Typhoid and invasive 9th Congress

Professor Gordon Dougan Wellcome Trust Sanger Institute Cambridge University

1 Origin of the typhoid and salmonellosis meetings

Carlos Hormaeche Tikki Pang Bernard Ivanoff

Split after first meeting into two, one became ASM meetings 2 26.9 million cases by region Mortality 1% or ~250,000 per annum

Paratyphi A

High incidence (>100 per 100 000 per year)

Medium incidence (10-100 per 100 000 Non-typhoidal per year) This map shows homogeneity Low incidence (<10 per 100 000 per salmonella year) whereas typhoid is a patchwork

Source: Crump et al, Bulletin WHO, 2004 and Buckle et al 2012 3 Salmonella typhi, the bacteria

• A monophyletic serovar of S. enterica that causes typhoid – Only emerged once several thousand years ago! • Produces Vi capsule and novel toxin • Human restricted, – Non-zoonotic! • Carrier state

Typhimurium

Paratyphi A a distinct monophyletic serovar 4 • First controlled challenge model for typhoid for ~50 years • Initially funded by The Wellcome Trust • Quailes strain of S. Typhi recovered from original studies and made at GMP • Investigating the pathogenesis, immunology, vaccinology of Typhi and Paratyphi A

5 A human challenge model for typhoid/paratyphoid

100 3 Fever profile TD 90 1-5x10 80 4 38.5 1-5x10 10-3 70 38.0 10-4 60 50 37.5 40 30 37.0 20 typhoid fever (%)typhoid 36.5

Participants without Participants 10 Proportion affected (%) Proportion affected 0 36.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 -6 -5 -4 -3 -2 -1 TD +1 +2 +3 +4 +5 +6 Day post challenge Day relative to typhoid diagnosis (TD)

• 1-5x103 Attack rate 10 9 Stool 55% 8 Blood culture 7 6 4 • 1-5x10 Attack rate 5 4 65% 3 2 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Still looking for volunteers! isolates of positive number Total 6 Days post challenge The emergence of typhoid symptoms in an individual challenged with Salmonella Typhi 40 180 Typhoid

39

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- reactiveprotein (mg/L)

C) 120 o 38 Differential response Early asymptomatic signature - TranscriptionalCdiffere signature - Cytokines - T cells (Regs) - Transcript?37 Temperature ( Temperature PCR + - Cytokines 60 - Antibody (B cell)

36

35 0 Da 00 11 22 33 44 55 66 7 7 8 8 9 9 1010 1111 1212 1313 1414 15 28 y Blood - - - - + + + - - - - Cx Stool - + - - - - + + + + - Cx QBC 6. CFU/mL 2 Wida 2 4 16 l O Wida N Christoph Blohmke 4 16 l H A 7 More persistent shedding of S. Typhi without symptoms or obvious blood carriage

Da 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Blooy ------d Sto - + - - - - + + + + + + ol 40 140 • How common is this in the field, is this the real typhoid? 120 • Typhoid39 is a stealth infection not always driving disease

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35 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Number days after 8 challenge Shedding of S. Typhi Ty2 ssaV (SPI-2) aroC and S. David Lewis Typhimurium TML ssaV aroC in the stools of volunteers TABLE 1 STOOL CULTURE

S. Typhi Ty2 ssaV aroC Days after oral challenge

Dose Subject ID -7 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 28

044 — — — + — — — — — — — — 107 009 — — — — — — — — — — — — 045 — — — — — — — — — — — — 048 — — + — — — — — — — — — 108 046 — — — — — — — — — — — — 035 — — — — — — — — — — — — 030 — — — — — — — — — — — — 109 023 — — + — + — — — + — — — 057 — BNO — BNO — — BNO BNO — — — —

008 — — + — + — — — — — — — 107 003 — — + — — — — + + — — — 010 — — — — + + + + + — — — 029 — — — + + + + + + + + + + + — — — — — — — — 108 011 — — — + + + + — + — — — — — — — — 050 — — — + + + + + + + + + — — + + — — — — — — — 012 — — + + + + + + + + + — — + + — — + — — + — — 109 062 — — + + + — — + — + + + + + + — + + — — — — — — — 063 — — + + + + + + + + + + + — — — + + + + + — — S. Typhimurium TML ssaV aroC

ACUTE PHASE PROTEINS UP ONLY IN - No Shedding + Shedding TYPHIMURIUM VOLUNTEERS 9 Theoretical differences in the pathogenicity of zoonotic non- typhoidal salmonellosis and typhoidal disease

Non-typhoidal Salmonella Salmonella Typhi

Luminal replication Shedding from and shedding gallbladder

M M

T Gallbladder Inflammation B T B Neutrophil infiltrate

Limited dissemination

10 Proposal at the last invasive Salmonellosis meeting in Dhaka, Bangladesh, 2013 • We form an international consortium to map and genotype S. Typhi/Paratyphi A across the world • We create a central web site based on free software to co- ordinate this • We design simple SNP-based assays for field testing • We use this to advocate typhoid control

Isolate organisms Sequence genomes Phylogenetic analysis

11 Organization of a global collection of S. Typhi for genome sequencing

2,000+ isolates 63+ countries 6 continents

12 Phylogeographic analysis of the dominant multidrug resistant H58 clade of Salmonella Typhi identifies unappreciated inter- and intra-continental transmission events

Vanessa K. Wong, Stephen Baker, Derek Pickard, Julian Parkhill, Andrew J. Page, Nicholas Feasey. Robert A. Kingsley, Nicholas R. Thomson, Jacqueline A. Keane, François-Xavier Weill, David J. Edwards, Jane Hawkey, Simon R. Harris, Alison E. Mather, Amy K. Cain, James Hadfield, Peter J. Hart, Nga Tran Vu Thieu, Elizabeth J. Klemm, Robert F. Breiman, Conall H. Watson, Samuel Kariuki, Melita Gordon, Robert S. Heyderman, Chinyere Okoro, Jan Jacobs, Octavie Lunguya, W. John Edmunds, Chisomo Msefula, Jose Alejandro Chabalgoity, Mike Kama, Kylie Jenkins, Shanta Dutta, Florian Marks, Josefina Campos, Corinne Thompson3, Stephen Obaro, Calman A. MacLennan, Christiane Dolecek, Karen H. Keddy, Anthony M. Smith, Christopher M. Parry, Abhilasha Karkey, E. Kim Mulholland, James I. Campbell, Sabina Dongol, Buddha Basnyat, Muriel Dufour, Don Bandaranayake, Take N. Toleafoa, Shalini Pravin Singh, Mochammad Hatta, Paul Newton, Robert S. Onsare, Lupeoletalalei Isaia, David Dance, Viengmon Davong, Guy Thwaites, Lalith Wijedoru, John A. Crump, Elizabeth De Pinna, Satheesh Nair, Eric J. Nilles, Duy Pham Thanh, Paul Turner, Sona Soeng, Mary Valcanis, Joan Powling, Karolina Dimovski, Geoff Hogg, Jeremy Farrar, Kathryn E. Holt† and Gordon Dougan Nature Genetics in press this week 13 S. Typhi Haplotype H58 has emerged over the past 20 years to dominate globally

AFRICA

OCEANIA

Ancestral root

ASIA H58 151 SNPs H58 cluster SOUTH Neighbouring ~150 SNPs AMERICA non-H58 cluster

14 H58 is associated with drug-resistant genotypes

H58

15 H58 is a hot spot for quinolone related mutations

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16 Like all Typhi, H58 is still undergoing genome streamlining, through transposition

Multidrug resistance transposon trimethoprim-sulfamethoxazole

Genomic signature of multidrug-resistant Salmonella enterica serovar typhi isolates related to a massive outbreakchloramphenicol in between 2010ampicillin and 2012. streptomycin Hendriksen RS, Leekitcharoenphon P, Lukjancenko O, Lukwesa-MusyaniHolt C, Tambatamba2007, J Bacteriol B, Mwaba J, Kalonda A, Nakazwe R, Kwenda G, Jensen JD, Svendsen CA, Dittmann KK, Kaas RS, Cavaco LM, Aarestruptransposition FM, Hasman H, MwansaTyphi cell JC. J Clin Microbiol. 2015 Jan;53(1):262-72

mutation

conjugation chromosome

IncHI1 plasmid 17 Holt 2011, PLoS Negl Trop Dis The dissemination of S. Typhi H58

Emerged ~30 years ago

18 Global dissemination of S. Typhi H58 as a timeline

India Africa

Kenya

Tanzania Kenya SE Asia

Africa

19 Phylogenetic relationship of African isolates within the H58 tree provides evidence of a recent expansion/epidemic

Key Kenya Tanzania Malawi

83 South Africa Africa 66 2004-9 58 Kenya (2004-9)

91 2008-9 Tanzania 2012 (2008-9) 96

66 2005 Malawi Malawi 2011-13 2012 (2011-13

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Need to fill in the gaps 20 Salmonella BSI at QECH, November Genotypes in 2010-August 2014 120 100 90 Malawi 100 80 70 80 60

60 50 /month 40 Caes 40 30 20 20

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21 Why is the H58 lineage so successful?

• Is it just antimicrobial resistance ? (what about other AMR lineages which are less successful) • H58 has a unique SNP repertoire with some interesting mutations e.g. effectors • How do you assess fitness in a host restricted pathogen? • Human challenge? • More in depth molecular clinical analysis

22 S. Typhi in the Pacific region are generally island-specific

Is this an opportunity for an eradication programme?

H58 not dominant yet

23 How did typhoid evolve?

• Single point of origin followed by global dissemination

• Human restriction

• Gene acquisition (e.g. Vi and typhoid toxin)

• Genome degradation (~300 pseudogenes)

• Change of niche from gut to systemic system & carriers

24 Invasive non-typhoidal Salmonellosis in Sub-Saharan Africa – does this involve human to human transmission?

Non-typhoidal Salmonella (NTS) common in blood stream infections in sub-Saharan Africa study site Dominant serotypes S. Typhimurium 50-80% S. Enteritiidis 10-40% West Africa

Not typhoid – Distinct syndrome Central Africa Non-specific fever East Diarrhea is infrequent Africa Rapid progression 24-51% mortality without ART

Host factors , anemia, malnutrition, HIV (Children) Southern Africa HIV (adults) Human to human transmission? Sam Kariuki Rob Kingsley Chinyere Okoro25 S. Typhimurium iNTS isolates are from two related clades ST313

! ! Lineage II ! 21 SNPs

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! ! ! ! ! ! ! •>90% of iNTS isolates ! ! ! ! ! ! ! in two clusters not ! ! ! ! found outside of Africa !

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! ! •One cluster is ! ! ! ! ! ! chloramphenicol ! "! ! # resistant 26 Time-dependent phylogeographic analysis using BEAST

Bayesian evolutionary analysis by sampling trees BEAST – integrates temporal, geographic and phylogenetic information

Okoro, Kingsley et al, Nature Genetics, 2012 27 Some S. Typhimurium ST313 have altered enteropathogenicity

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H 0 n n o s s a io ti e e m t a s g e ra tr s n d lt fil e a e fi n c h o in i b c l r r a e a a la t c s l lu p a o llu l ry rf c e Impaired inflammasome ce C u u c l S m l a b sa s u o co S c u u induction m 28 M b u S S. Typhimurium ST313 summary • Two clades emerged to spread across Sub-Saharan Africa associated with the HIV epidemic

• Is the ST313 wave over?

• ST313 have signatures of host adaptation

• ST313 was in Africa before HIV (in primates or children locally?)

• Is there human to human transmission of some sort?

Sam Kariuki was right, journal editors and referees were wrong!

29 Does remarkable pathogen evolution in an IL12/23 b1 receptor deficient individual throw light on how typhoid evolved?

IL12/23 b1 receptor deficient and chronically infected with recurrent S. Enteritidis infection • Age 12 presents with clinically severe bacteremia with Salmonella Enteritidis

• Treated with antibiotics and interferon g

Next decade

• >50 episodes of bacteremia becoming less clinically severe over time • No stool carriage just systemic site • Eventually no signs of chronic infection CRP, albumin, IgG

Bacteria originally typed as Salmonella enteritidis but became increasingly difficult30 Patient isolates show extensive mutation accumulation and genome degradation Maximum likelihood tree

Hypermutator phenotype caused by mutS mutation

DNA binding connector lever clamp lever ATPase

in-frame deletion in patient isolates

31

monomer dimer bound to mis-matched DNA Hallmarks of host-adaptation: genome degradation

Complete pseudogene analysis of four patient isolates sequenced by PacBio Molecular clock rate

Gastrointestinal serovars

Patient isolates

Extra-intestinal serovars

level comparable to host-adapted/restricted serovars 32 Convergent evolution with S. Typhi and S. Paratyphi A

Intestinal metabolome disrupted Adhesins and intestinal shedding genes gone SPI-1 dead SPI-2 stream lined O antigen ligase gone

Pseudogenes by pathway/ locus Patient isolates S. Typhi S. Paratyphi A Central anaerobic metabolism Nitrogen utilization narK narX narG narV narV narW narZ narW Tetrathionate utilization ttrB ttrR ttrS ttrB Vitamin B12 synthesis btuR cbiJ cbiK cbiL cbiN cbiC cbiJ cbiK cbiM cbiA Adhesion non-fimbrial adhesins shdA misL bigA shdA misL bigA shdA fimbrial sef locus sefC sefR sefA sefC sefD sefR sefD sefR fimbrial sth locus sthB sthC sthC sthE Type III secretion SPI-1 effectors sipA sopA sopD slrP sopE2 sopA slrP sopA SPI-2 effectors sseI sseJ sspH2 steC sseK1 steB sopD2 sseJ sseK2 steB sopD2 sseJ sseK2 sifB steC33 What does this study tell us, if anything?

• Microbes can evolve (mutate) at dramatically different rates in different settings • Compromised individuals or cohorts may be sources of adapted microbes. Can we spot adaptation to humans early? • Factors such as antimicrobial treatment are exerting unprecedented selective pressures (we see this in most pathogens)

Elizabeth Klemm 34 We have typhoid vaccines that work…..but….

Vi capsule, Paratyphi A O side chain • Length of polysaccharide ? • Typhi/Paratyphi carrier proteins? Vi-C Single • What is a correlate of protection oral (antibody, bactericidal ? dose

Clinical Clinical Trial Ty21a Project Phase Ty21a Licensed Vi Phase 2, M01ZH09 adults and children Phase 2, Ty800 adults (2008)

Phase I, CVD909 adults (2010) 35 What are the immediate challenges? Can we……

…get conjugate vaccines used effectively? …get any typhoid vaccine used broadly? …get diagnosis working at the bed-side? …track and define carriers more effectively …eradicate typhoid!

Advocacy and driving vaccines to WHO prequalification and licensing 36 The urgent need for new typhoid diagnostics

Metabolomics

PCR Serology, new antigens 37 What is a typhoid carrier in 2015 and where are they!

Gall bladder (2010) Acute typhoid (2004-2008)

38 Wellcome Trust Strategic Award on Typhoid

High incidence (>100 per 100 000 per year)

Medium incidence (10-100 per 100 000 per year)

Low incidence (<10 per 100 000 per year)

39 Acknowledgements Collaborators Collaborators

WTSI, Cambridge Gordon Dougan, Vanessa Wong, IVI, Korea Florian Marks Julian Parkhill, Nick Thomson, Derek Pickard, Robert Kingsley, Pasteur Institute, Paris Francois-Xavier Weill Andrew Page, Chinyere Okaro, Jacqueline Keane, David Harris, University of Otago John Crump Simon Harris, Amy Cain, Alison Mather, Elizabeth Klemm, LSTM Nick Feasey James Hadfield, Dafni Glinos, Rob Kingsley MLWT-CR Programme, University of Malawi Bio21, University of Melbourne Kathryn Holt, David Edwards, Rob Heyderman, Chisomo Msefula Jane Hawkey Institute of Infection & Global Health, OUCRU, Ho Chi Minh City Stephen Baker, Duy Pham Thanh, Melita Gordon Nga Tran Vu Thieu, Corinne Thompson, James Campbell, MDU, University of Melbourne Geoff Hogg, Mary Valcanis, Guy Thwaites, Jeremy Farrar Joan Powling, Karolina Dimovski Centre for Tropical Medicine & Global Health, LSHTM Christopher Parry, Kim Mulholland, W. John Edmunds, Christiane Dolecek, Paul Turner Conall Watson Laos-Oxford-Mahosot Hospital, WT Research Unit, Laos IBR, University of Birmingham Calman Maclennan, Peter Hart Paul Newton, David Dance, Viengmon Davong Centro de Referencia Regional PulseNet, Argentina NICD, South Africa Karen Keddy, Anthony Smith Josefina Campos COMRU, Cambodia Sona Soeng UNMC, University of Nebraska Stephen Obaro PAHS, Kathmandu Sabina Dongol, Buddha Basnyat, KEMRI, Kenya Sam Kariuki, Robert Onsare Abhilasha Karkey Institute of Hygiene, Uruguay Jose A. Chabalgoity NCBID, New Zealand Don Bandaranayake ESR, New Zealand Muriel Dufour Hasanuddin University, Indonesia Mohammad Hatta PHE, Colindale Elizabeth De Pinna, Satheesh Nair MOH, Samoa Take Toleafoa EGHI, Atlanta Robert Breiman MORU, Bangkok Lalith Wijedoru NHS, Samoa Lupeoletalalelei Isaia WHO, Pacific Islands Division Eric Nilles ITM, Belgium Jan Jacobs WHO, Fiji Shalini Singh INRB, DRC Octavie Lunguya MOH, Fiji Mike Kama Fiji Health Sector Support Program, Fiji Kylie Jenkins NICED, India Shanta Dutta 40