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MICROBIAL DISEASES LABORATORY​ BRANCH

​​​TB Whole Genome Sequencing (WGS) for Drug Susceptibility Testing (DST) and Genotyping FAQs


Q: Can I still order pyrosequencing for cultures?​

​​A: Yes, while pyrosequencing (PSQ) will be available through March 2024, we encourage submitters to additionally request TB WGS DST for more comprehensive molecular resistance detection from TB cultures.

If sediment, sterile body fluid, processed tissue, or mixed/non-viable culture are submitted, PSQ will continue to be the primary molecular DST method until tNGS test becomes available in March 2024. ​


​Q: Will WGS DST be performed on all isolates submitted for phenotypic DST regardless of if I request it on submittal form?

A: No, a submitter should request WGS DST specifically. The submitter must ensure appropriate test requisition to obtain timely TB WGS DST reports.

In specific circumstances, WGS DST reflex testing will be performed to confirm phenotypic resistance even if not requested by the submitter, but this will be done on a case-by-case basis. 


Q: What is the difference between MDL TB WGS-DST and CDC's MDDR service for molecular DST?

A: The main differences are:  ​

  1. ​Testing method/Acceptable sample types: The CDC’s MDDR service is a tNGS assay that can be performed on primary sediments and cultures, while MDL’s test is a WGS-based assay that requires pure culture.
  2. ​Covered gene loci: MDL’s TB WGS-DST test covers all targets available from CDC’s MDDR service and includes some additional loci.
    ​​
​​Q: Is there still utility in ordering phenotypic DST if I’m already getting WGS-DST for my isolate?

A: Yes. The phenotypic and WGS results are complementary. In particular, there may be cases for which a mutation is not detected in genes associated with resistance but the isolate is phenotypically resistant due to an unknown mechanism. Phenotypic DST is also important to clarify an effect of mutations of uncertain significance detected by WGS. Hence, if phenotypic DST has NOT been performed already at a clinical or public health laboratory, we recommend ordering first-line or the alternative 4-month regimen first-line drug panel for phenotypic DST, in parallel with WGS-DST.

In the future, MDL will evaluate modifying our testing algorithm to utilize WGS DST as the primary clinical test for first line drug resistance determination. In such workflow, samples would be only reflexed to phenotypic DST for the confirmation of resistance-conferring mutations, mutations with an uncertain effect, and upon submitter request. Submitters will be notified in the event of any service changes.


​​Q: Our lab performs phenotypic DST in-house. Should we send all new TB isolates to MDL for WGS DST since it covers so many different AMR genes, or only those isolates that we have problems with from our culture-based DST?

A: Once MDL starts submitting WGS data for national TB surveillance in March 2024, it is ideal to submit all new TB isolates to MDL for WGS DST. Sequences generated for DST will be also used for TB genotyping purposes. However, until we announce availability of TB genotyping, it is not necessary to send all isolates that you have tested by phenotypic DST to MDL for WGS DST unless otherwise indicated (e.g. evidence of resistance, provider request, etc.).


Q: What is the typical time frame for phenotypic versus WGS DST testing if both tests are ordered? If the phenotypic result becomes available later, does that require physicians to revisit both WGS and phenotypic results?

A: WGS results will normally be available and reported to the submitters before phenotypic results (WGS TAT 7-14 days, phenotypic DST TAT 19 days). We recommend reviewing and evaluating all molecular and phenotypic DST results, in addition to other clinical and laboratory data as results become available. Discrepancies between WGS and phenotypic results are possible and consultation is available by contacting MDL (Matthew.Sylvester@cdph.ca.gov, Varvara.Kozyreva@cdph.ca.gov, CDPHTBDST@cdph.ca.gov​) and MDR services (MDRTBService@cdph.ca.gov ​).


Q: If a culture is determined to be mixed by WGS, will it automatically be reflexed for tNGS, once that is a valid testing method?

A: Yes, cultures for which WGS results indicate contamination will be reflexed to tNGS automatically once the test is available; until that time it will be reflexed to pyrosequencing.


Q: Is false-resistance possible in WGS DST assay if MTb culture is contaminated with NTM?

A: In our validation study we saw no evidence of this being a concern. We evaluated the specificity of the WGS DST assay by analyzing sequences of various NTM species with our bioinformatics pipeline for resistance prediction and for those sample the pipeline did not return any results that could be mistaken for MTBC. Additionally, we performed in silico contamination study where we mixed MTBC and NTM DNA in different proportions and did not observe any interference with the WGS DST results from NTM contamination that would result in false-positive or false-negative resistance detection; however, above certain percentage, contamination resulted in assay failure. Therefore, even though TB WGS DST assay is tolerant to some level of contamination, we still require a pure culture.


Q: Will the WGS DST results be reported to the submitting labs, CalREDIE, or both?

A: Results will be reported to the submitting public health laboratories at this time. MDL is working to implement CalREDIE reporting in the near future; however, it is not available currently.


Q: Can WGS assay provide identification for all MTBC species?

A: Even though WGS technology has a potential to identify all MTBC species, the WGS DST assay validated by MDL only provides identification for Mycobacterium tuberculosis and M. bovis species and further differentiates BCG strain of M. bovis. In cases when DNA of MTBC organism is detected but cannot be identified as either M. tuberculosis or M. bovis, it will be reported as “DNA of Mycobacterium tuberculosis complex detected.” If no DNA of MTBC is detected, this will be also reflected on the report.


Q: If I request WGS-genotyping, should I expect a report with genotyping results from MDL?

A: For submitted pure cultures, WGS-genotyping data will be uploaded to the CDC for national surveillance. A report will be issued to the submitter only when MDL is unable to perform WGS genotyping (e.g. due to culture appearing mixed upon sequencing) and will include a request for the submitter to send an isolate. All genotyping results will be available in TB GIMS.


Q: If we submit an isolate for genotyping only, will we be notified of MDR cases?

​​A: If submitter did not request TB WGS DST on the requisition form and only requested WGS genotyping, we will not automatically perform analysis for resistance detection from the WGS data. However, once MDL starts submitting WGS data for national TB surveillance, we do recommend the following:

  • ​If you are submitting an isolate from a new patient, or if the previous tested sample for a patient was collected 3 months ago or more, request both WGS DST and WGS genotyping. This will allow you to receive a clinical report with predicted resistance results.
  • If clinician has concerns that justify WGS for an isolate from a patient who had previous testing on a sample collected <3 months ago (e.g., concern for acquired resistance, etc.), contact MDL for pre-approval, and WGS DST and/or WGS genotyping may be provided on case-by-case basis.

Q: Once MDL starts submitting WGS data for national TB surveillance, do LHJs need to submit isolates to Michigan State Public Health Laboratory still?​

​​A: No, submission of TB isolates to MDL for WGS genotyping will replace the need to send isolates to Michigan Genotyping lab.


​​Q: Do I need to send an Isolate Submission Form (ISF) with patient metadata to the MDL when requesting TB WGS genotyping (like what is done for the Michigan Genotyping lab submissions)? 

​​A: No, all patient data for TB GIMS uploads will be collected via the MDL Lab Web Portal (LWP/ETOR).

​​

​​ Q: What are the performance characteristics of the WGS-DST assay? 

A: Overall MTBC WGS-DST assay performance:


​​​Perfor​mance Characteristic​​​
​​​​​Evaluated aspect of the assay​​​​
​​​​Achieved values​​​
​Accuracy​​
​Predicted S/R profile
​​​98.16%​​
​​​​Detection of genomic variations in targeted loci​
​99.73%
​MTBC ID ​​
​100%​
​​​​Repeatability (Qualitative)​
​​Predicted S/R profile
​100%
​​​​​​Detection of genomic variations in targeted loci​
100%
​MTBC ID ​​​
​100%​
​Reproducibility ​(Qualitative)​​​
​Predicted S/R profile
​​100%

​ ​​​​Detection of genomic variations in targeted loci​​
​​99.8%

​ ​MTBC ID ​​​​
​100%
​​Diagnostic Sensitivity
​​Predicted S/R profile
​91.25%

​Detection of genomic variations in targeted loci​​
​99%

​​MTBC ID ​​​
​100%
​Diagnostic Specificity​​
​Predicted S/R profile​
​​99.4%

​Detection of genomic variations in targeted loci​​​
​​​​99.89%

​MTBC ID ​​​​
​100%
Definitions: 

  • ​ Predicted S/R profile- predicted susceptibility(S)/resistance(R) to drugs in MTBC organisms based on WGS-DST. 
  • Detection of genomic variations in targeted loci- detection of genomic variations (single nucleotide polymorphisms [SNPs], multi nucleotide polymorphisms [MNPs], and indels) throughout the established reporting range in the genetic targets included in this assay that are known or suspected of being responsible for drug resistance in MTBC. 
  • MTBC ID- identification of MTBC (based on lineage-specific SNPs) and differentiation of M. tuberculosis from M. bovis, and further delineation of M. bovis BCG strain. 
​​​​​​​

WGS-DST assay performance by drug:

​​​​​Drug​

​​​TP
​TN

​FN
​FP
​Accuracy
​Dx Sensitivity
Dx Specificity​​
PPV
​NPV
INH
85​
​86
​2
​0*
​98.84%
97.70%​
​100%
100%​
​​97.73%
​​ETA
44​
​114
​0
​3*
​98.14%
​100%
​97.445
​93.62%
​​100%
​​RIF
​18
171​ ​0
​2
​98.95%
100%​
98.84%​ ​90%
​100%
PZA
​26
​147
​16
​0
​91.53%
​61.90%
​100%
​100%
​​90.18%
​EMB
​11
​170
​0
​3*
​98.37%
​100%
​98.27%
​78.575
​​100%
​AMK
​5
​173
​0
​0
​100%​
​100%​
​100%
​100%​
​​100%​
​KAN
4​
​126
​0
​0
​100%​
​100%​
​100%​
​100%​
​100%​​
​CAP 6​ ​183
​1
​0
​​99.47%​
​85.71%​
​100%​
​100%​
​​99.46%​
MFX​
​17
​129
​2
​0
​98.65%​​
​89.47%​
​100%​
​100%​
​​98.47%​
LFX​​
1​​
​14
​0
​​0​
​100% ​​100%​ ​​100%​
​​100%​
​​​100%​
BDQ​​
0​ 5​
0​
0​ ​​​100%​ N/A​ 100%​ N/A​ ​​100%​
​CFZ​
0​
​5
​0
​0
​​100%​
N/A​ ​100%
N/A​ 100%​
LZD​
2​
​2
​0
​0
​​100%​
100%​ 100%​ 100%​ ​​100%
Overall
219​ ​1325
​21
​8
​98.16%
​91.25%
​99.40%
​96.48%
​​98.44%​

Footnotes: *Numbers after ​​discrepancies were resolved with gold standard method.


​​WGS-DST assay performance by gene target:

​Per​​formance
​pncA​​
​katG​​​​​
fabG1/
inhA
​rpoB
​g​yrA​
​​gyrB

​rrs
​embB
​rv0678
​atpE

pepQ
​mmpS5

mmpS5
rplC
​rrl
​Total
TP
​​27
​48
​43
30​
21​
​0
​5
​5
​12​
​3
​1
​1
​1
​2
​0
199
​TN​​
157​
123
​129
143​
98​
27​
114​
​​23
5​
15​ 16​ 15​
15​ 16​ 12​ 908​
​FP
​0
0​
0​
0​ 0​ 0​ 0​ 0​ 0​
0​
1​ 0​ 0​
0​ 0​ 1​
FN
​0
0​ 0​
0​ 2
0​
0​ 0​ 0​ 0​
0​ 0​ 0​ 0​
0​ 2
Accuracy
​100​​​%
​​​100%
​​​100%​
100%​
98.35%​ ​100%​​​
​100%​​​
100%​ 100%​ 100%​ 94.44%​ 100%​ ​100%
100%​ 100%​
99.73%​
​​Specificity
​100%
​100% ​100% ​100%
100%​ ​100% 100%​ ​100% 100%​ ​100% 94.12% ​100% ​100% ​100% ​100%
​99.89%
Sensitivity​​
​​100% ​100% ​100% 100%​ 91.30%​ N/A
​100% ​100% ​100% ​100% ​100%
​100% ​100%
​100% ​​N/A
​99.00%
​​PPV
​100%
​100% ​100% ​100% 100%​ N/A​
​100% ​100% ​100% 100%​ ​50%
​100% ​100% ​100% ​N/A
99.50%
NPV
​100% ​100% ​100% 100% ​98.00% ​100% ​100% ​100% ​100% 100%​ ​100% ​100% ​100%
100%​ 100%​ 99.78%

​​​​​​​​​​​​​​​​

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