title: Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.
abstract: Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients.
authors.affiliation: Department of Dermatology and Venereology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University.
authors.forename: Sha
authors.initials: S
authors.lastname: Lu
authors.affiliation: Department of Dermatology and Venereology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.
authors.forename: Xiqing
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, United States.
authors.forename: Richard
authors.initials: R
authors.lastname: Calderone
authors.affiliation: Department of Dermatology and Venereology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Dermatology and Venereology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.
authors.forename: Jianchi
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Dermatology and Venereology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.
authors.forename: Wenying
authors.initials: W
authors.lastname: Cai
authors.affiliation: Department of Dermatology and Venereology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China xiliyan@mail.sysu.edu.cn.
authors.forename: Liyan
authors.initials: L
authors.lastname: Xi
mesh_terms: D001769:Blood / Q000382:microbiology*; D004271:DNA, Fungal / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D016469:Fungemia / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D032901:Talaromyces / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004271:DNA, Fungal
keywords: T. marneffei; diagnosis; nested PCR; real-time PCR; whole blood