Suleiman Mzee Saidi

称号及び氏名博士（獣医学）

学位授与の日付

平成２６年２月２０日

論Dynamics of cholera outbreaks in endemic areas of Kenya

文

名(ケニアにおけるコレラアウトブレークの発生動態)

論文審査委員

主査

山崎伸二

副査

笹井 和美

副査

向本 雅郁

論文要旨
Introduction
Cholera is an acute secretory diarrhea, which may lead to loss of body fluids andeventually death. The causative agent of cholera is Gram-negative bacterium Vibrio cholerae.
There are more than 200 ‘O’ serogroups of V. cholerae but only O1 and O139 were responsible
for cholera epidemics and pandemics. Furthermore, V. cholerae O1 is classified into two
important biotypes, classical and El Tor. In the history of cholera, so far 7 cholera pandemics
were recorded. For first to sixth cholera pandemics V. cholerae O1 classical biotype wasthought to be responsible while the ongoing seventh pandemic, which was initiated fromIndonesia in 1961, was caused by O1 El Tor biotype. The seventh pandemic El Tor strains
reached African continent in 1970s. Virulence of V. cholerae associated with cholera isdepended on the production of cholera toxin and toxin co-regulated pilus proteins encoded bythe ctx and tcpA genes, respectively. The other serogroups referred to as non-O1/O139 normallylack ctx and tcpA genes but are associated with sporadic cases of diarrhea. Virulence in
non-O1/O139 is varying and attributed to type three secretion system (T3SS), heat-stableenterotoxin (also called nag-ST), cholix toxin (chxA), etc.
Recently, there has been an emergence of new V. cholerae O1 pathogenic variants which
posses traits of both classical and El Tor prototypes. These variants can be differentiated bygenetic comparative analysis of several genes such as ctxB, tcpA, rstC, rtxC, etc. The O1 El Tor
variant has been shown to be more virulent than the El Tor prototype and has been reported

from several Asian countries and a few from African. The highest number of cholera outbreaks
occurred in countries surrounded by the Bay of Bengal, which was considered the homeland ofcholera. However, since the beginning of the millennium, Africa has reported more than 90% of
the total global cholera cases and cholera related deaths, suggesting that cholera may havesettled in Africa. Kenya is one of the African countries, which experienced the increase ofcholera outbreaks. There is inadequate information on the dynamics of cholera in Kenya related
to continuous isolation of V. cholerae, the role of food handlers and index cases, phenotypic andgenotypic changes of isolates from consecutive cholera outbreaks as compared to studies carriedout in Asian endemic countries and more recently in Haiti. There is paucity of information on
the new pathogenic V. cholerae O1 El Tor variants in Africa including Kenya. Antimicrobials
are used to reduce transmission of V. cholerae and duration of diarrhea. However the increase ofmultidrug resistant V. cholerae strains has threatened the effective use of the recommended
antimicrobials such as tetracycline. Lack of systematic research on antimicrobial resistance hascreated gaps in dealing with cholera outbreaks in Africa including Kenya.
In this study, epidemiological survey of cholera endemic areas of Kenya was carried out
prior to the new millennium and between 2007 and 2009. Prevalence of V. cholerae wascompared with that of Salmonella spp. and Shigella spp. in patients with diarrhea, food handlers
and cholera index cases. By adopting new typing schemes V. cholerae isolates were identified
to their atypical levels. Prevalence of antimicrobial resistance in V. cholerae isolates wasdetermined. Molecular traits, presence of virulence factors and clonal relatedness of V. cholerae
isolates were assessed. The findings of this study illustrates that cholera in Kenya have notreached endemicity levels like those found in Asian countries, however serotype switching,extended multidrug resistance and replacement of V. cholerae O1 El Tor prototype by the V.
cholerae O1 El Tor variant have been observed.

Chapter 1. Epidemiology of cholera in endemic areas of Kenya
Kenya like other African countries experienced an increase in cholera outbreaks from theyear 2000 with surge in 2007 and 2009 and significant case fatality rate (CFR). In 2007, 1,206
cases were reported with CFR of 5.56%, whereas 11,475 cases were reported in 2009 with CFRof 2.31%. The highest CFR of 11% was reported in a cholera outbreak in 2008. Previous studies
focused on characterization of V. cholerae O1 isolates obtained from various outbreaks inKenya or risk factors involved in transmission. A holistic study to gauge the dynamics ofcholera has not been carried out in Kenya. This study was conducted to investigate
systematically the dynamics of cholera in endemic areas of Kenya in consecutive outbreaks.
The prevalence of enteropathogens in particular V. cholerae, Salmonella spp. andShigella spp. was studied in patients with gastroenteritis, food handlers, cholera index cases andcholera patients. The study areas were coastal regions of Malindi and Kwale, and an inland lake
region, Nyanza. Follow up studies in Nyanza were discontinued because of security problems
following a disputed presidential election. Among the 862 patients with diarrhea from theMalindi region between 1991 and 1993, V. cholerae was not isolated although Salmonella spp.
and Shigella spp. were isolated at a rate of 7.3% and 6.5%, respectively. A total of 3,006
specimens from Kwale were analyzed for enteropathogens between 2007 and 2009. The rate ofisolation of enteropathogens from the patients was Shigella spp. (11.0%), Salmonella spp.
(6.9%), V. cholerae O1 (2.8%), V. cholerae non-O1/O139 (0.9%) and Aeromonas spp. (0.1%),
while no pathogen was detected in 1,257 specimens (78.3%). Unlike Salmonella spp. andShigella spp., V. cholerae were isolated only during the cholera outbreak periods in 2007 and2009. V. cholerae O1 Ogawa and non-O1/O139 were isolated in 2007, whereas only V.
cholerae O1 Inaba was isolated in 2009. A total of 1,402 stool specimens from the foodhandlers analyzed yielded Salmonella spp. (1.6%) and Shigella spp. (0.7%). whereas 1,369(97.7%) were negative. V. cholerae was not isolated from the food handlers. Eighty-one V.
cholerae isolates were obtained from Nyanza in 2008 cholera outbreak. Nine cholera index
cases were investigated for V. cholerae. Six were positive for V. cholerae O1, two yielded
Aeromonas spp. and Salmonella spp. was isolated from one case.
Although Salmonella spp. and Shigella spp. were isolated throughout the study period, V.
cholerae was only isolated during outbreak periods. Misdiagnosis among index cases wascommon. Food handlers may not play an important role in transmission of cholera. This study
reports for the first time the serotype switch in V. cholerae from Ogawa to Inaba during two
consecutive cholera outbreaks not only in Kenya but also in the rest of Africa. It is important toconsider serotype switch when adopting new cholera vaccines. Cholera cases may haveincreased in Africa but more studies are required to confirm whether V. cholerae is establishedin this region. The dynamics of cholera outbreaks in Kenya are different from that observed in
Asian endemic countries.

Chapter 2. Prevalence of antimicrobial resistance in Vibrio cholerae isolates
Prevalence of multidrug resistant V. cholerae O1 has been increasingly recognized. The
guideline for antimicrobial use has been adopted mainly from studies carried out in endemic
Asian countries. Yet, one of the earliest reports on tetracycline resistant V. cholerae O1emanated from Africa. With the number of cholera outbreaks increasing in Africa then there is aneed to systematically monitor antimicrobial resistance among V. cholerae. This study wasdesigned to map prevalence of antimicrobial resistance among circulating strains from thedifferent endemic regions of Kenya and monitor development of resistance in consecutive
cholera outbreaks. A total of 130 V. cholerae isolates were analyzed for antimicrobial
susceptibity to 12 antimicrobial agents including trimethoprim sulfamethoxazole (SXT),
fosfomycin (FOF), ampicillin (AMP), imipenem (IPM), chloramphenicol (CHL), tetracycline(TET), norfloxacin (NOR), nalidixic acid (NAL), streptomycin (STR), kanamycin (KAN),
cefotaxime (CTX) and ciprofloxacin (CIP). The V. cholerae O1 isolated in Nyanza, 2008 and inKwale, 2007 were more susceptible to the tested antimicrobials. With the exception of a few
Nyanza isolates all these were susceptible to FOF, AMP, IPM, CHL, TET, NOR, KAN, CTX
and CIP. Most of the Nyanza isolates (88%) were resistant to CHL.
Interestingly, V. cholerae O1 isolated in Kwale, 2009 exhibited the highest level ofmultidrug resistance. The strain was resistant to 9 out of the 12 antimicrobials tested, with theexception of TET, NOR and CIP. This level of antimicrobial resistance in V. cholerae has notbeen reported previously from Kenya. Similarly the study presents first cholera outbreak inKenya caused by a toxigenic V. cholerae O1 showing resistance to third generation
cephalosporin. V. cholerae non-O1/O139 isolated in Nyanza, 2008 was susceptible to all
antimicrobials except AMP. On the other hand V. cholerae non-O1/O139 isolated in Kwale,2007 were susceptible to all the antimicrobials tested except for SXT and NAL. Multidrug
resistance pattern to the antimicrobials varied within the different endemic regions as well as in
the year of isolation of V. cholerae. It is important to carry out antimicrobial susceptibility test in
the course of each cholera outbreak and within each region in Kenya. The use of cephalosporins is
not recommended as an alternative to tetracycline. A larger national systematic study is required
to develop local policy on antimicrobial use for cholera management.

Chapter 3. Molecular characterization of Vibrio cholerae strains associated withcholera outbreak
New pathogenic variant of V. cholerae O1 have spread in not only Asia but also Africa.
However, only few African countries have reported emergency of the new variants. In Kenya
the prevalence of O1 El Tor variant has not been determined. This study proposed that one of
the contributing factors to the severity of cholera outbreaks in Kenya was due to the more
toxigenic V. cholerae O1 El Tor variant. As such molecular characterization of V. cholerae O1
isolated from outbreaks was carried out by employing several specific PCR, multiplex PCR andmismatch amplification (MAMA) PCR targeting rfb, ctxB, rstR, rstC, tcpA and toxR genes for V.
cholerae O1 and colony hybridization targeting heat stable toxin (nag-ST), T3SS related (vcsC2,vcsN2 and vopF) and chxA genes for V. cholerae non-O1/O139. All the V. cholerae O1
possessed the El Tor rstR and tcpA genes, rstC gene and all harbored the classical type ctxBallele. This study suggests that the O1 El tor variant has replaced the O1 El tor prototype inKenya. The V. cholerae O1 isolates were negative for nag-ST, T3SS related and chxA genes
except the only V. cholerae non-O1/O139 from Nyanza/2007 isolate which was positive forchxA gene. Analysis of NotI restriction enzyme digested PFGE profile of V. cholerae O1 strains
isolated from Nyanza province revealed that they were most likely of clonal origin. The PFGE
patterns of Kwale V. cholerae O1 isolates showed subtle differences with an insertion of asingle band (~90kb). Interestingly, the V. choleraeO1 from Kwale/2009 seems to be moreclosely related to the V. cholerae O1 strains isolated from Nyanza/2007.

Conclusions
V. cholerae O1 El tor variant has replaced the V. cholerae O1 El tor prototype inKenya and may be associated with severity of the disease. We are reporting for the firsttime in Kenya, serotype switch and replacement of less resistant strains by multidrug
resistant strains in the same area of Kenya during consecutive outbreaks. Unlike
endemic areas in South Asia V. cholerae is isolated only during outbreak periods andfood handler may not play a key role in transmission of V. cholerae. Cholera outbreaks
in different endemic areas in Kenya can be caused by V. cholerae O1, which share close
clonal relatedness. Cholera outbreak has increased in Africa but it has not reached toendemicity levels found in Asia.

審査結果の要旨
コレラは現在においても、南アジアやアフリカで猛威を振るっており、小児を中心に多くの命が失われている重要な感染症である。現在コレラ菌には 200 種類以上の O 血清型が知られているが、コレラの原因となるのは O1 と O139 コレラ菌のみである。一方、O1 と O139 以外のコ

レラ菌を一括して non-O1/non-O139 コレラ菌と呼び、重症下痢症患者から分離されるが、いわゆるコレラの原因とはならない。O1 コレラ菌は生物学的性状の違いから 2 種類に分類される。
1 つは第 5 次と 6 次の世界流行に関わり致命率が高い古典型、もう 1 つは現在も続く第７次の世界流行に関わり致命率は高くないが環境中での生存率が高いエルトール型である。O1 及び O139 コレラ菌の病原因子として、コレラ毒素（Cholera toxin: CT）や Toxin co-regulatedpilus (TCP) がある。通常、non-O1/non-O139 コレラ菌は、ctx や tcpA 遺伝子を持たず、３型分泌装置（T3SS）、耐熱性溶血毒（NAG-ST）、コリックス毒素（ChxA）等が病原因子として知られている。さらに近年、エルトール型と古典型の両方の性質を持ち合わせ、より病原性の強いハイブリッド型の O1 コレラ菌エルトールバリアントが出現し大きな問題となっている。今世紀に入る前はほとんどのコレラがベンガル湾周辺国で発生していたが、21 世紀に入り世界で発生するコレラの 90%以上はアフリカで起こっている。ケニアはアフリカの中でも最もコレラの発生が多い国の１つである。しかしながら、ケニアでのコレラの疫学情報や薬剤感受性を含む分離菌の細菌学的性状に関する情報はほとんどない。
申請者は、ケニアにおけるコレラの発生動態及び患者から分離されたコレラ菌の細菌学的性状を明らかにすること目的に、1) 1990 年前半と 2007 から 2009 年のケニアにおけるコレラ様下痢症の疫学的調査を行い、2) 分離されたコレラ菌の薬剤感受性、3)コレラ菌の病原因子プロファイルを含む分子疫学的解析を行った。
第１章では、コレラ様の下痢症が流行しているインド洋に面したマリンディとクワレ、ビクトリア湖に面したニャンザで疫学調査を行った。マリンディでの 1991 から 1993 年の調査では、下痢症患者からサルモネラ (7.3%) や赤痢菌 (6.5%) は分離されたがコレラ菌は分離されなかった。
2000 年代に入ると、下痢症の致命率が一気に上昇し、2007 年には 5.6%、2009 年は 2.3%、2008 年には 11%となった。2007 から 2009 年にクワレで行った調査では、赤痢菌 (11%)、サルモネラ (6.9%)、O1 コレラ菌 (2.8%) が主要な下痢原因菌として分離された。サルモネラや赤痢菌と異なり O1 コレラ菌はアウトブレーク時以外には分離されなかった。また、2007 年は稲葉型が流行し、2009 年は小川型が流行した。コレラ菌の血清型変換はケニアのみならずアフリカで初めての報告である。調理従事者からは O1 コレラ菌は分離されず、ケニアでは南アジアと異なり調理従事者がコレラの感染源となっていないことを明らかとした。
第２章では、コレラ様アウトブレーク時に分離された O1 コレラ菌の薬剤感受性について調べた。コレラの治療の第一選択薬として一般的にテトラサイクリンが挙げられる。しかし、アフリカにおいてテトラサイクリン耐性の O1 コレラ菌が原因のコレラアウトブレーク時にテトラサイクリンを使用したことで、多くの犠牲者を出した苦い経験がある。そこで、適切な抗菌薬の選択を行えるよう、分離した O1 コレラ菌 139 株について 12 種類の抗菌薬に対する薬剤感受性を調べた。その結果、2007 年にクワレ、2008 年にニャンザで分離した O1 コレラ菌は調べたほとんど全ての抗菌薬に感受性であったが、2009 年にクワレで分離した O1 コレラ菌はテトラサイクリン

とニューキノロン系の 3 種類を除く 9 種類で耐性を示す多剤耐性菌であった。また、薬剤耐性パターンは分離された年や都市で異なっていることを明らかとした。
第３章では、コレラ様アウトブレーク時に分離されたコレラ菌の病原因子プロファイルについて解析した。その結果、全ての O1 コレラ菌が ctx 遺伝子を保持し、ctx 遺伝子はエルトール型でなく古典型であった。その他は、エルトール型の性状を示す病原因子及び病原因子関連遺伝子を保持していた。よって、2007 年以降ケニアで発生したコレラ様アウトブレークは、いわゆる典型的なエルトール型 O1 コレラ菌によるものでなく、O1 コレラ菌エルトールバリアントによるものであることを明らかとした。一方、コレラ様アウトブレーク時に分離された
non-O1/non-O139 コレラ菌の 1 株で、近年新たな病原因子として見いだされた chxA II 遺伝子が陽性であった。それぞれのアウトブレークで分離された O1 コレラ菌の PFGE パターンの解析から、それぞれのアウトブレーク株はクローナルであること、また 2009 年のクワレ株が 2007年のニャンザ株と類似していることを明らかとした。
本研究において、ケニアでのコレラアウトブレークはエルトール型 O1 コレラ菌でなく、南アジアで流行し重症化率の高い O1 コレラ菌エルトールバリアントが原因であることを初めて明らかにした。また、ケニアのコレラアウトブレークで稲葉型から小川型への血清型変換が起こったことや薬剤感受性菌が多剤耐性菌に置き換わったことも初めて示した。コレラの流行時以外にコレラ菌は分離されず、調理従事者がコレラの感染に関わっていないことやアフリカにおけるコレラ流行様式は南アジアとは異なることを明らかにした。本研究で行なったコレラの疫学研究は、アフリカにおける致命率の高いコレラの原因の一端を明らかとするものであり、獣医学領域のみならず医学領域にも大きく貢献するものと評価できる。本論文の審査および学力確認の結果をあわせて博士（獣医学）の学位を授与することを適当と認める。