title: Evaluation of bleach-sedimentation for sterilising and concentrating Mycobacterium tuberculosis in sputum specimens.
abstract: BACKGROUND
  Bleach-sedimentation may improve microscopy for diagnosing tuberculosis by sterilising sputum and concentrating Mycobacterium tuberculosis. We studied gravity bleach-sedimentation effects on safety, sensitivity, speed and reliability of smear-microscopy.
  
  
  METHODS
  This blinded, controlled study used sputum specimens (n = 72) from tuberculosis patients. Bleach concentrations and exposure times required to sterilise sputum (n = 31) were determined. In the light of these results, the performance of 5 gravity bleach-sedimentation techniques that sterilise sputum specimens (n = 16) were compared. The best-performing of these bleach-sedimentation techniques involved adding 1 volume of 5% bleach to 1 volume of sputum, shaking for 10-minutes, diluting in 8 volumes distilled water and sedimenting overnight before microscopy. This technique was further evaluated by comparing numbers of visible acid-fast bacilli, slide-reading speed and reliability for triplicate smears before versus after bleach-sedimentation of sputum specimens (n = 25). Triplicate smears were made to increase precision and were stained using the Ziehl-Neelsen method.
  
  
  RESULTS
  M. tuberculosis in sputum was successfully sterilised by adding equal volumes of 15% bleach for 1-minute, 6% for 5-minutes or 3% for 20-minutes. Bleach-sedimentation significantly decreased the number of acid-fast bacilli visualised compared with conventional smears (geometric mean of acid-fast bacilli per 100 microscopy fields 166, 95%CI 68-406, versus 346, 95%CI 139-862, respectively; p = 0.02). Bleach-sedimentation diluted paucibacillary specimens less than specimens with higher concentrations of visible acid-fast bacilli (p = 0.02). Smears made from bleach-sedimented sputum were read more rapidly than conventional smears (9.6 versus 11.2 minutes, respectively, p = 0.03). Counting conventional acid-fast bacilli had high reliability (inter-observer agreement, r = 0.991) that was significantly reduced (p = 0.03) by bleach-sedimentation (to r = 0.707) because occasional strongly positive bleach-sedimented smears were misread as negative.
  
  
  CONCLUSIONS
  Gravity bleach-sedimentation improved laboratory safety by sterilising sputum but decreased the concentration of acid-fast bacilli visible on microscopy, especially for sputum specimens containing high concentrations of M. tuberculosis. Bleach-sedimentation allowed examination of more of each specimen in the time available but decreased the inter-observer reliability with which slides were read. Thus bleach-sedimentation effects vary depending upon specimen characteristics and whether microscopy was done for a specified time, or until a specified number of microscopy fields had been read. These findings provide an explanation for the contradictory results of previous studies.
authors: IFHAD: Innovation For Health And Development, London, UK. carlton.evans@imperial.ac.uk.
authors: Rusheng
authors: R
authors: Chew
authors: Carmen
authors: C
authors: Calderón
authors: Samuel G
authors: SG
authors: Schumacher
authors: Jonathan M
authors: JM
authors: Sherman
authors: Luz
authors: L
authors: Caviedes
authors: Patricia
authors: P
authors: Fuentes
authors: Jorge
authors: J
authors: Coronel
authors: Teresa
authors: T
authors: Valencia
authors: Beatriz
authors: B
authors: Hererra
authors: Mirko
authors: M
authors: Zimic
authors: Lucy
authors: L
authors: Huaroto
authors: Ivan
authors: I
authors: Sabogal
authors: A Rod
authors: AR
authors: Escombe
authors: Robert H
authors: RH
authors: Gilman
authors: Carlton A
authors: CA
authors: Evans
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D002498:Centrifugation / Q000379:methods*; D004202:Disinfectants / Q000494:pharmacology; D004203:Disinfection / Q000379:methods*; D006801:Humans; D008853:Microscopy / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000302:isolation & purification*; D015588:Observer Variation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012973:Sodium Hypochlorite / Q000494:pharmacology; D013048:Specimen Handling / Q000379:methods*; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004202:Disinfectants; D012973:Sodium Hypochlorite