Evaluation of a Multiplex Real-time PCR Targeting the β-tubulin Gene for the Detection and Differentiation of and

Overview

Journal Microbiol Spectr

Specialty Microbiology

Date 2024 Oct 22

PMID 39436137

Authors

Luisa F Lopez

Lalitha Gade

Anastasia P Litvintseva

D Joseph Sexton

Affiliations

Soon will be listed here.

Abstract

sp. is a thermally dimorphic genus of fungi known to cause subacute or chronic subcutaneous lesions in humans and animals and is the cause of increasing public health concern due to spread of feline-associated cases. Here, we adapted and evaluated a recently described real-time PCR assay targeting the β-tubulin gene to rapidly detect and differentiate two related species, and . The assay was tested with 55 . 19 . , and 85 isolates from other clinically relevant fungi, and showed 100% concordance with reference identification methods. The assay showed high analytical sensitivity with a limit of detection of 1 pg of DNA per microliter of sample for both targets. The assay was further evaluated with 11 fresh and 17 formalin-fixed, paraffin-embedded (FFPE) tissues. This multiplex real-time PCR assay successfully detected the DNA from both and isolates as well as from fresh and FFPE tissues. Our results demonstrate this assay performs well and could be a helpful molecular tool to support rapid species identification in cultures and primary specimens.IMPORTANCEHaving available molecular tools to identify and differentiate closely related species will allow clinical, veterinarians, and public health labs to provide diagnostic results with accuracy and short turnaround time for the routine and outbreak response activities.

References

Rodrigues A, de Hoog G, De Camargo Z . Sporothrix Species Causing Outbreaks in Animals and Humans Driven by Animal-Animal Transmission. PLoS Pathog. 2016; 12(7):e1005638. PMC: 4945023. DOI: 10.1371/journal.ppat.1005638. View

Ribeiro Dos Santos A, Gade L, Misas E, Litvintseva A, Nunnally N, Parnell L . Bimodal distribution of azole susceptibility in isolates in Brazil. Antimicrob Agents Chemother. 2024; 68(4):e0162023. PMC: 10989022. DOI: 10.1128/aac.01620-23. View

Lopez L, Tobon A, Caceres D, Chiller T, Litvintseva A, Gade L . Application of Real-Time PCR Assays for the Diagnosis of Histoplasmosis in Human FFPE Tissues Using Three Molecular Targets. J Fungi (Basel). 2023; 9(7). PMC: 10381543. DOI: 10.3390/jof9070700. View

Hu S, Chung W, Hung S, Ho H, Wang Z, Chen C . Detection of Sporothrix schenckii in clinical samples by a nested PCR assay. J Clin Microbiol. 2003; 41(4):1414-8. PMC: 153868. DOI: 10.1128/JCM.41.4.1414-1418.2003. View

Rees J, Pinner R, Hajjeh R, Brandt M, Reingold A . The epidemiological features of invasive mycotic infections in the San Francisco Bay area, 1992-1993: results of population-based laboratory active surveillance. Clin Infect Dis. 1998; 27(5):1138-47. View

Yu X, Wan Z, Zhang Z, Li F, Li R, Liu X . Phenotypic and molecular identification of Sporothrix isolates of clinical origin in Northeast China. Mycopathologia. 2013; 176(1-2):67-74. PMC: 3731519. DOI: 10.1007/s11046-013-9668-6. View

Etchecopaz A, Lanza N, Toscanini M, Devoto T, Pola S, Daneri G . Sporotrichosis caused by Sporothrix brasiliensis in Argentina: Case report, molecular identification and in vitro susceptibility pattern to antifungal drugs. J Mycol Med. 2019; 30(1):100908. DOI: 10.1016/j.mycmed.2019.100908. View

Kano R, Watanabe K, Murakami M, Yanai T, Hasegawa A . Molecular diagnosis of feline sporotrichosis. Vet Rec. 2005; 156(15):484-5. DOI: 10.1136/vr.156.15.484. View

Bass B, Engel K, Greytak S, Moore H . A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen?. Arch Pathol Lab Med. 2014; 138(11):1520-30. DOI: 10.5858/arpa.2013-0691-RA. View

10.

Kauffman C, Bustamante B, Chapman S, Pappas P . Clinical practice guidelines for the management of sporotrichosis: 2007 update by the Infectious Diseases Society of America. Clin Infect Dis. 2007; 45(10):1255-65. DOI: 10.1086/522765. View

11.

de Carvalho J, Monteiro R, Hagen F, De Camargo Z, Rodrigues A . Trends in Molecular Diagnostics and Genotyping Tools Applied for Emerging Species. J Fungi (Basel). 2022; 8(8). PMC: 9409836. DOI: 10.3390/jof8080809. View

12.

Almeida-Paes R, Pimenta M, Pizzini C, Monteiro P, Peralta J, Nosanchuk J . Use of mycelial-phase Sporothrix schenckii exoantigens in an enzyme-linked immunosorbent assay for diagnosis of sporotrichosis by antibody detection. Clin Vaccine Immunol. 2007; 14(3):244-9. PMC: 1828849. DOI: 10.1128/CVI.00430-06. View

13.

Gade L, Hurst S, Arunmozhi Balajee S, Lockhart S, Litvintseva A . Detection of mucormycetes and other pathogenic fungi in formalin fixed paraffin embedded and fresh tissues using the extended region of 28S rDNA. Med Mycol. 2016; 55(4):385-395. DOI: 10.1093/mmy/myw083. View

14.

Orofino-Costa R, de Macedo P, Rodrigues A, Bernardes-Engemann A . Sporotrichosis: an update on epidemiology, etiopathogenesis, laboratory and clinical therapeutics. An Bras Dermatol. 2017; 92(5):606-620. PMC: 5674690. DOI: 10.1590/abd1806-4841.2017279. View

15.

Rodrigues A, Teixeira M, de Hoog G, Schubach T, Pereira S, Fernandes G . Phylogenetic analysis reveals a high prevalence of Sporothrix brasiliensis in feline sporotrichosis outbreaks. PLoS Negl Trop Dis. 2013; 7(6):e2281. PMC: 3688539. DOI: 10.1371/journal.pntd.0002281. View

16.

Rodrigues A, de Hoog G, De Camargo Z . Molecular Diagnosis of Pathogenic Sporothrix Species. PLoS Negl Trop Dis. 2015; 9(12):e0004190. PMC: 4666615. DOI: 10.1371/journal.pntd.0004190. View

17.

Indoung S, Chanchayanon B, Chaisut M, Buapeth K, Morteh R, Jantrakajorn S . Feline sporotrichosis caused by Sporothrix schenckii sensu stricto in Southern Thailand: phenotypic characterization, molecular identification, and antifungal susceptibility. Med Mycol. 2022; 60(9). DOI: 10.1093/mmy/myac075. View

18.

Bernardes-Engemann A, Costa R, Miguens B, Penha C, Neves E, Pereira B . Development of an enzyme-linked immunosorbent assay for the serodiagnosis of several clinical forms of sporotrichosis. Med Mycol. 2005; 43(6):487-93. DOI: 10.1080/13693780400019909. View

19.

Della Terra P, Gonsales F, de Carvalho J, Hagen F, Kano R, Bonifaz A . Development and evaluation of a multiplex qPCR assay for rapid diagnostics of emerging sporotrichosis. Transbound Emerg Dis. 2021; 69(4):e704-e716. DOI: 10.1111/tbed.14350. View

20.

Wang H, Lu J, Chang C, Chou W, Hsieh J, Lin C . Development of a high sensitivity TaqMan-based PCR assay for the specific detection of Mycobacterium tuberculosis complex in both pulmonary and extrapulmonary specimens. Sci Rep. 2019; 9(1):113. PMC: 6331544. DOI: 10.1038/s41598-018-33804-1. View