Whole Genome Sequencing Investigation of a Tuberculosis Outbreak in Port-au-Prince, Haiti Caused by a Strain with a “Low-Level” <i>rpoB</i> Mutation L511P – Insights into a Mechanism of Resistance Escalation

<div><p>The World Health Organization recommends diagnosing Multidrug-Resistant Tuberculosis (MDR-TB) in high burden countries by detection of mutations in Rifampin (RIF) Resistance Determining Region of <i>Mycobacterium tuberculosis rpoB</i> gene with rapid molecular tests GeneXpert MTB/RIF and Hain MTBDR<i>plus</i>. Such mutations are found in >95% of <i>Mycobacterium tuberculosis</i> strains resistant to RIF by conventional culture-based drug susceptibility testing (DST). However routine diagnostic screening with molecular tests uncovered specific “low level” <i>rpoB</i> mutations conferring resistance to RIF below the critical concentration of 1 μg/ml in some phenotypically susceptible strains. Cases with discrepant phenotypic (susceptible) and genotypic (resistant) results for resistance to RIF account for at least 10% of resistant diagnoses by molecular tests and urgently require new guidelines to inform therapeutic decision making. Eight strains with a “low level” <i>rpoB</i> mutation L511P were isolated by GHESKIO laboratory between 2008 and 2012 from 6 HIV-negative and 2 HIV-positive patients during routine molecular testing. Five isolates with a single L511P mutation and two isolates with double mutation L511P&M515T had MICs for RIF between 0.125 and 0.5 μg/ml and tested susceptible in culture-based DST. The eighth isolate carried a double mutation L511P&D516C and was phenotypically resistant to RIF. All eight strains shared the same spoligotype SIT 53 commonly found in Haiti but classic epidemiological investigation failed to uncover direct contacts between the patients. Whole Genome Sequencing (WGS) revealed that L511P cluster isolates resulted from a clonal expansion of an ancestral strain resistant to Isoniazid and to a very low level of RIF. Under the selective pressure of RIF-based therapy the strain acquired mutation in the M306 codon of <i>embB</i> followed by secondary mutations in <i>rpoB</i> and escalation of resistance level. This scenario highlights the importance of subcritical resistance to RIF for both clinical management of patients and public health and provides support for introducing <i>rpoB</i> mutations as proxy for MICs into laboratory diagnosis of RIF resistance. This study illustrates that WGS is a promising multi-purpose genotyping tool for high-burden settings as it provides both “gold standard” sequencing results for prediction of drug susceptibility and a high-resolution data for epidemiological investigation in a single assay.</p>