研究目的は原因微生物が一般的な臨床検査で使用される培養法や核酸増幅法では特定されなかった不明熱原因疾患について、主治医と相談の上で患者同意のもと、無菌的に提出された血液、尿、髄液、組織等から核酸抽出を行い、細菌や真菌の16SリボソームRNAや18SリソボームRNA、その他のハウスキーピング遺伝子に共通するプライマーを用いて細菌や真菌由来の増幅産物の有無を確認する。増幅産物が確認された場合にはサンガー法あるいはナノポアシーケンサーによるシークエンスを行い、得られた配列をBlast等で検索することにより、無菌検体に含まれる病原微生物の同定を行うことである。今年度は該当検体が提出された場合の手順の整理およびPCRを行うための環境整備を行った。
実際には、術後CTにより膿瘍疑いを指摘された患者に対し穿刺を行い、一般細菌の塗抹・培養検査および抗酸菌培養が陰性だった検体について16SリボソームRNAの増幅産物の確認を行った。結果的に増幅産物は得られなかった。
This study aimed to explore unidentified febrile diseases for which the responsible microorganisms remain unrevealed by nucleic acid amplification or culture techniques commonly used in clinical laboratories. Nucleic acid is extracted from blood, urine, spinal fluid, and tissue specimens obtained from the patients after obtaining their consent following consultation with the attending physician. Further, the presence of amplification products derived from bacteria or fungi is confirmed using primers common to 16S ribosomal RNA, 18S lysosomal RNA, and other housekeeping genes in the bacteria or fungi. If the presence of the amplified product is verified, sequencing is performed using a nanopore sequencer or the Sanger method; subsequently, the obtained sequence is investigated using Basic Local Alignment Search Tool (BLAST) or other techniques to identify the pathogenic microorganisms in the sterile specimen. In this fiscal year, processes for sending the necessary specimens and settings for conducting the polymerase chain reaction test were primarily improved.
A general bacterial smear and culture as well as an acid-fast bacillus culture were performed on a puncture fluid specimen obtained from a patient with suspected abscess, but the results were negative. Therefore, in collaboration with the main department and the department of infection control, the amplification product of 16S ribosomal RNA was confirmed to have a therapeutic application. Notably, the results revealed no amplification products. A brain abscess specimen obtained from another patient revealed gram-positive bacillus on Gram staining, but no bacterial growth was seen on the culture; hence, the specimen was further examined to identify medical causes. Direct nucleic acid extraction was performed from the obtained material; subsequently, 16S ribosomal RNA was amplified, examined, and sequenced. A gram-negative bacillus, Porphyromonas gingivalis, was identified after extensive research using BLAST and other techniques. Further, the amplified product was re-examined with primers designed for gram-positive bacilli because the results of the above-mentioned tests were different from those for the target bacteria, but no amplification was noted.
Although only two cases were examined this year, we hope to examine more cases in the upcoming years. Moreover, we hope to develop a test that can help improve medical treatment by identifying more applicable diseases, target regions, and sequencing techniques.