Panfungal Polymerase Chain Reaction Research Articles

A Seminested PCR Method for the Diagnosis of Invasive Fungal Infections in Combat Injured.

Among combat injured, invasive fungal infections (IFIs) result in significant morbidity. Cultures and histopathology are the primary diagnostic methods for IFIs, but they have limitations. We previously evaluated a panfungal polymerase chain reaction assay, which was 83% sensitive and 99% specific for angioinvasive IFIs. Here, we evaluated 3 less resource-intensive seminested assays targeting clinically relevant fungi in the order Mucorales and genera Aspergillus and Fusarium. Formalin-fixed paraffin-embedded tissue specimens from a multicenter trauma IFI cohort (2009-2014) were used. Cases were US military personnel injured in Afghanistan with histopathologic IFI evidence. Controls were patients with similar injury patterns and no laboratory IFI evidence (negative culture and histopathology). Seminested assays specific to Mucorales (V4/V5 regions of 18S rDNA), Aspergillus (mitochondrial tRNA), and Fusarium (internal transcribed spacer [ITS]/28A regions of DNA) were compared with a panfungal assay amplifying the internal transcribed spacer 2 region of rDNA and to histopathology. Specimens from 92 injury sites (62 subjects) were compared with control specimens from 117 injuries (101 subjects). We observed substantial agreement between the seminested and panfungal assays overall, especially for the order Mucorales. Moderate agreement was observed at the genus level for Aspergillus and Fusarium. When compared with histopathology, sensitivity and specificity of seminested assays were 67.4% and 96.6%, respectively (sensitivity increased to 91.7% when restricted to sites with angioinvasion). Prior studies of seminested molecular diagnostics have focused on culture-negative samples from immunocompromised patients. Our findings underscore the utility of the seminested approach in diagnosing soft-tissue IFIs using formalin-fixed paraffin-embedded tissue samples, especially with angioinvasion.

Identification of Invasive Filamentous Mold Isolates Using DNA Sequencing: Experience of a Clinical Laboratory in a Resource-limited Setting

Background: Correct identification of clinically significant mold is becoming critical and cannot be relied only on phenotypic methods; hence, there is a dire need to develop an algorithm of workflows and capacity for molecular identification. This study shares the experience of DNA sequencing for invasive mold identification isolated and compared with phenotypic identification. Methods: This study was conducted at the microbiology laboratory, Aga Khan University, Karachi, Pakistan, and the Mycotic Disease Branch, Centers for Disease Control and Prevention, USA. Filamentous molds isolated from clinical specimens during January 2012–April 2013 were initially identified through phenotypic characteristics. Pan-fungal polymerase chain reaction targeting the internal transcribed spacer region of the ribosomal cistron and the D1/D2 domains of the 28S ribosomal cistron was performed. Sequencer version was utilized to edit and align the DNA sequences, and then sequences were identified using BLAST. The correlation between phenotypic and molecular identification was evaluated. Results: Gene sequencing identified 50% of clinical isolates as one of the Aspergillus species, followed by Mucorales 29%, Fusarium species 17%, and Cladosporium spp. 4%. Overall, 50% of clinical isolates were identified correctly till the species level by conventional methods. Phenotypic correlation with genotype till genus was 42%, while two isolates were wrongly identified phenotypically. Excellent agreement (100%) till species level between phenotypic and molecular identification for Aspergillus, while Mucorales had an agreement of 71%. Conclusion: Traditional phenotypic identification methods for filamentous molds had a good correlation with sequencing up to genus level identification; however, were not reliable up to species level.

P21 Facial papules in an immunocompromised patient

Abstract A 29-year-old man presented with a 6-week history of clustered facial papules. Of note, he had a diagnosis of HIV 5 months previously and was on maintenance antiretroviral treatment. His initial CD4 count was 5, which increased to 38, and his viral load was now no longer detectable. He had also been treated for cytomegalovirus reactivation, pneumocystis pneumonia and suspected mycobacterium infection for the previous 3 months. He had ongoing symptoms of malaise, fevers, weight loss and nausea, along with abnormal liver function tests and a raised C-reactive protein. A bone marrow trephine showed noncaseating discrete small granulomas; Grocott and periodic acid–Schiff (PAS) stains were negative. Prior to his dermatology review, the papules were initially perioral and he had been treated with empiric valaciclovir for suspected herpes simplex virus (HSV) but with no resolution. Testing for HSV DNA and syphilis was negative. On examination, there were clusters of small nontender, well-circumscribed papules (some umbilicated), distributed on the forehead, cheeks, and cutaneous and mucosal lips. A diagnostic punch biopsy was taken of one of the characteristic papules. Histological examination demonstrated a dense dermal infiltrate of histiocytes with focal non-necrotizing granuloma formation. Interspersed neutrophilic microabscesses and occasional eosinophils were present. PAS stain for fungi confirmed the presence of numerous thick-walled spherules, many with a single distinct nucleus, with some showing binucleation, consistent with fungal organisms located within the histiocytes in the dermis. Differential diagnosis included histoplasmosis, cryptococcosis, blastomycosis and coccidiomycosis. He was commenced on intravenous liposomal amphotericin B for disseminated endemic mycosis while awaiting definitive diagnosis. Formal identification by panfungal polymerase chain reaction of skin tissue was positive for Histoplasma capsulatum and itraconazole was added. The diagnosis was consistent with disseminated histoplasmosis with cutaneous involvement. The lesions resolved completely over 4 weeks, with some residual hyperpigmentation remaining. Histoplasma capsulatum is a dimorphic fungus found in soil and the droppings of bats and birds. Infection is through spore inhalation, with most patients being asymptomatic. It is more prevalent in temperate and tropical climates. Disseminated histoplasmosis occurs mostly in immunocompromised individuals and can be fatal if left untreated. Although endemic to specific world regions, disseminated histoplasmosis should be considered in immunocompromised patients presenting with atypical cutaneous findings, even in regions of low prevalence.

The first suspected disseminated Hormographiella aspergillata infection in China, diagnosed using metagenomic next-generation sequencing: a case report and literature review

ABSTRACT Hormographiella aspergillata is a rare and emerging cause of invasive mould infections in patients with haematological malignancies, with a mortality rate of approximately 70%. Here, we present the first reported case of suspected disseminated H. aspergillata infection in China. The patient experienced a second relapse of acute myeloid leukaemia and developed neutropenia, fever, discrepant blood pressure between limbs, and cutaneous lesions limited to the left upper extremity. Since lung tissue biopsy was not feasible, metagenomic next-generation sequencing (mNGS) and panfungal polymerase chain reaction (PCR) analysis of bronchoalveolar lavage fluid and blood samples were performed, which indicated probable H. aspergillata pulmonary infection. Histopathology of cutaneous lesions revealed numerous fungal hyphae within dermal blood vessels. mNGS of a skin biopsy sample identified H. aspergillata sequences, and the fungi was subsequently recovered from fungal culture, proving cutaneous H. aspergillata infection. Despite combined antifungal therapy, the patient died owing to disease progression. Additionally, 22 previously reported cases of invasive H. aspergillata infection were reviewed in patients with haematological malignancies. Thus, mNGS is a powerful diagnostic tool for the early and effective detection of invasive H. aspergillata infections, with the advantage of sequencing all potential pathogens, and providing results within 24 h.

Hyphae, pseudohyphae, yeasts, spherules, spores, and more: A review on the morphology and pathology of fungal and oomycete infections in the skin of domestic animals.

Fungi are among the most common infectious agents affecting the skin of animals. The skin can serve as a port of entry for fungal infections, which can eventually become disseminated. In some regions of the world, oomycetes, such as Pythium and Lagenidium, are also responsible for a significant number of severe cutaneous infections. Histologic evaluation of fungal morphology, including size, shape, septation, branching, and budding characteristics, combined with the distribution of inflammatory infiltrates within different skin layers can potentially identify etiologic agents, guiding selection of antifungals and additional diagnostics. Fungal infections of the skin surface are typically caused by Malassezia and rarely Candida, with opportunistic fungi also capable of colonizing the skin surface, especially when the barrier is broken. Folliculocentric infections, caused by dermatophytes, result in mild to severe inflammation and can occasionally penetrate deep into the skin. A wide range of fungi, including agents of hyalohyphomycosis, phaeohyphomycosis, and dimorphic fungal infections, as well as oomycetes, result in nodular cutaneous and subcutaneous lesions. With the occasional exception of dimorphic fungi, fungal speciation often requires cultures performed on fresh tissues. However, molecular techniques such as pan-fungal polymerase chain reaction on paraffin blocks is becoming an increasingly useful tool to distinguish between cutaneous fungal pathogens. This review focuses on describing the clinical and histologic features of the most common fungal and oomycete infections affecting the skin of animals, divided according to distribution patterns of lesions and fungal or oomycete morphology.

Role of Polymerase Chain Reaction in Peritoneal Dialysis Patients with Nonbacterial Peritonitis

Introduction: Culture-positive peritoneal dialysis (PD)-related peritonitis depends on proper sample collection and culture technique. Patients are investigated for fungal and tubercular peritonitis once the initial peritoneal effluent grows no bacteria and the patient is unresponsive to antimicrobials. However, they are diagnosed late in the course of illness and lead to irreversible peritoneal damage preventing reinsertion of PD catheter. The aim of the study was to assess the role of polymerase chain reaction (PCR) in diagnosis of fungal and Mycobacterium tuberculosis (MTB)-related peritonitis in patients on PD who presented with features of peritonitis. Materials and Methods: This was a prospective study and included patients who were diagnosed to have PD-related peritonitis as per the International Society of PD guidelines. All patients underwent panfungal and MTB complex (MTC) PCR of PD effluent on day 1 of admission. Results: Eighty-five patients with PD-related peritonitis were included in the study. In patients with no bacterial growth on PD fluid, panfungal PCR was positive in 22 (25.8%) patients and 2 (2.4%) patients had positive MTC PCR. Fungal organism could be isolated in 10 (11.7%) patients. Nine percent of patients with fungal peritonitis died. Conclusion: The use of PCR in patients with PD-related peritonitis will help in early diagnosis and management of fungal and tubercular peritonitis. It will also help in reducing mortality in these patients.

Infections by entomopathogenic fungi in common green iguanas (Iguana iguana) in captivity in Brazil

Entomopathogenic fungi, widely available biological agents used to control agricultural pests, are sporadically reported to cause focal or disseminated infection in reptiles and mammals, including humans. This study summarizes the clinical presentation, histopathological and molecular findings by panfungal polymerase chain reaction and sequencing of four cases of hypocrealean fungal infections in captive common green iguanas (Iguana, iguana). One case of granulomatous pneumonia, hepatitis and serositis was related to Metarhizium flavoviride complex infection. Two disseminated fungal infection cases, with scarce inflammatory cell infiltration, were caused by Beauveria bassiana while there was one case of multifocal granulomatous and necrotizing pneumonia by Purpureocillium spp. To the best of our knowledge, this is the first report of fatal mycosis infection due to entomopathogenic fungi in captive common green iguanas.

Molecular Identification of Fungi Causing Tissue Mycoses From Formalin Fixed Paraffin Embedded (FFPE) Archive Specimens

Introduction: Isolation of fungi from tissue specimens using conventional methods is time consuming. However, in some cases, the histopathological examination (HPE) of tissue alone is unable to provide a definite identity of the fungus. Alternatively, a non-culture method, such as polymerase chain reaction (PCR) detecting the internal transcribed spacer (ITS) rRNA genes of the fungi, is a promising diagnostic tool for rapid and accurate diagnosis of tissue mycoses. Methods: This work investigated the utility of panfungal PCR in identifying agents of tissue mycoses in 87 FFPE archive specimens. Deoxyribonucleic acid (DNA) extraction was performed on FFPE specimens by using QIAamp DNA FFPE Tissue Kit. The ITS2 region was amplified using ITS3/ITS4 primers. The PCR products were sequenced using the same primers and compared to the NCBI nucleotide database for species identification. Results: Fungal DNA was successfully amplified in 52 (59.8%) specimens, from which only 23 (44.0%) fungi were consistent with clinical/HPE findings. The identified fungi were Aspergillus spp., Candida spp., Penicillium spp., Cryptococcus neoformans, Talaromyces marneffei, and Rhizopus oryzae. A few rare fungi were also identified, such as Diaporthe longicolla and fungus-like oomycete such as Pythium insidiosum that are commonly associated with plant pathogens. Conclusion: Although PCR was able to offer accurate genus/species identification, utilising this method on paraffinised tissue specimens must be evaluated by considering many factors that will reduce its sensitivity and specificity. Therefore, it is important to correlate the PCR results with clinical and HPE findings to obtain a correct diagnosis and adequate treatment for tissue mycoses.

Evaluation of Panfungal Polymerase Chain Reaction in Early Detection of Invasive Fungal Infections in Egyptian Patients with Hematological Malignancies.

The incidence of invasive fungal infections (IFIs) has increased owing to the rising number of immunodeficient patients. A case-control study was performed at the Ain Shams University Hospitals, Cairo, Egypt. The case group (n = 80) included 80 patients diagnosed with hematological malignancies, and the control group (n = 20) included 20 patients. All patients were tested for the presence of fungal species using blood culture and panfungal real-time polymerase chain reaction (RT-PCR). Fungal species differentiation was performed using high-resolution melting (HRM) PCR. There were 39 suspected cases of IFIs among the 80 patients. The panfungal RT-PCR detection rate was 51.3% (41/80). HRM-PCR identified that 51.2% of the fungal species were Candida albicans, 44.0% were non-Candida albicans, and 4.9% were Mucor. The blood cultures were positive for the presence of fungi in two patients with acute myeloid leukemia. The fungal detection rate using the panfungal RT-PCR technique was significantly higher than that using the blood culture technique (P < 0.001). RT-PCR using panfungal markers is sensitive, rapid, and superior to the blood culture technique to detect IFIs. HRM-PCR is a specific test for species identification.

Changing Trends in the Survival of Immunosuppressed Children with Invasive Fungal Rhinosinusitis.

Acute invasive fungal rhinosinusitis has been associated with high mortality rates. We aimed to explore the contribution of novel detection and treatment methods on the outcome of immunosuppressed children with acute invasive fungal rhinosinusitis. The records of all children with a hematologic or oncologic disease who developed acute invasive fungal rhinosinusitis between 2005-2020 were reviewed. Thirty-four patients were included. Aspergillosis and mucormycosis were diagnosed in 20 patients (59%) and 12 patients (35%), respectively. Panfungal polymerase chain reaction (PCR) was associated with a change of treatment in 36% of patients. A more aggressive surgical approach as well as treatment with liposomal amphotericin B and novel antifungals were adopted in recent years. Overall, 26% of patients died of disease, however no disease-specific death occurred since 2012. Diagnosis using panfungal PCR (p = .04) and treatment with novel antifungal medications (p = .017) were significantly associated with disease-specific survival. Enhanced fungal detection using panfungal PCR and treatment with novel antifungal agents, combined with rapid diagnosis and treatment, aggressive surgical approach and better control over the underlying oncological disease, may significantly improve the outcome of immunosuppressed children with acute invasive fungal rhinosinusitis.

Talaromyces spp. infections in dogs from the Southern United States.

Talaromyces spp. are soil-dwelling fungi sporadically reported to cause disease in humans and dogs. This study summarized the clinical presentations, histologic findings, and Talaromyces sp. involved in 5 dogs diagnosed through the panfungal polymerase chain reaction service (PCR) at Texas A&M University, with a review of previously reported cases. Of the 5 cases, 3 were Labrador Retrievers, 2 were male, and 3 were female. Three of 5 involved the musculoskeletal or lymphatic systems, and 2 of 5 dogs presented with meningoencephalitis. Talaromyces helicus, Talaromyces aurantiacus, and Talaromyces boninensis were identified based on panfungal PCR, showing 99% to 100% sequence matches in combination with morphologic features. Three of 5 dogs had static disease at the time of publication, 1 was euthanized, and 1 was lost to follow-up. This study describes Talaromyces spp. as a cause of meningoencephalitis in dogs, identifies 2 novel Talaromyces spp. involved in infections, and adds to the existing knowledge of clinical presentations and outcomes.

Identification and Antifungal Drug Susceptibility Pattern of Candida auris in India.

Candida auris has turned up as a multidrug-resistant nosocomial agent with outbreaks reported worldwide. The present study was conducted to evaluate the antifungal drug susceptibility pattern of C. auris. Isolates of C. auris were obtained from clinically suspected cases of candidemia from January 2019 to June 2021. Identification was done with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and panfungal DNA polymerase chain reaction (PCR), followed by sequencing. Antifungal susceptibility testing was performed with broth microdilution method. Out of 50 isolates C. auris, 49 were identified by MALDI-TOF and one isolate was identified with panfungal DNA PCR followed by sequencing. For fluconazole, 84% (n = 42) isolates were found to be resistant and 16% (n = 8) isolates were susceptible (minimum inhibitory concentrations [MICs] range 0.5-16). Posaconazole exhibited potent activity, followed by itraconazole. For amphotericin B, only 6% (n = 3) isolates were resistant with MICs ≥2 μg/mL. Only 4% (n = 2) isolates exhibited resistance to caspofungin. No resistance was noted for micafungin and anidulafungin. One (2%) isolate was found to be panazole resistant. One (2%) isolate was resistant to fluconazole, amphotericin B, and caspofungin. Correct identification of C. auris can be obtained with the use of MALDI-TOF and sequencing methods. A small percentage of fluconazole-sensitive isolates are present. Although elevated MICs for amphotericin B and echinocandins are not generally observed, the possibility of resistance with the irrational use of these antifungal drugs cannot be denied. Pan azole-resistant and pan drug-resistant strains of C. auris are on rise.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides.

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

Outbreak of Paranannizziopsis australasiensis Infection in Captive African Bush Vipers (Atheris squamigera)

We report the epidemiological, clinical and pathological features of an outbreak of Paranannizziopsis australasiensis (order Onygenales) in captive African bush vipers (Atheris squamigera) (ABVs) that died suddenly. The snakes had multifocal, raised, white-grey to dark brown discoloured cutaneous patches. Microscopically, all had integumentary lesions characterized by multifocal to coalescent necroheterophilic epidermitis with superficial and intraepidermal fungal elements and bacteria. Concurrent epidermal hyperplasia, hyperkeratosis and intracellular and intercellular oedema, often leading to vesiculation, and fasciitis/superficial myositis, were consistent findings in all snakes, while ulceration (9/11) and dysecdysis (5/11) varied. A panfungal polymerase chain reaction targeting the internal transcribed spacer-2 region, and gene sequencing, confirmed P. australasiensis infection in three cases. This is the first report of P. australasiensis in the USA and the first record of paranannizziopsis infection in African bush vipers. P. australasiensis should be considered in the differential diagnosis of dermatomycosis in snakes and represents a potential threat to reptile conservation programmes.

Acute isolated Aspergillus appendicitis in pediatric leukemia

Aspergillus is a widespread fungus in the environment, usually invades through the respiratory tract. Invasive aspergillosis is a fatal disseminated infection in immunocompromised hosts. Appendicitis occurs scarcely in patients with leukemia. We report a case of Aspergillus appendicitis that underwent an urgent appendectomy. An 11-year-old boy received the diagnosis of acute myeloid leukemia, because of the bone pain and results of the bone marrow study. He obtained a complete remission after cancer chemotherapy and received peripheral blood stem cell transplantation from a histocompatible sibling. Leukemia relapsed 5 months post-transplant. Induction therapy with etoposide, cytarabine and mitoxantrone was started on Candida prophylaxis. Fifteen days after the end of chemotherapy, he presented with febrile neutropenia and abdominal pain, that did not respond to broad-spectrum antibiotics. Serum levels of C-reactive protein, β-D-glucan and procalcitonin were unremarkable. Computed tomography scan revealed a swollen appendix and the adjacent tissue inflammation. An urgent appendectomy led to a tentative diagnosis of Aspergillus appendicitis based on the histopathological findings of many fungal hyphal forms. Panfungal polymerase chain reaction using DNA extracted from the lesion determined the pathogen of Aspergillus niger. There was no evidence of invasive aspergillosis. During the prolonged anti-fungal therapy, he achieved a remission of leukemia and underwent the second hematopoietic cell transplantation. To our knowledge, Aspergillus appendicitis was reported to occur in 5 leukemia patients. Four of them survived after appendectomy and one died from intestinal perforation. Early surgical intervention is mandatory for a cure of Aspergillus appendicitis in neutropenic patients on Candida prophylaxis.

Identification of Fungal Species and Detection of Azole-Resistance Mutations in the Aspergillus fumigatus cyp51A Gene at a South Korean Hospital

PurposeWith changing fungal epidemiology and azole resistance in Aspergillus species, identifying fungal species and susceptibility patterns is crucial to the management of aspergillosis and mucormycosis. The objectives of this study were to evaluate performance of panfungal polymerase chain reaction (PCR) assays on formalin-fixed paraffin embedded (FFPE) samples in the identification of fungal species and in the detection of azole-resistance mutations in the Aspergillus fumigatus cyp51A gene at a South Korean hospital.Materials and MethodsA total of 75 FFPE specimens with a histopathological diagnosis of aspergillosis or mucormycosis were identified during the 10-year study period (2006–2015). After deparaffinization and DNA extraction, panfungal PCR assays were conducted on FFPE samples for fungal species identification. The identified fungal species were compared with histopathological diagnosis. On samples identified as A. fumigatus, sequencing to identify frequent mutations in the cyp51A gene [tandem repeat 46 (TR46), L98H, and M220 alterations] that confer azole resistance was performed.ResultsSpecific fungal DNA was identified in 31 (41.3%) FFPE samples, and of these, 16 samples of specific fungal DNA were in accord with a histopathological diagnosis of aspergillosis or mucormycosis; 15 samples had discordant histopathology and PCR results. No azole-mediating cyp51A gene mutation was noted among nine cases of aspergillosis. Moreover, no cyp51A mutations were identified among three cases with history of prior azole use.ConclusionPanfungal PCR assay with FFPE samples may provide additional information of use to fungal species identification. No azole-resistance mediating mutations in the A. fumigatus cyp51A gene were identified among FFPE samples during study period.

Retrospective Analysis of Aetiological Agents Associated with Pulmonary Mycosis Secondary to Enteric Salmonellosis in Six Horses by Panfungal Polymerase Chain Reaction

Pulmonary mycosis secondary to enterocolitis is an uncommon diagnosis in equine medicine, but is thought to result from mucosal compromise and translocation of enteric fungi. The aetiological agent associated with translocation is often identified based on fungal culture or hyphal features in histological sections. In order to understand better the aetiological agents involved, six horses diagnosed with Salmonella enteritis and concurrent pulmonary mycosis were identified retrospectively through a database search of veterinary teaching hospital records. Samples from these cases were subjected to polymerase chain reaction and sequencing of the internal transcribed spacer 2 (ITS-2) located between the 5.8S and 28S rRNA genes to identify the aetiological agent involved. Sequencing identified Aspergillus fumigatus, Aspergillus flavus, Fusarium spp., Cladosporium spp. and Curvularia spp. A single case had a dual infection with Fusarium spp. and A. fumigatus.

Evaluation of the diagnostic performance of panfungal polymerase chain reaction assay in invasive fungal diseases.

Timely diagnosis of invasive fungal diseases (IFDs) is important, as delays in treatment initiation are associated with increased mortality rates. However, early diagnosis of IFDs in immunocompromised patients remains difficult. The conventional diagnostic methods currently used for IFDs are not sufficiently effective. Molecular tests, such as polymerase chain reaction (PCR)-based assays, have great potential to improve the early diagnosis of IFDs due to their sensitivity and specificity. In the present study, the diagnostic performance of panfungal PCR assays in IFD patients who received bone marrow transplantation was evaluated. The results suggested that panfungal PCR assay offered a quick and convenient guide for clinical decision-making by identifying higher numbers of fungal species in comparison with the conventional blood culture method. Furthermore, panfungal PCR assay exhibited a sensitivity of 93% and a specificity of 71% in the diagnosis of IFD patients based on the EORTC/MSG criteria. Thus, the present study concluded that the reported PCR-based method was effective and sensitive in early IFD diagnosis and should be integrated into clinical decision-making for the treatment of IFDs in the future.

Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease.

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. 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. 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. 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.

Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues

Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.