title: Development and application of an in vitro susceptibility test for Acanthamoeba species isolated from keratitis to polyhexamethylene biguanide and chlorhexidine.
abstract: OBJECTIVE
  To develop a reliable in vitro drug susceptibility test against Acanthamoeba isolates and to determine the minimum cysticidal concentration (MCC) of the drug.
  
  
  METHODS
  Doubling dilutions of polyhexamethylene biguanide (PHMB) from 3,200 microg/mL to 3.125 microg/mL and chlorhexidine from 3,200 microg/mL to 1.5625 microg/mL were made in Durham tubes and tested against cysts of 19 Acanthamoeba isolates from keratitis. After the exposure to the drugs for 48 hours, the cysts were washed free of drugs by centrifugation. The deposit (cysts) was cultured on nonnutrient agar plates seeded with heat-killed Escherichia coli. The growth of trophozoites from cysts exposed to each of the dilution was recorded by microscopy to estimate the MCC of the drug.
  
  
  RESULTS
  The minimum cysticidal concentration of PHMB varied from 25 microg/mL to 100 microg/mL and that of chlorhexidine varied from 1.56 microg/mL to 100 microg/mL. The mean MCC value for PHMB was 55.26 microg/mL and that for chlorhexidine 32.81 microg/mL. Minimum cysticidal concentration 50 (MCC50) of PHMB and chlorhexidine on Acanthamoeba isolates was 50.0 microg/mL and 25.0 microg/mL, respectively. Anti-Acanthamoeba activity of chlorhexidine was higher than that of PHMB and this was statistically significant (p = 0.036). The end point of the results of this method was the detection of the viable cysts undergoing excystment and multiplication of trophozoites with a reproducible and clear-cut estimation of the MCC of PHMB and chlorhexidine.
  
  
  CONCLUSIONS
  The in vitro method described can be used as a standard test for assay of MCC of drugs on Acanthamoeba isolates and to study the susceptibility pattern of newer water-soluble anti-Acanthamoeba drugs.
authors.affiliation: Microbiology Research Center, Vision Research Foundation, Sankara Nehralaya, 18 College Road, Chennai 600 006, India.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Narasimhan
authors.forename: Hajib N
authors.initials: HN
authors.lastname: Madhavan
authors.forename: Lily Therese
authors.initials: LT
authors.lastname: K
mesh_terms: D000048:Acanthamoeba / Q000187:drug effects* / Q000302:isolation & purification; D015823:Acanthamoeba Keratitis / Q000469:parasitology*; D000818:Animals; D001645:Biguanides / Q000494:pharmacology*; D002710:Chlorhexidine / Q000494:pharmacology*; D004202:Disinfectants / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009994:Osmolar Concentration; D021261:Parasitic Sensitivity Tests; D013997:Time Factors
chemical_list: D001645:Biguanides; D004202:Disinfectants; C031233:polihexanide; D002710:Chlorhexidine