1. Evaluation of Tuberculosis Drug Resistance among Nigerian HIV+ Patients in the Harvard PEPFAR/APIN Plus Program Using the Genotype MTBDRplus Test Lana Dinic December 7 th 2011 ICASA, Addis Ababa, Ethiopia

2. Tuberculosis in Nigeria MDR-TB, Estimates among notified cases20082007 % of new TB cases with MDR-TB1.8 (0.0–4.3)1.8 % of retreatment TB cases with MDR-TB7.7 (0.0–18)9.4 Global Tuberculosis Control 2010 and 2009. WHO Population 2009 (millions) 155 Estimates of burden * 2009Number (thousands)Rate (per 100 000 pop) Mortality (excluding HIV)100 (82–130)67 (53-84) Prevalence (incl HIV)770 (360–1 300)497 (231-811) Incidence (incl HIV)460 (370–550)295 (240-356) Incidence (HIV-positive)120 (95–140)75 (62-91) Case detection, all forms (%)19 (16–24)

3. Tuberculosis in Nigeria MDR-TB, Estimates among notified cases20082007 % of new TB cases with MDR-TB1.8 (0.0–4.3)1.8 % of retreatment TB cases with MDR-TB7.7 (0.0–18)9.4 Global Tuberculosis Control 2010 and 2009. WHO Population 2009 (millions) 155 Estimates of burden * 2009Number (thousands)Rate (per 100 000 pop) Mortality (excluding HIV)100 (82–130)67 (53-84) Prevalence (incl HIV)770 (360–1 300)497 (231-811) Incidence (incl HIV)460 (370–550)295 (240-356) Incidence (HIV-positive)120 (95–140)75 (62-91) Case detection, all forms (%)19 (16–24)

4. Genotype MDRTBplus (HAIN’s test) Detect mutations in – rpoB gene – katG gene – inhA promoter 3 steps –DNA isolation –Amplification with biotinylated primers –Reverse hybridization ~ $1000 for 96 wells/stripes (Retail Price) Detects presence of MTb complex simultaneously

5. Genotype MDRTBplus (HAIN’s test) Detect mutations in – rpoB gene – katG gene – inhA promoter 3 steps –DNA isolation –Amplification with biotinylated primers –Reverse hybridization ~ $1000 for 96 wells/stripes (Retail Price) Detects presence of MTb complex simultaneously

6. Genotype MDRTBplus (HAIN’s test) Detect mutations in – rpoB gene – katG gene – inhA promoter 3 steps –DNA isolation –Amplification with biotinylated primers –Reverse hybridization ~ $1000 for 96 wells/stripes (Retail Price) Detects presence of MTb complex simultaneously

7. Genotype MDRTBplus (HAIN’s test) Detect mutations in – rpoB gene – katG gene – inhA promoter 3 steps –DNA isolation –Amplification with biotinylated primers –Reverse hybridization ~ $1000 for 96 wells/stripes (Retail Price) Detects presence of MTb complex simultaneously

8. Lagos, Lagos State Jos, Plateau State Treatment naïve Retreated Jos 13437 Lagos 90 17 Study setting and sample size Enrollment period: 12 mo. Map courtesy of Beth Chaplin

9. Characteristics of the patients enrolled Location Total patients Previous TB treatment exposure Median age (years) Male to female ratio Average length of symptoms Jos, Plateau state North Central region 13437 35.98 (sd 8.97) 1:1.16 7.36 weeks (CI 5.40, 9.33) 123 observations Lagos, Lagos state South West region 9017 35.62 (sd 7.94) 1:1.81 3.36 weeks (CI 2.78, 3.94) 86 observations

10. Characteristics of the patients enrolled Location Total patients Previous TB treatment exposure Median age (years) Male to female ratio Average length of symptoms Jos, Plateau state North Central region 13437 35.98 (sd 8.97) 1:1.16 7.36 weeks (CI 5.40, 9.33) 123 observations Lagos, Lagos state South West region 9017 35.62 (sd 7.94) 1:1.81 3.36 weeks (CI 2.78, 3.94) 86 observations

11. LocationINHRIFMDR-TBTotal Jos, Plateau state North Central region 3/127 2.36% 4/128 3.12% 2/123 1.62% 5/128 3.90% Lagos, Lagos state South West region 9/76 11.9% 11/70 15.7% 6/63 9.52% 14/76 18.42% Fisher’s exact (Jos vs. Lagos) (P-value) <0.014<0.005<0.025<0.001 Prevalence of drug resistant tuberculosis

12. LocationINHRIFMDR-TBTotal Jos, Plateau state North Central region 3/127 2.36% 4/128 3.12% 2/123 1.62% 5/128 3.90% Lagos, Lagos state South West region 9/76 11.9% 11/70 15.7% 6/63 9.52% 14/76 18.42% Fisher’s exact (Jos vs. Lagos) (P-value) <0.014<0.005<0.025<0.001 Prevalence of drug resistant tuberculosis The rates of drug resistant tuberculosis are much higher in Lagos than in Jos

13. Drug resistant tuberculosis in previously treated vs. treatment naïve patients TB treatment statusINHRIFMDR-TB Previously Treated 4/466 /443/39 8.69%*13.64%**7.69%*** Treatment naive 8 /1579 /1545 /147 5.09%*5.84%**3.40%***

14. Drug resistant tuberculosis in previously treated vs. treatment naïve patients TB treatment statusINHRIFMDR-TB Previously Treated 4/466 /443/39 8.69%*13.64%**7.69%*** Treatment naive 8 /1579 /1545 /147 5.09%*5.84%**3.40%*** Rifampicin resistance (indicative of MTB-DR) in treatment naïve patients exceeds the CI of modeled predictions Difference between groups not significantly different (* p<0.480, **p<0.124, ***p<0.374)

15. Gene mutations of drug resistant Mycobacterium tuberculosis (Genotype MTBDRplus) Genotype MTBDRplus pattern WT: missing MUT: present Codons analyzed by the Genotype MTBDRplus Number of isolates JosLagos rpoB*WT7526-529 2 WT7 & MUT2AH526Y 1 WT7, MUT2BH526D 1 WT8530-533 1 WT8, MUT3S531L 7 MUT3S531L 11 WT4 & WT8; MUT1516-519 & 530-533; D516V 1 katGWT, MUT1S315T1 24 inhAWT1, MUT1c −15 t 13 MUT1c −15 t 1 MUT2a −16 g 1

16. Gene mutations of drug resistant Mycobacterium tuberculosis (Genotype MTBDRplus) Genotype MTBDRplus pattern WT: missing MUT: present Codons analyzed by the Genotype MTBDRplus Number of isolates JosLagos rpoB*WT7526-529 2 WT7 & MUT2AH526Y 1 WT7, MUT2BH526D 1 WT8530-533 1 WT8, MUT3S531L 7 MUT3S531L 11 WT4 & WT8; MUT1516-519 & 530-533; D516V 1 katGWT, MUT1S315T1 24 inhAWT1, MUT1c −15 t 13 MUT1c −15 t 1 MUT2a −16 g 1

17. Gene mutations of drug resistant Mycobacterium tuberculosis (Genotype MTBDRplus) Genotype MTBDRplus pattern WT: missing MUT: present Codons analyzed by the Genotype MTBDRplus Number of isolates JosLagos rpoB*WT7526-529 2 WT7 & MUT2AH526Y 1 WT7, MUT2BH526D 1 WT8530-533 1 WT8, MUT3S531L 7 MUT3S531L 11 WT4 & WT8; MUT1516-519 & 530-533; D516V 1 katGWT, MUT1S315T1 24 inhAWT1, MUT1c −15 t 13 MUT1c −15 t 1 MUT2a −16 g 1

18. Gene mutations of drug resistant Mycobacterium tuberculosis (Genotype MTBDRplus) * Mutation appearing among codons 526-529 in the rpoB gene are associated with Jos while codons 530-533 are associated with Lagos (Fisher’s exact test p<0.033) Genotype MTBDRplus pattern WT: missing MUT: present Codons analyzed by the Genotype MTBDRplus Number of isolates JosLagos rpoB*WT7526-529 2 WT7 & MUT2AH526Y 1 WT7, MUT2BH526D 1 WT8530-533 1 WT8, MUT3S531L 7 MUT3S531L 11 WT4 & WT8; MUT1516-519 & 530-533; D516V 1 katGWT, MUT1S315T1 24 inhAWT1, MUT1c −15 t 13 MUT1c −15 t 1 MUT2a −16 g 1

19. Conclusions High rates of transmission of drug-resistant TB among the population – Transmitted RIF resistance higher than predicted [5.84% > (0 - 4.3%)] – Geographically distinct mutations in the rpoB gene Prevalence much higher in Lagos than Jos – Dense urban area, tight living quarters, public transportation – National reference laboratory (could be referrals)

20. Thank you All the patients for the participation in this study Co-authors: Akande, Patrick 2, Ani, Agatha 3, Akanbi, Maxwell 3, Adu, Rosemary 4, Agbaji, Oche 3, Onwujekwe, Dan 4, Adeniyi, Bukola 3, Nwosu, Rita 4, Lekuk, Chindak 3, Wahab, Maureen 4, Efere, Lauretta 4, Nwadike, Peter 4, Abiodu, Tope 4, Kunle-Ope, Chioma 4, Nwokoye, Nkiru 4, Onyejepu, Nneka 4, Idigbe, Emmanuel Oni 4, Kanki, Phyllis 1 – Institute(s): 1 Harvard School of Public Health, Immunology and Infectious Diseases, Boston, United States, 2 AIDS Prevention Initiative in Nigeria, Abuja, Nigeria, 3 Jos University Teaching Hospital, Jos, Nigeria, 4 Nigerian Institute of Medical Research (NIMR), Lagos, Nigeria Funders: – Harvard PEPFAR / APINPlus (patient support) – Uwe Brinkmann Travel Grant, Joyce and Zlatko Balokovic Scholarshipm, Science and Engineering Committee (LD research support) – ICASA Conference Scholarship Mentorship: – Prof. Kanki – Dr. Idigbe and Prof. Idoko – The Kanki lab – Prof. Lee, Hirsch, Rubin