Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Flowering levels, harvest season and yields of cupuassu (Theobroma grandiflorum)

Cupuassu (Theobroma grandiflorum), a specie native to Amazonia, has been planted commercially in Brazil to satisfy the demand for the flavorful juice obtained from the pulp around its seeds. The trees are notorious for low and irregular fruit production. Data gathered over two seasons from trees in a germplasm collection in Pará, Brazil, showed that some of them fruited more regularly than others. Differences in fruit production correlated to differences in flower production. Tree-to-tree variation in flower production, fruit production, and consistency of both over time suggest considerable scope for improving yields by selection. Hand pollinations resulted in a much higher frequency of fruit set than open pollinations, indicating that lack of effective pollination is also a reason for low yield. However, attempts to increase the level of effective pollination are handicapped by low knowledge about the pollinators of cupuassu and their behavior.

Effects of light and temperature on isoprene emission at different leaf developmental stages of eschweilera coriacea in central Amazon

Isoprene emission from plants accounts for about one third of annual global volatile organic compound emissions. The largest source of isoprene for the global atmosphere is the Amazon Basin. This study aimed to identify and quantify the isoprene emission and photosynthesis at different levels of light intensity and leaf temperature, in three phenological phases (young mature leaf, old mature leaf and senescent leaf) of Eschweilera coriacea (Matamatá verdadeira), the species with the widest distribution in the central Amazon. In situ photosynthesis and isoprene emission measurements showed that young mature leaf had the highest rates at all light intensities and leaf temperatures. Additionally, it was observed that isoprene emission capacity (Es) changed considerably over different leaf ages. This suggests that aging leads to a reduction of both leaf photosynthetic activity and isoprene production and emission. The algorithm of Guenther et al. (1999) provided good fits to the data when incident light was varied, however differences among E S of all leaf ages influenced on quantic yield predicted by model. When leaf temperature was varied, algorithm prediction was not satisfactory for temperature higher than ~40 °C; this could be because our data did not show isoprene temperature optimum up to 45 °C. Our results are consistent with the hypothesis of the isoprene functional role in protecting plants from high temperatures and highlight the need to include leaf phenology effects in isoprene emission models.

Yield of essential oil and safrole content based on fresh and dry biomass of long pepper in the Brazilian Amazon

Long pepper (Piper hispidinervum) is an Amazonian species of commercial interest due to the production of safrole. Drying long pepper biomass to extract safrole is a time consuming and costly process that can also result in the contamination of the material by microorganisms. The objective of this study was to analyze the yield of essential oil and safrole content of fresh and dried biomass of long pepper accessions maintained in the Active Germoplasm Bank of Embrapa Acre, in the state of Acre, Brazil, aiming at selecting genotypes with best performance on fresh biomass to recommend to the breeding program of the species. Yield of essential oil and safrole content were assessed in 15 long pepper accessions. The essential oil extraction was performed by hydrodistillation and analyzed by gas chromatography. A joint analysis of experiments was performed and the means of essential oil yield and safrole content for each biomass were compared by Student's t-test. There was variability in the essential oil yield and safrole content. There was no difference between the types of biomass for oil yield; however to the safrole content there was difference. Populations 9, 10, 12 and 15 had values of oil yield between 4.1 and 5.3%, and safrole content between 87.2 and 94.3%. The drying process does not interfere in oil productivity. These populations have potential for selection to the long pepper breeding program using oil extraction in the fresh biomass

Do Diabetic Patients with Acute Coronary Syndromes Have a Higher Threshold for Ischemic Pain?

Background: Data from over 4 decades have reported a higher incidence of silent infarction among patients with diabetes mellitus (DM), but recent publications have shown conflicting results regarding the correlation between DM and presence of pain in patients with acute coronary syndromes (ACS). Objective: Our primary objective was to analyze the association between DM and precordial pain at hospital arrival. Secondary analyses evaluated the association between hyperglycemia and precordial pain at presentation, and the subgroup of patients presenting within 6 hours of symptom onset. Methods: We analyzed a prospectively designed registry of 3,544 patients with ACS admitted to a Coronary Care Unit of a tertiary hospital. We developed multivariable models to adjust for potential confounders. Results: Patients with precordial pain were less likely to have DM (30.3%) than those without pain (34.0%; unadjusted p = 0.029), but this difference was not significant after multivariable adjustment, for the global population (p = 0.84), and for subset of patients that presented within 6 hours from symptom onset (p = 0.51). In contrast, precordial pain was more likely among patients with hyperglycemia (41.2% vs 37.0% without hyperglycemia, p = 0.035) in the overall population and also among those who presented within 6 hours (41.6% vs. 32.3%, p = 0.001). Adjusted models showed an independent association between hyperglycemia and pain at presentation, especially among patients who presented within 6 hours (OR = 1.41, p = 0.008). Conclusion: In this non-selected ACS population, there was no correlation between DM and hospital presentation without precordial pain. Moreover, hyperglycemia correlated significantly with pain at presentation, especially in the population that arrived within 6 hours from symptom onset.

O tratamento de pré-emergência com o 2, 4-D no milho

In order to study the action of herbicides - sodium salt, amine salt and ester of 2,4-D, TCA and 2,4,5-T a preliminary experiment for pre-emergence weed control was corried out, and the corresponding results are given in table I and II. The corn used in the experiments was of the flint type 1A 3531. The loam soil on which the experiment has been carried out is called "terra roxa". All treatments were highly significant when compared with the check plots, except the 2B one in the control of broad leaf weeds, and 4B in the control of grass weeds. Among these treatments there are no significant differences. But we note the following: (table I). a) treatments of higher concentrations were superior to lower ones. b) the treatments which gave the best control for broad leaf weeds were in the following decreasing order: 1A, 5A and 3A. For grass weeds, they were 5A, 1A and 3A. c) the amine 2,4-D (600 grs. per hectare) supplied very good control when we get into consideration that on the acid basis, it was in very low concentration. d) TCA in high concentration affected the germination, growth and yield, in the lower one it did not show good control of weeds, especially of grasses. It is not suitable for pre-emergence control in corn. e) 2,4,5-T was not better than the 2,4-D products. As it is much more expensive than the others, economically its use in pre-emergence weed control in corn is not praticable. f) all the products used controled grass weeds as well as broad leaf ones; this show the superiority of the pre-emergence treatment method over that of post-emergence. g) Even a dose as strong as the treatment 1A (3.400g. of 2,4-D acid per hectare) did not damage corn production (table II). h) the superiority noted in the production of all the treatments with the exception of 2A, which damaged the plants, we atribute to the lack of competion between corn and weeds; all chek-plots suffered this competition, because they were not Probably, there was, also, hormonial effect of 2,4-D on the corn plant. Not withstanding the fact that the present experiment has been successful, we think that new researches are necessary, especially with the purpose of studying factors as climate and soil which in other countries, interferred with the success of the pre-emergence weed control.

Arsenicais e inseticidas orgânicos sôbre o algodoeiro em terras roxa e arenosa

The authors carried out a series of pots and plots experiments applying arsenical and organic insecticides to cotton plants cultivated in "terra roxa" and in a sandy soil. The first results were presented in 1947, to the la. Reunião Brasileira de Ciência do Solo (First Brazilian Congress of Soil Science); they pointed out the danger resulting from the accumulation of arsenic in soils due to the constant applications of arsenicais to control cotton pests; in the course of the time, the amount of residual arsenic in the soil would determine a decrease in cotton yield caused by its toxic effect on the crop. The following conclusions were drawn from the last three experiments: 1) the field experiment conducted in a sandy soil to which lead arseniate was applied in increasing rates produced a reduction of 50 per cent in the yield (the three highest doses were responsible for this result); by this way, the pot experiment published in 1947 was confirmed); 2) in the pot experiment with "terra roxa" toxic effects appeared only in the plants receiving the last dosis of lead arsenate; this result is explained quite naturally by a considerable absorption of the AsO4 --- ion by "terra roxa" colloidal material; furthermore the CaO, P2O5 and Fe2O3 content and the pH value (higher) would decrease the arsenate solubilization in the soil considered; 3) the pot experiment with organic insecticides applied in the rates usually employed in the control of cotton pests, showed that 10% D.D.TD. and 2.5% Rotenone did not affect cotton plants cultivated in a sandy soil; however we agree with FOSTER (1951), in the point that both mineral and organic insecticides must be applied in the minimum amount as possible; we also think that experiments like those should be carried out with the known insecticides, in several soil conditions and with many crops in order to determine the maximum limits of tolerancy.

Efeitos do enxofre e do boro e da inoculação em alfafa (Medicago sativa L.)

This paper deals with the results of a pot and plot experiment which was carried out to determine the influence of sulphur and boron and the effect seed inoculation with Rhizobium meliloti in the yield of alfafa. Sulphur was applied as flower of sulphur at the rates of 1,000 and 2,000 kg por hectare; boron was employed in the proportion of 15 kg of borax per hectare; both sulphur and boron were distributed broadcast before planting; the experimental design chosen for the field trial was a latin square of 6 x 6 with the following treatments: Number Treatment 1 Control 2 One dosis S + inoculation 3 Two dosis S +inoculation 4 One dosis S + B + inoculation 5 B + inoculation 6 inoculation The crop supplied four cuttings in an eleven months period. The pot experiment nearly confirmed the plot one. The following conclusions can be drawn: 1. The classification of treatments in a decrescent order was: l.o - two doses S + inoculation; 2.o - one dosis S +inoculation, S + B + inoculation, and B + inoculation (these treatmente were not statistically different); 3.o - control; 4.o - inoculation; 2 The vield due to the treatment two dosis S + inoculation was 22 per cent higher than the control one, a fact that suggests that the S supply in the soil studied ("terra roxa misturada") is not sufficient for the total requirements of alfafa; 3. From an economical point of view the best treatment was: one dosis B + inoculation which permits a net gain of Cr$ 12.527,30 per hectare per year; 4. Based on the mentioned results we recommend in soils of same type the following fertilization for alfafa. 5 tons limestone/hectare 300 kg serranafosfato and 600 kg hiperfosfato/ha 300 kg muriate of potash/ha 15 kg borax/ha and a medium organic manuring if the soil is very poor in organic matter.

Estudos sobre o «vermelhão» do algodoeiro (III)

In several cotton crops areas of the State of S. Paulo it was observed, during the years of 1948, 1949, and 1951, the appearance of a purple color of the leaves; the color appears in the opening of the bolls and was correlated with a decrease of production. The opinions concerning the cause of such abnormality were very different and sometimes contradictory; certain investigators attributed the disease to insect attack, others to bad climatic conditions whereas others to a potassium deficiency now called "fome de potássio" (potash hunger); our ideas on the subject is another one. We think that the disease is caused by lack of a suitable supply of magnesium. This opinion is largely based on the syntomatology found in the literature. To study the problem, several experiments were carried out, namely: 1. pot experiments using soil collected in areas where the disorder had appeared; 2. pot experiments controlling the water supply; 3. sand culture experiments omitting either potassium or magnesium; 4. leaf analysis of plant matrial collected troughout the Piracicaba County; 5. plot experiments with the varieties Texas, Express, and I.A. 817 Campinas. The first four experiments were discussed elsewhere. To study the point 5 an experiment was carried out, with the following treatments : 1 - NPKCaMg (no K added) - Mg supplied as MgSO4 (a soluble form); 2 -NPKCa (no Mg added); 3 -NPKCaMg (complete) - Mg supplied as MgSO4; 4 - NPKCaMg (complete) - Mg supplied as dolomitic limestone (a slightly soluble form) as a rate 2.5 higher than in the treatment 1 and 3. Organic matter as cottonseed meal was applied in the proportion of 500 kg per hectare. The experimental design was randomized blocks with 4 replications and the results can be summarized as follows: 1 the I.A 817 variety was the most strongly affected by the physiological disorder, with severe decrease in yield; 2. the disease occurred more frequently in the minus magnesium treatment; 3. dolomitic limestone is so effective as magnesium sulfate in the control of the disease as well in the raising of the yield; 4. in the minus K treatment it was observed a marked occurrence of the typical symptoms of potassium deficiency (cotton rust); 5. magnesium was actually, in the experimental conditions the responsible for the purple color (vermelhão) of the cotton leaves.

Experiências de adubação mineral e orgânica com Capim Kikuyu (Pennisetum clandestinum Hochst.)

Kikuio grass (Pennisetum clandestinum Hochst) is beyond any doubt, a pasture very important for farm animals; since its chemical composition is very similar to that of alfalfa, the present field trial was carried out; a randomized block design with 8 treatments was selected as follows: 1 N - P - K - Ca - Mg (complete manuring) 2 N - P - K - Ca----- (without Mg) 3 N - P - K-------Mg (without Ca) 4 ----P - K - Ca - Mg (without N) 5 N------K - Ca Mg (without P) 6 N - P - Ca - Mg (without K) 7 organic matter (without mineral fertilizers) 8 control Nitrogen was applied as NaN03 (topdressed) and as ammonium sulfate; P2O5 was given as superphosphate associated to bonemeal; K2O was applied as muriate, CaO as "sambaquis" (oyster shells); MgO was given as MgSO4 (topdressed). The source of organic matter was farmyard manure. As far yields are concerned the following observations were made: 1. treatment n. 7 was superior to all others; 2. considering the mineral fertilizers, good responses were due to N and P2O5; 3. the control yield was exceedingly poor, being inferior to all the others treatments; The chemical analyses revealed that: 1. the protein content decreased accordingly to this order: 7, 6, 5 and 1; treatment 4 (without N) gave the lowest protein content; 2. treatment n. 4 produced the highest fat content; treatment no. 7 ranked second; no. 8 gave the lowest fat content; 3. crude fiber: highest - treatment 7; lowest - 8; 4. ashes: the ashes content was higher in treatment 5; proprobably because the most abundant element in the ashes is K, the ash content of treatment 6 (no K) was very low; 5. non nitrogenous substances (determined by difference) - high in treatment 8 and low in treatment 7; 6. mineral elements in the ashes - the element omitted from a given treatment was very low in the grasses therein obtained; this shows the relative poverty of the soil in that element. As general remark the Authors suggest the use of farmyard manure in the fertilization of Kikuio grass; farmyard manure could probably substitute wither green manure or compost.

Nitrificação e aproveitamento de alguns adubos nitrogenados

The rate of nitrification of several nitrogenous fertilizers (ammonium sulfate, nitre-chalk, ureia, and cottonseed meal) was studied in three soils, namely, "terra roxa legítima", a red soil derived from basalt, "terra roxa misturada", a soil also derived from basalt but with a higher proportion of sand, and "areito Corumbataí", a sandy soil. The effects of the following treatments on nitrification were considered: addition of limestone of micronutrients (Fe, Cu, Zn, Mn, and Mo), and inoculation with a suspension of spores of Aspergillus wentii, a heterotrophic nitrifier. The results showed that: in "terra roxa legítima" limestone had no influence on the nitrification rate, whereas the micronutrients estimulated the oxidation of nitre-chalk, cottonseed meal and urea; inoculation with A. wentii helped only the nitrification of ammonium sulfate and of the cottonseed meal; the latter, in all the treatments employed gave use to a smaller amount of nitrates; in "terra roxa misturada", all the fertilizers depending upon the treatments they were subjected to, presented maximum values for nitrification; limestone estimulated the oxidation of ammonium sulfate as well as the mineralization of the cottonseed meal; the addition of micronutrients helped the nitrification of all the fertilizers, except that of urea; inoculation showed a benefical influence on the nitrification of ammonium sulfate and cottonseed meal; in "arenito de Corumbatai", the amounts of nitrates produced was roughly the same for all the fertilizers investigated; limestone estimulated the nitrification of nitro-chalk, ammonium sulfate and cottonseed meal whilst the addition of micronutrients benefited only the latter two; the inoculation with A. wentii helped the oxidation of all the fertilizers. In order to study the availability of the various fertilizers above discussed, two plant growing experiments were carried cut, one in pots, using the three soil types and another one in the field, with "terra roxa misturada". In "arenito de Corumbatai" there was no significant difference in the yield both of straw and rice grains for none of the fertilizers: Chilean nitrate of soda was used as a control; ho marked agreement could be detected between the data concerning nitrification and the yield results. In "terra roxa legítima", ammonium sulfate won the competition and there was a good parallelism between nitrification and yield. In "terra roxa misturada", there was no statistical difference among the various fertilizers; the agreement between nitrification and yields was reasonable. In the field (corn), Chilean nitrate, ammonium sulfate and nitro-chalk were clearly beter than urea and cottonseed meal which did not differ from the minus nitrogen plots.

A disponibilidade do potássio da "leucita de Poços de Caldas", estudada por meio do arroz

In order to know potassium availability in "leucita de Poços de Caldas" from Poços de Caldas, Minas Gerais, a Mitscherlich pot experiment was set up. The pots were filled with 6 kilograms of sieved (4 mm) "Terra Roxa Misturada" soil. Rice (Oryza sativa, L.), Dourado Agulha variety was the testing plant. Doses of potassium referred to are 1,5 g (as K(2)0) from both KCl and "leucita de Poços de Caldas". There were 6 treatments, with 3 repetitions, as follows: 1) Control; 2) NP + 1 dose K (KCl); 3) NP + 2 doses K (KCl)); 4) NP + 3 doses K (KCl); 5) NP + 1 dose K (leucita); 6) NP + 2 doses K (leucita) and 7) NP + 3 doses K (leucita). Each pot received 50 seeds. Five days after germination the seedlings were thinned to 35. Harvesting took place 4 months after germination. Potassium (as KCl) promoted an increase in yield of both stalk and grain as compared with control. Potassium content in the leaves was also higher in all treatment in which KCl was supplied. Potassium, as "leucita de Poços de Caldas» did not show any favorable effect on both stalk and grain yield and on its content in the leaves.

Nutrient uptake and nutritional efficiency in sweet sorghum

Two sweet sorghum varieties, Brandes and Rio, were grown in full strenght and diluted nutrient solutions till completing the life cycle wherein mineral analyses were carried out. As a rule both varieties showed the same capacity to absorb nutrients in the two rates supplied. Dry matter yield, however was different in the dilute nutrient solution. The variety Brandes produced more fresh stalks in the full strength solution than Rio; under nutricional stress the yield was lower. Dry matter of stalks in the case of the variety Rio was consistently higher.

Higher genetic variation estimated by microsatellites compared to isoenzyme markers in Aedes aegypti from Rio de Janeiro

Aedes aegypti populations from five districts in Rio de Janeiro were analyzed using five microsatellites and six isoenzyme markers, to assess the amount of variation and patterns of gene flow at local levels. Microsatellite loci were polymorphic enough to detect genetic differentiation of populations collected at small geographic scales (e.g. within a city). Ae. aegypti populations were highly differentiated as well in the city center as in the outskirt. Thus, dengue virus propagation by mosquitoes could be as efficient in the urban area as in the outskirt of Rio de Janeiro, the main entry point of dengue in Brazil.