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In situ detection of Aspergillus 18S ribosomal RNA in invasive pulmonary aspergillosis
M Kobayashi1, H Sonobe, T Ikezoe
1Third Department of Internal Medicine, Kochi Medical School, Nankoku.
Objects:
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.
Materials:
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).
Conclusion:
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.
Insights
In situ hybridization (ISH) effectively diagnoses Aspergillus pulmonary infections using a specific probe. This method aids in differentiating Aspergillus from other fungi in lung tissue, crucial for invasive pulmonary aspergillosis (IPA).
Area of Science:
- Medical Mycology
- Pulmonary Pathology
- Diagnostic Microbiology
Background:
- Invasive pulmonary aspergillosis (IPA) diagnosis can be challenging.
- Accurate identification of Aspergillus species is critical for effective treatment.
- Existing diagnostic methods may lack specificity or sensitivity.
Purpose of the Study:
- To evaluate the utility of in situ hybridization (ISH) for diagnosing Aspergillus pulmonary infections.
- To assess the specificity of ISH in differentiating Aspergillus from other fungal pathogens in lung tissue.
- To explore the role of ISH combined with immunohistochemistry (IHC) in understanding host-pathogen interactions in IPA.
Main Methods:
- Developed a polymerase chain reaction (PCR)-based, digoxigenin-labeled probe targeting Aspergillus fumigatus 18S ribosomal RNA.
- Applied ISH technique using low-viscosity (LV) and high-viscosity (HV) hybridization buffers on formalin-fixed, paraffin-embedded lung tissues.
- Studied 12 cases of autopsy-confirmed IPA and 18 control cases with other pulmonary infections. Performed dual staining with ISH and IHC (anti-NE, anti-CD68).
Main Results:
- ISH with LV buffer correctly identified Aspergillus hyphae in all 12 IPA cases, while HV buffer identified them in 6 cases.
- ISH demonstrated Aspergillus elements within abscess centers (LV) and peripheries/blood vessels (HV), showing high specificity against other fungi.
- Immunohistochemistry revealed CD68+ cells in abscess centers and NE+ cells at the periphery, indicating distinct inflammatory responses.
Conclusions:
- In situ hybridization (ISH) is a valuable tool for the specific diagnosis of Aspergillus pulmonary infections.
- ISH, particularly with low-viscosity buffer, offers high sensitivity and specificity in detecting Aspergillus in lung tissues.
- Combined ISH and IHC provide insights into the host inflammatory response and aid in differentiating Aspergillus from other fungal infections.