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...an be proven by histopathology but species identification is limitedsThe demonstration of fungal elements in tissue is of major importance for establishing the diagnosissof invasive fungal infections =-=[8]-=-. Several morphologic characteristics allow for the differentiation ofsseveral groups of fungi such as yeasts, hyaline moulds, dematiaceous fungi and the mucorales [9●●].sHowever, as several fungi sha...

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...shing away unbound probe, hybridized cells can be identified by epifluorescence microscopy. Due to the high rRNA content of cells, a sensitive detection andsidentification of microbes can be achieved =-=[45]-=-.sIn the field of fungal diagnostics, FISH so far has been studied for the identification of agents ofscandidemia. DNA-probes have been described to differentiate between frequent isolates fromspatien...

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...8]. The extraction of fungal DNA from ffpe tissues ofspatients with proven IFD poses additional challenges, as documented by a lower amount of positivessamples when compared to fresh tissue specimens =-=[29,30]-=-. By increasing the amount of tissue usedsfor extraction, the detection of DNA of the mucorales from experimentally infected tissue could besimproved, demonstrating that amplifiable fungal DNA may be ...

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...sterile specimens. A distinction between colonization and IFD is often not possible. Culturessfrom involved tissues on the other hand, are only positive in 50% of cases with proven invasivesinfection =-=[5,6]-=-. Given these limitations in diagnostics, postmortem examinations classifying thesmorphology of fungal elements in tissues played a critical role in documenting changes in thesepidemiology of IFD, suc...

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...s in thesepidemiology of IFD, such as a decrease in Candida-infections and an increase in mould infectionssduring the 80´s and the increasing importance of mucormycosis in cancer patients in the 90´s =-=[5,7]-=-.sHowever, it is becoming increasingly recognized, that the classification of IFD into these broadscategories is not sufficient, as fungi with different in vitro susceptibilities can display identical...

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...pected in bronchial secretions and tissue specimens, the differentiationsbetween colonization and infection is becoming a critical issue when bronchial secretions arestargeted for diagnostic purposes =-=[25,26]-=-. In contrast, recent studies suggest examples where PCRsassays may already be successfully used to supplement conventional diagnostic tests in order tosbetter characterize the etiology of IFD. A rece...

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...re less likely to be recovered by culture from clinical specimens than other moulds. Thissmay be partly due to the tissue processing prior to cultivation and also due to subsequent culturesconditions =-=[6,17,18]-=-. Finally, fungi may be inhibited by co-cultured bacterial or fungal organisms,snecessitating the use of selective media for sensitive cultivation of the pathogen. For example,sScedosporium species ar...

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... and the emerging use ofsmolecular techniquessKeywords: invasive fungal diseases; aspergillosis; candidiasis; mucormycosis; PCR; FISH;shistopathology; culturesVolker Rickerts (1)*, David N. Fredricks =-=(2)-=-s* Corresponding authors1 Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germanysphone: +49-30-187542862, fax: +49-30-187542614, email: vrickerts@rki.des2 Vaccine and Infectious Disease Division, Fr...

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...didemia [64]. In polymicrobial communities,smicrobes interact, either directly, by secreted molecules or via competition for nutrients [65]. Thesesinteractions may modify antimicrobial susceptibility =-=[66]-=- and therefore have therapeutic orsprognostic implications. Deciphering the etiology may stimulate research on interactions betweensmicroorganisms [67]. Third, the etiology of IFD is difficult to asse...

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...sterile specimens. A distinction between colonization and IFD is often not possible. Culturessfrom involved tissues on the other hand, are only positive in 50% of cases with proven invasivesinfection =-=[5,6]-=-. Given these limitations in diagnostics, postmortem examinations classifying thesmorphology of fungal elements in tissues played a critical role in documenting changes in thesepidemiology of IFD, suc...

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... application of heat prior to DNAextraction has been shown to increase the amount of amplifiable human DNA from FFPE tissuessamples, probably by reversing cross linking of DNA with other biomolecules =-=[32]-=-. Recently, we havesshown that this is also relevant for the extraction of fungal DNA from FFPE tissue specimens.sImprovements in the amount of amplifiable human DNA up to 76-fold were documented, and...

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..., mucorales, Fusarium and Pseudallescheria. The designed Fusarium-probe was found tosspecifically stain hyphae from cultivated type strains and hyphal elements in tissues from animalsswith fusariosis =-=[58]-=-. In a comparable approach, we used a panel of type strains to generate DNAsprobes to identify Candida spp. and Aspergillus spp. in tissue samples by FISH and compared thesresults to those obtained by...

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..., the detection of DNA of the mucorales from experimentally infected tissue could besimproved, demonstrating that amplifiable fungal DNA may be retained below a detection limit insFFPE tissue samples =-=[31]-=-. However, increasing the amount of tissue is not always possible withsclinical samples that may only have a limited amount of fungal material. In addition, this strategysmay also inhibit the detectio...

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...R to identify fungal DNA fromsFFPE tissue specimens successfully identified fungal DNA in 70-80% of FFPE tissue samples insaccordance with culture results, suggesting that this is a feasible approach =-=[34,35]-=-.sThe target of PCR-assays used to amplify fungal DNA is critical for the identification of fungalspathogens. So far, multicopy genes such as the ribosomal rRNA genes have been used in order tosprovid...

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...R to identify fungal DNA fromsFFPE tissue specimens successfully identified fungal DNA in 70-80% of FFPE tissue samples insaccordance with culture results, suggesting that this is a feasible approach =-=[34,35]-=-.sThe target of PCR-assays used to amplify fungal DNA is critical for the identification of fungalspathogens. So far, multicopy genes such as the ribosomal rRNA genes have been used in order tosprovid...

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...uch as Candida nivariensis or Candida bacarensis were alsosstained with the probe aimed at the detection of C .glabrata, highlighting potential limitations in thesspecies resolution with short probes =-=[49]-=-. Additional PNA- probes have been evaluated to furthersbroaden the spectrum of cultivated yeasts (C. albicans, C. glabrata, C. tropicalis, C. krusei, C.sparapsilosis) identifiable by PNA-probes. Thes...

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...n reported in 50% of cases withsproven invasive fungal infection [6,14]. First, culture conditions such as temperature and duration ofscultivation affect the recovery of moulds from various specimens =-=[15]-=-. Second, antifungal treatmentsis known to impair the recovery of fungi in cultures [16]. Third, several studies suggest that thesmucorales are less likely to be recovered by culture from clinical spe...

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... Aspergillus. In these patients, the cultivation of Scedosporium from respiratory secretionssis much more likely when selective media are used that impair the growth of yeasts and Aspergillussspecies =-=[19]-=-.sBeside the lack of sensitivity of cultures in growing fungi from tissues of patients with proven IFD, it isspossible that current culture and identification methods underestimate the frequency of mi...

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...pected in bronchial secretions and tissue specimens, the differentiationsbetween colonization and infection is becoming a critical issue when bronchial secretions arestargeted for diagnostic purposes =-=[25,26]-=-. In contrast, recent studies suggest examples where PCRsassays may already be successfully used to supplement conventional diagnostic tests in order tosbetter characterize the etiology of IFD. A rece...

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...A probes with increased affinity forscomplementary nucleic acids, have been used to discriminate between cultivated yeasts. Closesrelatives such as C. albicans and C. dubliniensis were differentiated =-=[48]-=-. Also, the distinctionsbetween frequent agents of candidemia, requiring different antifungal therapies such as C. albicanssand C .glabrata was shown to be possible directly from positive blood cultur...

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...dsbe designed. Recently, online tools allowing the prediction of probes generating strong signals havesbeen introduced. These tools will help characterize probes to reduce time needed for experimentss=-=[61]-=-. Using such fungal probes with non-specific probes labeled with different fluorophores may helpsvisualize uncommon agents and localize them to the pathologic process in tissue. Improvements insmicros...

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...gic process in tissue. Improvements insmicroscopy may facilitate the application of different numbers of fluorescently labeled probessallowing for the detection of multiple species in a single sample =-=[62]-=-.sEmerging applications of molecular tissue diagnosissTissues samples from patients with invasive fungal infections are crucial for understanding thesetiology on IFD. Molecular tests are emerging as t...

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...do occursafter lung transplantation, that is an increasingly used therapeutic option to treat end stagespulmonary failure and that is associated with mortality rates of 29% despite antifungal therapy =-=[68]-=-.sConclusionsThe tissue diagnosis of IFD is important for understanding changes in the epidemiology ofsopportunistic fungal infections. The proper histopathologic description of fungal elements in tis...

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...ions: Current limitations and the emerging use ofsmolecular techniquessKeywords: invasive fungal diseases; aspergillosis; candidiasis; mucormycosis; PCR; FISH;shistopathology; culturesVolker Rickerts =-=(1)-=-*, David N. Fredricks (2)s* Corresponding authors1 Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germanysphone: +49-30-187542862, fax: +49-30-187542614, email: vrickerts@rki.des2 Vaccine and Infect...

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...published as:sRickerts, V., Fredricks, D.N.sTissue diagnosis of invasive fungal infections: Current limitations and the emerging use ofsmolecular techniquess(2012) Current Fungal Infection Reports, 6 =-=(3)-=-, pp. 221-228.sDOI: 10.1007/s12281-012-0098-6sThis is an author manuscript.sThe definitive version is available at: http://link.springer.com/s1sTissue diagnosis of invasive fungal infections: Current ...

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...th moulds, the validity on these classifications issdifficult to assess in the absence of alternative tests, such as culture, which may prove the presencesof mixed fungal pathogens in a tissue sample =-=[11]-=-.sThe British Society for Medical Mycology proposed standards of care for patients with invasive fungalsinfections. Included therein are recommendations on how tissues from immunocompromisedspatients ...

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...ed therein are recommendations on how tissues from immunocompromisedspatients with suspected IFD should be stained, how fungal elements may be classified and hows4spositive results should be reported =-=[12]-=-. A subsequent audit demonstrated that less than 50% ofspathology departments in the UK provided sufficient detailed descriptions of fungal morphology, asssuggested by the guidelines [13]. Comparable ...

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... be reported [12]. A subsequent audit demonstrated that less than 50% ofspathology departments in the UK provided sufficient detailed descriptions of fungal morphology, asssuggested by the guidelines =-=[13]-=-. Comparable data for other countries are not available.sCultivation of fungi from infected tissue is insensitivesAs the morphology of fungal elements in tissue is limited, while the diversity of fung...

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...ens is useful for identifying fungal species. Growth of fungi fromstissue specimens of patients with invasive fungal infections has been reported in 50% of cases withsproven invasive fungal infection =-=[6,14]-=-. First, culture conditions such as temperature and duration ofscultivation affect the recovery of moulds from various specimens [15]. Second, antifungal treatmentsis known to impair the recovery of f...

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...conditions such as temperature and duration ofscultivation affect the recovery of moulds from various specimens [15]. Second, antifungal treatmentsis known to impair the recovery of fungi in cultures =-=[16]-=-. Third, several studies suggest that thesmucorales are less likely to be recovered by culture from clinical specimens than other moulds. Thissmay be partly due to the tissue processing prior to culti...

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...re less likely to be recovered by culture from clinical specimens than other moulds. Thissmay be partly due to the tissue processing prior to cultivation and also due to subsequent culturesconditions =-=[6,17,18]-=-. Finally, fungi may be inhibited by co-cultured bacterial or fungal organisms,snecessitating the use of selective media for sensitive cultivation of the pathogen. For example,sScedosporium species ar...

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... for developing candidemia, such as bone marrowstransplant recipients and patients at surgical intensive care units, are colonized with more than onesyeast species as assessed by conventional culture =-=[20,21]-=-. As these colonizing strains form thesreservoir for endogenous infections in these patients, blood cultures may underestimate thesfrequency of mixed infections due to overgrowth with the dominant fun...

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... for developing candidemia, such as bone marrowstransplant recipients and patients at surgical intensive care units, are colonized with more than onesyeast species as assessed by conventional culture =-=[20,21]-=-. As these colonizing strains form thesreservoir for endogenous infections in these patients, blood cultures may underestimate thesfrequency of mixed infections due to overgrowth with the dominant fun...

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...bounds for a more common problem. Interestingly, a recent report described the cultivation ofsmore than one mould from respiratory specimens of patients with suspected invasive mouldsinfections in 7% =-=[22]-=-. However, as histopathology may not be able to differentiate between most ofsthese fungi and the cultivation of moulds may represent colonisation rather than infection, thesfrequency of mixed mold in...

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...identify the etiologic agents of invasive fungal diseasessfrom clinical samples. Studies using PCR to detect invasive mould infections from blood samples areslimited by the low fungal burden in blood =-=[23]-=-. This and technical issues such as a suboptimal DNAsextraction efficacy, contaminations by fungal DNA and the lack of a consensus on the optimalsdetection strategy limit the widespread use of this mo...

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...cal issues such as a suboptimal DNAsextraction efficacy, contaminations by fungal DNA and the lack of a consensus on the optimalsdetection strategy limit the widespread use of this molecular approach =-=[24]-=-. While higher mould DNAsconcentrations are expected in bronchial secretions and tissue specimens, the differentiationsbetween colonization and infection is becoming a critical issue when bronchial se...

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...tical for optimal DNA recovery from fungal pathogens. Usingsdifferent commercial extraction kits, up to a million-fold differences in the amount of fungal DNA cansbe documented using quantitative PCR =-=[28]-=-. The extraction of fungal DNA from ffpe tissues ofspatients with proven IFD poses additional challenges, as documented by a lower amount of positivessamples when compared to fresh tissue specimens [2...

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...8]. The extraction of fungal DNA from ffpe tissues ofspatients with proven IFD poses additional challenges, as documented by a lower amount of positivessamples when compared to fresh tissue specimens =-=[29,30]-=-. By increasing the amount of tissue usedsfor extraction, the detection of DNA of the mucorales from experimentally infected tissue could besimproved, demonstrating that amplifiable fungal DNA may be ...

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... amount of amplifiable human DNA up to 76-fold were documented, and thesnumber of samples from which fungal DNA was amplified by broad range PCR and successfullysidentified by sequencing was improved =-=[33]-=-. Recent studies using PCR to identify fungal DNA fromsFFPE tissue specimens successfully identified fungal DNA in 70-80% of FFPE tissue samples insaccordance with culture results, suggesting that thi...

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...t the price of an increased likelihood to detect contaminating DNA and a potentialsfor cross-amplification of human DNA. However, assays can be designed to detect fungal DNA in ansexcess of human DNA =-=[36]-=-. This might be critical in tissue biopsies where lots of human DNA can besexpected. Strategies to overcome contamination and adequate controls to address this issue havesbeen proposed previously [24]...

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...al DNA. An in-silico tool has been introduced to identify up to three bacteriasfrom mixed chromatograms of bacterial DNA amplified by broad range bacterial PCR from mixedsinfections such as abscesses =-=[37]-=-. This approach might be useful for unraveling mixedschromatograms from fungal PCR assays as well. Further options to decipher mixed amplicons includesthe generation of clone libraries. Using this app...

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...ications for PCRsand sequencing from tissue specimens may emerge. Vallor et al. successfully amplified the singlescopy FKS gene of A.fumigatus from tissue of an animal model of invasive aspergillosis =-=[40]-=-. Withsimprovements in DNA extraction, such genes may successfully be targeted in FFPE tissues. Targetingssingle-copy genes may allow for the identification of molecular siblings, fungi that cannot re...

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...bilities tosantifungal agents [41,42●].sIn addition, genetic elements related to in vitro resistance have beenssuccessfully detected directly from FFPE tissue of a patient with invasive Aspergillosis =-=[43]-=-. Suchsassays would be critical to use on FFPE tissue specimens to study reasons for antifungal treatmentsfailure.sSince its introduction in the late 1980´s fluorescence in situ hybridization (FISH) h...

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...introduction in the late 1980´s fluorescence in situ hybridization (FISH) has become a widelysused method for the identification and quantification of phylogenetically defined microbes inspopulations =-=[44]-=-. The basic steps of FISH include the fixation of samples to allow for the uptake ofslabeled synthetic DNA probes into target cells. Identification of microorganisms is achieved bysbinding of probes, ...

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...obes have been described to differentiate between frequent isolates fromspatients with candidemia such as C. albicans, C. krusei, C. glabrata and C. parapsilosis cultivated fromsblood culture bottles =-=[46]-=-. In addition, DNA probes targeting Cryptococcus neoformans ands8sCryptococcus gattii have been developed that differentiate these agents of cryptococcosis from othersascomycetes and basidiomycetous y...

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...of cryptococcosis from othersascomycetes and basidiomycetous yeasts. Using these probes allowed for the identification ofscryptococci directly from cerebrospinal fluid of patients with cryptococcosis =-=[47]-=-.sMore recently,speptide nucleic acid (PNA) probes, chemically modified DNA probes with increased affinity forscomplementary nucleic acids, have been used to discriminate between cultivated yeasts. Cl...

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...ated yeasts (C. albicans, C. glabrata, C. tropicalis, C. krusei, C.sparapsilosis) identifiable by PNA-probes. These yeast-species accounted for 99% of all yeastsscultivated in a diagnostic laboratory =-=[50]-=-.sThese studies document that FISH using DNA- or PNA probes targeting rRNA can be used tosdifferentiate between cultivated yeasts and suggest that their use may also be possible directly insuncultured...

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...s more successful in identifying the etiology ofsIFD. As in previous studies using ISH, the distinction of the probe signal was impaired in necroticstissue background in samples from mould infections =-=[34,51,52]-=-. This limitation may be overcome byssuperior probe chemistries such as PNA-, or LNA probes [45]. In addition, the hybridization signalsscan be restricted to parts of fungal elements in tissue [34,58,...

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...s more successful in identifying the etiology ofsIFD. As in previous studies using ISH, the distinction of the probe signal was impaired in necroticstissue background in samples from mould infections =-=[34,51,52]-=-. This limitation may be overcome byssuperior probe chemistries such as PNA-, or LNA probes [45]. In addition, the hybridization signalsscan be restricted to parts of fungal elements in tissue [34,58,...

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...,51,52]. This limitation may be overcome byssuperior probe chemistries such as PNA-, or LNA probes [45]. In addition, the hybridization signalsscan be restricted to parts of fungal elements in tissue =-=[34,58,59]-=-. This is in accordance with datasdemonstrating that rRNA signals obtained by PNA probes is most intense in the apical compartmentsof hyphae [60].sUltimately, a panel of probes with well-defined targe...

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...restricted to parts of fungal elements in tissue [34,58,59]. This is in accordance with datasdemonstrating that rRNA signals obtained by PNA probes is most intense in the apical compartmentsof hyphae =-=[60]-=-.sUltimately, a panel of probes with well-defined target species detecting frequent agents of IFD couldsbe designed. Recently, online tools allowing the prediction of probes generating strong signals ...

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...nts. Second,spolymicrobial infections in cancer patients are increasingly being recognized and can be a therapeuticschallenge. They may originate from abdominal infections such as neutropenic colitis =-=[63]-=-. Mixedsinfections are diagnosed in up to 23% of patients in Candidemia [64]. In polymicrobial communities,smicrobes interact, either directly, by secreted molecules or via competition for nutrients [...

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...ing recognized and can be a therapeuticschallenge. They may originate from abdominal infections such as neutropenic colitis [63]. Mixedsinfections are diagnosed in up to 23% of patients in Candidemia =-=[64]-=-. In polymicrobial communities,smicrobes interact, either directly, by secreted molecules or via competition for nutrients [65]. Thesesinteractions may modify antimicrobial susceptibility [66] and the...

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...]. Mixedsinfections are diagnosed in up to 23% of patients in Candidemia [64]. In polymicrobial communities,smicrobes interact, either directly, by secreted molecules or via competition for nutrients =-=[65]-=-. Thesesinteractions may modify antimicrobial susceptibility [66] and therefore have therapeutic orsprognostic implications. Deciphering the etiology may stimulate research on interactions betweensmic...

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...ractions may modify antimicrobial susceptibility [66] and therefore have therapeutic orsprognostic implications. Deciphering the etiology may stimulate research on interactions betweensmicroorganisms =-=[67]-=-. Third, the etiology of IFD is difficult to assess in patients colonized with multiplesfungi, such as patients with cystic fibrosis. While IFD may be rare in these patients, they do occursafter lung ...