Genotypic diversity of Acanthamoeba strains isolated from Chilean patients with Acanthamoeba keratitis.

BACKGROUND: Acanthamoeba spp. are the causative agents of a severe keratitis occurring mainly in contact lens wearers. The genus comprises more than 24 species that are currently divided into 20 different genotypes (T1-T20) according to sequence variations in the 18S rRNA gene. The objective of this study was to identify the genotypes and sub-genotypes of Acanthamoeba isolates collected at the Parasitology Laboratory of the Public Health Institute of Chile, the only laboratory in the country where Acanthamoeba screening is performed. This is the first report of genotypic identification of clinical isolates of Acanthamoeba in Chile and one of the few in South America. RESULTS: In this study, 114 Acanthamoeba isolates from 76 Acanthamoeba keratitis patients, obtained between 2005-2016, were genotyped. T4 was the predominant genotype; T2 and T11 genotypes, which are scarcely reported worldwide, were also identified in Chilean patients (one and two patients, respectively). This is the first report of T2 and T11 genotypes isolated from Acanthamoeba keratitis patients in South America. It is also the first report of the T2 genotype circulating in this continent. Analysis of the diagnostic fragment 3 region of the 18S rRNA gene showed 24 T4 variants, with a predominance of the sub-genotype T4/A, followed by T4/B, T4/G, T4/C and T4/D. Bayesian analysis revealed three groups among the T4 variants: two well supported groups that included 12 and 7 sub-genotypes, respectively, and a weakly supported group that included 5 sub-genotypes. Most of the predominant T4 sub-genotypes belonged to the same group, which included 71.3% of the patients, while some minority variants lied mainly in the other two clusters. CONCLUSIONS: T2, T4 and T11 genotypes were predominantly isolated from the Acanthamoeba keratitis patients in Chile. Chilean predominant T4 sub-genotypes, which have also been reported worldwide, formed a separate cluster of the minority T4 variants. This study provides useful information about the predominant genotypes and subgenotypes that would be useful in selecting suitable strains to develop immunological and/or molecular diagnostic assays in Chile.

Effectiveness of sampling methods employed for Acanthamoeba keratitis diagnosis by culture.

PURPOSE: This retrospective, observational study was designed to evaluate the effectiveness of the sampling methods commonly used for the collection of corneal scrapes for the diagnosis of Acanthamoeba keratitis (AK) by culture, in terms of their ability to provide a positive result. METHODS: A total of 553 samples from 380 patients with suspected AK received at the Parasitology Section of the Public Health Institute of Chile, between January 2005 and December 2015, were evaluated. A logistic regression model was used to determine the correlation between the culture outcome (positive or negative) and the method for sample collection. The year of sample collection was also included in the analysis as a confounding variable. RESULTS: Three hundred and sixty-five samples (27%) from 122 patients (32.1%) were positive by culture. The distribution of sample types was as follows: 142 corneal scrapes collected using a modified bezel needle (a novel method developed by a team of Chilean corneologists), 176 corneal scrapes obtained using a scalpel, 50 corneal biopsies, 30 corneal swabs, and 155 non-biological materials including contact lens and its paraphernalia. Biopsy provided the highest likelihood ratio for a positive result by culture (1.89), followed by non-biological materials (1.10) and corneal scrapes obtained using a modified needle (1.00). The lowest likelihood ratio was estimated for corneal scrapes obtained using a scalpel (0.88) and cotton swabs (0.78). CONCLUSION: Apart from biopsy, optimum corneal samples for the improved diagnosis of AK can be obtained using a modified bezel needle instead of a scalpel, while cotton swabs are not recommended.

Draft Genome Sequence of Bacillus sp. Strain K2I17, Isolated from the Rhizosphere of Deschampsia antarctica Desv.

We present here the draft genome sequence of Bacillus sp. strain K2I17, which was isolated from the rhizosphere of Deschampsia antarctica Desv. The genomic sequence contained 6,113,341 bp. This genome provides insights into the possible new biomedical and biotechnical applications of this specific Antarctic bacterium.

Draft Genome Sequence of a Copper-Resistant Marine Bacterium, Pantoea agglomerans Strain LMAE-2, a Bacterial Strain with Potential Use in Bioremediation.

Pantoea agglomerans LMAE-2 was isolated from seabed sediment moderately contaminated with Cu(2+) Here, we report its draft genome sequence, which has a size of 4.98 Mb. The presence of cop genes related with copper homeostasis in its genome may explain the resistance and strengthen its potential for use as bioremediation agent.

The MF6p/FhHDM-1 major antigen secreted by the trematode parasite Fasciola hepatica is a heme-binding protein.

Blood-feeding parasites have developed biochemical mechanisms to control heme intake and detoxification. Here we show that a major antigen secreted by Fasciola hepatica, previously reported as MF6p, of unknown function (gb|CCA61804.1), and as FhHDM-1, considered to be a helminth defense molecule belonging to the family of cathelicidin-like proteins (gb|ADZ24001.1), is in fact a heme-binding protein. The heme-binding nature of the MF6p/FhHDM-1 protein was revealed in two independent experiments: (i) immunopurification of the secreted protein·heme complexes with mAb MF6 and subsequent analysis by C8 reversed-phase HPLC and MS/MS spectrometry and (ii) analysis of the binding ability of the synthetic protein to hemin in vitro. By immunohistochemistry analysis, we have observed that MF6p/FhHDM-1 is produced by parenchymal cells and transported to other tissues (e.g. vitellaria and testis). Interestingly, MF6p/FhHDM-1 is absent both in the intestinal cells and in the lumen of cecum, but it can be released through the tegumental surface to the external medium, where it binds to free heme molecules regurgitated by the parasite after hemoglobin digestion. Proteins that are close analogs of the Fasciola MF6p/FhHDM-1 are present in other trematodes, including Clonorchis, Opistorchis, Paragonimus, Schistosoma, and Dicrocoelium. Using UV-visible spectroscopy and immunoprecipitation techniques, we observed that synthetic MF6p/FhHDM-1 binds to hemin with 1:1 stoichiometry and an apparent Kd of 1.14 × 10(-6) M(-1). We also demonstrated that formation of synthetic MF6p/FhHDM-1·hemin complexes inhibited hemin degradation by hydrogen peroxide and hemin peroxidase-like activity in vitro. Our results suggest that MF6p/FhHDM-1 may be involved in heme homeostasis in trematodes.

Closure of a lateral baffle leak in a Fontan patient with an amplatzer PDA II AS plug.

Cyanosis after Fontan surgery or surgery for total cavopulmonary connection (TCPC), due to different types of communications (fenestration, venovenous collaterals or fistula), is not uncommon. We present the case of an 8-year-old girl presenting with increasing cyanosis during exercise 4 years after an intracardiac TCPC with lateral tunnel. Angiography showed a fistulous trajectory originating at the superior vena cava towards the base of the right atrial appendage. Due to the difficult anatomy in our patient, closure with conventional devices was not possible. We finally used a new Amplatzer PDA II AS plug to successfully close the fistula. In conclusion, closure of lateral baffle leak and device choice in case of difficult anatomy can be cumbersome. The new PDA II AS type plug can offer an elegant alternative for successful closure of some fistula.

Development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fasciolosis.

BACKGROUND: Human fasciolosis is a re-emerging disease worldwide and is caused by species of the genus Fasciola (F. hepatica and F. gigantica). Human fasciolosis can be diagnosed by classical coprological techniques, such as the Kato-Katz test, to reveal parasite eggs in faeces. However, although 100% specific, these methods are generally not adequate for detection of acute infections, ectopic infections, or infections with low number of parasites. In such cases immunological methods may be a good alternative and are recommended for use in major hospitals where trained personnel are available, although they are not usually implemented for individual testing. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a new lateral flow test (SeroFluke) for the serodiagnosis of human fasciolosis. The new test was constructed with a recombinant cathepsin L1 from F. hepatica, and uses protein A and mAb MM3 as detector reagents in the test and control lines, respectively. In comparison with an ELISA test (MM3-SERO) the SeroFluke test showed maximal specificity and sensitivity and can be used with serum or whole blood samples. CONCLUSIONS/SIGNIFICANCE: The new test can be used in major hospitals in hypoendemic countries as well as in endemic/hyperendemic regions where point-of-care testing is required.

Association between anti-F. hepatica antibody levels in milk and production losses in dairy cows.

This study was conducted to determine: (1) the associations between anti-Fasciola hepatica antibody levels in milk and some productive and reproductive parameters in dairy cattle, and (2) the threshold antibody level associated with loss of productivity, at both herd and individual level. Anti-F. hepatica antibodies were analysed by MM3-SERO ELISA in milk samples from the bulk tanks of 490 dairy farms and from 686 lactating cows. The results of general linear model analysis revealed a significant (P<0.05) negative association between the F. hepatica infection status at herd level, determined by analysis of specific antibodies in bulk tank milk, and the average herd milk production. Highly positive herds (MM3-SERO ELISA result>0.405) produced an average of 1.5 kg milk/cow per day less than the negative herds. At cow-level, the mixed model analysis also revealed a significant (P<0.05) association between anti-F. hepatica antibody levels and milk yield. A significant (P<0.05) average reduction of 2 kg milk/day was observed in cows with highly positive ELISA results (>0.762) in relation to cows with negative results. The results of the study led us to conclude that MM3-SERO ELISA is a powerful tool that can be successfully applied, if appropriate "economic thresholds" are established, to identify herds and cows suffering from milk production losses associated with natural infection by F. hepatica.

MISS-Prot: web server for self/non-self discrimination of protein residue networks in parasites; theory and experiments in Fasciola peptides and Anisakis allergens.

Infections caused by human parasites (HPs) affect the poorest 500 million people worldwide but chemotherapy has become expensive, toxic, and/or less effective due to drug resistance. On the other hand, many 3D structures in Protein Data Bank (PDB) remain without function annotation. We need theoretical models to quickly predict biologically relevant Parasite Self Proteins (PSP), which are expressed differentially in a given parasite and are dissimilar to proteins expressed in other parasites and have a high probability to become new vaccines (unique sequence) or drug targets (unique 3D structure). We present herein a model for PSPs in eight different HPs (Ascaris, Entamoeba, Fasciola, Giardia, Leishmania, Plasmodium, Trypanosoma, and Toxoplasma) with 90% accuracy for 15 341 training and validation cases. The model combines protein residue networks, Markov Chain Models (MCM) and Artificial Neural Networks (ANN). The input parameters are the spectral moments of the Markov transition matrix for electrostatic interactions associated with the protein residue complex network calculated with the MARCH-INSIDE software. We implemented this model in a new web-server called MISS-Prot (MARCH-INSIDE Scores for Self-Proteins). MISS-Prot was programmed using PHP/HTML/Python and MARCH-INSIDE routines and is freely available at: . This server is easy to use by non-experts in Bioinformatics who can carry out automatic online upload and prediction with 3D structures deposited at PDB (mode 1). We can also study outcomes of Peptide Mass Fingerprinting (PMFs) and MS/MS for query proteins with unknown 3D structures (mode 2). We illustrated the use of MISS-Prot in experimental and/or theoretical studies of peptides from Fasciola hepatica cathepsin proteases or present on 10 Anisakis simplex allergens (Ani s 1 to Ani s 10). In doing so, we combined electrophoresis (1DE), MALDI-TOF Mass Spectroscopy, and MASCOT to seek sequences, Molecular Mechanics + Molecular Dynamics (MM/MD) to generate 3D structures and MISS-Prot to predict PSP scores. MISS-Prot also allows the prediction of PSP proteins in 16 additional species including parasite hosts, fungi pathogens, disease transmission vectors, and biotechnologically relevant organisms.

Using entropy of drug and protein graphs to predict FDA drug-target network: theoretic-experimental study of MAO inhibitors and hemoglobin peptides from Fasciola hepatica.

There are many drugs described with very different affinity to a large number of receptors. In this work, we selected Drug-Target pairs (DTPs/nDTPs) of drugs with high affinity/non-affinity for different targets like proteins. Quantitative Structure-Activity Relationships (QSAR) models become a very useful tool in this context to substantially reduce time and resources consuming experiments. Unfortunately, most QSAR models predict activity against only one protein. To solve this problem, we developed here a multi-target QSAR (mt-QSAR) classifier using the MARCH-INSIDE technique to calculate structural parameters of drug and target plus one Artificial Neuronal Network (ANN) to seek the model. The best ANN model found is a Multi-Layer Perceptron (MLP) with profile MLP 32:32-15-1:1. This MLP classifies correctly 623 out of 678 DTPs (Sensitivity = 91.89%) and 2995 out of 3234 nDTPs (Specificity = 92.61%), corresponding to training Accuracy = 92.48%. The validation of the model was carried out by means of external predicting series. The model classifies correctly 313 out of 338 DTPs (Sensitivity = 92.60%) and 1411 out of 1534 nDTP (Specificity = 91.98%) in validation series, corresponding to total Accuracy = 92.09% for validation series (Predictability). This model favorably compares with other LDA and ANN models developed in this work and Machine Learning classifiers published before to address the same problem in different aspects. These mt-QSARs offer also a good opportunity to construct drug-protein Complex Networks (CNs) that can be used to explore large and complex drug-protein receptors databases. Finally, we illustrated two practical uses of this model with two different experiments. In experiment 1, we report prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of 10 rasagiline derivatives promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, SEC and 1DE sample preparation, MALDI-TOF MS and MS/MS analysis, MASCOT search, MM/MD 3D structure modeling, and QSAR prediction for different peptides of hemoglobin found in the proteome of the human parasite Fasciola hepatica; which is promising for anti-parasite drug targets discovery.

MM3-ELISA detection of Fasciola hepatica coproantigens in preserved human stool samples.

In this study, we evaluate the MM3-COPRO method for detection of Fasciola coproantigens in human fecal samples, and the usefulness of a new preservative/diluent, CoproGuard, developed for preservation of Fasciola coproantigens. The MM3-COPRO assay was evaluated with 213 samples from healthy patients, 30 Fasciola positive fecal samples (according to the Kato-Katz method), and 83 samples from patients with other parasitic infections. All Fasciola positive specimens were detected with the MM3-COPRO assay (100% sensitivity) and there was no cross-reactivity with other common parasites present in the clinical specimens analyzed (100% specificity). The use of CoproGuard enhanced coproantigen extraction without affecting the detection limit of the assay, and the antigenicity of Fasciola coproantigens in fecal samples stored at 37 degrees C was retained throughout the entire observation period (120 days). We concluded that the MM3-COPRO ELISA combined with the use of CoproGuard may be a very useful tool for the diagnosis of human fascioliasis.

MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica.

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4-7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3-6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33-66 adults), while it was for F. gigantica infection (burden of 17-69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.