Acanthamoeba Polyphaga Cysts Research Articles

Riboflavin and ultraviolet A as adjuvant treatment against Acanthamoeba cysts.

Experimental studies have shown that the standard dose of riboflavin (R) or R + ultraviolet-A (UVA) as solo treatment are not able to exterminate Acanthamoeba cysts or even trophozoites. The purpose of this study is to determine whether the application of R + UVA can enhance the cysticidal effects of cationic antiseptic agents in vitro. The log of either polyhexamethylene biguanide or chlorhexidine minimal cysticidal concentration in solutions containing riboflavin (concentrations 0.1, 0.05 and 0.025%) plus either Acanthamoeba castellanii cysts or Acanthamoeba polyphaga cysts was determined and compared in groups treated with UVA 30 mW/cm(2) for 30 min and in control groups (with no exposure to UVA). A permutation test was used to determine the P value associated with treatment. Regardless of the riboflavin concentration and UVA treatment condition, no trophozoites were seen in plates where the cysts were previously exposed to cationic antiseptic agent concentrations ≥200 µg/mL for Acanthamoeba castellanii samples and ≥100 µg/mL for A. polyphaga samples. There was no statistical evidence that R + UVA treatment was associated with minimal cysticidal concentration (P = 0.82). R + UVA in doses up to 10 times higher than recommended for corneal crosslinking does not enhance the cysticidal effect of either polyhexamethylene biguanide or chlorhexidine in vitro.

The fine structure of theAcanthamoeba polyphagacyst wall

Members of the genus Acanthamoeba are present in diverse environments, from freshwater to soil, and also in humans, causing serious brain and corneal infections. Their life cycle presents two stages: the dividing trophozoite and the quiescent cyst. The structures of these life stages have been studied for many years, and structural data have been used for taxonomy. The ultrastructural work on Acanthamoeba cysts was carried out previously by routine transmission electron microscopy (TEM), a process that requires the use of chemical fixation, a procedure that can cause serious artifacts in the ultrastructure of the studied material. In order to prevent fixation artifacts, we processed Acanthamoeba polyphaga cysts by ultrarapid freezing, followed by freeze-fracturing and deep-etching, in order to obtain a 3D visualization of the arrangements of the cyst wall. The exocyst presented an irregular surface, with vesicles located within or near this layer. The endocyst, instead, showed a biphasic arrangement with a more compact district in its innermost part, and a more loosened outer layer. For this reason, it was difficult to distinguish the filaments present in the intercyst space from those forming the endocyst. Surprisingly, the intercyst space was thinner when compared with samples processed by conventional TEM, evidencing the possible damage consequent to the use of chemical fixation.

EmergingMycobacteriaspp. in Cooling Towers

To the Editor: The importance of nontuberculous mycobacteria (NTM) in various clinical situations recently has increased. Members of the Mycobacterium avium complex (MAC) cause a high percentage of infections in persons with acquired immunodeficiency syndrome. Some species are considered emerging pathogens, particularly those of the M. chelonae–M. abscessus group. They occur not only in immunocompromised persons but also in persons without predisposing conditions. Although sources of infection are considered to originate from the human environment, until now cooling towers were not clearly demonstrated to be one of these contamination sources, despite being suspected (1). Natural streams, groundwater, brook waters, and swamps already were reported to contain different species of NTM. Constructed environments, such as hospital hot water systems, aerosols from showers, ice machines, swimming pools, dental unit water, endoscopes, and bronchoscopes are other sources (2). Other studies have shown members of MAC in drinking-water distribution systems (3). A 1999 study in South Africa reported the presence of mycobacteria in cooling towers but reported no details about species (4). In 2003, cooling towers were reported to be a potential source of slow-growing mycobacterial species (1). Some mycobacterial species recently were shown to survive within amebae (5). We aimed to study the population of ameba-associated mycobacterial species in 3 cooling towers using a co-coculture method. These cooling towers (E, H, and O), located in downtown Paris, France, were sampled in May 2006. Water was taken in a sample point of the cooling circuit located just before the entrance of the tower basins. Because these cooling towers are regularly treated by oxidizing biocide BCDMH (1-bromo-3-chloro-5,5-dimethylhydantoin) to prevent development of Legionella spp., no Legionella spp. or Legionella-like amebal pathogens were isolated. Two liters of water samples were filtrated through 0.22-µm pore-sized filters that were injected onto amebal microplates as previously described (6) and incubated at 32°C. We screened amebal microplates by examination under inverted microscope and by Ziehl-Nielsen, Gram, and Gimenez staining. Positive wells were subcultured on axenic medium and incubated at 32°C for 10 days. Acid-fast isolates were identified by using partial rpoB gene amplification and sequencing with Myco-F and Myco-R primers (7). These primers did not allow amplification of partial rpoB gene for the 6 M. phocaicum strains. We thus used Myco-F/Myco-Rbon (5′-AGCGGCTGCTGGGTGATCAT-3′) primer pair for M. phocaicum (8). We compared these sequences with the rpoB gene sequence of Mycobacteria type strains available in the GeneBank database by using BLASTn on the NCBI website (www.ncbi.nlm.nih.gov). We observed bacteria growing in amebae. Subculture on axenic media led to polymicrobial cultures and allowed isolation of 33 mycobacterial strains (Table). All these strains were submitted to molecular identification. The 33 isolates corresponded to 5 mycobacterial species: M. fortuitum, M. conceptionense, M. chelonae, M. chimaera, and M. phocaicum (Table). Some of these mycobacteria already had been shown to survive in free-living amebae and to be implicated in human diseases, such as M. chelonae (5). The same author demonstrated recently that 26 environmental mycobacteria survived in the trophozoites and cysts of Acanthamoeba polyphaga (5). The recently described species M. phocaicum was isolated only in samples from humans and was associated with chronic pneumonia (9). However, the natural source of this species is still unknown. M. fortuitum was described as an opportunistic Mycobacterium associated with disseminated infections in a leukemia patient or in furunculosis after footbath in nail salons. M. conceptionense belongs to the M. fortuitum group and was isolated in a posttraumatic osteitis inflammation (10). M. chimaera belongs to MAC and has been isolated only in patients with pulmonary disorders but not from immunocompromised patients. The authors reported that the isolate showed unusually high virulence. We isolated this species for the first time in an environmental sample. It could belong to the transitory flora of the H cooling tower because it was isolated only once in our samples. Cooling towers already had been investigated for slow-growing mycobacteria (1), and our study showed they also can be an environmental source of rapidly growing NTM pathogens. In our procedure for amebal co-cultures, incubation cannot last >10 days because amebae do not survive longer in Page’s amebal saline buffer. Moreover, agar plates were incubated for 10 days only, which explains why we could recover only rapidly growing mycobacteria. The cooling towers, already known as a source of dissemination of Legionella spp., may disseminate mycobacteria in aerosols and be a previously nonrecognized source of infection. Table Identification of isolated nontuberculous mycobacteria according to their rpoB gene sequence similarity and GenBank accession numbers

Survival of environmental mycobacteria in Acanthamoeba polyphaga.

Free-living amoebae in water are hosts to many bacterial species living in such an environment. Such an association enables bacteria to select virulence factors and survive in adverse conditions. Waterborne mycobacteria (WBM) are important sources of community- and hospital-acquired outbreaks of nontuberculosis mycobacterial infections. However, the interactions between WBM and free-living amoebae in water have been demonstrated for only few Mycobacterium spp. We investigated the ability of a number (n = 26) of Mycobacterium spp. to survive in the trophozoites and cysts of Acanthamoeba polyphaga. All the species tested entered the trophozoites of A. polyphaga and survived at this location over a period of 5 days. Moreover, all Mycobacterium spp. survived inside cysts for a period of 15 days. Intracellular Mycobacterium spp. within amoeba cysts survived when exposed to free chlorine (15 mg/liter) for 24 h. These data document the interactions between free-living amoebae and the majority of waterborne Mycobacterium spp. Further studies are required to examine the effects of various germicidal agents on the survival of WBM in an aquatic environment.

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

To determine the efficacy of solar disinfection (SODIS) in disinfecting water contaminated with poliovirus and Acanthamoeba polyphaga cysts. 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). 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. This research presents the first SODIS inactivation curve for poliovirus and provides further evidence that batch SODIS provides effective protection against waterborne protozoan cysts.

Experimental Acanthamoeba keratitis: I. Preliminary findings.

Experimental Acanthamoeba keratitis was induced in Wistar rats by intrastromal inoculation of Acanthamoeba polyphaga cysts. Keratitis developed in all corneas injected and was characterised by a diffuse granular opacity. The animals were examined for 70 days and a gradual reduction of keratitis was observed. Pathological sections showed liquefactive stromal necrosis and Acanthamoeba localised in deep stroma persisting to 70 days. A brisk inflammatory infiltrate of neutrophils and macrophages was seen in the early days of infection. Severity of keratitis was found to correlate with inflammatory activity in pathological sections. The uses of an animal model of Acanthamoeba keratitis are described.

Acanthamoeba Keratitis and Contact Lens Wear

The avoidance of nonsterile solutions is important to curtailing Acanthamoeba keratitis, a serious infection that has been found to occur with all types of contact lens (CL) wear. Increased patient education, revised recommendations regarding the use of tap and distilled water, and improved disinfecting systems are vital to preventing infection. These precautions are particularly important since it appears that Acanthamoeba, unlike Pseudomonas, may not require an epithelial defect to establish corneal infection and, once in the cornea, may not respond to drug therapy or surgical extirpation. Unfortunately, many patients receive poor lens care instructions or cannot be relied on to follow appropriate routines. Finding a foolproof means of lens disinfection for them is critical. Recently, several disinfection systems were tested against Acanthamoeba castellanii and Acanthamoeba polyphaga cysts and trophozoites to see which might prove most effective. Effective systems included heat disinfection at 70 to 80 degrees C for 10 min, 3% hydrogen peroxide for 2-3 h, 0.001% thimerosal with edetate for 4 h, 0.005% benzalkonium chloride with edetate and reagent for 4 h, and either 0.001% chlorhexidine for 4 h or 0.004% chlorhexidine for 1 h. Thus, for patients who are careless or persist in using nonsterile rinsing solutions, it appears that at least some methods of disinfection will help prevent Acanthamoeba infection.