Acanthamoebae are the causative agents of an often seriously progressing keratitis (AK) occurring predominantly in contact lens wearers and can cause several disseminating infections potentially resulting in granulomatous amoebic encephalitis (GAE) in the immunocompromised host. Our institution is the Austrian reference laboratory for Acanthamoeba diagnostics and the aim of this study was to give an overview of proven cases of Acanthamoeba infections in Austria during the past 20 yr. All samples of patients with suspected AK or GAE were screened for Acanthamoeba spp. by culture and/or PCR and the detected amoebae were genotyped. Altogether, 154 cases of AK and three cases of GAE were diagnosed. Age of the AK patients ranged from 8 to 82 yr (mean 37.8) and 58% of the patients were female. Approximately 89% of the AK patients were contact lens wearers, almost all cases were unilateral and 19% of the patients required a keratoplasty. Age of the GAE patients ranged from 2 to 25 yr (mean 14.7), all were HIV-negative, but two were severely immunosuppressed at the time of diagnosis. The predominant genotype in the AK cases was T4, other genotypes found were T3, T5, T6, T10 and T11. The three GAE cases involved genotypes T2, T4 and T5.
characterisation; contact lens; infection; protozoa; SSU; typing
Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. In view of the devastating nature of this disease, here we present our current understanding of Acanthamoeba keratitis and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be potential targets for improved diagnosis, therapeutic interventions and/or for the development of preventative measures. Novel molecular approaches such as proteomics, RNAi and a consensus in the diagnostic approaches for a suspected case of Acanthamoeba keratitis are proposed and reviewed based on data which have been compiled after years of working on this amoebic organism using many different techniques and listening to many experts in this field at conferences, workshops and international meetings. Altogether, this review may serve as the milestone for developing an effective solution for the prevention, control and treatment of Acanthamoeba infections.
Acanthamoeba; keratitis; diagnosis; therapy; pathogenesis
Legionella pneumophila is known as the causative agent of Legionnaires’ disease and free-living amoebae (FLA) can serve as vehicles for legionellae. The aim of this study was to screen industrial waters for the occurrence of FLA and their co-occurrence with legionellae. A total of 201 water samples, including 129 cooling waters and 72 process waters, and 30 cooling lubricants were included in the study. Treated waters were screened periodically, pre and post treatment. Altogether, 72.6% of the water samples were positive for FLA, acanthamoebae being most prevalent (in 23.9% of the samples) followed by Vermamoeba vermiformis (19.4%). Only one cooling lubricant was positive (Acanthamoeba genotype T4). Legionella spp. were detected in 34.8% of the water samples and in 15% in high concentrations (>1000 CFU/100 ml). Altogether, 81.4% of the Legionella-positive samples were positive for FLA by standard methods. By applying a highly sensitive nested PCR to a representative set of random samples it was revealed that Legionella spp. always co-occurred with Acanthamoeba spp. Although the addition of disinfectants did influence amoebal density and diversity, treated waters showed no difference concerning FLA in the interphases of disinfection. It appears that FLA can re-colonize treated waters within a short period of time.
Acanthamoeba; Free-living amoebae; Industrial water; Legionella; PCR
The giant liver fluke Fascioloides magna, an invasive species originating from North America, was recorded in Austria in the wild for the first time in 2000. Since then, various data concerning the epidemiology in snail intermediate hosts and cervid final hosts have been reported. Galba truncatula acts as snail intermediate host, and red deer, roe deer and fallow deer act as final hosts. G. truncatula is abundant throughout the region, especially along muddy shores of slow-flowing branches of the river system. Prevalence in deer (20–100 %) is much higher than in snails (0.03–0.2 %). Despite medical treatment of parts of the deer population, the parasite has successfully established itself on both sides of the Danube floodplain environments southeast of Vienna. Genetic analysis revealed that the infection of Austrian deer populations apparently originated from foci in the Czech Republic or from populations of Danube tributaries. Areas adjacent southwards, which will soon be joined through wildlife crossings, have not yet evidenced F. magna. Nonetheless, these environments are inhabited by host snails and deer and therefore constitute suitable habitats for F. magna. Invading alien parasites not only threaten native individual hosts but also influence host populations, thus potentially also modifying parasite communities and interactions. The host range of F. magna includes a variety of potential hosts, notably other Lymnaeidae as potential intermediate hosts and various ungulates, including sheep and cattle, as final hosts. Because eradication after medical treatment was unsuccessful, and due to the risk of further spread of the parasite into unaffected regions, enhanced control strategies need to be developed. We recommend assessment of introduction pathways and dispersal, continuous monitoring of host abundance and distribution and the prevalence of flukes in intermediate and final hosts, as well as coordinated and concerted actions with neighbouring countries. This strategy could help to reduce potential negative impacts of this and other invasive parasites on host populations in Europe.
Digenea; Trematoda; Invader; Dispersal; Austria; Digenea; Trematoda; Neobiota; Ausbreitung; Österreich
The possible existence of autochthonous sandfly populations in Central Europe north of the Alps has long been excluded. However, in the past years, sandflies have been documented in Germany, Belgium, and recently, also in Austria, close to the Slovenian border. Moreover, autochthonous human Leishmania and Phlebovirus infections have been reported in Central Europe, particularly in Germany. From 2010 to 2012, sandfly trapping (740 trap nights) was performed at 53 different capture sites in Austria using battery-operated CDC miniature light traps. Sites were chosen on the basis of their climate profile in the federal states Styria, Burgenland, and Lower Austria. Sandfly specimens found were transferred to 70 % ethanol for conservation. Identification was based on morphological characters of the male genitalia and the female spermathecae, respectively. Altogether, 24 specimens, 22 females and 2 males, all identified as Phlebotomus (Transphlebotomus) mascittii Grassi, 1908, were found at six different sampling sites in all three federal states investigated. The highest number of catches was made on a farm in Lower Austria. Altogether, the period of sandfly activity in Austria was shown to be much longer than presumed, the earliest capture was made on July 3rd and the latest on August 28th. Sandflies have been autochthonous in Austria in small foci probably for long, but in the course of global warming, further spreading may be expected. Although P. mascittii is only an assumed vector of Leishmania spp.—data on its experimental transmission capacity are still lacking—the wide distribution of sandflies in Austria, a country thought to be free of sandflies, further supports a potential emergence of sandflies in Central Europe. This is of medical relevance, not only with respect to the transmission of Leishmania spp. for which a reservoir is given in dogs, but also with respect to the phleboviruses.
Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data has been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and HPLC data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man5GlcNAc2) with α1,2-mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species.
trichomonads; N-glycan; mass spectrometry; pentose; phosphoethanolamine
The authors report the first indigenous case of Plasmodium ovale infection from Bangladesh. The diagnosis was confirmed by PCR and sequence analysis. The patient had neither been outside of the country nor ever received blood transfusions. The authors concluded that there was evidence for a local transmission of P ovale malaria in Bangladesh. P ovale malaria should therefore always be considered a potential differential diagnosis in the indigenous population as well as travellers and migrants returning from South Asia, possibly up to years after their return.
Leishmania guyanensis; Leishmania; Viannia; Phlebotomus; Lutzomyia; Sandfly; leishmaniasis; parasites; Austria
A patient presenting with an atypical manifestation of cutaneous leishmaniasis after travel to Cyprus was successfully treated with miltefosine. The K26 typing revealed a hitherto undescribed strain of the Leishmania donovani/infantum complex as the causing agent.
In spite of the high prevalence of malaria in Southeastern Bangladesh, there remains a significant shortage of information regarding the presence of three of five human malaria parasites: Plasmodium ovale, P. malariae, and P. knowlesi. The presence of P. ovale and P. knowlesi has previously never been reported from Bangladesh. We used a genus- and species-specific nested polymerase chain reaction, targeting highly conserved regions of the small subunit ribosomal RNA (SSU rRNA) gene, to investigate the presence of malaria parasites in a total number of 379 patient samples in a survey of patients with febrile illnesses in the Chittagong Hill Tracts in Southeastern Bangladesh. We identified the first cases of P. ovale in Bangladesh. They were confirmed by sequence analysis; 189 of 379 samples (49.9%; 95% confidence interval = 44.9–54.9%) were positive for Plasmodium sp. by PCR. P. falciparum monoinfections accounted for 68.3% (61.3–74.5%), followed by P. vivax (15.3%; 10.9–21.2%), P. malariae (1.6%; 0.5–4.6%), P. ovale (1.6%; 0.5–4.6%), and mixed infections (13.2%; 9.1–18.8%). We found no evidence of P. knowlesi in this region.
Linguatula serrata, the so-called tongue worm, is a worm-like,
bloodsucking parasite belonging to the Pentastomida group. Infections with
L. serrata tongue worms are rare in Europe. We describe a
case of ocular linguatulosis in central Europe and provide molecular data on
L. serrata tongue worms.
Linguatula; Pentastomida; anterior chamber; uveitis; 18S ribosomal DNA; tongue worm; parasites; molecular data; Austria; dispatch
A 49-year-old immunocompetent white man had a painful ulcer (1.5 cm in diameter) on the left ventrolateral surface of a grossly enlarged tongue. The ulcer was present for two months. Impaired swallowing resulted in substantial weight loss and fatigue. Histopathologic analysis of a punch biopsy specimen indicated numerous Leishman Donovan bodies within macrophages. A polymerase chain reaction confirmed the presence of L. donovani. Therapy with two cycles of liposomal amphotericin B over a three-month period was administered. Four months after discharge, the ulcer had healed completely and the tongue returned to its normal size and function.
Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is usually treated with metronidazole, however resistance is on the rise. In this study, N-chlorotaurine (NCT), a new endogenous mild active chlorine compound for topical use, killed T. vaginalis in vitro within 15 min of treatment at a concentration of 55 mM (1%), which is well tolerated by human tissue. The activity of NCT was further enhanced by addition of ammonium chloride (NH4Cl). A combination of 5.5 mM (0.1%) NCT plus 19 mM (0.1%) NH4Cl killed 100% of trichomonads within 5 min.
Trichomonas vaginalis; Susceptible; N-Chlorotaurine; Oxidant; In vitro
Between May 2007 and April 2009, 29 falcons with identically localized, yellowish discolored cutaneous lesions in the thigh and lateral body wall region were presented at Abu Dhabi Falcon Hospital. Out of 18 falcons integrated in this study, 16 tested positive to Mycobacterium. avium complex. The 2 negative falcons tested positive in the Mycobacterium genus PCR. Moreover, 1 falcon tested positive to M. avium. paratuberculosis in tissue samples by PCR. In all cases, blood and fecal samples tested negative. In the acid-fast stain, all samples showed the for mycobacteriosis typical rods. Moreover, in 13 samples Acinetobacter baumannii was detected by PCR and proven by DNA sequencing. Clinical features included highly elevated WBCs, heterophilia, lymphocytopenia, monocytosis, severe anemia and weight loss. A. baumannii, a gram-negative bacillus with the ability to integrate foreign DNA, has emerged as one of the major multidrug resistant bacteria. In veterinary medicine, it has so far been detected in dogs, cats, horses and wild birds. To the authors' knowledge, this is the first report of an A. baumannii infection in falcons and of a veterinary Mycobacterium-Acinetobacter coinfection.
Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in A. castellanii is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.
Babesiosis is a tick-transmitted disease of veterinary and medical importance. The first Austrian case of human babesiosis was recently recorded. In the current study, ticks at all life cycle stages (instars), including 853 Ixodes ricinus and 11 Haemaphysalis concinna ticks, from sampling sites throughout Austria were examined for the presence of Babesia spp. by using 18S rRNA gene PCR and sequencing, and the overall mean infection rate was 51.04%. The infection rates for sampling sites were highly variable, ranging from 0% to almost 100%. Different instars and different sexes were infected almost equally. Babesia isolates occurring in Austrian ticks were identified as Babesia divergens, Babesia divergens-like, and Babesia sp. strain DD by sequencing a fragment of the heat shock protein 70 gene and internal transcribed spacer regions 1 and 2. To our knowledge, this is the first investigation of Babesia spp. in Austrian ticks.
We report on an HIV-negative but immunocompromised patient with disseminated acanthamoebiasis, granulomatous amoebic encephalitis, and underlying miliary tuberculosis and tuberculous meningitis. The patient responded favorably to treatment with miltefosine, an alkylphosphocholine. The patient remained well with no signs of infection 2 years after treatment cessation.
Acanthamoeba; granulomatous Acanthamoeba encephalitis (GAE); tuberculosis; immunocompromised non–HIV-infected patient; miltefosine; dispatch
Acanthamoeba spp. are the causative agents of Acanthamoeba keratitis (AK), which mainly occurs in contact lens wearers, and of skin lesions, granulomatous amoebic encephalitis (GAE), and disseminating diseases in the immunocompromised host. AK therapy is complex and irritating for the eye, skin lesions are difficult to treat, and there is no effective treatment for GAE. Therefore, new anti-Acanthamoeba drugs are needed. We investigated the anti-Acanthamoeba activity of N-chlorotaurine (NCT), an endogenous mild antiseptic. It was shown that NCT has amoebicidal qualities, both in phosphate-buffered saline (PBS) and in amoebic culture medium. After 6 h of treatment with 10 mM NCT in PBS, the levels of trophozoites of all strains investigated already showed at least a 2-log reduction. When the trophozoites were treated with 20 mM NCT in culture medium, they showed a 2-log reduction after 24 h. The addition of NH4Cl to NCT led to a faster decrease in the numbers of living cells, if tests were carried out in PBS. A delay of excystation was observed when cysts were treated with 55 mM (1%) NCT in culture medium. A complete failure of excystment was the result of treatment with 1% NCT plus 1% NH4Cl in PBS. Altogether, NCT clearly demonstrated amoebicidal activity at concentrations well tolerated by human tissues and might be useful as a topical drug for the treatment of Acanthamoeba infections. The addition of ammonium chloride can be considered to enhance the activity.
Acanthamoeba amoebae of genotype T2 were identified as the causative agent of Acanthamoeba skin lesions and granulomatous amoebic encephalitis (GAE) in a human immunodeficiency virus-negative patient with underlying tuberculosis. To our knowledge this, is the first case of GAE involving genotype T2.
We identified Onchocerca jakutensis as the causative agent of an unusual human filariasis in a patient with lupus erythematosus. To our knowledge, this is the first case of human infection with O. jakutensis and the first human case of zoonotic onchocercosis involving >1 worm.
Onchocerca jakutensis; zoonosis; filariasis; PCR; paraffin-embedded tissue; lupus erythematosus; dispatch
We describe a 66-year-old woman with therapy-refractory cryoglobulinemia treated with rituximab, plasmapheresis, and steroids; a case of fatal meningoencephalitis caused by Acanthamoeba spp. then developed. Such infections are rare and show an unusually rapid course (possibly related to rituximab).
Acanthamoeba; encephalitis; immunosuppression; rituximab; dispatch
Early identification of Acanthamoeba in cerebrospinal fluid is mandatory to prevent fatal granulomatous amebic encephalitis. In the case presented here amebic trophozoites were detected in a routine cerebrospinal fluid sample. The antibiotic treatment and the apparently low virulence of this isolate were responsible for the benign progression of the infection.
Free-living amoebae of the genus Acanthamoeba are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against Acanthamoeba spp. The in vitro activities of eight different APCs against three Acanthamoeba strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 μM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of Acanthamoeba keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.
Eleven Acanthamoeba isolates, obtained from Acanthamoeba keratitis patients, from contact lens cases of non-Acanthamoeba keratitis patients, from asymptomatic individuals, from necrotic tissue, and from tap water and two reference strains were investigated by morphological, molecular biological, and physiological means in order to discriminate clinically relevant and nonrelevant isolates. All clinically relevant isolates showed Acanthamoeba sp. group II morphology. 18S ribosomal DNA sequencing revealed sequence type T4 to be the most prevalent group among the isolates and also the group recruiting most of the pathogenic strains. Interestingly, within T4 the strains of no clinical relevance clustered together. Moreover, physiological properties appeared to be highly consistent with initial pathogenicity and with sequence clustering. Altogether, the results of our study indicate a correlation between the phylogenetic relationship and pathogenicity.
The free-living, but potentially pathogenic, bacteriovorous amoebae of the genus Acanthamoeba can be easily grown axenically in a laboratory culture. This, however, often leads to considerable losses in virulence, and encystment capacity, and to changes in drug susceptibility. We evaluated potential options for a reactivation of a number of physiological properties, attenuated by prolonged axenic laboratory culture, including encystment potential, protease activity, heat resistance, growth rates and drug susceptibility against N-chlorotaurine (NCT). Toward this end, a strain that had been grown axenically for 10 years was repeatedly passaged on human HEp-2 cell monolayers or treated with 5′-azacytidine (AzaC), a methyltransferase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, in order to uplift epigenetic gene regulation. Culture on human cell monolayers resulted in significantly enhanced encystment potentials and protease activities, and higher susceptibility against NCT, whereas the resistance against heat shock was not altered. Treatment with AzaC/TSA resulted in increased encystment rates and protease activities, indicating the participation of epigenetic mechanisms. However, lowered resistances against heat shock indicate that possible stress responses to AzaC/TSA have to be taken into account. Repeated growth on human cell monolayers appears to be a potential method to reactivate attenuated characteristics in Acanthamoeba.
Acanthamoeba; encystment; protease; axenic culture; N-chlorotaurine