Real-Time PCR in the early detection of invasive fungal infection in immunodeficient infants and children

Abstract

Background: Crucial to the diagnosis and effective therapy of invasive fungal infection (IFI) in the immunodeficient is the early identification of the causative agent especially in patients who lack clinical evidence of the disease. The standard methods for the detection of fungi in clinical specimens are direct microscopy and mycological culture. Microscopy often lacks a satisfactory sensitivity, whereas diagnosis by mycological culture often requires a long growth period. Studies have demonstrated the feasibility of detecting molds and yeast in a single reaction using the universal fungal primer. Objective: Evaluation of the role of real-time PCR in the early detection of fungal infection in immunodeficient patients with suspected IFI, who lack clinical evidence of the disease. Methods: This study included 30 immunodeficiency patients suspected of having IFI; 9 with primary and 21 with secondary immunodeficiency. All patients had at least one host factor, but no clinical criteria according to the EORTC-MSG definition of IFI. Twenty seven had fever and 3 had bronchopneumonia, both not responding to broad spectrum antibiotics for 96 hrs. or more. Blood samples were cultured for fungi and were analyzed with real-time PCR using universal fungal primers. For positive samples of fungal infection, aspergillus-specific primers were used for detection of aspergillus. Results: Seventeen patients (56.7%) proved to have IFI. Blood culture detected Candida in 2 patients only, while PCR detected Candida in another 9 and Aspergillus in 6, thus 15/17 patients with IFI (88%) were missed by blood culture. Blood culture for IFI diagnosis had a very low sensitivity (12%) but had a 100% specificity and positive predictive value. The results PCR did not vary with gender, degree of fever, immunodeficiency type, clinical presentation or current intake of antifungal treatment. Patients with proven IFI showed significantly increased CRP levels as compared to those without infection. Conclusion: Real-time PCR proved superior to culture in early diagnosis of IFI in patients with immunodeficiency before the appearance of the characteristic clinical and imaging signs. Reliance on blood culture alone at that stage would result in missing most of the positive cases with consequent delay in the initiation of specific treatment.

Keywords: Invasive fungal infection, immunodeficiency, blood culture, real-time PCR, candida, aspergillus

Egypt J Pediatr Allergy Immunol 2012;10(2):67-74