The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome

Abstract We report an Acanthamoeba keratitis case associated with the use of contact lens in a 28-year-old female from Brasília, Brazil. Samples from corneal scraping and contact lens case were used for culture establishment, PCR amplification, and partial sequencing (fragments of ~400kb) of small subunit rDNA; both culture and PCR were positive. The sequence analyses of the cornea and of isolates from the contact lens case showed similarity with the T4 genotype. To the best of our knowledge, this is the first report of T4 Acanthamoeba keratitis case from the Midwest region of Brazil.

Potentially pathogenic Acanthamoeba genotype T4 isolated from dental units and emergency combination showers

BACKGROUND Acanthamoeba is the genus of free-living amoebae that is most frequently isolated in nature. To date, 20 Acanthamoeba genotypes have been described. Genotype T4 is responsible for approximately 90% of encephalitis and keratitis cases. Due to the ubiquitous presence of amoebae, isolation from environmental sources is not uncommon; to determine the clinical importance of an isolation, it is necessary to have evidence of the pathogenic potential of amoebae. OBJECTIVE The aim of this study was to physiologically characterise 8 Acanthamoeba T4 isolates obtained from dental units and emergency combination showers and to determine their pathogenic potential by employing different laboratory techniques. METHODS Eight axenic cultures of Acanthamoeba genotype T4 were used in pathogenic potential assays. Osmotolerance, thermotolerance, determination and characterisation of extracellular proteases and evaluation of cytopathic effects in MDCK cells were performed. FINDINGS All of the isolates were osmotolerant, thermotolerant and had serine proteases from 44-122 kDa. Two isolates had cytopathic effects on the MDCK cell monolayer. MAIN CONCLUSION The presence of Acanthamoeba T4 with pathogenic potential in areas such as those tested in this study reaffirms the need for adequate cleaning and maintenance protocols to reduce the possibility of infection with free-living amoebae.

Presencia de Acanthamoeba spp. en agua para consumo ganadero en la provincia de La Pampa, Argentina / Presence of Acanthamoeba spp. in watering trough in La Pampa province, Argentina

Las amebas de vida libre del género Acanthamoeba incluyen cepas patógenas y no patógenas que actualmente se encuentran clasificadas en 18 genotipos diferentes (T1-T18). Se llevó a cabo un relevamiento para determinar la presencia de cepas de Acanthamoeba en agua de bebida para consumo ganadero en la provincia de La Pampa, Argentina. Las muestras fueron sembradas en agar no nutritivo. La identificación de cepas de Acanthamoeba se realizó mediante polymerase chain reaction con los primers JDP1/JDP2 específicos para género. De las 65 muestras tomadas, 13 fueron positivas a Acanthamoeba spp. Estas fueron caracterizadas a nivel de genotipo mediante la secuenciación del fragmento DF3. Los resultados de la secuenciación revelaron la presencia de los genotipos T4, T5 y T15 dentro de las muestras estudiadas, siendo la más frecuente la T4. Nuestro estudio revela importancia de la presencia de Acanthamoeba en el ambiente ganadero y la necesidad de realizar más estudios, para asociar la presencia de estos organismos y el papel que cumplen en patologías veterinarias. Este es el primer estudio en la provincia de La Pampa que demuestra la presencia de Acanthamoeba y el primero estudiado a nivel de genotipo de Argentina.

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 inwatering trough sample in La Pampa province, Argentina. Sample were inoculated onto non-nutrient agar plates and were checked for the presence of Acanthamoeba. Polymerase chain reaction was performed with genus-specific primers JDP1/JDP2, followed by direct sequencing of the polymerase chain reaction product for molecular identification. Sequencing results revealed the presence of T4, T5 and T15 genotypes within the studied samples. Sequencing revealed presence of T4, T5 and T15 in the samples studied genotypes, the most frequent T4. Our study reveals importance of the presence of Acanthamoeba in the livestock environment and the need for further studies to associate the presence of these organisms and the role in veterinary pathology. To the best of our knowledge, this is the first report demonstrating the presence Acanthamoeba in La Pampa province and the first study at the genotype level in Argentina.

Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

Construction of EST Database for Comparative Gene Studies of Acanthamoeba

The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported cDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www.amoeba.or.kr).

Molecular characterization of bacterial endosymbionts of Acanthamoeba isolates from infected corneas of Korean patients

The endosymbionts of 4 strains of Acanthamoeba (KA/E9, KA/E21, KA/E22, and KA/E23) isolated from the infected corneas of Korean patients were characterized via orcein stain, transmission electron microscopic examination, and 16S rDNA sequence analysis. Double membrane-bound, rod-shaped endosymbionts were distributed randomly throughout both the trophozoites and cysts of each of Acanthamoeba isolates. The endosymbionts of KA/E9, KA/E22, and KA/E23 were surrounded by electron-translucent areas. No lacunae-like structures were observed in the endosymbionts of KA/E21, the bacterial cell walls of which were studded with host ribosomes. Comparative analyses of the 16S rDNA sequences showed that the endosymbionts of KA/E9, KA/E22 and KA/E23 were closely related to Caedibacter caryophilus, whereas the KA/E21 endosymbiont was assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum. In the 4 strains of Acanthamoeba, the hosts of the endosymbionts were identified as belonging to the Acanthamoeba castellanii complex, which corresponds to the T4 genotype. Acanthamoeba KA/E21 evidenced characteristics almost identical to those of KA/E6, with the exception of the existence of endosymbionts. The discovery of these endosymbionts from Acanthamoeba may prove essential to future studies focusing on interactions between the endosymbionts and the amoebic hosts.

Natural occurrence of Mycobacterium as an endosymbiont of Acanthamoeba isolated from a contact lens storage case

Recent in vitro studies have revealed that a certain Mycobacterium can survive and multiply within freeliving amoebae. It is believed that protozoans function as host cells for the intracellular replication and evasion of Mycobacterium spp. under harmful conditions. In this study, we describe the isolation and characterization of a bacterium naturally observed within an amoeba isolate acquired from a contact lens storage case. The bacterium multiplied within Acanthamoeba, but exerted no cytopathic effects on the amoeba during a 6-year amoebic culture. Trasnmission electron microscopy showed that the bacteria were randomly distributed within the cytoplasm of trophozoites and cysts of Acanthamoeba. On the basis of the results of 18S rRNA gene analysis, the amoeba was identified as A. lugdunensis. A 16S rRNA gene analysis placed this bacterium within the genus Mycobacterium. The bacterium evidenced positive reactivity for acid-fast and fluorescent acid-fast stains. The bacterium was capable of growth on the Middlebrook 7H11-Mycobacterium-specific agar. The identification and characterization of bacterial endosymbionts of free-living protozoa bears significant implications for our understanding of the ecology and the identification of other atypical mycobacterial pathogens.

Molecular and biochemical characterization of a novel actin bundling protein in Acanthamoeba

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 micrometer of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.

Identification of differentially expressed cDNAs in Acanthamoeba culbertsoni after mouse brain passage

Free-living amoebae of the genus Acanthamoeba are causative agents of granulomatous amebic encephalitis and amebic keratitis. Because the virulence of Acanthamoeba culbertsoni cultured in the laboratory is restored by consecutive brain passages, we examined the genes induced in mouse brain-passaged A. culbertsoni by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Enhanced A. culbertsoni virulence was observed during the second mouse brain passage, i.e., infected mouse mortality increased from 5% to 70%. Ten cDNAs induced during mouse brain passage were identified by DDRT-PCR and this was confirmed by northern blot analysis. BlastX searches of these cDNAs indicated the upregulations of genes encoding predictive NADH-dehydrogenase, proteasomal ATPase, and GDP-mannose pyrophosphorylase B, which have previously been reported to be associated with A. culbertsoni virulence factors.

Genetic diversity of Acanthamoeba isolated from ocean sediments

Genetic diversity of 18 Acanthamoeba isolates from ocean sediments was evaluated by comparing mitochondrial (mt) DNA RFLP, 18S rDNA sequences and by examining their cytopathic effects on human corneal epithelial cells versus reference strains. All isolates belonged to morphologic group II. Total of 16 restriction phenotypes of mtDNA from 18 isolates demonstrated the genetic diversity of Acanthamoeba in ocean sediments. Phylogenetic analysis using 18s rDNA sequences revealed that the 18 isolates were distinct from morphological groups I and III. Fifteen isolates showed close relatedness with 17 clinical isolates and A. castellanii Castellani and formed a lineage equivalent to T4 genotype of Byers' group. Two reference strains from ocean sediment, A. hatchetti BH-2 and A. griffini S-7 clustered unequivocally with these 15 isolates. Diversity among isolates was also evident from their cytopathic effects on human corneal cells. This is the first time describing Acanthamoeba diversity in ocean sediments in Korea.

Random amplified polymorphic DNA profiles as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba

The genus Acanthamoeba comprises free-living amebae identified as opportunistic pathogens of humans and other animal species. Morphological, biochemical and molecular approaches have shown wide genetic diversity within the genus. In an attempt to determine the genetic relatedness among isolates of Acanthamoeba we analyzed randomly amplified polymorphic DNA (RAPD) profiles of 11 Brazilian isolates from cases of human keratitis and 8 American type culture collection (ATCC) reference strains. We found that ATCC strains belonging to the same species present polymorphic RAPD profiles whereas strains of different species show very similar profiles. Although most Brazilian isolates could not be assigned with certainty to any of the reference species, they could be clustered according to pattern similarities. The results show that RAPD analysis is a useful tool for the rapid characterization of new isolates and the assessment of genetic relatedness of Acanthamoeba spp. A comparison between RAPD analyses and morphological characteristics of cyst stages is also discussed (au)

Subgenus classification of Acanthamoeba by riboprinting

Subgenus classification of Acanthamoeba remains uncertain. Twenty-three reference strains of Acanthamoeba including 18 (neo)type-strains were subjected for classification at the subgenus level by riboprinting. PCR/RFLP analysis of 18S rRNA gene (rDNA). On the dendrogram reconstructed on the basis of riboprint analyses, two type-strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged early from the other strains and were quite distinct from each other. Four type-strains of morphological group 3, A. culbertsoni, A. palestinensis, A. healyi were considered taxonomically valid, but A. pustulosa was regarded as an invalid synonym of A. palestinensis. Strains of morphological group 2 were classified into 6 subgroups. Among them, A. griffini which has an intron in its 18S rDNA was the most divergent from the remaining strains. Acanthamoeba castellanii Castellani, A. quina Vil3, A. lugdunensis L3a, A. polyphaga Jones, A. triangularis SH621, and A. castellanii Ma strains belonged to a subgroup, A. castellanii complex. However, A. quina and A. lugdunensis were regarded as synonyms of A. castellanii. The Chang strain could be regarded as A. hatchetti. Acanthamoeba mauritaniensis, A. divionensis, A. paradivionensis could be considered as synonyms of A. rhysodes. Neff strain was regarded as A. polyphaga rather than as A. castellanii. It is likely that riboprinting can be applied for rapid identification of Acanthamoeba isolated from the clinical specimens and environments.