In situ detection of Aspergillus 18S ribosomal RNA in invasive pulmonary aspergillosis

Objects We attempted to evaluate the usefulness of in situ hybridization (I SH) in the specific diagnosis of Aspergillus pulmonary infection. Methods W e used an ISH technique using a multiple digoxigenin-incorporating probe, w hich was constructed by means of the polymerase chain reaction (PCR) from t he 18S ribosomal RNA of Aspergillus fumigatus. Materials We studied twelve formalin-fixed, paraffin-embedded lung tissue sections from autopsy-confirm ed invasive pulmonary aspergillosis (IPA) (5 acute myelocytic leukemias, 2 acute lymphocytic leukemias, 2 chronic myelocytic leukemias, 1 adult T-cell leukemia, 1 non-Hodgkin's lymphoma and 1 chronic obstructive pulmonary dis ease.), and 18 sections from other pulmonary infections as control. Results ISH using the probe and a low-viscosity hybridization buffer solution (LV) positively stained hyphal elements in 12 of 12 autopsy lung tissue specime ns from subjects with IPA, while ISH using the probe and a high viscosity h ybridization buffer solution (HV) positively stained the hyphal elements in 6 of 12. Specifically, ISH (LV) demonstrates hyphal elements of Aspergillu s spp. in the center of Aspergillus abscess. While, ISH (HV) can detect hyp hal elements located in the periphery of a suppurative abscess as well as t hose in the blood vessel. Conversely, ISH did not show positive results for any of the autopsy tissue specimens from subjects with other fungal pneumo nia infections (Candida n=5, Mucor n=2, Cryptococcus n=2, and Pseudallesche ria n=1), Pneumocystis carinii pneumonia (n=5), and cytomegalovirus pneumon ia (n=3). Dual staining by means of ISH and immunohistochemistry (IHC) usin g anti-neutrophil elastase (NE) and anti-CD68 monoclonal antibodies showed that NE positive cells were localized at the edge of the radial growth of t he organism, but CD68 positive cells were located around the center of the abscess. The accumulation of NE positive cells was rarely seen in half of t he cases (6/12). In contrast, CD68 positive cells were routinely present in the center of the abscess (12/12). Conclusion ISH in conjunction with IHC is a useful tool for differentiating Aspergillus spp. from other fungal gen era in tissue sections from patients with IPA and may have a certain role i n the evaluation of the interactions between organisms and recruiting infla mmatory cells.