Cryptic Microheteroresistance Explains Mycobacterium tuberculosis Phenotypic Resistance.
Am J Respir Crit Care Med
; 196(9): 1191-1201, 2017 11 01.
Article
en En
| MEDLINE
| ID: mdl-28614668
ABSTRACT
RATIONALE Minority drug-resistant Mycobacterium tuberculosis subpopulations can be associated with phenotypic resistance but are poorly detected by Sanger sequencing or commercial molecular diagnostic assays. OBJECTIVES:
To determine the role of targeted next-generation sequencing in resolving these minor variant subpopulations.METHODS:
We used single molecule overlapping reads (SMOR), a targeted next-generation sequencing approach that dramatically reduces sequencing error, to analyze primary cultured isolates phenotypically resistant to rifampin, fluoroquinolones, or aminoglycosides, but for which Sanger sequencing found no resistance-associated variants (RAVs) within respective resistance-determining regions (study group). Isolates also underwent single-colony selection on antibiotic-containing agar, blinded to sequencing results. As a positive control, isolates with multiple colocalizing chromatogram peaks were also analyzed (control group). MEASUREMENTS AND MAINRESULTS:
Among 61 primary culture isolates (25 study group and 36 control group), SMOR described 66 (49%) and 45 (33%) of 135 total heteroresistant RAVs at frequencies less than 5% and less than 1% of the total mycobacterial population, respectively. In the study group, SMOR detected minor resistant variant subpopulations in 80% (n = 20/25) of isolates with no Sanger-identified RAVs (median subpopulation size, 1.0%; interquartile range, 0.2-3.9%). Single-colony selection on drug-containing media corroborated SMOR results for 90% (n = 18/20) of RAV-containing specimens, and the absence of RAVs in 60% (n = 3/5) of isolates. Overall, Sanger sequencing was concordant with SMOR for 77% (n = 53/69) of macroheteroresistant (5-95% total population), but only 5% of microheteroresistant (<5%) subpopulations (n = 3/66) across both groups.CONCLUSIONS:
Cryptic minor variant mycobacterial subpopulations exist below the resolving capability of current drug susceptibility testing methodologies, and may explain an important proportion of false-negative resistance determinations.Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Análisis de Secuencia
/
Farmacorresistencia Bacteriana
/
Mycobacterium tuberculosis
Idioma:
En
Revista:
Am J Respir Crit Care Med
Asunto de la revista:
TERAPIA INTENSIVA
Año:
2017
Tipo del documento:
Article