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Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden
M Chabé1, S L Vargas2, I Eyzaguirre2
1Ecology of Parasitism, EA-3609-IFR17, Pasteur Institute of Lille, 1 rue du Professeur-Calmette BP245, 59019 Lille, France.
Abstract:
Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived PNEUMOCYSTIS: Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.
Insights
Researchers identified Pneumocystis jirovecii in sudden infant death (SID) victims using an optimized DNA extraction method from lung tissue. This study clarifies the Pneumocystis species linked to SID in Chile.
Area of Science:
- Medical Microbiology
- Pathology
- Molecular Biology
Background:
- Histological studies suggest a link between Pneumocystis infection and Sudden Infant Death Syndrome (SIDS).
- Identifying the specific Pneumocystis species in Sudden Infant Death (SID) victims is crucial for understanding its role.
Purpose of the Study:
- To determine the species of Pneumocystis in Chilean Sudden Infant Death (SID) victims.
- To develop and optimize a DNA extraction method for Pneumocystis detection in formalin-fixed paraffin-embedded (FFPE) lung tissues.
Main Methods:
- Optimized the QIAamp DNA Isolation from Paraffin-Embedded Tissue method for FFPE lung sections.
- Validated DNA extraction using rat-derived Pneumocystis and PCR amplification of the mitochondrial large subunit rRNA gene.
- Applied the optimized method to FFPE lung tissue sections from Chilean SID victims, followed by DNA sequencing.
Main Results:
- Successfully extracted and amplified Pneumocystis DNA from FFPE lung tissue sections.
- Confirmed the presence of Pneumocystis carinii in rat models.
- Identified Pneumocystis jirovecii in all tested Chilean SID victims' lung tissue samples.
Conclusions:
- An efficient protocol for isolating PCR-ready DNA from FFPE lung tissues was established.
- Pneumocystis jirovecii was identified as the species present in the lungs of Chilean SID victims.
- This research contributes to understanding the etiology of Sudden Infant Death Syndrome.
