Enrique Martínez Carretero - Academia.edu (original) (raw)

Papers by Enrique Martínez Carretero

The American Journal of Tropical Medicine and Hygiene, 2007

We surveyed 236 potentially pathogenic Acanthamoeba strains, isolated from water sources in the C... more We surveyed 236 potentially pathogenic Acanthamoeba strains, isolated from water sources in the Canary Islands, for the presence of human adenoviruses (HAdV) using a polymerase chain reaction (PCR)-based typing assay. A total of 34 of these strains were found to be positive for adenovirus belonging to four different HAdV serotypes (HAdV-1, 2, 8, and 37). We found that HAdV-2 was the most frequently encountered serotype amongst the Acanthamoeba strains, and their identification was confirmed by a nested PCR specific for this serotype. We showed that Acanthamoeba genotype T4 was highly associated with serotype HAdV-2, whereas Acanthamoeba genotype T3 was most often associated with adenovirus serotypes related to ocular diseases. Based on these data, we suggest that Acanthamoeba should be considered as a potential reservoir and perhaps even a transmitter of adenoviruses to human and other secondary hosts.

Journal of Clinical Microbiology, 1996

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is... more A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obt...

Experimental Parasitology, 2017

Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They ... more Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They are frequently found in water and soil samples, but they have also been reported to be associated with bacterial human pathogens such as Legionella spp., Campylobacter spp or Vibrio cholerae among others. Including within Vibrio spp., V. harveyi (Johnson and Shunk, 1936) is a bioluminescent marine bacteria which has been found swimming freely in tropical marine waters, being part of the stomach and intestine microflora of marine animals, and as both a primary and opportunistic pathogen of marine animals. Our aim was to study the interactions between Vibrio harveyi and Acanthamoeba castellanii Neff. Firstly, in order to analyze changes in it cultivability, V. harveyi was coincubated with A. castellanii Neff axenic culture and with Acanthamoeba Conditioned Medium (ACM) at different temperatures in aerobic conditions. Interestingly, at 4°C and 18-20°C bacteria were still cultivable in marine agar, at 28°C, in aerobic conditions, but there weren´t significant differences comparing with the controls. We also noted an enhanced migration of Acanthamoeba toward V. harveyi on non-nutrient agar plates compared to controls with no bacteria.

Expert Opinion on Orphan Drugs, 2016

ABSTRACT Introduction: Free Living Amoebae (FLA) are unicellular protozoa common to most soil and... more ABSTRACT Introduction: Free Living Amoebae (FLA) are unicellular protozoa common to most soil and aquatic environments. Only members of the genus Acanthamoeba and the species Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea are known to infect humans and other animals, often with fatal consequences. Acanthamoeba species are the causative agents of a sight threating infection of the cornea known as Acanthamoeba keratitis (AK) that mainly affects immunocompetent contact lens wearers and has shown an increased incidence in the recent years worldwide. To date, most of the available treatments and contact lens maintenance solutions are not fully effective against these pathogens. Areas covered: There is a clear need for novel fully effective therapeutic agents against AK. In the recent years the application of ‘phylopharmacology’ and RNAi has shown to be a reliable tool for the elucidation of novel drug targets and therapeutic agents. Current therapeutic options for AK as well as ongoing research advances are presented and discussed. Expert opinion: The available AK treatments are not fully effective so far mostly due to the existence of a cyst stage in this pathogen. Moreover, most of the first line treatment options are not available in all countries. There is an urgent need to validate novel drug targets and therapeutic agents against AK. Recent approaches have allowed the validation of drug targets and novel compounds that could become fully effective therapies against these emerging pathogens.

Acta Parasitologica, 2016

Pathogenic strains of

Parasitology Research, 2016

The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and en... more The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, forty soil samples were collected in the island of El Hierro, Canary Islands, Spain, and checked for the presence of Acanthamoeba. Samples were cultivated onto 2 % non-nutrient agar plates seeded with a layer of heat killed Escherichia coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 47.5 % of soil samples. Moreover, genotypes T2, T4, and T11 were identified in these samples. To the best of our knowledge, this is the first study to establish genotypes T2, T4, and T11 in soil sources from El Hierro island.

Protist, 2015

RNA helicases are ubiquitous enzymes that participate in almost all aspects of RNA processing, in... more RNA helicases are ubiquitous enzymes that participate in almost all aspects of RNA processing, including RNA and RNA-protein complex remodelling. In trypanosomatids, which post-transcriptionally regulate gene expression, the formation of different kinds of ribonucleoprotein granules under stress conditions modulates the parasite's RNA metabolism. This paper describes the isolation of a putative DEVH-box RNA helicase produced by promastigotes of Leishmania braziliensis. Using a Cy3-labelled dT 30 oligo, FISH showed the localization of this protein to mRNA granules under starvation stress conditions. The central region of the protein was shown to be responsible for this behaviour.

Journal of Medical Microbiology, 2015

Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the c... more Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis, which is often associated with the misuse of contact lenses. However, there is still a question remaining to be answered, which is whether these microorganisms are present on the ocular surface of healthy individuals. Therefore, the aim of this study was to determine the presence of Acanthamoeba on the ocular surface in healthy patients and also in those with other ocular surface infections. Sterile Schirmer test strips were used to collect samples from a group of patients who attended an ophthalmology consultation at the Hospital del Norte, Icod de los Vinos, Tenerife, Canary Islands. Most of the patients (46 individuals, 79.31 %) presented ocular surface pathologies such as blepharitis or conjunctivitis; the rest did not present any pathology. None of the patients included in the study wore contact lenses. The collected samples were cultured in 2 % non-nutrient agar plates and positive plates were then cultured in axenic conditions for further analyses. Molecular analysis classified all isolated strains as belonging to Acanthamoeba genotype T4, and osmotolerance and thermotolerance assays revealed that all strains were potentially pathogenic. Furthermore, all strains were assayed for sensitivity against voriconazole and chlorhexidine. Assays showed that both drugs were active against the tested strains. In conclusion, the Schirmer strip test is proposed as an effective tool for the detection of Acanthamoeba on the ocular surface.

Antimicrobial Agents and Chemotherapy, 2015

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatenin... more Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba . Here, we study the processes of programmed cell death in Acanthamoeba , induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC 50 s) and IC 90 s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with ...

Archives of Microbiology, 2015

Free-living amoebae (FLA) of the genus Acanthamoeba are opportunistic pathogenic agents able to c... more Free-living amoebae (FLA) of the genus Acanthamoeba are opportunistic pathogenic agents able to cause life-threatening infections in immunosuppressed patients. Chronic kidney disease impairs adaptive and innate immunity. Thus, patients with chronic kidney disease are prone to opportunistic infections by potentially pathogenic FLA. Therefore, in the present study, the investigation of Acanthamoeba genotypes isolated from the oral cavity of hemodialysis patients of reference hospitals in Iran was aimed, using both morphology and molecular (sequence-based analysis) tools. Furthermore, classification of the strains at the genotype level was performed on the basis of differences in the diagnostic fraction 3 (DF3) region of the 18S rRNA gene. The pathogenic potential of the isolated amoebae was also determined using thermotolerance and osmotolerance assays. Out of the 187 oral cavity samples investigated, nine (4.8%) were positive for FLA. DNA sequencing of the ASA.A1 region of the 18S rRNA gene revealed that the isolated strains belonged to the Acanthamoeba T1 and T4 genotypes. Genotype T1 was isolated for the first time from a patient in Iran. Interestingly, the T1 strain (AN2 strain) exhibits a high pathogenic potential in tolerance assays. The pathogenicity assay revealed that five strains were able to grow at high temperatures (37-40°C) and high osmolarity (0.5 and 1 M D-mannitol) conditions; thus, they were considered as potentially pathogenic strains. Moreover, two of the patients were positive for Vermamoeba genus. The present study is the first report of genotype T1 isolation in Iran and the first to identify the occurrence of Acanthamoeba and Vermamoeba genera in patients undergoing hemodialysis worldwide. Monitoring hemodialysis and renal failure patients should be a priority for possible control of Acanthamoeba and other FLA-related diseases.

Non-Flavivirus Encephalitis, 2011

Pathogens and Global Health, 2014

Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia m... more Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia mandrillaris, and the genera Sappinia and Acanthamoeba. In this study, a survey was conducted in order to evaluate the presence of potentially pathogenic amoebic strains in water samples collected from wells located in the western part of Guinea-Bissau. The samples were left to precipitate for 48 hours and then the sediments were seeded on non-nutrient agar plates containing Escherichia coli spread and cultures were checked daily for the presence of FLA. Identification of FLA strains was based on the morphological and polymerase chain reaction (PCR) using the 18S rDNA or 16S mitochondrial rDNA genes in the case of Naegleria and Balamuthia genera, respectively. In the case of positive samples of Acanthamoeba, strains were further classified at the genotype level by sequencing the diagnostic fragment 3 (DF3) region located in the 18S rDNA gene as previously described. Sappinia sp. was not isolated during the study and thus, no molecular analysis was performed for this genus. The obtained results revealed the presence of Acanthamoeba (genotypes T3 and T4), Naegleria fowleri, and Balamuthia mandrillaris. To the best of our knowledge, this is the first report demonstrating the presence of FLA in water bodies from Guinea-Bissau and the first report on the isolation of Balamuthia mandrillaris from environmental sources in Africa.

Epidemiology and Infection, 2014

SUMMARYBalamuthia mandrillaris is an emerging cause of encephalitis in humans. The transmission d... more SUMMARYBalamuthia mandrillaris is an emerging cause of encephalitis in humans. The transmission dynamics are poorly understood due to the high fatality rate and the sporadic nature of cases. Seventy-two soil samples were collected from beaches and the banks of lagoons, rivers, ponds, mineral springs and streams from across Jamaica and assayed for the presence of B. mandrillaris. Seventy-nine sites were sampled and the mitochondrial 16S rDNA gene of B. mandrillaris was amplified and sequenced to confirm the presence of the amoeba. One isolate of B. mandrillaris was recovered from soil from mineral spring which hosts an informal therapeutic mud bath business. Although B. mandrillaris is less frequently isolated from soil than other free-living amoebae, rubbing mud containing the organism onto the skin increases the likelihood of exposure and infection. This first report on the isolation of B. mandrillaris in the Caribbean and its presence in soil where human contact is likely warrants...

Veterinary Parasitology, 2014

A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fe... more A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fever and movement difficulty. After 24 h, the dog was found to have a high fever (39.5 • C) and was treated empirically with doxycycline/ciprofloxacin. At 48 h, after submission the fever rose to 41 • C and the animal presented with a stiff neck and dehydration. Peripheral blood and cerebrospinal fluid (CSF) were sampled and trophozoites with an Acanthamoeba-like morphology were observed in the CSF. PCR specific for Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris were performed and the CSF sample found positive for Acanthamoeba. Lungs, kidney, liver and spleen samples were collected post mortem. All collected organ samples were positive for Acanthamoeba by PCR, thus confirming a multisystemic infection. Water samples taken at a suspected site of infection yielded an almost identical PCR fragment to those of the clinical samples, indicating that this was probably where the infection originated. This is the first report of a fatal case of Acanthamoeba disseminated infection in a dog in Spain.

Trends in Parasitology, 2013

Parasitology Research, 2007

A mixed keratitis due to Acanthamoeba and Hartmannella species is reported. The patient was a sof... more A mixed keratitis due to Acanthamoeba and Hartmannella species is reported. The patient was a soft contact lens wearer. Early diagnosis was achieved by polymerase chain reaction and culture. The pathogenic potential of the isolated amoebae was proven using cytotoxicity assays. The reported case underlines the difficulties in identifying a corneal amoebic infection. In our case, the early diagnosis of a mixed infection allowed a proper antiamoebic treatment in an early stage of infection. This may have been the reason of a successful outcome after therapy. Free-living amoebae of the genera Acanthamoeba and Hartmannella are ubiquitous in nature. Acanthamoeba spp. are well known as causative agents of severe eye infections,

Parasitology Research, 2013

The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or ... more The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or postmortem features, in humans and other animals. The only diagnostic test described so far for the serological diagnosis of Toxoplasma gondii in marine mammals is the modified agglutination test (Dubey et al., Am J Vet Res 48(8):1239-1243, 1987). The development of more sensible and specific immunological techniques requires specific antibodies, which are currently unavailable in the scientific market. Indirect immunofluorescence (IIF) is one of the most widely used methods for the diagnosis of toxoplasmosis in humans (Auer et al., Parasitol Res 12:965-970, 2000). In order to develop and apply this technique to the bottlenose dolphin (Tursiops truncatus), immunoglobulins were firstly purified using ion-exchange chromatography. The purified immunoglobulins were then injected in New Zealand rabbits in order to obtain polyclonal antibodies. These antisera were validated by the IIF technique, using as controls serum samples of dolphins infected by Toxoplasma. The results were visualized using antirabbit IgG labeled with fluorescein. This newly developed and specific serological assay was then tested with the dolphin collection of Loro Parque, Tenerife, Spain (group I), and L'Oceanogràfic of Valencia, Spain (group II). The obtained results in this study showed that none of the dolphins from group 1 were infected by T. gondii and two animals were positive in group 2. Furthermore, we conclude that this study has produced antibodies with high specificity against dolphin immunoglobulins and an IIF method which may be used as immunological diagnostic tools, especially for the serological diagnosis of toxoplasmosis.

Parasite, 2006

Sera from individuals suffering from leishmaniasis have been shown to strongly react against cons... more Sera from individuals suffering from leishmaniasis have been shown to strongly react against conserved proteins from the parasite, such as ribosomal, histones and heat-shock proteins. Some of these proteins have also been described as immunogenic in several auto-immune syndromes, and the detection of antibodies against them is considered to be indicative of disorder of the immune system. In this paper, we investigate whether there is any relationship between the recognition of some conserved proteins from Leishmania braziliensis by individuals suffering from cutaneous (CL) and mucocutaneous (MCL) leishmaniasis, and the recognition of the human homologues of these antigens found in sufferers from autoimmune diseases. Our findings reveal that the immune response generated during CL and MCL is elicited specifically by the parasitic histone H1 and Hsp70, since the CL and MCL sera do not react against their human counterparts. In addition, evidence is presented showing the specific recognition of human proteins by the autoimmune sera, showing only a weak crossreaction with the most divergent regions of the parasitic proteins.

Journal of Parasitology, 2004

The American Journal of Tropical Medicine and Hygiene, 2007

We surveyed 236 potentially pathogenic Acanthamoeba strains, isolated from water sources in the C... more We surveyed 236 potentially pathogenic Acanthamoeba strains, isolated from water sources in the Canary Islands, for the presence of human adenoviruses (HAdV) using a polymerase chain reaction (PCR)-based typing assay. A total of 34 of these strains were found to be positive for adenovirus belonging to four different HAdV serotypes (HAdV-1, 2, 8, and 37). We found that HAdV-2 was the most frequently encountered serotype amongst the Acanthamoeba strains, and their identification was confirmed by a nested PCR specific for this serotype. We showed that Acanthamoeba genotype T4 was highly associated with serotype HAdV-2, whereas Acanthamoeba genotype T3 was most often associated with adenovirus serotypes related to ocular diseases. Based on these data, we suggest that Acanthamoeba should be considered as a potential reservoir and perhaps even a transmitter of adenoviruses to human and other secondary hosts.

Journal of Clinical Microbiology, 1996

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is... more A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obt...

Experimental Parasitology, 2017

Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They ... more Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They are frequently found in water and soil samples, but they have also been reported to be associated with bacterial human pathogens such as Legionella spp., Campylobacter spp or Vibrio cholerae among others. Including within Vibrio spp., V. harveyi (Johnson and Shunk, 1936) is a bioluminescent marine bacteria which has been found swimming freely in tropical marine waters, being part of the stomach and intestine microflora of marine animals, and as both a primary and opportunistic pathogen of marine animals. Our aim was to study the interactions between Vibrio harveyi and Acanthamoeba castellanii Neff. Firstly, in order to analyze changes in it cultivability, V. harveyi was coincubated with A. castellanii Neff axenic culture and with Acanthamoeba Conditioned Medium (ACM) at different temperatures in aerobic conditions. Interestingly, at 4°C and 18-20°C bacteria were still cultivable in marine agar, at 28°C, in aerobic conditions, but there weren´t significant differences comparing with the controls. We also noted an enhanced migration of Acanthamoeba toward V. harveyi on non-nutrient agar plates compared to controls with no bacteria.

Expert Opinion on Orphan Drugs, 2016

ABSTRACT Introduction: Free Living Amoebae (FLA) are unicellular protozoa common to most soil and... more ABSTRACT Introduction: Free Living Amoebae (FLA) are unicellular protozoa common to most soil and aquatic environments. Only members of the genus Acanthamoeba and the species Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea are known to infect humans and other animals, often with fatal consequences. Acanthamoeba species are the causative agents of a sight threating infection of the cornea known as Acanthamoeba keratitis (AK) that mainly affects immunocompetent contact lens wearers and has shown an increased incidence in the recent years worldwide. To date, most of the available treatments and contact lens maintenance solutions are not fully effective against these pathogens. Areas covered: There is a clear need for novel fully effective therapeutic agents against AK. In the recent years the application of ‘phylopharmacology’ and RNAi has shown to be a reliable tool for the elucidation of novel drug targets and therapeutic agents. Current therapeutic options for AK as well as ongoing research advances are presented and discussed. Expert opinion: The available AK treatments are not fully effective so far mostly due to the existence of a cyst stage in this pathogen. Moreover, most of the first line treatment options are not available in all countries. There is an urgent need to validate novel drug targets and therapeutic agents against AK. Recent approaches have allowed the validation of drug targets and novel compounds that could become fully effective therapies against these emerging pathogens.

Acta Parasitologica, 2016

Pathogenic strains of

Parasitology Research, 2016

The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and en... more The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, forty soil samples were collected in the island of El Hierro, Canary Islands, Spain, and checked for the presence of Acanthamoeba. Samples were cultivated onto 2 % non-nutrient agar plates seeded with a layer of heat killed Escherichia coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 47.5 % of soil samples. Moreover, genotypes T2, T4, and T11 were identified in these samples. To the best of our knowledge, this is the first study to establish genotypes T2, T4, and T11 in soil sources from El Hierro island.

Protist, 2015

RNA helicases are ubiquitous enzymes that participate in almost all aspects of RNA processing, in... more RNA helicases are ubiquitous enzymes that participate in almost all aspects of RNA processing, including RNA and RNA-protein complex remodelling. In trypanosomatids, which post-transcriptionally regulate gene expression, the formation of different kinds of ribonucleoprotein granules under stress conditions modulates the parasite's RNA metabolism. This paper describes the isolation of a putative DEVH-box RNA helicase produced by promastigotes of Leishmania braziliensis. Using a Cy3-labelled dT 30 oligo, FISH showed the localization of this protein to mRNA granules under starvation stress conditions. The central region of the protein was shown to be responsible for this behaviour.

Journal of Medical Microbiology, 2015

Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the c... more Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis, which is often associated with the misuse of contact lenses. However, there is still a question remaining to be answered, which is whether these microorganisms are present on the ocular surface of healthy individuals. Therefore, the aim of this study was to determine the presence of Acanthamoeba on the ocular surface in healthy patients and also in those with other ocular surface infections. Sterile Schirmer test strips were used to collect samples from a group of patients who attended an ophthalmology consultation at the Hospital del Norte, Icod de los Vinos, Tenerife, Canary Islands. Most of the patients (46 individuals, 79.31 %) presented ocular surface pathologies such as blepharitis or conjunctivitis; the rest did not present any pathology. None of the patients included in the study wore contact lenses. The collected samples were cultured in 2 % non-nutrient agar plates and positive plates were then cultured in axenic conditions for further analyses. Molecular analysis classified all isolated strains as belonging to Acanthamoeba genotype T4, and osmotolerance and thermotolerance assays revealed that all strains were potentially pathogenic. Furthermore, all strains were assayed for sensitivity against voriconazole and chlorhexidine. Assays showed that both drugs were active against the tested strains. In conclusion, the Schirmer strip test is proposed as an effective tool for the detection of Acanthamoeba on the ocular surface.

Antimicrobial Agents and Chemotherapy, 2015

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatenin... more Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba . Here, we study the processes of programmed cell death in Acanthamoeba , induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC 50 s) and IC 90 s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with ...

Archives of Microbiology, 2015

Free-living amoebae (FLA) of the genus Acanthamoeba are opportunistic pathogenic agents able to c... more Free-living amoebae (FLA) of the genus Acanthamoeba are opportunistic pathogenic agents able to cause life-threatening infections in immunosuppressed patients. Chronic kidney disease impairs adaptive and innate immunity. Thus, patients with chronic kidney disease are prone to opportunistic infections by potentially pathogenic FLA. Therefore, in the present study, the investigation of Acanthamoeba genotypes isolated from the oral cavity of hemodialysis patients of reference hospitals in Iran was aimed, using both morphology and molecular (sequence-based analysis) tools. Furthermore, classification of the strains at the genotype level was performed on the basis of differences in the diagnostic fraction 3 (DF3) region of the 18S rRNA gene. The pathogenic potential of the isolated amoebae was also determined using thermotolerance and osmotolerance assays. Out of the 187 oral cavity samples investigated, nine (4.8%) were positive for FLA. DNA sequencing of the ASA.A1 region of the 18S rRNA gene revealed that the isolated strains belonged to the Acanthamoeba T1 and T4 genotypes. Genotype T1 was isolated for the first time from a patient in Iran. Interestingly, the T1 strain (AN2 strain) exhibits a high pathogenic potential in tolerance assays. The pathogenicity assay revealed that five strains were able to grow at high temperatures (37-40°C) and high osmolarity (0.5 and 1 M D-mannitol) conditions; thus, they were considered as potentially pathogenic strains. Moreover, two of the patients were positive for Vermamoeba genus. The present study is the first report of genotype T1 isolation in Iran and the first to identify the occurrence of Acanthamoeba and Vermamoeba genera in patients undergoing hemodialysis worldwide. Monitoring hemodialysis and renal failure patients should be a priority for possible control of Acanthamoeba and other FLA-related diseases.

Non-Flavivirus Encephalitis, 2011

Pathogens and Global Health, 2014

Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia m... more Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia mandrillaris, and the genera Sappinia and Acanthamoeba. In this study, a survey was conducted in order to evaluate the presence of potentially pathogenic amoebic strains in water samples collected from wells located in the western part of Guinea-Bissau. The samples were left to precipitate for 48 hours and then the sediments were seeded on non-nutrient agar plates containing Escherichia coli spread and cultures were checked daily for the presence of FLA. Identification of FLA strains was based on the morphological and polymerase chain reaction (PCR) using the 18S rDNA or 16S mitochondrial rDNA genes in the case of Naegleria and Balamuthia genera, respectively. In the case of positive samples of Acanthamoeba, strains were further classified at the genotype level by sequencing the diagnostic fragment 3 (DF3) region located in the 18S rDNA gene as previously described. Sappinia sp. was not isolated during the study and thus, no molecular analysis was performed for this genus. The obtained results revealed the presence of Acanthamoeba (genotypes T3 and T4), Naegleria fowleri, and Balamuthia mandrillaris. To the best of our knowledge, this is the first report demonstrating the presence of FLA in water bodies from Guinea-Bissau and the first report on the isolation of Balamuthia mandrillaris from environmental sources in Africa.

Epidemiology and Infection, 2014

SUMMARYBalamuthia mandrillaris is an emerging cause of encephalitis in humans. The transmission d... more SUMMARYBalamuthia mandrillaris is an emerging cause of encephalitis in humans. The transmission dynamics are poorly understood due to the high fatality rate and the sporadic nature of cases. Seventy-two soil samples were collected from beaches and the banks of lagoons, rivers, ponds, mineral springs and streams from across Jamaica and assayed for the presence of B. mandrillaris. Seventy-nine sites were sampled and the mitochondrial 16S rDNA gene of B. mandrillaris was amplified and sequenced to confirm the presence of the amoeba. One isolate of B. mandrillaris was recovered from soil from mineral spring which hosts an informal therapeutic mud bath business. Although B. mandrillaris is less frequently isolated from soil than other free-living amoebae, rubbing mud containing the organism onto the skin increases the likelihood of exposure and infection. This first report on the isolation of B. mandrillaris in the Caribbean and its presence in soil where human contact is likely warrants...

Veterinary Parasitology, 2014

A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fe... more A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fever and movement difficulty. After 24 h, the dog was found to have a high fever (39.5 • C) and was treated empirically with doxycycline/ciprofloxacin. At 48 h, after submission the fever rose to 41 • C and the animal presented with a stiff neck and dehydration. Peripheral blood and cerebrospinal fluid (CSF) were sampled and trophozoites with an Acanthamoeba-like morphology were observed in the CSF. PCR specific for Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris were performed and the CSF sample found positive for Acanthamoeba. Lungs, kidney, liver and spleen samples were collected post mortem. All collected organ samples were positive for Acanthamoeba by PCR, thus confirming a multisystemic infection. Water samples taken at a suspected site of infection yielded an almost identical PCR fragment to those of the clinical samples, indicating that this was probably where the infection originated. This is the first report of a fatal case of Acanthamoeba disseminated infection in a dog in Spain.

Trends in Parasitology, 2013

Parasitology Research, 2007

A mixed keratitis due to Acanthamoeba and Hartmannella species is reported. The patient was a sof... more A mixed keratitis due to Acanthamoeba and Hartmannella species is reported. The patient was a soft contact lens wearer. Early diagnosis was achieved by polymerase chain reaction and culture. The pathogenic potential of the isolated amoebae was proven using cytotoxicity assays. The reported case underlines the difficulties in identifying a corneal amoebic infection. In our case, the early diagnosis of a mixed infection allowed a proper antiamoebic treatment in an early stage of infection. This may have been the reason of a successful outcome after therapy. Free-living amoebae of the genera Acanthamoeba and Hartmannella are ubiquitous in nature. Acanthamoeba spp. are well known as causative agents of severe eye infections,

Parasitology Research, 2013

The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or ... more The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or postmortem features, in humans and other animals. The only diagnostic test described so far for the serological diagnosis of Toxoplasma gondii in marine mammals is the modified agglutination test (Dubey et al., Am J Vet Res 48(8):1239-1243, 1987). The development of more sensible and specific immunological techniques requires specific antibodies, which are currently unavailable in the scientific market. Indirect immunofluorescence (IIF) is one of the most widely used methods for the diagnosis of toxoplasmosis in humans (Auer et al., Parasitol Res 12:965-970, 2000). In order to develop and apply this technique to the bottlenose dolphin (Tursiops truncatus), immunoglobulins were firstly purified using ion-exchange chromatography. The purified immunoglobulins were then injected in New Zealand rabbits in order to obtain polyclonal antibodies. These antisera were validated by the IIF technique, using as controls serum samples of dolphins infected by Toxoplasma. The results were visualized using antirabbit IgG labeled with fluorescein. This newly developed and specific serological assay was then tested with the dolphin collection of Loro Parque, Tenerife, Spain (group I), and L'Oceanogràfic of Valencia, Spain (group II). The obtained results in this study showed that none of the dolphins from group 1 were infected by T. gondii and two animals were positive in group 2. Furthermore, we conclude that this study has produced antibodies with high specificity against dolphin immunoglobulins and an IIF method which may be used as immunological diagnostic tools, especially for the serological diagnosis of toxoplasmosis.

Parasite, 2006

Sera from individuals suffering from leishmaniasis have been shown to strongly react against cons... more Sera from individuals suffering from leishmaniasis have been shown to strongly react against conserved proteins from the parasite, such as ribosomal, histones and heat-shock proteins. Some of these proteins have also been described as immunogenic in several auto-immune syndromes, and the detection of antibodies against them is considered to be indicative of disorder of the immune system. In this paper, we investigate whether there is any relationship between the recognition of some conserved proteins from Leishmania braziliensis by individuals suffering from cutaneous (CL) and mucocutaneous (MCL) leishmaniasis, and the recognition of the human homologues of these antigens found in sufferers from autoimmune diseases. Our findings reveal that the immune response generated during CL and MCL is elicited specifically by the parasitic histone H1 and Hsp70, since the CL and MCL sera do not react against their human counterparts. In addition, evidence is presented showing the specific recognition of human proteins by the autoimmune sera, showing only a weak crossreaction with the most divergent regions of the parasitic proteins.

Journal of Parasitology, 2004