Gas chromatography-mass spectrometry (GC/MS) reveals urine metabolites associated to light and heavy infections by Schistosoma mansoni in mice.

High-throughput profiling of metabolites has been used to identify metabolic changes in murine models as a response to the infection by the parasitic trematode Schistosoma. These investigations have contributed to our understanding on the pathogenesis of this tropical neglected disease, with a potential of helping diagnosis. Here, our study aimed to investigate the application of gas chromatography-mass spectrometry (GC/MS) on the profiling of urine metabolites from mice carrying infections by Schistosoma mansoni. Two larval infection doses created distinctive infection intensities in mice, whereby the heavily infected animals were found to release 25 times more eggs in faeces than lightly infected animals. Over 200 urine metabolites were identified from these animals by GC/MS, following two complementary derivatisation methods. A list of 14 individual metabolites with altered relative abundances between groups were identified. Most of the altered metabolites showed a trend of increased abundances in response to infection intensity, indicating host-specific metabolic alterations as a result of the disease. Hippurate, a metabolite which concentration is intimately modulated by the gut microbiota, was found to be highly correlated to infection intensity. Our study showed that urine metabolic profiling by GC/MS can distinguish non-infected animals from those carrying light and heavy infections by S. mansoni, revealing metabolites associated to the infection and providing insights on the pathogenesis of schistosomiasis.

Cooperative Interactions between Trichomonas vaginalis and Associated Bacteria Enhance Paracellular Permeability of the Cervicovaginal Epithelium by Dysregulating Tight Junctions.

The human protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection, which is accompanied by a species-diversified vaginal microbiota named community state type IV (CST-IV). Coincidently, CST-IV includes species associated with bacterial vaginosis (e.g. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia). Both diseases are linked to the transmission of human immunodeficiency virus (HIV) and preterm birth, which complications are likely to result from the disruption of the cervicovaginal epithelial barrier. Here, we show that paracellular permeability of fluorescein isothiocyanate (FITC)-dextran through a monolayer of human ectocervical cells (hECs) is increased as a consequence of the activity of T. vaginalis and the aforementioned species of CST-IV bacteria cooperatively. T. vaginalis enhances paracellular permeability of hECs two times more than the individual bacterial species, by up to ∼10% versus ∼5%, respectively. However, any two or all three bacterial species are capable of synergizing this effect. T. vaginalis and the bacteria together increase the paracellular permeability of hECs by ∼50%, which is 5 to 10 times more than the results seen with the protozoan or bacteria alone. This effect is accompanied by enhancement of phosphatase activity, while phosphatase inhibition results in preservation of the integrity of the ectocervical cell monolayer. In addition, these microorganisms induce changes in the expression of tight junction proteins, particularly occludin, and of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). Together, our findings establish that cooperative interactions between CST-IV bacteria and T. vaginalis enhance the paracellular permeability of the cervicovaginal epithelium by disturbing the integrity of the tight junction complex. Our study results highlight the importance of understanding the contribution of the vaginal microbiota to trichomoniasis.

A Cell Surface Aggregation-Promoting Factor from Lactobacillus gasseri Contributes to Inhibition of Trichomonas vaginalis Adhesion to Human Vaginal Ectocervical Cells.

Trichomoniasis, a prevalent sexually transmitted infection, is commonly symptomatic in women. The causative agent is Trichomonas vaginalis, an extracellular protozoan parasite. The host-protective mechanisms and molecules of vaginal lactobacilli that counteract this pathogen are largely unknown. This study examines the inhibition promoted by Lactobacillus gasseri against the adhesion of T. vaginalis to host cells, a critical virulence aspect of this pathogen. We observed that the vaginal strain L. gasseri ATCC 9857 is highly inhibitory by various contact-dependent mechanisms and that surface proteins are largely responsible for this inhibitory phenotype. We found that the aggregation-promoting factor APF-2 from these bacteria significantly contributes to inhibition of the adhesion of T. vaginalis to human vaginal ectocervical cells. Understanding the molecules and mechanisms used by lactobacilli to protect the host against T. vaginalis might help in the development of novel and specific therapeutic strategies that take advantage of the natural microbiota.

The adherence of Trichomonas vaginalis to host ectocervical cells is influenced by lactobacilli.

OBJECTIVES: Trichomoniasis is a common sexually transmitted disease, and adhesion of the pathogen Trichomonas vaginalis to the host vaginal cells is the first step in establishing infection. For this to happen, the pathogen has to overcome a natural protective barrier composed mostly of lactobacilli. The objective of this study was to understand the role of lactobacilli in the adhesion of T vaginalis to host cells. METHODS: Adhesion assays were carried out by incubating vaginal epithelial cells (VECs) with T vaginalis and lactobacilli together and compared with non-lactobacilli recipient controls. By varying incubation parameters and testing several microbial isolates, the number of pathogens that adhered to the VECs was determined by flow cytometry. RESULTS: Overall, but with few exceptions, lactobacilli caused inhibition of T vaginalis adhesion to a variable degree. Lactobacillus gasseri ATCC 9857 and CBI3 (ambiguous Lactobacillus plantarum or Lactobacillus pentosus) caused the highest level of parasite adhesion inhibition and enhancement, respectively. These isolates of Lactobacillus can profoundly alter the adhesive properties of low-adherent and high-adherent strains of T vaginalis in a dose-dependent manner. Additionally, the effects of lactobacilli on T vaginalis adhesion are strictly contact-dependent, and surface lipoglycans of T vaginalis are most likely not involved in this modulation of adhesion mediated by the bacteria. CONCLUSIONS: Lactobacilli can modulate adhesion of T vaginalis by significantly modifying the natural adhesive properties of various T vaginalis strains. This study highlights the importance of considering the role of the vaginal microbiota in the pathogenesis of trichomoniasis.

An improved quantitative method to assess adhesive properties of Trichomonas vaginalis to host vaginal ectocervical cells using flow cytometry.

Microbial adhesion is a critical step for infection and colonization of the host. Trichomonas vaginalis, a human urogenital extracellular parasite, relies on host cell adhesion for infection and pathogenesis. Although host cell adhesion of T. vaginalis is strain-dependent and it may be influenced by many environmental factors, a technical limitation to quantify T. vaginalis adhesion falls upon a laborious and time-consuming protocol of fluorescent microscopy. This technical limitation reduces the ability of screening multiple parameters or detecting multiple cell types simultaneously. Here we tested the capability of using flow cytometry as a qualitative and quantitative method to measure adhesion of this human infectious microorganism to vaginal ectocervical cells. Various strains of T. vaginalis with different adhesion properties were stained with CellTracker Orange (CMTMR) prior to incubation with host cells. Analyses by flow cytometry revealed that adhered CMTMR-stained parasites were clearly distinguishable from the host cells and also enabled absolute cell counts to be determined. This method was validated with the comparison of parasite strains that display variable degrees of host cell adhesion. This assay can now be applied to test many variables and environmental factors simultaneously that may affect T. vaginalis adhesion.

Novel core promoter elements and a cognate transcription factor in the divergent unicellular eukaryote Trichomonas vaginalis.

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5' untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif.

A metazoan/plant-like capping enzyme and cap modified nucleotides in the unicellular eukaryote Trichomonas vaginalis.

The cap structure of eukaryotic messenger RNAs is initially elaborated through three enzymatic reactions: hydrolysis of the 5'-triphosphate, transfer of guanosine through a 5'-5' triphosphate linkage and N7-methylation of the guanine cap. Three distinctive enzymes catalyze each reaction in various microbial eukaryotes, whereas the first two enzymes are fused into a single polypeptide in metazoans and plants. In addition to the guanosine cap, adjacent nucleotides are 2'-O-ribose methylated in metazoa and plants, but not in yeast. Analyses of various cap structures have suggested a linear phylogenetic trend of complexity. These findings have led to a model in which plants and metazoa evolved a two-component capping apparatus and modification of adjacent nucleotides while many microbial eukaryotes maintained the three-component system and did not develop modification of adjacent nucleotides. Here, we have characterized a bifunctional capping enzyme in the divergent microbial eukaryote Trichomonas vaginalis using biochemical and phylogenetic analyses. This unicellular parasite was found to harbor a metazoan/plant-like capping apparatus that is represented by a two-domain polypeptide containing a C-terminus guanylyltransferase and a cysteinyl phosphatase triphosphatase, distinct from its counterpart in other microbial eukaryotes. In addition, T. vaginalis mRNAs contain a cap 1 structure represented by m(7)GpppAmpUp or m(7)GpppCmpUp; a feature typical of metazoan and plant mRNAs but absent in yeast mRNAs. Phylogenetic and biochemical analyses of the origin of the T. vaginalis capping enzyme suggests a complex evolutionary model where differential gene loss and/or acquisition occurred in the development of the RNA capping apparatus and cap modified nucleotides during eukaryote diversification.

The divergent eukaryote Trichomonas vaginalis has an m7G cap methyltransferase capable of a single N2 methylation.

Eukaryotic RNAs typically contain 5' cap structures that have been primarily studied in yeast and metazoa. The only known RNA cap structure in unicellular protists is the unusual Cap4 on Trypanosoma brucei mRNAs. We have found that T. vaginalis mRNAs are protected by a 5' cap structure, however, contrary to that typical for eukaryotes, T. vaginalis spliceosomal snRNAs lack a cap and may contain 5' monophophates. The distinctive 2,2,7-trimethylguanosine (TMG) cap structure usually found on snRNAs and snoRNAs is produced by hypermethylation of an m(7)G cap catalyzed by the enzyme trimethylguanosine synthase (Tgs). Here, we biochemically characterize the single T. vaginalis Tgs (TvTgs) encoded in its genome and demonstrate that TvTgs exhibits substrate specificity and amino acid requirements typical of an RNA cap-specific, m(7)G-dependent N2 methyltransferase. However, recombinant TvTgs is capable of catalysing only a single round of N2 methylation forming a 2,7-dimethylguanosine cap (DMG) as observed previously for Giardia lamblia. In contrast, recombinant Entamoeba histolytica and Trypanosoma brucei Tgs are capable of catalysing the formation of a TMG cap. These data suggest the presence of RNAs with a distinctive 5' DMG cap in Trichomonas and Giardia lineages that are absent in other protist lineages.

Diagnosis of human papillomatosis by polymerase chain reaction in cases of divergence between results of hybrid capture and papanicolaou cytology

As various types of human papillomavirus (HPV) are involved in the pathogenesis of cervical cancer, correct diagnosis is of fundamental importance for screening programs. We evaluated the divergence of results between Papanicolaou cytology and hybrid capture by PCR detection of HPV DNA . A transversal study was conducted on 70 women attending private gynecological clinics in Brasilia, Brazil. PCRs were conducted with specific primers for general and high-risk HPV DNA. Based on the PCR results, hybrid capture was a superior diagnostic technique. When Papanicolaou was compared with the molecular biology methods, it was found that a positive Papanicolaou result does not necessarily indicate the presence of HPV. The agreement between PCR and hybrid capture results can be attributed to the fact that both methods detect latent infection, while Papanicolaou detects only microscopic cellular alterations.

Diagnosis of human papillomatosis by polymerase chain reaction in cases of divergence between results of hybrid capture and papanicolaou cytology.

As various types of human papillomavirus (HPV) are involved in the pathogenesis of cervical cancer, correct diagnosis is of fundamental importance for screening programs. We evaluated the divergence of results between Papanicolaou cytology and hybrid capture by PCR detection of HPV DNA . A transversal study was conducted on 70 women attending private gynecological clinics in Brasilia, Brazil. PCRs were conducted with specific primers for general and high-risk HPV DNA. Based on the PCR results, hybrid capture was a superior diagnostic technique. When Papanicolaou was compared with the molecular biology methods, it was found that a positive Papanicolaou result does not necessarily indicate the presence of HPV. The agreement between PCR and hybrid capture results can be attributed to the fact that both methods detect latent infection, while Papanicolaou detects only microscopic cellular alterations.

Influence of the technique of re-educating thoracic and abdominal muscles on respiratory muscle strength in patients with cystic fibrosis.

OBJECTIVE: To determine the effect that re-education of the thoracic and abdominal muscles has on the respiratory muscle strength of patients with cystic fibrosis evaluated over time at the Cystic Fibrosis Outpatient Clinic of the Universidade Católica de Brasília (Catholic University of Brasília). METHODS: The sample consisted of 29 cystic fibrosis patients, characterized based on anthropometric, genetic and bacterial colonization data. The patients were submitted to physical therapy sessions, involving re-education of the respiratory muscles, twice a week for four months. Spirometry, pressure manometry and anthropometry were performed before and after each session. RESULTS: Comparing baselines values to those obtained after physical therapy, increases in maximum inspiratory pressure and maximum expiratory pressure were observed in all patients, those without any obstructive respiratory disease and those with mild obstructive respiratory disease (p < 0.05). A positive correlation between age and maximum expiratory pressure was observed for most of the patients. Maximum inspiratory pressure correlated positively with age only in the group with mild obstructive respiratory disease (p = 0.012; r = 0.817). In female patients and in the group of patients without obstructive respiratory disease, a negative correlation was observed between maximum expiratory pressure and colonization with Pseudomonas aeruginosa (p = 0.036; r = -0.585). CONCLUSION: Use of the thoracic and abdominal muscle re-education technique increased respiratory muscle strength in the cystic fibrosis patients studied, a finding that underscores the importance of including physical therapy in the treatment of these patients.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon.

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Trichomonas vaginalis: intrastrain polymorphisms within the ribosomal intergenic spacer do not correlate with clinical presentation.

Trichomoniasis presents a broad spectrum of clinical patterns ranging from asymptomatic to severe vaginitis and cervicitis. Despite its importance, very little is known about the genetic relatedness of its causative agent, Trichomonas vaginalis, and the clinical phenotypes. To address this question, analysis of restriction length polymorphism (RFLP) within the intergenic spacer of the ribosomal DNA (IGS) from 60 clinically defined isolates of T. vaginalis was performed. This is the first description of the IGS polymorphism of T. vaginalis. As expected, a considerable number of patients were asymptomatic (28%) while only 12% presented both leukorrhea and macular colpitis, the most evident symptoms of trichomoniasis. The IGS-RFLP with the use of eight restriction enzymes showed absence of correlation between the genetic relatedness of the isolates and symptomatology. Further studies are necessary to evaluate the importance of the IGS polymorphism to the parasite virulence and clinical phenotype.

A comparative evaluation of the Papanicolaou test for the diagnosis of trichomoniasis.

BACKGROUND: Trichomoniasis is the most prevalent nonviral sexually transmitted disease in humans worldwide. In addition to its pathologic implications, trichomoniasis is a risk factor for the transmission of the HIV and is associated with reproductive complications in females. Diagnosis of the disease is problematic due to inadequate accuracy of current diagnostic methods. Recently developed DNA-based techniques for detection of Trichomonas vaginalis seem to be promising alternatives. GOAL: The goal of this study was to evaluate the accuracy of the Papanicolaou test for the diagnosis of trichomoniasis by comparing polymerase chain reaction (PCR) with other current diagnostic methods. STUDY DESIGN: A total of 1008 cervicovaginal swab specimens from a randomized population attending a gynecological service were analyzed in this study. In addition to current diagnostic methods, two sets of specific primers were used for PCR detection of T vaginalis in the cervicovaginal DNA samples, with a PCR quality control. Different examiners conducted PCR and Papanicolaou analyses in a double-blind trial. RESULTS: The prevalence of trichomoniasis in this population was 6%. A considerable number of diagnostic results of the Papanicolaou test were false negative or false positive. Compared with PCR, specificity of the Papanicolaou test was 97.6%, whereas sensitivity was only 60.7%. The positive predictive value of the Papanicolaou smear was 61.7%. These results suggest that irregularly shaped parasites without clearly defined nuclei and flagella and bacteria-induced focal cytolysis limit the ability of the Papanicolaou test to detect T vaginalis. CONCLUSION: The Papanicolaou test, the most readily available cytologic method for screening sexually transmitted pathogens and cellular abnormalities in most developing countries, is inadequate for the diagnosis of trichomoniasis due to its inherent limitations. However, PCR is a highly sensitive and specific test for the diagnosis of trichomoniasis.

Six-year follow-up survey of sexually transmitted diseases in Brasilia, the Capital of Brazil.

The notification of sexually transmitted diseases (STD) is a prime component of well-designed public health policy. However, peculiar aspects of STD must be taken into account for the correct management of surveillance activities. Here, we describe the distribution of the most common sexually transmitted diseases among patients attended by the gynecological clinics of the principal public hospitals of Brasilia and the Federal District, Brazilian capital, during six years. A total of 142,158 patients had their cervicovaginal samples collected for Papanicolaou preparations and eventual biopsies. Diagnosis was made according to cytological and histological alterations, distinguishing among vaginal infections, and pre-cancerous and cancerous cervical lesions. We also looked at the annual prevalence of the various types of infections and alterations. There was a high prevalence of bacterial vaginosis, trichomoniasis and candidiasis, with suggestive changes over the years. Pre-cancerous and cancerous lesions increased 2.2 fold during the six years. A large proportion of the cases involved late stages of cervical cancer, indicating the necessity of prompt attendance of the population in a routine gynecological prevention program.

Six-year follow-up survey of sexually transmitted diseases in Brasilia, the Capital of Brazil

The notification of sexually transmitted diseases (STD) is a prime component of well-designed public health policy. However, peculiar aspects of STD must be taken into account for the correct management of surveillance activities. Here, we describe the distribution of the most common sexually transmitted diseases among patients attended by the gynecological clinics of the principal public hospitals of Brasilia and the Federal District, Brazilian capital, during six years. A total of 142,158 patients had their cervicovaginal samples collected for Papanicolaou preparations and eventual biopsies. Diagnosis was made according to cytological and histological alterations, distinguishing among vaginal infections, and pre-cancerous and cancerous cervical lesions. We also looked at the annual prevalence of the various types of infections and alterations. There was a high prevalence of bacterial vaginosis, trichomoniasis and candidiasis, with suggestive changes over the years. Pre-cancerous and cancerous lesions increased 2.2 fold during the six years. A large proportion of the cases involved late stages of cervical cancer, indicating the necessity of prompt attendance of the population in a routine gynecological prevention program.

A historical perspective and prospects of biomedical research on parasitic diseases

We all hope that biotechnology will answer some social and economical unavoidable requirements of the modern life. It is necessary to improve agriculture production, food abundance and health quality in a sustainable development. It is indeed a hard task to keep the progress on taking into account the rational use of genetic resources and the conservation of biodiversity. In this context, a historical perspective and prospects of the biomedical research on parasitic diseases is described in a view of three generations of investigators. This work begins with a picture of the scientific progress on biomedical research and human health over the last centuries. This black-and-white picture is painted by dissecting current advancements of molecular biology and modern genetics, which are outlined at the meaning of prospecting achievements in health science for this new millenium

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.