A study of rapid diagnosis for invasive pulmonary aspergillosis by molecular biological technique

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We developed a nested PCR method for detecting DNA specific for Aspergillus species in serum samples of animal medels of aspergillosis and patients with invasive pulmonary aspergillosis.1)Nested PCR : Fourteen strains of clinically important fungi and 10 strains of bacteria were used for examination of specificity of the PCR.Two sets of oligonucleotide primers were derived from the sequence of the variable regions V7 and V9 of the 18S rRNA genes of Aspergillus fumigatus. The specific fragment was amplified from five strains of Aspergillus species in the single and nested PCR but not from other microorganisms. Target DNA was detected by the nested PCR with as little as 50 fg of the extracted DNA of A.fumigatus.2)Detection of DNA specific for Aspergillus species in serum of animal models. : We investigated the detection of DNA specific for Aspergillus species in serum of murine and rats with experimental aspergillosis. The specific fragment was detected by the nested PCR in 71% of serum samples of infected mice, while galactomannan antigen was detected in 43% of them. In rats model, we have also shown enhanced sensitivity of the nested PCR over galactomannan antigenemia assay and (1*3) beta-D-glucan assay.Detection of Aspergillus DNA in clinical samples. : The nested PCR was positive for 14 of 20 patients (70%) with a histological diagnosis of invasive pulmonary aspergillosis, while galactomannan antigen was positive 12 (60%).In conclusion, the high sensitivity and specificity of the nested PCR indicate that the assay can provide early diagnosis with sufficient accuracy to be clinically useful for immunocompromised patients with invasive pulmonary aspergillosis.