ABSTRACT
Aims @#DNA sequencing is a powerful tool and less time-consuming for bacterial detection and identification. The aim of this study was to compare the application of the Oxford Nanopore MinION™ sequencing device for direct DNA sequencing from clinical specimens with the routine workup. @*Methodology and results @#We used conventional bacteriological-based methods to detect and identify bacterial pathogens in 10 clinical specimens. In addition, the 16S metagenomic sequencing was performed by using a MinION™sequencing device with barcoded primers of a 16S Barcoding kit (Code N° SQK-RAB204, Oxford Nanopore Technologies, UK). The DNA was amplified by PCR using specific 16S primers (27F and 1492R) that contain barcodes and 5' tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters of the 16S Barcoding kit. Data wasanalyzed with WIMP and EPI2ME to classify and identify species in real-time. Ten clinical specimens were processed for bacterial isolation. A total of 8 urine samples were subjected to culture-dependent methods, successfully identifying the presence of pathogenic bacteria. Out of the total eight urine samples, both methods successfully identified six bacterial pathogens. Escherichia coli were identified, and the others were detected as Salmonella enterica, Veillonella parvula and Streptococcus anginosus using MinION™ sequencing. Two urine samples had different results. Escherichia coli was detected directly through MinION™ sequencing, bypassing the need for culture results.@*Conclusion, significance and impact of study@#MinION™ sequencing of 16S rRNA genes could accurately detect diverse bacterial pathogens in clinical specimens. Additionally, the bacterial species classification generated by analyzing 16S rRNA gene sequences can be helpful for rapid identification. The whole procedure takes less than 8 h to complete; same-day diagnosis can be completed.
ABSTRACT
Objectives@#Tuberculosis (TB) and drug-resistant TB (DR-TB) are national health burdens in Vietnam. In this study, we investigated the prevalence of rifampicin (RIF) and/or isoniazid (isonicotinic acid hydrazide, INH) resistance in patients with suspected TB, and applied appropriate techniques to help rapidly target DR-TB. @*Methods@#In total, 1,547 clinical specimens were collected and cultured using the BACTEC MGIT system (Becton Dickinson and Co.). A resazurin microtiter assay (REMA) was used to determine the proportions of RIF and/or INH resistance. A real-time polymerase chain reaction panel with TaqMan probes was employed to identify the mutations of rpoB and katG associated with DR-TB in clinical isolates. Genotyping of the identified mutations was also performed. @*Results@#A total of 468 Mycobacterium tuberculosis isolates were identified using the REMA. Of these isolates, 106 (22.6%) were found to be resistant to 1 or both antibiotics. Of the resistant isolates, 74 isolates (69.8%) were resistant to isoniazid (INH) only, while 1 isolate (0.94%) was resistant to RIF only. Notably, 31 isolates (29.24%) were resistant to both antibiotics. Of the 41 phenotypically INH-resistant isolates, 19 (46.3%) had the Ser315Thr mutation. There were 8 different rpoB mutations in 22 (68.8%) of the RIF-resistant isolates. The most frequently detected mutations were at codons 531 (37.5%), 526 (18.8%), and 516 (6.3%). @*Conclusion@#To help prevent new cases of DR-TB in Vietnam, it is crucial to gain a comprehensive understanding of the genotypic DR-TB isolates.