DNA FINGERPRINTING. Barry N. Kreiswirth, PhD Director, PHRI TB Center

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1 DNA FINGERPRINTING Barry N. Kreiswirth, PhD Director, PHRI TB Center

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3 Molecular Epidemiology Local Epidemiology Are M. tuberculosis isolates recovered from localized cases of disease the same or different strains? Global Epidemiology Are strains causing disease in one geographic area related to those isolates world-wide?

4 Molecular Tools - Genotyping Methods Primary Genotyping Method IS6110 Southern blot hybridization Secondary Genotyping Methods Spoligotyping Binary typing, DR region PGRS Southern blot hybridization VNTR, MIRU PCR, multiple targets IS6110 mapping Southern blot hybridization DNA sequencing Resistance targets, SNP Array analysis Deletion mapping

5 Genotyping Targets to Discriminate M. tuberculosis Strain 210 Strain HN5 Barnes et al. NEJM 2003;349:1149

6 Biology of IS6110 Unique to M. tuberculosis complex Copy number from 1-26 insertions Insertions dispersed around genome Chromosomal hot-spots identified Insertions stable over time Movement is a replicative process

7 Insertion Sequence IS6110

8 IS6110 DNA Fingerprinting Standardized methodology Southern blot hybridization PvuII restriction digest Common right-side hybridization probe Common molecular weight standards Digitized patterns Pattern matching software

9 DNA Fingerprints of M. tuberculosis Strains

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11 Searching the Database for Strain W4

12 IS6110 DNA Fingerprint

13 IS6110 Genotyping Limitations Not able subtype low copy number strains (<6 IS6110 insertions) Not able to determine strain relatedness Turn-around time is too slow

14 Secondary Typing Method Spoligotyping PCR-based, binary hybridization method DR-region: 36 bp repeats / bp spacers Rapid turn-around time, objective data Does not provide IS6110-like discrimination It s a grouper

15 W-Beijing

16 Genotyping Data Public Health Issues Evaluate nosocomial and community transmission Evaluate suspected cases of laboratory contamination Distinguish relapse vs. re-infection Genotype drug resistance genes to distinguish spread vs. acquisition Distinguish recent transmission and endemic strains

17 SRO Outbreak in San Francisco

18 Molecular Epidemiology Stamp collecting building a strain repository Outbreak investigations Population based studies NYC TB program Targeted studies Geography Drug Resistance Pulmonary / Extra-pulmonary Pediatric / Adult Nosocomial / Community

19 Molecular Epidemiology PHRI TB CENTER >23,000 M. tuberculosis isolates Diverse geography MDR isolates All fingerprinted using IS % spoligotyped >2000 ssnp analysis Patient and strain database Archived fingerprint library

20 Exogenous Reinfection Exogenous reinfection with multidrug-resistant Mycobacterium tuberculosis in patients with advanced HIV infection. Small, PM, Shafer, RW, Hopewell, PC, Singh, SP, Murphy, MJ, Desmond, E., Sierra, MF, Schoolnik, GK. N Engl J Med 1993;328:

21 P Strain MDR Causing Re-Infections in AIDS Patients

22 Tuberculosis in New York City

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24 WHEN AND WHERE

25 March 1992, 168 th Street, NYC

26 C Strain - Outbreak Widespread dissemination of a drug-susceptible strain of Mycobacterium tuberculosis. Friedman, CR, Quinn, GC, Perlman, DC, Salomon, N, Schluger, N, Lutfey, M, Berger, J, Poltoratskaia, N, Riley, LW. J Infect Dis 1997;176:

27 Case Study

28 Background - 1 8/5/05 Patient KD admitted to hospital A with diagnosis of suspected pulmonary tuberculosis CXR abnormal/non-cavitary Sputum smear positive (4+) Final culture M. tb Resistant INH 8/10/05 patient threatens to leave hospital AMA Visit to hospital to attempt problem identification/problem resolution No health officer restraining order requested 1:1 monitoring in place 8/11/05 patient leaves hospital AMA Numerous attempts to locate patient prove futile Fictitious identity and locating information provided to hospital by patient Cell phone conversation with index patient indicates on way to Florida with pregnant girlfriend Case dispositioned as lost to follow-up

29 Background - 2 4/5/06 Patient CB admitted to hospital B with diagnosis of suspected pulmonary tuberculosis CXR abnormal/non-cavitary Sputum smear positive (4+) Final culture MTB Resistant INH, EMB, and PZA 4/6/06 patient threatens to leave hospital AMA Visit to hospital to attempt problem identification/problem resolution Patient uncooperative Legal intervention requested Health Officer hospital restraining order served Despite completely different demographic information subtle physical similarities existed between patients KD and CB CB denies any knowledge of KD Upon hospital discharge CB placed on directly observed therapy and followed up as an out-patient at local TB clinic where he completed treatment

30 Questions For Discussion Were patients KD and CB the same? KD was never located CB completed treatment As the TB Control staff member responsible for the on-going review of these two cases what would be some of your thoughts?

31 Advantages Of TB Genotyping Improve contact investigations by identifying epidemiological links among cases and in the process potentially identify new and unusual settings of transmission Provide an epidemiological analysis of potential outbreaks Detect cross-jurisdictional transmission Distinguish between reactivation and re-infection Identify and confirm laboratory crosscontamination Assess patterns and prevalence of MTB strains Evaluate TB prevention and control efforts Confirm patient identity

32 1 2 3 JI strains isolated from patient CB/KD

33 Genotyping Data Public Health Issues Evaluate nosocomial and community transmission Evaluate suspected cases of laboratory contamination Distinguish relapse vs. re-infection Genotype drug resistance genes to distinguish spread vs. acquisition Distinguish recent transmission and endemic strains

34 Laboratory Contamination of Mycobacterium tuberculosis Cultures in New Jersey

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36 Patient JH: Image # 1 JH - 51 y/o male referred to a local NJ health department clinic with symptoms of TB. A sputum sample was collected and was 4+ on smear for AFB and grew M.tb that was resistant to INH and SM DOT was started with RIPE, daily However he did not complete his treatment in NJ patient relocated to Orange County, California and in February 05 to Illinois

37 Patient JH: Bacteriology Date Smear Culture Specimen Sp# Resistance M.tb Sputum >10 M.tb Sputum S M.tb Sputum Neg. M.tb Sputum Neg. M.tb Sputum Neg. M.tb Sputum INH, SM

38 A View of Laboratory Cross-Contamination * * * * * * Lane 1 Patient JH (5) Lane 2 Patient TA (4) Lane 3 Patient WS (3) Lane 4 Patient WS (3) Lane 5 Patient LH (2) Lane 6 Patient LH (2) Lane 7 Patient LH (2) Lane 8 - Patient PS (1) Lane 9 - Patient PS (1)

39 Patient LH: Images # 5, 6 and 7 LH 42 y/o diabetic H/F born in DR and immigrated to the US in Feb Sept she c/o wt. loss and cough and was admitted to JCMC from where she was diagnosed with pulmonary TB. A CXR revealed cavitary infiltrate in the RUL consistent with active TB DOT started with RIPE, daily for 8 weeks followed by RI, daily for 16 weeks. Her sputum was initially thought to convert to negative on culture for M.tb at 4 weeks sputum specimen collected and processed and on the lab grew M.tb. This sequence of events, if true, could have caused an extension of treatment

40 Patient LH: Bacteriology Date Smear Culture Specimen Sp# Sensitivity M.tb Sputum M.tb Sputum M.tb Sputum Neg. M.tb Sputum Neg. Neg. Sputum Neg. M.tb Sputum S Neg. Neg. Sputum Neg. Neg. Sputum Neg. Neg. Sputum

41 A View of Laboratory Cross-Contamination * * * * * * Lane 1 Patient JH (5) Lane 2 Patient TA (4) Lane 3 Patient WS (3) Lane 4 Patient WS (3) Lane 5 Patient LH (2) Lane 6 Patient LH (2) Lane 7 Patient LH (2) Lane 8 - Patient PS (1) Lane 9 - Patient PS (1)

42 Patient PS: Images # 8 & 9 PS 32 y/o H/M born in Ecuador and immigrated to the US in June 2002 May 2003 admitted to Hospital with c/o cough, fever, night sweats, wt. loss, and hemoptysis. A chest CT scan revealed LUL cavitary lesions associated with pleural thickening and fibrotic changes. Sputum smears were + for AFB and cultures grew pan-susceptible M.tb DOT started with RIPE, daily for 8 weeks, followed by RI, BIW for 18 weeks

43 Sputum converted to negative on smear and culture for M.tb at 6 weeks DOT was discontinued (26 weeks) when RI were stopped. The patient felt well and had a normal ESR (8 mm/hr) when treatment was stopped he was seen in clinic for routine followup visit and remained well; his sputum was smear and culture negative for AFB patient presented for routine clinic visit, felt well and was w/o complaints. A sputum sample (S ) collected at this visit grew M.tb on culture

44 Laboratory Contamination 10/1/10: patient MJ, 12 month old infant brought to clinic with respiratory distress X-ray abnormal; suspected pneumoniae Sent for BAL to check for a foreign object No foreign objects; BAL cultured Infant hospitalized; treated for pneumoniae 10/6/10: Infant sent home and symptoms resolved in two weeks 10/30/10: Laboratory report BAL was culture positive for M. tuberculosis

45 Laboratory Contamination 11/4/10: Infant and parents advised of the tuberculosis finding and the baby, although well, was placed on four tuberculosis drugs IREZ Case reported to the NJ DOH Infant to treated for 6-9 months Monthly clinic visits Contact investigation 11/26/10: M. tuberculosis fingerprint identified the strain as H37Ra the laboratory control strain used in susceptibility testing. The laboratory contaminated the BAL. The infant does not have tuberculosis. 11/27/10: Infant taken off drugs; case removed; no investigation

46 Summary The integration of molecular typing of M. tuberculosis with traditional TB control programs improves public health Cluster analysis helps to prioritize resources and provides a direct measurement of transmission The most significant clinical and public health role for genotyping M. tuberculosis is to rapidly identify cases of laboratory cross-contamination

47 Treating Tuberculosis Limited arsenal of antitubercular drugs Combination therapy to prevent resistance Acquired vs. primary resistance Drug susceptibility testing ~ 1 month Drug resistance targets identified Rapid genotyping of drug resistance possible

48 Combination Therapy Streptomycin failed as a single drug Combination therapy required Two to four drugs six to nine months Never add a single agent to a failing regimen Adherence to therapy improved by DOTS

49 Acquired vs Primary Resistance Acquired resistance Nonadherence to therapy Inappropriate therapy due to poor regimen selection, erratic drug supply Primary resistance Nosocomial transmission Community transmission

50 Drug Susceptibility Testing M. tuberculosis slow growth confounds classical drug susceptibility testing One month turnaround time is common Second line drug testing to determine XDR only done on MDR strains

51 Antitubercular Antibiotics ANTIBIOTICS YEAR Streptomycin 1944 PAS 1949 Isoniazid 1952 Pyrazinamide 1954 Cycloserine 1955 Capreomycin Ethionamide Ethambutol 1962 Rifampin 1963 Kanamycin Fluoroquinolones

52 Genotyping Drug Resistance Nearly all drug resistance target genes identified With two exceptions, non-synonymous mutations in drug resistance target genes predicts resistance Molecular approaches are able to genotype resistance in less than 24 hrs too costly even for developed countries

53 Drug Resistance Target Genes

54 Rifampin Resistance & rpob Mutations

55 Tuberculosis in New York City

56 Tuberculosis Cases and Rates New York City, Cases in 2006 Number of Cases 4,000 Case Rate 55.0 Rate/100, ,500 # Cases 50 3,000 2,500 2, , , Year

57 Causes of Resurgent Tuberculosis in NYC Poverty, homelessness, crowding, substance abuse HIV/AIDS epidemic Decline of public health infrastructure Marked reduction in TB control program staff and clinic facilities Lack of accessible health care TB abroad on the rise; immigration from high prevalence countries Poor infection control practices in hospitals Poor treatment practices No susceptibility results for most patients Bad regimens By 1989, less than half of patients who began treatment were cured

58 W Strain MDR Outbreak in NYC January August Months - 8,021 Cases 357 Patients with W strain tuberculosis Spread in NYC hospitals and state prisons All resistant to first line drugs 86% HIV infected; >90% Mortality 160 Patients identified since study 22 Patients identified outside of NYC Bifani et al., JAMA 1996:275;452. Munsiff et al., JID 2003:188;356.

59 Outbreak of the Multidrug Resistant W Tuberculosis Clone

60 W MDR Outbreak: Isoniazid (100%) katg - 315:AGC>ACA; Ser>Thr Rifampin (100%) rpob - 526:CAC>TAC; His>Tyr Streptomycin (100%) rpsl - 43:AAG>AGG: Lys>Arg Ethambutol (100%) embb - 306:ATG>GTG; Met>Val Pyrazinamide (55%) pnca - 139:ACC>GCC; Thr>Ala Kanamycin (92%) rrs - nucleotide 1400 Fluoroquinolones (0%)

61 15 YEARS LATER The New face of TB in South Africa: XDR and HIV

62 MDR and XDR M. tuberculosis Multidrug Resistance (MDR) Resistance to at least isoniazid (INH) and rifampin (RIF) Extensively Drug Resistance (XDR) Resistance to at least isoniazid and rifampin plus resistance to fluoroquinolones AND one of the second line injectable aminoglycoside drugs (amikacin, kanamycin or capreomycin)

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64 KwaZulu Natal, South Africa Jan 2005 March ,539 TB Diagnosed 542 Culture Positive Cases 168 MDR Cases 53 XDR Cases 52 / 53 Died 44 Patients Tested All HIV+ MEDIAN SURVIVAL OF 16 DAYS FROM THE TIME OF DIAGNOSIS

65 Lessons Learned and Unlearned Infection control essential Rapid diagnostic and 2 nd line susceptibility testing Ensure adequate supply of drugs New drugs and more effective regimens needed REBUILDING NYC TB CONTROL $1,000,000,000

66 As we learned in NYC during our HIV MDR outbreak, it will take strong political will, dedicated medical and public health teams and a great deal of money to deal with this emerging epidemic If not, XDR and all its baggage will appear at your doorsteps