Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture

Wagner, Karoline; Springer, Burkard; Pires, V.P.; Keller, Peter M.

doi:10.1038/s41598-018-25129-w

Cited by 54 publications

(45 citation statements)

References 26 publications

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“…cfDNA sequencing identified the same pathogen in four of the six proven IFD cases with fungi detected by invasive biopsy (skin, lung, pseudocyst fluid drainage) and blood culture (Table ). One proven IFD case with negative cfDNA NGS involved a 15‐month old female with acute lymphoblastic leukemia (ALL) who had a cheek lesion with central eschar, which revealed Rhizopus oryzae on fungal culture and 18S rDNA targeted sequencing on sterile biopsy. The other discrepant case was documented in a 21‐year‐old male with ALL with persistent fevers, cough, hypoxia, radiologic evidence of extensive bilateral nodular opacities with regions of cavitation, and an elevated BG at >500 pg/mL.…”

Section: Resultsmentioning

confidence: 99%

Cell‐free DNA next‐generation sequencing successfully detects infectious pathogens in pediatric oncology and hematopoietic stem cell transplant patients at risk for invasive fungal disease

Armstrong

Rossoff

Hollemon³

et al. 2019

Pediatric Blood & Cancer

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Background We sought to determine if next‐generation sequencing (NGS) of microbial cell‐free DNA (cfDNA) in plasma would detect pathogens in pediatric patients at risk for invasive fungal disease (IFD). Procedures Pediatric hematology, oncology, and stem cell transplant patients deemed at risk for new IFD had blood samples drawn at three time‐points separated by 1‐month intervals. The primary outcome measure was detection of fungal pathogens compared to standard clinical testing. Secondary outcomes included identification of other infectious pathogens, relationship to European Organization for Research and Treatment of Cancer's Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases’ Mycoses Study Group (EORTC/MSG) guidelines, and assessment of antifungal therapy. Results NGS identified fungal pathogens in seven of 40 at‐risk patients for IFD and results were identical in four of six proven cases, including Aspergillus fumigatus by lung biopsy, Candida albicans by blood or pancreatic pseudocyst cultures, and Rhizopus delemar by skin biopsy. Rhizopus oryzae identified on skin biopsy and A. fumigatus isolated on day 27 of 28 of culture from lung biopsy were not detected by cfDNA NGS, possibly due to lack of bloodstream penetration and questionable pathogenicity, respectively. Numerous DNA viruses were detected in patients with prolonged febrile neutropenia or abnormal imaging. Extended antifungal therapy was used in 73% of patients. Follow‐up cfDNA sequencing in patients who were positive at enrollment was negative at 1 and 2 months. Conclusions cfDNA NGS detected fungal pathogens from blood confirming its potential to guide treatment decisions in pediatric patients at risk for IFD and limit excessive empiric antifungal use. Future studies are needed to better understand the sensitivity and specificity of this approach.

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Section: Resultsmentioning

confidence: 99%

Cell‐free DNA next‐generation sequencing successfully detects infectious pathogens in pediatric oncology and hematopoietic stem cell transplant patients at risk for invasive fungal disease

Armstrong

Rossoff

Hollemon³

et al. 2019

Pediatric Blood & Cancer

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“…Based on the data presented, ITS-sequencing has been established at our center as a routine investigation in addition to all the other diagnostic tools, like culture, biomarker testing including recently released Aspergillus lateral flow device tests and PCRs. In some studies, the sequencing of amplicons of 18S or ITS2 genes outperformed the results created by using ITS1 targets, as used in our study [34,35].…”

Section: Discussionmentioning

confidence: 68%

Impact of ITS-Based Sequencing on Antifungal Treatment of Patients with Suspected Invasive Fungal Infections

Guenter

Gorkiewicz

Halwachs

et al. 2020

JoF

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Molecular techniques including the sequencing of fungal-specific DNA targets are increasingly used in the diagnosis of suspected invasive fungal infections. In contrast to established biomarkers like galactomannan or 1-3-β-d-glucan, the clinical impact of these methods remains unknown. We retrospectively investigated the impact of ITS1-sequencing on antifungal treatment strategies in 71 patients (81 samples) with suspected invasive fungal infections. ITS-sequencing either confirmed already ongoing antifungal therapy (19/71 patients, 27%), led to a change in antifungal therapy (11/71, 15%) or supported the decision to withhold antifungal treatment (34/71, 48%) (in seven of 71 patients, ITS-sequencing results were obtained postmortem). ITS-sequencing results led to a change in antifungal therapy in a relevant proportion of patients, while it confirmed therapeutic strategies in the majority. Therefore, ITS-sequencing was a useful adjunct to other fungal diagnostic measures in our cohort.

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“…In order to obtain reliable and faster results, methods based on sequence analysis of ITS regions have been developed and used in routine laboratories [11] . ITS region is accepted and frequently used as a genetic marker for the identification of fungi [12,13] . It is known that ITS regions 1 and 2 are more variable than the other subunit genes among fungi [14,15] .…”

Section: Discussionmentioning

confidence: 99%

Evcil Hayvanlardan İzole Edilen Mantarların Teşhisinde Kullanılan Konvansiyonel ve ITS Dizi Analizi Metotlarının Karşılaştırılması

Müştak¹,

Sariçam²,

Müştak³

2019

Kafkas Univ Vet Fak Derg

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How to Cite This ArticleMüştak İB, Sarıçam S, Müştak HK: Comparison of internal transcribed spacer region sequencing and conventional methods used in the identification of fungi isolated from domestic animals. Kafkas Univ Vet Fak Derg, 25 (5): 639-643, 2019. AbstractThe aim of this study was to compare gold standard conventional culture method and internal transcribed spacer (ITS) sequence-based analysis in the identification of fungi isolated from domestic animals. A total of 35 animals (15 cats, 11 dogs, 4 horses and 5 chickens) suspected for fungal infection were examined. Of the 35 samples, 20 were found to be positive for fungal culture. Among positive samples 8 (40%) were predominantly found to be dermatophyte species by conventional methods. The ITS regions (ITS1, ITS2 and complete ITS) of fungal isolates were also amplified, sequenced and the results were compared with conventional culture method. The identification results of 18 (90%) fungal species were found to be compatible with both conventional culture and sequencing methods. Comparison of the results demonstrated that, complete ITS regions gene sequencing could be used for the identification of medically important fungi rapidly. Since the results of complete ITS regions gene sequencing were found to be compatible with the results of phenotypic identification, it can be concluded that ITS regions gene sequencing of fungal isolates can be also used as a confirmative tool of conventional culture methods. ÖzBu çalışmanın amacı, evcil hayvanlardan izole edilen mantar türlerinin identifikasyonunda altın standart olan konvansiyonel kültür yöntemi ve Internal Ara Bölgeler (ITS) dizi analizi yönteminin karşılaştırılmasıdır. Çalışmada, mantar enfeksiyonlarından şüphelenilen toplam 35 hayvan (15 kedi, 11 köpek, 4 at ve 5 tavuk) incelendi. 35 örnek içinde 20 tanesinin mantar kültürleri pozitif bulundu. Pozitif örnekler arasında 8 (%40) tanesi konvansiyonel yöntemlerle ağırlıklı olarak dermatofit türleri olarak bulundu. İzolatların ITS bölgeleri (ITS1, ITS2 ve ITS bölgesinin tamamı) amplifiye edildi, sekanslandı ve elde edilen sonuçlar kültür yöntemiyle karşılaştırıldı. Hem konvansiyonel hem de dizi analizi yöntemiyle elde edilen identifikasyon sonuçları 18 (%90) örnekte birbiri ile uyumlu bulundu. Bu sonuçlar doğrultusunda, ITS bölgesinin tamamının dizi analizi, mantarların hızlı teşhisi için kullanılabileceğini ortaya koydu. ITS gen bölgesinin tamamına ait dizi analizi sonuçlarının fenotipik identifikasyon sonuçları ile uyumlu olması, ITS gen bölgesi dizi analizinin konvansiyonel yöntemlerin doğrulanmasında bir araç olarak kullanılabileceğini ortaya koymuştur.

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Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture

Cited by 54 publications

References 26 publications

Cell‐free DNA next‐generation sequencing successfully detects infectious pathogens in pediatric oncology and hematopoietic stem cell transplant patients at risk for invasive fungal disease

Cell‐free DNA next‐generation sequencing successfully detects infectious pathogens in pediatric oncology and hematopoietic stem cell transplant patients at risk for invasive fungal disease

Impact of ITS-Based Sequencing on Antifungal Treatment of Patients with Suspected Invasive Fungal Infections

Evcil Hayvanlardan İzole Edilen Mantarların Teşhisinde Kullanılan Konvansiyonel ve ITS Dizi Analizi Metotlarının Karşılaştırılması

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