Davi Josué Marcon ^1,2* Abhinav Sharma ³ Alex Brito Souza ^1,2* Rafaella Bonfim Barros ^4* Valnete das Graças Dantas Andrade ^4* Ricardo Jose de Paula Souza e Guimaraes ⁵ Luana Nepomucemo Gondim Lima ^1,2* Lúcia Helena Martins Tavares Monteiro ^6* Ana Judith Pires Garcia Quaresma ^1,2* Layana Rufino Ribeiro ^2* PHILIP NOEL SUFFYS ⁷ Robin Mark Warren ³ Carlos Augusto Abreu Albério ⁸ Karla Valéria Batista Lima ^1,2* Emilyn Costa Conceição ^3*

• ¹ Programa de Pós graduação em Biologia Parasitária na Amazônia, Belém, Pará, Brazil
• ² Seção de Bacteriologia do Instituto Evandro Chagas, Anaindeua, Pará, Brazil
• ³ DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
• ⁴ Laboratório Central do Estado do Pará, Belém, Brazil
• ⁵ Seção de Epidemiologia do Instituto Evandro Chagas, Ananindeua, Pará, Brazil
• ⁶ Secretaria de Estado da Saúde do Pará, Belém, Pará, Brazil
• ⁷ Laboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
• ⁸ Hospital Universitário João de Barros Barreto (HUJBB), Belém, Pará, Brazil

Bedaquiline, an antimicrobial used to treat drug-resistant tuberculosis (DR-TB), was introduced in Brazil in October 2021. Monitoring the emergence and transmission of DR-TB is crucial for implementing public health to control the spread of DR strains of Mycobacterium tuberculosis. We aimed to conduct genomic surveillance of DR-TB after bedaquiline was introduced in Brazil, to measure its impact on the multi-drug treatment scheme in the state of Pará. Individuals treated for DR-TB between October/2021 and December/2022, in the reference hospital to treat DR-TB cases from the state of Pará, were included in the study. Clinical and bacteriological information was obtained from the National Laboratory Management Environment and the Special TB Treatment Information System. Genomic DNA was extracted from bacterial cultures performed at the Pará Central Laboratory (LACEN-PA). Whole-genome sequencing (WGS) was obtained using Illumina Nextera-XT and NextSeq 550 and genomes were analyzed using the MAGMA and TB-Profiler pipelines interpreted according to the World Health Organization (WHO) mutations catalogue 2nd edition. Geoprocessing was performed based on the patient's residences. Cutoffs of 5-12 Single Nucleotide Polymorphisms (SNPs) were used for transmission analysis. From the 103 patients reported as DR-TB, viable cultures were obtained from 67. Forty isolates were selected randomly for WGS. Among these, a mixed infection of M. tuberculosis L1 and L4 and a co-infection of M. tuberculosis and Mycobacterium chelonae was observed. The genotypic drug susceptibility profile of TB stains (39/40) was as follows: sensitive (1/2,5%), rifampicin mono-resistant (RR) (4/10%), isoniazid mono-resistant (1/2%), multidrug-resistant (MDR) (21/52%), extensively drug-resistant (XDR) (3/7%), pre-XDR (8/20%) and other (1/2%). Among the 38 isolates M. tuberculosis strains without mixed infection, using a cut-off of 12 SNPs and suggestive of recent TB transmission, 14 (37%) were grouped into five clusters (C1-C5), and included RR (C5), MDR (C3, C4, C5), pre-XDR and XDR (C2) strains. We recommend greater attention from the regional public health authorities to detect and track resistance to new drugs, especially in areas with pre-XDR and XDR cases. This is the first report on the detection and transmission of XDR-TB in Pará, Brazil after the recent re-definition of XDR-TB by the WHO in 2021.