Solar disinfection of poliovirus and Acanthamoeba polyphaga cysts in water - a laboratory study using simulated sunlight.

AIMS: To determine the efficacy of solar disinfection (SODIS) in disinfecting water contaminated with poliovirus and Acanthamoeba polyphaga cysts. METHODS AND RESULTS: Organisms were subjected to a simulated global solar irradiance of 850 Wm(-2) in water temperatures between 25 and 55 degrees C. SODIS at 25 degrees C totally inactivated poliovirus after 6-h exposure (reduction of 4.4 log units). No SODIS-induced reduction in A. polyphaga cyst viability was observed for sample temperatures below 45 degrees C. Total cyst inactivation was only observed after 6-h SODIS exposure at 50 degrees C (3.6 log unit reduction) and after 4 h at 55 degrees C (3.3 log unit reduction). CONCLUSIONS: SODIS is an effective means of disinfecting water contaminated with poliovirus and A. polyphaga cysts, provided water temperatures of 50-55 degrees C are attained in the latter case. SIGNIFICANCE AND IMPACT OF THE STUDY: This research presents the first SODIS inactivation curve for poliovirus and provides further evidence that batch SODIS provides effective protection against waterborne protozoan cysts.

Efficacy of multipurpose solutions against Acanthamoeba species.

AIM: The disinfection efficacy of contact lens multipurpose solutions (MPSs) against Acanthamoeba polyphaga (Ros) and Acanthamoeba castellanii (ATCC30868) cysts and trophozoites was determined by both biocidal and manufacturer-recommended no-rub/rinse regimen testing. METHODS: A biocidal assay using four MPSs (ReNu with MoistureLoc, Opti-free Express, Solo-care Plus, and Complete MoisturePlus) was conducted with or without the presence of organic soil. A second test procedure compared the ability of five MPSs (ReNu with MoistureLoc MPS, ReNu MultiPlus, Opti-free Express, Solo-care Aqua, and Complete MoisturePlus) to remove and kill Acanthamoeba species cysts and trophozoites from SofLens 38 and Surevue conventional hydrogel lenses, and Focus Night & Day silicone hydrogel lenses using the manufacturer-recommended regimen. RESULTS: In the biocidal assay, only ReNu with MoistureLoc successfully killed both trophozoites and cysts (>3 log) within the manufacturer-recommended soak time. A >3 log decrease in trophozoites, but not cysts, was reported for Opti-free Express; however, Solo-care Plus and Complete MoisturePlus did not reduce the number of cysts or trophozoites by >3 log during the manufacturer-recommended soak time. In the no-rub/rinse regimen tests, only ReNu with MoistureLoc removed an inoculum of 2 x 10(5) trophozoites or cysts from SofLens 38 and Surevue hydrogel lenses, as well as Focus Night & Day silicone hydrogel lenses. Less than 10 viable organisms were recovered from the lenses after the 10s rinse and 4h soak. Opti-free Express, Solo-care Aqua, and ReNu MultiPlus were effective at removing trophozoites and cysts from SofLens 38 and Surevue conventional hydrogel lenses, but not from Focus Night & Day silicone hydrogel lenses. In excess of 10 viable organisms were recovered from all lenses after the manufacturer-recommended regimen using Complete MoisturePlus. CONCLUSIONS: These data suggest that some MPSs, when used as recommended by the manufacturer, are more effective at killing representative strains of Acanthamoeba than others.

Solar and photocatalytic disinfection of protozoan, fungal and bacterial microbes in drinking water.

The ability of solar disinfection (SODIS) and solar photocatalytic (TiO(2)) disinfection (SPC-DIS) batch-process reactors to inactivate waterborne protozoan, fungal and bacterial microbes was evaluated. After 8 h simulated solar exposure (870 W/m(2) in the 300 nm-10 microm range, 200 W/m(2) in the 300-400 nm UV range), both SPC-DIS and SODIS achieved at least a 4 log unit reduction in viability against protozoa (the trophozoite stage of Acanthamoeba polyphaga), fungi (Candida albicans, Fusarium solani) and bacteria (Pseudomonas aeruginosa, Escherichia coli). A reduction of only 1.7 log units was recorded for spores of Bacillus subtilis. Both SODIS and SPC-DIS were ineffective against the cyst stage of A. polyphaga.

Comparison of hydrogen peroxide contact lens disinfection systems and solutions against Acanthamoeba polyphaga.

Acanthamoeba is a free-living amoeba causing a potentially blinding infection of the cornea. Contact lens wearers are most at risk and account for some 95% of cases. Hydrogen peroxide is used for contact lens disinfection due to its broad antimicrobial activity. Lenses must be neutralized before use to avoid pronounced stinging and possible corneal damage. Neutralization is achieved by adding a catalyst during the disinfection process (one-step) or afterwards (two-step). Here, the activities of commercial peroxide systems and individual solutions against trophozoites and cysts of Acanthamoeba polyphaga were compared. All disinfection systems were active against trophozoites, giving a > or = 3-log (99.9%) kill within 1 h. Of the four one-step systems, only one showed some cysticidal activity, giving a 1.28 +/- 0.41-log reduction. Both two-step systems were cysticidal, giving a > or = 3-log kill at 4 h. All system peroxide solutions were cysticidal, giving a > or = 3-log kill by 4 to 6 h. Variation in the cysticidal rate was observed with two solutions that gave a 1.8- to 2.1-log kill at 4 h compared with 3.0 to 4.0 for the rest (P < 0.05). No cysticidal activity was found with the peroxigen sodium perborate or the contact lens protein remover subtilisin A. Two-step systems are cysticidal providing contact times of at least 4 h are employed. Variation in cyst killing occurs between peroxide solutions, possibly due to formulation differences. One-step systems are less effective against Acanthamoeba cysts due to rapid peroxide neutralization. The cysticidal activity of one-step systems could be improved if neutralization rates were retarded.

A comparison of cyst age and assay method of the efficacy of contact lens disinfectants against Acanthamoeba.

AIMS: (i) To determine effect of Acanthamoeba cyst age, method of production, and (ii) to assay technique on the efficacy of multipurpose solutions (MPS) and hydrogen peroxide based contact lens disinfectants. (iii) To establish if MPS can remove mature cysts from contact lenses according to the ISO/DIS 14729 regimen test for microbe removal. METHODS: Immature and mature cysts of A polyphaga were tested against the MPS Opti-Free express and the hydrogen peroxide based solutions Oxysept 1Step and Oxysept 1 using two assay methods. Simulated patient regimen testing was performed with the Opti-Free express and Complete using mature cysts inoculated on to group I or group IV lenses. RESULTS: Immature cysts were sensitive to disinfection by all solutions. No killing was observed with mature cysts with Opti-Free express, while immature cysts yielded a 1-2 log reduction in viability. Oxysept 1Step gave a 1.1 (SD 0.3) log reduction in mature cysts after 6 hours. Oxysept 1 gave a 2.4 (0.3) log reduction in mature cysts after 4 hours and a 3.8 (0.5) log reduction after 6 hours. Patient regimen testing using Opti-Free express and Complete resulted in no recovery of viable mature cysts from the contact lenses or from the soaking solutions. CONCLUSION: Cyst age but not method of production used in this study influences the efficacy of contact lens disinfectants against Acanthamoeba. MPS are effective in removing cysts from contact lens surfaces and may have a role in the prevention of acanthamoeba keratitis.

Polymerase chain reaction analysis of corneal epithelial and tear samples in the diagnosis of Acanthamoeba keratitis.

PURPOSE: Acanthamoeba is an uncommon cause of corneal infection in which the best visual outcome follows prompt diagnosis and a long course of appropriate antimicrobial therapy. Because conventional detection techniques for Acanthamoeba have certain limitations, we investigated the ability of the polymerase chain reaction (PCR) to confirm the clinical diagnosis of Acanthamoeba keratitis, with the ultimate aim of achieving early diagnosis. METHODS: Using two different pairs of primers, PCR was performed on representative cultured Acanthamoeba isolates to confirm the assay's ability to amplify Acanthamoeba DNA from a wide range of acanthamoebae. Subsequently, corneal epithelial samples from 19 patients and tear samples from 12 patients with Acanthamoeba keratitis were analyzed by PCR for the presence of Acanthamoeba DNA. RESULTS: Acanthamoeba DNA was amplified by PCR from 16 (84%) of 19 corneal epithelial samples, whereas Acanthamoeba was cultured from 10 samples (53%), all of which were PCR positive. Tear samples from 8 (66%) of 12 patients were positive on PCR testing, and one tear sample was PCR positive, whereas the corresponding epithelial biopsy had yielded a negative PCR result. Samples from culture-positive patients were positive on PCR testing more frequently than those from culture-negative patients (10/10 culture-positive corneal epithelial and 5/7 [71%] culture-positive initial tear samples versus 6/9 [66%] culture-negative corneal epithelial and 2/5 [40%] culture-negative tear samples). All control epithelial (n = 15) and tear (n = 15) samples yielded negative results. CONCLUSIONS: PCR was a more sensitive diagnostic test than a culture for Acanthamoeba keratitis, and the use of two different primers achieved better sensitivity than a single set. A PCR of a tear sample also may be a useful complementary test and, in combination with PCR of epithelial samples, would prove particularly helpful in confirming the clinical diagnosis in culture-negative cases.

Molecular data suggest an early acquisition of the mitochondrion endosymbiont.

The three deepest branching eucaryotic lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microspora, Metamonada and Parabasala. They are followed by either the Euglenozoa (e.g. Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eucaryotes. To investigate the hypothesis of an even earlier timing of the mitochondrion endosymbiosis we have amplified a partial cpn-60 coding region from the parabasalid Trichomonas vaginalis and the first such sequence from a percolozoan, Naegleria fowleri. Analysis of predicted protein sequences reveals a high degree of sequence similarity (> or = 40%) with a selection of published bacterial and mitochondrial cpn-60s for both taxa. Both sequences were recovered within a strongly supported monophyletic group, otherwise defined by mitochondrial sequences, which systematically clustered with alpha-proteobacteria. These results provide compelling evidence that the ancestor of T. vaginalis once contained the endosymbiont which gave rise to mitochondria, and suggest that this symbiosis probably occurred before the Trichomonas lineage diverged from the main eukaryote trunk. It also makes feasible the published hypothesis that the Trichomonas hydrogenosome might represent a biochemically modified mitochondrion. Analysis of the N. fowleri cpn-60 did not support the hypothesis that the mitochondrion-containing Percolozoa represent an earlier branch in the cpn-60 tree than Trichomonas or Trypanosoma.

Development of a PCR for identification of Naegleria fowleri from the environment.

A species-specific PCR for the identification of Naegleria fowleri was developed. In sensitivity studies, 10 trophozoites or cysts and 1 trophozoite or cyst could be detected after 35 and 45 cycles, respectively. In conjunction with a rapid DNA isolation method, this PCR was used to identify N. fowleri directly from primary cultures of environmental samples.

Identification and epidemiological typing of Naegleria fowleri with DNA probes.

Naegleria fowleri is a small free-living amoeboflagellate found in warm water habitats worldwide. The organism is pathogenic to humans, causing fatal primary amoebic meningoencephalitis. When monitoring the environment for the presence of N. fowleri, it is important to reliably differentiate the organism from other closely related but nonpathogenic species. To this end, we have developed species-specific DNA probes for use in the rapid identification of N. fowleri from the environment. Samples were taken from the thermal springs in Bath, England, and cultured for amoebae. Of 84 isolates of thermophilic Naegleria spp., 10 were identified as N. fowleri by probe hybridization. The identity of these isolates was subsequently confirmed by their specific whole-cell DNA restriction fragment length polymorphisms (RFLPs). One DNA clone was found to contain a repeated element that detected chromosomal RFLPs that were not directly visible on agarose gels. This enabled the further differentiation of strains within geographically defined whole-cell DNA RFLP groups. N. fowleri DNA probes represent a specific and potentially rapid method for the identification of the organism soon after primary isolation from the environment.

Automated molecular design: a new fragment-joining algorithm.

A popular first step in the problem of structure-based, 'de novo' molecule design is to identify regions where specific functional groups or chemical entities would be expected to interact strongly. When the three-dimensional structure of the receptor is not available, it may be possible to derive a pharmacophore giving the three-dimensional relationships between such chemical groups. The task then is to design synthetically feasible molecules which not only contain the required groups, but which can also position them in the desired relative orientation. One way to do this is to first link the groups using an acyclic chain. We have investigated the application of the 'tweak' algorithm [Shenkin, P.S. et al., Biopolymers, 26 (1987) 2053] for generating families of acyclic linkers. These linking structures can subsequently be 'braced' using a ring-joining algorithm [Leach, A.R. and Lewis, R.A., J. Comput. Chem., 15 (1994) 233], giving rise to an even wider variety of molecular skeletons for further studies.

A clinicopathologic study of in vitro sensitivity testing and Acanthamoeba keratitis.

PURPOSE: To examine the extent of any correlation between the in vitro sensitivity and the clinical outcomes of Acanthamoeba keratitis. METHODS: The clinical outcomes were correlated with the in vitro sensitivity of 23 isolates of 23 patients with culture-positive Acanthamoeba keratitis. The laboratory assay assessed the amoebicidal and cysticidal efficacy of 13 drugs. RESULTS: Most agents were effective against the trophozoites in vivo. Polyhexamethylene biguanide (PHMB) and chlorhexidine were the most successful cysticidal agents, followed by sepazonium and propamidine. Clotrimazole, paramomycin, and ketoconazole were cysticidal in a few specimens, but usually in high concentrations. Neomycin was ineffective against cysts in vivo. Nineteen patients were treated with topical propamidine and neomycin, and a medical cure was obtained in nine (47%). There was poor correlation between the clinical outcomes of individual cases and the in vitro sensitivity testing. The medical failures were treated with topical PHMB and propamidine and eight of ten (80%) of these were medically cured. Two patients, however, were still culture positive after 28 and 41 weeks of treatment. PHMB has an excellent in vitro sensitivity profile, but the two cases of failure were sensitive to the drug and resistance had not developed. CONCLUSIONS: In vitro sensitivity testing has been important in the screening of new agents, although disappointing in the management of individual cases in this set of studies.

Acanthamoeba trophozoite and cyst adherence to four types of soft contact lens and removal by cleaning agents.

Trophozoite and cyst adherence of two Acanthamoeba keratitis strains (PHL/530 and PHL/978) to four types of unworn soft contact lens and their removal by cleaning agents were studied. Greater adherence of the trophozoites compared with the cysts was recorded for both strains. Trophozoites of PHL/530 adhered in greater numbers to type I lenses (61.4% poly[2-hydroxyethyl methacrylate-38.6% water), with no differences between type II (30% lidofilcon A-70% water), III (55% bufilcon A-45% water) and IV lenses (42% etafilcon A-58% water). Adherence of PHL/978 trophozoites to type II lenses was decreased compared with their adherence to the other lenses. Cysts of both strains showed greater adherence to type I and III lenses. Interstrain differences in trophozoite adherence occurred, with PHL/530 showing greater adherence to type I and II lenses. Recommended cleaning procedures using three commercial solutions were effective in removing Acanthamoeba from the lenses. This study indicates the possible role of adherence to contact lenses in the acquisition of Acanthamoeba keratitis, but shows that correct use of commercial cleaning agents may be important in the prevention of infection.

Treatment of Acanthamoeba keratitis with polyhexamethylene biguanide.

Polyhexamethylene biguanide (PHMB) is a polymeric biguanide disinfectant that has not previously been used in the treatment of infection. Six patients with confirmed Acanthamoeba keratitis were treated with PHMB 0.02%. All patients had uncontrolled keratitis refractory to therapy with multiple conventional antiamebic agents. The rationale for use and the dose of PHMB was determined by in vitro sensitivity testing of the Acanthamoeba corneal isolates to the drugs available for use. Trophozoite forms were sensitive to most agents. Only PHMB was cysticidal at low concentrations in all cases. Sensitivity to the other drugs, including propamidine, showed wide variation. In 5 of 6 cases, complete resolution of inflammation followed the introduction of PHMB. Toxicity to the ocular surface was not evident with PHMB, unlike propamidine or neomycin. The reasons for the treatment failure in one case, despite cyst sensitivity to both PHMB and propamidine, are not clear. PHMB is a promising new treatment for this infection.

Differentiation of Acanthamoeba strains from infected corneas and the environment by using restriction endonuclease digestion of whole-cell DNA.

Restriction endonuclease digestion of Acanthamoeba whole-cell DNA was used to study the relationship between 33 morphologically identical strains from keratitis cases (30 strains), contact lens storage containers (2 strains), and soil (1 strain). Samples digested with BglII, EcoRI, or HindIII and separated by agarose gel electrophoresis contained detectable mitochondrial DNA restriction fragment length polymorphisms (RFLPs). By comparing RFLPs, the strains could be assigned to seven multiple-strain and three single-strain groups. The largest of these contained nine strains, eight of which were isolated in keratitis cases in various locations worldwide and may indicate a group particularly associated with keratitis. Restriction endonuclease analysis of whole-cell DNA is proposed as a valuable technique for detecting mitochondrial DNA RFLPs in the differentiation of morphologically identical Acanthamoeba strains and may therefore be useful in resolving the complex taxonomy of the genus, which has hitherto been founded on subjective morphological criteria.