1. TB in Nebraska, New Challenges & Solutions

2. Mycobacterium

3. TB in Nebraska, New Challenges & Solutions Mycobacterium

4. Challenges: 1.Accurately diagnose infections 2.Prevent transmission 3.Provide appropriate treatment 4.Correctly classify the organism 1.Accurately diagnose infections 2.Prevent transmission 3.Provide appropriate treatment 4.Correctly classify the organism

5. 1. Accurately diagnose infections Tuberculosis (TB) is a serious re-emerging bacterial illness that usually affects the lungs. TB bacteria are spread from person to person through the air. –Mycobacterium tuberculosis complex Tuberculosis (TB) is a serious re-emerging bacterial illness that usually affects the lungs. TB bacteria are spread from person to person through the air. –Mycobacterium tuberculosis complex

6. There are two forms of TB: –TB infection (latent TB) – not contagious –TB disease (active TB) – are contagious People with TB infection (latent TB) can take drugs to prevent them from getting TB disease (active TB). There are two forms of TB: –TB infection (latent TB) – not contagious –TB disease (active TB) – are contagious People with TB infection (latent TB) can take drugs to prevent them from getting TB disease (active TB).

7. Therefore: Prevention of TB involves: –Identification of latent TB infections Prevention of TB involves: –Identification of latent TB infections

8. However: People with latent TB infection: –Have no symptoms –Don’t feel sick –Have a normal chest x-ray –Have a negative sputum smear –Have circulating blood cells (lymphocytes) that recognize mycobacterial proteins (antigens) People with latent TB infection: –Have no symptoms –Don’t feel sick –Have a normal chest x-ray –Have a negative sputum smear –Have circulating blood cells (lymphocytes) that recognize mycobacterial proteins (antigens)

9. QuanFERON –TB Gold Assay Alternative to tuberculin skin test (TST) in vitro vs. in vivo M. tb complex-specific antigens used 1 visit to clinic Less subject to errors Fewer false-positives Alternative to tuberculin skin test (TST) in vitro vs. in vivo M. tb complex-specific antigens used 1 visit to clinic Less subject to errors Fewer false-positives

10. Principle: –Tests for infected lymphocyte’s ability to respond to mycobacterial antigens ESAT-6 (Early Secretory Antigenic Target – 6) CFP-10 (Culture Filtrate Protein – 10) –By secreting a cytokine IFN-γ (interferon-gamma) –And measured by ELISA (Enzyme-Linked Immunosorbent Assay) Principle: –Tests for infected lymphocyte’s ability to respond to mycobacterial antigens ESAT-6 (Early Secretory Antigenic Target – 6) CFP-10 (Culture Filtrate Protein – 10) –By secreting a cytokine IFN-γ (interferon-gamma) –And measured by ELISA (Enzyme-Linked Immunosorbent Assay) QuanFERON –TB Gold Assay

11. Stage 1 – Incubation of Blood

12. Stage 2 – Detection of IFN-γ -Negative control (not stimulated) -ESAT-6 stimulated - CFP-10 stimulated - Positive control (mitogen)

13. Recommended for: Groups more likely to be exposed to TB –People from countries where TB is common –People in close contact with active TB case –People with HIV –People in nursing homes, prisons or homeless shelters –Laboratory personnel Groups more likely to be exposed to TB –People from countries where TB is common –People in close contact with active TB case –People with HIV –People in nursing homes, prisons or homeless shelters –Laboratory personnel

14. 2. Prevent transmission Identifying suspected sources Understanding transmission patterns Identifying suspected sources Understanding transmission patterns Genotyping provides tool

15. Genotyping Analysis Isolate AIsolate B Likely Related

16. Genotyping Analysis Isolate AIsolate B Not Related

17. Genotyping Methods Two PCR-based methods: –Spoligotyping –MIRU-VNTR Results converted to numeric code Matches can be further investigated by other technologies Two PCR-based methods: –Spoligotyping –MIRU-VNTR Results converted to numeric code Matches can be further investigated by other technologies

18. Spoligotyping Spacer Oligonucleotide Typing Presence or absence of 43 spacer regions found in the Direct Repeat region of M. tb genome. Results converted to 15 digit code Spacer Oligonucleotide Typing Presence or absence of 43 spacer regions found in the Direct Repeat region of M. tb genome. Results converted to 15 digit code

19. Original banding pattern Binary code 14 + 1 grouping Designation (15 digits) Original banding pattern Binary code 14 + 1 grouping Designation (15 digits) Spoligotyping 1 1 1 1 0 0 1 1 0 0 1 1 1 111-100-110-011-1….. 7 4 6 3

20. MIRU-VNTR Mycobacterial Interspersed Repetitive Units – Variable Number of Tandem Repeats Identifies strains by the difference in copy number of tandem repeats at 12 different locations of the genome Mycobacterial Interspersed Repetitive Units – Variable Number of Tandem Repeats Identifies strains by the difference in copy number of tandem repeats at 12 different locations of the genome

21. MIRU-VNTR MIRU locus name 02, 04, 10, 16, 20, 23, 24, 26… # of repeats 2 3 2 2 3 4 2 5 MIRU designation (12 digits) 23223425…. MIRU locus name 02, 04, 10, 16, 20, 23, 24, 26… # of repeats 2 3 2 2 3 4 2 5 MIRU designation (12 digits) 23223425….

22. Genotyping Results M909641604L1037 Nebraska Pub HealthNE776137607760771224226133323 T8037041404L1035 Nebraska Pub HealthNE776377777760771233326163224 T1196240804L1038 Nebraska Pub HealthNE776137607760771224226133323 M1003443705L2767 Nebr. Pub. Health Lab.NE777777777413731254225223533 Submitter Number RVCT Number Accession_ noSubmitting Labstate_idspoligotypeMiru

23. Genotyping Program: Laboratory component –Specimen submission to genotyping lab Atlanta, GA Richmond, CA Ann Arbor, MI –Tests performed in reference lab > 32,000 isolates tested –Results sent back to state lab Laboratory component –Specimen submission to genotyping lab Atlanta, GA Richmond, CA Ann Arbor, MI –Tests performed in reference lab > 32,000 isolates tested –Results sent back to state lab

24. Program component –Share patient information with laboratory –Receive and interpret genotyping reports –Decision to act on genotyping results Program component –Share patient information with laboratory –Receive and interpret genotyping reports –Decision to act on genotyping results Genotyping Program:

25. Genotyping results: Identifying suspected sources –Some “source cases” identified by epi investigation have been different strains Understanding transmission patterns –Unsuspected sources have been identified Identifying suspected sources –Some “source cases” identified by epi investigation have been different strains Understanding transmission patterns –Unsuspected sources have been identified

26. 3. Provide appropriate treatment People infected with TB can take medications to prevent active TB disease. People with active TB disease can usually be cured with anti-TB drugs. The drugs must be taken exactly as prescribed. Some new TB strains are resistant to anti- TB drugs. People infected with TB can take medications to prevent active TB disease. People with active TB disease can usually be cured with anti-TB drugs. The drugs must be taken exactly as prescribed. Some new TB strains are resistant to anti- TB drugs.

27. M. Tb Treatment Long term treatment Multi-drug regimen –Primary drugs Rifampin, Isoniazid, pyrazinamide, ethambutol –Secondary drugs Streptomycin, cycloserine, macrolides, quinolones Long term treatment Multi-drug regimen –Primary drugs Rifampin, Isoniazid, pyrazinamide, ethambutol –Secondary drugs Streptomycin, cycloserine, macrolides, quinolones

28. Drug resistance MDR TB: Multi - drug resistant –Resistant to Rifampin & Isoniazid –About 5% of all TB infections (average) –Highest rate in former Soviet republics XDR TB: Extensively drug resistant –Resistant to all primary and at least one secondary –45 countries report at least one case MDR TB: Multi - drug resistant –Resistant to Rifampin & Isoniazid –About 5% of all TB infections (average) –Highest rate in former Soviet republics XDR TB: Extensively drug resistant –Resistant to all primary and at least one secondary –45 countries report at least one case

29. Drug resistance testing Antimycobacterial Susceptibility Tests (ASTs) Two methods –Agar based –Broth based Creighton University does NE surveillance Antimycobacterial Susceptibility Tests (ASTs) Two methods –Agar based –Broth based Creighton University does NE surveillance

30. ASTs by Agar proportion method Gold standard Dilutions of standardized inoculum onto control and drug containing agar Compare growth in absence or presence of drug >1% colony growing on the drug containing agar suggests resistance Gold standard Dilutions of standardized inoculum onto control and drug containing agar Compare growth in absence or presence of drug >1% colony growing on the drug containing agar suggests resistance

31. Limitations of method Organism must be identified to the species level before reporting AST data Results take about 3 weeks Organism must be identified to the species level before reporting AST data Results take about 3 weeks

32. 4. Correctly classify organism Non-TB mycobacteria are cause of disease –Mycobacterium avium - respiratory disease –M. kansasii – respiratory / cutaneous disease –M. marinum – “fish tank granuloma” –M. leprae – skin disease –M. gordonae - contaminate Non-TB mycobacteria are cause of disease –Mycobacterium avium - respiratory disease –M. kansasii – respiratory / cutaneous disease –M. marinum – “fish tank granuloma” –M. leprae – skin disease –M. gordonae - contaminate

33. Greater than 90 species known to exist Treatments vary by species Conventional methods of ID can be lengthy Molecular methods can be utilized Greater than 90 species known to exist Treatments vary by species Conventional methods of ID can be lengthy Molecular methods can be utilized

34. MycoAlign Developed as a collaboration between UNO and UNMC Combination molecular and web-based computational system ID of Mycobacterium spp. Developed as a collaboration between UNO and UNMC Combination molecular and web-based computational system ID of Mycobacterium spp.

35. Molecular Target – rDNA gene Composed of multiple genes that code for ribosomes Bacteria16S and 23S Contains a variable region to discriminate among species Internal transcribed spacer regions (ITS) Composed of multiple genes that code for ribosomes Bacteria16S and 23S Contains a variable region to discriminate among species Internal transcribed spacer regions (ITS)

36. Stable within species Contains conserved sequence areas –Create universal primer sets –Small sequence is manageable Stable within species Contains conserved sequence areas –Create universal primer sets –Small sequence is manageable Molecular Target – rDNA gene

37. rDNA Complex PCR Product

38. 600 bp→ M 1 2 3 4 5 Gel electrophoresis

40. MycoAlign

42. MycoAlign results MycoAlign ResultMycoAlign Score TOT MycoAlign LabTB Lab ResultProbe TOT M.intercellulare Mac B98.84M.avium7 M.fortuitum1004M.fortuitum/chelonae complex10 M.avium1007MycoAlign usedn/a M.intercellulare1003M.avium complex13 M.tbcomplex1003M.tbcomplex9 M.intercellulare97.64M.avium complex3 M.gordonae1004M.gordonae10 M.gordonae1004M.gordonae6 M.tbcomplex98.244M.tbcomplex1 M.chimerae/avium1004M.avium complex3 M.gordonae1004M.gordonae2 M.tbcomplex1002M.tbcomplex2 1002M.tbcomplex20 3.777.17

43. Challenges: 1.Accurately diagnose infections Solution: Use QuantiFERON-Gold Assay Solution: Use QuantiFERON-Gold Assay

44. Challenges: 2.Prevent transmission Solution: Use National Genotyping Program Solution: Use National Genotyping Program

45. Challenges: 3.Provide appropriate treatment Solution: Conduct AST Surveillance Solution: Conduct AST Surveillance

46. Challenges: 4.Correctly classify the organism Solution: Use MycoAlign software Solution: Use MycoAlign software