Detection and quantification of hepatitis E virus in the absence of IgG and IgM anti-HEV in HIV-positive patients.

AIMS: To improve RT-qPCR with an internal control and a synthetic standard curve to detect HEV in HIV co-infected patients. METHODS AND RESULTS: A single-stranded RNA (ssRNA) and a double-stranded DNA (dsDNA) synthetic curve were designed, compared to the international reference panel for HEV genotypes, and tested to quantify and detect a reference panel for HEV genotypes. The detection limit of the RNA synthetic curve (50 copies per ml) was better than the DNA synthetic curve (100 copies per ml) and the WHO standard curve (250 copies per ml). Then, 280 serum samples from HIV-positive patients were tested for HEV RNA, which was detected in 3·6% of serum samples. The viral load ranged from 2 × 102 copies per ml to 4·78 × 108 copies per ml. HEV IgM/IgG antibodies were not detected in the RNA-positive patients. Sequencing analysis of HEV showed that the virus belongs to genotype 3 (HEV GT3). CONCLUSIONS: Real-time PCR was a useful tool to estimate co-infection with HEV/HIV, even in patients with low viral loads and undetectable anti-HEV IgG and IgM antibodies. SIGNIFICANCE AND IMPACT OF THE STUDY: Hepatitis E virus genotype 3 (HEV GT3) has been associated with silent chronic hepatitis and cirrhosis in HIV-positive subjects worldwide, but there is a lack of data on this co-infection in Brazil.

Enteric viruses' dissemination in a private reserve of natural heritage.

This study aimed to assess anthropogenic impact of surrounding population in the Private Reserve of Natural Heritage at Pantanal, the world's largest freshwater wetland ecosystem located in the centre of South America. Viral aetiological agents of acute gastroenteritis as rotavirus A (RVA), noroviruses, human adenoviruses, klassevirus and of hepatitis, as hepatitis A virus, were investigated in different aquatic matrices. Annual collection campaigns were carried out from 2009 to 2012, alternating dry and rainy seasons. Viral particles present in the samples were concentrated by the adsorption-elution method, with negatively charged membranes, and detected by qualitative and quantitative PCR. From a total of 43 samples at least one virus was detected in 65% (28) of them. Viruses were detected in all matrices with concentrations ranging from 2 × 102 to 8·3 × 104 genome copies per litre. A significant higher RVA frequency was observed in the dry season. Our data revealing dissemination of human enteric viruses in water matrices both inside and outside the reserve could be useful to trace faecal contamination in the environment and to minimize the risk of infection by exposure of susceptible individuals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is part of a collaborative project designed to investigate the environmental and health conditions of the Private Reserve of Natural Heritage at Pantanal, the largest seasonally flooded wetland in the world. The project aimed to promote health and quality of human and wildlife extending technical-scientific knowledge about pathogens present in the region. By assessing the occurrence of human enteric viruses in different water matrices we demonstrated the anthropogenic impact of surrounding population and pointed out the potential risk of infection by exposure of susceptible individuals.

Tomato plants use non-enzymatic antioxidant pathways to cope with moderate UV-A/B irradiation: A contribution to the use of UV-A/B in horticulture.

Plants developed receptors for solar UV-A/B radiation, which regulate a complex network of functions through the plant's life cycle. However, greenhouse grown crops, like tomato, are exposed to strongly reduced UV radiation, contrarily to their open-field counterparts. A new paradigm of modern horticulture is to supplement adequate levels of UV to greenhouse cultures, inducing a positive mild stress necessary to stimulate oxidative stress pathways and antioxidant mechanisms. Protected cultures of Solanum (cv MicroTom) were supplemented with moderate UV-A (1h and 4h) and UV-B (1min and 5min) doses during the flowering/fruiting period. After 30days, flowering/fruit ripening synchronization were enhanced, paralleled by the upregulation of blue/UV-A and UV-B receptors' genes cry1a and uvr8. UV-B caused moreover an increase in the expression of hy5, of HY5 repressor cop1 and of a repressor of COP1, uvr8. While all UV-A/B conditions increased SOD activity, increases of the generated H2O2, as well as lipid peroxidation and cell mebrane disruption, were minimal. However, the activity of antioxidant enzymes downstream from SOD (CAT, APX, GPX) was not significant. These results suggest that the major antioxidant pathways involve phenylpropanoid compounds, which also have an important role in UV screening. This hypothesis was confirmed by the increase of phenolic compounds and by the upregulation of chs and fls, coding for CHS and FLS enzymes involved in the phenylpropanoid synthesis. Overall, all doses of UV-A or UV-B were beneficial to flowering/fruiting but lower UV-A/B doses induced lower redox disorders and were more effective in the fruiting process/synchronization. Considering the benefits observed on flowering/fruiting, with minimal impacts in the vegetative part, we demonstrate that both UV-A/B could be used in protected tomato horticulture systems.

The influence of Citrate or PEG coating on silver nanoparticle toxicity to a human keratinocyte cell line.

Surface coating of silver nanoparticles may influence their toxicity, in a way yet to decipher. In this study, human keratinocytes (HaCaT cells) were exposed for 24 and 48h to well-characterized 30nm AgNPs coated either with citrate (Cit30 AgNPs) or with poly(ethylene glycol) (PEG30 AgNPs), and assessed for cell viability, reactive oxygen species (ROS), cytokine release, apoptosis and cell cycle dynamics. The results showed that Cit30 AgNPs and PEG30 AgNPs decreased cell proliferation and viability, the former being more cytotoxic. The coating molecules per se were not cytotoxic. Moreover, Ag(+) release and ROS production were similar for both AgNP types. Cit30 AgNPs clearly induced apoptotic death, while cells exposed to PEG30 AgNPs appeared to be at an earlier phase of apoptosis, supported by changes in BAX, BCL2 and CASP-3 expressions. Concerning the impact on cell cycle dynamics, both Cit30 and PEG30 AgNPs affected cell cycle regulation of HaCaT cells, but, again, citrate-coating induced more drastic effects, showing earlier downregulation of cyclin B1 gene and cellular arrest at the G2 phase. Overall, this study has shown that the surface coating of AgNPs influences their toxicity by differently regulating cell-cycle and cell death mechanisms.

Renal hemodynamic response to an angiotensin II antagonist, eprosartan, in healthy men.

In view of the vasodilator potential of angiotensin-converting enzyme (ACE) inhibition via prostaglandins and kinins, we asked why renin inhibition induces a larger renal vasodilator response than ACE inhibitors in healthy humans in earlier studies. One possibility was that there was a more complete blockade of the renin system, which could also be achieved by an angiotensin II antagonist, eprosartan. We measured the hormonal and renal hemodynamic responses to eprosartan doses, from 10 to 400 mg in 9 healthy young men in balance on a 10-mmol/d sodium intake. The threshold eprosartan dose to influence renal perfusion was <10 mg, and the 100-mg dose induced a near-maximal vasodilator response of 135+/-19.7 mL x min(-1) x 1.73 m2. When the dose was increased to 400 mg, there was a modest additional increase of 147+/-57 mL x min(-1) x 1.73 m(-2). A highly significant dose-related fall in arterial blood pressure occurred (r=-.97; P<.001), with no indication of a maximal response at 400 mg. In 6 additional subjects, we compared responses to eprosartan on a high salt and a low salt diet. The renal response to 200 mg eprosartan on a high salt diet, 26.0+/-6.6 mL x min(-1) x 1.73 m(-2), was significantly less than that seen with the low salt diet (P<.001). There was no renal partial agonist angiotensin-like effect of eprosartan. Eprosartan reduced sharply the pressor, renal vascular, and hormonal responses to exogenous angiotensin II. The renal vasodilator response to the angiotensin II antagonist eprosartan closely resembles responses to renin inhibition and exceeds previously reported responses to ACE inhibitors. Thus, eprosartan probably exerted its effect via the angiotensin receptor. More complete blockade of the renin system can be achieved by pharmacological interruption at this level, a finding that could have therapeutic implications.

In vitro effect of fibrinogen on Candida albicans germ tube formation.

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.

The state and responsiveness of the renin-angiotensin-aldosterone system in patients with type II diabetes mellitus.

We have recently reported a combination of renal features that suggests independent angiotensin-mediated control of the renal circulation in the majority of hypertensive patients with type II diabetes. To ascertain whether other tissue elements of the renin-angiotensin-aldosterone system (RAAS) also were activated, we examined the adrenal response to angiotensin II (AngII) infusion on a low salt diet. We assessed also the renin response to the upright position in patients on a low salt diet and renin suppression in patients on a high salt diet. We compared responses in 42 hypertensive patients with type II diabetes (53.1 +/- 1.4 years, mean +/- SEM), 25 healthy controls (52.6 +/- 4.4 years); and 137 essential hypertensives without diabetes (43.3 +/- 1.2 years). A low renin state, defined as a plasma renin activity (PRA) <2.5 ng angiotensin I (AI)/mL/h after 5 to 7 days on a 10-mmol Na diet and 2 h of upright posture, was found in 21% of the essential hypertensives, but in only 14% of patients with type II diabetes. On this diet, PRA increased from 2.7 +/- 0.4 supine to 10.1 +/- 1.3 ng AI/mL/h upon standing in healthy subjects. In patients with type II diabetes, PRA was 3.6 +/- 0.4 and increased to 9.1 +/- 1.0 ng AI/mL/h. On a high salt (200 mmol) diet, healthy subjects showed the expected PRA suppression (0.3 +/- 0.1), but in patients with type II diabetes the PRA was less suppressed (1.2 +/- 0.3 ng AI/mL/h; P = .003). Thus, in most hypertensive patients with type II diabetes the RAAS shows normal activation, but is poorly suppressible. AngII infused intravenously to assess adrenal responsiveness in patients on a low salt diet caused an essentially identical increase in aldosterone concentration in patients with type II diabetes (21.1 +/- 1.7 to 44.0 +/- 5.9 ng/dL) and in essential hypertension (20.6 +/- 1.4 to 43.7 +/- 2.8 ng/ dL). The frequency of nonmodulation assessed as a blunted adrenal response to AngII infusion was identical in type II diabetes (47%) and in essential hypertension (46%) after exclusion of the low renin patients. Thus, at the level of one tissue renin system, the adrenal glomerulosa, responses were unaltered in patients with type II diabetes. The relative unresponsiveness of the renal blood supply to infused AngII in type II diabetes in association with an enhanced renal vasodilator response to angiotensin converting enzyme inhibition probably reflects local, intrarenal actions secondary to the diabetic state. The infrequency of a low renin state, and the inappropriately high renin levels on a high salt intake, provide a rational basis for pharmacologic interruption of the renin system to treat patients with type II diabetes.

Hepatitis A virus infection in hepatitis C Brazilian patients.

OBJECTIVE: HAV infection in patients with pre-existing chronic liver disease has been associated with increased rate of fulminant hepatitis and mortality. The aim of this study was to investigate the presence of serological and molecular HAV markers in a population of HCV infected patients. PATIENTS AND METHODS: The presence of total and IgM anti-HAV antibodies was investigated in 197 patients (mean age 44.8+/-12.5 years) referred to the Brazilian Reference Center for Viral Hepatitis and who tested positive for anti-HCV antibodies and HCV RNA. HAV RNA was investigated by reverse transcription-nested PCR in these patients.Results. One hundred seventy patients (86%) had total, but not IgM anti-HAV antibodies, being therefore, immune to hepatitis A, while 27 (14%) were not. A high proportion (6/27, 22%) of the susceptible patients presented markers of recent HAV infection: One patient was IgM anti-HAV positive, three were HAV RNA positive, and two presented both markers. By nucleotide sequencing, it was demonstrated that the HAV isolates infecting these patients belonged to subgenotypes 1A and 1B. CONCLUSIONS: Superinfection with HAV was a common event in the group of HCV infected patients under study. Implementation of hepatitis A vaccination should be considered for this population.

Staged carotid and coronary surgery for concomitant carotid and coronary artery disease.

OBJECTIVE: To demonstrate that staged, consecutive, carotid endarterectomy (CEA) and coronary artery bypass grafting (CABG) are safe, perhaps preferable, alternative for the treatment of patients with severe carotid and coronary artery disease. METHODS: During an 8-year period ending December 1999, 77 (2.1%) of 3633 consecutive patients who were referred for isolated coronary surgery were found to have significant carotid disease and underwent CEA, and subsequently, CABG. The mean age was 65.2 +/- 5.9 years and 66 (85.7%) were males. The majority (84.4%) had triple vessel and 19.4% had left main disease. Carotid disease was unilateral in 71 patients (92.2%) and bilateral in six (7.8%), and 57 (74.0%) were neurologically asymptomatic. Only obstructions >70% were considered for endarterectomy. RESULTS: Eighty-three isolated CEAs were performed with direct clamping of the artery (mean 20.1 +/- 5.9 min) in all but one. There were no deaths. There were two strokes (2.4%) and three (3.6%) myocardial infarctions (MI). The mean admission time was 6.0 +/- 3.5 days. The staging interval was 32.4 days. During coronary surgery, a mean of 2.9 coronary grafts/patient was performed and all but one patient received at least one IMA graft. One patient (1.3%) died. There were two cases (2.6%) of MI and three patients (3.9%) had a stroke. Hence, the overall rates of perioperative mortality, MI and stroke were 1.3, 6.3 and 6.3%, respectively. The mean admission time was 8.3 +/- 6.0 days. CONCLUSIONS: Staging of carotid and coronary operations resulted in low global perioperative mortality and morbidity rates in these high-risk patients and is a good alternative therapeutic option.

Involucrin expression in vulvar lesions.

Involucrin, a precursor of the envelope protein present in human stratum corneum, may be used as a histologic marker. Involucrin expression was studied in biopsy specimens from different types of vulvar lesions. In general, the pattern of staining for involucrin has been the one described for similar pathologies of the integument in other bodily areas. However, an unexpected pattern was noted in lichen sclerosus of the vulva.

Prevalence of Candida albicans in vaginal fluid of asymptomatic Portuguese women.

Candida albicans is by far the most frequent agent of genital candidosis. We studied the prevalence of C albicans in normal asymptomatic women attending primary health care centers throughout Portugal. The overall prevalence of C albicans in the vaginal fluid of 1,004 women studied was 10.4%. Interestingly, the prevalence rates were lower (6.8%) in women taking combination oral contraceptives and higher (13.0%) in those using intrauterine devices.

PIP: In Portugal, a physician from a rural primary health care center and one from an urban primary health care center from each of 18 districts took vaginal swabs from 1004 14-to-80-year-old asymptomatic women to send to a laboratory at the University of Porto School of Medicine, where staff examined 1 week old cultures for yeast cells. This vaginal fluid collection was part of a research project of the Portuguese Group for the Study of Pelvic Inflammatory Disease and Sexually Transmitted Diseases based at the University's Departments of Microbiology, Biostatistics, and Hygiene and Social Medicine. They confirmed 104 positive Candida albicans cultures (10.4%). C. albicans prevalence was considerably higher in women using an IUD than it was in women using an oral contraceptive (13% vs. 6.8%; p = .03). Prevalence was also greater among women whose vaginal fluid had a macroscopic appearance suspicious of candidosis than among those whose vaginal fluid did not have such an appearance (p .05). Pregnant women had the highest colonization rates. The various means for daily genital care (i.e., soap vs. antiseptics) did not influence C. albicans prevalence. These results were probably representative of the female population in Portugal.

[Prevalence of serological markers of hepatitis B in a small rural community of São Paulo State, Brazil]. / Prevalência de marcadores sorológicos de hepatite B numa pequena comunidade rural do Estado de São Paulo, Brasil.

Prevalence of three hepatitis B markers was measured by immunoassay techniques in small rural community of the State of S. Paulo, Brazil. Total prevalence was 7.74%, corresponding to values of 0.10%, 1.69% and 7.74% for HBsAg, anti-HBs and anti-HBc, respectively. The importance of anti-HBc determination in such studies is stressed. Comparisons between the low viral circulation observed in this area and high prevalences described in other rural communities may contribute to the raising of new hypothesis concerning alternative transmission mechanisms of hepatitis B.

[Influence of migration on the prevalence of serologic hepatitis B markers in a rural community. 2. Comparative analysis of various characteristics of the population studied]. / Influência da migração na prevalência de marcadores sorológicos de hepatite B em comunidade rural. 2-Análise comparativa de algumas caracteristicas das populações estudadas.

The association between prevalence of hepatitis B serological markers and birthplace, in a study carried out in a small rural county of S. Paulo State, Brazil, suggests different risk factors for hepatitis B between migrants and nonmigrant populations. These two groups were compared with regard to the following variables: level of education, professional occupation, number of previous hospitalizations, past history of blood transfusions and type of dental treatment. Migrants, mainly those from other states of Brazil, showed a low-level of education, a high proportion of people employed in agricultural activities, a higher number of past hospitalizations and higher exposure to blood transfusion and to more aggressive dental procedures. Associations were observed between the prevalence of serological markers and the following variables: level of education, professional occupation, number of previous hospitalizations and type of dental procedures, even though the last two associations did not justify the higher prevalences observed among migrants. The different distribution of hepatitis B markers seems to be dependent on the migrants' worse socio-economic condition, demonstrated by their lower level of education and by the predominance of secondary occupations.

[Influence of migration on the prevalence of serologic hepatitis B markers in a rural community. 1. Analysis of prevalence by birthplace]. / Influência da migração na prevalência de marcadores sorológicos de hepatite B em comunidade rural. 1-Análise da prevalência segundo local de nascimento.

Distribution of hepatitis B serological markers according to people's birthplaces and area of residence (urban/rural) was studied as a part of a field epidemiological research project carried out in Cássia dos Coqueiros, a small rural community of S. Paulo State, Brazil. The total prevalence of HBV markers was 7.7%, with rural showing a higher risk than urban inhabitants (9.8% as against 4.9%, respectively). Analysis of prevalence according to people's birthplaces revealed the highest value among those from other Brazilian states (15.8%), followed by people from other municipalities of the State of S. Paulo (9.2%). Those born in Cássia dos Coqueiros and particularly in Ribeirão Preto (the main city of the area, located 80 kilometers away), showed the lowest values of prevalence (5.2% and 2.5%, respectively). The importance of studying people's birthplaces when field epidemiological studies on hepatitis B are carried out is stressed. This variable is considered to be capable of exercising an influence on the natural history of the disease in a community, and may even explain differences in the distribution of markers in apparently similar populations.

[The surgical treatment of acute myocardial infarct in the 90s]. / O tratamento cirúrgico do enfarte agudo do miocárdio nos anos noventa.

At present, the treatment for acute myocardial infarction is revascularization during the critical initial period of six hours after the beginning of coronary occlusion. Despite the fact that surgery performed within this time period presents a hospital of mortality around 2%, and with excellent results in the long term, it is seldom used due to logistic limitations and capabilities of hospital infrastructures, high costs and the possibility of the surgical team initiating surgery inside the useful time period. Surgery is thus limited to the patients with suitable anatomy, who are not candidates or had failure of thrombolytic/angioplasty therapy and are in the six-hour period after initiation of symptoms. Surgery performed at a later stage has good results if performed in a non emergency situation, specially after the first 72 hours. Surgery continues to be the only treatment for the mechanical complications of infarction, and good results have recently been shown in ventricular septal ruptures, with hospital mortality of 14%, due to the use of an endoventricular patch in patients operated early, before the consequences of low cardiac output develop at systemic level. In the surgical treatment of mitral regurgitation, the tendency has been to use repair techniques whenever possible, but still with hospital mortality up to 15%. The recent advances of the techniques and tactics of myocardial preservation during surgery have made a very significant contribution to the better results we see today.

Development and validation study for the chromatographic purification process for tetanus anatoxin on Sephacryl S-200 High Resolution.

The tetanus purified anatoxin is used in the preparation of multiple immunoprophylactics. WHO (World Health Organization) specifies that the tetanus anatoxin must exhibit a degree of purity greater than or equal to 1,000 Lf/mg protein nitrogen (PN). Today liquid chromatography is a well established technique for the purification of tetanus anatoxin and several different methods are used in production scale. On a small scale, we purified tetanus anatoxin on Sephacryl S-200 High Resolution (gel filtration) and we obtained a successful high-yield purification. On the basis of these results, by combining conventional tangential flow filtration (TFF) at 50,000 N.M.W.L. (Nominal Molecular Weight Limit) ultrafiltration membrane with gel filtration on Sephacryl S-200 High Resolution, we have been able to purify 14 lots of tetanus anatoxin using the Bioprocess System (Amersham Pharmacia Biotech) to a large scale operation. Using this method, 77,401,332 doses of tetanus toxoid were prepared in 14 consecutive lots, supporting the reproducibility and reliability of the method presented here.

Use of sephacryl high resolution (HR) in tetanus anatoxin purification.

The tetanus purified anatoxin is used in the preparation of the tetanus toxoid and multiple vaccines (dT, DT and DTP), all of them strictly following specifications established by the WHO with a minimum antigenic purity equal to 1,000 Lf/mgPN. Aiming to establish more sensitive and accurate methods for purification, samples from four different lots of tetanus anatoxin were submitted to gel filtration in twenty independent trials using the Sephacryl S-100 HR and S-200 HR resins. The Authors were careful to optimize their parameters of performance as to sample volume, elution and selectivity flow for tetanus anatoxin purification, allowing their use in industrial scale. The Sephacryl S-100 HR resin presented the best selectivity, that is, the best separation, allowing a greater linear-flow and, consequently, the best purity index. Satisfactory results were also achieved with the Sephacryl S-200 HR resin after optimization of chromatographic parameters for elution flow and volume of the sample applied. The good results of purification obtained, as well as the high chemical stability, have pointed out both the Sephacryl S-100 HR and S-200 HR resins as equally efficient for industrial production.

A data mining system for providing analytical information on brain tumors to public health decision makers.

Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. Malignant brain neoplasms are among the most devastating and incurable forms of cancer, and their treatment may be excessively complex and costly. Public health decision makers require significant amounts of analytical information to manage public treatment programs for these patients. Data mining, a technology that is used to produce analytically useful information, has been employed successfully with medical data. However, the large-scale adoption of this technique has been limited thus far because it is difficult to use, especially for non-expert users. One way to facilitate data mining by non-expert users is to automate the process. Our aim is to present an automated data mining system that allows public health decision makers to access analytical information regarding brain tumors. The emphasis in this study is the use of ontology in an automated data mining process. The non-experts who tried the system obtained useful information about the treatment of brain tumors. These results suggest that future work should be conducted in this area.