1. A novel ciprofloxacin resistant subclade of H58 Salmonella Typhi is associated with fluoroquinolone treatment failure
Dr. Abhilasha Karkey

2. Study site: Patan Hospital
One of three general hospitals
90% patients are from surrounding Patan area
2003: Clinical assessment of enteric fever patients
Randomised control trials
Epidemiological studies

4. Randomised control trials
Gatifloxacin versus Cefixime: PLoS One 2007 (Stopped by DSMB)
Gatifloxacin versus Chloramphenicol: Lancet Infectious Diseases 2011
Gatifloxacin versus Ofloxacin: PLoS Neglected
Tropical Disease 2013
Gatifloxacin versus Ceftriaxone: Lancet Infectious Diseases 2016 (Stopped by DSMB)
Azithromycin versus Chloramphenicol
Because treatment with third generation cephalosporins is associated with slow clinical improvement and high relapse burden for enteric fever, whereas the fluoroquinolone gatifloxacin is associated with rapid fever clearance and low relapse burden, we postulated that gatifloxacin would be superior to the cephalosporin ceftriaxone in treating enteric fever

5. ANTIMICROBIAL RESISTANCE IN PATAN HOSPITAL
Chung The, Karkey et al, EMBO Molecular Medicine, 2015

6. Pham Thanh, Karkey et al, eLife 2016
A novel ciprofloxacin resistant sub-clade of H58 Salmonella Typhi is associated with fluoroquinolone treatment failure
Pham Thanh, Karkey et al, eLife 2016

7. Study setting: Gatifloxacin vs Cefrtriaxone
Patan Hospital
4 kilometers apart. Provide for Patan and Kathmandu areas with the Valley of Kathmandu
Civil Services Hospital

8. Randomised control trials
Gatifloxacin versus Cefixime: PLoS One 2007
Stopped by DSMB
Gatifloxacin versus Chloramphenicol: Lancet Infectious Diseases 2011
Gatifloxacin versus Ofloxacin: PLoS Neglected Tropical Disease 2013
Gatifloxacin versus Ceftriaxone: Lancet Infectious Diseases 2016
(Stopped by DSMB)
Because treatment with third generation cephalosporins is associated with slow clinical improvement and high relapse burden for enteric fever, whereas the fluoroquinolone gatifloxacin is associated with rapid fever clearance and low relapse burden, we postulated that gatifloxacin would be superior to the cephalosporin ceftriaxone in treating enteric fever

9. Study design (2011-2014) Randomly assigned to 7 days of
Oral gatifloxacin (400mg tablets) at a dose of 10mg/kg once daily
Intravenous ceftriaxone (1000 mg injection vial) injected over 10 minutes at a dose of 60 mg/kg up to a maximum of 2 grams (2-13 years) or two grams (≥ 14 years) once daily
Arjyal et al 2016; Lancet Infectious Disease

10. Role of Community Medical Assistants

11. The primary endpoint Treatment failure
Fever clearance time (FCT) >7 days post treatment initiation
Requirement for rescue treatment
Microbiological failure: blood culture positivity on day 8 of treatment
Culture confirmed or syndromic enteric fever relapse within 28 days of initiation of treatment
Development of enteric fever related complication within 28 days after the initiation of treatment
time from the first dose of a study drug until the temperature dropped to ≤37.5°C and remained there for at least 2 days

12. Further…………
Time to treatment failure: time from the first dose of treatment until the date of the earliest failure event
FCTS were calculated using twice daily recorded temperatures and treated as interval censored outcomes
Patients without fever clearance or relapse, respectively, were censored at the time of their last follow up visit.
Arjyal et al 2016; Lancet Infectious Disease

13. Microbiology Blood (3 ml if aged <14 years; 8 ml if aged ≥14 years)
Adult blood samples conventional culture while Bactec Peds Plus bottles for paediatric samples.
Culture results were reported for up to seven days, positive bottles were sub cultured onto blood, chocolate and MacConkey agar
Identification through standard biochemical tests and serotype specific anti sera
Kirby Bauer disc diffusion method with CLSI guidelines
Etests were used to determine MICs
inoculated into media containing tryptone soya broth ad sodium polyethanl sulphonate, upto a total volume of 50mL.

14. Previous RCT findings
Protracted FCT associated with higher MIC against FQs in patients treated with ciprofloxacin and ofloxacin
Clinical efficacy with older FQs contentious
Fourth generation FQ, gatifloxacin, remained efficacious for uncomplicated disease,
including patients with reduced susceptibility to ciprofloxacin (MIC≥0.125μg/mL)

15. Latest RCT observations
Increased number of treatment failures associated with FQ resistant (MIC>32μg/mL) S. Typhi
Data safety and monitoring board stopped the trial
How had a drug lost its efficacy so quickly?

16. Aim Assess molecular epidemiology of infecting isolates
Investigate how genotype may be related to treatment outcome
Whole genome sequencing (WGS) on S. Typhi
Stratified by genotype
Assessed clinical presentation and outcome

17. Whole genome sequencing
Genomic DNA from S. Typhi (78)
2μg of genomic DNA subjected to WGS on an Illumina MiSeq platform to generate 250bp/100 bp paired end reads
All reads mapped to the reference sequence of S. Typhi CT18
Candidate single neucleotide polymorphism (SNPs) were called against the reference sequence using SAM tools
Li et al: Reference sequencing
Wong et al: SNPs
Emary, Holt, Parkhill: H58 definiteion
Li et al 2009, Bioinformatics; Wong et al 2015, Nature Genetics; Emory et al 2012,Transactions
of the Royal Society; Holt et al, 2008 Nature Genetics; Parkhill et al 2001, Nature

18. Emergence of novel ciprofloxacin-resistant H58 sub-clade in Nepal
This figure showing the phylogenetic structure of all Typhi isolates from the clinical trial with a majority of strains fell into H58 lineages, the most dominant genotype worldwide and a number of non-H58 lineages. The tree was mapped against various mutations on gyrA, parC, parE genes, these genes confer resistance to fluoroquinolone, this bar show ciprofloxacin sensitivity phenotype, dark blue=resistance, light blue intermediate resistance and sensitivity. What you can notice is a subclade of H58 nested within H58 lineage and separated from the rest of the group by a branch defined by 30 SNPs and having triple mutations, 2 on gyra and 1 in parC gene and are fully resistance to fluoroquinolones

19. Emergence of novel ciprofloxacin resistant H58 sub-clade in Nepal
This figure showing the phylogenetic structure of all typhi isolates from the clinical trial with a majority of strains fell into H58 lineages, the most dominat genotype worldwide and a number of non-H58 lineages. The tree was mapped against various mutaions on gyrA, parC,ParE genes, these genes confer resistance to fluoquinolone, this bar show cipro sensitivity phenotype, dark blue=resistance, light blue intermidiate resistance and sensitvie. What you can notice is a subclade of H58 nested within H58 lineage and separated from the rest of the group by a branch defined by 30 SNPs and having triple mutations, 2 on gyraa and 1 in oarC gnee and are fully resistance to fluoroquinolone,

20. Whole genome sequencing
Majority H58 lineage (65/78; 83.3%)
61 common DNA gyrase (gyrA) mutation in codon 83 (S83F)
Sub clade of 12 isolates: a mutation in gyrA at codon 87 (D87N) and 83 (S83F), and additionally in the topoisomerase gene parC (S801)
High MIC against ciprofloxacin (≥ 24 μg/mL)
Remaining 13 (16.7%) represented eight different lineages
2 non H58 had MIC for ciprofloxacin ≥24μg/mL had the S83F gyrA mutation, an alternative mutation at codon 87 (D87V), the S801 parC mutation, and an A364V mutation in parE
None harboured plasmid mediated quinolone resistance genes (PMQR) or contained additional AMR genes within the IncH1 family of plasmids
H58 triple mutant nested within, separated by 30 SNPs,

21. Clinical presentation of S. Typhi infections
Clinical data stratified by H58 status and baseline characteristics compared:
No significant difference in the demographics, and no association between disease severity at presentation between the group
Baseline characteristics of patients stratified by ciprofloxacin susceptibility
No difference in disease severity or demographics on presentation
FQ resistant more from adults

22. Clinical presentation
Significantly lower proportion of H58 S. Typhi (4/65) were susceptible to FQs compared to non H58 isolates (6/13)
H58 isolates had significantly higher MICs against the majority of tested antimicrobials than non H-58 isolates

23. Treatment failure and FCT
Treatment failure with H58 significantly less in the ceftriaxone group
In the gatifloxacin arm, H58 tended to be associated with a higher risk of treatment failure (p=0.06) and a longer FCT (p=0.013)
FCT differed significantly between the two treatment groups in H58 infections (5.03 vs 3.07 days)
No significant difference in treatment failure in the non H58 isolates
Time to FCT was not mirrored in the non H58 (2.87 to 3.12)

24. 2 non H58 isolates FQ resistance
Stratified outcome for the gatifloxacin arm (40 patients) by FQ susceptibility
Those infected with FQ resistant S. Typhi failed gatifloxacin treatment more frequently (8/10) than those infected with intermediately resistant or susceptible strains
They had significantly higher median FCTs (2.96, 4.01, 8.2 versus 3.83 days)

25. Effects of S. Typhi genotype and ciprofloxacin susceptibility on clinical outcomes
Ciprofloxacin sensitivity
Treatment failure
Fever clearance time
Intermediate
Susceptible
Resistant
H58
Non-H58
After stratification of the isolates by genotype H58 versus non-H58 and ciprofloxacin susceptibility we compare the treatment outcome between treatment arms. We found that treatment failure is significantly higher and fever clearance time is significantly longer in H58 infected patients between treatment arms. Figure a is kaplan meier curve for time to treatment failure by H58 and non-H58 Salmonella Typhi in the gatifloxacin arm. C is Non-parametric maximum likelihood estimators for interval-censored fever clearance time (see methods) by H58 and non-H58. Moreover in the gatifloxacin arm, we found that Those infected with FQ-resistant S. Typhi failed gatifloxacin treatment more frequently and with significantly higher median FCTs than those infected with an intermediately resistant organism or a susceptible organism. Kaplan-Meier curve for time to treatment failure by Salmonella Typhi susceptibility group (susceptible, intermediate, resistant) against ciprofloxacin. interval-censored fever clearance time by Salmonella Typhi susceptibility group

26. Koirala et al 2012, Antimicrobial agents and chemotherapy
Single ancestor carrying triple gyrA/parC mutant
Treatment failure
Wong et al 2015, Nature Genetics
Same combination of triple FQ resistant mutations
Equivalently high MICs against ciprofloxacin
Isolated in India from 2008 to 2012
Introduction from India or elsewhere in South Asia within the last 4-5 years
Unfortunately has entered into an endemic transmission cycle in Kathmandu

27. The phylogenetic structure of fluoroquinolone resistant Salmonella Typhi H58 in a regional context
In order to understand the origin of fully ciprofloxacin resistance sub-clade, we hypothesize that this sub-clade originated from another country given the unusual long genetic distance from all other isolate during the same period, we therefore look for the strain with the same resistance mutation and resistance profile from a repository of 2000 Typhi genomes published previously, we found a subset of 20 strains from India with match the profiles isolated prior to our study period and clustered tightly to the Nepalese H58 sub-clade of triple mutation. This phylo tree is exactly the same with the one I shown you but with the addition of Indian strains basal to the Nepalese sub-clade with the nearest neighbor isolated in 2010.

28. Conclusion Link phylogeny, microbiological phenotype and outcome
Emergent FQ resistant strain in South Asia
FQs should no longer be used for empirical therapy
World Health Organisation (2003)
National guideline in Nepal (2015)
Model for how genomics and clinical studies can be used for understanding the impact of AMR
Point 3: Particularly in the Indian subcontinent

29. Next step Randomised vaccination trial Salmonella Paratyphi
Ty21a (live ,administered orally)
Vi capsular polysaccharide (injectable)
Salmonella Paratyphi

30. Acknowledgements
Stephen Baker Buddha Basnyat Amit Arjyal Patan Hospital Typhoid group Archie Clemens Kathryn Holt Christiane Dolecek Gordon Dougan Jeremy Farrar
Chris Parry
Renee Tsolis
Li Liang
Phil Felgner
Tran Thuy Chau
Sabina Dongol
Jim Campbell
Tran Vu Thieu Nga
Duy Pham Thanh
Sally Whitehead
Fernanda Schreiber
Michael Favorov
Nick Thomson
Julian Parkhill
Katie Anders
Maciek Boni
Mark Achtman
Philippe Roumagnac