A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients

BMC Infectious Diseases

Gilda Maria Barbaro Del Negro²  Margarete Teresa Gottardo de Almeida³  Maria Esther Jurfest Rivero Ceccon¹  Artur Figueiredo Delgado¹  Thelma Suely Okay⁴  Cleison Ledesma Taira² 

[1] Pediatric and Neonatal Intensive Care Units, Instituto da Criança, HCFMUSP, São Paulo, SP, Brazil;Laboratory of Medical Mycology (LIM-53), Clinical Dermartology Division, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) and Instituto de Medicina Tropical da Universidade de São Paulo (IMT-USP), Av. Dr. Enéas Carvalho de Aguiar, 500, Andar térreo, Predio 2, CEP, 05403-900 São Paulo, SP, Brazil;Microbiology Laboratory, Department of Dermatologic, Infectious and Parasitic Diseases, Faculdade de Medicina de São José do Rio Preto (FAMERP), São José do Rio Preto, SP, Brazil;Laboratory of Seroepidemiology and Immunobiology, IMT-USP, São Paulo, SP, Brazil

关键词: Diagnosis;    Paediatric;    ICU;    Multiplex PCR;    Candida spp;    Candidaemia;   

Others  :  1127322
DOI  :  10.1186/1471-2334-14-406

 received in 2014-03-21, accepted in 2014-07-10,  发布年份 2014

【 摘 要 】

Background

Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients.

Methods

DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures.

Results

The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients.

Conclusions

Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.

【 授权许可】

   
2014 Taira et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Arendrup MC: Epidemiology of invasive candidiasis. Curr Opin Crit Care 2010, 16(5):445-452.
• [2]Avni T, Leibovici L, Paul M: PCR diagnosis of invasive candidiasis: systematic review and meta-analysis. J Clin Microbiol 2011, 49(2):665-670.
• [3]Benjamin DK Jr, Poole C, Steinbach WJ, Rowen JL, Walsh TJ: Neonatal candidemia and end-organ damage: a critical appraisal of the literature using meta-analytic techniques. Pediatrics 2003, 112(3 Pt1):634-640.
• [4]Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB: Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 2004, 39:309-317.
• [5]Zaoutis TE, Prasad PA, Localio AR, Coffin SE, Bell LM, Walsh TJ, Gross R: Risk factors and predictors for candidemia in pediatric intensive care unit patients: implications for prevention. Clin Infect Dis 2010, 51(5):e38-e45.
• [6]Kaufman DA: Challenging issues in neonatal candidiasis. Curr Med Res Opin 2010, 26(7):1769-1778.
• [7]Chai ALY, Denning DW, Warn P: Candida tropicalis in human disease. Crit Rev Microbiol 2010, 36(4):282-298.
• [8]Nucci M, Queiroz-Telles F, Alvarado-Matute T, Tiraboschi IN, Cortes J, Zurita J, Guzman-Blanco M, Santolaya ME, Thompson L, Sifuentes-Osornio J, Echevarria JI, Colombo AL: Epidemiology of candidemia in Latin America: a laboratory-based survey. PLoS One 2013, 8(3):e59373.
• [9]See LLC: Bloodstream infection in children. Pediatr Crit Care Med 2005, 6:S42-44.
• [10]Lau A, Sorrell TC, Chen S, Stanley K, Iredell J, Halliday C: Multiplex tandem PCR: a novel platform for rapid detection and identification of fungal pathogens from blood culture specimens. J Clin Microbiol 2008, 46(9):3021-3027.
• [11]Zilberberg MD, Kollef MH, Arnold H, Labelle A, Micek ST, Kothari S, Shorr AF: Inappropriate empiric antifungal therapy for candidemia in the ICU and hospital resource utilization: a retrospective cohort study. BMC Infect Dis 2010, 10:150.
• [12]Ahmad S, Khan Z, Mustafa AS, Khan ZU: Seminested PCR for diagnosis of candidemia: comparison with culture, antigen detection, and biochemical methods for species identification. J Clin Microbiol 2002, 40:2483-2489.
• [13]Alam FF, Mustafa AS, Khan ZU: Comparative evaluation of (1,3)-β-D-glucan, mannan and anti-mannan antibodies, and Candida species-specific snPCR in patients with candidemia. BMC Infect Dis 2007, 7:103-111.
• [14]Carvalho A, Costa-De-Oliveira S, Martins ML, Pina-Vaz C, Rodrigues AG, Ludovico P, Rodrigues F: Multiplex PCR identification of eight clinically relevant Candida species. Med Mycol 2007, 45:619-627.
• [15]Goldstein B, Giroir B, Randolph A: International pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics. Pediatr Crit Care Med 2005, 6(1):2-8.
• [16]Löffler J, Hebart H, Schumacher U, Reitze H, Einsele H: Comparison of different methods for extraction of DNA of fungal pathogens from cultures and blood. J Clin Microbiol 1997, 35:3311-3312.
• [17]Van Burik J, Myerson D, Schreckhise RW, Bowden R: Panfungal PCR assay for detection of fungal infection in human blood specimens. J Clin Microbiol 1998, 36:1169-1175.
• [18]Luo G, Mitchell TG: Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR. J Clin Microbiol 2002, 40:2860-2865.
• [19]Bougnoux M, Dupont C, Mateo J, Saulnier P, Faivre V, Payen D, Nicolas-Chanoine M: Serum is more suitable than whole blood for diagnosis of systemic candidiasis by nested PCR. J Clin Microbiol 1999, 37:925-930.
• [20]Rolfs A, Schuller I, Finckh U, Weber-Rolfs I: PCR contamination and falsely interpreted results. In PCR: clinical diagnostics and research. Berlin: Springer; 1992:61-67.
• [21]Riggsby WS, Torres-Bauza LJ, Wills JW, Townes TM: DNA content, kinetic complexity, and the ploid question in Candida albicans. Mol Cell Biol 1982, 2:853-862.
• [22]Colombo AL, Nucci M, Park BJ, Nouér SA, Arthington-Skaggs B, da Matta DA, Warnock D, Morgan J, Brazilian Network Candidemia Study: Epidemiology of candidemia in Brazil: a nationwide sentinel surveillance of candidemia in eleven medical centers. J Clin Microbiol 2006, 44(8):2816-2833.
• [23]Pfaller MA, Diekema DJ: Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2007, 20:133-163.
• [24]Blinkhorn RJ, Adelstein D, Spagnuolo PJ: Emergence of a new opportunistic pathogen: Candida lusitaniae. J Clin Microbiol 1989, 27(2):236-240.
• [25]Aragão PA, Oshiro IC, Manrique EI, Gomes CC, Matsuo LL, Leone C, Moretti-Branchini ML, Levin AS, IRIS Study Group: Pichia anomala outbreak in a nursery: exogenous source? Pediatr Infect Dis J 2001, 20(9):843-848.
• [26]White PL, Barton R, Guiver M, Linton CJ, Wilson S, Smith M, Gomez BL, Carr MJ, Kimmitt PT, Seaton S, Rajakumar K, Holyoake T, Kibbler CC, Johnson E, Hobson RP, Jones B, Barnes RA: A consensus on fungal polymerase chain reaction diagnosis? A United Kingdom-Ireland evaluation of polymerase chain reaction methods for detection of systemic fungal infections. J Molec Diagn 2006, 8(3):376-384.
• [27]Tirodker UH, Nataro JP, Smith S, LasCasas L, Fairchild KD: Detection of fungemia by polymerase chain reaction in critically ill neonates and children. J Perinatol 2003, 23(2):117-122.
• [28]Del Negro GM, Delgado AF, Manuli ER, Yamamoto L, Okay TS: Dual candidemia detected by nested polymerase chain reaction in two critically ill children. Med Mycol 2010, 48(8):1116-1120.
• [29]Boktour M, Kontoyiannis DP, Hanna HA, Hachem RY, Girgawy E, Bodey GP, Raad II: Multiple-species candidemia in patients with cancer. Cancer 2004, 101(8):1860-1865.
• [30]Jensen J, Muñoz P, Guinea J, Rodríguez-Créixems M, Peláez T, Bouza E: Mixed fungemia: incidence, risk factors, and mortality in a general hospital. Clin Infect Dis 2007, 44(12):e109-114.
• [31]Pulimood S, Ganesan L, Alangaden G, Chandrasekar P: Polymicrobial candidemia. Diagn Microbiol Infect Dis 2002, 44(4):353-357.
• [32]Colombo AL, Guimarães T, Camargo LF, Richtmann R, Queiroz-Telles FD, Salles MJ, Cunha CA, Yasuda MA, Moretti ML, Nucci M: Brazilian guidelines for the management of candidiasis - a joint meeting report of three medical societies: Sociedade Brasileira de Infectologia, Sociedade Paulista de Infectologia and Sociedade Brasileira de Medicina Tropical. Braz J Infect Dis 2013, 17(3):283-312.
• [33]Santolaya ME, Alvarado T, Queiroz-Telles F, Colombo AL, Zurita J, Tiraboschi IN, Cortes JA, Thompson L, Guzman M, Sifuentes J, Echevarría JI, Nucci M: Active surveillance of candidemia in children from Latin America: a key requirement for improving disease outcome. Pediatr Infect Dis J 2014, 33(2):e40-44.
• [34]Clancy CJ, Nguyen MH: Finding the “missing 50%” of invasive candidiasis: how nonculture diagnostics will improve understanding of disease spectrum and transform patient care. Clin Infect Dis 2013, 56(9):1284-1292.
• [35]Montagna MT, Caggiano G, Borghi E, Morace G: The role of the laboratory in the diagnosis of invasive candidiasis. Drugs 2009, 69(Suppl 1):59-63.
• [36]Nguyen MH, Wissel MC, Shields RK, Salomoni MA, Hao B, Press EG, Shields RM, Cheng S, Mitsani D, Vadnerkar A, Silveira FP, Kleiboeker SB, Clancy CJ: Performance of Candida real-time polymerase chain reaction, β-D-glucan assay, and blood cultures in the diagnosis of invasive candidiasis. Clin Infect Dis 2012, 54(9):1240-1248.
• [37]Neely LA, Audeh M, Phung NA, Min M, Suchocki A, Plourde D, Blanco M, Demas V, Skewis LR, Anagnostou T, Coleman JJ, Wellman P, Mylonakis E, Lowery TJ: T2 magnetic resonance enables nanoparticle-mediated rapid detection of candidemia in whole blood. Sci Transl Med 2013, 5(182):182ra54.
• [38]Lau A, Halliday C, Chen SC, Playford EG, Stanley K, Sorrell TC: Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR. J Clin Microbiol 2010, 48(3):811-816.