High silent prevalence of human herpesvirus 1 (HSV-1) infection affecting the indigenous reservation of the municipality of Dourados, Central-West Brazil.

BACKGROUND: The indigenous population located in the central region of Brazil, is the second largest in terms of population size in the country. The Indigenous Reserve of Dourados has risk factors that increase the vulnerability of the indigenous population to infectious diseases, especially Human alphaherpesvirus (HSV-1), a neglected disease with high prevalence in priority populations in developing countries. The virus can also cause many more severe diseases, including widespread neonatal infections, herpetic keratitis, and herpes encephalitis, which can be fatal if left untreated. We estimated the prevalence of anti-HSV-1 antibodies and correlated it with the demographic and behavioral characteristics of the Indigenous population of the Jaguapirú and Bororó villages (Dourados, Mato Grosso do Sul (MS), Brazil). METHODS: Our approach was cross-sectional. From March 2017 to November 2018. Using anti-HSV-1 (Gg1) IgM and anti-HSV-1 (gG1) IgG Euroimmun and the detection and quantification of HSV-1 viral load in plasma samples, through real-time PCR. The maps were constructed using QGIS and the statistical analyses using R Studio software. RESULTS: A total of 1138 individuals (> 18 years old) were enrolled. The prevalence of anti-HSV-1 IgM and IgG were 20% and 97.5%, respectively. The prevalence of anti-HSV-1 antibodies for IgG was higher in both sexes. Anti-HSV-1 IgM antibodies were present in 17.1%, 21.2%, 12.5%, and 22% of the participants with urinary problems, genital wounds, genital warts, and urethral discharge, respectively. Real-time PCR was used for confirmatory testing; HSV-1 DNA was detected in 25.6% (54/211) of anti-HSV1 IgM-positive samples. Viral loads ranged from 5.99E + 02 to 3.36E + 13. CONCLUSIONS: The seroprevalence of HSV-1 IgM and detection of HSV-1 DNA in the Indigenous population confirmed high silent prevalence. Furthermore, the seroprevalence of HSV-1 in the Indigenous population was higher than that reported in the general adult Brazilian population. Various socioeconomic factors, drug use, and health and sexual behaviors could contribute to the facilitation of HSV-1 transmission in the Indigenous population. Our results may help develop culturally appropriate intervention programs that eliminate health access barriers and improve the implementation of public health policies aimed at promoting information regarding the prevention, treatment, and control of HSV-1 infection in Brazilian Indigenous populations.

Trypanosoma cruzi strains in the Calomys callosus: parasitemia and reaction of intracellular forms with stage-specific antibodies in the acute and chronic phase of infection and after immunosuppression.

An experimental model for chronic Chagas disease was developed to investigate whether reactivation is influenced by the genetic origin of Trypanosoma cruzi isolates. In addition, we examined whether the distribution of T. cruzi stage-specific epitopes, as defined by monoclonal antibodies (Mab), raised against mammalian-stage parasite forms, exhibited comparable distribution patterns in Calomys callosus myocardium during the acute phase and after reactivation of the infection. Animals were infected with parasites of the G (T. cruzi I), Y (T.cruzi II) or CL strains (T. cruzi VI). Heart sections were labelled with the Mabs 2C2, 1D9, 2B7, 3B9 and 4B9, which react with carbohydrate epitopes on Ssp-4, a major amastigote surface glycoprotein. Mab 1D9 and 2B7 showed polymorphic distributions over amastigotes among animals infected with the G, Y or CL strains. Mab 3B2, which recognises a non-carbohydrate epitope in flagellated forms, showed an active state of parasite dissemination in the myocardium of C. callosus that were infected with Y or CL strains and then immunosuppressed after 6 or 12 months. C. callosus infected with the G strain (T. cruzi I) displayed absence of amastigote nests in the heart after immunosuppression. Our results permit us to suggest that parasites of the G strain may be more sensitive to the immune response, since we could not find either evidence of parasitemia or amastigote nests. Conversely, parasites from the Y and CL strains appeared able to escape the immune response, as evidenced by an inflammatory infiltrate and disseminated infection after immunosuppression.

Host cell actin remodeling in response to Trypanosoma cruzi: trypomastigote versus amastigote entry.

Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a highly prevalent vector-borne disease in Latin America. Chagas' disease is a major public health problem in endemic regions with an estimated 18 million people are infected with T. cruzi and another 100 million at risk (http://www.who.int/ctd/chagas/disease.htm). During its life cycle, T. cruzi alternates between triatomine insect vectors and mammalian hosts. While feeding on host's blood, infected triatomines release in their feces highly motile and infective metacyclic trypomastigotes that may initiate infection. Metacyclic trypomastigotes promptly invade host cells (including gastric mucosa) and once free in the cytoplasm, differentiate into amastigotes that replicate by binary fission. Just before disruption of the parasite-laden cell, amastigotes differentiate back into trypomastigotes which are then released into the tissue spaces and access the circulation. Circulating trypomastigotes that disseminate the infection in the mammalian host may be taken up by feeding triatomines and may also transform, extracellularly, into amastigote-like forms. Unlike their intracellular counterparts, these amastigote-like forms, henceforth called amastigotes, are capable of infecting host cells. Studies in which the mechanisms of amastigote invasion of host cells have been compared to metacyclic trypomastigote entry have revealed interesting differences regarding the involvement of the target cell actin microfilament system.

Distribution of Trypanosoma cruzi stage-specific epitopes in cardiac muscle of Calomys callosus, BALB/c mice, and cultured cells infected with different infective forms.

To examine whether distinct parasite infective forms or the mammalian host could affect the distribution of Trypanosoma cruzi stage-specific epitopes defined by monoclonal antibodies (Mabs) raised against mammalian-stage parasite forms, immunofluorescence studies followed the intracellular life cycle of the parasite in the cardiac muscle of Calomys callosus and BALB/c mice in the acute phase of the disease and in LLC-MK(2) cultured cells. Animals and cells were infected either with tissue-culture derived trypomastigotes (TCT) or bloodstream trypomastigotes (BT) from the Y strain of T. cruzi. Samples were examined under confocal fluorescence microscopy after labeling with Mabs 2C2, 1D9, 2B7, 3G8, 3B9, and 4B9 that react with carbohydrate epitopes on Ssp-4, a major amastigote surface glycoprotein; Mab 4B5 that identifies a noncarbohydrate epitope on all intracellular parasites stages, and Mab 3B2 that also recognizes a noncarbohydrate epitope expressed only in flagellated forms. Samples were double labeled with DAPI to visualize parasites' kinetoplasts and nuclei. Most of the Mabs used in this work displayed a surface labeling pattern on amastigotes present in Calomys and mice hearts, and in LLC-MK(2) cultured cells infected with BT or TCT. Mab 2B7, however, displayed a marked polymorphic distribution in antigen expression between both mammalian hosts, independent on the infective form. Beyond the polymorphic distribution of amastigote surface epitopes, Calomys, and mice heart sections presented several inflammatory cells around amastigotes and trypomastigotes nests.

Disruption of myofibrillar proteins in cardiac muscle of Calomys callosus chronically infected with Trypanosoma cruzi and treated with immunosuppressive agent.

Calomys callosus (Rodentia: Cricetidae) chronically infected with CL strain of Trypanosoma cruzi undergo recrudescence of the acute phase when treated with the immunosuppressor cyclophosphamide. The distribution of cytoskeletal proteins in cardiac tissue of immunosuppressed animals was mapped by immunofluorescence and electron microscopy to evaluate myofibrillar distribution during the intracellular life cycle of T. cruzi. Cardiac muscle sections showed enhancement of myocarditis and parasite proliferation after immunosuppression. Immunofluorescence using monoclonal antibodies against myosin, actin, desmin, titin, tropomyosin, and troponin T demonstrated disruption and loss of contractile proteins, such as myosin and actin. Desmin and titin were irregularly distributed in close proximity to parasite nests. Ultrastructural observations confirmed alterations of cardiac cells with Z-line fragmentation, indistinguishable I-bands and A-bands, and loss of myofibrillar elements. The disruption of the muscle cell architecture was greater as infection progressed, probably as a result of increased myocarditis and physical displacement due to the activity of flagellated parasites.

Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi.

The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 M, these compounds displayed a strong lytic activity. It ranged from 88.4 0.6 to 99.0 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 M and 41-87 M, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 M. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 M. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.

Towards a more precise assay of sperm function in egg binding.

Historically, the treatment of severe male factor infertility has relied on donor sperm insemination. A decade ago the option of treating severe male factor infertility with partner sperm became a viable alternative. With the introduction of intracytoplasmic sperm injection (ICSI) in conjunction with in vitro fertilization (IVF), only men who produce no sperm are denied the option of fathering their own children. The use of ICSI has been extended to couples with mild male factors. Despite the known genetic risks (both inherent and de novo) of ICSI to offspring, couples with male factors as part of their infertility problem often prefer ICSI to standard IVF, due to apprehension that their sperm might not otherwise succeed in fertilization. This apprehension would be alleviated if an assay for the egg binding capability of human sperm were available. We examine here the possibility that recombinant human zona pellucida 3 (rec hZP3), the primary sperm receptor sulfoglycoprotein of the egg zona pellucida (ZP), be used as a human ZP surrogate for assessing sperm ability to bind to the ZP. Unlike human eggs, which cannot be obtained for this purpose, rec hZP3 can be produced in quantity. An efficient assay can be established by incubating sperm with rec hZP3 coated to a microwell plate. Infertile men with sperm having ability to bind to rec hZP3 can be advised to select standard IVF or intrauterine insemination, which have fewer genetic and medical risks.

Acquisition of arylsulfatase A onto the mouse sperm surface during epididymal transit.

Arylsulfatase A (AS-A) is localized to the sperm surface and participates in sperm-zona pellucida binding. We investigated how AS-A, usually known as an acrosomal enzyme, trafficked to the sperm surface. Immunocytochemistry of the mouse testis confirmed the existence of AS-A in the acrosomal region of round and elongating spermatids. However, immunofluorescence and flow cytometry indicated the absence of AS-A on the surface of live testicular sperm. In contrast, positive AS-A staining was observed in the heads of live caudal epididymal and vas deferens sperm. The results suggested that acquisition of AS-A on the sperm surface occurred during epididymal transit. Immunocytochemistry of the epididymis revealed AS-A in narrow and apical cells in the initial segment and in clear cells in all epididymal regions. However, these epithelial cells are in the minority and are not involved in secretory activity. In the caudal epididymis and vas deferens, AS-A was also localized to principal cells, the major epithelial cells. Because principal cells have secretory activity, they may secrete AS-A into the epididymal fluid. This hypothesis was supported by our results revealing the presence of AS-A in the epididymal and vas deferens fluid (determined by immunoblotting and ELISA) and an AS-A transcript in the epididymis (by reverse transcription polymerase chain reaction). Alexa-430 AS-A bound to epididymal sperm with high affinity (Kd = 46 nM). This binding was inhibited by treatment of sperm with an antibody against sperm surface sulfogalactosylglycerolipid. This finding suggests that AS-A in the epididymal fluid may deposit onto sperm via its affinity to sulfogalactosylglycerolipid.

Cytotoxic and genotoxic effects of megazol, an anti-Chagas' disease drug, assessed by different short-term tests.

Cyto- and genotoxicity induced by drugs can limit the dose and duration of treatment, can adversely affect patient quality of life, and may be life-threatening. Two drugs are currently being used for treatment of the acute phase of Chagas' disease and both have serious undesirable effects. In this research, cyto- and genotoxic activity of the nitroimidazole-tiadiazole derivative CL 64855 2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole (megazol), a promising alternative drug, was evaluated in vitro with different short-term tests: (a) induction of recombination events and mutation in the yeast Saccharomyces cerevisiae D7 strain, with and without induction of cytochrome P-450; DNA damage (single and double strand breaks, alkali-labile sites, etc.) by the Comet assay in different mammalian cells. S. cerevisiae did not show a significant increase of mutant and recombinant event frequency, both with and without cytochrome P-450. On the other hand, the cytochrome complex appeared to detoxify the drug with respect to cytotoxicity. Results in rat and mouse fresh leukocytes showed a dose-response relation of drug-induced DNA damage. Findings in treated VERO cells suggested a complex treatment time-DNA damage relationship and the possible induction of repair mechanisms. Furthermore, bleomycin effects were increased in rat cells by simultaneous administration of megazol. Megazol shows different biological activity in relation to cellular types and experimental conditions (with or without cytochrome P-450, short/long time of exposure, with or without other genotoxins), thus suggesting a modulation of effectiveness by different physiological/biochemical conditions of cells. The findings could be useful to evaluate new megazol-derived compounds and to assess the risks/benefits relationship for each drug.