Specific detection of Aspergillus fumigatus in sputum sample of pulmonary tuberculosis patients by two-step PCR

Abstract

The incidence of life-threatening systemic fungal infections has been increasing in recent years, and the increasing incidence has been correlated with increasing numbers of immunocompromised
patients. We developed a two-step PCR assay that specifically amplifies a region of the 18S rRNA gene that is highly conserved in Aspergillus fumigatus. This assay allows direct and rapid detection of down to 10 fg of Aspergillus fumigatus DNA corresponding to 1 to 5 colony forming unit (CFU) per ml of sputum sample of pulmonary tuberculosis patients. The two primer pairs that produced PCR products
with the highest sensitivity and species specificity were the Aspergillus fumigatus primers AFU7S and AFU7AS, which amplified a fragment of 405 bp, followed by AFU5S and AFU5AS, which produced an
internal fragment of 236 bp. PCR has been shown to be a highly sensitive diagnostic tool for the detection of infectious fungi specimens. Our point to the considerable potential clinical value of this
simple, specific, rapid, and inexpensive PCR assay is for improving the means of early diagnosis of systemic aspergillosis tuberculosis patients.