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Acanthamoeba contamination of hemodialysis and dental units in Alexandria, Egypt: A neglected potential source of infection
Journal of Infection and Public Health (2012) 5, 304—310
Acanthamoeba contamination of hemodialysis and dental units in Alexandria, Egypt: A neglected potential source of infection Azza Hassan a, Hanan Farouk a, Faika Hassanein a, Rashad Abdul-Ghani b,c,∗, Ahmed H. Abdelhady d a
Tropical Health Department, High Institute of Public Health, Alexandria University, Alexandria, Egypt Parasitology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt c Department of Medical Parasitology, Faculty of Medicine and Health Sciences, Sana’a University, P.O. Box 1247, Sana’a, Yemen d Military Medical Academy, Alexandria, Egypt b
Received 5 April 2012 ; received in revised form 26 May 2012; accepted 1 June 2012
KEYWORDS Acanthamoeba; Free-living amoeba; Hemodialysis; Dental unit; Alexandria
Summary Ubiquitous free-living amoebae of the genus Acanthamoeba can be pathogenic and can serve as carriers of other pathogenic organisms. These amoebae are potentially dangerous when they contaminate health facilities, and these organisms may act as a source of infection for medical personnel and those seeking medical care. The purpose of the present study was to evaluate the extent to which Acanthamoeba species contaminate hemodialysis and dental units in Alexandria, Egypt. Seventy samples were collected aseptically from these systems and cultivated on non-nutrient agar at room temperature, followed by morphological confirmation of the identity of Acanthamoeba using trichrome-stained smears. This study revealed that 42.9% of water samples from the hydraulic systems of both hemodialysis and dental units were positive for Acanthamoeba, with no statistically significant difference between the two unit types or between pre- and post-disinfection samples for each type of unit. The surgical category of dental clinics had the highest contamination rate (72.7%), whereas no contamination was observed for water samples from pediatric dentistry clinics. In conclusion, the hydraulic systems of hemodialysis and dental units in Alexandria are contaminated with Acanthamoeba species, and to minimize the risk of human infections, there is an urgent need to implement effective preventative measures, such as the installation of water filtration units. © 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding author at: Parasitology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt. Tel.: +20 3 591 8407. E-mail address: [email protected] (R. Abdul-Ghani).
1876-0341/$ — see front matter © 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jiph.2012.06.001
Acanthamoeba contamination of hemodialysis and dental units
Introduction Free-living amoebae (FLA) of the genus Acanthamoeba are ubiquitous and inhabit a variety of water, air, and soil environments [1,2]. These amoebae have been isolated from many ecological habitats, including the air, natural thermal waters, and eyewash stations [3—6]. They have been isolated from fresh, brackish, and salt water; soil samples ranging from the Antarctic to arid desertlike areas; sewage dump sites; and household garden and flowerpot soil, water taps, humidifiers, and aquaria [7]. Acanthamoeba spp. have also been recovered from shower heads, ventilators, hydrotherapy baths and heating, ventilation, and air conditioning units in the hospital environment and dental irrigation systems [8]. Dendana et al. [8] reported that water at the entrance of hemodialysis apparatus contained FLA, with many FLA of the genus Acanthamoeba. These authors attributed this contamination to the long stagnation of water, which could lead to the development of biofilms, providing favorable conditions for the growth and proliferation of many microbes and FLA [8]. In addition, these amoebae have been found in dental water lines [9,10]. In addition to disease caused by direct exposure to Acanthamoeba spp., these amoebae can act as vehicles for many pathogenic microorganisms with high virulence and resistance to antibiotics [11]. The report by Huws et al. [11] on the enhancement of methicillinresistant Staphylococcus aureus (MRSA) survival, replication and virulence in association with Acanthamoeba polyphaga provides evidence for this role of amoebae. Cirillo et al. [12] reported enhanced invasion of Legionella pneumophila grown in Acanthamoeba relative to those grown under standard laboratory conditions. A high level resistance of L. pneumophila released from A. polyphaga to disinfectants and antimicrobials has also been reported [13,14]. Many clinical studies have been carried out to assess the pathologic outcomes of amoebal infections in humans. The first human infection by Acanthamoeba, previously reported as Hartmannella, was described after the discovery of amoebae and cysts in brain sections from a patient with brain abscesses in the early 1970s [15]. In addition to causing chronic granulomatous amoebic encephalitis, keratitis, and lung and skin infections, Acanthamoeba spp. also serve as hosts for bacterial pathogens, such as Legionella spp., and for this reason, Acanthamoeba spp. have recently received a lot of attention [16]. Many pathogenic microorganisms that replicate inside free-living amoebae, such as MRSA, are more virulent and more
305
resistant, complicating the situation when FLA and other pathogens co-exist in health care settings [11]. Fukumoto et al. [17] demonstrated the co-existence of Parachlamydia acanthamoebae, a potential pathogen causing hospital-acquired pneumonia, with Acanthamoeba in an actual hospital environment. These authors suggested that the significant impact of Acanthamoeba on the longterm survival of this pathogen could facilitate the pathogen’s spread through a hospital environment [17]. The situation could be dangerous in health care settings visited by immunocompromised patients, including those on hemodialysis. Patients in hospitals may be exposed to Acanthamoebaassociated nosocomial infections, as documented in previous reports [18—21]. In Egypt, only a few studies on Acanthamoeba have been published. The presence of Acanthamoeba spp. in freshwater sources associated with human activities in the Nile Delta region was revealed using morphologic and molecular parameters [22]. In Alexandria, Acanthamoeba spp. were isolated from canals and drains [23]. In addition, A. gruberi and A. rhysodes were isolated from the nasal passages of six healthy children living near the contaminated canals [23]. However, no previously published appraisal of the hydraulic systems of hemodialysis and dental units for the presence of Acanthamoeba spp. exist. Therefore, the present study assessed the hydraulic systems of hemodialysis and dental units in Alexandria to determine whether these systems were contaminated with Acanthamoeba spp. The efficacy of the disinfection processes to eliminate acanthamoebal contamination was also assessed.
Materials and methods The present study is a descriptive, cross-sectional study conducted in selected hemodialysis and dental units in the Alexandria governorate, Egypt, from June to August 2011. This study was carried out in governmental hemodialysis and dental centers in Alexandria. Seventy water samples were collected from hemodialysis and dental units. All 15 hemodialyzers in the hemodialysis center and 20 machines in the dental center were included in the study. Both virus-contaminated and non-virus-contaminated hemodialyzers were included in the sample because patients infected with hepatitis B and/or hepatitis C undergo hemodialysis on separate hemodialyzers from those used by uninfected patients. 2 samples, pre- and post-disinfection, were collected from each machine. The pre-disinfection samples
306 were collected one day before disinfection, and the post-disinfection samples were taken one night collected after disinfection. Water samples (500 ml) were collected from the inlets and outlets of the hemodialyzers and from the hand-pieces of dental machines before and after disinfection using sterile 500-ml containers. The samples were labeled with the site and time of sampling and the number of the machine. The water samples were filtered through sterile 0.45 m membrane filters (Sartorius Stedim Biotech, Germany) within 2 h of collection. Acanthamoeba spp. were monoxenically isolated by inverting the membrane filters on 1.5% non-nutrient agar (NNA) seeded with inactivated Escherichia coli in glass Petri dishes [24]. The culture plates were then incubated at room temperature and monitored for the presence of trophozoites and/or cysts daily for up to two weeks in the Laboratory of Tropical Health Department of the High Institute of Public Health (HIPH), Alexandria University. The presence of Acanthamoeba spp. was investigated by examining the NNA culture plates by inverted phase-contrast microscope (CK; Olympus, Tokyo, Japan) using 10× and 40× objectives. Confirmation of Acanthamoeba, based on size and the presence of acanthopodia and a large central karyosome, was performed using trichrome-stained smears. Smears were examined microscopically for Acanthamoeba trophozoites and/or cysts at 1000× magnification under a light microscope [8]. The data were entered, verified and analyzed using version 15.0 of SPSS for Windows® (SPSS Inc., Chicago, IL, USA). 2 and Fisher’s exact tests were used to assess the differences in the proportions.
Results Thirty (42.9%) out of the 70 samples collected from hemodialysis and dental hydraulic systems, both pre- and post-disinfection, were positive, as determined by inverted microscopy analysis of NNA culture plates followed by microscopic examination of stained smears (Fig. 1). No statistically significant difference (P = 0.791) regarding acanthamoebal contamination of water samples was found between hemodialysis and dental units, with contamination rates of 42.9% (12/30) and 45.0% (18/40), respectively (Table 1). No statistically significant differences were found between the pre- and post-disinfection contamination rates in the water samples from both hemodialysis units [42.9% (6/14) vs. 37.5% (6/16), respectively, P > 0.05] and dental units [47.6% (10/21) vs. 42.1% (8/19), respectively, P > 0.05] (Table 2).
A. Hassan et al.
Fig. 1 Acanthamoeba spp. trophozoites on a trichromestained smear (1000×).
Acanthamoebal contamination of hemodialyzers was related to the viral contamination status. The virus-contaminated hemodialyzers had a higher contamination rate of 61.5% (8/13) compared with only 23.5% (4/17) for the non-virus-contaminated hemodialyzers, as revealed by NNA cultivation and inverted microscopy; this difference was not statistically significant (P > 0.05) (Table 3). The contamination of the dental hydraulic systems was analyzed based on the different dentistry categories, i.e., preventive and conservative, surgical, cosmetic and pediatric. A statistically significant difference (2 -test; P < 0.05) was observed among different dental clinic categories regarding the level of acanthamoebal contamination. The surgical category had the highest contamination rate at 72.7% (8/11), followed by contamination rates of 57.1% (4/7) for the cosmetic category and 33.3% (6/18) for the preventive and conservative category. No contamination was observed for water samples (0/4) from pediatric dentistry clinics using NNA culture plates with an inverted microscope followed by microscopical examination of stained smears (Table 4).
Discussion Because Acanthamoeba spp. are ubiquitously distributed and adapted to many types of environments and conditions, the contamination of hemodialysis and dental units with Acanthamoeba spp. could be a global issue. To our knowledge, the present study is the first study to investigate Acanthamoeba contamination of hemodialysis and dental units in Alexandria. The present study uncovered the potential contamination of hemodialysis and dental units with Acanthamoeba, which has gained increasing interest as a contaminant of
Acanthamoeba contamination of hemodialysis and dental units Table 1
Contamination of the hydraulic systems of haemodialysis and dental units with Acanthamoeba spp.
Type of unit
Non—nutrient agar followed by stained smear examination
Hemodialysis Dental Total a
307
P-value for
No. examined
No. (%) contaminated
P-valuea
30 40
12 (42.9) 18 (45.0)
0.791
70
30 (42.9)
2 -test.
Table 2 Contamination of pre- and post-disinfection water samples with Acanthamoeba spp. according to the type of unit. Type of unit
Non—nutrient agar followed by stained smear examination
Hemodialysis Pre-disinfection Post-disinfection Total Dental Pre-disinfection Post-disinfection Total a
P-values for
Table 3
No. examined
No. (%) contaminated
P-valuea
14 16
6 (42.9) 6 (37.5)
0.940
30
12 (40.0)
21 19
10 (47.6) 8 (42.1)
40
18 (45.0)
0.975
2 -test.
Contamination of water with Acanthamoeba spp. according to the viral contamination of hemodialyzers.
Status of haemodialyzers
Non-nutrient agar followed by stained smear examination No. examined
No. (%) contaminated
P-valuea
Virus-contaminated Non-virus-contaminated
13 17
8 (61.5) 4 (23.5)
0.061
Total
30
12 (40.0)
a
P-value for Fischer’s exact test.
hospital environments. Dental and hemodialysis units showed comparable contamination rates of 45.0% (18/40) and 42.9% (12/30), respectively. Bagheri et al. [25] reported the presence of
Acanthamoeba in approximately 48.0% (45/94) of cold and warm tap water samples collected from different wards of hospitals in 13 cities in Iran. More recently, FLA were detected in 52.9% (37/70)
Table 4 Contamination of dental hydraulic systems’ water with Acanthamoeba spp. according to dentistry clinic categories. Dentistry clinic category
Non-nutrient agar followed by stained smear examination No. examined
No. (%) contaminated
P-valuea
Preventive and conservative Surgical Cosmetic Pediatric
18 11 7 4
6 (33.3) 8 (72.7) 4 (57.1) 0 (00.0)
0.044
Total
40
18 (45.0)
a
P-value for
2 -test.
308 of dust and biofilm samples from hospital wards serving immunocompromised patients in Tehran, Iran [26]. Patients in hemodialysis units are also immunocompromised and therefore, more attention should be paid to the possible contamination of hemodialysis units with Acanthamoeba spp. In Africa, the majority of authors have been interested in detecting and identifying Acanthamoeba species in clinical samples rather than environmental samples [8]. Hamadto et al. [27] reported nonpathogenic Acanthamoeba spp. in 25% (4/16) of swimming pool samples, 40% (4/10) of surface water and canal samples and in all (2/2) tap water samples taken from different Egyptian governorates. The detection of Acanthamoeba in dialysis units is not a new issue, as the presence of FLA was reported in a home dialysis unit in the late 1970s [28]. In Tunisia, Dendana et al. [8] reported Acanthamoeba spp. in 32.6% (15/46) of water samples collected from the hydraulic system of a hemodialysis unit. In the present study, no significant difference in the contamination rate was observed after disinfection of the hydraulic systems of both types of units. This lack of a difference may be due to the low concentrations of chlorine that are usually used for the disinfection of hemodialyzer inlets and outlets and the disinfection of handpiece tubing. These low concentrations are used to prevent corrosion of the machines, according to the instructions from the machines’ distributors (personal communication). In addition, cysts of these amoebae are resistant to changes in temperature and pH and exposure to chlorine, detergents and common disinfectants [29]. The contamination of hemodialysis hydraulic systems could be partly attributed to biofilm formation as a result of water stagnation in the long bending drain connecting the water treatment unit to the hemodialysis unit [8]. As a common practice, in Egypt, hemodialysis patients are allocated to different machines according to their initial screening results for hepatitis B and/or C viruses. A higher percentage of virus-contaminated hemodialyzers were contaminated with Acanthamoeba spp. than non-virus-contaminated hemodialyzers. However, the statistical analysis demonstrated that this difference was not statistically significant. The reason for the greater frequency of acanthamoebal contamination of the virus-contaminated machine could not be determined in the present study. However, there are two water treatment units in the hemodialysis center: one for the viruscontaminated machines and the other for the non-virus-contaminated ones. In addition, little attention is given to virus-contaminated machines
A. Hassan et al. during disinfection and between sessions as a result of the overuse of the machines reserved for HCV-infected patients. This lack of appropriate disinfection might contribute, in part, to the higher level of contamination of these machines (personal communication). Acanthamoeba spp. may pose a direct risk to patients. Tilak et al. [30] first reported an unusual case of Acanthamoeba peritonitis in a malnourished patient on continuous ambulatory peritoneal dialysis for whom cultures for bacteria and fungi were negative. Acanthamoeba spp. have also been reported in dental unit water lines [9,10]. Importantly, the endosymbiotic pathogenic bacteria within amoebae could be a source of microbiological risk for patients with deep dental work or immunosuppression [10]. Legionella spp., Pseudomonas spp. and other amoeba-associated pathogens are colonizers of hospital water supplies and have been found to be causative agents of hospital-acquired pneumonia [31]. It was found that the concentration of FLA is up to 300 times higher in dental unit water samples than tap water despite having the same source [9]. Water used as a coolant and irrigant during dental procedures could be heavily contaminated with microorganisms [32]. In the present study, the hydraulic systems of surgical dentistry clinics were found to be the most frequently contaminated (72.7%; 8 out of 11) with Acanthamoeba spp. This high rate of contamination requires high attention to prevent infection with Acanthamoeba endosymbionts. The high prevalence of FLA, including Acanthamoeba spp., in the hydraulic systems of dental units could be attributed to the formation of biofilms inside some dental instruments; these biofilms would augment the growth and proliferation of FLA [9,33,34]. The FLA in dental water lines provide a reservoir for many pathogenic bacteria, including L. pneumophila and Pseudomonas aeruginosa [33], and thus, an ecological loop is established [9]. Dental water lines could be a potential source of infection for both patients and dental personnel. Higher antibody titers to L. pneumophila have been reported in the sera of dental personnel than the sera of other groups [35—37]. Therefore, immunocompromised patients visiting dental units would be expected to be at risk of acquiring an infection. Microbial aerosols generated during dental treatments may represent an important source of infection [38], and mucosal contact or inhalation of amoebae or cysts by dental workers and patients cannot be ruled out [9]. Acanthamoeba spp. were isolated from the nasopharyngeal and oral regions of male patients with dental caries at an odontological clinic [39]. In addition, eye infection with Acanthamoeba spp.
Acanthamoeba contamination of hemodialysis and dental units after accidental splatter has been reported [40]. A serious ocular infection, possibly linked to a water splash from a dental hand-piece, was reported in a woman who visited her dentist for the replacement of a bridge; this infection resulted in a lawsuit against the dentist [40]. In conclusion, Acanthamoeba spp. were found to be a major contaminant of water in hemodialysis and dental units in Alexandria, Egypt. Therefore, it is recommended that filters with suitable pore sizes be added to filter water in the hydraulic systems of hemodialysis units and dental irrigation water lines. Previous studies have also recommended using filters with a small pore size (0.2 m) at the entrances of hemodialysis machines [8] and the hand-pieces of dental units [10]. The results of the present study are limited by its preliminary nature, as only morphological criteria were employed. Further studies to investigate the potential pathogenicity of Acanthamoeba spp. based on pathogenicity tests and molecular methods are also encouraged. The evaluation of the entire environment, including the air and dust, of these units is also of paramount importance to make a comprehensive appraisal of the situation. Water treatment plants for hemodialysis units and water supplies of dental units should be investigated. The relationship between the contamination of these units and direct or indirect disease acquisition among those seeking healthcare should be the focus of subsequent epidemiological studies. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required.
Acknowledgements The authors would like to thank Prof. Dr. Medhat Ashour, Head of the Microbiology Department, HIPH, Alexandria University and the staff who helped them with the inverted microscopy analysis. They also thank the department heads and nurses of the hemodialysis and dental centers from which water samples were collected. The authors also gratefully thank Prof. John C. Beier, Professor of Epidemiology and Public Health, Miller School of Medicine, University of Miami, for his critical comments and in-depth insight during the preparation of this manuscript.
References [1] Mergeryan H. The prevalence of Acanthamoeba in the human environment. Reviews of Infectious Diseases 1991;13:S390—1.
309
[2] Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clinical Microbiology Reviews 2003;16:273—307. [3] Kingston D, Warhurst DC. Isolation of amoebae from the air. Journal of Medical Microbiology 1969;2:27—36. [4] Rivera F, Roy-Ocotla G, Rosas I, Ramirez E, Bonilla P, Lares F. Amoebae isolated from the atmosphere of Mexico City and environs. Environmental Research 1987;42:149—54. [5] Rivera F, Lares F, Gallegos E, Ramirez E, Bonilla P, Calderon A, et al. Pathogenic amoebae in natural thermal waters of three resorts of Hidalgo, Mexico. Environmental Research 1989;50:289—95. [6] Paszko-Kolva C, Yamamoto H, Shahamat M, Sawyer TK, Morris G, Colwell RR. Isolation of amoebae and Pseudomonas and Legionella spp. from eyewash stations. Applied and Environment Microbiology 1991;57:163—7. [7] Schuster FL, Visvesvara GS. Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease. Veterinary Parasitology 2004;126:91—120. [8] Dendana F, Sellami H, Jarraya F, Sellami A, Makni F, Cheikhrouhou F, et al. Free-living amoebae (FLA): detection, morphological and molecular identification of Acanthamoeba genus in the hydraulic system of an haemodialysis unit in Tunisia. Parasite 2008;15:137—42. [9] Barbeau J, Buhler T. Biofilms augment the number of free-living amoebae in dental unit waterlines. Research in Microbiology 2001;152:753—60. [10] Trabelsi H, Sellami A, Dendena F, Sellami H, Cheikh-Rouhou F, Makni F, et al. Free-living Amoebae (FLA): morphological and molecular identification of Acanthamoeba in dental unit water. Parasite 2010;17:67—70. [11] Huws SA, Smith AW, Enright MC, Wood PJ, Brown MR. Amoebae promote persistence of epidemic strains of MRSA. Environmental Microbiology 2006;8:1130—3. [12] Cirillo JD, Falkow S, Tompkins S. Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infection and Immunity 1994;62:3254—61. [13] Kilvington S, Price J. Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. Journal of Applied Bacteriology 1990;68: 519—25. [14] Barker J, Scaife H, Brown MR. Intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila. Antimicrobial Agents and Chemotherapy 1995;39:2684—8. [15] Jager BV, Stamm WP. Brain abscesses caused by free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin’s disease. Lancet 1972;2:1343—5. [16] Visvesvara GS, Moura H, Schuster FL. Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunology and Medical Microbiology 2007;50:1—26. [17] Fukumoto T, Matsuo J, Hayashi M, Oguri S, Nakamura S, Mizutani Y, et al. Impact of free-living amoebae on presence of Parachlamydia acanthamoebae in the hospital environment and its survival in vitro without requirement for amoebae. Journal of Clinical Microbiology 2010;48:3360—5. [18] Marrie TJ, Raoult D, La Scola B, Birtles RJ, de Carolis E, Canadian Community-Acquired Pneumonia Study Group. Legionella-like and other amoebal pathogens as agents of community-acquired pneumonia. Emerging Infectious Diseases 2001;7:1026—9. [19] La Scola B, Mezi L, Auffray JP, Berland Y, Raoult D. Patients in the intensive care unit are exposed to amoeba-associated pathogens. Infection Control and Hospital Epidemiology 2002;23:462—5.
310
A. Hassan et al.
[20] La Scola B, Boyadjiev I, Greub G, Khamis A, Martin C, Raoult D. Amoeba-resisting bacteria and ventilator-associated pneumonia. Emerging Infectious Diseases 2003;9:815—21. [21] Berger P, Papazian L, Drancourt M, La Scola B, Auffray JP, Raoult D. Ameba-associated microorganisms and diagnosis of nosocomial pneumonia. Emerging Infectious Diseases 2006;12:248—55. [22] Lorenzo-Morales J, Ortega-Rivas A, Martínez E, Khoubbane M, Artigas P, Periago MV, et al. Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt. Acta Tropica 2006;100:63—9. [23] Sadaka HA, el-Nassery SF, Abou Samra LM, Awadalla HN. Isolation and identification of free-living amoebae from some water sources in Alexandria. Journal of the Egyptian Society of Parasitology 1994;24:247—57. [24] Schuster FL. Cultivation of pathogenic and opportunistic free-living amebas. Clinical Microbiology Reviews 2002;15:342—54. [25] Bagheri HR, Shafiei R, Shafiei F, Sajjadi SA. Isolation of Acanthamoeba spp. from drinking waters in several hospitals of Iran. Iranian Journal of Parasitology 2010;5:19—25. [26] Lasjerdi Z, Niyyati M, Haghighi A, Shahabi S, Biderouni FT, Taghipour N, et al. Potentially pathogenic free-living amoebae isolated from hospital wards with immunodeficient patients in Tehran, Iran. Parasitology Research 2011;109:575—80. [27] Hamadto HH, Aufy SM, el-Hayawan IA, Saleh MH, Nagaty IM. Study of free living amoebae in Egypt. Journal of the Egyptian Society of Parasitology 1993;23:631—7. [28] Casemore DP. Free-living amoebae in home dialysis unit. Lancet 1977;2:1078. [29] Teixeira HL, Rocha S, Pinto RMF, Caseiro MM, da Costa SOP. Prevalence of potentially pathogenic free-living amoebae from Acanthamoeba and Naegleria genera in non-hospital, public, internal environments from the city of Santos, Brazil. Brazilian Journal of Infectious Diseases 2009;13:395—7. [30] Tilak R, Singh RG, Wani IA, Parekh A, Prakash J, Usha U. An unusual case of Acanthamoeba peritonitis in a
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
malnourished patient on continuous ambulatory peritoneal dialysis (CAPD). Journal of Infection in Developing Countries 2008;2:146—8. Rutala WA, Weber DJ. Water as a reservoir of nosocomial pathogens. Infection Control and Hospital Epidemiology 1997;18:609—16. Depaola LG, Mangan D, Mills SE, Costerton W, Barbeau J, Shearer B, et al. A review of the science regarding dental unit waterlines. Journal of the American Dental Association 2002;133:1199—206. Barbeau J. Waterborne biofilms and dentistry: the changing face of infection control. Journal of Canadian Dental Association 2000;66:539—41. Walker JT, Bradshaw DJ, Bennett AM, Fulford MR, Martin MV, Marsh PD. Microbial biofilm formation and contamination of dental-unit water systems in general dental practice. Applied and Environment Microbiology 2000;66: 3363—7. Fotos PG, Westfall HN, Snyder IS, Miller RW, Mutchler BM. Prevalence of Legionella-specific IgG and IgM antibody in a dental clinic population. Journal of Dental Research 1985;64:1382—5. Reinthaler FF, Mascher F, Stünzner D. Serological examinations for antibodies against Legionella species in dental personnel. Journal of Dental Research 1988;67: 942—3. Szymanska J. Risk of exposure to Legionella in dental practice. Annals of Agricultural and Environmental Medicine 2004;11:9—12. Grenier D. Quantitative analysis of bacterial aerosols in two different dental clinic environments. Applied and Environment Microbiology 1995;61:3165—8. Rivera F, Rosas I, Castillo M, Chávez M, Gómez R, Chío RE, et al. Pathogenic and free-living protozoa cultured from the nasopharyngeal and oral regions of dental patients. Environmental Research 1986;39:364—71. Barbeau J. Lawsuit against a dentist related to serious ocular infection possibly linked to water from a dental handpiece. Journal of Canadian Dental Association 2007;73:618—22.
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Acanthamoeba contamination of hemodialysis and dental units in Alexandria, Egypt: A neglected potential source of infection Azza Hassan a, Hanan Farouk a, Faika Hassanein a, Rashad Abdul-Ghani b,c,∗, Ahmed H. Abdelhady d a
Tropical Health Department, High Institute of Public Health, Alexandria University, Alexandria, Egypt Parasitology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt c Department of Medical Parasitology, Faculty of Medicine and Health Sciences, Sana’a University, P.O. Box 1247, Sana’a, Yemen d Military Medical Academy, Alexandria, Egypt b
Received 5 April 2012 ; received in revised form 26 May 2012; accepted 1 June 2012
KEYWORDS Acanthamoeba; Free-living amoeba; Hemodialysis; Dental unit; Alexandria
Summary Ubiquitous free-living amoebae of the genus Acanthamoeba can be pathogenic and can serve as carriers of other pathogenic organisms. These amoebae are potentially dangerous when they contaminate health facilities, and these organisms may act as a source of infection for medical personnel and those seeking medical care. The purpose of the present study was to evaluate the extent to which Acanthamoeba species contaminate hemodialysis and dental units in Alexandria, Egypt. Seventy samples were collected aseptically from these systems and cultivated on non-nutrient agar at room temperature, followed by morphological confirmation of the identity of Acanthamoeba using trichrome-stained smears. This study revealed that 42.9% of water samples from the hydraulic systems of both hemodialysis and dental units were positive for Acanthamoeba, with no statistically significant difference between the two unit types or between pre- and post-disinfection samples for each type of unit. The surgical category of dental clinics had the highest contamination rate (72.7%), whereas no contamination was observed for water samples from pediatric dentistry clinics. In conclusion, the hydraulic systems of hemodialysis and dental units in Alexandria are contaminated with Acanthamoeba species, and to minimize the risk of human infections, there is an urgent need to implement effective preventative measures, such as the installation of water filtration units. © 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding author at: Parasitology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt. Tel.: +20 3 591 8407. E-mail address: [email protected] (R. Abdul-Ghani).
1876-0341/$ — see front matter © 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jiph.2012.06.001
Acanthamoeba contamination of hemodialysis and dental units
Introduction Free-living amoebae (FLA) of the genus Acanthamoeba are ubiquitous and inhabit a variety of water, air, and soil environments [1,2]. These amoebae have been isolated from many ecological habitats, including the air, natural thermal waters, and eyewash stations [3—6]. They have been isolated from fresh, brackish, and salt water; soil samples ranging from the Antarctic to arid desertlike areas; sewage dump sites; and household garden and flowerpot soil, water taps, humidifiers, and aquaria [7]. Acanthamoeba spp. have also been recovered from shower heads, ventilators, hydrotherapy baths and heating, ventilation, and air conditioning units in the hospital environment and dental irrigation systems [8]. Dendana et al. [8] reported that water at the entrance of hemodialysis apparatus contained FLA, with many FLA of the genus Acanthamoeba. These authors attributed this contamination to the long stagnation of water, which could lead to the development of biofilms, providing favorable conditions for the growth and proliferation of many microbes and FLA [8]. In addition, these amoebae have been found in dental water lines [9,10]. In addition to disease caused by direct exposure to Acanthamoeba spp., these amoebae can act as vehicles for many pathogenic microorganisms with high virulence and resistance to antibiotics [11]. The report by Huws et al. [11] on the enhancement of methicillinresistant Staphylococcus aureus (MRSA) survival, replication and virulence in association with Acanthamoeba polyphaga provides evidence for this role of amoebae. Cirillo et al. [12] reported enhanced invasion of Legionella pneumophila grown in Acanthamoeba relative to those grown under standard laboratory conditions. A high level resistance of L. pneumophila released from A. polyphaga to disinfectants and antimicrobials has also been reported [13,14]. Many clinical studies have been carried out to assess the pathologic outcomes of amoebal infections in humans. The first human infection by Acanthamoeba, previously reported as Hartmannella, was described after the discovery of amoebae and cysts in brain sections from a patient with brain abscesses in the early 1970s [15]. In addition to causing chronic granulomatous amoebic encephalitis, keratitis, and lung and skin infections, Acanthamoeba spp. also serve as hosts for bacterial pathogens, such as Legionella spp., and for this reason, Acanthamoeba spp. have recently received a lot of attention [16]. Many pathogenic microorganisms that replicate inside free-living amoebae, such as MRSA, are more virulent and more
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resistant, complicating the situation when FLA and other pathogens co-exist in health care settings [11]. Fukumoto et al. [17] demonstrated the co-existence of Parachlamydia acanthamoebae, a potential pathogen causing hospital-acquired pneumonia, with Acanthamoeba in an actual hospital environment. These authors suggested that the significant impact of Acanthamoeba on the longterm survival of this pathogen could facilitate the pathogen’s spread through a hospital environment [17]. The situation could be dangerous in health care settings visited by immunocompromised patients, including those on hemodialysis. Patients in hospitals may be exposed to Acanthamoebaassociated nosocomial infections, as documented in previous reports [18—21]. In Egypt, only a few studies on Acanthamoeba have been published. The presence of Acanthamoeba spp. in freshwater sources associated with human activities in the Nile Delta region was revealed using morphologic and molecular parameters [22]. In Alexandria, Acanthamoeba spp. were isolated from canals and drains [23]. In addition, A. gruberi and A. rhysodes were isolated from the nasal passages of six healthy children living near the contaminated canals [23]. However, no previously published appraisal of the hydraulic systems of hemodialysis and dental units for the presence of Acanthamoeba spp. exist. Therefore, the present study assessed the hydraulic systems of hemodialysis and dental units in Alexandria to determine whether these systems were contaminated with Acanthamoeba spp. The efficacy of the disinfection processes to eliminate acanthamoebal contamination was also assessed.
Materials and methods The present study is a descriptive, cross-sectional study conducted in selected hemodialysis and dental units in the Alexandria governorate, Egypt, from June to August 2011. This study was carried out in governmental hemodialysis and dental centers in Alexandria. Seventy water samples were collected from hemodialysis and dental units. All 15 hemodialyzers in the hemodialysis center and 20 machines in the dental center were included in the study. Both virus-contaminated and non-virus-contaminated hemodialyzers were included in the sample because patients infected with hepatitis B and/or hepatitis C undergo hemodialysis on separate hemodialyzers from those used by uninfected patients. 2 samples, pre- and post-disinfection, were collected from each machine. The pre-disinfection samples
306 were collected one day before disinfection, and the post-disinfection samples were taken one night collected after disinfection. Water samples (500 ml) were collected from the inlets and outlets of the hemodialyzers and from the hand-pieces of dental machines before and after disinfection using sterile 500-ml containers. The samples were labeled with the site and time of sampling and the number of the machine. The water samples were filtered through sterile 0.45 m membrane filters (Sartorius Stedim Biotech, Germany) within 2 h of collection. Acanthamoeba spp. were monoxenically isolated by inverting the membrane filters on 1.5% non-nutrient agar (NNA) seeded with inactivated Escherichia coli in glass Petri dishes [24]. The culture plates were then incubated at room temperature and monitored for the presence of trophozoites and/or cysts daily for up to two weeks in the Laboratory of Tropical Health Department of the High Institute of Public Health (HIPH), Alexandria University. The presence of Acanthamoeba spp. was investigated by examining the NNA culture plates by inverted phase-contrast microscope (CK; Olympus, Tokyo, Japan) using 10× and 40× objectives. Confirmation of Acanthamoeba, based on size and the presence of acanthopodia and a large central karyosome, was performed using trichrome-stained smears. Smears were examined microscopically for Acanthamoeba trophozoites and/or cysts at 1000× magnification under a light microscope [8]. The data were entered, verified and analyzed using version 15.0 of SPSS for Windows® (SPSS Inc., Chicago, IL, USA). 2 and Fisher’s exact tests were used to assess the differences in the proportions.
Results Thirty (42.9%) out of the 70 samples collected from hemodialysis and dental hydraulic systems, both pre- and post-disinfection, were positive, as determined by inverted microscopy analysis of NNA culture plates followed by microscopic examination of stained smears (Fig. 1). No statistically significant difference (P = 0.791) regarding acanthamoebal contamination of water samples was found between hemodialysis and dental units, with contamination rates of 42.9% (12/30) and 45.0% (18/40), respectively (Table 1). No statistically significant differences were found between the pre- and post-disinfection contamination rates in the water samples from both hemodialysis units [42.9% (6/14) vs. 37.5% (6/16), respectively, P > 0.05] and dental units [47.6% (10/21) vs. 42.1% (8/19), respectively, P > 0.05] (Table 2).
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Fig. 1 Acanthamoeba spp. trophozoites on a trichromestained smear (1000×).
Acanthamoebal contamination of hemodialyzers was related to the viral contamination status. The virus-contaminated hemodialyzers had a higher contamination rate of 61.5% (8/13) compared with only 23.5% (4/17) for the non-virus-contaminated hemodialyzers, as revealed by NNA cultivation and inverted microscopy; this difference was not statistically significant (P > 0.05) (Table 3). The contamination of the dental hydraulic systems was analyzed based on the different dentistry categories, i.e., preventive and conservative, surgical, cosmetic and pediatric. A statistically significant difference (2 -test; P < 0.05) was observed among different dental clinic categories regarding the level of acanthamoebal contamination. The surgical category had the highest contamination rate at 72.7% (8/11), followed by contamination rates of 57.1% (4/7) for the cosmetic category and 33.3% (6/18) for the preventive and conservative category. No contamination was observed for water samples (0/4) from pediatric dentistry clinics using NNA culture plates with an inverted microscope followed by microscopical examination of stained smears (Table 4).
Discussion Because Acanthamoeba spp. are ubiquitously distributed and adapted to many types of environments and conditions, the contamination of hemodialysis and dental units with Acanthamoeba spp. could be a global issue. To our knowledge, the present study is the first study to investigate Acanthamoeba contamination of hemodialysis and dental units in Alexandria. The present study uncovered the potential contamination of hemodialysis and dental units with Acanthamoeba, which has gained increasing interest as a contaminant of
Acanthamoeba contamination of hemodialysis and dental units Table 1
Contamination of the hydraulic systems of haemodialysis and dental units with Acanthamoeba spp.
Type of unit
Non—nutrient agar followed by stained smear examination
Hemodialysis Dental Total a
307
P-value for
No. examined
No. (%) contaminated
P-valuea
30 40
12 (42.9) 18 (45.0)
0.791
70
30 (42.9)
2 -test.
Table 2 Contamination of pre- and post-disinfection water samples with Acanthamoeba spp. according to the type of unit. Type of unit
Non—nutrient agar followed by stained smear examination
Hemodialysis Pre-disinfection Post-disinfection Total Dental Pre-disinfection Post-disinfection Total a
P-values for
Table 3
No. examined
No. (%) contaminated
P-valuea
14 16
6 (42.9) 6 (37.5)
0.940
30
12 (40.0)
21 19
10 (47.6) 8 (42.1)
40
18 (45.0)
0.975
2 -test.
Contamination of water with Acanthamoeba spp. according to the viral contamination of hemodialyzers.
Status of haemodialyzers
Non-nutrient agar followed by stained smear examination No. examined
No. (%) contaminated
P-valuea
Virus-contaminated Non-virus-contaminated
13 17
8 (61.5) 4 (23.5)
0.061
Total
30
12 (40.0)
a
P-value for Fischer’s exact test.
hospital environments. Dental and hemodialysis units showed comparable contamination rates of 45.0% (18/40) and 42.9% (12/30), respectively. Bagheri et al. [25] reported the presence of
Acanthamoeba in approximately 48.0% (45/94) of cold and warm tap water samples collected from different wards of hospitals in 13 cities in Iran. More recently, FLA were detected in 52.9% (37/70)
Table 4 Contamination of dental hydraulic systems’ water with Acanthamoeba spp. according to dentistry clinic categories. Dentistry clinic category
Non-nutrient agar followed by stained smear examination No. examined
No. (%) contaminated
P-valuea
Preventive and conservative Surgical Cosmetic Pediatric
18 11 7 4
6 (33.3) 8 (72.7) 4 (57.1) 0 (00.0)
0.044
Total
40
18 (45.0)
a
P-value for
2 -test.
308 of dust and biofilm samples from hospital wards serving immunocompromised patients in Tehran, Iran [26]. Patients in hemodialysis units are also immunocompromised and therefore, more attention should be paid to the possible contamination of hemodialysis units with Acanthamoeba spp. In Africa, the majority of authors have been interested in detecting and identifying Acanthamoeba species in clinical samples rather than environmental samples [8]. Hamadto et al. [27] reported nonpathogenic Acanthamoeba spp. in 25% (4/16) of swimming pool samples, 40% (4/10) of surface water and canal samples and in all (2/2) tap water samples taken from different Egyptian governorates. The detection of Acanthamoeba in dialysis units is not a new issue, as the presence of FLA was reported in a home dialysis unit in the late 1970s [28]. In Tunisia, Dendana et al. [8] reported Acanthamoeba spp. in 32.6% (15/46) of water samples collected from the hydraulic system of a hemodialysis unit. In the present study, no significant difference in the contamination rate was observed after disinfection of the hydraulic systems of both types of units. This lack of a difference may be due to the low concentrations of chlorine that are usually used for the disinfection of hemodialyzer inlets and outlets and the disinfection of handpiece tubing. These low concentrations are used to prevent corrosion of the machines, according to the instructions from the machines’ distributors (personal communication). In addition, cysts of these amoebae are resistant to changes in temperature and pH and exposure to chlorine, detergents and common disinfectants [29]. The contamination of hemodialysis hydraulic systems could be partly attributed to biofilm formation as a result of water stagnation in the long bending drain connecting the water treatment unit to the hemodialysis unit [8]. As a common practice, in Egypt, hemodialysis patients are allocated to different machines according to their initial screening results for hepatitis B and/or C viruses. A higher percentage of virus-contaminated hemodialyzers were contaminated with Acanthamoeba spp. than non-virus-contaminated hemodialyzers. However, the statistical analysis demonstrated that this difference was not statistically significant. The reason for the greater frequency of acanthamoebal contamination of the virus-contaminated machine could not be determined in the present study. However, there are two water treatment units in the hemodialysis center: one for the viruscontaminated machines and the other for the non-virus-contaminated ones. In addition, little attention is given to virus-contaminated machines
A. Hassan et al. during disinfection and between sessions as a result of the overuse of the machines reserved for HCV-infected patients. This lack of appropriate disinfection might contribute, in part, to the higher level of contamination of these machines (personal communication). Acanthamoeba spp. may pose a direct risk to patients. Tilak et al. [30] first reported an unusual case of Acanthamoeba peritonitis in a malnourished patient on continuous ambulatory peritoneal dialysis for whom cultures for bacteria and fungi were negative. Acanthamoeba spp. have also been reported in dental unit water lines [9,10]. Importantly, the endosymbiotic pathogenic bacteria within amoebae could be a source of microbiological risk for patients with deep dental work or immunosuppression [10]. Legionella spp., Pseudomonas spp. and other amoeba-associated pathogens are colonizers of hospital water supplies and have been found to be causative agents of hospital-acquired pneumonia [31]. It was found that the concentration of FLA is up to 300 times higher in dental unit water samples than tap water despite having the same source [9]. Water used as a coolant and irrigant during dental procedures could be heavily contaminated with microorganisms [32]. In the present study, the hydraulic systems of surgical dentistry clinics were found to be the most frequently contaminated (72.7%; 8 out of 11) with Acanthamoeba spp. This high rate of contamination requires high attention to prevent infection with Acanthamoeba endosymbionts. The high prevalence of FLA, including Acanthamoeba spp., in the hydraulic systems of dental units could be attributed to the formation of biofilms inside some dental instruments; these biofilms would augment the growth and proliferation of FLA [9,33,34]. The FLA in dental water lines provide a reservoir for many pathogenic bacteria, including L. pneumophila and Pseudomonas aeruginosa [33], and thus, an ecological loop is established [9]. Dental water lines could be a potential source of infection for both patients and dental personnel. Higher antibody titers to L. pneumophila have been reported in the sera of dental personnel than the sera of other groups [35—37]. Therefore, immunocompromised patients visiting dental units would be expected to be at risk of acquiring an infection. Microbial aerosols generated during dental treatments may represent an important source of infection [38], and mucosal contact or inhalation of amoebae or cysts by dental workers and patients cannot be ruled out [9]. Acanthamoeba spp. were isolated from the nasopharyngeal and oral regions of male patients with dental caries at an odontological clinic [39]. In addition, eye infection with Acanthamoeba spp.
Acanthamoeba contamination of hemodialysis and dental units after accidental splatter has been reported [40]. A serious ocular infection, possibly linked to a water splash from a dental hand-piece, was reported in a woman who visited her dentist for the replacement of a bridge; this infection resulted in a lawsuit against the dentist [40]. In conclusion, Acanthamoeba spp. were found to be a major contaminant of water in hemodialysis and dental units in Alexandria, Egypt. Therefore, it is recommended that filters with suitable pore sizes be added to filter water in the hydraulic systems of hemodialysis units and dental irrigation water lines. Previous studies have also recommended using filters with a small pore size (0.2 m) at the entrances of hemodialysis machines [8] and the hand-pieces of dental units [10]. The results of the present study are limited by its preliminary nature, as only morphological criteria were employed. Further studies to investigate the potential pathogenicity of Acanthamoeba spp. based on pathogenicity tests and molecular methods are also encouraged. The evaluation of the entire environment, including the air and dust, of these units is also of paramount importance to make a comprehensive appraisal of the situation. Water treatment plants for hemodialysis units and water supplies of dental units should be investigated. The relationship between the contamination of these units and direct or indirect disease acquisition among those seeking healthcare should be the focus of subsequent epidemiological studies. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required.
Acknowledgements The authors would like to thank Prof. Dr. Medhat Ashour, Head of the Microbiology Department, HIPH, Alexandria University and the staff who helped them with the inverted microscopy analysis. They also thank the department heads and nurses of the hemodialysis and dental centers from which water samples were collected. The authors also gratefully thank Prof. John C. Beier, Professor of Epidemiology and Public Health, Miller School of Medicine, University of Miami, for his critical comments and in-depth insight during the preparation of this manuscript.
References [1] Mergeryan H. The prevalence of Acanthamoeba in the human environment. Reviews of Infectious Diseases 1991;13:S390—1.
309
[2] Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clinical Microbiology Reviews 2003;16:273—307. [3] Kingston D, Warhurst DC. Isolation of amoebae from the air. Journal of Medical Microbiology 1969;2:27—36. [4] Rivera F, Roy-Ocotla G, Rosas I, Ramirez E, Bonilla P, Lares F. Amoebae isolated from the atmosphere of Mexico City and environs. Environmental Research 1987;42:149—54. [5] Rivera F, Lares F, Gallegos E, Ramirez E, Bonilla P, Calderon A, et al. Pathogenic amoebae in natural thermal waters of three resorts of Hidalgo, Mexico. Environmental Research 1989;50:289—95. [6] Paszko-Kolva C, Yamamoto H, Shahamat M, Sawyer TK, Morris G, Colwell RR. Isolation of amoebae and Pseudomonas and Legionella spp. from eyewash stations. Applied and Environment Microbiology 1991;57:163—7. [7] Schuster FL, Visvesvara GS. Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease. Veterinary Parasitology 2004;126:91—120. [8] Dendana F, Sellami H, Jarraya F, Sellami A, Makni F, Cheikhrouhou F, et al. Free-living amoebae (FLA): detection, morphological and molecular identification of Acanthamoeba genus in the hydraulic system of an haemodialysis unit in Tunisia. Parasite 2008;15:137—42. [9] Barbeau J, Buhler T. Biofilms augment the number of free-living amoebae in dental unit waterlines. Research in Microbiology 2001;152:753—60. [10] Trabelsi H, Sellami A, Dendena F, Sellami H, Cheikh-Rouhou F, Makni F, et al. Free-living Amoebae (FLA): morphological and molecular identification of Acanthamoeba in dental unit water. Parasite 2010;17:67—70. [11] Huws SA, Smith AW, Enright MC, Wood PJ, Brown MR. Amoebae promote persistence of epidemic strains of MRSA. Environmental Microbiology 2006;8:1130—3. [12] Cirillo JD, Falkow S, Tompkins S. Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infection and Immunity 1994;62:3254—61. [13] Kilvington S, Price J. Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. Journal of Applied Bacteriology 1990;68: 519—25. [14] Barker J, Scaife H, Brown MR. Intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila. Antimicrobial Agents and Chemotherapy 1995;39:2684—8. [15] Jager BV, Stamm WP. Brain abscesses caused by free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin’s disease. Lancet 1972;2:1343—5. [16] Visvesvara GS, Moura H, Schuster FL. Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunology and Medical Microbiology 2007;50:1—26. [17] Fukumoto T, Matsuo J, Hayashi M, Oguri S, Nakamura S, Mizutani Y, et al. Impact of free-living amoebae on presence of Parachlamydia acanthamoebae in the hospital environment and its survival in vitro without requirement for amoebae. Journal of Clinical Microbiology 2010;48:3360—5. [18] Marrie TJ, Raoult D, La Scola B, Birtles RJ, de Carolis E, Canadian Community-Acquired Pneumonia Study Group. Legionella-like and other amoebal pathogens as agents of community-acquired pneumonia. Emerging Infectious Diseases 2001;7:1026—9. [19] La Scola B, Mezi L, Auffray JP, Berland Y, Raoult D. Patients in the intensive care unit are exposed to amoeba-associated pathogens. Infection Control and Hospital Epidemiology 2002;23:462—5.
310
A. Hassan et al.
[20] La Scola B, Boyadjiev I, Greub G, Khamis A, Martin C, Raoult D. Amoeba-resisting bacteria and ventilator-associated pneumonia. Emerging Infectious Diseases 2003;9:815—21. [21] Berger P, Papazian L, Drancourt M, La Scola B, Auffray JP, Raoult D. Ameba-associated microorganisms and diagnosis of nosocomial pneumonia. Emerging Infectious Diseases 2006;12:248—55. [22] Lorenzo-Morales J, Ortega-Rivas A, Martínez E, Khoubbane M, Artigas P, Periago MV, et al. Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt. Acta Tropica 2006;100:63—9. [23] Sadaka HA, el-Nassery SF, Abou Samra LM, Awadalla HN. Isolation and identification of free-living amoebae from some water sources in Alexandria. Journal of the Egyptian Society of Parasitology 1994;24:247—57. [24] Schuster FL. Cultivation of pathogenic and opportunistic free-living amebas. Clinical Microbiology Reviews 2002;15:342—54. [25] Bagheri HR, Shafiei R, Shafiei F, Sajjadi SA. Isolation of Acanthamoeba spp. from drinking waters in several hospitals of Iran. Iranian Journal of Parasitology 2010;5:19—25. [26] Lasjerdi Z, Niyyati M, Haghighi A, Shahabi S, Biderouni FT, Taghipour N, et al. Potentially pathogenic free-living amoebae isolated from hospital wards with immunodeficient patients in Tehran, Iran. Parasitology Research 2011;109:575—80. [27] Hamadto HH, Aufy SM, el-Hayawan IA, Saleh MH, Nagaty IM. Study of free living amoebae in Egypt. Journal of the Egyptian Society of Parasitology 1993;23:631—7. [28] Casemore DP. Free-living amoebae in home dialysis unit. Lancet 1977;2:1078. [29] Teixeira HL, Rocha S, Pinto RMF, Caseiro MM, da Costa SOP. Prevalence of potentially pathogenic free-living amoebae from Acanthamoeba and Naegleria genera in non-hospital, public, internal environments from the city of Santos, Brazil. Brazilian Journal of Infectious Diseases 2009;13:395—7. [30] Tilak R, Singh RG, Wani IA, Parekh A, Prakash J, Usha U. An unusual case of Acanthamoeba peritonitis in a
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
malnourished patient on continuous ambulatory peritoneal dialysis (CAPD). Journal of Infection in Developing Countries 2008;2:146—8. Rutala WA, Weber DJ. Water as a reservoir of nosocomial pathogens. Infection Control and Hospital Epidemiology 1997;18:609—16. Depaola LG, Mangan D, Mills SE, Costerton W, Barbeau J, Shearer B, et al. A review of the science regarding dental unit waterlines. Journal of the American Dental Association 2002;133:1199—206. Barbeau J. Waterborne biofilms and dentistry: the changing face of infection control. Journal of Canadian Dental Association 2000;66:539—41. Walker JT, Bradshaw DJ, Bennett AM, Fulford MR, Martin MV, Marsh PD. Microbial biofilm formation and contamination of dental-unit water systems in general dental practice. Applied and Environment Microbiology 2000;66: 3363—7. Fotos PG, Westfall HN, Snyder IS, Miller RW, Mutchler BM. Prevalence of Legionella-specific IgG and IgM antibody in a dental clinic population. Journal of Dental Research 1985;64:1382—5. Reinthaler FF, Mascher F, Stünzner D. Serological examinations for antibodies against Legionella species in dental personnel. Journal of Dental Research 1988;67: 942—3. Szymanska J. Risk of exposure to Legionella in dental practice. Annals of Agricultural and Environmental Medicine 2004;11:9—12. Grenier D. Quantitative analysis of bacterial aerosols in two different dental clinic environments. Applied and Environment Microbiology 1995;61:3165—8. Rivera F, Rosas I, Castillo M, Chávez M, Gómez R, Chío RE, et al. Pathogenic and free-living protozoa cultured from the nasopharyngeal and oral regions of dental patients. Environmental Research 1986;39:364—71. Barbeau J. Lawsuit against a dentist related to serious ocular infection possibly linked to water from a dental handpiece. Journal of Canadian Dental Association 2007;73:618—22.
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