GENETIC DIVERSITY OF MDR TB: IMPLICATIONS FOR DIAGNOSTICS AND EVOLUTION

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1 GENETIC DIVERSITY OF MDR TB: IMPLICATIONS FOR DIAGNOSTICS AND EVOLUTION Megan Murray, MD, MPH, ScD Harvard Medical School Brigham and Women s Hospital Harvard School of Public Heath

2 INH Resistance All Happy Families are Alike. Each Unhappy family is unhappy in it own way. Tolstoy, from Anna Karenina

3 Comparative Species Diversity: Bacteria S. typhi H. pylori S. Aureus MONOMORPHIC SPECIES DIVERSE LOCI MTB =.01% SPECIES DEFINITION DIVERSE LOCI<=30%

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5 Mechanisms of Rifampin Resistance Rifampin Rifampin Binding Site rpob homology model target of Rifampin rpob homolog from Thermus aquaticus with rifampicin bound (crystal structure)

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8 Activation and Binding of Isoniazid Derivatives S315T Isoniazid Activation Activation of Isoniazid by KatG Codon S315T Isoniazid-NADH Isoniazid-NADH bound to InhA

9 DR Mutation Database project Gates-funded initiative to identify mutations and their frequency in drug resistance genes in M. tuberculosis. Developed archive of 1800 well-characterized strains from 9 countries and 6 contributing labs. Sequenced 28 genes and promoters. Created public database. Used machine learning approach to identify optimal set of snps to diagnose resistance by drug.

10 Molecular Inversion Probes

11 Epidemiologic studies Epidemic modeling Within-host modeling Sequencing Molecular Inversion Probes High specificity Scalability for high-throughput, multiplexed analysis Exploits high-throughput platforms developed for WGS Allows simultaneous DR testing and genotyping Very high coverage enables detection of minority populations of strains in mixed samples

12 Preliminary Results

13 Gaps 4 Es Errors Epistasis Efflux Exotic or rare variants

14 PZA Discrepant DST ph of media can inhibit growth PZA MIC 55 μg/ml at ph 5.5 MIC 200 at ph 6.1 Large innocula reduces PZA growth Error (in DST) contributes to low sensitivity of genotypic PZA tests.

15 Additional mutations modulate levels of drug resistance Meacci F et al. J Clin Micro 2005, 2 µg/ml MIC katg 234 ahpc -9 P01 P02 P03 P04 P05 P06 P07 P08 P09 P10 Time Epistasis, interaction of multiple mutations to alter DR levels.

16 RIF EXPOSURE IN RESISTANT STRAINS LEADS TO OFLOX EFFLUX Efflux may contribute to modulation of levels of resistance.

17 2/50 Rif Resistant clinical strains had no RRDR mutation. Contribution of novel rpob mutations shown by modeling and transformation. Exotic mutations or rare variants.

18 Some mutations that lead to drug resistance do not impair fitness Wildtype KatG S315T INH resistant mutant Other INH resistant KatG mutants The white bars correspond to the CFU after 1 day, and the gray bars correspond to the CFU after 40 days in the lungs. Pym A et al, Inf Imm 2002

19 Suggests exotic mutations more common in mono-r and that they exert a higher fitness cost. Hazbon et al. 2007

20 Evidence for low fitness costs of INH resistance mutation katg S315T Laboratory evidence Pym Infection and Immunity 2002 S315T mutants retain catalase & peroxidase activity, virulent in mouse model Epidemiological evidence Van Soolingen JID 2000 & Gagneux PLoS Pathogens 2008 Increased molecular clustering of 315 mutants compared with other isoniazid resistant mutants 315 mutants over-represented among MDR cases Ecological evidence Cohen Microbial Drug Resistance 2005 Proportion of INH resistance due to 315 may be positively correlated with the amount of transmitted resistance Proportion of isoniazid-resistant mutant isolates with 315 mutations in katg i id TB incidence (cases/100k) r s =

21 Specific rif resistance mutations affect bacterial reproductive fitness. Lab-derived resistance S531L other rpob mutants Resistance arising in a host Gagneux Science 2006

22 Evidence for compensatory mutations affecting fitness of Rif resistant strains Comas, Nature Genetics, 2012

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26 Genes with mutations conferring resistance or a growth/fitness advantage in the presence of drug will be under strong positive selection.

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28 Methods for detecting positive selection dn/ds Detects a preponderance of protein altering versus neutral mutations Convergence Detects repeated acquisition of changes in unrelated branches of a phylogeny Differential Clustering Detects high density of changes in specific loci differentially distributed in DR versus DS strains Dr. Jesse Shapiro and Dr. Pardis Sabetti Method # Known DR Genes Detected Convergence 11/11 Differential density 10/11 dn/ds 5/11

29 Convergence of changes along the phylogenetic tree

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31 Mycobacterial Cell Wall Among 30 novel genes under positive selection in DR, preponderance of cell wall components and genes associated with permeability.

32 Team Murray Lab Maha Farhat Razvan Sultana Hanna Guimaraes Karen Jacobsen Chuan-Chin Chang Broad Institute Eric Lander Pardis Sabeti Jesse Shapiro Mark Borowsky James Galagan Brian Weiner Bruce Birren Peter Sisk Partners Genomics Oleg Iartchouck Alison Brown Collaborators Jen Gardy, Jay Johnson. British Columbia CDC Midori Kato, UCSF Alex Sloutsky, MSLI PIH Tommie Victor and Rob Warren, Stellenbosch Bonnie Plikyatis and Jamie Posey, US CDC Marco Oggiono, Sienna Sebastien Gagneux Funders Ellison Foundation NIAID Broad Institute Midas (NIGMS) Gates Foundation