Parasitol Res DOI 10.1007/s00436-014-4083-6

ORIGINAL PAPER

Isolation and molecular characterization of Acanthamoeba and Balamuthia mandrillaris from combination shower units in Costa Rica Lissette Retana-Moreira & Elizabeth Abrahams-Sandí & Alfonso Martín Cabello-Vílchez & María Reyes-Batlle & Basilio Valladares & Enrique Martínez-Carretero & José E. Piñero & Jacob Lorenzo-Morales

Received: 17 May 2014 / Accepted: 11 August 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Free living amoebae (FLA) are ubiquitous protozoa, which may behave as parasites under certain conditions. Four genera are recognized as causal agents of infections in humans and animals: Naegleria, Sappinia, Acanthamoeba and Balamuthia. This work determines the presence of FLA in combination shower units and employs molecular biology for the characterization of isolates. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 30 % of the units sampled. In addition to Acanthamoeba cysts, trophozoites with morphological characteristics similar to Balamuthia were identified. PCR assay using the mitochondrial 16S rRNA gene as a target confirmed the identification of the amoeba as Balamuthia mandrillaris. Up to date, this is the first report of the isolation of B. mandrillaris in Central America and the fifth report worldwide.

L. Retana-Moreira : E. Abrahams-Sandí (*) Department of Parasitology, University of Costa Rica, San Pedro, Montes de Oca, Costa Rica e-mail: [email protected] L. Retana-Moreira : E. Abrahams-Sandí Centro de Investigación en Enfermedades Tropicales, CIET, University of Costa Rica, San Pedro, Montes de Oca, Costa Rica A. M. Cabello-Vílchez Laboratory of Clinical Microbiology, Tropical Medicine Institute “Alexander von Humboldt” IMT-AvH, Universidad Peruana Cayetano Heredia, Lima, Peru M. Reyes-Batlle : B. Valladares : E. Martínez-Carretero : J. E. Piñero : J. Lorenzo-Morales University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Avda. Astrofísico Fco. Sánchez, S/N, 38203 La Laguna, Tenerife, Canary Islands, Spain

Keywords Free-living amoebae . Acanthamoeba . Balamuthia mandrillaris . Costa Rica

Introduction Free-living amoebae (FLA) are protozoa widely distributed in nature, which can be found in a variety of environments such as water, soil, dust, air, air conditioning units, contact lenses, eyewash solutions and dental treatment units (Trabelsi et al. 2012; Nacapunchai et al. 1999; Niyyati et al. 2009; Khan 2006; Michel and Just 1984). Four genera are recognized as causal agents of infections in humans and animals: Naegleria, Sappinia, Acanthamoeba and Balamuthia. Besides being considered causative agents of diseases, FLA may also be hosts for intracellular pathogenic bacteria (Matin et al. 2008). Among the 30 species of Naegleria described, only Naegleria fowleri is known to cause primary amoebic meningoencephalitis (PAM) in both healthy children and young adults, considered as a fatal infection of the central nervous system (Trabelsi et al. 2012). On the other hand, Sappinia diploidea has been isolated only once, from a brain infection in a healthy person (Schuster and Visvesvara 2004; Gelman et al. 2001). Acanthamoeba spp. and Balamuthia mandrillaris cause granulomatous amoebic encephalitis (GAE) primarily in immunocompromised patients, although B. mandrillaris has been found in immunocompetent individuals (Trabelsi et al. 2012; Matin et al. 2008; Visvesvara et al. 2007). To date, more than 200 cases of Balamuthia encephalitis (BAE) have been reported worldwide, with the majority of cases reported in Latin America and the USA (Matin et al. 2008; CabelloVílchez et al. 2014). Of these, 55 cases were reported in

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Peru, 4 cases in Argentina, 4 cases in Mexico, 1 case in Brazil and 1 case in Venezuela, with no cases reported so far in Central America. Besides clinical manifestations in the central nervous system, both Acanthamoeba and Balamuthia have been isolated from different organs such as skin, lungs and kidneys (Trabelsi et al. 2012; Matin et al. 2008; CabelloVílchez et al. 2014). Moreover, Acanthamoeba spp. is also the causative agent of amoebic keratitis in immunocompetent persons, mostly contact lens wearers. The pathophysiology of the diseases they cause, as well as the information provided by studies based on 16S RNA sequencing of these two amoebic genera, demonstrates that they are closely related and belong taxonomically to the same super group (Amebozoa:Acanthamoebidae) (Amaral Zettler et al. 2000; Adl et al. 2005). Of the four genera mentioned above, Acanthamoeba is believed to be the most common amoeba (Trabelsi et al. 2012). More than 24 species were identified and initially classified into three groups, based on morphological criteria and cyst size, but, due to the morphological changes in cysts caused by culture conditions (Trabelsi et al. 2012), this classification is not recommended. The identification of isolates has been improved with the description of a genotyping method based on sequencing of the diagnostic fragment 3 (DF3) of the 18S ribosomal RNA (rRNA) gene (Booton et al. 2002; De Jonckheere 2007). Today, 18 genotypes of Acanthamoeba have been established and named from T1 to T18, but only some of them are considered to be pathogenic (Stothard et al. 1998; Horn et al. 1999; Gast 2001; Hewett et al. 2003; Corsaro and Venditti 2010; Nuprasert et al. 2010; Qvarnstrom et al. 2013). Furthermore, it has also been reported that the majority of human infections have been caused by Acanthamoeba genotype T4 (Trabelsi et al. 2012). So far, there are numerous reports of isolation of Acanthamoeba in environmental samples, being especially important those reports of isolation of amoebae in places that could serve as direct sources of infection for humans (i.e. swimming pools, contact lenses, air conditioning units, different kind of surfaces at hospitals and clinics, dialysis and dental units, public baths, water reservoirs etc.) (Lorenzo-Morales et al. 2013; Tanveer et al. 2013; Kiss et al. 2014; Lass et al. 2014). In the present study, combination shower units installed in the research and teaching laboratories of a public university in Costa Rica were checked for the presence of the four genera FLA of clinical importance mentioned above. Moreover, to the best of our knowledge, this is the first investigation employing molecular biology tools for the characterization of environmental FLA of Acanthamoeba and Balamuthia genera in Costa Rica.

Material and methods Sample collection Thirty six dust (24) and water (12) samples were collected from 12 combined emergency units, located in different laboratories at the School of Chemistry and the Faculty of Microbiology, in the University of Costa Rica (San Pedro de Montes de Oca, Costa Rica). The dust samples were collected from shower head and wash bowl of the combination units using sterile cotton swabs. Swabs were submersed in 3 ml of sterile Page solution and washed using shaking. Then, the solution was centrifuged at 1,500 rpm for 10 min. The supernatant was removed and 200 μl of each sample was inoculated onto 1.5 % nonnutrient agar plates seeded with an Escherichia coli suspension. The samples were incubated at 30 °C and examined daily after 3 days, up to 7 days. After that, plates without amoebic growth were discarded. Regarding the water samples, stagnant water samples from spray heads were collected using sterile Pasteur pipettes. Each sample was centrifuged at 1,500 rpm for 10 min and approximately 120 μl from the resulting sediment was inoculated in non-nutrient agar plates as described above. Microscopic examination The presence of FLA was observed by using an inverted microscope (Olympus CK30). The initial identification of potentia lly pathogenic species (Acanthamo eba, Balamuthia and Naegleria) was based on the observation of the shape and size of cysts, characteristics of the endocysts, appearance of trophozoites and type of pseudopods (Page 1988). DNA extraction and isolates genotyping DNA from the positive culture was extracted using the method described by Reyes-Batlle et al. (2014). DNA concentration was determined using a Nano Drop 1000 spectrophotometer (Fisher Scientific, Spain). Amplifications of 18S ribosomal DNA (rDNA) gene (DF3 region, 500 bp) from Acanthamoeba were performed as previously described by Booton et al. in 2005. The PCR amplification reaction was performed in a final volume of 50 μL, using an Arktik thermal cycler (Thermo Scientific). The reaction mixture contained 40 ng/μl of DNA, 200 μL of each of the four deoxynucleoside triphosphates (dNTP), 10 mM of each primer (JDP1 and JDP2) (Invitrogen, Life Technologies), 3.5 mM of MgCl2 (AmpONE, GeneALL®, Vitro, Madrid, Spain), 1X of PCR buffer (AmpONE, GeneALL®, Vitro, Madrid, Spain) and 5 U of Taq polymerase (AmpONE, GeneALL®, Vitro, Madrid, Spain), The thermal cycling

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conditions were as follows: an initial incubation at 95 °C for 5 min, followed by 35 cycles of denaturing at 95 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s and a final extension at 72 °C for 7 min. Acanthamoeba castellanii Neff American Type Culture Collection (ATCC) 30010 strain and Acanthamoeba T4 strain SGC-3 (Reyes-Batlle et al. 2014) were used as positive controls and distilled water added to the reaction mixture (instead of DNA) as the negative control. A m p l i f i c a t i o n o f th e 1 6 S r D N A g e n e f r o m Balamuthia was performed using the method described by Niyyati et al. in 2009. This reaction generates a 1,075-bp PCR product with B. mandrillaris DNA. The amplification reaction was performed as described above, but with the forward primer Balspec16S (5′CGCATGTATGAAGAAGACCA-3′) and the reverse primer Balspec16S (5′-TTACCTATATAATTGTCGATAC CA-3′). Amplification started with an initial incubation at 95 °C for 5 min, followed by 40 cycles of denaturing at 95 °C for 60 s, annealing at 48 °C for 60 s and extension at 72 °C for 60 s, with a final extension at 72 °C for 7 min. Balamuthia strain ID-19 (Niyyati et al. 2009) and ATCC B. mandrillaris CDC:V451 (ATCC PRA-291) strain were used as positive control and distilled water as the negative control. PCR products were purified using the QIAquick PCR purification kit (QIAGEN, Germany), according to the manufacturer’s instructions and sequenced in both directions. The sequencing was done in a MegaBACE 1000 automatic sequencer (Healthcare Biosciences, Spain) using the University of La Laguna sequencing services (Servicio de Secuenciación SEGAI, University of La Laguna). Sequences were edited and aligned using Mega 5.0 software program (Tamura et al. 2011). Phylogenetic analyses were carried out using the methodology described by Reyes-Batlle et al. (2014), with Mega 5.0 software program.

Results and discussion From the 36 dust samples, five samples (three from shower heads and two from wash bowls) were positive for Acanthamoeba, based on morphological characteristics (Fig. 1) and PCR results. This represents a 14 % of positive samples, which correspond to units 2, 3, 5 and 7. Phylogenetic analysis indicated that all isolates belonged to Acanthamoeba genotype T4 (Fig. 2). In addition to Acanthamoeba cysts, trophozoites with morphological characteristics similar to Balamuthia genus were identified in one of the dust samples (Fig. 3). The PCR reaction using the mitochondrial 16S rRNA gene as a target confirmed the identification of the amoeba as B. mandrillaris. Moreover, the obtained sequence of the PCR product revealed a homology of 97 % with the available sequences for other B. mandrillaris strains from GenBank. Furthermore, amoebae with morphology similar to Naegleria or Sappinia were not found in this study. Until now, there are only few investigations about FLA in Costa Rica. The first two studies reported the presence of Acanthamoeba in stool samples and nasal mucus from healthy individuals, pointing that some of these isolates induced cerebral lesions in laboratory animals (Chinchilla et al. 1979; Echandi et al. 1994). In 2006, Morales et al. reported the first and only known case of amoebic encephalitis in Costa Rica, in a cow. Nevertheless, in 2011, Wagner et al. described a clinical case of encephalitis in a patient in Venezuela who suggested that the infection may have occurred in a Costa Rican forest. The present study is the first about isolation of FLA from environmental samples in Costa Rica and one of the few studies published about isolation of these amoebae from combination shower units as sampling sites. The obtained results indicate that 14 % of the samples, representing 30 % of the combination shower units included in this study, were positive for Acanthamoeba genus. The isolation of potentially pathogenic FLA in emergency

Fig. 1 Cysts of amoebae isolated from dust samples. Morphological characteristics suggest that cysts belong to Acanthamoeba spp.

Parasitol Res Fig. 2 Phylogenetic analysis of Acanthamoeba isolates from dust samples. All of the isolates from Costa Rica belong to genotype T4 (Sed2: clones a, b, c, d; Sed5: clone a; Sed7: clone a; Tor3: clones a, c and Tor7: clone a)

eyewash and shower equipment has been reported before. Tynd all et al. (19 87) fo und Aca nthamo eba a nd Hartmannella in portable and sanitary stations; Bier and Sawyer (1990) demonstrated the presence of amoebae in 31 of 56 eyewash stations, reporting a higher prevalence in units with water reservoirs. Finally, Paszko-Kolva et al. (1991) informed about the presence of FLA in 47.5 % of these units, including Acanthamoeba and also demonstrating the presence

of Pseudomonas and Legionella sp. These authors suggest that contaminated units could be the source of severe infections if these potentially pathogenic organisms are introduced in traumatized eyes and even consider this equipment a potential environmental hazard. In the case of our study and through the use of molecular technique, it was determined that the isolated Acanthamoeba belonged to genotype T4. This genotype is the most common isolate in environmental

Fig. 3 a Sed2 sample showing a mixed population of Acanthamoebae (cysts) and Balamuthia mandrillaris trophozoites (magnification of ×10). b Sed 2 sample at ×100 (arrows indicate Balamuthia mandrillaris trophozoites within the agar)

A

B

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samples worldwide and most of the Acanthamoeba associated with central nervous system and eye infection cases (keratitis) also belong to this genotype. Additional tests are required to determine if our isolates could be considered as potentially pathogenic (e.g., determination of proteases, thermotolerance, osmotolerance, among others). Many other authors have reported of the presence of pathogenic genotypes of Acanthamoeba in water and dust related sources including household drinking water, natural water reservoirs and public baths among others (Tanveer et al. 2013; Kiss et al. 2014; Lass et al. 2014). It is important to highlight in this study, the isolation and identification of B. mandrillaris. Up to date, this species has been isolated four times: three times from soil sources (Dunnebacke et al. 2004; Schuster et al. 2003; CabelloVílchez et al. 2014) and once from dust samples (Niyyati et al. 2009). Only in one of these cases, which occurred in the USA (Schuster et al. 2003), the isolate was associated with the death of a child due to BAE. Just recently, Cabello-Vílchez et al. (2014) reported the environmental isolation of four B. mandrillaris strains from soil sources in Peru, the second country worldwide in number of BAE reported cases. Interestingly, these soil samples were collected from areas where clinical cases of BAE were reported previously and therefore soil could be a source of infection at least in Peru (Cabello-Vílchez et al. 2014). The environmental isolate of B. mandrillaris included in the present study represents the fifth time that this species is isolated from the environment and the first report of this amoebic genus in Costa Rica and Central America. According to Niyyati et al., the isolation of this species from dust samples indicates that the risk of infection with this amoeba is not limited to soil, an aspect that must be considered when evaluating the epidemiology of a clinical case. In Costa Rica, there are no reports of infections due to Balamuthia; this fact implies that the impact of this agent and other FLA on public health in this country is unknown. Nonetheless, Costa Rica was suggested as the infection source in a case of GAE reported in Venezuela (Wagner et al. 2011) and thus awareness should be raised towards a better knowledge and control of this emerging pathogenic amoeba. Although the number of infections due to B. mandrillaris is relatively low, the estimated frequency of cases reported so far is likely to be underestimated (Cabello-Vílchez et al. 2014). Among other issues, difficulties in isolation due to its fastidious growth at laboratory conditions, limits the knowledge in terms of biology and geographical distribution of this organism (Lorenzo-Morales et al. 2013; Cabello-Vílchez et al. 2014). To date, there are reports of a significant number of people with antibodies against this amoeba (Schuster et al. 2001, 2009), but it is unknown if antibody response is due to continuous exposure with non-pathogenic Balamuthia-like amoebae, as reported in the case of Acanthamoeba.

Finally, the findings that we report in this study, reiterate the need of an appropriate control and maintenance of combination shower units, to prevent the establishment of possible infection sources in people who, at the time of using these units, may have a lesion which could facilitate the entrance of pathogenic species. Acknowledgments This study was supported by project 803-B4-050, Vicerrectoría de Investigación, University of Costa Rica. This work was supported by the grants RICET (project no. RD12/0018/0012of the programme of Redes Temáticas de Investigación Cooperativa, FIS), Spanish Ministry of Health, Madrid, Spain and the Project FIS PI10/01298 “Protozoosis emergentes por amebas de vida libre: aislamiento y caracterización molecular, identificación de cepas transportadoras de otros agentes patógenos y búsqueda de quimioterapias efectivas”, PI13/00490 “Protozoosis Emergentes por Amebas de Vida Libre: Aislamiento, Caracterización, Nuevas Aproximaciones Terapéuticas y Traslación Clínica de los Resultados” from the Instituto de Salud Carlos III and Project ref. AGUA3 “Amebas de Vida Libre como Marcadores de Calidad del Agua” from CajaCanarias Fundación. MRB was funded by CEI Canarias, Campus Atlántico Internacional and Becas Fundación Cajacanarias para Postgraduados 2014. JLM was supported by the Ramón y Cajal Subprogramme from the Spanish Ministry of Economy and Competivity RYC-2011-08863.

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