Chagas infection transmission control: situation of transfusional transmission in Brazil and other countries of Latin America

The transmission of the transfusion-associated Chagas disease is an important mechanism of its dissemination in several Latin American countries. The transmission risk depends on five factors: prevalence of infection in blood donors, degree of serological coverage, sensibility of used tests, safety of obtained results and infection risk. The Southern Cone Iniciative set off by the Pan-American Health Organization, in 1991, is contributing to the implementation of blood law in each endemic country, and to reduce the risk of transfusional transmission of this horrible disease. Despite the clear improvement of Brasilian hemotherapy after 1980 (with the creation of the Blood National Program - Pró-Sangue) and the significant reduction of the chagasic infection among its blood donors; socio-economic, politic and cultural unlevels, prevent it from reaching the necessary universality and security. In order to assure both, the Brazilian Ministry of Health decided to restructure its blood system. In May, 1998, a great program was launched, to reach a specific goal: Blood - 100% with quality safety in all its process until 2003. It was divided in 12 projects, intends to guarantee the quality and self sufficiency in blood and hemoderivates.

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: Psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

Experimental toxoplasmosis in Balb/c mice. Prevention of vertical disease transmission by treatment and reproductive failure in chronic infection

In a study of congenital transmission during acute infection of Toxoplasma gondii, 23 pregnant Balb/c mice were inoculated orally with two cysts each of the P strain. Eight mice were inoculated 6-11 days after becoming pregnant (Group 1). Eight mice inoculated on the 10th-15th day of pregnancy (Group 2) were treated with 100 mg/kg/day of minocycline 48 h after inoculation. Seven mice inoculated on the 10th-15th day of pregnancy were not treated and served as a control (Group 3). Congenital transmission was evaluated through direct examination of the brains of the pups or by bioassay and serologic tests. Congenital transmission was observed in 20 (60.6%) of the 33 pups of Group 1, in one (3.6%) of the 28 pups of Group 2, and in 13 (54.2%) of the 24 pups of Group 3. Forty-nine Balb/c mice were examined in the study of congenital transmission of T. gondii during chronic infection. The females showed reproductive problems during this phase of infection. It was observed accentuated hypertrophy of the endometrium and myometrium. Only two of the females gave birth. Our results demonstrate that Balb/c mice with acute toxoplasmosis can be used as a model for studies of congenital T. gondii infection. Our observations indicate the potential of this model for testing new chemotherapeutic agents against congenital toxoplasmosis.

Sand fly evolution and its relationship to Leishmania transmission

The evolutionary relationships of sand flies and Leishmania are discussed in this report, which draws distinctions between co-association, co-evolution and co-speciation (or co-cladogenesis). Examples focus on Phlebotomus vectors of Le. infantum and Le. major in the Mediterranean subregion.

Vertical human immunodeficiency virus type 1 - HIV-1 -transmission - a review

Several factors appear to affect vertical HIV-1 transmission, dependent mainly on characteristics of the mother (extent of immunodeficiency, co-infections, risk behaviour, nutritional status, immune response, genetical make-up), but also of the virus (phenotype, tropism) and, possibly, of the child (genetical make-up). This complex situation is compounded by the fact that the virus may have the whole gestation period, apart from variable periods between membrane rupture and birth and the breast-feeding period, to pass from the mother to the infant. It seems probable that an extensive interplay of all factors occurs, and that some factors may be more important during specific periods and other factors in other periods. Factors predominant in protection against in utero transmission may be less important for peri-natal transmission, and probably quite different from those that predominantly affect transmission by mothers milk. For instance, cytotoxic T lymphocytes will probably be unable to exert any effect during breast-feeding, while neutralizing antibodies will be unable to protect transmission by HIV transmitted through infected cells. Furthermore, some responses may be capable of controlling transmission of determined virus types, while being inadequate for controlling others. As occurence of mixed infections and recombination of HIV-1 types is a known fact, it does not appear possible to prevent vertical HIV-1 transmission by reinforcing just one of the factors, and probably a general strategy including all known factors must be used. Recent reports have brought information on vertical HIV-1 transmission in a variety of research fields, which will have to be considered in conjunction as background for specific studies.

Role of two Triatoma (Hemiptera: Reduviidae: Triatominae) species in the transmission of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) to man in the West Coast of Mexico

From August 1997 to August 1998, 334 specimens of Triatoma longipennis and 62 of T. picturata were collected in four groups of localities placed in the zone from Guadalajara, Jalisco to Tepic, Nayarit, in the West Coast of Mexico. Most T. longipennis were collected outdoors (69.2%) while most T. picturata (58.1%) were collected indoors. All collected specimens were examined for Trypanosoma cruzi infection, which was detected on 98 (29.3%) T. longipennis and 17 (27.4%) T. picturata. This study confirms the role of T. longipennis and T. picturata as some of the main T. cruzi vectors to humans in Mexico. Habitation Infestation Rate with T. longipennis was of 0.09 and with T. picturata was of 0.03 and the predominating ecotopes were pile of blocks, chicken coops, pigsties, wall crawls and beds.

Domestic and peridomestic transmission of American cutaneous leishmaniasis: changing epidemiological patterns present new control opportunities

Predictions that deforestation would reduce American cutaneous leishmaniasis incidence have proved incorrect. Presentations at a recent international workshop, instead, demonstrated frequent domestication of transmission throughout Latin America. While posing new threats, this process also increases the effectiveness of vector control in and around houses. New approaches for sand fly control and effective targeting of resources are reviewed.

In vitro neuroendocrine effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the AhR-expressing hypothalamic rat GnV-3 cell line.

The aryl hydrocarbon receptor (AhR) is involved in a wide variety of biological and toxicological responses, including neuroendocrine signaling. Due to the complexity of neuroendocrine pathways in e.g. the hypothalamus and pituitary, there are limited in vitro models available despite the strong demand for such systems to study and predict neuroendocrine effects of chemicals. In this study, the applicability of the AhR-expressing rat hypothalamic GnV-3 cell line was investigated as a novel model to screen for neuroendocrine effects of AhR ligands using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as reference compound. The qRT-PCR analyses demonstrated the presence of several sets of neurotransmitter receptors in the GnV-3 cells. TCDD (10nM) altered neurotransmitter signaling by up-regulation of glutamate (Grik2), gamma-amino butyric acid (Gabra2) and serotonin (Ht2C) receptor mRNA levels. However, no significant changes in basal and serotonin-evoked intracellular Ca(2+) concentration ([Ca(2+)]i) or serotonin release were observed. On the other hand, TCDD de-regulated period circadian protein homolog 1 (Per1) and gonadotropin releasing hormone (Gnrh) mRNA levels within a 24-h time period. Both Per1 and Gnrh genes displayed a similar mRNA expression pattern in GnV-3 cells. Moreover, the involvement of AhR in TCDD-induced alteration of Neuropeptide Y (Npy) gene expression was found and confirmed by using siRNA targeted against Ahr in GnV-3 cells. Overall, the combined results demonstrate that GnV-3 cells may be a suitable model to predict some mechanisms of action and effects of AhR ligands in the hypothalamus.

Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55%) had unique IS6110 restriction fragment length polymorphism (RFLP) patterns, while isolates from 23 others (45%) had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

Scanning and transmission electron microscopy of the tegument of Paranaella luquei Kohn, Baptista-Farias & Cohen, 2000 (Microcotylidae, Monogenea), parasite of a Brazilian catfish, Hypostomus regani

The surface topography and ultrastructure of the tegument of Paranaella luquei Kohn, Baptista-Farias & Cohen, 2000, a microcotylid monogenean parasite from the gills of Hypostomus regani (Ihering, 1905) (Loricariidae) was studied by scanning (SEM) and transmission electron microscopy (TEM). By SEM, it was observed that the tegument presents transversal ridges, forming folds in the ventral and dorsal surfaces and microvillous-like tegumental projections in the anterior and median regions of body. These projections were also observed by TEM. The tegument is made up of a syncytium delimited by apical and basal plasma membranes, containing inclusion bodies and mitochondria, connected to the nucleated region by means of cytoplasmatic processes. The tegumental cells present a well developed nucleus and cytoplasm containing inclusion bodies, similar to those found on the external layer, mitochondria, rough endoplasmatic reticulum and free ribossomes.

Statistical evaluation of the influence of writing postures on on-line signatures. Study of the impact of time.

The aim of this study is to investigate the influence of unusual writing positions on a person's signature, in comparison to a standard writing position. Ten writers were asked to sign their signature six times, in each of four different writing positions, including the standard one. In order to take into consideration the effect of the day-to-day variation, this same process was repeated over 12 sessions, giving a total of 288 signatures per subject. The signatures were collected simultaneously in an off-line and on-line acquisition mode, using an interactive tablet and a ballpoint pen. Unidimensional variables (height to width ratio; time with or without in air displacement) and time-dependent variables (pressure; X and Y coordinates; altitude and azimuth angles) were extracted from each signature. For the unidimensional variables, the position effect was assessed through ANOVA and Dunnett contrast tests. Concerning the time-dependent variables, the signatures were compared by using dynamic time warping, and the position effect was evaluated through classification by linear discriminant analysis. Both of these variables provided similar results: no general tendency regarding the position factor could be highlighted. The influence of the position factor varies according to the subject as well as the variable studied. The impact of the session factor was shown to cover the impact that could be ascribed to the writing position factor. Indeed, the day-to-day variation has a greater effect than the position factor on the studied signature variables. The results of this study suggest guidelines for best practice in the area of signature comparisons and demonstrate the importance of a signature collection procedure covering an adequate number of sampling sessions, with a sufficient number of samples per session.

Experimental transmission of Trypanosoma cruzi through the genitalia of albino mice

Trypanosoma cruzi is usually transmitted by contact with the excreta of infected Triatominae; among non-vectorial infections, direct transmission through coitus has been proposed. We investigated this possibility by instilling, through the external meatus of the vagina and the penis of previously anesthetized NMRI albino mice, blood of mice infected with strains isolated from Didelphis marsupialis (opossum, strain CO57), Rattus rattus (rat, strain CO22) and human (strain EP). Some animals were allowed to copulate the same day of the instillation. In other experiments, the strains were inoculated in the scrotum. To determine the effect of immunosuppression, some mice were treated with cyclophosphamide 30 days post-instillation. Controls were instilled orally and ocularly. Vaginal instillation with strain CO22 produced systemic infection with tropism to the heart, skeletal muscle, skin, duodenum, pancreas, ovary and sternum. Scrotal inoculation with strain EP likewise invaded liver, spleen, lung, lymph nodes and urogenital organs; while strain CO57 invaded skeletal and cardiac muscle, pancreas, testis, and vas deferens. Penile infection with strain CO22 was detected by xenodiagnosis. Immunosuppression did not increase parasitemia of vaginally infected mice or controls. Mating did not produce infection. Our results show that contact of blood trypomastigotes of T. cruzi with genital mucosa can produce blood and tissue infections. These results are discussed in relation to reports of frequent experimental tropism of T. cruzi toward urogenital organs.

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiânia city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.

A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.

Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.