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Development and Application of an In Vitro Susceptibility Test for Acanthamoeba Species Isolated From Keratitis to Polyhexamethylene Biguanide and Chlorhexidine

Narasimhan, Sandhya M.S.-MLT.; Madhavan, Hajib N. M.D., Ph.D.; K, Lily Therese Ph.D.

Basic Investigations

Purpose. 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 μg/mL to 3.125 μg/mL and chlorhexidine from 3,200 μg/mL to 1.5625 μg/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 μg/mL to 100 μg/mL and that of chlorhexidine varied from 1.56 μg/mL to100 μg/mL. The mean MCC value for PHMB was 55.26 μg/mL and that for chlorhexidine 32.81μg/mL. Minimum cysticidal concentration 50 (MCC50) of PHMB and chlorhexidine on Acanthamoeba isolates was 50.0 μg/mL and 25.0 μg/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.

Conclusion. 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.

From Vision Research Foundation, Chennai, India.

Submitted April 25, 2001.

Revision received September 27, 2001.

Accepted October 3, 2001.

Address correspondence and reprint requests to Dr. H.N. Madhavan, Microbiology Research Center, Vision Research Foundation, Sankara Nethralaya, 18 College Road, Chennai 600 006, India. E-mail:

© 2002 Lippincott Williams & Wilkins, Inc.