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Canine protothecosis is a rare disease caused by saprophytic unicellular achlorophyllous aerobic algae that are ubiquitous in the environment. We report a novel case of neurological and cardiological manifestations associated with disseminated protothecosis. An adult spayed female Boxer dog was presented with a 1-week history of anorexia, progressive central vestibular signs, and a Grade III/VI systolic heart murmur. Magnetic resonance (MR) imaging revealed obstructive hydrocephalus at the level of the mesencephalic aqueduct, while echocardiography and elevated troponin levels suggested an infiltrative cardiomyopathy. No obvious cause was identified. Cerebrospinal fluid (CSF) collection was not performed due to associated procedural risks. Despite receiving symptomatic treatment and maintaining stability for 3 weeks, the dog eventually suffered cardiorespiratory arrest. Postmortem examination revealed disseminated protothecosis, predominantly affecting the heart and brain. We recommend that in cases where the cause of obstructive hydrocephalus is unclear, especially when CSF collection is not feasible, a comprehensive diagnostic method should be implemented. This includes meticulous investigations to identify infected tissues, followed by sampling and performing cytology/histology and culture tests to confirm the presence of the algal organism. Early diagnosis may allow early treatment, although long-term prognosis remains largely unfavorable due to the absence of effective treatments.

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Invasive fungal disease (IFD) presents a life-threatening condition in immunocompromised patients, thus often prompting empirical administration of antifungal treatment, without adequate mycological evidence. Over the past years, wide use of antifungal prophylaxis resulted in decreased occurrence of IFD but has contributed to changes in the spectrum of fungal pathogens, revealing the occurrence of previously rare fungal genera causing breakthrough infections. The expanding spectrum of clinically relevant fungal pathogens required the implementation of screening approaches permitting broad rather than targeted fungus detection to support timely onset of pre-emptive antifungal treatment. To address this diagnostically important aspect in a prospective setting, we analyzed 935 serial peripheral blood (PB) samples from 195 pediatric and adult patients at high risk for IFD, involving individuals displaying febrile neutropenia during treatment of hematological malignancies or following allogeneic hematopoietic stem cell transplantation. Two different panfungal-PCR-screening methods combined with ensuing fungal genus identification by Sanger sequencing were employed. In the great majority of PB-specimens displaying fungal DNAemia, the findings were transient and revealed fungi commonly regarded as non-pathogenic or rarely pathogenic even in the highly immunocompromised patient setting. Hence, to adequately exploit the diagnostic potential of panfungal-PCR approaches for detecting IFD, particularly if caused by hitherto rarely observed fungal pathogens, it is necessary to confirm the findings by repeated testing and to identify the fungal genus present by ensuing analysis. If applied appropriately, panfungal-PCR-screening can help prevent unnecessary empirical therapy, and conversely, contribute to timely employment of effective pre-emptive antifungal treatment strategies.

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Fungal infections can be challenging to diagnose in returning travellers due to their non-specific clinical manifestations and changing epidemiology. We present a case of progressive disseminated histoplasmosis in a returning traveller from Bangladesh. The patient had a progressive and prolonged respiratory illness necessitating mechanical ventilatory support. The clue to potential fungal aetiology was provided by serum fungal markers - 1-3-ß-D-glucan and Aspergillus galactomannan. Diagnosis was eventually made using panfungal PCR on bronchioalveolar lavage fluid.

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Performance of panfungal PCR-DNA sequencing assays for diagnosis of invasive fungal disease on formalin-fixed, paraffin-embedded tissue (FFPE) is influenced by many variables. Interpretation of a positive result can be challenging due to the need to differentiate colonisers and contaminants from clinically significant pathogens. We conducted a retrospective audit on FFPE tissue specimens that underwent panfungal PCR from January 2021 to August 2022. Panfungal PCR results from samples where fungal elements were visualised on histopathology were compared with results from samples where no fungal elements were visualised. The cost per clinically significant positive sample in each group was calculated. Of the 248 FFPE tissues sampled, 18.1% (45/248) had fungal forms seen on histopathology. Panfungal PCR was positive in 22/45 samples (48.9%), with 16 (35.6%) results deemed clinically significant. For the remaining 203 specimens, panfungal PCR was positive in 19 (9.4%) samples with only six (3.0%) clinically significant. The average cost per clinically significant result was AUD 258.13 in the histopathology positive group and AUD 3,105.22 in the histopathology negative group. Our data suggest panfungal PCR has limited clinical utility in FFPE tissue when no fungal elements are seen. Restricting the assay to only those samples that are positive on histopathological examination aids interpretation of PCR positive results and conserves laboratory resources.

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Background: Angioinvasive Rhizomucor pusillus infection with dissemination to the liver and spleen is exceedingly uncommon, representing less than 1% of reported cases of mucormycosis. Methods: Diagnosis of mucormycosis is often difficult using conventional methods that rely on broad-based non-septate hyphae present on histologic examination and morphological identification of the cultured organism. Our laboratory also uses an in-house panfungal molecular assay to rapidly diagnose invasive fungal infection when conventional methods do not provide definitive results. Results: Herein we present a case of disseminated mucormycosis with hepatosplenic involvement in a 49-year-old female with acute myelogenous leukemia following induction chemotherapy. But in this case repeated tissue biopsy cultures were negative. R. pusillus infection was diagnosed using an in-house panfungal PCR/sequencing assay based on dual priming oligonucleotide primers. Conclusions: New molecular assays facilitate prompt diagnosis of invasive fungal infections.

Historique: L'infection à Rhizomucor pusillus angio-invasive avec dissémination au foie et à la rate est très peu courante, puisqu'elle représente 1% des cas de mucormycose déclarés. Méthodologie: Le diagnostic de mucormycose est souvent difficile à poser au moyen des méthodes habituelles, qui reposent sur la présence d'hyphes non cloisonnées généralisées à l'examen histologique et sur l'identification morphologique de l'organisme mis en culture. Le laboratoire recourt également à un dosage moléculaire panfongique maison pour diagnostiquer rapidement l'infection fongique invasive lorsque les méthodes habituelles ne fournissent pas de résultats définitifs. Résultats: Les chercheurs présentent un cas de mucormycose disséminée avec atteinte hépatosplénique chez une femme de 49 ans atteinte de leucémie myélogène aiguë après une chimiothérapie d'induction. Dans ce cas, les résultats des biopsies tissulaires répétées étaient négatifs. L'infection à R. pusillus a été diagnostiquée au moyen d'un dosage maison par séquençage ou PRC panfongique d'après des amorces oligonucléotidiques à double usage. Conclusions: Les nouveaux dosages moléculaires facilitent un diagnostic rapide d'infection fongique invasive.

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We reviewed the performance of a panfungal ITS-2 PCR and Sanger sequencing assay performed on 88 FFPE specimens at The Hospital for Sick Children (Toronto, Canada) in 2019. A potential fungal pathogen was identified by ITS PCR in 62.7 and 2.9% of positive and negative direct slide examination of tissue specimens, respectively. ITS amplicons were detected in 87/88 specimens, with 53/88 (60.2%) considered as 'positive-contaminants' and 34/88 (38.6%) as 'positive-potential pathogen' upon sequencing. Potential pathogens included Blastomyces dermatitidis (17.1%), Cryptococcus neoformans (17.1%), Histoplasma capsulatum (14.3%) and Mucormycetes (11.4%). Laboratories should only perform ITS PCR on FFPE tissues if fungal elements have been confirmed on histopathology slides. LAY SUMMARY: In this study, we examined how well a DNA-based test could detect DNA from fungi in archived human biopsy tissues. The best performance was achieved if fungi were seen in the tissue under a microscope before being tested. Our results indicate that we should only use this test if these conditions are met.

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BACKGROUND: The prevalence of invasive fungal infections (IFIs) is increasing due to the increasing population of immunocompromised patients. Fungal culture is the gold standard for diagnosis but not sensitive and the turnaround time is long. Samples for histopathology are difficult to obtain because of profound cytopenias. We conducted this study with the aim to evaluate panfungal PCR for the diagnosis of IFIs in patients of febrile neutropenia. METHODS: This was a single-centre, cross-sectional observational study. Patients of febrile neutropenia suspected of having IFI were included in the study. Panfungal PCR was performed on the blood of included patients along with other investigations for diagnosis of IFI. The sensitivity, specificity, positive predictive value, and negative predictive value of panfungal PCR were calculated using EORTC/MSG 2008 criteria as the gold standard. RESULTS: Fifty patients of febrile neutropenia were included in the study, of which 52% were diagnosed positive by panfungal PCR assay. The sensitivity, specificity, positive predictive value, and negative predictive value of panfungal PCR assay was found to be 82.76%, 90.48%, 92.31% and 79.17% respectively. CONCLUSION: Panfungal PCR is a promising and highly sensitive diagnostic test for screening at-risk patients suspected to have IFIs. The use of panfungal PCR assay in combination with other diagnostic modalities and clinical judgment can be very helpful in the early diagnosis of IFI.

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Introduction. Invasive mucormycosis (IM) is a life-threatening infection caused by fungi belonging to the order Mucorales. Histopathology, culture and radiology are the mainstay of diagnosis but lack sensitivity, leading to a delay in timely diagnosis and intervention. Recently, PCR-based approaches have been shown to be a promising method in diagnosing IM.Hypothesis/Gap Statement. Molecular-based approaches may be a valuable adjunct to standard conventional methods for diagnosing IM, especially among culture negatives and patients on antifungal therapy.Aim. In the present study we aimed to evaluate the clinical utility of panfungal and Mucorales-specific PCR for diagnosing IM from various clinical specimens.Methodology. This was a prospective study in which 239 clinically suspected cases of IM attending our tertiary care hospital from August 2015 to March 2018 were enrolled. All the cases were defined as 'proven', 'probable' or 'possible' based on EORTC/MSGERC guidelines. In addition to conventional diagnostics (KOH-calcofluor stain and culture), panfungal and Mucorales-specific PCR assays were also performed. The amplified products were sequenced for species identification. In vitro antifungal susceptibility was performed on all the culture-positive isolates.Results. Among 239 clinically suspected cases of IM, only 140 cases were diagnosed by the demonstration of aseptate ribbon-like hyphae on direct microscopy. Culture was positive in 35.7 % (54/140) of direct microscopy-positive samples. Among the proven cases (n=11), the sensitivity for both Mucorales-specific nested PCR and panfungal PCR was 100 %, but specificity was 91.9 and 73.7% respectively. In probable cases (n=129), the sensitivity of both the PCRs was 98.5 % and specificity for panfungal PCR was 73.7 and 91.9 % for Mucorales-specific PCR.Conclusion. Pan fungal PCR in combination with Mucorales-specific PCR, followed by sequencing, may play a significant role in IM diagnosis especially among those negative for both direct microscopy and culture.

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The diagnostic utility and costs of panfungal PCR assays for invasive fungal disease (IFD) from bronchoalveolar lavage fluid (BALF) specimens are incompletely defined. In a retrospective audit, panfungal PCR results from 2014-2018 were matched with information on request forms and the registrar/microbiologist diary of clinical liaison. Identification of a single fungus other than a commensal was considered potentially clinically significant, and assessed for clinical relevance. Of 1002 specimens tested, an estimated 90% were requested in patients without clinical suspicion of IFD. There were 530 (52.9%) PCR-positive results of which 485/530 (91.5%) identified multiple fungal species or commensal fungi; 45 (8.5%) were clinically significant but only in 12 (1.2%) was panfungal PCR the sole diagnostic test leading to IFD diagnosis, all in immunocompromised patients with clinical suspicion of IFD. Costs of panfungal PCR tests averaged AUD 133 per test, or AUD 26,767/annum. However, the average cost-per-diagnosis achieved was AUD 15,978/annum. Limiting testing to patients at risk and with clinical suspicion of IFD, may save over AUD 13,383/annum (assuming 50-90% reduction in testing). The value-added utility of panfungal PCR on BALF is 1.2% (12/1002). We have since introduced pre-analytical stewardship limiting routine panfungal PCR testing of BALF to high-risk patients in our hospital.

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A 21-year-old man developed infectious keratitis after swimming in Spain whilst wearing contact lenses. Mycelial growth from a corneal sample suggested keratomycosis, but a drastic worsening of the patient's condition was observed on antifungal drugs. On day 38, panfungal PCR identified Pythium insidiosum, which is an aquatic organism belonging to the oomycete family. Based on the recent literature, this patient was promptly prescribed a systemic and local antibiotic regimen and cure was ultimately achieved. In order to facilitate P. insidiosum identification in future cases, we have generated the first matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) reference spectrum for P. insidiosum. It is planned to deposit this MALDI-TOF MS reference spectrum on an open-access platform and this should allow immediate identification of the pathogen. Finally, this case report also demonstrates that P. insidiosum is emerging outside tropical and subtropical areas. Clinicians and microbiologists should have better knowledge to accurately manage and diagnose this sight-threatening infection.

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Molecular techniques in fungal detection and identification represent an efficient complementary diagnostic tool which is increasingly used to overcome limitations of routinely used culture techniques. The aim of this study was to characterize Candida sp. representation in samples from urine, urinary catheter, and ureteral stent biofilm using ITS2 ribosomal DNA (rDNA) amplification followed by fluorescent capillary electrophoresis (f-ITS2-PCR-CE) and to compare the results with those obtained by culture. A total of 419 samples were analyzed, and 106 (25.2%) were found positive, out of which 17 (16%) were polyfungal. The positivity rate did not differ between samples from catheters and stents (23.6% versus 20.9%) or between catheter and stent corresponding urine samples (40.2% versus 30.2%). Ten different Candida species were detected, with Candida parapsilosis (31.4%), Candida albicans (26.5%), and Candida tropicalis (12.4%) predominating. f-ITS2-PCR-CE was evaluated as substantially less time-consuming and 8.3 times more sensitive than the routinely applied culture technique with 1 µl of urine/sonicated fluid inoculated, detecting 67 (19.9%) versus 8 (2.4%) positive samples out of 337 initially analyzed samples. The culture sensitivity considerably improved to 1.7 times lower than that of f-ITS2-PCR-CE after the inoculation volume was increased to 100 µl in the additional 82 samples. Moreover, the molecular technique, unlike routine cultivation, enabled precise pathogen composition determination in polymicrobial samples. In conclusion, the f-ITS2-PCR-CE method was shown to be a quick and efficient tool for culture-independent detection and identification of fungi in urinary tract-related samples, demonstrating a higher sensitivity than culture.

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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. 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.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. CONCLUSION: 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.

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BACKGROUND: Accurate diagnosis of invasive mold diseases (IMD) remains challenging. Here, the performance of panfungal PCR, Aspergillus and MucoralesPCR in bronchoalveolar lavage (BAL) was evaluated. METHODS: We conducted a single-center study including 167 hematologic patients at risk for IMD with BAL performed 2011-2014. Diagnostic performance of single tests (Aspergillus-, Mucorales-, and panfungal PCR, galactomannan (GM)≥0.5 and ≥1, culture/cytology) or in combination was calculated for predicting IMD comparing proven/probable or proven/probable/possible IMD vs no IMD, respectively. RESULTS: IMD was classified as proven (n = 6), probable (n = 31), possible (n = 29) and no IMD (n = 101) according to EORTC/MSG criteria. GM ≥ 0.5 in BAL showed the highest sensitivity with 81% for diagnosing IMD whereas the other tests only 5%-35%. By contrast, specificity was highest for panfungal PCR with 99% and GM ≥ 1, Mucorales and AspergillusPCR reached specificity ≥91%. When combining the tests, GM ≥ 0.5 and panfungal PCR show a sensitivity and specificity of 87% and 78% for IMD or with AspergillusPCR a sensitivity and specificity of 88% and 72% for invasive pulmonary aspergillosis, respectively. Including possible IMD patients did not improve the sensitivity of PCRs. In probable/proven IMD patients, the addition of panfungal PCR resulted further in detection of Fusarium species and Alternaria species, and the MucoralesPCR was positive in 2 probable IMD cases. CONCLUSION: This study illustrates that the diagnosis of IMD is still very problematic and lacks objectivity. Together with GM in BAL, the PCRs may prove an addition to the current available diagnostic armamentarium in IMD because of their ability to identify molds on a species level.

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BACKGROUND: Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD. METHODS: Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified. RESULTS: Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases. CONCLUSIONS: The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing.

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Over the past decade, the incidence of life-threatening invasive fungal infections has dramatically increased. Infections caused by hitherto rare and emerging fungal pathogens are associated with significant morbidity and mortality among immunocompromised patients. These observations render the coverage of a broad range of clinically relevant fungal pathogens highly important. The so-called panfungal or, perhaps more correctly, broad-range nucleic acid amplification techniques do not only facilitate sensitive detection of all clinically relevant fungal species but are also rapid and can be applied to analyses of any patient specimens. They have therefore become valuable diagnostic tools for sensitive screening of patients at risk of invasive fungal infections. This chapter summarizes the currently available molecular technologies employed in testing of a wide range of fungal pathogens, and provides a detailed workflow for patient screening by broad-spectrum nucleic acid amplification techniques.

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Despite advances in the treatment of invasive fungal diseases (IFD), mortality rates remain high. Moreover, due to the expanding spectrum of causative agents, fast and accurate pathogen identification is necessary. We designed a panfungal polymerase chain reaction (PCR), which targets the highly variable ITS2 region of rDNA genes and uses high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of this method was tested on a broad spectrum of the most clinically important fungal pathogens including Aspergillus spp., Candida spp. and mucormycetes. Despite the fact that fluid from bronchoalveolar lavage (BAL) is one of the most frequently tested materials there is a lack of literature sources aimed at panfungal PCR as an IFD diagnostic tool from BAL samples. The applicability of this method in routine practice was evaluated on 104 BAL samples from immunocompromised patients. Due to high ITS region variability, we obtained divergent melting peaks for different fungal species. Thirteen out of 18 patients with proven or probable IFD were positive. Therefore, the sensitivity, specificity, positive predictive value and negative predictive value of our method were 67%, 100%, 100%, and 94%, respectively. In our assay, fungal pathogens identification is based on HRM, therefore omitting the expensive and time consuming sequencing step. With the high specificity, positive and negative predictive values, short time needed to obtain a result, and low price, the presented assay is intended to be used as a quick screening method for patients at risk of IFD.

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The role of panfungal polymerase chain reaction (PCR) assays for diagnosis of invasive fungal disease (IFD) is inadequately defined. We describe the use of an internal transcribed spacer 1 (ITS-1) region-directed panfungal PCR in this context at a tertiary referral transplant center. A retrospective review of patients at Alfred Health, Melbourne, Australia (2009-2014) who had clinical samples referred for panfungal PCR testing was conducted. Baseline patient characteristics, antifungal drug history, fungal culture/histopathology, and radiology results were recorded. For bronchoalveolar lavage (BAL) fluid samples, identification of a fungus other than a Candida spp. was defined as a potential pathogen.Of 138 panfungal PCR tests (108 patients), 41 (30%) were positive for a fungal product. Ninety-seven percent (134/138) of specimens were from immunocompromised hosts. Thirteen percent (19/138) of panfungal PCR positive results were for potential pathogens and potential pathogens were detected more frequently in tissue as compared with BAL (12/13 vs. 6/26; P = .0001). No positive panfungal PCR results were obtained from CSF specimens. If histopathology examination was negative, panfungal PCR identified a potential pathogen in only 12% (11/94) of specimens. For the 20 culture negative/histopathology positive specimens, diagnosis of IFD to causative species level by panfungal PCR occurred in 35% (6/20).Sterile site specimens, in particular tissue, were more frequently panfungal PCR positive for potential pathogens than BAL. The utility of panfungal PCR appears greatest in tissue specimens, as an adjunct to histopathology to improve diagnostic sensitivity and specificity. Based on the results of this study we are now only testing tissue specimens by panfungal PCR.

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Early antifungal therapeutic strategies are proposed during invasive fungal infection (IFI), but antifungal stewardship programs should institute a systematic reevaluation of prescriptions, particularly in the context of empirical treatment. Here, we aimed to evaluate the performances and particularly the negative predictive value (NPV) of diagnostic strategies, including a whole blood panfungal quantitative PCR assay (PF-qPCR) in a high risk population for IFI. The first step was to standardize and optimize a new PF-qPCR targeting ITS2 region. Then, this method was evaluated in a multicenter prospective study including 313 patients with suspected IFI for whom an early antifungal treatment was prescribed. All patients enrolled at day 0 of their treatment benefited from serum Aspergillus galactomannan (GM) antigen detection twice a week, weekly PF-qPCR assay, and when indicated and feasible, CT-scan and mycological sampling. In total, 125 of 313 patients were diagnosed with IFI: 68 invasive aspergillosis (eight proven, 48 probable and 12 possible), one fusariosis, 47 candidemia, three disseminated candidiasis and six cryptococcosis. Globally, the sensitivity of the PF-qPCR assay was only 40%, but the specificity, PPV and NPV were 96%, 88% and 69%, respectively. In the population of patients at high risk for invasive aspergillosis who also benefited from Aspergillus GM detection, the sensitivity and the NPV of the combined detection reached to 78% and 84%, respectively. Even higher NPV were obtained when combining negative PF-qPCR and CT scan (95%) as well as negative GM and CT scan (93%), thus allowing to rationalize and re-evaluate the prescription of empirical treatment in such highly selected population.

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Invasive fungal disease (IFD) has high mortality rate, especially in the growing population of immunocompromised patients. In spite of introduction of novel diagnostic approaches, the intravital recognition of IFD is challenging. Autopsy studies remain a key tool for assessment of epidemiology of visceral mycoses. We aimed to determine species distribution and trends of IFD over the last 10 years in unselected autopsy series from a large university hospital. Forty-five cases of visceral mycoses, confirmed by histopathology and panfungal PCR, were found in 587 consecutive autopsies. Major underlying diseases were diabetes mellitus (20%), hematologic malignancies (15.6%) and systemic lupus erythematosus (15.6%). There was a high risk for disseminated IFD in immunocompromised patients stayed in the hospital over 1 month with a fever longer than 3 weeks. The most common fungi were Aspergillus spp. (58%), Candida spp. (16%), Mucorales (14%) and Fusarium spp. (10%). We found significant increase in Aspergillus flavus (P = 0.04) and Mucorales (P < 0.01) infections over the last 5 years. Concordance rate between histopathology and panfungal PCR was 89.5% to the genus level. All 6 cases of fusariomycosis were misinterpreted as aspergillosis by histology alone. The precise species identification, necessary for targeted antifungal treatment, was rendered only by the molecular technique. Panfungal PCR showed high performance on formalin-fixed paraffin-embedded specimens, providing important epidemiological data in retrospective autopsy series. Rapid detection of fungi by panfungal PCR assay has high potential for intravital diagnostics of IFD in surgical and biopsy specimens.

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