title: Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay.
abstract: The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.
authors.affiliation: Laboratory of Molecular Biology and Diagnosis of Infectious Diseases, Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Avenida Brasil 4365, Manguinhos 21045-900, Rio de Janeiro, Brazil.
authors.forename: Martha M
authors.initials: MM
authors.lastname: de Oliveira
authors.forename: Adalgiza
authors.initials: A
authors.lastname: da Silva Rocha
authors.forename: Maraníbia
authors.initials: M
authors.lastname: Cardoso Oelemann
authors.forename: Harisson M
authors.initials: HM
authors.lastname: Gomes
authors.forename: Leila
authors.initials: L
authors.lastname: Fonseca
authors.forename: Angela M
authors.initials: AM
authors.lastname: Werneck-Barreto
authors.forename: Andréia Moura
authors.initials: AM
authors.lastname: Valim
authors.forename: Maria L
authors.initials: ML
authors.lastname: Rossetti
authors.forename: Rudi
authors.initials: R
authors.lastname: Rossau
authors.forename: Wouter
authors.initials: W
authors.lastname: Mijs
authors.forename: Bart
authors.initials: B
authors.lastname: Vanderborght
authors.forename: Philip
authors.initials: P
authors.lastname: Suffys
mesh_terms: D000904:Antibiotics, Antitubercular / Q000494:pharmacology; D001938:Brazil; D024881:Drug Resistance, Bacterial; D005838:Genotype; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014376:Tuberculosis / Q000188:drug therapy* / Q000382:microbiology
chemical_list: D000904:Antibiotics, Antitubercular; D012293:Rifampin