African Ints Disease

African iNTS disease

Melita Gordon

Malawi-Liverpool-Wellcome Trust (MLW) Clinical Research Programme & University of Liverpool, UK ESCMID eLibrary © by author 2 Salmonella invasive syndromes

Salmonella enterica

Typhimurium Enteritidis Paratyphi Typhi

Non-typhoid, iNTS Typhoid, enteric fever

Immunocompromised Healthy population Extremes of age Pre-school – young adults Zoonotic? Human-restricted 20% case fatality 1% case fatality ESCMID eLibrary © by author African invasive nontyphoidal Salmonella (iNTS) disease

. Burden – how much, and who

. Epidemiology and transmission – humans or animals?

. Host-adaptation of ST313 strains

. Strategies for the future ...... ESCMID eLibrary © by author Burden of disease of iNTS in Africa

Typhoid 33 Rotavirus 119

Tuberculosis 266

Invasive NTS 388

Pneumococcus 412

Malaria 626

DeathsDeaths x1000 pa, x sub 1000-Saharan pa Africa Sub-Saharan Africa ESCMID eLibraryAo et al, Emerg Infect Dis 2015 © by author Incidence of iNTS disease in Africa

Study Details All ages Under 5y Under 2y Under 1y comments /100,000 /100,000 /100,000 /100,000 pyo pyo pyo pyo Ao et al Syst review 227 - - - 68% of cases in under 2015 & global (152-341) 5’s modelling MacLennan RTSS, - 461 776 7-fold > typhoid et al 2017 7 sites (402-526) link to frequent malaria Marks et al TSAP 0-237 291-1733 - Variability between 2017 10 sites sites ESCMID eLibrary © by author 250 NTS in South Africa Unknown gender 200 Male Female 150

100

Case CountCase 50

Age distribution 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81

NTS in Malawi 1000

pyo 800 600 400

Case countCase 200

0 Cases per 100,000 Cases 100,000 per 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81

Age in months Age in years S. Typhi in South Africa 8 7 Feasey et al, 2010 MacLennan et al, 2017 6 ESCMID 5 eLibrary 4 3

Case CountCase 2 1 0 © by author1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81

S. Typhi in Malawi 7 6 5 4 3

2 Case CountCase 1 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 Age in years 1200 1200 1200

1000 1000 1000 MDR Epidemics 1200 800 800 800

1000 600 600 600 800

400 400 600 400

400 200 200 200 Number cases of Number 200

0 0 0 0

EnteritidisSalmonella Typhi Salmonella Typhimurium Salmonella Enteritidis Salmonella Typhi Salmonella Typhimurium100% EnteritidisSalmonellaSalmonella Enteritidis Typhi Salmonella Typhimurium SalmonellaSalmonella Typhi Enteritidis Salmonella Typhimurium Salmonella Enteritidis 90% Enteritidis Typhimurium Typhi 100% 80% 90% Enteritidis Typhimurium Typhi 100%70%Enteritidis Typhimurium100% Typhi100% 80% 60% 100%70% 80% 100% 80% 100% 80% 60% 50% 80% 60% 80% 60% 80% 60% 50% 40% 60% Proportion 60% 60%

40% 40%30% 40% 40% Proportion 40%30% Proportion 20% 40% 40%

Proportion 20% 20% 20% 20% 20% 10% 20% 20% 10% 0% 0% Proportion Proportion MDR 0% 0% 0% 0%0% 0%

Year Year SusceptibleSusceptibleSusceptibleSusceptibleResistantResistant toResistant 1-2 agents Resistantto to 1-2 MDR1-2 agents agents to 1-2MDR agents MDR = start of epidemic Feasey et al, ESCMID eLibraryClin Infect Dis 2015 © by author Feasey al et Nature Genetics 2016 Okoro ST313 Transmission Transcontinental

ESCMID et Nature al Genetics 2012 eLibrary S. © by author Typhimurium

Median HIV prevalence (%)

Lineage 2 Lineage 1 MDR HIV

Transcontinental Transmission Lineage 1 Lineage ST313 S. Typhimurium

Genomic degradation

Acquisition of accessory genes

Lineage 2 Lineage Extended multidrug resistance

Okoro et al Nature Genetics 2012 Feasey etESCMID al Nature Genetics 2016 eLibrary © by author Human or animal reservoirs of iNTS?

Kariuki et al, Kenya, J Med Micro 2006 127 index invasive disease iNTS 32/467 family contacts carried Salmonella, 65% matched index by PFGE 4 environmental isolates also similar, but very few animal isolates

Dione et al, Gambia PLOS NTD 2011 GEMS 14 NTS cases (8 diarrhoea, 6 healthy controls) 210 household animals (chicken, sheep goat) 21 animal NTS isolates (10%), MLST and serotyping no sequence type overlap between human enteric and animal isolates ESCMID eLibrary © by author Case control study of cases and controls

Visit case Index Case household Conduct identification within 2 weeks Sampling at at QECH Homes Culture and (Typhi + NTS) Select Control identification household by Family bottle-spin and 60 cases 100 yard walk Animals WGS 60 controls Environment URBAN Obtain ESCMIDConsents eLibrary © by author Sampling

• Stool (Human, adults and children) • Animal stool/rectal swabs (chickens, pigs, cows, goats, sheep, cats, dogs, gecko) • EnvironmentESCMID sterile boot sox (perimeter, eLibrary latrine, rubbish, bedroom, cooking areas) © by author Human or Environmental Sample animal stool Enrichment BPW @ 37ᴼC 18 – 24h

Selective Selenite Broth @ 37ᴼC 18 – 24h

XLD @ 37ᴼC 18 – 24h

Colonies ID’d Serology & API 20E

Confirmed Salmonella ESCMID eLibraryfor WGS © by author Total samples Case No. of % Control No. of % 1510 Total Salmonella Positive Total Salmonella Positive Samples Isolated Samples Isolated

Human Stool 273 11 4 282 4 4 Animal Rectal Swab 17 0 0 9 1 11 Animal Stool 32 2 6 34 1 6 Bootsocks 305 6 2 297 24 8

Total 802 19 2.4 708 30 4.2 ESCMID eLibrary © by author Invasive NTS cases S. Typhimurium ST313

“non-invasive” NTS serovars

Illumina sequencing ESCMID eLibraryEnterobase phylogenetic trees © by author ESCMID eLibrary © by author * Environmental **** * samples *** ** * * *** ** *** ** ESCMID eLibrary** © by author Adult stool Child stool

ESCMID eLibrary © by author *Animal isolate *

* * ESCMID eLibrary* © by author * Adult stool ** Child stool ** Animal isolate ** *Boot sox isolates * * * ** * * *** *** ** *** *** ESCMID eLibrary* © by author Household matches between non-invasive strains

healthy humans animals

14 4

healthy humans animals

13 0 2

0 1 2

healthy humans animals 8 0 5 2

0 1 2

healthy humans animals 8 0 3 2 2 ST313 19 0 invasive 1 2 cases

What are the immune defects? Are the bacteria adapted to humans?

Pseudogenised enteric metabolic lifestyle genes Altered microbiota ↓CD4 IL-17 production in HIV ↓PMN recruitment Less colitis Loss of gut barrier function InvasionESCMIDmucosal eLibraryhumoral © by author Innate inflammatory milieu cellular Decreased NfKB in advanced HIV Genetic defects in TH1, IL- 123//17 and IL12/γIFN pathways (including STAT4 in African children) Malaria-dependent ↑ IL10

HIV - loss of CD4 cognate responses

Reduced flagellin expression by ST313 alters inflammation conflicting results? ESCMIDmucosal eLibraryhumoral © by author D23580 ST313 strain vs global LT2 diarrhoeal strain FACS & single cell transcriptomics ST313 more heterogeneous response over time

ST313 bystander MoDCs - PAMPS or engulfed and cleared → hyperactivated state, diverting immune system away

ST313 infected MoDCs → increased IL-10 and MARCH1 and decreased CD83, CD40 and HLA expression – immune evasion

Resulted in reduced activation and IFN-γ production ESCMID by eLibrarySalmonella-specific CD4 clones © by author cellular

Deficient antibody in young children Dysregulated excess anti- LPS antibody in HIV- infected adults Complement depletion in ESCMIDmucosal eLibraryhumoral malaria and malnutrition © by author cellular

Single C→T base-change in noncoding pgtE promotor gene causes serum complement- resistance in African clades ESCMIDmucosal humoral eLibraryHammarlöf et al, PNAS, 2017 © by author Humoral and cellular host defects both targeted by bacterial evolution

High incidence High case fatality Large disease burden – lives, DALYs, economic Antimicrobial resistance - ESBL and fluoroquinolones Vulnerable people Economic, political and climate instability Multiple serovars ESCMID eLibrary © by author Malaria Cells

Relevant vaccine

Young Humoral age

How will we design and evaluate an iNTS vaccine that is effective for susceptible children? Which populations will we give it to? At what age? ESCMIDMultivalent – NTS serovars? eLibrary Typhoid? © by author ESCMID eLibrary © by author Thank you

MALAWI LIVERPOOL OXFORD James Meiring Shaun Pennington Alison Simmons Nick Feasey Paul Wigley Lorena Preciado-Llanes Chisomo Msefula Jay Hinton Andrew Pollard Ben Kumwenda Sian Owen Vincenzo Cerundolo Robert Heyderman Miren Iturriza Gomara Reenesh Prakash SANGER INSTITUTE Leonard Koolman Gordon Dougan Ndaru Jambo Nick Thomson Angeziwa Chunga-Chirambo ESCMIDInnocent Nyirenda eLibrary © by author