCR, as well to perform a comparative study between those molecular results and those obtained by mycological culture.
METHODS
From 2015 (January) to 2017 (February), biopsied tissue samples were collected from patients with clinical suspicion of deep or subcutaneous fungal infections. The patient population was not selected with regard to any risk factor or group. Enrolled patients were from several Portuguese districts (continental area) as well as from Cape Verde, S. Tome and Principe Islands and Guinea. The biopsied tissues were sent to the Portuguese Reference Mycology Laboratory and included samples from colon, liver, brain, skin, nose, eye, lung, bone, wrist, stomach, lip, mediastinum, palate, tonsil, kidney, and also inguinal, cervical and occipital ganglia.
METHODS
The samples were collected under sterile conditions according to the protocols established by each hospital, and were then sent to the Mycology Reference Laboratory within a 24 h period. Fresh specimens were sent in sterile containers with a small amount of sterile, preservative-free saline solution. Whenever possible, tissue samples were divided in two: one for culture and a second for PCR testing (Fig. 1) . Fresh tissue samples were always recommended by the laboratory as first choice for diagnosing invasive and subcutaneous fungal infections. In cases where fresh tissue was not available, the study was performed using paraffined tissue samples. Data on these patients (if available) were collected from the requisition form, including age, gender, sample type, date of sample collection, underlying disease, other laboratory tests and histological information on the sample.
METHODS
Cases with positive microscopic analysis or fungi in culture from specimens obtained by biopsy were considered as having a proven fungal infection, while those with a host factor, mycological evidence and clinical features were considered as probable, according to De Pauw et al. [2] .
Cultures of fresh tissues
Tissue fragments were sliced in smaller fragments for culture, as recommended by CLSI guidelines for the diagnosis of fungal infections [3] . An initial report was issued 30 days after incubation and a final report 60 days after incubation (Fig. 1) . Positive cultures were observed according to their macro-morphological characteristics, and microscopic examination was performed with lactophenol cotton blue stain and identified using an identification atlas [4] .
Molecular analysis of tissue samples
Paraffined tissue biopsy samples were de-paraffined by lavage with xylene (100 %) followed by serial washes with ethanol (40-100 %).
Molecular analysis of tissue samples
DNA was extracted from fresh tissue (whenever possible, !25 mg of tissue) and de-paraffined samples (~3-10 (Fig. 1) . For that purpose, the universal fungal primers ITS1 (5¢-TCCGTAGGTGAACCTGCGG-3¢) and ITS2 (5¢-GC TGCGTTCTTCATCGATGC-3¢) were used to amplify DNA, as described previously [5] . Amplifications were performed in a 25 µl volume reaction of PCR beads (Illustra PuReTaq Read-to-Go; GE Healthcare, Buckinghamshire, UK), containing 15 pmol of each primer and 20-50 ng of genomic DNA.
Molecular analysis of tissue samples
In order to establish the detection limit of this methodology, a reference DNA (Histoplasma capsulatum INSA-IFI04) was quantified using Qubit Fluorometric Quantitation (Thermo Scientific, Waltham, MA, USA) and then a serial tenfold dilution was performed.
Molecular analysis of tissue samples
For molecular identification of fungal cultures, DNA extraction was performed using the same extraction kit. The universal fungal primers ITS1 and ITS4 (5¢-TCCTCCGCTTA TTGATATGC-3¢) were used to amplify DNA from all fungal isolates, as described previously [6] .
Molecular analysis of tissue samples
Amplicons were purified using the ExoSAP-IT enzyme system (USB Corporation, Cleveland, OH, USA), according to the manufacturer's instructions.
Molecular analysis of tissue samples
Sequencing of both strands was performed with the BigDye terminator v 1.1 cycle sequencing kit (Applied Biosystems) in the thermal cycler using the same primers as were used in PCR amplification (ITS1 and ITS2 primers for sequence amplicons from tissue samples, and ITS1 and ITS4 for sequence amplicons from fungal cultures).
Molecular analysis of tissue samples
The resultant nucleotide sequences were edited using the program Chromas Lite v 2.01 and aligned with the program CLUSTALX v 2.1 [7] . The obtained sequences were compared with those deposited in the databases of GenBank (Bethesda, MD, USA) and the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands) to achieve identification of the aetiological agent. The sequences obtained were further deposited in the GeneBank database.
Molecular analysis of tissue samples
In cases of suspicion of Aspergillus infection, a specific PCR directed to Aspergillus was performed using the AsperGenius multiplex real-time PCR assay (PathoNostics, Maastricht, the Netherlands) on the Qiagen RotorGene Q instrument (Qiagen, Hilden, Germany), following the manufacturer's instructions. An internal control was included to monitor for inhibition or manual handling errors.
RESULTS
Forty-six tissue biopsy samples were analysed. These samples were collected from 39 patients (27 male and 12 female) with ages ranging 1 to 85 years. These tissues were collected by biopsy from the following: skin (N=17; 40.0 %), liver (N=6; 13.0 %), ganglia (N=4; 8. Regarding the molecular detection of fungal DNA, 30 (65.2 %) samples gave negative results whereas 16 (34.8 %) were positive. From these, 12 cases were detected by panfungal PCR and six by Aspergillus PCR (in two cases, both panfungal and Aspergillus PCR were performed). Detection limit of fungal DNA in tissue samples by panfungal PCR using the primers ITS1/ITS2 was determined as described in Methods, and was estimated to be~12 pg µl
À1
.
À1
In all panfungal PCR-positive cases it was possible to identify the aetiological agent by further sequencing. Filamentous fungi were identified in 10 cases (seven Aspergillus fumigatus, one Alternaria infectoria, one Alternaria alternata, one Trichophyton rubrum); dimorphic fungi were identified in two cases (one Histoplasma duboisii and one Paracoccidioides brasiliensis) and yeasts were identified in four cases (one Candida albicans and three C. tropicalis) ( Table 1 ) (GeneBank accession numbers MH707360-MH707371).
À1
Culture was performed in 36 (78.3 %) samples (Table 2 ). In the other 10 cases, tissue samples were not cultured due either to their having been immersed in paraffin or formolsaline solution, or to the lack of biological material available to perform both cultural and molecular methods (in such cases, priority was given to what was requested by the clinician). From those 36 samples, 29 (80.5 %) cultures remained negative after the recommended incubation period whereas seven (19.4 %) gave positive results ( Table 2) . Two of those were positive only for yeasts (Candida spp. and Trichosporon montevideense) and three were positive only for filamentous fungi (Alternaria spp. and Exophiala sp.). Mixed cultures were obtained in two cases: C. albicans plus Histoplasma sp. (colon tissue) and C. albicans plus Trichoderma sp. (nose biopsy) ( Table 1) .
À1
Agreement for results obtained by both molecular and cultural methods was 61 %. From the 36 samples analysed, in 18 both cultures and PCRs were negative while in four cases both were positive (Table 2) .
DISCUSSION
According to our study, 13 patients had proven invasive fungal infection since a positive culture and/or positive histology for fungi were obtained from a normally sterile sample.
DISCUSSION
When comparing positive results obtained by cultural and molecular methods, PCR showed a higher frequency of positivity since 16 out of 19 (84 %) positive results were PCR positive ( Table 2 ). This value is similar to that recorded by Babouee et al. [8] . These authors used a molecular strategy similar to ours, but did not utilize PCR directed toward Aspergillus. In a study performed by Buitrago et al. [5] , the sensitivity of microbiological culture was 56 % and the the panfungal real-time PCR-based technique was positive in 89.3 % of patients. This variation can be explained by the different molecular methodologies used, since real-time PCR is described as being a more sensitive method.
DISCUSSION
In three cases there was a negative PCR result and positive culture; this may have been due to inefficient DNA extraction. The fungal cell wall structure and composition poses difficulties in regard to cell disruption and lysis of fungal organisms.
DISCUSSION
Overall, the concordance between the PCR and the culture results was 61 %, a value lower than that obtained by both Ala-Houhala et al. (86 %) [9] and Trubiano et al. (80 %) [10] . This discrepancy can be explained by the high number of cases studied by these authors. In six cases, both panfungal PCR and real-time PCR directed toward Aspergillus allowed the detection and identification of Aspergillus fumigatus sensu stricto. Since Aspergillus is one of the major agents of invasive fungal infection, the use of real-time PCR directed at this genus enabled a more rapid diagnosis of aspergillosis (1-2 days earlier).
DISCUSSION
In many of the cases in the present study, the hospital did not report whether the patient was being administered antifungal therapy when the biopsy was performed, which would have helped the laboratory in the evaluation of results. The potential administration of antifungal therapy/ prophylaxis may explain the low number of positive cultures obtained. In specific cases where the culture was negative and PCR gave a positive result, the laboratory was in possession of information that the patient was being administered azole therapy (e.g. samples #14, #15 and #27).
DISCUSSION
In the absence of fungal culture, the PCR results allowed a provisional diagnosis and enabled correct therapeutic management: examples included the administration of voriconazole to two immunosuppressed patients [one with a nasal lesion (sample #14) and one with a bone lesion (sample #15) (PCR positive for Aspergillus)]; and a change in therapy from amphotericin and fluconazole to itraconazole (PCR positive for Paracccidioides brasililensis; sample #27).
DISCUSSION
In this study, in two cases both culture and PCR were positive and sequencing led to the identification of only one fungal species, whereas more than one species grew in culture.
DISCUSSION
The culture of tissue sample #1 resulted in C. albicans plus Histoplasma sp. The purification and subsequent molecular identification of culture for Histoplasma sp. identification was not possible given the overgrowth of the yeast colony. DNA extraction and PCR performed directly from the tissue sample allowed the amplification and identification to species level of the infecting agent (H. duboisii). In tissue sample #24, the culture resulted in identification of C. albicans and Trichoderma sp. This latter was probably the aetiological agent of the infection but when PCR and sequencing were performed directly from the tissue sample, only the DNA from C. albicans amplified, showing a preferential amplification when both DNAs are present, which may be due to very dissimilar amounts of both.
DISCUSSION
Our results showed that the proposed polyphasic approach (culture together with panfungal and directed PCR) appears to be a practical strategy in the detection of fungi from tissue biopsy samples. Each method has its advantages and disadvantages, and their combined use may overcome the majority of the disadvantages. Furthermore, the suggested use of a real-time PCR directed toward Aspergillus increases the efficiency of this approach. Antifungal susceptibility data are not in included. However, due to international recommendations to perform antifungal susceptibility testing in invasive fungal infections [11] , the suggested algorithm for the detection of deep fungal infections (Fig. 1) includes a second step advising the determination of antifungal susceptibility (performed following clinical evaluation and request).
DISCUSSION
In future studies, prospective work using this approach applied to specific groups of patients (e.g. post-transplant, or under antifungal prophylaxis or therapy) would be helpful in order to reinforce this strategy. We also intend to implement further methods based on genus-specific PCRs in order to increase the specificity of this methodology, namely for Candida spp. and to the Mucorales group. Due to the difficulty in interpretating positive results, panfungal PCR has limited use in the testing of fungal infections from non-sterile clinical material (e.g. BAL, sputum). Nevertheless, together with culture and genus-targeted PCR and when applied to tissue samples collected from sterile sites, this methodology proved to be adequate and allowed a better diagnosis of deep and subcutaneous fungal infections.

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