Parasitol Res (2014) 113:2509–2513 DOI 10.1007/s00436-014-3900-2
ORIGINAL PAPER
The isolation of Balamuthia mandrillaris from environmental sources from Peru Alfonso Martín Cabello-Vílchez & María Reyes-Batlle & Esmelda Montalbán-Sandoval & Carmen Mª Martín-Navarro & Atteneri López-Arencibia & Rafaela Elias-Letts & Humberto Guerra & Eduardo Gotuzzo & Enrique Martínez-Carretero & José E. Piñero & Sutherland K. Maciver & Basilio Valladares & Jacob Lorenzo-Morales
Received: 18 February 2014 / Accepted: 9 April 2014 / Published online: 30 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Balamuthia mandrillaris is an opportunistic freeliving amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely A. M. Cabello-Vílchez : M. Reyes-Batlle : C. M. Martín-Navarro : A. López-Arencibia : E. Martínez-Carretero : J. E. Piñero : B. Valladares : J. Lorenzo-Morales (*) University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Avenida Astrofísico Francisco Sánchez SN, 38203, La Laguna Tenerife, Canary Islands, Spain e-mail: [email protected] A. M. Cabello-Vílchez : H. Guerra : E. Gotuzzo Universidad Peruana Cayetano Heredia, Laboratory of Clinical Microbiology, Tropical Medicine Institute “Alexander von Humboldt” IMT-AvH, Lima, Peru E. Montalbán-Sandoval Asociación Civil Impacta Perú Salud y Educación, Lima, Peru C. M. Martín-Navarro : S. K. Maciver Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, Scotland, UK R. Elias-Letts Laboratorio de Ecotoxicología, Laboratorios de Investigación y Desarrollo (LID), Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru
encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillarislike trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding. Keywords Balamuthia mandrillaris . Emerging pathogen . Encephalitis . Skin . Therapy . Diagnosis . Peru
Introduction Since the first description of a human infection being caused by an otherwise free-living amoeba (Delgado and AguilaPardo 1957), a number of amoebic species have been found to have the capacity to cause fatal infections in humans. Amongst these are Acanthamoeba, Naegleria and Sapinia, but more recently another amoebic genus, Balamuthia was added to this list (Visvesvara et al. 2007). Balamuthia mandrillaris was formerly described as a leptomyxid amoeba (Visvesvara et al. 1990, 1993) and was first discovered from the brain of a mandrill baboon that died of meningoencephalitis. Currently, B. mandrillaris causing
2510
Balamuthia amoebic encephalitis (BAE) is recognized as being an emerging disease, but this amoeba is also believed to cause skin lesions in both immunocompetent and immunocompromised individuals (Maciver 2007; Matin et al. 2008; Bravo and Seas 2012). Since, the description of B. mandrillaris as the causative agent of BAE around 200 cases have been reported worldwide (Maciver 2007; Matin et al. 2008; Diaz 2011; Bravo and Seas 2012; Jackson et al. 2014). The Americas have reported the highest number of infected patients, mainly in the southwest of the USA (Arizona, Texas and California) and also in Latin America with cases reported in Mexico, Peru, Venezuela, Argentina, Brazil and Chile (Lorenzo-Morales et al. 2013). In the last 29 years, around 55 cases of B. mandrillaris infections have been reported in Peru (Bravo and Seas 2012). It is worth mentioning that a higher frequency of cutaneous involvement (usual location in the central face area) has been reported from the Peruvian cases compared to the pure neurological involvement seen in many of the North American cases (Moser 2011; Bravo and Seas 2012; Lorenzo-Morales et al. 2013). Investigation of these cases have shown that general symptoms are absent during the cutaneous stage of BAE (Moser 2011; Bravo and Seas 2012). Skin lesion is present in almost all of the affected Peruvian patients reported so far, and these lesions have been reported to form a thin, painless plaque of one to several centimeters in diameter, commonly located around the nose, although other locations such as the knee, the chest and the elbow have also been observed (Bravo and Seas 2012; Lorenzo-Morales et al. 2013). While a single lesion is present on most patients, satellite lesions sometimes appear at the periphery of the main lesion on the face and extremities as well as the oral cavity. After a variable period of time, from weeks to years, the infection progresses to the brain forming BAE that often results in death. Available data for BAE cases indicate that it is less likely to be diagnosed correctly due to the lack of standard diagnostic tools and the unfamiliarity of clinicians with this amoebic pathogen (Schuster et al. 2009; Bravo and Seas 2012; Lobo et al. 2013; Lorenzo-Morales et al. 2013). Thus, BAE is gaining a reputation as a significant threat to human health; the most distressing aspect is the limited availability of fully effective treatments against these infections. So far, only ten BAE survivors have been recorded worldwide, and unfortunately in most of these cases, the patients survives with permanent impairment (Bravo and Seas 2012; LorenzoMorales et al. 2013). Even though the risk factors for BAE are still largely unknown, environmental exposure to soil (i.e. during the performance of activities such as agricultural work or gardening) has been reported as the main potential source for B. mandrillaris infections worldwide (Schuster et al. 2003;
Parasitol Res (2014) 113:2509–2513
Dunnebacke et al. 2004; Maciver 2007; Matin et al. 2008; Schuster et al. 2003, 2009). Often the infection has occurred in association with a break in the skin. To the best of our knowledge, this is the first time that B. mandrillaris has been isolated from the environment in Peru. There is an urgent need to identify the environmental habitats of these deadly pathogens and to understand the risk factors involved for its thriving in order to take measures to prevent and control populations of this amoeba.
Material and methods Sample sites and culture of amoebae In this study, 21 samples were collected in two different locations in Peru in order to check for the presence of Balamuthia-like amoebae. A group of five samples were collected in the city of Lima from different areas of the garden of the house of a patient who previously died of BAE (PS-1 to PS-5, Table 1). The second area checked for the presence of these amoebae was the region of Piura. In this region, 13 soil samples were collected from the desert located in the north coast of Peru (5, PS-16 to PS-21, Table 1) (around 7 km from the city of Catacaos, Piura) and a dried forest outside the coast of Piura (11, PS-6 to PS-15, Table 1). All samples were kept at room temperature until processed in the laboratory. Samples were collected with a sterile cotton swab humidified in Page’s saline and were cultured in 2 % non-nutrient agar (NNA) plates with a layer of Escherichia coli. Plates were incubated at room temperature and at 30ºC and were checked daily for the presence of Balamuthia-like amoebae for up to 30 days. DNA extraction DNA from cultures suspicious of Balamuthia-like amoebae by microscopy was extracted by placing 1–2 ml of amoebic cultures directly into the Maxwell\ 16 Tissue DNA Purification Kit sample cartridge (Promega, Madrid, Spain). Amoebic genomic DNA was purified using the Maxwell\ 16 Instrument as described in the Maxwell\ 16 DNA Purification Kits Technical Manual #TM284 (Promega, Madrid, Spain). DNA yield and purity were determined using the NanoDrop\ 1000 spectrophotometer (Fisher Scientific, Madrid, Spain). PCR amplification and DNA sequencing For the identification of the putative B. mandrillaris strains, the 16S ribosomal DNA (rDNA) gene was amplified and sequenced as previously described (Booton et al. 2003; Niyyati et al. 2009). PCR products were purified using the
Parasitol Res (2014) 113:2509–2513 Table 1 Soil samples and positive locations for Balamuthia mandrillaris in this study
PS Peru soil a
Samples that were positive for B. mandrillaris by both culture and PCR
2511
Sample code
Location
Culture/PCRa Balamuthia mandrillaris
Strain code
Source
PS-1 PS-2 PS-3 PS-4 PS-5 PS-6 PS-7 PS-8 PS-9 PS-10
City of Lima, Lima City of Lima, Lima City of Lima, Lima City of Lima, Lima City of Lima, Lima Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura
+ − − − − − − − − −
CG-13 − − − − − − − − −
Garden Garden Garden Garden Garden Soil Soil Soil Soil Soil
PS-11 PS-12 PS-13 PS-14 PS-15 PS-16 PS-17 PS-18 PS-19 PS-20 PS-21
Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura 7 km from Catacaos City, Piura 7 km from Catacaos City, Piura 7 km from Catacaos City, Piura 7 km from Catacaos City, Piura 7 km from Catacaos City, Piura
− − − − + − − + + − −
− − − − REL/C-13 − − MC-13A MC-13B − −
Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil
Qiaquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced using a MEGABACE 1000 automatic sequencer (Healthcare Biosciences, Barcelona, Spain) in the University of La Laguna Sequencing Services (Servicio de Secuenciación SEGAI, University of La Laguna). The Fig. 1 Trophozoites (a, c and d) and cysts (b) of CG-13 strain. Magnifications of×20 for a and b and×40 for b
obtained sequences were aligned using Mega 5.0 software program (Tamura et al. 2011). These sequences were compared to sequences available in GenBank database. The sequences for the new isolates are deposited in the GenBank database under the accession numbers KJ439568–KJ439571.
A
B
C
D
2512 Fig. 2 Trophozoites of REL/C13 strain a and b (×20) feeding on other amoebae in NNA plates
Parasitol Res (2014) 113:2509–2513
A
Results and Discussion Four samples of the 21 included in this study were positive for Balamuthia-like amoebae by microscopy. Furthermore, PCR of the 16S rDNA mitochondrial gene of B. mandrillaris confirmed the culture observations in all four cases (Table 1). The PCR products of these strains named REL/C-13, CG-13, MC13A and MC-13B (Table 1), showed sequence homology between 97 and 99 % when compared to the B. mandrillaris sequences available in GenBank. It is interesting to mention that Balamuthia amoebae were observed around day 25 after inoculation of NNA plates and disappeared around day 35 (Figs. 1 and 2). Unfortunately, axenification of the four isolates in 2 % bactocasitone or BM-3 medium was not possible. The strain classified as CG-1 was isolated from soil collected from the rose garden of a patient from Lima that previously died of BAE (Paucar et al. 2010). In the clinical report, the patient manifested skin lesion in the right knee and consistent with the observation that the patient habitually rested on his knees while working in the rose garden. It is suspected that the patient became infected with the strain that we have subsequently isolated during this study. The other three positives samples (PS-15, PS-18 and PS19) were collected in the region of Piura. The samples classified as PS-18 and PS-19 (strains MC-13A and MC-13B) were collected in the desert area of the north coast of Peru nearby the city of Catacaos. The PS-15 sample (strain REL/C-13) was collected from the dried forest located nearby the city of Piura. This second region has also reported clinical cases of BAE in the recent years in this country. To the best of our knowledge, this is the first time that B. mandrillaris has been isolated from the environment in Peru and the second time as the environmental source of an infection because this pathogen has been identified worldwide. Peru ranks second in the number of BAE cases worldwide after the USA (Bravo and Seas 2012; Lorenzo-Morales et al. 2013). Interestingly, the main affected areas in both countries are surrounded by dust and soil-related environments that might be the source of these pathogens. However, even in
B
the present study, environmental isolation of B. mandrillaris seems to be a low frequency occurrence. In addition to the present study, researchers have only been able to isolate B. mandrillaris from the environment on four occasions. The development of improved culture techniques to allow a fast, reliable and repetitive approach to isolate and grow these amoebae in axenic conditions is urgently required. Despite all the recent advances in the study of B. mandrillaris, several questions about this organism and BAE remain unanswered. 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. Previous reports have accounted for approximately 0.1 % of total encephalitis cases in otherwise healthy individuals to be caused by Naegleria fowleri or B. mandrillaris (Maciver 2007; Lorenzo-Morales et al. 2013). Therefore, BAE cases should be considered as possible causes of encephalitis when patients are displaying general nonspecific encephalitis symptoms. In conclusion, understanding the pathogenesis and pathophysiology of BAE at the molecular, cellular and clinical level are of primary importance. Further knowledge about this pathogen will allow a development of more effective preventive and therapeutic interventions for those afflicted with BAE. Acknowledgments This work was supported by the grants RICET (project no. RD12/0018/0012 of 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. ALA was funded by the grant “Ayudas del Programa de Formación de Personal Investigador, para la realización de Tesis Doctorales” from the Agencia Canaria de Investigación, Innovación y Sociedad de la Información from the Canary Islands Government. CMMN was
Parasitol Res (2014) 113:2509–2513 supported by a postdoctoral grant from the Fundación Canaria Manuel Morales, La Palma, Canary Islands. MRB was funded by 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. AMCV was funded by Agencia Española de Cooperación Internacional y Desarrollo (AECID) and CEI Canarias, Campus Atlántico Internacional.
References Booton GC, Carmichael JR, Visvesvara GS, Byers TJ, Fuerst PA (2003) Genotyping of Balamuthia mandrillaris based on nuclear 18S and mitochondrial 16S rRNA genes. Am J Trop Med Hyg 68:65–69 Bravo FG, Seas C (2012) Balamuthia mandrillaris amoebic encephalitis: an emerging parasitic infection. Curr Infect Dis Rep 14:391–396 Delgado J, Aguila-Pardo E (1957) Abscesos amebianos cerebrales y meningoencefalitis. Arch Per Pat y Clin 9(1–4):21–26 Diaz JH (2011) The public health threat from Balamuthia mandrillaris in the southern United States. J La State Med Soc 163:197–204 Dunnebacke TH, Schuster FL, Yagi S, Booton GC (2004) Balamuthia mandrillaris from soil samples. Microbiology 150:2837–2842 Jackson BR, Kucerova Z, Roy SL, Aguirre G, Weiss J, Sriram R, Yoder J, Foelber R, Baty S, Derado G, Stramer SL, Winkelman V, Visvesvara GS (2014) Serologic survey for exposure following fatal Balamuthia mandrillaris infection. Parasitol Res 2014 Jan 24. [Epub ahead of print] Lobo SA, Patil K, Jain S, Marks S, Visvesvara GS, Tenner M, Braun A, Wang G, El Khoury MY (2013) Diagnostic challenges in Balamuthia mandrillaris infections. Parasitol Res 112(12):4015–4019 Lorenzo-Morales J, Cabello-Vílchez AM, Martín-Navarro CM, Martínez-Carretero E, Piñero JE, Valladares B (2013) Is Balamuthia mandrillaris a public health concern worldwide? Trends Parasitol 29(10):483–488 Maciver SK (2007) The threat from Balamuthia mandrillaris. J Med Microbiol 56:1–3
2513 Matin A, Siddiqui R, Jayasekera S, Khan NA (2008) Increasing importance of Balamuthia mandrillaris. Clin Microbiol Rev 21:435–448 Moser MA (2011) A descriptive review of Balamuthia and non-keratitis Acanthamoeba cases in the United States, 1955–2009. Public Health Theses Paper 162 Niyyati M, Lorenzo-Morales J, Rezaeian M, Martin-Navarro CM, Haghi AM, Maciver SK, Valladares B (2009) Isolation of Balamuthia mandrillaris from urban dust, free of known infectious involvement. Parasitol Res 106:279–281 Paucar K, Del Solar M, Bravo F, Salomón M, Puell L, Feria K, Ramos C, Giglio P (2010) Balamutiasis primaria cutánea: primary cutaneous balamuthiasis Folia dermatol Peru 21 (2): 105-109 Schuster FL, Dunnebacke TH, Booton GC, Yagi S, Kohlmeier CK, Glaser C, Vugia D, Bakardjiev A, Azimi P, Maddux-Gonzalez M, Martinez AJ, Visvesvara GS (2003) Environmental isolation of Balamuthia mandrillaris associated with a case of amebic encephalitis. J Clin Microbiol 41:3175–3180 Schuster FL, Yagi S, Gavali S, Michelson D, Raghavan R, Blomquist I, Glastonbury C, Bollen AW, Scharnhorst D, Reed SL, Kuriyama S, Visvesvara GS, Glaser CA (2009) Under the radar: Balamuthia amebic encephalitis. Clin Infect Dis 48:879–887 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 28(10):2731–2739. doi:10.1093/molbev/ msr121 Visvesvara GS, Martinez AJ, Schuster FL, Leitch GJ, Wallace SV, Sawyer TK, Anderson M (1990) Leptomyxid ameba, a new agent of amebic meningoencephalitis in humans and animals. J Clin Microbiol 28:2750–2756 Visvesvara GS, Schuster FL, Martinez AJ (1993) Balamuthia mandrillaris, N. G., N. sp., agent of amebic meningoencephalitis in humans and other animals. J Eukaryot Microbiol 40:504–514 Visvesvara GS, Moura H, Schuster FL (2007) Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol 50:1–26
ORIGINAL PAPER
The isolation of Balamuthia mandrillaris from environmental sources from Peru Alfonso Martín Cabello-Vílchez & María Reyes-Batlle & Esmelda Montalbán-Sandoval & Carmen Mª Martín-Navarro & Atteneri López-Arencibia & Rafaela Elias-Letts & Humberto Guerra & Eduardo Gotuzzo & Enrique Martínez-Carretero & José E. Piñero & Sutherland K. Maciver & Basilio Valladares & Jacob Lorenzo-Morales
Received: 18 February 2014 / Accepted: 9 April 2014 / Published online: 30 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Balamuthia mandrillaris is an opportunistic freeliving amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely A. M. Cabello-Vílchez : M. Reyes-Batlle : C. M. Martín-Navarro : A. López-Arencibia : E. Martínez-Carretero : J. E. Piñero : B. Valladares : J. Lorenzo-Morales (*) University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Avenida Astrofísico Francisco Sánchez SN, 38203, La Laguna Tenerife, Canary Islands, Spain e-mail: [email protected] A. M. Cabello-Vílchez : H. Guerra : E. Gotuzzo Universidad Peruana Cayetano Heredia, Laboratory of Clinical Microbiology, Tropical Medicine Institute “Alexander von Humboldt” IMT-AvH, Lima, Peru E. Montalbán-Sandoval Asociación Civil Impacta Perú Salud y Educación, Lima, Peru C. M. Martín-Navarro : S. K. Maciver Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, Scotland, UK R. Elias-Letts Laboratorio de Ecotoxicología, Laboratorios de Investigación y Desarrollo (LID), Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru
encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillarislike trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding. Keywords Balamuthia mandrillaris . Emerging pathogen . Encephalitis . Skin . Therapy . Diagnosis . Peru
Introduction Since the first description of a human infection being caused by an otherwise free-living amoeba (Delgado and AguilaPardo 1957), a number of amoebic species have been found to have the capacity to cause fatal infections in humans. Amongst these are Acanthamoeba, Naegleria and Sapinia, but more recently another amoebic genus, Balamuthia was added to this list (Visvesvara et al. 2007). Balamuthia mandrillaris was formerly described as a leptomyxid amoeba (Visvesvara et al. 1990, 1993) and was first discovered from the brain of a mandrill baboon that died of meningoencephalitis. Currently, B. mandrillaris causing
2510
Balamuthia amoebic encephalitis (BAE) is recognized as being an emerging disease, but this amoeba is also believed to cause skin lesions in both immunocompetent and immunocompromised individuals (Maciver 2007; Matin et al. 2008; Bravo and Seas 2012). Since, the description of B. mandrillaris as the causative agent of BAE around 200 cases have been reported worldwide (Maciver 2007; Matin et al. 2008; Diaz 2011; Bravo and Seas 2012; Jackson et al. 2014). The Americas have reported the highest number of infected patients, mainly in the southwest of the USA (Arizona, Texas and California) and also in Latin America with cases reported in Mexico, Peru, Venezuela, Argentina, Brazil and Chile (Lorenzo-Morales et al. 2013). In the last 29 years, around 55 cases of B. mandrillaris infections have been reported in Peru (Bravo and Seas 2012). It is worth mentioning that a higher frequency of cutaneous involvement (usual location in the central face area) has been reported from the Peruvian cases compared to the pure neurological involvement seen in many of the North American cases (Moser 2011; Bravo and Seas 2012; Lorenzo-Morales et al. 2013). Investigation of these cases have shown that general symptoms are absent during the cutaneous stage of BAE (Moser 2011; Bravo and Seas 2012). Skin lesion is present in almost all of the affected Peruvian patients reported so far, and these lesions have been reported to form a thin, painless plaque of one to several centimeters in diameter, commonly located around the nose, although other locations such as the knee, the chest and the elbow have also been observed (Bravo and Seas 2012; Lorenzo-Morales et al. 2013). While a single lesion is present on most patients, satellite lesions sometimes appear at the periphery of the main lesion on the face and extremities as well as the oral cavity. After a variable period of time, from weeks to years, the infection progresses to the brain forming BAE that often results in death. Available data for BAE cases indicate that it is less likely to be diagnosed correctly due to the lack of standard diagnostic tools and the unfamiliarity of clinicians with this amoebic pathogen (Schuster et al. 2009; Bravo and Seas 2012; Lobo et al. 2013; Lorenzo-Morales et al. 2013). Thus, BAE is gaining a reputation as a significant threat to human health; the most distressing aspect is the limited availability of fully effective treatments against these infections. So far, only ten BAE survivors have been recorded worldwide, and unfortunately in most of these cases, the patients survives with permanent impairment (Bravo and Seas 2012; LorenzoMorales et al. 2013). Even though the risk factors for BAE are still largely unknown, environmental exposure to soil (i.e. during the performance of activities such as agricultural work or gardening) has been reported as the main potential source for B. mandrillaris infections worldwide (Schuster et al. 2003;
Parasitol Res (2014) 113:2509–2513
Dunnebacke et al. 2004; Maciver 2007; Matin et al. 2008; Schuster et al. 2003, 2009). Often the infection has occurred in association with a break in the skin. To the best of our knowledge, this is the first time that B. mandrillaris has been isolated from the environment in Peru. There is an urgent need to identify the environmental habitats of these deadly pathogens and to understand the risk factors involved for its thriving in order to take measures to prevent and control populations of this amoeba.
Material and methods Sample sites and culture of amoebae In this study, 21 samples were collected in two different locations in Peru in order to check for the presence of Balamuthia-like amoebae. A group of five samples were collected in the city of Lima from different areas of the garden of the house of a patient who previously died of BAE (PS-1 to PS-5, Table 1). The second area checked for the presence of these amoebae was the region of Piura. In this region, 13 soil samples were collected from the desert located in the north coast of Peru (5, PS-16 to PS-21, Table 1) (around 7 km from the city of Catacaos, Piura) and a dried forest outside the coast of Piura (11, PS-6 to PS-15, Table 1). All samples were kept at room temperature until processed in the laboratory. Samples were collected with a sterile cotton swab humidified in Page’s saline and were cultured in 2 % non-nutrient agar (NNA) plates with a layer of Escherichia coli. Plates were incubated at room temperature and at 30ºC and were checked daily for the presence of Balamuthia-like amoebae for up to 30 days. DNA extraction DNA from cultures suspicious of Balamuthia-like amoebae by microscopy was extracted by placing 1–2 ml of amoebic cultures directly into the Maxwell\ 16 Tissue DNA Purification Kit sample cartridge (Promega, Madrid, Spain). Amoebic genomic DNA was purified using the Maxwell\ 16 Instrument as described in the Maxwell\ 16 DNA Purification Kits Technical Manual #TM284 (Promega, Madrid, Spain). DNA yield and purity were determined using the NanoDrop\ 1000 spectrophotometer (Fisher Scientific, Madrid, Spain). PCR amplification and DNA sequencing For the identification of the putative B. mandrillaris strains, the 16S ribosomal DNA (rDNA) gene was amplified and sequenced as previously described (Booton et al. 2003; Niyyati et al. 2009). PCR products were purified using the
Parasitol Res (2014) 113:2509–2513 Table 1 Soil samples and positive locations for Balamuthia mandrillaris in this study
PS Peru soil a
Samples that were positive for B. mandrillaris by both culture and PCR
2511
Sample code
Location
Culture/PCRa Balamuthia mandrillaris
Strain code
Source
PS-1 PS-2 PS-3 PS-4 PS-5 PS-6 PS-7 PS-8 PS-9 PS-10
City of Lima, Lima City of Lima, Lima City of Lima, Lima City of Lima, Lima City of Lima, Lima Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura
+ − − − − − − − − −
CG-13 − − − − − − − − −
Garden Garden Garden Garden Garden Soil Soil Soil Soil Soil
PS-11 PS-12 PS-13 PS-14 PS-15 PS-16 PS-17 PS-18 PS-19 PS-20 PS-21
Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura Dried forest, Piura 7 km from Catacaos City, Piura 7 km from Catacaos City, Piura 7 km from Catacaos City, Piura 7 km from Catacaos City, Piura 7 km from Catacaos City, Piura
− − − − + − − + + − −
− − − − REL/C-13 − − MC-13A MC-13B − −
Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil
Qiaquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced using a MEGABACE 1000 automatic sequencer (Healthcare Biosciences, Barcelona, Spain) in the University of La Laguna Sequencing Services (Servicio de Secuenciación SEGAI, University of La Laguna). The Fig. 1 Trophozoites (a, c and d) and cysts (b) of CG-13 strain. Magnifications of×20 for a and b and×40 for b
obtained sequences were aligned using Mega 5.0 software program (Tamura et al. 2011). These sequences were compared to sequences available in GenBank database. The sequences for the new isolates are deposited in the GenBank database under the accession numbers KJ439568–KJ439571.
A
B
C
D
2512 Fig. 2 Trophozoites of REL/C13 strain a and b (×20) feeding on other amoebae in NNA plates
Parasitol Res (2014) 113:2509–2513
A
Results and Discussion Four samples of the 21 included in this study were positive for Balamuthia-like amoebae by microscopy. Furthermore, PCR of the 16S rDNA mitochondrial gene of B. mandrillaris confirmed the culture observations in all four cases (Table 1). The PCR products of these strains named REL/C-13, CG-13, MC13A and MC-13B (Table 1), showed sequence homology between 97 and 99 % when compared to the B. mandrillaris sequences available in GenBank. It is interesting to mention that Balamuthia amoebae were observed around day 25 after inoculation of NNA plates and disappeared around day 35 (Figs. 1 and 2). Unfortunately, axenification of the four isolates in 2 % bactocasitone or BM-3 medium was not possible. The strain classified as CG-1 was isolated from soil collected from the rose garden of a patient from Lima that previously died of BAE (Paucar et al. 2010). In the clinical report, the patient manifested skin lesion in the right knee and consistent with the observation that the patient habitually rested on his knees while working in the rose garden. It is suspected that the patient became infected with the strain that we have subsequently isolated during this study. The other three positives samples (PS-15, PS-18 and PS19) were collected in the region of Piura. The samples classified as PS-18 and PS-19 (strains MC-13A and MC-13B) were collected in the desert area of the north coast of Peru nearby the city of Catacaos. The PS-15 sample (strain REL/C-13) was collected from the dried forest located nearby the city of Piura. This second region has also reported clinical cases of BAE in the recent years in this country. To the best of our knowledge, this is the first time that B. mandrillaris has been isolated from the environment in Peru and the second time as the environmental source of an infection because this pathogen has been identified worldwide. Peru ranks second in the number of BAE cases worldwide after the USA (Bravo and Seas 2012; Lorenzo-Morales et al. 2013). Interestingly, the main affected areas in both countries are surrounded by dust and soil-related environments that might be the source of these pathogens. However, even in
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the present study, environmental isolation of B. mandrillaris seems to be a low frequency occurrence. In addition to the present study, researchers have only been able to isolate B. mandrillaris from the environment on four occasions. The development of improved culture techniques to allow a fast, reliable and repetitive approach to isolate and grow these amoebae in axenic conditions is urgently required. Despite all the recent advances in the study of B. mandrillaris, several questions about this organism and BAE remain unanswered. 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. Previous reports have accounted for approximately 0.1 % of total encephalitis cases in otherwise healthy individuals to be caused by Naegleria fowleri or B. mandrillaris (Maciver 2007; Lorenzo-Morales et al. 2013). Therefore, BAE cases should be considered as possible causes of encephalitis when patients are displaying general nonspecific encephalitis symptoms. In conclusion, understanding the pathogenesis and pathophysiology of BAE at the molecular, cellular and clinical level are of primary importance. Further knowledge about this pathogen will allow a development of more effective preventive and therapeutic interventions for those afflicted with BAE. Acknowledgments This work was supported by the grants RICET (project no. RD12/0018/0012 of 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. ALA was funded by the grant “Ayudas del Programa de Formación de Personal Investigador, para la realización de Tesis Doctorales” from the Agencia Canaria de Investigación, Innovación y Sociedad de la Información from the Canary Islands Government. CMMN was
Parasitol Res (2014) 113:2509–2513 supported by a postdoctoral grant from the Fundación Canaria Manuel Morales, La Palma, Canary Islands. MRB was funded by 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. AMCV was funded by Agencia Española de Cooperación Internacional y Desarrollo (AECID) and CEI Canarias, Campus Atlántico Internacional.
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