Abstract
OBJECTIVE: To investigate potential molecular evidence of the presence of infuenza A in a patient who died in 1912. MATERIALS: Two to three paraffin curls from a formalin-fixed paraffin-embedded (FFPE) lung sample from an autopsy case of a one-and-a-half-year-old girl who reportedly died of acute pneumonia at the Royal London Hospital on February 1, 1912. METHODS: RNA was extracted from the sample. Real-time reverse transcription PCR (RT-PCR) was performed to screen for influenza A virus, followed by total RNA library preparation and high-throughput sequencing on the Illumina NextSeq platform. Bioinformatic analysis was conducted on the obtained reads, which included metagenomic classification and sequence alignment to reference genomes. RESULTS: Real-time RT-PCR and next-generation sequencing both revealed no evidence of influenza A virus infection. However, metagenomic analysis identified a significant number of reads matching Rickettsia felis, with 233 unique reads specifically aligning to its OmpB gene, and over 4 million fungal reads classified within the Mucor genus, although the exact species could not be determined. These findings provide suggestive molecular evidence of co-infection with R. felis and a Mucor species. CONCLUSIONS: This study demonstrates the utility of modern molecular techniques in evaluating early diagnoses. Despite records attributing the cause of death to acute pneumonia, no influenza A virus RNA was detected. The identification of R. felis and Mucor sequences suggests that this rare co-infection may have contributed to the infant's death. SIGNIFICANCE: Retrospective diagnosis is enhanced by the use of modern molecular and bioinformatic techniques. This case study provides important paleopathological insight into the etiology of recorded "acute pneumonia", indicating the presence of pathogens other than influenza A leading up to the 1918 pandemic. LIMITATIONS: Contamination during autopsy, tissue processing, or storage cannot be ruled out. Due to the lack of remaining material, histopathological confirmation of Mucor or Rickettsia infection was not possible. SUGGESTIONS FOR FURTHER RESEARCH: Molecular and bioinformatic research into other cases of suspected influenza A.