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大学・研究所にある論文を検索できる 「Molecular epidemiology of spotted fever group rickettsiae in Zambia and development of multiplex LAMP for simultaneous detection of the rickettsiae and malaria parasites」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

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Molecular epidemiology of spotted fever group rickettsiae in Zambia and development of multiplex LAMP for simultaneous detection of the rickettsiae and malaria parasites

Moonga, Lavel Chinyama 北海道大学

2021.03.25

概要

紅斑熱群リケッチアは吸血性節足動物が保有する細菌群であり、そのうち一部の種がヒトに感染し、発熱や発疹などの症状を引き起こす。これまでにマダニによって媒介される疾病がいくつか報告されてきたが、近年ノミが保有するRickettsia felisやR. asembonensisのヒトからの遺伝子の検出例や分離例が相次いで報告され、これらノミ媒介性紅斑熱リケッチアの新興感染症起因菌としての可能性が議論されつつある。しかし、その分布域や自然界における存続様式、ヒトへの病原性についてはほとんど明らかにされていない。特にサブサハラアフリカにおける知見は乏しく、これら地方における感染の実態調査が急務であった。そこで本研究では分子遺伝学的手法を用いて、ザンビア共和国の動物と節足動物、ヒトにおける感染率調査を行い、ノミ媒介性紅斑熱群リケッチア症の流行疫学の基礎的知見を得ることを目的とした。加えてリケッチア症はマラリアと類似した症状を示すため、マラリアと紅斑熱群リケッチアを鑑別診断可能な迅速診断法の開発を目指した。

 第一章では、ザンビア共和国各地より収集したノミ・蚊・犬血液・げっ歯類臓器より抽 出したDNAを試料として、紅斑熱群リケッチア特異的なPCRを行った。陽性検体について、サンガーシーケンス法、リアルタイムPCR法、および次世代シーケンサーを用いたアンプ リコン解析によって種の同定を行った。その結果、犬血液150検体中7検体(4.6%)、ネコ ノミ48検体中7検体(14.5%)、マストミス属ネズミ77検体中12検体(15.5%)からR. felis の遺伝子が検出された。一方で、R. felisの媒介昆虫として報告のある蚊について172検体調 査したが、本研究では紅斑熱群リケッチアは検出されなかった。さらに、ネコノミでは22 検体(45.8%)からR. asembonensisの遺伝子が検出され、R. felisが陽性であった7検体は 全てR. asembonensisとの共感染が認められた。これらの結果から、ザンビア共和国では犬 やネズミとネコノミの間でR. felisが、ネコノミ間でR. asembonensisの生活環が維持されて いる可能性が示唆された。これは同国においてR. felisとR. asembonensisが分布しているこ とを示した初めての報告であり、ヒトへの感染の有無についての調査が急務であると考えら れた。

 第二章ではノミ媒介性紅斑熱群リケッチアのヒトにおける感染実態調査を行った。調査試料として、ザンビアの首都ルサカとその近郊都市の2箇所で採取した血液検体、および地方部における健常人の血液濾紙検体の合計1,153検体の既存試料を使用した。これらから DNAを抽出した後に、紅斑熱群リケッチア特異的PCRによる検出を行い、陽性検体についてサンガーシーケンス法による遺伝子配列解析を行った。その結果、ルサカの病院で採取された2名の検体が紅斑熱群リケッチア特異的PCRで陽性を示した。これら2検体からそれぞれ4遺伝子の増幅と解析を行ったところ、いずれもR. asembonensisの既報配列と100%の相同性を示した。うち1遺伝子(gltA)は第一章でのネコノミからの配列を含む、アフリカ大陸に固有の一塩基変異を有する配列であった。2名のR. asembonensis陽性患者のうち 1名では貧血と体重減少が認められ、もう1名の臨床症状は不明であった。貧血とR. asembonensis感染との相関は不明であり、R. asembonensisのヒトへの感染性およびその病原性について、さらなる探索が必要であると考えられた。

 第三章では、紅斑熱群リケッチアとマラリア原虫を同時に検出可能な迅速診断法の開発を行った。紅斑熱群リケッチアとヒト感染性マラリア原虫を特異的に検出する2つのLAMP増幅プライマーを混合し、増幅後の反応液を核酸クロマトグラフィーに展開した。本反応系ではマラリア原虫とリケッチアのそれぞれ各100コピー相当の遺伝子を60分以内に検出し、その反応性は培養細菌・原虫を用いた試験によっても確認できた。ザンビアを含むサブサハラ地方は高度なマラリア流行地域であり、症状の類似する紅斑熱群リケッチア症が広く見過ごされている可能性がある。本法はこれら地域において使用可能な、迅速診断法としての普及が期待される。

 以上のように本研究によって、ザンビア共和国にノミ媒介性の紅斑熱群リケッチアが動 物と節足動物内に存在することを初めて明らかにした。そのうちR. asembonensisはヒトに も感染し、疾病を起こしている可能性があることを明らかにした。また、マラリアと同時検 出可能な紅斑熱群リケッチアの迅速診断法を開発した。これらの研究成果は、これまで顧み られてこなかったノミ媒介性の紅斑熱群リケッチアのサブサハラにおける存続様式の解明と、その診断治療に貢献するものである。

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