Fungal Integrated Histomolecular Diagnosis Using Targeted Next-Generation Sequencing on Formalin-Fixed Paraffin-Embedded Tissues

被引：10

作者：

Trecourt, Alexis ^{[1
,2
]}

Rabodonirina, Meja ^{[3
,4
]}

Mauduit, Claire ^{[1
,4
,5
]}

Traverse-Glehen, Alexandra ^{[1
,4
,6
]}

Devouassoux-Shisheboran, Mojgan ^{[1
,7
]}

Meyronet, David ^{[7
,8
]}

Dijoud, Frederique ^{[7
,8
]}

Ginevra, Christophe ^{[9
,10
,11
]}

Chapey-Picq, Emmanuelle ^{[3
,4
]}

Josse, Emilie ^{[3
]}

Martins-Simoes, Patricia ^{[9
,11
]}

Bentaher, Abderrazzak ^{[2
,4
]}

Dupont, Damien ^{[3
,7
]}

Miossec, Charline ^{[3
]}

Persat, Florence ^{[2
,3
,7
]}

Wallon, Martine ^{[3
,4
]}

Ferry, Tristan ^{[7
,12
]}

Pham, Felix ^{[13
]}

Simon, Bruno ^{[9
,14
]}

Menotti, Jean ^{[2
,3
,7
,9
]}

机构：

[1] Hosp Civils Lyon, Ctr Hosp Lyon Sud, Serv Pathol Multis Site Site Sud, Lyon, France

[2] Univ Claude Bernard Lyon 1, Fac Medecine Lyon Sud Charles Merieux, CICLY Equipe Inflammat & Immun Epithelium Resp UR, Lyon, France

[3] Hosp Civils Lyon, Hop Croix Rousse, Inst Agents Infect, Serv Parasitol & Mycol Med, Lyon, France

[4] Univ Claude Bernard Lyon 1, Fac Med Lyon Sud Charles Merieux, Lyon, France

[5] Inst Natl St & Rech Medicale, Ctr Mediterraneen Med Mol C3M, Unite 1065, Nice, France

[6] Univ Claude Bernard Lyon 1, Fac Med Lyon Sud Charles Merieux, Ctr Rech Cancerol Lyon, INSERM U1052 CNRS UMR5286, Lyon, France

[7] Univ Claude Bernard Lyon 1, Fac Med Lyon Est, Lyon, France

[8] Hosp Civils Lyon, Ctr Hosp Lyon Est, Serv Pathol Multis Est S, Lyon, France

[9] Hosp Civils Lyon, Hop Croix Rousse, Inst Agents Infect, Gen Epidemiol Malad Infectieuses GENEPII, Lyon, France

[10] Hosp Civils Lyon, Hop Croix Rousse, Inst Agents Infect, Ctr Natl Reference Legionelles, Lyon, France

[11] Hosp Civils Lyon, Hop Croix Rousse, Inst Agents Infect, Ctr Natl Reference Staphyloccoques, Lyon, France

[12] Hosp Civils Lyon, Hop Croix Rousse, Serv Malad Infectieuses & Trop, Lyon, France

[13] Hosp Civils Lyon, Ctr Hosp Lyon Sud, Serv Dermatol, Lyon, France

[14] Hosp Civils Lyon, Hop Croix Rousse, Inst Agents Infect, Serv Virol, Lyon, France

来源：

JOURNAL OF CLINICAL MICROBIOLOGY | 2023年 / 61卷 / 03期

关键词：

integrated histomolecular diagnosis; histopathology; next-generation sequencing; fungal identification; formalin-fixed paraffin-embedded tissues; massive parallel sequencing; MOLECULAR-DETECTION; MURINE MODEL; IDENTIFICATION; PCR; DNA; ASPERGILLOSIS; MUCORMYCOSIS;

D O I：

10.1128/jcm.01520-22

中图分类号：

Q93 [微生物学];

学科分类号：

071005 ; 100705 ;

摘要：

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

引用

页数：14

共 48 条

[1] Circulating Aspergillus fumigatus DNA Is Quantitatively Correlated to Galactomannan in Serum [J].