This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423-to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culturenegative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.The demonstrated pathogenicity for humans and animals of organisms belonging to the genus Acanthamoeba (17, 26), coupled with the difficulty of using morphological criteria for subgeneric identification of isolates (30,38), has stimulated a number of laboratories to pursue molecular methods for detection and identification. The objective is to develop methods that are suitable for both clinical and environmental applications. The identification of amoebic isolates should be very reliable and, at least for clinical use, the detection system should be very sensitive. Several research groups, including our own, have demonstrated the usefulness of PCR methods for detection of acanthamoebae (10,15,21,25,27,40). As few as 1 to 10 trophozoites can be detected. It also is possible to enhance detection of individual amoeba in very dilute liquid clinical samples with fluorescent in situ hybridization (FISH) (36). Several molecular approaches increase the reliability of specimen identification, but the use of DNA sequence variation appears to be the most promising. The variation is observed in restriction fragment length polymorphisms of complete or partial nuclear 18S rRNA genes (8,20,21,22), of complete mitochondrial 16S rRNA genes (7, 46), and of the complete mitochondrial genome (3,7,13,18,22,45). It also is observed in the DNA seq...

Purpose To establish the incidence, etiology and risk factors for microbial keratitis (MK) in Hong Kong. Methods Two hundred and twenty-three new cases of presumed MK were recruited over a period of 17 months and comprehensive microbiologic studies performed. A nested case-control study was pursued for patients wearing contact lenses (CLW) to determine risk factors for MK with regards to types of CLW and hygiene practice. Results Of the 223 patients recruited, 59 (26%) wore contact lenses. Corneal scrapes yielded positive cultures from 77 patients (35% overall, 56 non-CLW, 21 CLW). Two hundred and six CLW volunteers were recruited to participate in the case-control study, of whom 135 were matched with 45 CLW patients. The annual incidence of MK was 0.63 per 10 000 population and 3.4 per 10 000 CLW with rates for daily, extended and rigid lens wear of 3.09, 9.30 and 0.44 per 10 000 CLW respectively. Pseudomonas aeruginosa was the dominant bacterial pathogen. Six cases of Acanthamoeba keratitis occurred, five in CLW (incidence 0.33 per 10 000 CLW) and one following corneal abrasion. Non-CLW developed MK at a peak age of 73, which is 10 years younger than expected for Scotland and USA. Conclusions Previous ocular surface disease and trauma were the main risk factors for MK in Hong Kong. CLW appears at least as safe as that found in Scotland and the USA. Acanthamoeba keratitis was detected but with an incidence rate five times lower than Scotland. Factors predisposing hydrogel CLWs to MK, that were statistically significant, included overnight wear, poor hygiene and smoking.

We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of approximately 113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections.

A reliable figure for the expected incidence of

Intracameral cefuroxime administered at the time of surgery significantly reduced the risk for developing endophthalmitis after cataract surgery.

Microbial keratitis has been studied in Hong Kong as a representative sub-tropical climate of south China. An 18-month investigation in 1997/98 of 223 cases of ulcerative keratitis (presumed microbial) was conducted in the 2 million population of Shatin and Kowloon at the Prince of Wales and Hong Kong Eye Hospitals respectively with comprehensive microbiology. A case-control study was pursued at the same time between 45 contact-lens wearers (CLW) developing microbial keratitis and 135 lens-wearing volunteers matched for age, sex, educational status and visual acuity. Home water supplies were sampled for Acanthamoeba. Previous ocular surface disease and trauma (preventable by wearing goggles for grinding) were common predisposing causes while cosmetic wear of contact lenses was responsible for 26% of cases overall. Pseudomonas aeruginosa was the commonest bacterium isolated, from both CLW and non-CLW, with infection being acquired within the community. These 28 pseudomonads remained fully sensitive to the third-generation cephalosporins, aminoglycosides and quinolone antibiotics, which is very encouraging. Fungi were isolated, predominantly Fusarium sp., but less commonly than expected. A fungal/bacterial ratio was obtained of 1/17, while in comparison, the expected ratio for a tropical climate ranges from 1/5 (Singapore) to 1/2 (South India). Acanthamoeba was the second commonest microbe isolated from keratitis of CLW. The domestic water environment of 8% of homes of both patients and controls wearing contact lenses was colonized with Acanthamoeba. Lack of hygiene, use of tap water for storing lenses, failure to air-dry lens-storage cases or use of one-step hydrogen peroxide disinfectant were identified as risk factors for keratitis in CLW. The study results commend use of multipurpose solutions by CLW in Hong Kong to achieve the lowest expected rates of infection.

SUMMARYTrophozoites and cysts of 20 isolates of Acanthamoeba from the cornea and five from related samples were tested in vitro for sensitivity to ten drugs (three aromatic dia midines, two aminoglycosides, two macrolides, a polyene macrolide antibiotic, an organoarsenical and an anti metabolite) and two cationic antiseptics (chlorhexidine and polyhexamethylene biguanide, PHMB). Keratitis associated with Acanthamoeha infection is a relatively rare, sight-threatening condition occurring most often in contact lens wearers, I where there has been inappropriate or inadequate disinfection of contact lens systems." The clinical presentation of the disease is often mistakenly diagnosed as herpes simplex or fungal infec tion.3 This results in inappropriate anti-microbial agents being administered. Early features4-6 such as pain, photo phobia and recurrent epithelial breakdown with little infil-

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