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.

Nonsmall cell lung cancer: evaluation of 737 consecutive patients in a single institution

OBJECTIVE: To analyze surgical and pathological parameters and outcome and prognostic factors of patients with nonsmall cell lung cancer (NSCLC) who were admitted to a single institution, as well as to correlate these findings to the current staging system. METHOD: Seven hundred and thirty seven patients were diagnosed with NSCLC and admitted to Hospital do Cancer A. C. Camargo from 1990 to 2000. All patients were included in a continuous prospective database, and their data was analyzed. Following staging, a multidisciplinary team decision on adequate management was established. Variables included in this analysis were age, gender, histology, Karnofsky index, weight loss, clinical stage, surgical stage, chemotherapy, radiotherapy, and survival rates. RESULTS: 75.5% of patients were males. The distribution of histologic type was squamous cell carcinoma 51.8%, adenocarcinoma 43.1%, and undifferentiated large cell carcinoma 5.1%. Most patients (73%) presented significant weight loss and a Karnofsky index of 80%. Clinical staging was IA 3.8%, IB 9.2%, IIA 1.4%, IIB 8.1%, IIIA 20.9%, IIIB 22.4%, IV 30.9%. Complete tumor resection was performed in 24.6% of all patients. Surgical stage distribution was IA 25.3%, IB 1.4%, IIB 17.1%, IIIA 16.1%, IIIB 20.3%, IV 11.5%. Chemotherapy and radiotherapy were considered therapeutic options in 43% and 72%, respectively. The overall 5-year survival rate of nonsmall cell lung cancer patients in our study was 28%. Median survival was 18.9 months. CONCLUSIONS: Patients with NSCLC who were admitted to our institution presented with histopathologic and clinical characteristics that were similar to previously published series in cancer hospitals. The best prognosis was associated with complete tumor resection with lymph node dissection, which is only achievable in earlier clinical stages.

Five egg-laying queens in a single colony of brazilian stingless bees (Melipona scutellaris Latreille

Polygyny, characterized by the presence of several egg-laying queens, is considered as a temporary colony status. In stingless bees it is rarely observed. This paper reports the first case of natural polygyny in Melipona scutellaris colony, with five egg-laying queens.

Exercise stress testing in healthy subjects during cholinergic stimulation after a single dose of pyridostigmine

OBJETIVE: The evaluation, by exercise stress testing, of the cardiorespiratory effects of pyridostigmine (PYR), a reversible acetylcholinesterase inhibitor. METHODS: A double-blind, randomized, cross-over, placebo-controlled comparison of hemodynamic and ventilation variables of 10 healthy subjects who underwent three exercise stress tests (the first for adaptation and determination of tolerance to exercise, the other two after administration of placebo or 45mg of PYR). RESULTS: Heart rate at rest was: 68±3 vs 68±3bpm before and after placebo, respectively (P=0.38); 70±2 vs 59±2bpm, before and after pyridostigmine, respectively (P<0.01). During exercise, relative to placebo: a significantly lower heart rate after PYR at, respectively, 20% (P=0.02), 40% (P=0.03), 80% (P=0.05) and 100% (P=0.02) of peak effort was observed. No significant differences were observed in arterial blood pressure, oxygen consumption at submaximal and maximal effort, exercise duration, respiratory ratio, CO2 production, ventilation threshold, minute ventilation, and oxygen pulse. CONCLUSION: Pyridostigmine, at a dose of 45mg, decreases heart rate at rest and during exercise, with minimal side effects and without interfering with exercise tolerance and ventilation variables.

Block of the Mitral-pulmonary Isthmus During Ablation of a Single Left-sided Accessory Pathway Causing Different Patterns of Retrograde Atrial Activation

The case of a 16-year-old patient with atrioventricular tachycardia caused by a single left anterolateral accessory pathway is reported. When the patient underwent radiofrequency ablation, a lesion on the mitral annulus lateral wall produced changes in the retrograde atrial activation pattern determined by that pathway; changes ranged from a delay in depolarization of the annulus posterior portions to full left atrium counterclockwise activation. Such phenomena were probably caused by a block in the isthmus between the annulus and the lower left pulmonary vein ostium. This case illustrates the importance of the mitral-pulmonary isthmus in the process of left atrium activation, an alert to changes induced by its unintentional block during accessory pathway ablation.

Prognostic value of technetium-99m-labeled single-photon emission computerized tomography in the follow-up of patients after their first myocardial revascularization surgery

OBJECTIVE: To assess the prognostic value of Technetium-99m-labeled single-photon emission computerized tomography (SPECT) in the follow-up of patients who had undergone their first myocardial revascularization. METHODS: We carried out a retrospective study of 280 revascularized patients undergoing myocardial scintigraphy under stress (exercise or pharmacological stress with dipyridamole) and at rest according to a 2-day protocol. A set of clinical, stress electrocardiographic and scintigraphic variables was assessed. Cardiac events were classified as "major" (death, infarction, unstable angina) and "any" (major event or coronary angioplasty or new myocardial revascularization surgery). RESULTS: Thirty-six major events occurred as follows: 3 deaths, 11 infarctions, and 22 unstable anginas. In regard to any event, 22 angioplasties and 7 new surgeries occurred in addition to major events, resulting a total of 65 events. The sensitivity of scintigraphy in prognosticating a major event or any event was, respectively, 55% and 58%, showing a negative predictive value of 90% and 83%, respectively. Diabetes mellitus, inconclusive stress electrocardiography, and a scintigraphic visualization of left ventricular enlargement were significant variables for the occurrence of a major event. On multivariate analysis, abnormal myocardial scintigraphy was a predictor of any event. CONCLUSION: Myocardial perfusion tomography with Technetium-99m may be used to identify high-risk patients after their first myocardial revascularization surgery.

Comparison of immediate results and follow-up of patients with single-vessel and multivessel coronary artery disease younger than 50 years of age undergoing coronary stent implantation

OBJECTIVE: To assess the in-hospital results and clinical follow-up of young patients (< 50 years) with multivessel coronary artery disease undergoing stent implantation in native coronary arteries and to compare their results with those of patients with single-vessel coronary artery disease. METHODS: We retrospectively studied 462 patients undergoing coronary stent implantation. Patients were divided into 2 groups: group I (G-I) - 388 (84%) patients with single-vessel coronary artery disease; and group II (G-II) - 74 (16%) patients with multivessel coronary artery disease. RESULTS: The mean age of the patients was 45±4.9 years, and the clinical findings at presentation and demographic data were similar in both groups. The rate of clinical success was 95% in G-I and 95.8% in G-II (P=0.96), with no difference in regard to in-hospital evolution between the groups. Death, acute myocardial infarction, and the need for myocardial revascularization during clinical follow-up occurred in 10.1% and 11.2% (P=0.92) in G-I and G-II, respectively. By the end of 24 months, the actuarial analysis showed an event-free survival of 84.6 % in G-I and 81.1% in G-II (P=0.57). CONCLUSION: Percutaneous treatment with coronary stent implantation in young patients with multivessel disease may be safe with a high rate of clinical success, a low incidence of in-hospital complications, and a favorable evolution in clinical follow-up.

Use of Short-term Circulatory Support as a Bridge in Pediatric Heart Transplantation

Background:Heart transplantation is considered the gold standard therapy for the advanced heart failure, but donor shortage, especially in pediatric patients, is the main limitation for this procedure, so most sick patients die while waiting for the procedure.Objective:To evaluate the use of short-term circulatory support as a bridge to transplantation in end-stage cardiomyopathy.Methods:Retrospective clinical study. Between January 2011 and December 2013, 40 patients with cardiomyopathy were admitted in our Pediatric Intensive Care Unit, with a mean age of 4.5 years. Twenty patients evolved during hospitalization with clinical deterioration and were classified as Intermacs 1 and 2. One patient died within 24 hours and 19 could be stabilized and were listed. They were divided into 2 groups: A, clinical support alone and B, implantation of short-term circulatory support as bridge to transplantation additionally to clinical therapy.Results:We used short-term mechanical circulatory support as a bridge to transplantation in 9. In group A (n=10), eight died waiting and 2 patients (20%) were transplanted, but none was discharged. In group B (n=9), 6 patients (66.7%) were transplanted and three were discharged.The mean support time was 21,8 days (6 to 984h). The mean transplant waiting list time was 33,8 days. Renal failure and sepsis were the main complication and causeof death in group A while neurologic complications were more prevalent en group B.Conclusion:Mechanical circulatory support increases survival on the pediatric heart transplantation waiting list in patients classified as Intermacs 1 and 2.

Comportamento dos cromossômios na meiose de Euryophthalmus rufipennis Laporte (Hemiptera Pyrrhocoridae)

In this paper an account is given of the principal facts observer in the meiosis of Euryophthalmus rufipennis Laporte which afford some evidence in favour of the view held by the present writer in earlier publications regarding the existence of two terminal kinetochores in Hem ip ter an chromosomes as well as the transverse division of the chromosomes. Spermatogonial mitosis - From the beginning of prophase until metaphase nothing worthy of special reference was observed. At anaphase, on the contrary, the behavior of the chromosomes deserves our best attention. Indeed, the chromoso- mes, as soon as they begin to move, they show both ends pronouncedly turned toward the poles to which they are connected by chromosomal fibres. So a premature and remarkable bending of the chromosomes not yet found in any other species of Hemiptera and even of Homoptera points strongly to terminally localized kinetochores. The explanation proposed by HUGHES-SCHRADER and RIS for Nautococcus and by RIS for Tamalia, whose chromosomes first become bent late in anaphase do not apply to chromosomes which initiate anaphase movement already turned toward the corresponding pole. In the other hand, the variety of positions assumed by the anaphase chromosomes of Euryophthalmus with regard to one another speaks conclusively against the idea of diffuse spindle attachments. First meiotic division - Corresponding to the beginning of the story of the primary spermatocytes cells are found with the nucleus entirelly filled with leptonema threads. Nuclei with thin and thick threads have been considered as being in the zygotente phase. At the pachytene stage the bivalents are formed by two parallel strands clearly separated by a narrow space. The preceding phases differ in nothing from the corresponding orthodox ones, pairing being undoubtedly of the parasynaptic type. Formation of tetrads - When the nuclei coming from the diffuse stage can be again understood the chromosomes reappear as thick threads formed by two filaments intimately united except for a short median segment. Becoming progressively shorter and thicker the bivalents sometimes unite their extremities forming ring-shaped figures. Generally, however, this does not happen and the bivalents give origin to more or less condensed characteristic Hemipteran tetrads, bent at the weak median region. The lateral duplicity of the tetrads is evident. At metaphase the tetrads are still bent and are connected with both poles by their ends. The ring-shaped diakinesis tetrads open themselves out before metaphase, showing in this way that were not chiasmata that held their ends together. Anaphase proceeds as expected. If we consider the median region of the tetrads as being terminalized chiasmata, then the chromosomes are provided with a single terminal kinetochore. But this it not the case. A critical analysis of the story of the bivalents before and after the diffuse stage points to the conclusion that they are continuous throughout their whole length. Thence the chromosomes are considered as having a kinetochore at each end. Orientation - There are some evidences that Hemipteran chromosomes are connected by chiasmata. If this is true, the orientation of the tetrads may be understood in the following manner: Chiasmata being hindered to scape by the terminal kinetochores accumulate at the ends of the tetrads, where condensation begins. Repulsion at the centric ends being prevented by chiasmata the tetrads orient themselves as if they were provided with a single kinetochore at each extremity, taking a position parallelly to the spindle axis. Anaphase separation - Anaphase separation is consequently due to a transverse division of the chromosomes. Telophase and secund meiotic division - At telophase the kinetochore repeli one another following the moving apart of the centosomes, the chiasmata slip toward the acentric extremities and the chromosomes rotate in order to arrange themselves parallelly to the axis of the new spindle. Separation is therefore throughout the pairing plane. Origin of the dicentricity of the chromosomes - Dicentricity of the chromosomes is ascribed to the division of the kinetochore of the chromosomes reaching the poles followed by separation and distension of the chromatids which remain fused at the acentric ends giving thus origin to terminally dicentric iso-chromosomes. Thence, the transverse division of the chromosomes, that is, a division through a plane perpendicular to the plane of pairing, actually corresponds to a longitudinal division realized in the preceding generation. Inactive and active kinetochores - Chromosomes carrying inactive kinetochore is not capable of orientation and active anaphasic movements. The heterochromosome of Diactor bilineatus in the division of the secondary spermatocytes is justly in this case, standing without fibrilar connection with the poles anywhere in the cell, while the autosomes are moving regularly. The heterochromosome of Euryophthalmus, on the contrary, having its kinetochores perfectly active ,is correctly oriented in the plane of the equator together with the autosomes and shows terminal chromosomal connection with both poles. Being attracted with equal strength by two opposite poles it cannot decide to the one way or the other remaining motionless in the equator until some secondary causes (as for instances a slight functional difference between the kinetochores) intervene to break the state of equilibrium. When Yiothing interferes to aide the heterochromosome in choosing its way it distends itself between the autosomal plates forming a fusiform bridge which sometimes finishes by being broken. Ordinarily, however, the bulky part of the heterochromosome passes to one pole. Spindle fibers and kinetic activity of chromosomal fragments - The kinetochore is considered as the unique part of the chromosome capable of being influenced by other kinetochore or by the poles. Under such influence the kinetochore would be stimulated or activited and would elaborate a sort of impulse which would run toward the ends. In this respect the chromosome may be compared to a neüròn, the cell being represented by the kinetochore and the axon by the body of the chromosome. Due to the action of the kinetochore the entire chromosome becomes also activated for performing its kinetic function. Nothing is known at present about the nature of this activation. We can however assume that some active chemical substance like those produced by the neuron and transferred to the effector passes from the kinetochore to the body of the chromosome runing down to the ends. And, like an axon which continues to transmit an impulse after the stimulating agent has suspended its action, so may the chromosome show some residual kinetic activity even after having lost its kinetochore. This is another explanation for the kinetic behavior of acentric chromosomal fragmehs. In the orthodox monocentric chromosomes the kinetic activity is greater at the kinetochore, that is, at the place of origin of the active substance than at any other place. In chromosomes provided with a kinetochore at each end the entire body may become active enough to produce chromosomal fibers. This is probably due to a more or less uniform distribution and concentration of the active substance coming simultaneously from both extremities of the chromosome.

Distinct ultrastructural aspects in different biopsies of a single patient with diffuse cutaneous leishmaniasis

The authors investigated the relation between parasites and host-cells in active and regressed lesions of a patient with diffuse cutaneous leishmaniasis, evaluating the frequency of different cell types, and the location and integrity of amastigotes. No correlation was found between parasite integrity and size of parasitophorous vacuoles. They observed ultrastructural findings characterizing a cell mediated immune response: macrophages lysis, parasitic destruction inside macrophages, close contact between parasitized macrophages and lymphocytes and between parasites and lymphocytes, lymphocytic infiltration and fibrosis. They suggest that in DCL there is a limited cellular immune response, although insufficient to control infection.

Pharmacokinetic profile of tert-butylaminoethanethiol

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethanethiol (TBAESH) was performed after administration of a single dose (35 mg/kg) either orally or intravenously. Plasma or blood samples were treated with dithiothreitol, perchloric acid and, after filtration, submitted to further purification with anionic resin. In the final step the drug was retained on a cationic resin column, eluted with NaCl lM and detected according to the method of Ellman (1958). The results suggested a pharmacokinetic behavior related to a one open compartment model with the following values for the total drug: area under the intravenous curve (AUC i.v.): 443(+ ou -) 24.0; AUC oral: 85.5(+ ou -) 14.5 ug min.ml(elevado a -1); elimination rate constant: 0.069(+ ou -) 0.0055 min(elevado a -1), biological half-life: 10.0(+ ou -) 0.80 min; distribution volume 1.15(+ ou -) 0.15 ml/g; biodisponibility: 0.19(+ ou -) 0.02. From a pharmacokinetic standpoint, TBAESH seems to have no advantage over the analogous disulfide compound.

Identification of snails within the Bulinus africanus group from East Africa by multiplex SNaPshotTManalysis of single nucleotide polymorphisms within the cytochrome oxidase subunit I

Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI) as detected by a novel multiplexed SNaPshotTM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshotTM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host species.