Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood
- 804 Downloads
Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5–10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.
KeywordsCandidiasis Caspofungin Invasive Candidiasis Polymerase Chain Reaction Test Conventional Polymerase Chain Reaction
We thank Randy Jirtle and David Skaar for the use of their ABI 7900 real-time thermocycler and Alejandro Escalante-Flores for technical assistance. This project was supported by USA Public Health Service grants from the National Institutes of Health, U01 AI 066590 and K24 AI 072522 (B.D.A.).
Conflict of interest
Co-authors Benton, Poore, Rouse, Boles, V. K. Pamula, Eckhardt, and Pollack are employed by Advanced Liquid Logic, Inc. Co-authors Smith, Johnson, Alexander, Benjamin, Perfect, and Mitchell declare no conflict of interest. Co-author Schell has a small equity ownership in Advanced Liquid Logic, Inc.
- 6.Benjamin DK Jr, Stoll BJ, Fanaroff AA, McDonald SA, Oh W, Higgins RD, Duara S, Poole K, Laptook A, Goldberg R; National Institute of Child Health and Human Development Neonatal Research Network (2006) Neonatal candidiasis among extremely low birth weight infants: risk factors, mortality rates, and neurodevelopmental outcomes at 18 to 22 months. Pediatrics 117:84–92PubMedCrossRefGoogle Scholar
- 9.Mitchell TG, Verweij PE, Hoepelman AIM (2010) Opportunistic and systemic fungi. In: Cohen J, Powderly WG, Opal SM (eds) Infectious diseases, 3rd edn. Mosby Elsevier, London, pp 1823–1852Google Scholar
- 13.Chen Y-C, Eisner JD, Kattar MM, Rassoulian-Barrett SL, LaFe K, Yarfitz SL, Limaye AP, Cookson BT (2000) Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes. J Clin Microbiol 38:2302–2310PubMedGoogle Scholar
- 15.Wulff-Burchfield E, Schell WA, Eckhardt AE, Pollack MG, Hua Z, Rouse JL, Pamula VK, Srinivasan V, Benton JL, Alexander BD, Wilfret DA, Kraft M, Cairns CB, Perfect JR, Mitchell TG (2010) Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens. Diagn Microbiol Infect Dis 67:22–29PubMedCrossRefGoogle Scholar
- 18.Cowen LE, Singh SD, Köhler JR, Collins C, Zaas AK, Schell WA, Aziz H, Mylonakis E, Perfect JR, Whitesell L, Lindquist S (2009) Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc Natl Acad Sci USA 106:2818–2823PubMedCrossRefGoogle Scholar
- 19.White PL, Perry MD, Loeffler J, Melchers WJG, Klingspor L, Bretagne S, McCulloch E, Cuenca-Estrella M, Finnstrom N, Donnelly JP, Barnes RA; European Aspergillus PCR Initiative (2010) Critical stages of extracting DNA from Aspergillus fumigatus in whole-blood specimens. J Clin Microbiol 48:3753–3755PubMedCrossRefGoogle Scholar
- 22.McMullan R, Metwally L, Coyle PV, Hedderwick SA, McCloskey BV, O’Neill HJ, Patterson CC, Thompson G, Webb CH, Hay RJ (2008) A prospective clinical trial of a real-time polymerase chain reaction assay for the diagnosis of candidemia in nonneutropenic, critically ill adults. Clin Infect Dis 46:890–896PubMedCrossRefGoogle Scholar
- 32.Srinivasan V, Pamula VK, Pollack MG, Fair RB (2003) Clinical diagnostics on human whole blood, plasma, serum, urine, saliva, sweat, and tears on a digital microfluidic platform. In: Proceedings of MicroTAS 2003, Squaw Valley, CA, October 2003, pp 1287–1290Google Scholar
- 34.Sista RS, Eckhardt AE, Wang T, Graham C, Rouse JL, Norton SM, Srinivasan V, Pollack MG, Tolun AA, Bali D, Millington DS, Pamula VK (2011) Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns. Clin Chem 57:1444–1451PubMedCrossRefGoogle Scholar