Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran

In this study, 15 Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of the Diagnostic Fragment 3 (DF3) of Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of Acanthamoeba through animal faeces in Iran.Overall, the widespread distribution of pathogenic Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.     

Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method

A method employing loop-mediated isothermal amplification (LAMP) of 18S ribosomal RNA gene was developed to detect Acanthamoeba in contact lens cases. A prevalence of 7% (10/150) was detected, with 100% sensitivity and 100% specificity when compared with the standard culture technique. Using visual inspection of turbidity a minimum of 10 pg of Acanthamoeba DNA could be detected, 10 times more sensitive than quantitative PCR employing two of the LAMP primers. The production of LAMP amplicons was confirmed by gel-electrophoresis and ethidium bromide staining. The LAMP procedure takes less than 2 h to perform and will be useful for incorporation into a point-of-care screening of suspected Acanthamoeba infection.     

Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City

A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis.     

Acanthamoeba spp.: Efficacy of Bioclen FR One Step®, a povidone-iodine based system for the disinfection of contact lenses

Pathogenic strains of Acanthamoeba are causative agents of a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. The prevalence of this infection has risen in the past 20 years, mainly due to the increase in number of contact lens wearers. Bioclen FR One Step® (Ophtecs Corporation) is the only available povidone-iodine based system for the disinfection of silicone hydrogel lenses and soft contact lenses on the market. Bioclen FR has been proven to be highly effective against bacteria and fungi that can cause problems for contact lens users. In this study, Bioclen FR One Step® was tested against three clinical Acanthamoeba isolates from contact lens cases. The results demonstrated that the tested Acanthamoeba clinical strains were sensitive to Bioclen FR One Step®.     

Novel in vitro and in vivo models to study central nervous system infections due to Acanthamoeba spp.

Acanthamoeba granulomatous encephalitis is a serious human infection with fatal consequences. The most distressing aspect of Acanthamoeba granulomatous encephalitis is the limited improvement in mortality. The underlying neurobiology is at present not well understood and treatment options are often of limited efficacy. There is therefore a real need to obtain more knowledge regarding the pathogenesis and pathophysiology of Acanthamoeba granulomatous encephalitis and to develop new chemotherapeutic approaches. However, the difficulties in using mammalian models to study this infection have hindered our search for therapeutic interventions. Recent availability of the blood-brain barrier, in vitro and use of locust as an in vivo model will undoubtedly allow us to investigate disease pathogenesis, mechanisms of parasite traversal across the blood-brain barrier and new drug therapies. It is argued that the models described here can offer several advantages in terms of speed, cost, technical convenience, and ethical acceptance. Furthermore, they are extremely valuable tools to discriminate molecules participating from both sides of the host-parasite interaction and will generate potentially useful leads in the identification of new potential drugs, as well as testing drug toxicity.     

First report on isolation of Leishmania tropica from sandflies of a classical urban Cutaneous leishmaniasis focus in southern Iran

Shiraz district in south of Iran is a classical focus of Cutaneous leishmaniasis (CL) and previous research has consistently documented the etiologic agent to be Leishmania tropica and Leishmania major in urban and rural areas, respectively. However, none of the Phlebotomus sergenti, a known vector for L. tropica, of the region has been found infected. We report the first isolation of L. tropica from sandflies in urban community of southern part of Shiraz city. Parasite polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses indicate CL cases in this community were caused by either L. major or L. tropica. Sandflies of P. sergenti were infrequent, however, three out of 10 (30.0%) females captured in urban area were found infected with L. tropica. But, no human cases were found to be infected with L. tropica. Phlebotomus papatasi were found the most dominant and infected species where 41 out of 207 (20%) tested individuals harboring L. major in suburb area of the city. Patients have been lived in the suburb area of the city where people keep normally domestic animals in their houses which provide appropriate environment for completion of sandfly life cycle and expansion of CL disease in the region.     

Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp. isolated from tap water in Brazil

Acanthamoeba spp., known to cause keratitis and granulomatous encephalitis in humans, are frequently isolated from a variety of water sources. Here we report for the first time the characterization of an Acanthamoeba sp. (ACC01) isolated from tap water in Brazil. This organism is currently being maintained in an axenic growth medium. Phylogenetic analysis based on SSU rRNA gene sequences positioned the new isolate in genotype T4, closest to the keratitis-causing isolate, A. polyphaga ATCC 30461 (∼99% similarity). Acanthamoeba ACC01 and A. polyphaga 30461 both grew at 37 °C and were osmotically resistant, multiplying in hyperosmolar medium. Both isolates secreted comparable amounts of proteolytic enzymes, including serine peptidases that were optimally active at a near neutral/alkaline pH and resolved identically in gelatin gels. Incubation of gels at pH 4.0 with 2 mM DTT also indicated the secretion of similar cysteine peptidases. Altogether, the results point to the pathogenic potential of Acanthamoeba ACC01.     

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 °C, subsequently displayed a high thermotolerance, being able to grow at 42 °C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.     

Drug target identification, validation, characterisation and exploitation for treatment of Acanthamoeba (species) infections

New more efficacious antimicrobials as required for the treatment of Acanthamoeba infections as those currently available require arduous treatment regimes, are not always effective and are poorly active against the cystic stages. Herein, we review potential drug targets including tubulin, alternative oxidase, amino acid biosynthesis and myosin. In addition, we review the literature for current missing tools and resources for the identification, validation and development of new antimicrobials for this organism. Additional targets should come to light through a concerted genome sequencing effort.     

Acanthamoeba spp.: In vitro effects of clinical isolates on murine macrophages, osteosarcoma and HeLa cells

Three different cell lines (murine macrophages, HeLa and osteosarcoma cells) were assayed in order to check for the manifestation of the cytopathic effects of three strains of Acanthamoeba recently isolated in our laboratory from contact lens cases: CLC-16, CLC-41.r and CLC-51-l. Adhesion and cytotoxicity assays were carried out with these strains and the type strain Acanthamoeba castellanii Neff as a control. Briefly, the ability of these amoebae to bind to the three cell lines was calculated and supernatants were examined for cytotoxicity by measuring lactate dehydrogenase released as an estimate of cytotoxicity using a commercial detection kit. The three strains showed high adhesion and cytotoxicity levels when tested in the three cell lines. This study demonstrates the ability of these amoebae to degrade any of the tested cell lines. To the best of our knowledge, this is the first report of the in vitro effects of acanthamoebae on osteosarcoma cells.     

Echinococcus granulosus genotypes in livestock of Iran indicating high frequency of G1 genotype in camels

In this study, 112 Echinococcus granulosus isolates from different livestock of Iran were genotyped by PCR amplification of ribosomal DNA-internal transcribed spacer 1 (rDNA-ITS1) region followed by restriction fragment length polymorphism (RFLP) with the enzyme RsaI. The possibility of intra-genotype variation was also investigated using randomly amplified polymorphic DNA (RAPD) analysis. Isolates from sheep, goats, cattle and the majority of camels (12 of 18; 66.7%) were identified as the G1 genotype and a few camel isolates (6 of 18; 33.3%) belonged to the G6 genotype. Overall G1 and G6 genotypes were identified in 94.6% (106 of 112) and 5.3% (6 of 112) of all isolates, respectively. RAPD analysis based on 15 separate primers showed 7-14 bands of 200-3000 bp for strain G1. Considering each individual primer, no differences observed among isolates from different hosts and between livers and lungs. This study confirmed the existence of G1 and G6 genotypes in Iran. Moreover, G1 is much more prevalent even in camels, indicating the importance of sheep-dog cycle in public health. Studying intra-genotypic variation of E. granulosus warrants more research using other primers and methods.     

Balamuthia and Acanthamoeba-binding antibodies in West African human sera

Little is known about the prevalence of Balamuthia mandrillaris amoebae and Balamuthia amoebic encephalitis in Africa. As an approach, relative concentrations of amoebae-binding serum antibodies (Ab) were assessed by flow cytometry using formaldehyde-fixed B. mandrillaris, Acanthamoeba lenticulata 72-2 and Acanthamoeba castellanii 1BU amoebae for specific Ab capture (B.m.-Ab, A.l.-Ab, A.c.-Ab). One hundred and ninety-two sera from West African (Côte d’Ivoire) donors aged 11-95 years (mean 38 a; 51% males), and living in villages surrounded by rainforest near the Liberian border, were tested and related to reference sera from Berlin. While B.m.-Ab tended to increase with donor age, A.l.-Ab and A.c.-Ab did not. Accordingly, B.m.-Ab correlated only weakly with A.l.-Ab or A.c.-Ab. Of the nine individuals with the highest B.m.-Ab concentrations, most were elderly (mean 58 a), male (78%), and professed intensive outdoor activity (hunting, farming). Only three of these sera also showed elevated A.l.-Ab, and none elevated A.c.-Ab.     

Fruit anatomy of the genus Pimpinella L. (Apiaceae) in Iran

The genus Pimpinella L., with about 170–180 species in the world, is one of the largest genera of the family Apiaceae (Umbelliferae). Based on the Flora Iranica treatment, this genus has 25 species in the Iranian plateau, including 19 species in Iran, and six of those (P. tragioides, P. deverroides, P. pastinacifolia, P. anisactis, P. khorasanica and P. khayyamii) are endemic.     

Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran

Leishmania major is the causative agent and Phlebotomus papatasi is the main vector of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran and elsewhere. Nested PCR protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene) in female P. papatasi. In the current investigation, L. major was found in Natanz, Isfahan province in centre of Iran, in a focus of rural ZCL. Ten (1.8%) out of 549 female P. papatasi was found to be infected with L. major based on the PCR detection and sequencing of parasite ITS-rDNA.Nine (1.8%) out of 498 female P. papatasi infected with L. major came from animal shelters, inside houses and yards. And one (1.9%) out of 51 female P. papatasi infected with L. major came from gerbil borrows. Infection rates were higher for females containing red blood meals, large eggs (semi-mature and mature) than for those without either blood meals or eggs. From the 10 infections detected three different haplotypes of L. major were identified. Two haplotypes were found to be novel. The other haplotypes of L. major was found to be identical to that of isolates of L. major from Iran and in elsewhere using GenBank data. Comparisons of infection rates between habitats will be inaccurate when the proportions of blood-fed and gravid flies differ among sandfly samples.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Pollen morphology of Campanula (Campanulaceae) and allied genera in Iran with special focus on its systematic implication

Pollen grains of 47 taxa of Campanulaceae including 35 taxa of Campanula that represent its five subgenera and nine sections are investigated. Moreover, five species and three subspecies representing three sections in Asyneuma and one species of each genera Legousia, Michauxia, Zeugandra and Theodorovia were studied by light and scanning electron microscopy. The basic shape of the pollen grains was spheroidal. The apertures vary from tri- to hexa-porate. The sculpturing pattern of exine was rugulate-echinate, rugulate-microechinate or in few species rugulate-microreticulate and microechinate. The most valuable characters for subgeneric classification were the length and density of echini. The length of echini were significantly long (> 2 μm) in C. sclerotricha, Legousia falcata and Michauxia laevigata. Pollen grains show low variation in different species of subgen. Rapunculus, but were variable among different species of some groups, such as sect. Rupestres, probably indicating their non-monophyly despite homogeneity with respect to other morphological characters. Pollen morphology does not support recognition of Asyneuma, Legousia, Michauxia, Symphyandra, Theodorovia, and Zeugandra as separated from Campanula, since none of them exhibit any unique feature.     

Acanthamoeba castellanii: High antibody prevalence in racially and ethnically diverse populations

Acanthamoeba is an opportunistic protozoan pathogen that can produce keratitis and rare but fatal encephalitis. In the present study, we examined secretory IgA antibody to Acanthamoeba castellanii of the T4 genotype in mucosal secretions from 114 individuals of 37 countries, inhabitants and/or visitors, aged 16-65 years in London, UK. Acanthamoeba antibody prevalence rate was more than 85%, without any significant differences between males (86.2%) and females (89.2%). Some epidemiological factors contributing to the high prevalence of antibody to Acanthamoeba in surveyed population are discussed further.     

The role of domestic tap water in Acanthamoeba contamination in contact lens storage cases in Korea

A survey was carried out from August to December 2004 in Pusan, Korea to document the presence of free-living amoeba (FLA), including the genus Acanthamoeba, in both contact lens storage cases and domestic tap water. Acanthamoeba was isolated from 5 (4.2%) in 120 contact lens storage cases. Four house tap water samples from residents, whose contact lens storage cases had been contaminated by Acanthamoeba, were also found to be contaminated with Acanthamoeba. Therefore, the contamination rate of FLA and Acanthamoeba in domestic tap water was investigated in order to examine the role of domestic tap water in Acanthamoeba contamination of contact lens storage cases. FLA and Acanthamoeba were identified in 97 (46.8%) and 16 (7.7%) of the 207 domestic tap water samples, respectively. There were no significant differences between the contamination rates of FLA in tap water according to the filtration plant of origin. No FLA was detected in the tap water directly supplied by the water purification plants. Water storage tanks appear to promote FLA colonization, including Acanthamoeba, in domestic tap water. This increases the risk of Acanthamoeba contamination in contact lens storage cases as well as increasing the risk of Acanthamoeba keratitis.