Detection and identification of Candida spp. in human serum by LightCycler real-time polymerase chain reaction.

The aim of this work was to develop LightCycler real-time polymerase chain reaction method to allow rapid detection and identification of Candida spp. in human serum with panfungal primers (internal transcribed spacer [ITS] and L18). Melting-curve analysis of the ITS sequences showed that each amplicon presented a specific melting point and enabled identification of 5 Candida spp. After parameters optimization, 58 sera were preliminary analyzed from 23 patients. For L18 primers, the LightCycler system enabled detection of DNA in 92% of patients with positive blood culture. These primers were not able to differentiate between species of Candida. By using ITS primers, the LightCycler system enabled detection of DNA in sera from 76.9% of patients with positive blood culture. With ITS primers, the species responsible for the infection was identified for 11 patients. These data revealed the LightCycler as a potential tool for early detection and identification of Candida.

[1]  L. Mele,et al.  PCR-Restriction Enzyme Analysis for Detection ofCandida DNA in Blood from Febrile Patients with Hematological Malignancies , 1999, Journal of Clinical Microbiology.

[2]  P. White,et al.  Detection of seven Candida species using the Light-Cycler system. , 2003, Journal of medical microbiology.

[3]  C. Heath,et al.  Real-time automated polymerase chain reaction (PCR) to detect Candida albicans and Aspergillus fumigatus DNA in whole blood from high-risk patients. , 2003, Diagnostic microbiology and infectious disease.

[4]  A. Francesconi,et al.  Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans, with Implications for Diagnosis of Invasive Candidiasis , 2006, Journal of Clinical Microbiology.

[5]  D. Denning,et al.  Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. , 2002, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[6]  R. Auckenthaler,et al.  Comparison of BacT/Alert with Signal blood culture system , 1995, Journal of clinical microbiology.

[7]  M. Adelson,et al.  Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing. , 2005, Molecular and cellular probes.

[8]  David W Williams,et al.  Detection of Candida in Concentrated Oral Rinse Cultures by Real-Time PCR , 2004, Journal of Clinical Microbiology.

[9]  Y. Senda,et al.  Multiplex PCR Using Internal Transcribed Spacer 1 and 2 Regions for Rapid Detection and Identification of Yeast Strains , 2001, Journal of Clinical Microbiology.

[10]  S. Fridkin,et al.  Epidemiology and Predictors of Mortality in Cases of Candida Bloodstream Infection: Results from Population-Based Surveillance, Barcelona, Spain, from 2002 to 2003 , 2005, Journal of Clinical Microbiology.

[11]  R. Wenzel,et al.  Current trends in the epidemiology of nosocomial bloodstream infections in patients with hematological malignancies and solid neoplasms in hospitals in the United States. , 2003, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[12]  Suhail Ahmad,et al.  Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification , 2002, Journal of Clinical Microbiology.

[13]  P. Schnitzler,et al.  Simple and Rapid Detection of Candida albicans DNA in Serum by PCR for Diagnosis of Invasive Candidiasis , 2000, Journal of Clinical Microbiology.

[14]  Y. Tanaka,et al.  Use of real-time PCR on blood samples for diagnosis of invasive aspergillosis. , 2001, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[15]  S. Bretagne,et al.  Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis. , 2005, FEMS immunology and medical microbiology.

[16]  A. Hirschl,et al.  Development of Novel Real-Time PCR Assays for Detection and Differentiation of Eleven Medically Important Aspergillus and Candida Species in Clinical Specimens , 2007, Journal of Clinical Microbiology.

[17]  R. Borrow,et al.  Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR , 2001, Journal of Clinical Microbiology.

[18]  H. Einsele,et al.  Detection of PCR-amplified fungal DNA by using a PCR-ELISA system. , 1998, Medical mycology.

[19]  R. Bowden,et al.  Panfungal PCR Assay for Detection of Fungal Infection in Human Blood Specimens , 1998, Journal of Clinical Microbiology.

[20]  M. Ruhnke,et al.  Epidemiology of Candida albicans infections and role of non-Candida-albicans yeasts. , 2006, Current drug targets.

[21]  K. Bhatia,et al.  Monochrome LightCycler PCR assay for detection and quantification of five common species of Candida and Aspergillus. , 2005, Journal of medical microbiology.

[22]  M. Pfaller,et al.  Epidemiology of Invasive Candidiasis: a Persistent Public Health Problem , 2007, Clinical Microbiology Reviews.

[23]  E. Anaissie,et al.  The epidemiology of hematogenous candidiasis caused by different Candida species. , 1997, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[24]  A. Huang,et al.  Rapid Identification of Yeasts in Positive Blood Cultures by a Multiplex PCR Method , 2001, Journal of Clinical Microbiology.

[25]  M. Nicolas-Chanoine,et al.  Serum Is More Suitable than Whole Blood for Diagnosis of Systemic Candidiasis by Nested PCR , 1999, Journal of Clinical Microbiology.

[26]  S. Sanche,et al.  Rapid Identification of Fungi by Using the ITS2 Genetic Region and an Automated Fluorescent Capillary Electrophoresis System , 2000, Journal of Clinical Microbiology.

[27]  R. Auckenthaler,et al.  Candida Colonization and Subsequent Infections in Critically III Surgical Patients , 1994, Annals of surgery.

[28]  H. Einsele,et al.  Quantification of Fungal DNA by Using Fluorescence Resonance Energy Transfer and the Light Cycler System , 2000, Journal of Clinical Microbiology.

[29]  M. Kaku,et al.  Quantitative evaluation of cytomegalovirus DNA in infantile hepatitis , 2001, Journal of viral hepatitis.

[30]  A. Heim.  From poliovirus surveillance to enterovirus surveillance: a complete picture? , 2005, Journal of medical microbiology.

[31]  C. Cordonnier,et al.  Real-Time PCR Coupled with Automated DNA Extraction and Detection of Galactomannan Antigen in Serum by Enzyme-Linked Immunosorbent Assay for Diagnosis of Invasive Aspergillosis , 2002, Journal of Clinical Microbiology.