Prevalence of Acanthamoeba and other naked amoebae in South Florida domestic water.

The purpose was to identify the prevalence of naked amoebae in tap water in south Florida to ascertain the risk of amoebal infections of the cornea in contact lens wearers. Over the course of a 2-year period, water samples were collected from sites throughout Broward, Palm Beach, and Dade counties, Florida. The presence of amoebae in samples was based on an enrichment cultivation method appropriate for Acanthamoeba. Amoebae were identified using diagnostic features discernable by light microscopy. A total of 283 water samples were processed and amoebae were noted in 80 of these. Acanthamoeba were found on 8 occasions (2.8%). The genera Hartmannella and Vahlkampfia, rarely involved in keratitis cases, were found in 3.5% and 2.8% of samples, respectively. A total of 19 different naked amoebae were recorded and amoebae (regardless of genus) were present in 19.4% of all samples. Previous surveys in England and Korea have shown that acanthamoebae are found in 15 to 30% of tap water samples in the home and have been associated with corneal infection in contact lens wearers. The incidence of acanthamoebae infection in the USA (2.8%) has been found to be lower than that in the UK and it has been postulated that this is related to the lack of a storage water tank in the roof loft space. However, the level of treatment of municipal water is clearly not effective at killing amoebal cysts (or trophozoites) as evidenced by the high occurrence of amoebae (19.4%) in this study.

Acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment.

A reliable figure for the expected incidence of Acanthamoeba keratitis of one per 30000 contact lens wearers per year has now been obtained from a combination of three cohort and three Questionnaire Reporting Surveys; 88% of cases wore hydrogel lenses and 12% wore rigid lenses. This figure now provides a basis for the expected number of cases against which to judge either epidemic outbreaks or effects of prevention with disinfecting solutions, better hygiene, or the use of disposable lenses. Molecular biology of Acanthamoeba has advanced considerably in the last 10 years with new automated sequencing technology. This has allowed the construction of a genotype identification scheme with 13 different genotypes against which to compare clinical isolates for epidemiological investigations or pathogenicity markers. So far, only four genotypes have been associated with keratitis of which the majority have been T4 but T3, T6, and T11 have each caused individual cases. Each genotype is heterogenous and can be further subdivided by comparison of sequences of diagnostic fragments of 18S rDNA, riboprinting by PCR-RFLP of 18S rDNA, or by mitochondrial DNA RFLP. Drug therapy has been revolutionised with the introduction of the biguanides-chlorhexidine or polyhexamethylene biguanide-with most but not all infections quickly resolving. Failure can still occur occasionally and further research is needed on more effective combination chemotherapy. A number of guanidines have been identified in this paper that could be usefully pursued as part of combination chemotherapy along with the alkylphosphocholines.

18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong.

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.

Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

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 culture-negative 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.

Necrotizing fasciitis.

Necrotizing fasciitis continues to occur due to beta-haemolytic streptococci but is now also recognized as being due to Vibrio spp. in fishermen and those in contact with warm water in the Gulf of Mexico and South-East Asia, including Hong Kong. Magnetic resonance image scanning has identified the extent of fasciitis and soft tissue oedema infiltrating fascial planes prior to necrosis presenting clinically and is a useful tool in early diagnosis. Surgical debridement or incisional drainage remains essential. An enhanced bactericidal response against beta-haemolytic streptococci has been found with a combination of penicillin and clindamycin. Intravenous immunoglobulin has been shown to reduce mortality if the necrotizing fasciitis is associated with the toxic shock syndrome, by decreasing the superantigen activity of the beta-haemolytic streptococci on cytokine release by T cells.

Salicylate inhibition of Acanthamoeba attachment to contact lenses: a model to reduce risk of infection.

PURPOSE: Acanthamoeba attachment (adsorption) to hydrogel contact lenses is enhanced by Pseudomonas aeruginosa biofilm. The effect of sodium salicylate on Acanthamoeba attachment to biofilm-coated and uncoated hydrogel lenses was investigated. DESIGN: Experimental study. PARTICIPANTS AND CONTROLS: A minimum of 16 replicates were used for each test condition; a control condition using clean lenses without biofilm was included. METHODS: Four groups of hydrogel contact lenses (etafilcon A) were pretreated with P. aeruginosa to form a biofilm. In addition, two more groups remained untreated. Quartered lenses of all six groups were then incubated in a suspension of A. castellanii trophozoites. Two batches of lenses had either 3 or 30 mM sodium salicylate added to the bacterial suspension (stage 1 intervention). Two other batches of lenses had salicylate added to the amoebal suspension (stage 2 intervention). One of the batches, which had a stage 1 intervention, had salicylate added at the second stage as well. The remaining batches received no salicylate exposure and included lenses with and without biofilm coating. MAIN OUTCOME MEASURE: The outcome measure in this study was the number of Acanthamoeba trophozoites attached, per square centimeter, to the hydrogel surfaces. RESULTS: Biofilm coating from P. aeruginosa gave a significantly increased attachment of A. castellanii trophozoites to the contact lens. When introduced at a first (biofilm) stage, second (trophozoite attachment) stage, or with intervention at both stages, 30 mM sodium salicylate reduced amoebal attachment to the hydrogel lens. When applied to both stages and when applied at stage 2 to the biofilm coated contact lenses, 3 mM sodium salicylate reduced amoebal attachment. The 3 mM concentration was not effective for the lower level of amoebae attachment to uncoated (nonbiofilm) lenses. CONCLUSIONS: Sodium salicylate successfully reduced amoebal trophozoite attachment to hydrogel lenses. This was the result of one of the following possibilities or a combination thereof: inhibition of biofilm formation; a direct effect on the amoebae; an alteration in the biofilm-amoebal attachment and resulting modification of the hydrogel lens surface. The results of this study suggest the major action is at stage 2 (on amoebal attachment to lenses) and favors alteration of the biofilm-amoebal attachment mechanism. This study demonstrates salicylate's potential benefit as a component of contact lens care solutions, designed to reduce microbial attachment and the risk of infection.

Disinfection of contact lenses without tap water rinsing: is it effective?

PURPOSE: To establish the efficacy of the two most popular contact lens disinfecting systems--one-step hydrogen peroxide and multipurpose disinfecting solution--for 1 month's use in practice in the absence of tap water rinsing. METHODS: This was a descriptive, prospective microbiological study of contact lens contamination with ideal hygiene compliance and new lenses and storage cases. One hundred and fifty contact lens wearers were instructed to avoid risk factors identified for Acanthamoeba infection. They were randomly assigned to use one of three disinfecting systems and taught to follow manufacturers' instructions. In addition, they were taught to avoid all use of tap water for contact lens hygiene, except for hand washing. RESULTS: There was no isolation of Acanthamoeba from any lens storage case, precluding the chance of amoebic infection. The multi-purpose solution gave the lowest rate of bacterial contamination, with 78% sterility and 15% of cases with < 10(4) bacteria/ml. For both one-step peroxide and multi-purpose solutions, Gram-negative bacteria were reduced in frequency compared with values expected historically, while Bacillus sp. were found more frequently. Storage cases of both one-step peroxide systems leaked fluid. CONCLUSIONS: On the basis of contamination in previous studies, when hydrogen peroxide and other chemical disinfectants were used together with tap water washing, it was expected that approximately 40% of lens storage cases would yield bacteria, often with a high count, and that up to 8% would yield Acanthamoeba. Such contamination did not occur, however, in this study. The multipurpose solution, for 1 month's use, gave the lowest rate of bacterial contamination with only 7% of storage cases harbouring bacteria at > 10(4)/ml and with 78% sterility. One of the two one-step hydrogen peroxide systems performed equally well. Importantly, Acanthamoeba was not isolated from any of the 150 storage cases. Whether lens storage cases need to be sterile or contain < 10(3) bacteria/ml solution within them is debatable, but it is essential that Acanthamoeba be absent from them.

Fluorescent oligonucleotide probes for clinical and environmental detection of Acanthamoeba and the T4 18S rRNA gene sequence type.

The first genus- and subgenus-specific fluorescent oligonucleotide probes for in situ staining of Acanthamoeba are described. Sequences of these phylogeny-based probes complement the 18S rRNA and the gene encoding it (18S rDNA). The genus-specific probe (GSP) is a fluorescein-labeled 22-mer specific for Acanthamoeba as shown here by its hybridization to growing trophozoites of all 12 known Acanthamoeba 18S rDNA sequence types and by its failure to hybridize with amoebae of two other genera (Hartmannella vermiformis and Balamuthia mandrillaris), two human cell lines, and two bacteria (Pseudomonas aeruginosa and Escherichia coli). The sequence type T4-specific probe (ST4P) is a rhodamine-labeled 30-mer specific for Acanthamoeba 18S rDNA sequence type T4, as shown here in hybridization tests with trophozoites of all 12 sequence types. T4 is the subgenus group associated most closely with Acanthamoeba keratitis (AK). GSP also was tested with corneal scrapings from 17 patients with a high index of clinical suspicion of AK plus 5 patient controls. GSP stained both trophozoites and cysts, although nonspecific cyst wall autofluorescence also was observed. Results could be obtained with GSP in 1 to 2 days, and based on results from cell culture tests, the probe correctly detected the presence or absence of Acanthamoeba in 21 of 24 specimens from the 22 patients. The use of GSP with cultured trophozoites and cysts from corneal scrapings has illustrated the suitability of using fluorescent oligonucleotide probes for identification of the genus Acanthamoeba in both environmental and clinical samples. In addition, the use of ST4P with cultured amoebae has indicated the potential of oligonucleotide probes for use in subgenus classification.

Population-based cohort study of microbial keratitis in Scotland: incidence and features.

OBJECTIVE: To investigate the incidence and features of bacterial, fungal and protozoal keratitis in Scotland. DESIGN: Prospective, population-based cohort study of all persons who developed culture proven microbial keratitis over an 8 month period. SETTING: West of Scotland, UK. SUBJECTS: Approximately 3,000,000 population. MAIN OUTCOME MEASURES: Incidence and risk factors for microbial keratitis. METHODS: All patients were included who had presumed microbial keratitis from which bacteria, fungi or Acanthamoeba was isolated from the corneal scraping by the hospital laboratory using a standardised protocol. In addition, contact lens wearing patients with pathognomonic features of Acanthamoeba keratitis, who yielded a negative culture result when referred on chlorhexidine therapy, were included if Acanthamoeba could be cultured from their lens storage case. RESULTS: The overall annual incidence of culture-proven microbial keratitis was 0.26 per 10,000 with a rate of 1.8 per 10,000 for contact lens wearers (all types, soft and rigid). Based on a previous pilot study of 'presumed' microbial keratitis in Glasgow, it was possible to estimate the incidence of expected 'presumed' microbial keratitis as 0.36 per 10,000 overall and 2.44 per 10,000 for contact lens wearers (all types). The incidence for Acanthamoeba keratitis was 1.49 per 10,000 soft contact lens wearers; this infection was not detected in the absence of contact lens wear nor with use of gas permeable or rigid contact lenses. CONCLUSIONS: 'Presumed' microbial keratitis from all causes, in the adult population, was approximately three times less common in the West of Scotland (0.36 per 10,000) than would be expected from a comparable retrospective study from Minnesota, USA for the years 1980-1988 (1.1 per 10,000). It was rare (approximately one case expected in 2 million per year) in the absence of pre-existing corneal disease, cosmetic contact lens wear or trauma. Ocular surface disease was the underlying cause predisposing to infection in 58% of cases, with an incidence of 'presumed' keratitis of 0.21 per 10,000 population; the highest incidence was found in the elderly population. Contact lens wear was responsible for 38% of cases, emphasising the importance of preventive hygiene and effective disinfection in this group. The estimated incidence of 'presumed' microbial keratitis in the West of Scotland associated with cosmetic wear (daily and extended use) of soft contact lenses was significantly less (P<0.05) than that expected from a prospective study in New England, America in 1985 (266 per 10,000, rather than 8.05 per 10,000). However, the estimated incidence for presumed microbial keratitis for the West of Scotland associated with wearing soft contact lenses for cosmetic purposes in the daily wear modality (266 per 10,000) was less, but not significantly less, than that found in the prospective American study (4.20 per 10,000). The daily wear mode for contact lenses is almost universal in the West of Scotland, where extended wear has never been recommended. Extended wear has been shown in the USA to be associated with an incidence of presumed microbial keratitis between five and ten times higher than that associated with daily wear. This explains the lower incidence we have observed and a difference with the US study for overall infection rates but not when associated with daily wear alone. The incidence of proven Acanthamoeba keratitis found in the Scottish study among wearers of soft contact lenses for daily wear cosmetic purposes was exceptionally high at 1.49 per 10,000.

Acanthamoeba keratitis in Scotland: risk factors for contact lens wearers.

OBJECTIVE: To investigate risk factors for Acanthamoeba keratitis amongst contact lens wearers in Scotland. DESIGN: Patients with Acanthamoeba keratitis in the Scottish study, all of whom wore contact lenses, were compared with 46 healthy asymptomatic contact lens-wearing controls. They were all visited at home for contact lens and environmental microbiological sampling. In addition, all 288 optical practices in the West of Scotland were polled for contact lens types and disinfecting solutions sold in 1995, and a sample, each of whom fitted more than 500 contact lenses per year, were polled for a second time. Independently, a poll was commissioned by the Eyecare Information Service in July/August 1995 to estimate the numbers of contact lens wearers in Scotland and the UK. Industry was polled for numbers of each contact lens disinfecting regimen sold in Scotland in 1995. SETTING: West of Scotland, UK. SUBJECTS: All contact lens wearers among the 3 million population of the West of Scotland Health Board Areas. MAIN OUTCOME MEASURES: Risk factors for Acanthamoeba infection and recommendations for its prevention. RESULTS: When Acanthamoeba infection occurred, patients' home water systems were frequently (54%) found to be colonised by this amoeba. Patients more frequently washed their storage cases in tap water than controls (P<0.05) with resulting contamination, kept storage cases wet rather than air drying them (P<0.05), and had coliform bacteria cultured from the storage case (P<0.05) and had viable Acanthamoeba within the storage case (P<0.0001). Overall, patients were found to have significantly more risk factors than controls (P<0.0001). The noncompliant use of chlorine tablet disinfection, or failure to disinfect contact lenses at all, was associated with increased risk (P<0.05). Ionic high water content contact lenses (FDA group 4 material), when used without disinfection or with non-compliant use of low chlorine (Soflab) tablet-based disinfection, were associated with increased risk of Acanthamoeba infection (P<0.05). In log-linear modelling of risky hygiene behaviours associated with contamination of storage cases with Acanthamoeba, the most significant behaviour was found to be use of the less effective disinfection methods (chlorine tablets or no disinfection). However further investigation showed that these methods were associated with an increased probability of rinsing the storage case in tap water, so that these two behaviours are confounded in the group studied. CONCLUSIONS: Failure to disinfect contact lenses, non-compliant use of chlorine tablets and/or introduction of tap water rinsing of storage cases were associated with increased risk of Acanthamoeba infection. New multipurpose solutions and hydrogen peroxide gave the lowest risk of Acanthamoeba infection, with no statistically significant difference between them. Ionic high-water content (FDA group 4) contact lenses were at increased risk of being associated with Acanthamoeba keratitis if used without effective disinfection (multipurpose solutions or hydrogen peroxide). The use of domestic tap water for contact lens and storage case hygiene must be avoided, as a chain-of-causation' was identified from the home water supply.

Confirmatory evidence from 18S rRNA gene analysis for in vivo development of propamidine resistance in a temporal series of Acanthamoeba ocular isolates from a patient.

DNA sequences of three 18S rRNA gene alleles present in trophozoites obtained before and after therapy for Acanthamoeba keratitis substantiate a previous report that the infection was due to a single Acanthamoeba strain. Thus, the possibility that propamidine resistance which developed during therapy was due to a mixed infection was ruled out.

Antimicrobial management of presumed microbial keratitis: guidelines for treatment of central and peripheral ulcers.

AIMS: To determine the quantitative relation between the major risk factors for microbial keratitis of previous ocular surface disease and contact lens wear and central and peripheral infiltration, often associated with ulceration, in order to establish a rational chemotherapeutic management algorithm. METHODS: Data from 55 patients were collected over a 10 month period. All cases of presumed microbial keratitis where corneal scrapes had been subjected to microbiological examination were included. Risk factor data and laboratory outcome were recorded. Antimicrobial regimens used to treat each patient were documented. RESULTS: 57 episodes of presumed microbial keratitis were identified from 55 patients, 24 male and 31 female. There were 30 central infiltrates and 27 peripheral infiltrates of which 28 were culture positive (73% of central infiltrates, 22% of peripheral infiltrates). 26 patients had worn contact lenses of whom 12 had culture positive scrapes (9/14 for central infiltrates, 3/12 for peripheral infiltrates). 31 patients had an ocular surface disease of whom five previous herpes simplex virus keratitis patients developed secondary bacterial infection. Anterior chamber activity and an infiltrate size > or = 4 mm2 were more common with culture positive central infiltrates than peripheral infiltrates (chi 2 test = 11.98, p < 0.001). CONCLUSIONS: Predisposing factors for "presumed" microbial keratitis, either central or peripheral, were: ocular surface disease (26/57 = 45.6%), contact lens wear (26/57 = 45.6%), and previous trauma (5/57 = 8.8%). Larger ulceration (> or = 4 mm2) with inflammation was more often associated with positive culture results for central infiltration. None of these four variables (contact lens wear, ocular surface disease, ulcer size, anterior chamber activity) were of intrinsic value in predicting if a peripheral infiltrate would yield identifiable micro-organisms. Successful management of presumed microbial keratitis is aided by a logical approach to therapy, with the use of a defined algorithm of first and second line broad spectrum antimicrobials, for application at each stage of the investigative and treatment process considering central and peripheral infiltration separately.

Lack of evidence for systemic toxicity following topical chloramphenicol use.

There has been considerable controversy regarding the safety of topical chloramphenicol in ophthalmic practice. The evidence for associated haematopoietic toxicity in idiosyncratic and dose-dependent forms was reviewed. The 7 cases of idiosyncratic haematopoietic reactions associated with topical chloramphenicol reported in the literature are refutable evidence for the existence of such a response. In Scotland, despite extensive prescription of topical chloramphenicol, the incidence of acquired aplastic anaemia was found to be low, as were associated reports of blood dyscrasias throughout the UK. The epidemiology of acquired aplastic anaemia failed to make an association with topical chloramphenicol use. High-performance liquid chromatography (minimum detection limit 1 mg/l) was used to investigate whether serum accumulation of chloramphenicol occurred after topical therapy in 40 patients. The mean dose of chloramphenicol eye drops used after 1 week of treatment was 8.0 mg, and after 2 weeks, 15.3 mg. As expected, chloramphenicol failed to accumulate to detectable levels. This supported the view that topical chloramphenicol was not a risk factor for inducing dose-related bone marrow toxicity. Calls for the abolition of treatment with topical chloramphenicol based on current data are not supported.