1. TB Genotyping and Whole Genome Sequencing in California
CTCA Educational Conference
March 7, 2017
Martin Cilnis, MPH, MS
Tuberculosis Control Branch
California Department of Public Health

2. Potential Uses of Genotyping
Confirm or refute outbreaks
Identify lab cross-contamination
Identifying possible sources of TB in children
Distinguish TB relapse from reinfection
Uncover linkages and settings not found by traditional contact investigations (CI)
Measure success of TB control
Define accuracy of contact and source case investigations
Many uses of genotyping

3. TB Genotyping Coverage in California, 2011-2016
2015 National Objective 94%
High genotyping coverage in CA, exceeds national objective, high confidence that genotyping clusters are capturing culture-positive CA cases, 90% in 2016 is because 2016 isolates are still being submitted by local health departments

4. How Do We Learn About Outbreaks?
CDC Outbreak Detection Using TB Genotyping
TB genotype cluster alerts:
Generated when a genotype has a higher than expected concentration within a county compared to other counties in the US in a 3-year window
Large outbreak surveillance:
≥10 TB cases in a 3-year period related by recent transmission
Notifications from local health departments
Several ways CA TBCB learns about possible outbreaks

5. CDC TB Genotype Cluster Alerts in CA, 2011-2016
The number of genotype alerts have increased since Some of these alerts were indeed incident or prevalent outbreaks, however, some also are not outbreaks or other recent transmission events. We take each genotype alert seriously and, depending on our outbreak team’s assessment, minimal to extensive resources could be expended to investigate a cluster. WGS can help prioritize which genotype clusters to investigate.
Potential question: Why has there an increase in cluster alerts? We don’t have a definitive answer: some genotype clusters alert more than once, some of the clusters are recent transmission or incident outbreaks, some are prevalent outbreaks occurring over multiple years, some are common genotypes that are not recent transmission. Improved genotyping coverage in CA might explain a small percent of the increase.
We also learn about clusters or outbreaks through communication with local health departments and monitoring of genotypes via our watch lists.
Note: a genotype can have more than one alert. The graph includes the multiple alerts because the outbreak response team examines each alert.

6. Steps of a TB Genotype Cluster Investigation
TB genotype cluster alert received by TB outbreak team
Prioritize genotype clusters
Higher priority
Lower/no priority
Notify local health departments
Monitor genotype
Investigation can be a variety of things, e.g., review patient charts, discuss cluster with case managers, re-interview cases, expand contact investigations.
In a time of diminishing public health resources, it is important to prioritize the clusters that are important to investigate and not spend time on false positive alerts. Whole genome sequencing could help with the cluster prioritization.
WGS could help with cluster prioritization and investigation.
Investigation
Implement interventions

7. Cluster investigation ≠ “Just another TB contact investigation”
Cluster investigations more challenging
Aim: Interrupt transmission by identifying contacts AND where, when and from whom patient was infected
Scope: Multiple patients; often multiple LHDs
Timeframe of interest: 2-3 years
Methods: Not yet well-established; tools not widely available
Training: Not yet readily available; few SMEs; not yet seen as core competency for staff
Differences between genotype cluster investigation and a contact investigation

8. Outcomes for Alerted TB Genotype Clusters, 2016
47 alerts for 40 county-clusters
26 (55%) higher priority clusters
27 outcomes
Prior outbreaks with new cases: 13 (48%)
New outbreaks: 9 (33%)
Confirmed/probable outbreaks: 3 (33%)
Suspected outbreaks: 6 (67%)
No evidence of an outbreak: 3 (11%)
Limited transmission: 5 (100%)
Cross-contamination: 2 (7%)
Outcomes of genotype clusters that were alerted in 2016: (outcomes based on cluster investigation findings before WGS was available)
Prior outbreaks with new cases (i.e., growth of genotype clusters associated with prior outbreaks; new cases assessed by CA TBCB and cluster re-prioritized)
New outbreaks (i.e., genotype clusters alerted in that were determined to be suspected, probable, or confirmed outbreaks)
No evidence of an outbreak (i.e., cluster investigation found some epi links among cases but not enough to reach the defined threshold of a suspected, probable, or confirmed outbreak)
Cross-contamination (i.e., the alerted genotype cluster was due to lab cross-contamination not previously known by the LHD or local PHL)
For reference only: WGS was requested for 17 of the higher priority county-clusters; WGS results were reported for 12, outcomes were reaffirmed by WGS except for 4 clusters where WGS refined possible transmission links (i.e., excluded clustered cases, included cluster cases, identified sub-clusters)

9. Genotyping Report No epi/clinical data Key data fields:
Case name and DOB
Specimen collection date
Genotype
Accession Number
Genotyping Lineage
Comments
Genotype report is sent by encrypted from CA TBCB to local health departments, Excel spreasheet contains data for your jurisdiction since the beginning of the genotyping service in 2004, has many data fields, however, there are some key data fields, contain no epi or clinical data, however, the report itself can be useful (examples on next slides).

10. Example: Refute suspected transmission
Suspected 5-person outbreak among foreign-born adults living in same city
3 with culture-confirmed TB
2 with suspected TB and pending cultures
All diagnosed within 2-month period
4 attend the same school, 2 are friends, and 2 lived at same apartment complex
Genotypes for 3 culture-pos patients did not match!!
Can stand down from outbreak investigation unless new genotype matches found
The report can be used to look-up case names genotypes to determine if whether transmission has occurred

11. Example: Evaluate outbreak containment3 6 4 2 3 4
Outbreak Contained?
New Transmission?
The report can be used to monitor for new cases in a genotype associated with an outbreak

12. Methods Used in TB Molecular Analysis
Whole Genome Sequencing
Various molecular analysis methods have been used for TB, MIRU-VNTR analyze a small portion of the Mtb genome (~1%) while WGS analyzes over 90% of the genome (more discriminatory)
Current genotyping methods
Source: A brief primer on genomic epidemiology: lessons learned from Mycobacterium tuberculosis. Guthrie JL, Gardy JL. Ann N Y Acad Sci Dec 23. doi: /nyas.13273

13. TB WGS in California, 2013-2016 41 clusters
32 (78%) clusters with complete WGS results
(35 outcomes)
9 (22%) clusters with pending WGS results
14 (40%) refuted as an outbreak
21 (60%) outbreak not refuted
WGS outcomes for 41 clusters.
40% of clusters were refuted as an outbreak but in most of these clusters, sub-clusters of cases were identified which suggest transmission (the focus of investigation can be on the sub-cluster of cases rather than the entire cluster)
60% of clusters were not refuted as outbreaks, most were confirmed and probable outbreaks indicating that WGS and the cluster investigation findings aligned
11 (79%) limited transmission
14 (67%) confirmed and probable outbreak

14. Summary
State TB Control Branch prioritizes alerted genotype clusters for investigation
Genotype cluster investigation are different from contact investigations
Most of the prioritized county-clusters were known new outbreaks or prior outbreaks with new cases
WGS can help elucidate chains of transmission and help focus on cases with closely-related WGS

15. Thank you! Martin Cilnis, MPH, MS Epidemiologist,
Outbreak Prevention and Control Section,
Tuberculosis Control Branch,
California Department of Public Health