Assessment of the lightcycler PCR assay for diagnosis of invasive aspergillosis in paediatric patients with onco-haematological diseases S. Cesaro, 1 C. Stenghele, 1 E. Calore, 1 E. Franchin, 2 I. Cerbaro, 2 R. Cusinato, 2 G. Tridello, 1 R. Manganelli, 2 M. Carli 1 and G. Palu ` 2 1 Pediatric Hematology Oncology, Department of Pediatrics, University of Padua, Padova, Italy and 2 Department of Histology, Microbiology and Medical Biotechnology, University of Padua, Padova, Italy Summary A reliable diagnosis of invasive aspergillosis (IA) is hampered by the difficulty in obtaining suitable tissue samples. To evaluate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the LightCycler PCR for the diagnosis of IA, 536 blood samples were collected over a 22-month period from 62 paediatric patients (median age 10 years, range 1–18) considered at risk of IA. The galactomannan antigen (GM) and fungal DNA were assessed on serial blood samples. IA was diagnosed in eight of 62 patients (13%): proven, five, probable, three. Sensitivity, specificity, PPV and NPV of LightCycler PCR varied according to the number of positive samples used to define positivity: 88%; 37%; 17% and 95% for single sample positivity; and 63%, 81%, 33% and 94% for serial sample positivity respectively. The concordance between positivity of LightCycler PCR assay and the diagnosis of IA was 79%. The single positivity of LightCycler PCR assay showed a good sensitivity for the diagnosis of IA in paediatric patients. The high NPV makes LightCycler PCR a promising tool in addition to GM testing to design a strategy of pre- emptive antifungal therapy, although further validation studies are needed. Key words: Paediatric malignancy, invasive aspergillosis, LightCycler PCR, galactomannan antigen. Introduction Aspergillus is a saprophytic fungus whose natural ecological niche is the soil and whose conidia are inhaled into the lower respiratory tract. 1 Aspergillus spp. causes severe and often fatal disease in immunocom- promised hosts. 2,3 The incidence of invasive aspergillosis (IA) appears to be increasing, the annual figures being 7.3–10.5%. 4,5 Early and prompt diagnosis of IA still represents a challenge today because results from culture methods are delayed and biopsy is often hindered by the difficulty in performing invasive procedures in critically ill patients; moreover, clinical and radiological signs do not allow a diagnosis of certainty. 6–8 Recently, the detection of the galactomannan antigen (GM test) in serum has raised much interest as a non- invasive tool for the diagnosis of IA, although the sensitivity and specificity of this test may vary consid- erably according to the patient population and the cut- off level used. 8–10 To date, IA has been less frequently investigated in paediatric patients than in adults, and interestingly, differences have been reported for paediatric IA con- cerning the radiological presentation (frequency of halo sign at lung CT scan) or GM test. 11 Herbrecht et al. found a higher rate of false positive GM results in children than in adults with an overall specificity of 94.8% for the GM test in adults compared with only 47.6% for the group of 42 children analysed. 12 Lately, the determination of fungal DNA in the blood by qualitative and quantitative PCR has shown prom- ising results as an alternative or complementary non- invasive method to the GM test for the diagnosis of IA. Correspondence: Simone Cesaro, Pediatric Hematology Oncology, Depart- ment of Pediatrics, University of Padua, Via Giustiniani 3, 35128 Padova, Italy. Tel.: +39 49 821 3579 ⁄ 1461. Fax: +39 49 821 3510 ⁄ 1462. E-mail: simone.cesaro@unipd.it Accepted for publication 29 January 2008 Original article Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd doi:10.1111/j.1439-0507.2008.01512.x