The use of polymerase chain reaction for early diagnosis of tuberculosis in Mycobacterium tuberculosis culture

Early diagnosis plays a vital role in controlling tuberculosis. The conventional methodology is slow, with results taking several weeks, in addition to having low sensitivity, especially in clinical paucibacillary samples. The objective of this study was to evaluate the use of polymerase chain reaction (PCR) on solid medium culture for a rapid diagnosis of tuberculosis, mainly in cases of negative sputum smears. Forty sputum samples were collected from inpatients with tuberculosis treated for less than 2 days. Bacilloscopy, PCR for sputum, culture on Löwestein-Jensen (LJ) solid medium, and daily PCR from culture were performed on each sample. DNA extracted from the BCG vaccine, which contains attenuated bacillus Calmette-Guérin, was used as the positive control. Smear microscopy showed 68.6% sensitivity, 80% specificity, 96% positive predictive value, and 26.7% negative predictive value, with culture on LJ medium as the gold standard. Culture at day 28 showed 74.3% sensitivity and 100% specificity. PCR of DNA extracted from sputum amplified a 1027-bp fragment of the 16s RNA gene, showing 22.9% sensitivity and 60% specificity. PCR performed with DNA extracted from daily culture showed that, from the 17th to the 40th day, the sensitivity (85.7%) and specificity (60%) were constant. We conclude that a 17-day culture is a good choice for rapid diagnosis and to interfere with the transmission chain of tuberculosis.

A note on the Pazos-Simonsen mechanism and Kaldor's early research on Latin American inflation

In contrast with the inflationary finance story, inflation acceleration in Latin America has been explained as the result of the interaction of inflation dynamics and the frequency of wage adjustments. Accordingly, small inflation disturbances are connected with a shift from moderate to high inflation (or beyond to hyperinflation) though a mechanism that makes adjustment intervals in wage contracts endogenous. Rudiger Dornbusch (1986) labeled this process the "Pazos-Simonsen mechanism". In this note we summarize the basic contribution of both Felipe Pazos (1978) and Mario Henrique Simonsen (1983) and find crucial differences between their views on wage dynamics, specifically regarding the endogeneity of the time interval between wage readjustments. A remarkable affinity with Pazos's view on wage dynamics and inflation is found in an early and almost unknown essay written by Nicholas Kaldor in 1957 (inspired in his brief experience in Latin America).

Trypanosoma cruzi isolated from a triatomine found in one of the biggest metropolitan areas of Latin America

Abstract: INTRODUCTION: To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. METHODS: The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in São Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. RESULTS: After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. CONCLUSIONS: Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite.

Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens

The present report describes an alternative method for in vitro detection of HIV-1 -specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analized. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection.

The early use of yellow fever virus strain 17D for vaccine production in Brazil - a review

The use of yellow fever (YF) virus 17D strain for vaccine production adapted in Brazil since its introduction in 1937 was reviewed. This was possible due to the availability of official records of vaccine production. The retrieved data highlight the simultaneous use of several serially passaged 17D substrain viruses for both inocula and vaccine preparation that allowed uninterrupted production. Substitution of these substrain viruses became possible with the experience gained during quality control and human vaccination. Post-vaccinal complications in humans and the failure of some viruses in quality control tests (neurovirulence for monkeys) indicated that variables needed to be reduced during vaccine production, leading to the development of the seed lot system. The 17DD substrain, still used today, was the most frequently used substrain and the most reliable in terms of safety and efficacy. For this reason, it is possible to derive an infectious cDNA clone of this substrain combined with production in cell culture that could be used to direct the expression of heterologous antigens and lead to the development of new live vaccines.

Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

The parasitic protozoan Leishmania (Leishmania) amazonensis alternates between mammalian and insect hosts. In the insect host, the parasites proliferate as procyclic promastigotes andthen differentiate into metacyclic infective forms. The meta 1 gene is preferentially expressed during metacyclogenesis. Meta 1 expression profile determination along parasite growth curves revealed that the meta 1 mRNA level peaked at the early stationary phase then decreased to an intermediate level. No correlation was observed between meta 1 expression and infectivity. Conversely, infectivity correlated with the increase of apoptotic cells in the late stationary phase.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Role of cyclooxygenase-2 in Trypanosoma cruzisurvival in the early stages of parasite host-cell interaction

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

Advances in apple culture worldwide

Over the last 60 years, planting densities for apple have increased as improved management systems have been developed. Dwarfing rootstocks have been the key to the dramatic changes in tree size, spacing and early production. The Malling series of dwarfing rootstocks (M.9 and M.26) have been the most important dwarfing rootstocks in the world but are poorly adapted in some areas of the world and they are susceptible to the bacterial disease fire blight and the soil disease complex, apple replant disease which limits their uses in some areas. Rootstock breeding programs in several parts of the world are developing improved rootstocks with resistance to fire blight, and replant disease, and improved cold hardiness and yield efficiency. A second important trend has been the increasing importance of new cultivars. New cultivars have provided opportunities for higher prices until they are over-produced. A new trend is the "variety club" in which variety owners manage the production and marketing of a new unique cultivar to bring higher prices to the growers and variety owners. This has led to many fruit growers being unable to plant or grow some new cultivars. Important rootstock and cultivar genes have been mapped and can be used in marker assisted selection of future rootstock and cultivar selections. Other important improvements in apple culture include the development of pre-formed trees, the development of minimal pruning strategies and limb angle bending which have also contributed to the dramatic changes in early production in the 2nd-5th years after planting. Studies on light interception and distribution have led to improved tree forms with better fruit quality. Simple pruning strategies and labor positioning platform machines have resulted in partial mechanization of pruning which has reduced management costs. Improved plant growth regulators for thinning and the development of a thinning prediction model based on tree carbohydrate balance have improved the ability to produce the optimum fruit size and crop load. Other new plant growth regulators have also allowed control of shoot growth, control of preharvest fruit drop and control of fruit softening in storage after harvest. As we look to the future, there will be continued incremental improvement in our understanding of plant physiology that will lead to continued incremental improvements in orchard management but there is likely to be dramatic changes in orchard production systems through genomics research and genetic engineering. A greater understanding of the genetic control of dwarfing, precocity, rooting, vegetative growth, flowering, fruit growth and disease resistance which will lead to new varieties and rootstocks which are less expensive to grow and manage.

The role of arbuscular mycorrhizal fungi on the early-stage restoration of seasonally dry tropical forest in Chamela, Mexico

It was evaluated the effect of two different sources of local inocula from two contrasting sites (mature forest, pasture) of arbuscular mycorrhizae fungi (AMF) and a non-mycorrhizal control on the plant growth of six woody species differing in functional characteristics (slow-, intermediate- and fast-growth), when introduced in a seasonally tropical dry forest (STDF) converted into abandoned pasture. Six plots (12 X 12m) were set as AMF inoculum source. Six replicates of six different species arranged in a Latin Square design were set in each plot. Plant height, cover area and the number of leaves produced by individual plant was measured monthly during the first growing season in each treatment. Species differed in their ability to benefit from AMF and the largest responsiveness in plant height and leaf production was exhibited by the slow-growing species Swietenia humilis, Hintonia latiflora and Cordia alliodora. At the end of the growing season (November), the plant height of the fast growing species Tabebuia donnel-smithii, Ceiba pentandra and Guazuma ulmifolia were not influenced by AMF. However, inocula of AMF increased leaf production of all plant species regardless the functional characteristics of the species, suggesting a better exploitation of above-ground space and generating a light limited environment under the canopy, which contributed to pasture suppression. Inoculation of seedlings planted in abandoned pasture areas is recommended for ecological restoration due to the high responsiveness of seedling growth in most of species. Use of forest inoculum with its higher diversity of AMF could accelerate the ecological restoration of the above and below-ground comunities.

Floristics, structure and soil of insular vegetation in four quartzite-sandstone outcrops of "Chapada Diamantina", Northeast Brazil

Soil islands on rocky surfaces often harbor aggregated vegetation that consists of insular plant communities. These islands are typical of the rocky outcrops and in various parts of Brazil form the so-called "campos rupestres" vegetation. Four of such sites have been selected in the state of Bahia, Northeast Brazil, for this comparative study on floristics and vegetation structure: three areas situated inside the "Parque Nacional da Chapada Diamantina" (Guiné, Fumaça and "Gerais da Fumaça") and one is at the border of the Environmental Protection Area of "Marimbus-Iraquara" ("Mãe Inácia"). All occurring vegetation islands were studied in four random plots of 10 × 10 m per site. Soil was often shallow, sandy and acidic. Vascular plant species were determined, with respective life forms and canopy coverage areas. The total number of species when all four sites were added was 135, and the number of species per island varied from 2 to 32. The areas of the 214 soil islands varied from 0.015 to 91.9 m², totaling 568 m² in the four sites. Monocotyledon families were dominant, essentially Velloziaceae, as well as Orchidaceae, Bromeliaceae, Amaryllidaceae and Cyperaceae. Among the eudicotyledons, dominant families were mainly Clusiaceae, Asteraceae and Melastomataceae. The biological spectra revealed that phanerophytes and hemicryptophytes predominated among the life forms, while chamaephytes had the largest coverage area. Epilithic and desiccant chamaephytes composed the most conspicuous interspecific associations, and were probably related to early successional processes. Sites closest to one another were not the most similar in structure, indicating that other factors more relevant than distance might be involved in the abundance of species in space.

Effect of actinomycin D on simian rotavirus (SA11) replication in cell culture

Rotaviruses are the major cause of viral diarrhea in humans and animals. Actinomycin D (Act D) is an antibiotic that intercalates DNA and therefore inhibits DNA-dependent transcription. The current study was carried out to assess the influence of Act D on the replication of simian rotavirus (SA11) in cell culture. Virus-infected MA-104 cell cultures were studied in the presence of Act D at concentrations of 1.25 and 2.5 µg/ml. Treatment of rotavirus-infected cells with 2.5 µg/ml Act D 48 h post-infection reduced the cytoplasmic metachromasia after staining with acridine orange by 25%. Viral RNA labeled with ³H-uridine in the presence of the drug was separated by polyacrylamide gel electrophoresis. Viral RNA replication was not affected by Act D, but increased ³H-uridine uptake was demonstrable by infected cells in the presence of the drug. This possibly was due to the inhibition of cellular RNA synthesis by Act D, which thus enhances incorporation of the radionuclide into the viral RNA. Act D reduced the number of infected cells presenting virus-specific fluorescence 48 h post-infection by more than 50%. These data suggest that Act D may have complexed with viral RNA and prevented newly synthesized mRNA from being translated, but may not have prevented early replication.

Cholesterol induces fetal rat enterocyte death in culture

The effect of cholesterol on fetal rat enterocytes and IEC-6 cells (line originated from normal rat small intestine) was examined. Both cells were cultured in the presence of 20 to 80 µM cholesterol for up to 72 h. Apoptosis was determined by flow cytometric analysis and fluorescence microscopy. The expression of HMG-CoA reductase and peroxisome proliferator-activated receptor gamma (PPARgamma) was measured by RT-PCR. The addition of 20 µM cholesterol reduced enterocyte proliferation as early as 6 h of culture. Reduction of enterocyte proliferation by 28 and 41% was observed after 24 h of culture in the presence and absence of 10% fetal calf serum, respectively, with the effect lasting up to 72 h. Treatment of IEC-6 cells with cholesterol for 24 h raised the proportion of cells with fragmented DNA by 9.7% at 40 µM and by 20.8% at 80 µM. When the culture period was extended to 48 h, the effect of cholesterol was still more pronounced, with the percent of cells with fragmented DNA reaching 53.5% for 40 µM and 84.3% for 80 µM. Chromatin condensation of IEC-6 cells was observed after treatment with cholesterol even at 20 µM. Cholesterol did not affect HMG-CoA reductase expression. A dose-dependent increase in PPARgamma expression in fetal rat enterocytes was observed. The expression of PPAR-gamma was raised by 7- and 40-fold, in the presence and absence of fetal calf serum, respectively, with cholesterol at 80 mM. The apoptotic effect of cholesterol on enterocytes was possibly due to an increase in PPARgamma expression.

Role of a GenoType MTBDRplus line probe assay in early detection of multidrug-resistant tuberculosis at a Brazilian reference center

Resistance to Mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. To face this challenge, the World Health Organization has recommended the use of rapid molecular tests. We evaluated the routine use (once a week) of a line probe assay (Genotype MTBDRplus) for early diagnosis of resistance and for assessment of the main related risk factors over 2 years. A total of 170 samples were tested: 15 (8.8%) were resistant, and multidrug resistance was detected in 10 (5.9%). The sensitivity profile took 3 weeks (2 weeks for culture and 1 week for rapid testing). Previous treatment for tuberculosis and the persistence of positive acid-fast smears after 4 months of supervised treatment were the major risk factors observed. The use of molecular tests enabled early diagnosis of drug-resistant bacilli and led to appropriate treatment of the disease. This information has the potential to interrupt the transmission chain of resistant M. tuberculosis.

Early changes in serum albumin: impact on 2-year mortality in incident hemodialysis patients

AbstractIntroduction/objective:We evaluated the predictability of early changes in serum albumin (sAlb) on the two-year mortality of incident hemodialysis patients.Methods:Observational, longitudinal retrospective study using the database of Fresenius Medical Care of Latin America. Adult patients starting dialysis from January/2000 to June/2004, from 25 centers were included. Changes in sAlb during the first 3 months on hemodialysis were used as the main predictor. The outcome was death from any cause.Results:1,679 incident patients were included. They were 52 ± 15 years old, 58.7% male and 21.5% diabetic, with a median sAlb of 38 g/L (bromocresol green). 923 patients had sAlb < 38 g/L (Low sAlb Group) and 756 ones had sAlb > 38.0 g/L (Adequate sAlb Group). The mortality was significantly higher in Low sAlb Group (17% vs. 11%, p < 0.001). Early changes in sAlb significantly affected two-year mortality. Factoring the Kaplan Meier curve of Low sAlb Group by the presence of an increase in sAlb uncovered of a statistically significant difference in mortality favoring the ones whose sAlb went up (19% vs. 15%, p = 0.043). Differently, patients from Adequate sAlb Group with a decrease in their sAlb had a statistically higher mortality rate (13% vs. 8%, p = 0.029).Conclusions:Early sAlb changes showed a significant predictive power on mortality at 2 years in incident hemodialysis patients. Those with low initial sAlb may have a better prognosis if their sAlb rises. In contrast, patients with satisfactory initial levels can have a worsening of their prognosis in the case of an early reduction in sAlb.