Enrique Martínez-Carretero

Early diagnosis of amoebic keratitis due to a mixed infection with Acanthamoeba and Hartmannella

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.

Glycogen Phosphorylase in Acanthamoeba spp.: Determining the Role of the Enzyme during the Encystment Process Using RNA Interference

ABSTRACT Acanthamoeba infections are difficult to treat due to often late diagnosis and the lack of effective and specific therapeutic agents. The most important reason for unsuccessful therapy seems to be the existence of a double-wall cyst stage that is highly resistant to the available treatments, causing reinfections. The major components of the Acanthamoeba cyst wall are acid-resistant proteins and cellulose. The latter has been reported to be the major component of the inner cyst wall. It has been demonstrated previously that glycogen is the main source of free glucose for the synthesis of cellulose in Acanthamoeba, partly as glycogen levels fall during the encystment process. In other lower eukaryotes (e.g., Dictyostelium discoideum), glycogen phosphorylase has been reported to be the main tool used for glycogen breakdown in order to maintain the free glucose levels during the encystment process. Therefore, it was hypothesized that the regulation of the key processes involved in the Acanthamoeba encystment may be similar to the previously reported regulation mechanisms in other lower eukaryotes. The catalytic domain of the glycogen phosphorylase was silenced using RNA interference methods, and the effect of this phenomenon was assessed by light and electron microscopy analyses, calcofluor staining, expression zymogram assays, and Northern and Western blot analyses of both small interfering RNA-treated and control cells. The present report establishes the role of glycogen phosphorylase during the encystment process of Acanthamoeba. Moreover, the obtained results demonstrate that the enzyme is required for cyst wall assembly, mainly for the formation of the cell wall inner layer.

Acanthamoeba keratitis due to genotype T11 in a rigid gas permeable contact lens wearer in Spain

A case of a 59-year-old Spanish patient who presented with severe ocular pain, blurred vision, eyelid swelling and foreign body sensation in the right eye is reported. She was a regular gas permeable contact lens [corrected] wearer who initially claimed to maintain standard lens care. After exploration, conjunctival injection, dendritiform corneal ulcers and stromal edema were observed. She was initially treated for a possible viral keratitis due to herpes simplex virus using 3% topical acyclovir and 0.1% dexamethasone eye drops 5 times a day. The patient did not respond to this treatment and six weeks later, corneal scrapings were positive for Acanthamoeba genotype T11. She was then treated with chlorhexidine 0.02%, propamidine 0.1% and 1% cycloplegic eye drops hourly which resulted in a significant improvement. After a month, ocular pain decreased and the clinical signs of keratitis ameliorated observed as a diminution of the size of the ulcer and also in the extension and opacity of the corneal infiltrates. The patient has been following this treatment for 3 months and it is possible that she will have to carry on with it for a whole year. To the best of our knowledge, this is the first case of severe keratitis due to Acanthamoeba genotype T11 in Spain .

Is Balamuthia mandrillaris a public health concern worldwide

Balamuthia mandrillaris is an opportunistic, free-living amoeba that can cause skin lesions and the typically fatal Balamuthia amoebic encephalitis (BAE) both in immunocompromised and immunocompetent individuals. Available data for BAE cases indicate that this disease is difficult to detect because knowledge of predisposing factors is lacking, causing a challenge for diagnosing BAE. The number of reported BAE cases is increasing worldwide, and this is a major concern because little is known about the pathogen, no standardized detection tools are available, and most of the treatments are almost empirical. The recently reported cases, novel diagnostics tools, and successful therapeutic approaches against BAE infections are reviewed here.

Successful Monitoring and Treatment of Intraocular Dissemination of Acanthamoeba

I ntraocular spread of Acanthamoeba after keratitis is rare, being previously documented and microbiologically confirmed in 1 case of chorioretinitis and 1 case of endophthalmitis. Both occurred several months following penetrating keratoplasty (PK), were initially treated as suspected sterile inflammations, did not receive specific oral or intraocular treatment for Acanthamoeba, and finally required evisceration. We report the first documented case, to our knowledge, of successful management of intraocular Acanthamoeba dissemination. Intraocular and systemic treatment was monitored through microbiological testing and polymerase chain reaction analysis of serial aqueous taps.

Therapeutic Potential of a Combination of Two Gene-Specific Small Interfering RNAs against Clinical Strains of Acanthamoeba

ABSTRACT Pathogenic strains of the genus Acanthamoeba are causative agents of severe infections, such as fatal encephalitis and a sight-threatening amoebic keratitis. Antimicrobial therapy for these infections is generally empirical, and patient recovery is often problematic, due to the existence of a highly resistant cyst stage in these amoebae. In previous studies, small interfering RNAs (siRNAs) against the catalytic domains of extracellular serine proteases and glycogen phosphorylase from Acanthamoeba were designed and evaluated for future therapeutic use. The silencing of proteases resulted in Acanthamoeba failing to degrade human corneal cells, and silencing of glycogen phosphorylase caused amoebae to be unable to form mature cysts. After the siRNA design and concentration were optimized in order to avoid toxicity problems, cultures of Acanthamoeba were treated with a combination of both siRNAs, and cells were evaluated under an inverted microscope. This siRNA-based treatment dramatically affected the growth rate and cellular survival of the amoebae. These results were observed less than 48 h after the initiation of the treatment. In order to check possible toxic effects of the siRNA combination, three eukaryotic cell lines (HeLa, murine macrophages, and osteosarcoma cells) were treated with the same molecules, and cytotoxicity was examined by measuring lactate dehydrogenase release. The future use of the combination of these siRNAs is proposed as a potential therapeutic approach against pathogenic strains of Acanthamoeba.

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.

Microscopy and molecular biology for the diagnosis and evaluation of malaria in a hospital in a rural area of Ethiopia

BackgroundMalaria is a leading public health problem in Ethiopia. Accurate diagnosis of Plasmodium infections is crucial for the reduction of malaria in tropical areas and for epidemiological studies. The role of light microscopy (LM) as gold standard has been questioned and, therefore, new molecular methods have been developed for the detection of Plasmodium species. The aim of the present work was to compare different malaria diagnostic methods in order to detect the most common species of Plasmodium and to broaden the knowledge of malaria prevalence in a hospital in a rural area in Ethiopia.MethodsA cross-sectional survey of 471 individuals was carried out in a hospital in the rural area of Gambo (Ethiopia). Blood samples were prepared for microscopic observation and collected in filter paper for Seminested-Multiplex PCR (SnM-PCR) and real time PCR (qPCR) testing. The SnM-PCR was considered as the gold standard technique and compared with the rest. Thus, agreement between SnM-PCR and LM was determined by calculating Kappa Statistics and correlation between LM and qPCR quantification was calculated by pair-wise correlation co-efficient.ResultsSamples analysed by LM and SnM-PCR were positive for Plasmodium sp. 5.5% and 10.5%, respectively. Sensitivity was 52.2% by LM and 70% by qPCR. Correlation co-efficient between microscopy counts and qPCR densities for Plasmodium vivax was R2 = 0.586. Prevalence was estimated at 7% (95% CI: 4.7–9.3). Plasmodium vivax was the dominant species detected and the difference was statistically significant (χ2 = 5.121 p < 0.05). The highest prevalence of the parasite (10.9%) was observed in age groups under 15 years old.ConclusionAccurate malaria diagnostic methods have a great effect in the reduction of the number of malaria-infected individuals. SnM-PCR detection of malaria parasites may be a very useful complement to microscopic examination in order to obtain the real prevalence of each Plasmodium species. Although SnM-PCR shows that it is a good tool for the determination of Plasmodium species, today light microscopy remains the only viabletool for malaria diagnosis in developing countries. Therefore, re-inforcement in the training of microscopists is essential for making the correct diagnosis of malaria. Plasmodium vivax was the predominant species in Gambo, a meso-endemic area for this species.

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.