Evaluation of a real-time PCR assay based on the repetitive B1 gene for the detection of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in human peripheral blood

In this paper, we examined the diagnostic value of a real-time polymerase chain reaction (PCR) using fluorescence resonance energy transfer (TaqMan assay) with a new set of primers and probe targeting the B1 gene to reproducibly detect and quantify Toxoplasma gondii in human blood. A total of 183 buffy coat samples from patients serologically classified as recent toxoplasmosis (immunoglobulin M (IgM)+, n = 35) or chronic infection (IgM- and immunoglobulin G (IgG)+, n = 110), and seronegative individuals (n = 38) was investigated. Of the IgM seropositive patients, 17:35 (48.6%) presented parasitaemia, whereas 3.6% positivity was achieved in those individuals that theoretically corresponded to chronic infection (4:110). In the seronegative group, the assay provided 7.9% (3/38) of positive results. Interestingly, one of them was confirmed as positive in a conventional PCR targeting the Toxoplasma B1 gene after hybridization with an internal probe. Real-time PCR was able to accurately quantify the parasite load when concentrations of T. gondii DNA are low, revealing a parasite burden ranged from 9.92 x 10(-3) to 8.73 x 10(-1) tachyzoites genome per milliliter of blood. The chance of an IgM+ patient to present parasitemia detected by the TaqMan procedure was 19.02 times greater than in IgM- individuals (P < 0.05). It was observed a positive association between the optical density values of the IgM serological tests and the number of circulating parasites in the acute patients (P < 0.0001). The specificity of the molecular test was 95.3% when calculated using IgM+ patients as disease group and IgM- as nondisease group. The low sensitivity observed in the IgM seropositive group (48.6%) could be due to the use of buffy coat as clinical material for DNA extraction. An amplification control based on the human beta-actin gene was used in parallel to monitor PCR inhibition and to control for DNA integrity.     

Vampire bat reproductive control by a naturally occurring phytooestrogen

Rabies transmission by wild animals has not being controlled satisfactorily. One major rabies vector to humans and cattle is the hematophagous vampire bat Desmodus rotundus whose distribution is still increasing in the Americas. Of all of the strategies currently in place to control this vector, none of them are really specific and some have ecological impacts. In the present study we used a naturally occurring phytoestrogen on a small vampire bat colony. After collection, bats were fed bovine blood containing 200 microg coumestrol for a 30-day period. After treatment, gonads were excised and processed for histological evaluation. Data indicate that coumestrol adversely affects gonad histology and has a possible impact on the fertility of both male and female vampire bats.     

Risk of domiciliation of Triatoma williami Galvão,Souza e Lima, 1965 in a municipality of Brazilian Legal Amazon region

ObjectiveTo notify the positivity and presence of vectors in natural and artificial ecotopes and analyze the basic knowledge of the Chagas disease vectors among population of amazon legal municipality.MethodsThe molecular confirmation of the parasite species was by PCR using species-specific markers. Data collection was conducted using semi-structured interviews.ResultsAll of 34 specimens collected were classified to be a single species, Triatoma williami. The natural infection rate of vectors in the specimens by T. cruzi was 30%. Most interviewees recognized adult triatomines. For 24.43% of respondents who had found the vector inside the house the main practice reported was killing the insects by crushing.ConclusionsDespite the knowledge shown by the residents, educational measures are needed to improve entomological surveillance of Chagas disease into enzootic amazon area.     

Low accumulation of L90M in protease from subtype F HIV-1 with resistance to protease inhibitors is caused by the L89M polymorphism

BACKGROUND: This work evaluates the role of subtype F human immunodeficiency virus type 1 (HIV-1) protease (PR) substitutions L89M and L90M in viral replication and resistance to PR inhibitors (PIs). METHODS: Subtype B and F PR genes were subjected to site-directed mutagenesis, to create and reverse the methionine at positions 89 and 90. Viruses were re-created in cell culture, and their replicative capacity was assessed by fitness assay. Generated viruses were also phenotyped for PI resistance. RESULTS: The subtype F clone (89M90L) showed a replicative capacity comparable to that of the PI-susceptible subtype B clone (89L90L) and was more fit than the L89M mutated subtype B clone (89M90L). Both 89M90M subtype B and F clones presented the lowest fitness s values. The L89M mutation impacted phenotypic resistance to all PIs in half of the subtype F isolates but not in the subtype B isolates. Subtype F isolates presented a phenotypic profile similar to that of subtype B isolates when the M89L mutation was introduced. CONCLUSION: The L89M mutation in subtype F viruses is a high genetic barrier to the accumulation of the L90M resistance mutation and can function as a resistance mutation, depending on the presence of other polymorphisms in the subtype F PR backbone.     

Nefrectomía laparoscópica simple con asistencia de notes transvaginal y uso de instrumentos laparoscópicos estándar

IntroductionNatural orifice transluminal endoscopic surgery (NOTES) is an emerging technique, which allows to performed surgical procedures avoiding any surgical scars. However there are some problems due to the lack of equipment available for these procedures. The aim of these study is to present our initial experience with the transvaginal nephrectomy NOTES using standard laparoscopic instruments.Material and MethodsTwo female patients 23 and 29 years old, both of them with diagnosis of recurrent urinary tract infection and renal atrophy. A transvaginal simple nephrectomy was performed using a transvaginal Access for the camera port and two abdominal work ports of 10 and 3mm.ResultsTotal operation room time was 120 min in the first case and 40 min. in the second. with an average blood loss of 200 cc. There were no perioperative complications, and both patients was discharged 36 hours after the surgeryConclusionLaparoscopic nephrectomy with transvaginal NOTES assistance is technically feasible with the use of standard laparoscopic instruments. Special Access trocars and instruments development for this procedure will allow to perform a pure technique without the use of abdominal incisions.     

Higher cytotoxic activity and increased levels of IL-1β, IL-6, and TNF-α in patients undergoing cardiopulmonary bypass

Extracorporeal circulation (EC) by surgical bypass is often associated with a systemic inflammatory response. The purpose of this study was to assess the effect of EC on the serum levels of pro-inflammatory mediators Interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and soluble CD-23. Peripheral blood lymphocyte subsets and natural killer cell (NK) cytotoxic activity were also analyzed before and after the bypass process. The results from eight patients who underwent cardiac surgery showed a significant increase in the levels of IL-1β, IL-6, and TNF-α, a decrease in CD4⁺/CD8⁺ lymphocyte ratio, and an overstimulated NK cytotoxic activity. These changes on serum cytokine levels and cellular immunology parameters could play an important role in the development of adverse effects associated with EC. © 1995 Wiley-Liss, Inc.