1. Risk of acquired drug resistance during DOTS tuberculosis treatment in a setting of high drug resistance Helen S. Cox 1,3, Stefan Niemann 2, Gabit Ismailov 3, Daribay Doshetov 4, Juan Daniel Orozco 3, Lucie Blok 5,6, Sabine Rüsch- Gerdes 2, Yared Kebede 5 1 Australian International Health Institute, Melbourne, Australia, 2 Forchungszentrum Borstel, National Reference Center for Mycobacterium, Borstel, Germany, 3 Médecins Sans Frontières, Karakalpakstan, Uzbekistan, 4 Ministry of Health, Nukus, Karakalpakstan, Uzbekistan, 5 Médecins Sans Frontières, Amsterdam, Holland. 6 Royal Tropical Institute, Holland (current

3. Background  2 regions:Karakalpakstan (Uzbekistan) & Dashoguz (Turkmenistan).  Combined population of 2.8 million.  DOTS implemented from 1998-2003  Case notification in 2002 (462/100,000 & 213/100,000 in Karak. and Dash. respectively)

4. (Background) DOTS outcomes for all SS (+) cases Karakalpakstan, 1998-2003 Success Rate 62%

5. (Background) DST survey : July 2001-Jan 2002 Karakalpakstan Type of Resistance New cases (%) (N=106) Re-treatment cases (%) (N=107) TOTAL (N= 213) Mono-resist.14.117.715.9 PDR18.822.421.6 MDR13.240.226.8 Any resistance 48.180.464.3 Dashoguz: MDR rates 4% in new and 18% in re-treatment cases

6. (Background) Drug resistance and treatment outcome in 6 former Soviet Union countries TOT n Success % Failure % MDR % Abkhasia 112657.1%18.7% Karabagh 307717%16% Kemerovo prison 3045231.6%40% Kazakhstan 1155619%37% Karakalpakstan 64555.315%40% Doshoguz 5056912.4%18% *M. Bonnet, V. Sizaire, Y. Kebede et al; Does one size fit all? Drug resistance and standard treatments: results of six tuberculosis programs in former Soviet countries. Int J Tuberc Lung Dis 2005 9(10):1147-1154.

7. Study Objective Quantify the extent of acquired drug resistance during standardized chemotherapy among a cross-sectional sample of patients enrolled in a DOTS TB program in a high resistance setting in Uzbekistan and Turkmenistan

8. Study Design Samples taken from four districts in each region between July 2001 and March 2002. Sputum smear +ve patients were enrolled. Repeat samples for smear positives at end of intensive phase and 2 months into cont. phase obtained Written informed consent was obtained. Patients were started on Cat I (intermittent cont.) & Cat II regimen. Hospitalized during intensive phase DOT by health worker in cont. phase.

9. Laboratory testing and statistical analysis Sputum samples sent to SRL, Borstel, Germany for analysis. DST for H,R,E,S,Z by LJ proportion method and BACTEC 460TB Fingerprinting done using IS6110 All isolates were analyzed by spoligotyping technique. Molecular typing data analyzed with bio- numeric software Epi-info used for data entry and analysis

10. Results Both DNA fingerprinting and DST results were available from 382 patients. Repeat samples obtained from 82 patients. –20 excluded (10 different strain, 5 double infection, 5 lab contamination) 62 infected with same strain as at diagnosis

11. Results Of the 382 patients at diagnosis > 50% of were infected with strains with some resistance A third were infected with strains resistant to 2 or more first line drugs. Overall, 19 of the 62 identical strains (31%) developed new drug resistance during treatment.

12. (Results) Amplification of drug resistance First-line drug resistance profile Diagnosis, number of strains Repeat testing, number of identical strains (%) Amplification detected among identical strains (%, 95% confidence interval) Pan- susceptible 17712 (7%)2 (1.1%, 0.1-4.0) Mono- resistant 7210 (14%)1 (1.4%, 0.0-7.5) Poly- resistant 6516 (25%)11 (17%, 9-28) MDR 6824 (35%)5 (7%, 2-16)

13. Amplification of drug resistance during treatment and treatment outcomes for 19 patients in which amplification was detected. New cases : Category I DOTS treatment 2HREZ(S)/ 4H 3 R 3 PatientDrug R at diagnosis Resistance at end of intensive phase Resistance 2 months into the continuation phase Treatment outcome Amplified drug resistance during treatment. 1SUSCSSFailureS 2HS HRSFailureR 3HS HRSFailureR 4HSHRS FailureR 5HSHRS DefaultR 6HESHRESHRSFailureR 7HSZHESZ CompE 8HRSHRESZ FailureEZ 9HRSHRSZ DiedZ 10HRESHRESZ CuredZ

14. Amplification of drug resistance during treatment and treatment outcomes for 19 patients in which amplification was detected. P reviously treated: Category II DOTS treatment 2HRZES/ 1HREZ/ 5H 3 R 3 E 3 PatientDrug R at diagnosis Resistance at end of intensive phase Resistance 2 months into the continuation phase Treatment outcome Amplified drug resistance during treatment. 11SUSC HRSFailureHRS 12SHS FailureH 13HSHRES FailureRE 14HSZHRESZ DiedRE 15HESHRES FailureR 16HESHRESZ FailureRZ 17HESZ HRSZFailureR 18HR HRESFailureES 19HRESHRESZHRZSDiedZ

15. Reversion of drug resistance Some strains became susceptible to drugs that were initially resistant. 5 strains lost resistance to E 3 strains lost resistance to S

16. Discussion 19 out of 62 patients with identical strains developed additional drug resistance. 17% patients with PDR-TB  amplified resistance further  MDR 7% MDR-TB  developed further 1 st line resistance. Overall 11/314 (3.5%) not initially infected with MDR  developed MDR after SCC. Majority with amplification  poor outcome.

17. Discussion Limited study on drug amplification in SCC Large scale study done in Tomsk (Russia)* –42% not initially infected with MDR-TB and have failed treatment, acquired MDR-TB. –H or R resistance  highest rate of resistance amplification (71% developed MDR-TB) Tomsk study didn’t include molecular typing (DNA finger printing) *Seung KJ et al. The effect of Initial Drug resistance on treatment Response and Acquired Drug Resistance during Standardized Short-Course Chemotherapy for Tuberculosis. CID 2004;39:1321-1328.

18. Discussion Present study included mol. Genotyping Highest rate of resistance amplification among H and S resistant isolates. Beijing genotype* highly prevalent in FSU regions  more susceptible to amplify resistance * Cox HS, Kubica T, Male R, Doshetov D, Kebede Y, Rüsch-Gerdes S, Niemann S. The Beijing genotype and drug resistant tuberculosis in the Aral Sea region of Central Asia. Resp Res 2005, 6:134.

19. Discussion Amplification level under estimated  only smear +ve cases were re-tested. If all samples cultured  additional amplification might have been detected Reversion of resistance to susceptible –Less accurate DST for E and S –DST for E is often problematic –Different resistance profile within same bacillary population

20. Summary Demonstrated high level of drug resistance amplification after DOTS SCC in settings with underlying resistance to first line drugs. Need for projects to weigh additional cost for DST against management of newly created MDR-TB  systematic DST vital. Standard regimen using 2 nd line drugs for patients at risk of MDR amplification required. Further determine, what level of drug resistance prevalence would require routine DST to avoid creation of unnecessary and costly drug resistance. Strategy to reduce risk of amplification through integration of DR-TB management

21. Acknowledgements We would like to thank MSF national and expatriate staff, MOH staff of Karakalpakstan and Doshoguz; and the SRL in Borstel, Germany.