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Journal of clinical laboratory analysis
Mar, 2019;33 (3): e22716.

Direct detection from clinical sputum samples to differentiate live and dead Mycobacterium Tuberculosis.

Jie Lu, Huiwen Zheng, Ping Chu, Shujing Han, Hui Yang, Zhongdong Wang, Jin Shi, Zuosen Yang
Author Information
  1. Jie Lu: Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children's Hospital, National Center for Children's Health, Capital Medical University, Beijing, China. ORCID
  2. Huiwen Zheng: Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children's Hospital, National Center for Children's Health, Capital Medical University, Beijing, China.
  3. Ping Chu: Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children's Hospital, National Center for Children's Health, Capital Medical University, Beijing, China.
  4. Shujing Han: Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children's Hospital, National Center for Children's Health, Capital Medical University, Beijing, China.
  5. Hui Yang: Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children's Hospital, National Center for Children's Health, Capital Medical University, Beijing, China.
  6. Zhongdong Wang: Qingdao Center for Disease Control and Prevention, Qingdao, China.
  7. Jin Shi: Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children's Hospital, National Center for Children's Health, Capital Medical University, Beijing, China.
  8. Zuosen Yang: Liaoning Provincial Center for Disease Control and Prevention, Shenyang, China.
PMID: 30461054 DOI: 10.1002/jcla.22716

Abstract

BACKGROUND: In this study, we aimed to optimize the condition of propidium monoazide (PMA) treatment for direct detection of Mycobacterium tuberculosis (MTB) from clinical specimens.
METHODS: The light exposure time, dark incubation time, bacterial load, and PMA concentration were varied to determine the optimal condition of PMA treatment.
RESULTS: Overall, the maximum ΔCq value was observed in the group receiving a light exposure time of 20 minutes, which was significantly higher than the others (P < 0.05). The prolongation of dark incubation time seemed more likely to result in greater ΔCq value, and the ΔCq values were 2.0, 4.1, 6.5, 10.1, and 12.7 cycles under dark incubation time of 10, 20, 40, 60, and 120 minutes, respectively. Alternatively, the 4+ samples exhibited favorable detection results at the application of 10 -fold dilution by PMA assay with Cq values higher than 35 cycles. Further evaluation revealed that the PMA assay showed an accordance rate of 98.0% (98/100) among clinical sputa.
CONCLUSIONS: we develop an acceptable method to directly identify the live bacteria from sputum samples. Our data demonstrate that the dark incubation plays a crucial role in the efficacy of PMA treatment for MTB.

Keywords

Mycobacterium tuberculosis dead MTB diagnosis live MTB propidium monoazide

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Grants

  1. 2016-4-1141/Capital's Funds for Health Improvement and Research
  2. 20153079/Beijing Health System Top Level Health Technical Personnel Training Plan

MeSH Term

Bacterial Load
Bacteriological Techniques
Humans
Microbial Viability
Mycobacterium tuberculosis
Sputum
Tuberculosis
Journal Article
Article has an altmetric score of 1

Word Cloud

Created with Highcharts 10.0.0PMAtimeMTBdarkincubationtreatmentdetectionMycobacteriumclinicalΔCq10samplesliveconditionpropidiummonoazidetuberculosislightexposurevaluehighervalues1cyclesassaysputumdeadBACKGROUND:studyaimedoptimizedirectspecimensMETHODS:bacterialloadconcentrationvarieddetermineoptimalRESULTS:Overallmaximumobservedgroupreceiving20 minutessignificantlyothersP < 005prolongationseemedlikelyresultgreater20465127204060120 minutesrespectivelyAlternatively4+exhibitedfavorableresultsapplication-folddilutionCq35evaluationrevealedshowedaccordancerate980%98/100amongsputaCONCLUSIONS:developacceptablemethoddirectlyidentifybacteriadatademonstrateplayscrucialroleefficacyDirectdifferentiateTuberculosisdiagnosis

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