Protein Phosphatase PP2C Identification in Entamoeba spp.

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

Evaluation of molecular characterization and phylogeny for quantification of Acanthamoeba and Naegleria fowleri in various water sources, Turkey.

Free-living amoeba (FLA) is widely distributed in the natural environment. Since these amoebae are widely found in various waters, they pose an important public health problem. The aim of this study was to detect the presence of Acanthamoeba, B. mandrillaris, and N. fowleri in various water resources by qPCR in Izmir, Turkey. A total of (n = 27) 18.24% Acanthamoeba and (n = 4) 2.7% N. fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers. The resulting concentrations varied in various water samples for Acanthamoeba in the range of 3.2x105-1.4x102 plasmid copies/l and for N. fowleri in the range of 8x103-11x102 plasmid copies/l. The highest concentration of Acanthamoeba and N. fowleri was found in seawater and damp samples respectively. All 27 Acanthamoeba isolates were identified in genotype level based on the 18S rRNA gene as T4 (51.85%), T5 (22.22%), T2 (14.81%) and T15 (11.11%). The four positive N. fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers. Four N. fowleri isolates were genotyped (three isolate as type 2 and one isolate as type 5) and detected for the first time from water sources in Turkey. Acanthamoeba and N. fowleri genotypes found in many natural environments are straightly related to human populations to have pathogenic potentials that may pose a risk to human health. Public health professionals should raise awareness on this issue, and public awareness education should be provided by the assistance of civil authorities. To the best of our knowledge, this is the first study on the quantitative detection and distribution of Acanthamoeba and N. fowleri genotypes in various water sources in Turkey.

A Targeted Mass Spectrometric Analysis Reveals the Presence of a Reduced but Dynamic Sphingolipid Metabolic Pathway in an Ancient Protozoan, Giardia lamblia.

Giardia lamblia, a single-celled eukaryote, colonizes and thrives in the small intestine of humans. Because of its compact and reduced genome, Giardia has adapted a "minimalistic" life style, as it becomes dependent on available resources of the small intestine. Because Giardia expresses fewer sphingolipid (SL) genes-and glycosphingolipids are critical for encystation-we investigated the SL metabolic cycle in this parasite. A tandem mass spectrometry (MS/MS) analysis reveals that major SLs in Giardia include sphingomyelins, sphingoid bases, ceramides, and glycosylceramides. Many of these lipids are obtained by Giardia from the growth medium, remodeled at their fatty acyl chains and end up in the spent medium. For instance, ceramide-1-phosphate, a proinflammatory molecule that is not present in the culture medium, is generated from sphingosine (abundant in the culture medium) possibly by remodeling reactions. It is then subsequently released into the spent medium. Thus, the secretion of ceramide-1-phospate and other SL derivatives by Giardia could be associated with inflammatory bowel disease observed in acute giardiasis. Additionally, we found that the levels of SLs increase in encysting Giardia and are differentially regulated throughout the encystation cycle. We propose that SL metabolism is important for this parasite and, could serve as potential targets for developing novel anti-giardial agents.

Primary amoebic meningoencephalitis in an infant.

Primary amoebic meningoencephalitis is rare but fatal disease encountered in immunocompetent individuals. Here, we present a case of a previously healthy 8-month-old female child, who presented with features of meningoencephalitis of 2 days' duration. Rapidly moving trophozoites of amoeba were observed in cerebrospinal fluid, which were confirmed to be Naegleria fowleri on polymerase chain reaction. Broad-spectrum antimicrobial therapy with ceftriaxone, vancomycin, amphotericin B and acyclovir was initiated. However, the patient deteriorated and left the hospital against medical advice. The isolation of N. fowleri in this case demands for increased awareness for prompt diagnosis and management in view of its high mortality.

Entamoeba chiangraiensis n. sp. (Amoebozoa: Entamoebidae) isolated from the gut of Asian swamp eel (Monopterus albus) in northern Thailand.

The genus Entamoeba comprises mostly gut parasites and commensals of invertebrate and vertebrate animals including humans. Herein, we report a new species of Entamoeba isolated from the gut of Asian swamp eels (Monopterus albus) in northern Thailand. Morphologically, the trophozoite is elongated and has a single prominent pseudopodium with no clear uroid. The trophozoite is actively motile, 30-50 µm in length and 9-13 µm in width. Observed cysts were uninucleate, ranging in size from 10 to 17.5 µm in diameter. Chromatin forms a fine, even lining along the inner nuclear membrane. Fine radial spokes join the karyosome to peripheral chromatin. Size, host and nucleus morphology set our organism apart from other members of the genus reported from fish. The SSU rRNA gene sequences of the new isolates are the first molecular data of an Entamoeba species from fish. Phylogenetic analysis places the new organism as sister to Entamoeba invadens. Based on the distinct morphology and SSU rRNA gene sequence we describe it as a new species, Entamoeba chiangraiensis.

Lophomonas blattarum Associated Broncho-Pulmonary Infection After Immunotherapy: A Case Report and A Smart-phone Based Video of Trophozoite

The frequency of bronchopulmonary protozoan infections has raised due to increased number of immunosuppressed patients in recent years. One of them is Lophomonas blattarum which is a multi-flagellated protozoan parasite of termites and several cockroach species. The drug regimens commonly used in bronchopulmonary infections are not effective against L. blattarum. Therefore, rapid and accurate diagnosis of L. blattarum infection is of great importance in the treatment success. The laboratory diagnosis of L. blattarum infection is made on the basis of observation of the characteristic trophozoite in various samples. It is of a great importance to distinguish the protozoon from ciliated respiratory epithelium to avoid wrong positivity. The presented case developed an acute respiratory distress syndrome a short while after taking nivolumab immunotherapy. The morphological features of L. blattarum were demonstrated by examining the bronchoalveolar lavage fluid of the patient under light microscopy. Additionally, URL (https://youtu.be/EQIAsFl6AJY) of a smart-phone based video of trophozoite of this patient was added into this report.

Another case of coincidental Giardia infection and pancreatic cancer.

Until now, few cases of coincidental giardiasis and pancreatic tumors have been described. Among these cases, three described giardiasis cases coincided with confirmed pancreatic cancer. We present another case of Giardia infection coexisting with pancreatic cancer in a 67-year-old man who suffered from stenosis of the distal ductus choledochus combined with a hypoechoic mass in the head of the pancreas. The diagnostic conclusion of suspicious adenocarcinoma was based on endoscopic ultrasound fine-needle aspiration (EUS-FNA) biopsy and confirmed by a partial duodenopancreatectomy. On bloody cytology smears prepared from the EUS-FNA specimen, trophozoites of Giardia intestinalis accompanying an inflammatory background and features that fulfilled the morphological criteria of malignancy were observed. In histological sections from the duodenopancreatectomy specimens, the parasites were observed attached to the epithelium, but individual Giardia parasites were also observed beneath the epithelial lining. According to conventional genotyping, the infecting Giardia belonged to sub-assemblage AII.

Malaria Detection by Third-Harmonic Generation Image Scanning Cytometry.

Despite global efforts aimed at its elimination, malaria is still a significant health concern in many countries across the world. The disease is caused by blood-borne parasites, Plasmodium species, and is transmitted by female Anopheles mosquitoes and presents with generic febrile symptoms that are challenging to diagnose clinically. To adequately tackle this issue, an effective detection method is required for screening potential malaria patients for infection. To this day, the gold standard for malaria detection remains basic light microscopy of Giemsa-stained patient blood smears to first enable detection and manual counting to determine the parasite density by a microscopist. While effective at detecting parasites, this method requires both significant time and skilled personnel. As an alternate approach, we propose a new malaria detection method that we call third-harmonic generation image scanning cytometry (THGISC) based on the combination of third-harmonic generation imaging, high-speed motorized scanning, and automated software processing. Third-harmonic generation (THG) is a nonlinear optical process in which the frequency of incident photons is tripled within the sample material. We have previously demonstrated that hemozoin, a metabolic byproduct of the malaria parasite, presents a significant THG signal. We now present a practical approach that uses the selectivity of this contrast mechanism to perform label-free image scanning cytometry of patient blood smears for automated malaria detection. In this work, we applied this technique to lab-cultured parasites and parasites in whole blood obtained from malaria patients. We also compared its effectiveness to parasite counts obtained by classical methods. The ability to easily and rapidly determine parasitemia by THG offers potential not only for the easy confirmation of malaria diagnoses following symptoms, but also the tracking of treatment progress in existing patients, potentially allowing physicians to adjust medication and dosage for each individual.

Identification of free-living amoebae isolated from tap water in Istanbul, Turkey.

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul.

Effect of Riboflavin/Rose Bengal-Mediated PACK-CXL on Acanthamoeba Trophozoites and Cysts in Vitro.

PURPOSE: To evaluate the antiamoebic properties of photo-activated chromophore for keratitis (PACK)-corneal cross-linking (CXL) (PACK-CXL), in combination with riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%), for treatment of Acanthamoeba trophozoites and cysts. MATERIALS AND METHODS: Cultures of Acanthamoeba castellanii were grown in a fluid medium at a concentration of 2.7 × 105 cell/ml. PACK-CXL was used on A. castellani cells in combination with either riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%). Riboflavin-containing wells were irradiated with ultraviolet-A (UVA) light (365-nm wavelength). Rose bengal-containing wells were irradiated with green light (523-nm wavelength). A power density of 9 mW/cm2 for 10 min and total irradiation dose of 5.4 J/cm2 was used for both riboflavin and rose bengal. After UVA and green light irradiation, cell viabilities were evaluated, and percentage of dead cells calculated. RESULTS: No significant amoebicidal activity was observed following PACK-CXL/riboflavin at either concentration. PACK-CXL/rose bengal, however, was observed to be highly effective in eradicating Acanthamoeba cells at either concentration, with no significant difference observed between the two concentrations. The percentage of dead cells was 63% following treatment at either rose bengal concentration. CONCLUSION: PACK-CXL with rose bengal demonstrated pronounced antiamoebic activity against A.castellanii. Further in vitro and in vivo studies are needed to confirm this finding.

Balantioides coli: morphological and ultrastructural characteristics of pig and non-human primate isolates.

Balantioides coli is a ciliated protozoon that inhabits the intestine of pigs, non-human primates and humans. Light microscopy studies have described over 50 species of the genus Balantioides but their validity is in doubt. Due to the limited information about this genus, this study is aimed to identify morphological characteristics of Balantioides coli isolated using fluorescence microscopy and both scanning (SEM) and transmission electron microscopy (TEM). Trophozoites isolated from the feces of pig and macaque were washed and subjected to centrifugation. These cells were fixed with paraformaldehyde for immunofluorescence. Other aliquots of these trophozoites were fixed with glutaraldehyde, post fixed with osmium tetroxide and processed for SEM and TEM. Immunofluorescence studies revealed microtubules with a longitudinal distribution to the main axis of the parasite and in the constitution of cilia. SEM demonstrated a high concentration of cilia covering the oral apparatus and a poor presence of such structures in cytopyge. TEM revealed in the plasma membrane, several associated structures were observed to delineate the cellular cortex and mucocysts. The cytoskeleton of the oral region was observed in detail and had an organization pattern consisting of microtubules, which formed files and nematodesmal networks. Organelles such as hydrogenosomes like and peroxisomes were observed close to the cortex. Macronuclei were observed, but structures that were consistent with micronuclei were not identified. Ultrastructural morphological analysis of isolates confirms its similarity to Balantioides coli. In this study were identified structures that had not yet been described, such as hydrogenosomes like and cytoskeletal structures.

Molecular Phylogenetic Positions and Ultrastructure of Marine Gregarines (Apicomplexa) Cuspisella ishikariensis n. gen., n. sp. and Loxomorpha cf. harmothoe from Western Pacific scaleworms (Polynoidae).

Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts.

In Vitro Evaluation of Adhesion of Two Acanthamoeba Strains to Cosmetic Contact Lenses.

PURPOSE: To evaluate the factors affecting the adhesion of Acanthamoeba trophozoites to the surface of cosmetic contact lenses (CCLs). METHODS: Acanthamoeba castellanii and A. hatchetti trophozoites were inoculated onto CCLs (hema copolymer [HM] [38.5% H2O], phemfilcon [PF] [55% H2O], polymacon [PM] [38% H2O], polyhema [PH] [%42 H2O], and hema [HM55] [55% H2O]), and the number of trophozoites adhered to the lens surfaces was assessed over time, that is, at 15 min, 1, and 24 hr. In addition, scanning electron microscopy (SEM) analysis of the lens surfaces was performed to evaluate the effect of lens surface topology on adhesion. RESULTS: The number of amoeba adhered to the contact lens surface was found lower with PF and PH production materials, than lenses with HM, PM, and HM55 production materials (P<0.05). No significant difference was detected in amoebic strains adhered in all the contact lens types (P>0.05). No significant difference was found on average amoeba adhesion between contact lenses with hema production material but with different water contents (45%, 55%), to see the effect of water content on amoebic adhesion (P>0.05). As a result of SEM analysis, surface topology showed no effect on adhesion. CONCLUSION: (1) Chemical composition of lenses seemed to be mostly responsible for the adhesion of Acanthamoeba. (2) Different numbers of trophozoites, obtained after the adhesion experiment, could also indicate that adherence capacity can also differ among Acanthamoeba species.

Canine amoebic meningoencephalitis due to Balamuthia mandrillaris.

A 1-year-old Siberian Husky dog with acute-onset of seizures, recumbency, paddling, and muscular fasciculations was autopsied. A locally extensive hemorrhagic and malacic focus was noted in the right cerebral frontal cortex, and severe necrotizing and hemorrhagic, neutrophilic meningoencephalitis was diagnosed microscopically. Amoebic trophozoites and cysts were identified within the affected cerebral parenchyma and confirmed by indirect immunofluorescence assay and real-time PCR as Balamuthia mandrillaris. B. mandrillaris is found in soil and water and the infection has been reported in both immunocompromised and immunocompetent humans and rarely in the dog.

Primary care challenges of an obscure case of "Alice in Wonderland" syndrome in a patient with severe malaria in a resource-constrained setting: a case report.

BACKGROUND: "Alice in Wonderland" syndrome (AIWS) is a rare neurological abnormality characterized by distortions of visual perceptions, body schema and experience of time. AIWS has been reported in patients with various infections such as infectious mononucleosis, H1N1 influenza, Cytomegalovirus encephalitis, and typhoid encephalopathy. However, AIWS occurring in a patient with severe malaria is less familiar and could pose serious primary care challenges in a low-income context. CASE PRESENTATION: A 9-year-old male of black African ethnicity was brought by his parents to our primary care hospital because for 2 days he had been experiencing intermittent sudden perceptions of his parents' heads and objects around him either "shrinking" or "expanding". The visual perceptions were usually brief and resolved spontaneously. One week prior to the onset of the visual problem, he had developed an intermittent high grade fever that was associated with other severe constitutional symptoms. Based on the historical and clinical data that were acquired, severe malaria was suspected and this was confirmed by hyperparasitaemia on blood film analysis. The patient was treated with quinine for 10 days. Apart from a single episode of generalized tonic-clonic seizures that was observed on the first day of treatment, the overall clinical progress was good. The visual illusions completely resolved and no further abnormalities were recorded during 3 months of follow-up. CONCLUSION: Symptoms of AIWS usually resolve spontaneously or after treatment of an underlying cause. In our case, the successful treatment of severe malaria coincided with a complete regression of AIWS whose aetiology was poorly-elucidated given the resource constraints. In any case, the good outcome of our patient aligns with previous reports on acute AIWS that highlight a limited need for excessive investigation and treatment modalities which are, in passing, predominantly unaffordable in resource-limited primary care settings.

Case Series of Naegleria fowleri Primary Ameobic Meningoencephalitis from Karachi, Pakistan.

Naegleria fowleri causes primary amoebic meningoencephalitis (PAM) which is almost always fatal. Naegleria fowleri is waterborne, and its infections are usually associated with aquatic activities but it can also be transmitted via the domestic water supply. An increasing number of N. fowleri cases have been reported from Pakistan. Improved methods for diagnosis are required. We report the utility of polymerase chain reaction (PCR) for the diagnosis of N. fowleri in patients suspected of PAM. One hundred and sixteen cases suspected of having PAM were examined. Cerebrospinal fluid (CSF) specimens were tested at the Aga Khan University Hospital, Karachi. Nineteen CSF specimens were positive for N. fowleri using PCR. Naegleria fowleri positive patients had a median age of 28 years and were 84% male and 16% female. Overall, CSF wet preparation microscopy was performed in 85 (73%) cases and identified that seven specimens were positive for motile trophozoites. The CSF wet preparation results were available for 15 of the 19 N. fowleri PCR positive CSF samples; seven (40%) wet preparations were positive. Our data highlight the threat of N. fowleri infection as a cause of PAM. It also emphasizes the utility of the PCR-based diagnosis of the amoeba for early diagnosis and management of the disease.

Molecular identification of Vermamoeba vermiformis from freshwater fish in lake Taal, Philippines.

Free Living Amoebae (FLA) are considered ubiquitous. FLAs may infect various biological organisms which act as reservoir hosts. Infected freshwater fishes can pose a public health concern due to possible human consumption. This study aims to identify possible pathogenic FLAs present in freshwater fishes. Seventy five (75) Oreochromis niloticus were studied for the presence of FLAs. Fish organs were suspended in physiologic saline pelleted and cultured in non-nutrient agar (NNA) lawned with Escherichia coli and were incubated in 33 °C for 14 days. Eighteen (18) fish gills and nineteen (19) fish intestine samples presented with positive growth. Trophozoites and cystic stages of FLAs were subcultured until homogenous growth was achieved. Cells were harvested from cultured plates and DNA was extracted using Chelex resin. DNA was subjected to polymerase chain reaction using universal forward primer EukA and reverse primer EukB targeting the 18s RNA. Of the 37 plates that presented with positive amoebic growth, 9 samples showed the presence of DNAs and were sent for further purification and sequencing. Basic Local Alignment Search Tool (BLAST) results showed that protists isolated from fish organs in Lake Taal include: Eocercomonas (HM536152), Colpoda steinii (KJ607915) and Vermamoeba vermiformis (KC161965). The results showed that fresh-water fishes can harbour FLAs in the gut. It is proposed that freshwater reservoirs utilized for aquaculture be monitored for the presence of FLAs and extensive study be conducted on the pathogenicity of bacterial endosymbionts and infecting viruses to its mammalian and non-mammalian host.

A case of amebiasis with negative serologic markers that caused intra-abdominal abscess.

A 23-year-old Japanese woman presented with abdominal distention following fever, diarrhea, and abdominal pain during a stay in Taiwan. Serology for the detection of amebic-antibodies and stool microscopic examination were both negative. A computed tomography scan showed a 13 cm diameter abscess spreading from the lower abdominal wall to the pelvic retroperitoneal space. Needle aspiration of the abscess was done under computed tomography guidance, and microscopy of the aspirated fluid revealed trophozoites of Entamoeba. The patient was diagnosed as amebiasis with negative serologic markers that caused intra-abdominal abscess. Intravenous metronidazole treatment for two weeks did not result in any improvement of the abscess. After irrigation and drainage of the abscess, her symptoms resolved. This case report highlights that amebiasis should be considered when indicated by patient history, including travelers returning from endemic areas, and that further evaluation is necessary for diagnosis, even if the serology and stool test are negative.