Genetic Variability of the Internal Transcribed Spacer and Pyruvate:Ferredoxin Oxidoreductase Partial Gene of Trichomonas vaginalis from Female Patients.

In the present study, we evaluated the genetic variability of the internal transcribed spacer (ITS) region and the pyruvate:ferredoxin oxidoreductase (pfor) A gene of Trichomonas vaginalis from female patients and its possible implications in the host-parasite relationship. Phylogenetic and genetics of populations analyses were performed by analyzing sequences of the ITS region and partial pfor A gene of clinical samples with T. vaginalis, as previously documented. Alignments of protein sequences and prediction of three-dimensional structure were also performed. Although no correlation between the main clinical characteristics of the samples and the results of phylogeny was found, a median-joining analysis of ITS haplotypes showed two main clusters. Also, pfor A, due to its phylogenetic divergence, could be used as a marker to confirm the genus and species of trichomonads. Alignment of protein sequences and prediction of three-dimensional structure showed that PFOR A had a highly conserved structure with two synonymous mutations in the PFOR domain, substituting a V for a G or a S for a P. Our results suggest that the role of genetic variability of PFOR and ITS may not be significant in the symptomatology of this pathogen; however, their utility as genus and species markers in trichomonads is promising.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3.

Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1-3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS-PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1-3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3

ABSTRACT Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1-3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS-PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1-3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

An Unusual Case of Extra-Enteric Blastocystosis in the Uterine Cervix.

Extra-enteric infections by Blastocystis spp. have rarely been documented. Here, we report a case of extra-enteric blastocystosis in a patient with minimal cervicitis symptoms. A 47-year-old Hispanic female patient was attended in a primary health centre in Michoacan state, Mexico, for her routine gynaecological medical examination. As only symptom, she referred to a slight vaginal itching. The presence of several vacuolar-stages of Blastocystis spp. were identified by Papanicolaou staining; molecular identification was attempted by culture-PCR sequencing of a region of 18S gene from cervical and faecal samples obtained 2 months after cytological examination, even when patient declared that she tried self-medicating with vaginal ovules. Blastocystis ST1 was identified only in the faecal sample. The presence of Blastocystis spp. in the cervix of a patient with scarce symptomatology, demonstrates the extraordinary flexibility of this microorganism to adapt to new environments and niches.

Interaction between human mucins and parasite glycoproteins: the role of lectins and glycosidases in colonization by intestinal protozoa.

Intestinal mucins are the first line of defense against microorganisms. Although knowledge about the mechanisms involved in the establishment of intestinal protozoa is limited, there is evidence that these parasites produce lectin-like molecules and glycosidases, that exert both, constitutive and secretory functions, promoting the establishment of these microorganisms. In the present review, we analyse the main interactions between mucins of the host intestine and the four main protozoan parasites in humans and their implications in intestinal colonization. There are lectin-like molecules that contain complex oligosaccharide structures and N-acetylglucosamine (GlcNAc), mannose and sialic acid as main components, which are excreted/secreted by Giardia intestinalis, and recognized by the host using mannose-binding lectins (MBL). Entamoeba histolytica and Cryptosporidium spp. express the lectin galactose/N-acetyl-D-galactosamine, which facilitates their adhesion to cells. In Cryptosporidium, the glycoproteins gp30, gp40/15 and gp900 and the glycoprotein lectin CpClec are involved in protozoan adhesion to intestinal cells, forming an adhesion-attack complex. G. intestinalis and E. histolytica can also produce glycosidases such as ß-N-acetyl-D-glucosaminidase, α-d-glucosidase, ß-d-galactosidase, ß-l-fucosidase, α-N-acetyl-d-galactosaminidase and ß-mannosidase. In Blastocystis, α-D-mannose, α-D-glucose, GlcNAc, α-D-fucose, chitin and sialic acid that have been identified on their surface. Fucosidases, hexosaminidases and polygalacturonases, which may be involved in the mucin degradation process, have also been described in the Blastocystis secretoma. Similarly, symbiotic coexistence with the intestinal microbiota promotes the survival of parasites facilitating cell invasion and nutrients obtention. Furthermore, it is necessary to identify and characterize more glycosidases, which have been only partially described by in silico analyses of the parasite genome.

Blastocystis Isolates from Patients with Irritable Bowel Syndrome and from Asymptomatic Carriers Exhibit Similar Parasitological Loads, but Significantly Different Generation Times and Genetic Variability across Multiple Subtypes.

Blastocystis spp is a common intestinal parasite of humans and animals that has been associated to the etiology of irritable bowel syndrome (IBS); however, some studies have not found this association. Furthermore, many biological features of Blastocystis are little known. The objective of present study was to assess the generation times of Blastocystis cultures, from IBS patients and from asymptomatic carriers. A total of 100 isolates were obtained from 50 IBS patients and from 50 asymptomatic carriers. Up to 50 mg of feces from each participant were cultured in Barret's and in Pavlova's media during 48 h. Initial and final parasitological load were measured by microscopy and by quantitative PCR. Amplicons were purified, sequenced and submitted to GenBank; sequences were analysed for genetic diversity and a Bayesian inference allowed identifying genetic subtypes (ST). Generation times for Blastocystis isolates in both media, based on microscopic measures and molecular assays, were calculated. The clinical symptoms of IBS patients and distribution of Blastocystis ST 1, 2 and 3 in both groups was comparable to previous reports. Interestingly, the group of cases showed scarce mean nucleotide diversity (π) as compared to the control group (0.011±0.016 and 0.118±0.177, respectively), whilst high gene flow and small genetic differentiation indexes between different ST were found. Besides, Tajima's D test showed negative values for ST1-ST3. No statistical differences regarding parasitological load between cases and controls in both media, as searched by microscopy and by qPCR, were detected except that parasites grew faster in Barret's than in Pavlova's medium. Interestingly, slow growth of isolates recovered from cases in comparison to those of controls was observed (p<0.05). We propose that generation times of Blastocystis might be easily affected by intestinal environmental changes due to IBS probably because virulent strains with slow growth may be selected, reducing their genetic variability.

Plasma cytokine levels and cytokine gene polymorphisms in Mexican patients during the influenza pandemic A(H1N1)pdm09.

BACKGROUND: In Mexico, the initial severe cases of the 2009 influenza pandemic virus A (H1N1) [A(H1N1)pdm09] were detected in early March. The immune mechanisms associated with the severe pneumonia caused by infection with this new virus have not been completely elucidated. Polymorphisms in interleukin genes have previously been associated with susceptibility to infectious diseases due to their influence on cytokine production. OBJECTIVES: The present case-control study was performed to compare several immunologic and genetic parameters of patients and controls during the initial phase of the pandemic. STUDY DESIGN: Sixty-five patients who were hospitalized due to infection with the influenza A(H1N1)pdm09 virus and 46 healthy controls were studied. A hemagglutination inhibition assay (HIA) was performed to measure anti-influenza antibody titers in these subjects. Protein levels of the cytokines interleukin (IL)-4, IL-6, IL-8, IL-10, tumor necrosis factor-α (TNFα), interferon gamma (IFNγ), transforming growth factor beta (TGFß)1 and TGFß2 were quantified in plasma. Single nucleotide polymorphisms in IL6, IL10 and TNFα were also assessed. RESULTS: Influenza patients had lower antibody titers and produced significantly higher levels of IL-6, IL-10 and TNFα than healthy controls. The frequencies of the TNFα -308G, IL-10 -592C and IL-10 -1082A alleles and the IL10 -1082(A/A) genotype were associated with susceptibility to severe disease, while the haplotypes TNFα AG and IL-10 GTA and GCA were associated with protection from severe disease [P=0.016, OR (CI)=0.11 (0.01-0.96); P=0.0187, OR (CI)=0.34 (0.13-0.85); P=0.013, OR (CI)=0.39 (0.18-0.83)]. CONCLUSIONS: This study demonstrates that the influenza A(H1N1)pdm09 patients and healthy controls have different profiles of immune parameters and that there is an association between IL-10 and TNFα polymorphisms and the outcome of this disease.

Chinchilla laniger can be used as an experimental model for Taenia solium taeniasis.

Chinchilla laniger has been reported as an experimental definitive host for Taenia solium; however no information about its suitability and yield of gravid tapeworm proglottids containing viable and infective eggs has been published. In total 55 outbred female chinchillas were infected with 4 cysticerci each; hosts were immunodeppressed with 6 or 8 mg of methyl-prednisolone acetate every 14 days starting the day of infection and their discomfort was followed. Kinetics of coproantigen ELISA or expelled proglottids was used to define the infection status. Efficiency of tapeworm establishment was 21% and of parasite gravidity was 8%; chinchillas showed some degree of suffering along the infection. Viability of eggs obtained from gravid proglottids was tested comparing methods previously published, our results showed 62% viability with propidium iodide, 54% with trypan blue, 34% with neutral red, 30% by oncosphere activation and 7% with bromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio (MTT) reduction; no statistical differences were obtained between most techniques, except activation. Four piglets were infected with 50,000 eggs each, necropsy was performed 3 months later and, after counting the number of cysticerci recovered, the percentage of infection was similar to data obtained with T. solium eggs recovered from humans. Our results demonstrate that the experimental model of T. solium taeniasis in C. laniger is a good alternative for providing eggs and adult tapeworms to be used in different types of experiments; optimization of the model probably depends on the use of inbred hosts and on the reduction of infected animals' suffering.

Comparison of hemodynamic, biochemical and hematological parameters of healthy pregnant women in the third trimester of pregnancy and the active labor phase.

BACKGROUND: Pregnancy is accompanied by several hemodynamic, biochemical and hematological changes which revert to normal values after labor. The mean values of these parameters have been reported for developed countries, but not for Mexican women. Furthermore, labor constitutes a stress situation, in which these factors may be altered. It is known that serologic increase of heat shock protein (Hsp) 70 is associated with abnormal pregnancies, presenting very low level in normal pregnant women. Nevertheless, there are no studies where these measurements are compared in healthy pregnant women at their third trimester of pregnancy (3TP) and the active labor phase (ActLP). METHODS: Seventy five healthy Mexican pregnant women were included. Hemodynamic, biochemical and hematological parameters were obtained in all cases, and serum Hsp70 levels were measured in a sample of 15 women at 3TP and at ActLP. RESULTS: Significant differences were found in most analysis performed and in Hsp70 concentration at 3TP as compared to ActLP, however all were within normal range in both conditions, supporting that only in pathological pregnancies Hsp70 is drastically increased. CONCLUSION: Results obtained indicate that 3TP and ActLP have clinical similarities in normal pregnancies, therefore if abnormalities are found during 3TP, precautions should be taken before ActLP.

Immunolocalization of TSOL18 and TSOL45-1A, the successful protective peptides against porcine cysticercosis, in Taenia solium oncospheres.

Taenia solium life cycle includes humans as definitive hosts and pigs as intermediate hosts. One of the measures to stop the life cycle of this parasite is by vaccination of pigs. In experiments performed in pigs with TSOL18 and TSOL45-1A, two recombinant T. solium proteins, 99.5% and 97.0% protection was induced, respectively. The purpose of this paper was to localize these antigens in all stages of the parasite (adult worms, oncospheres and cysticerci) by immunofluorescence, with the use of antibodies against TSOL18 and TSOL45-1A that were obtained from the pigs used in the vaccination experiment. Results show that TSOL18 and TSOL45-1A are expressed on the surface of T. solium oncospheres and not in tapeworms or cysticerci, indicating that they are stage-specific antigens. This, therefore, might explain the high level of protection these antigens induce against pig cysticercosis.

Short report: evaluation of a self-detection tool for tapeworm carriers for use in public health.

The current study was designed to evaluate a tool for the self-identification of tapeworm carriers. Clinical and animal health care practitioners and schoolteachers were trained regarding the life cycle, risk factors, and control measures related to infection with Taenia solium. More than 120 small glass bottles with a few tapeworm segments fixed in formaldehyde and an instructional guide were distributed among all clinical practitioners (physicians and nurses) working in health centers. The guide contained 10 key points on how to ask questions about tapeworm infections. Information on taeniosis and cysticercosis was also provided to the general population via different media. Seven tapeworm carriers were recorded in the official epidemiology surveillance system the year previous to the study, compared with the year after the study, when 41 tapeworm carriers (37 Taenia saginata; 4 Taenia solium) were recorded. Six times more tapeworm carriers were notified after the study. All four persons with Taenia solium were treated, thereby eliminating the parasite and subsequently preventing any new cases of human and swine cysticercosis that might have arisen from them.

Cloning, characterization, and functional expression of Taenia solium calreticulin.

Calreticulin is an endoplasmic reticulum protein involved in the homeostasis of intracellular Ca++ and other physiological processes. A complementary DNA clone containing the complete coding sequence of Taenia solium calreticulin (TsCRT) was isolated and characterized. Recombinant TsCRT was expressed in bacteria as a 50-kDa protein that specifically bound calcium when tested in a radioassay. The deduced amino acid sequence has 47-50% identity with other reported calreticulins. Poor recognition of TsCRT by human and pig sera with confirmed cysticercosis discourages its use for diagnosis of the disease. However, further characterization and localization studies could provide insights into the role of TsCRT in T. solium physiology and host-parasite interactions.

Induction of protection against porcine cysticercosis by vaccination with recombinant oncosphere antigens.

Two recombinant Taenia solium oncosphere antigens, designated TSOL18 and TSOL45-1A, were investigated as vaccines to prevent transmission of the zoonotic disease cysticercosis through pigs. Both antigens were effective in inducing very high levels of protection (up to 100%) in three independent vaccine trials in pigs against experimental challenge infection with T. solium eggs, which were undertaken in Mexico and Cameroon. This is the highest level of protection that has been achieved against T. solium infection in pigs by vaccination with a defined antigen. TSOL18 and TSOL45-1A provide the basis for development of a highly effective practical vaccine that could assist in the control and, potentially, the eradication of human neurocysticercosis.