A genotyping study of human immunodeficiency virus type-1 drug resistance in a small Brazilian municipality

In Brazil, surveillance studies on antiretroviral drug resistance among drug-naïve and treatment-experienced patients have focused primarily on patients living in large urban centers. As the epidemic spreads towards small municipalities and the innermost parts of the country, it will be essential to monitor the prevalence of antiretroviral drug resistance in these areas. We report the first survey on the prevalence of antiretroviral drug resistance in a small Brazilian municipality. Between July 1999 and March 2005, 72 adult human immunodeficiency virus type-1(HIV-1)-infected patients received care at the Municipal HIV/AIDS Program of the small, southeastern municipality of Miracema, state of Rio de Janeiro. A genotyping study of antiretroviral drug resistance was performed in 54 patients. Among 27 samples from treatment-experienced patients, 9 (33.3%) harbored strains with reduced drug susceptibility. Among these, 6 had reduced susceptibility to reverse transcriptase (RT) inhibitors and 3 to both RT and protease inhibitors. No primary antiretroviral drug resistance was recorded among 27 drug-naïve subjects. The relatively low prevalence of resistance mutations in the Miracema cohort argues against the concern that resource-poor settings should not implement widespread accessibility to standard of care antiretroviral combinations due to the possibility of sub-optimal adherence leading to the emergence and spread of drug-resistant strains.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Expression of neuronal nitric oxide synthase in the hippocampal formation in affective disorders

Hippocampal output is increased in affective disorders and is mediated by increased glutamatergic input via N-methyl-D-aspartate (NMDA) receptor and moderated by antidepressant treatment. Activation of NMDA receptors by glutamate evokes the release of nitric oxide (NO) by the activation of neuronal nitric oxide synthase (nNOS). The human hippocampus contains a high density of NMDA receptors and nNOS-expressing neurons suggesting the existence of an NMDA-NO transduction pathway which can be involved in the pathogenesis of affective disorders. We tested the hypothesis that nNOS expression is increased in the human hippocampus from affectively ill patients. Immunocytochemistry was used to demonstrate nNOS-expressing neurons in sections obtained from the Stanley Consortium postmortem brain collection from patients with major depression (MD, N = 15), bipolar disorder (BD, N = 15), and schizophrenia (N = 15) and from controls (N = 15). nNOS-immunoreactive (nNOS-IR) and Nissl-stained neurons were counted in entorhinal cortex, hippocampal CA1, CA2, CA3, and CA4 subfields, and subiculum. The numbers of Nissl-stained neurons were very similar in different diagnostic groups and correlated significantly with the number of nNOS-IR neurons. Both the MD and the BD groups had greater number of nNOS-IR neurons/400 µm² in CA1 (mean ± SEM: MD = 9.2 ± 0.6 and BD = 8.4 ± 0.6) and subiculum (BD = 6.7 ± 0.4) when compared to control group (6.6 ± 0.5) and this was significantly more marked in samples from the right hemisphere. These changes were specific to affective disorders since no changes were seen in the schizophrenic group (6.7 ± 0.8). The results support the current view of the NMDA-NO pathway as a target for the pathophysiology of affective disorders and antidepressant drug development.

VP22 herpes simplex virus protein can transduce proteins into stem cells

The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.

Preliminary antigenic characterisation of an adult worm vomit preparation of Fasciola hepatica by infected human sera

Fascioliasis is an emerging/re-emerging vector-borne disease with the widest known distribution. Approximately 17 million people are infected around the world, being the Andean region the most affected area. There is an important necessity to develop sensitive and specific diagnostic tools to treat patients early and to avoid complications. In this paper we evaluated the immune response of infected humans against two antigenic preparations: the total soluble extract (FhTSE) and the adult worm vomit (FhAWV) in order to identify antigenic fractions specific for Fasciola hepatica. Both preparations were processed by SDS-PAGE and Western blot with human sera with fascioliasis (F), other parasitosis and healthy individuals. In the immunoblot of FhTSE, sera F recognised 16 bands with MW between eight and 110 kDa, from which those of 8, 9, 10, 38, 45 and 57 kDa were specific. In the preparation FhAWV, sera F recognised nine bands with MW from eight to 85 kDa, from which those of 8, 12, 15 and 24 kDa were specific. Some bands of cross-reaction were evident with sera from patients with other parasitoses, more frequent with the FhTSE. Bands within the MW mentioned, particularly that of eight kDa, have been shown to be specific by others, and deserve additional characterisation for their potential use in immunodiagnosis.

Characterization of a clone from an adult worm cDNA library selected with anti-Schistosoma mansoni human antibodies dissociated from immune complexes: a preliminary report

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

Schistosoma mansoni Sambon, 1907: morphometric differences between adult worms from sympatric rodent and human isolates

A computer software for image analysis (IMAGE PRO PLUS, MEDIA CYBERNETICS) was utilized in male and females adult worms, aiming the morphological characterization of Schistosoma mansoni samples isolated from a slyvatic rodent, Nectomys squamipes, and humans in Sumidouro, Rio de Janeiro, Brazil and recovered from Mus musculus C3H/He. The following characters for males's testicular lobes were analyzed: number, area, density, larger and smaller diameter, longer and shorter axis and perimeter and extension; for females: area, longer and shorter axis, larger and smaller diameter and perimeter of the eggs and spine; oral and ventral suckers area and distance between them in both sex were determined. By the analysis of variance (one way ANOVA) significant differences (p<0.05) were observed in all studied characters, except for the density of testicular lobes. Significant differences (p<0.05) were detected for all characters in the female worms. Data ratify that sympatric isolates present phenotypic differences and the adult female characters are useful for the proper identification of S. mansoni isolates.

Identification of candidate antigens from adult stages of Toxocara canis for the serodiagnosis of human toxocariasis

In the present work, we identified adult Toxocara canis antigens through sodium dodecyl sulfate-polyacrylamide gel electrophoresis for potential use in human toxocariasis immunodiagnosis. The sensitivity and specificity of several semi-purified antigens, as well as their cross-reactivity with other parasitic infections, were assessed by IgM and IgG-enzime linked immunosorbent assay. Whilst we found that the crude extract of the parasite presented limited sensitivity, specificity and high cross-reactivity against other parasites, we identified 42, 58, 68 and 97-kDa semi-purified antigens as the most promising candidates for immunodiagnosis. Moreover, the 58 and 68-kDa antigens presented the lowest IgM cross-reactivity. When tested as a combination, a mixture of the 58 and 68-kDa antigens presented 100% sensitivity and specificity, as well as minor cross-reactivity. Although the combination of the 42, 58, 68 and 97-kDa antigens presented 100% sensitivity at a dilution of 1:40, the low specificity and high cross-reactivity observed suggested a limited use for diagnostic purposes. Our data suggested that the 58 and 68-kDa antigens might be most suitable for the immunodiagnosis of human toxocariasis.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

Studies on mosquitoes (Diptera: Culicidae) and anthropic environment: 8- Survey of adult behaviour of Spissipes Section species of Culex (Melanoconion) in South-Eastern Brazil

A study of Spissipes Section of Culex (Melanoconion) adults behaviour was carried out from August 1992 through December 1993 in human dominated (anthropic) environment in the Ribeira Valley, S.Paulo State, Brazil. By sampling at several sites it the dominance of Culex ribeirensis and Cx. sacchettae became evident even through a total number of ten species was recorded. Those two mosquitoes showed a clear tendency to frequent the domiciliary environment where they were caught, both indoor and outdoor, through the use of the human bait. In the outside environments, the residual patchy forests seems to display a concentration role, from which these adults spread to the open land and reach the dwellings. As their vector competence has been demonstrated through the virus isolations in natural conditions, it is advisable to pay attention to the presence of these mosquitoes in the man-made environment.

Studies on mosquitoes (Diptera: Culicidae) and anthropic environment: 10- survey of adult behaviour of Culex nigripalpus and other species of Culex (Culex) in South-Eastern Brazil

A survey of adult behaviour of Culex (Culex) species was carried out from August 1992 through December 1993 in a human modified (anthropic) environment in the Ribeira Valley, S.Paulo State, Brazil. Culex nigripalpus dominated the catches at several sites and it's tendency to increase in the anthropic environment became quite clear. Nevertheless no high level of synanthropy was demonstrated. So it seems that the mosquito may have a restricted role in natural arbovirus cycles. Nonetheless, Cx. nigripalpus must be considered a potential vector of arboviruses, especially St. Louis encephalitis virus outside dwellings.

New cases of human diphyllobothriosis in Patagonia, Argentine

The purpose of the paper is to describe 4 new cases of human diphyllobothriosis in Patagonia, Argentine. Adult parasites recovered were submitted to morphological and histological analyses for taxonomic identification. The etiological agent found was always Diphyllobothrium latum and all the cases were autochthonous. These data combined with previous information make the number of autochthonous human cases of diphyllobothriosis registered in Argentina increase to 18.

Aedes albopictus may not be vector of dengue virus in human epidemics in Brazil

Over 60,500 dengue cases were reported in the state of Espírito Santo (ES), Brazil, between 1995 and 1998. The study's purpose was to identify whether Aedes albopictus was transmitting the dengue virus during an epidemic in the locality of Vila Bethânia (Viana County),Vitória, ES. From April 3 to 9, 1998, blood and serum samples were collected daily for virus isolation and serological testing. Four autochthonous cases were confirmed through DEN 1 virus isolation and two autochthonous cases through MAC ELISA testing. Of 37 Ae. aegypti and 200 Ae. albopictus adult mosquitoes collected and inoculated, DEN1 virus was isolated only from a pool of two Ae. aegypti female mosquitoes. The study results suggest that Ae. albopictus still cannot be considered an inter-human vector in dengue epidemics in Brazil.

Immunopathology of human schistosomiasis mansoni: I. Immunomodulatory influences on T cell function

Cell mediated immune response was studied in patients with recent and chronic Schistosoma mansoni infection. Precultured peripheral mononuclear cells showed significantly higher responses to S. mansoni adult worm antigen (SAWA) when compared to fresh cell preparations. The addition of each patient serum to the precultured cells reactions to SAWA or recall antigens demonstrated a strong inhibitory serum action, which was also noted on allogeneic cells derived from healthy subjects. The CD4 subset was the main responding cell to SAWA being this reactivity highly suppressed by the presence of the monocyte macrophage accessory cells. We stressed the simultaneous inhibitory action of humoral and cellular factors on the specific cell response to S. mansoni.

Immunopathology of human schistosomiasis mansoni: II. NK activity and stimulation by specific antigen

Sixteen S. mansoni infected and untreated patients (5 with recent infection and 11 with chronic disease) were evaluated for their in vitro natural killer (NK) activity against the NK sensitive target K562 cell line. NK levels in 9 out of 11 patients (82%) with chronic disease were significantly lower (mean = 15 ± 6%),compared with patients recently infected (mean = 41 ± 9% p < 0.001) and with the control group (mean = 38 ± 13% p < 0.001). However, both patients and controls NK activity was stimulated by soluble adult worm antigens (SAWA), indicating that NK function even in the chronic stage of the infection is able to respond to the parasite antigens. These results suggest the possibility of NK cell participation as effector mechanism against S. mansoni.