In situ detection of Aspergillus 18S ribosomal RNA in invasive pulmonary aspergillosis.
OBJECTIVE
We attempted to evaluate the usefulness of in situ hybridization (ISH) in the specific diagnosis of Aspergillus pulmonary infection.


METHODS
We used an ISH technique using a multiple digoxigenin-incorporating probe, which was constructed by means of the polymerase chain reaction (PCR) from the 18S ribosomal RNA of Aspergillus fumigatus.


METHODS
We studied twelve formalin-fixed, paraffin-embedded lung tissue sections from autopsy-confirmed 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 disease.), 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 specimens from subjects with IPA, while ISH using the probe and a high viscosity hybridization buffer solution (HV) positively stained the hyphal elements in 6 of 12. Specifically, ISH (LV) demonstrates hyphal elements of Aspergillus spp. in the center of Aspergillus abscess. While, ISH (HV) can detect hyphal elements located in the periphery of a suppurative abscess as well as those in the blood vessel. Conversely, ISH did not show positive results for any of the autopsy tissue specimens from subjects with other fungal pneumonia infections (Candida n=5, Mucor n=2, Cryptococcus n=2, and Pseudallescheria n=1), Pneumocystis carinii pneumonia (n=5), and cytomegalovirus pneumonia (n=3). Dual staining by means of ISH and immunohistochemistry (IHC) using anti-neutrophil elastase (NE) and anti-CD68 monoclonal antibodies showed that NE positive cells were localized at the edge of the radial growth of the 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 the cases (6/12). In contrast, CD68 positive cells were routinely present in the center of the abscess (12/12).


CONCLUSIONS
ISH in conjunction with IHC is a useful tool for differentiating Aspergillus spp. from other fungal genera in tissue sections from patients with IPA and may have a certain role in the evaluation of the interactions between organisms and recruiting inflammatory cells.
Kobayashi
M
M
Third Department of Internal Medicine, Kochi Medical School, Nankoku.
Sonobe
H
H
Ikezoe
T
T
Hakoda
E
E
Ohtsuki
Y
Y
Taguchi
H
H
D000328:Adult; D001228:Aspergillosis / Q000175:diagnosis* / Q000382:microbiology*; D001232:Aspergillus fumigatus / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D008172:Lung Diseases, Fungal / Q000175:diagnosis* / Q000382:microbiology*; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000235:genetics* / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D014783:Viscosity
D017931:DNA Primers; D012331:RNA, Fungal; D012337:RNA, Ribosomal, 18S