María Reyes-Batlle

Isolation and characterization of Acanthamoeba strains from soil samples in Gran Canaria, Canary Islands, Spain.

Free-living Amoebae of Acanthamoeba genus include non-pathogenic and pathogenic strains that are currently classified in 18 different genotypes, T1–T18. In this study, a survey was carried out to evaluate the presence of Acanthamoeba strains in soil samples collected between 2012 and 2013 in Gran Canaria Island, Canary Islands, Spain. Samples were inoculated onto non-nutrient agar (NNA) plates and were checked for the presence of Acanthamoeba. Identification of Acanthamoeba strains was based on the morphology of the cyst and trophozoite forms. Subsequently, positive samples were cloned for their molecular characterization at the genotype level by sequencing the DF3 region located in the 18S rDNA gene of Acanthamoeba as previously described. Sequencing results revealed the presence of T2, T5 and T4 genotypes within the studied samples. To the best of our knowledge, this is the first report demonstrating the presence of Acanthamoeba in Gran Canaria Island and the first study at the genotype level in the Canary Islands.

Genotyping of potentially pathogenic Acanthamoeba strains isolated from nasal swabs of healthy individuals in Peru.

Free Living Amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can be found in the air, soil and water; and have also been isolated from air-conditioning units. In humans, they are causative agents of a sight-threating infection of the cornea, Acanthamoeba keratitis (AK) and a fatal infection of the central nervous system known as Granulomatous Amoebic Encephalitis (GAE). In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in nasal swabs from individuals in two regions of Peru. Identification of isolates was based on cyst morphology and PCR-sequencing of the Diagnostic Fragment 3 to identify strains at the genotype level. The pathogenic potential of the isolates was also assayed using temperature and osmotolerance assays and extracellular proteases zymograms. The obtained results revealed that all isolated strains exhibited pathogenic potential. After sequencing the highly variable DF3 (Diagnostic Fragment 3) region in the 18S rRNA gene as previously described, genotype T4 was found to be the most common one in the samples included in this study but also genotype T15 was identified. To the best of our knowledge, this is the first study on the characterization of Acanthamoeba strains at the genotype level and the first report of genotype T4 and T15 in Peru.

Voriconazole as a first-line treatment against potentially pathogenic Acanthamoeba strains from Peru

Pathogenic strains of Acanthamoeba genus are the causative agents of fatal granulomatous amoebic encephalitis and a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. In a previous study, Acanthamoeba strains were isolated from nasal swabs collected from healthy individuals in Peru. In the present study, the pathogenic potential of the isolated strains was established based on temperature and osmotolerance assays as well as the secretion rate of extracellular proteases. Based on these experiments, four strains that showed the highest pathogenic potential were selected for sensitivity assays against two molecules (voriconazole and chlorhexidine) which are currently used for the treatment of Acanthamoeba infections. After performing sensitivity and activity assays, it was found that both drugs were active against the tested strains. However, voriconazole showed higher activity against the studied strains compared to chlorhexidine. Therefore, voriconazole should be established as a first-line treatment against Acanthamoeba infections at least in the studied region of Peru.

Statins and Voriconazole Induce Programmed Cell Death in Acanthamoeba castellanii

ABSTRACT 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 (IC50s) and IC90s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba. All the drugs assayed in this study induced PCD in Acanthamoeba. To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba.

In vitro effects of triterpenic acids from olive leaf extracts on the mitochondrial membrane potential of promastigote stage of Leishmania spp.

Protozoan diseases, such as leishmaniasis, are a cause of considerable morbidity throughout the world, affecting millions every year. In this study, two triterpenic acids (maslinic and oleanolic acids) were isolated from Tunisian olive leaf extracts and their in vitro activity against the promastigotes stage of Leishmania (L.) infantum and Leishmania (L.) amazonensis was investigated. Maslinic acid showed the highest activity with an IC50 of 9.32 ± 1.654 and 12.460 ± 1.25 μg/ml against L. infantum and L. amazonensis, respectively. The mechanism of action of these drugs was investigated by detecting changes in the phosphatidylserine (PS) exposure, the plasma membrane permeability, the mitochondrial membrane potential and the ATP level production in the treated parasites. By using the fluorescent probe SYTOX® Green, both triterpenic acids showed that they produce a time-dependent plasma membrane permeabilization in the treated Leishmania species. In addition, spectrofluorimeteric data revealed the surface exposure of PS in promastigotes. Both molecules reduced the mitochondrial membrane potential and decreased the ATP levels to 15% in parasites treated with IC90 for 24h. We conclude that the triterpenic acids tested in this study, show potential as future therapeutic alternative against leishmaniasis. Further studies are needed to confirm this.

The isolation of Balamuthia mandrillaris from environmental sources from Peru

Balamuthia mandrillaris is an opportunistic free-living 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 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. mandrillaris-like 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.

Presence of potentially pathogenic free-living amoebae strains from well water samples in Guinea-Bissau

Abstract 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.

Fatal meningoencephalitis in child and isolation of Naegleria fowleri from hot springs in Costa Rica.

Asian lineage and had 99% identity with sequences of ZIKV isolated in French Polynesia in 2013 (10). Serum specimens from both patients were cultured on Vero cells, and supernatants were evaluated by RT-PCR. Each specimen was positive only for DENV, which was probably caused by low viral loads for ZIKV. We report co-infection of 2 patients with DENV and ZIKV; each patient was infected with a different DENV serotype. No synergistic effects of the 2 viral infections were observed because both patients were not hospitalized and recovered after a mild clinical course. During this outbreak, patients in New Caledonia were tested for DENV, chikungunya virus, and ZIKV within the framework of the arboviruses sentinel network, which enabled detection of co-infections. Thus, clinicians should be aware of infections with multiple pathogens in the differential diagnosis of dengue-like illness, especially in patients who returned from tropical regions. This diagnostic procedure could be improved by using multiplex RT-PCR for travelers, given the frequent co-circulation of multiple arboviruses in tropical regions.

Bioassay guided isolation and identification of anti-Acanthamoeba compounds from Tunisian olive leaf extracts

Pathogenic Acanthamoeba strains are causative agents of Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide. The existence of the cyst stage complicates Acanthamoeba therapy as it is highly resistant to antibiotics and physical agents. The aim of this study was to investigate the activity of Limouni olive leaf cultivar against the trophozoite stage of Acanthamoeba. The ethyl acetate and methanol extracts of this variety were tested against Acanthamoeba castellanii Neff. The ethyl acetate extract of olive leaf was the most active showing an IC50 of 5.11±0.71μg/ml of dry extract. Bio-guided fractionation of this extract was conducted and led to the identification of three active compounds namely oleanolic and maslinic acids and oleuropein which could be used for the development of novel therapeutic approaches against Acanthamoeba infections.

Activity of olive leaf extracts against the promastigote stage of Leishmania species and their correlation with the antioxidant activity.

Leishmaniasis is one of the neglected tropical diseases in terms of drug discovery and development. Furthermore, the chemotherapy used to treat this disease has been proved to be highly toxic and to present resistance issues. As consequent, the need for novel leishmanicidal molecules has notably increased in the recent years. In the present work an attempt was made to evaluate the antioxidant and leishmanicidal activities besides presence of compounds in leaf extracts of 5 different Tunisian olive tree varieties, used as traditional medicine in this country. The concentration of extracts needed to inhibit 50% of the parasitic growth (IC50) was estimated using different Leishmania strains. All tested extracts showed an inhibitory effect on the parasite growth with IC50 ranging from 2.130±0.023 to 71.570±4.324μg/ml, respectively for the methanolic extracts of Limouni and Zarrazi against Leishmania donovani. In fact, this activity was significantly affected by the olive cultivar and the tested Leishmania strain. Furthermore, the activities against both Leishmania tropica and major species were correlated to the total phenolic compounds. These results could suggest that olive leaf extract could carry potential new compounds for the development of novel drugs against Leishmaniasis.