Typhoid and invasive salmonellosis 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 Salmonella meetings 2 26.9 million cases Typhoid fever 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 C - reactive protein (mg/L) protein reactive 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 100 C) o 38 80 60 37 Oral Temperature ( Oral Temperature reactive protein (mg/L) protein reactive - 40 C 36 20 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 related quinolone spot for a hot is H58 50 H58 non-H58 40 s e 30 t a l o s i f o % 20 10 0 I * L F F F Y Y Y K N R N G 0 Y 5 3 4 6 3 4 7 4 5 7 0 4 8 7 6 8 6 1 8 6 8 8 6 8 2 8 S 8 4 4 4 H58 4 S 4 4 S E D E D L D S S Q C D Q A A r A C A C r r r E r A r B r B B a B E y r r y r r r y y a r a r p g y y a g y y g g p y p a g g p g g g p Amino acid substitution 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 Zambia 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 Malawi 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 77 2010-11 2008-9 Tanzania 0.003 (2008-9) South Africa (2010-12) 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 % of Typhi which S. are MDR H58 10 0 2.3.2 0 0 2.3.6 4 2.5.1 Month 3.4.1 0 3 4.1.1 Salmonella typhi Salmonella4.3.1 enteritidis Salmonella Typhimurium % of STy MDR 0 2 0 1 0 2004 2006 2008 2010 2012 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 Malaria, anemia, malnutrition, HIV (Children) Southern Africa HIV (adults) Human to human transmission? Sam Kariuki Rob Kingsley Chinyere Okoro25 S.