Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

Efficacy and tolerability of two single-day regimens of triclabendazole for fascioliasis in Peruvian children

Abstract:INTRODUCTION:The therapeutic scheme of triclabendazole (TCBZ), the recommended anthelmintic against Fasciola hepatica , involves 10mg/kg of body weight administered in a single dose; however, clinical trials in children are scarce. We evaluated the efficacy and tolerability of 2 schemes of TCBZ.METHODS: Eighty-four Peruvian children with F. hepatica eggs in their stools were allocated into 2 groups: 44 received 2 dosages of 7.5mg/kg each with a 12-h interval (Group I), and 40 received a single 10-mg/kg dose (Group II). Evaluation of efficacy was based on the presence of eggs in stools, and tolerability was based on the presence of symptoms and signs post-treatment.RESULTS: A parasitological cure was obtained in 100% of individuals from Group I and 95% of individuals from Group II. The most common adverse event was biliary colic.CONCLUSIONS: The tested scheme was efficacious and tolerable, and it might be an optimal scheme in the region. To the best of our knowledge, this represents the largest series of children treated with TCBZ in a non-hospital setting.

Reliability of the information about the history of diagnosis and treatment of hypertension. Differences in regard to sex, age, and educational level. The pró-saúde study

OBJECTIVE: To assess the intraobserver reliability of the information about the history of diagnosis and treatment of hypertension. METHODS: A multidimensional health questionnaire, which was filled out by the interviewees, was applied twice with an interval of 2 weeks, in July '99, to 192 employees of the University of the State of Rio de Janeiro (UERJ), stratified by sex, age, and educational level. The intraobserver reliability of the answers provided was estimated by the kappa statistic and by the coefficient of intraclass correlation (CICC). RESULTS: The general kappa (k) statistic was 0.75 (95% CI=0.73-0.77). Reliability was higher among females (k=0.88, 95% CI=0.85-0.91) than among males (k=0.62, 95% CI=0.59-0.65).The reliability was higher among individuals 40 years of age or older (k=0.79; 95% CI=0.73-0.84) than those from 18 to 39 years (k=0.52; 95% CI=0.45-0.57). Finally, the kappa statistic was higher among individuals with a university educational level (k=0.86; 95% CI=0.81-0.91) than among those with high school educational level (k=0.61; 95% CI=0.53-0.70) or those with middle school educational level (k=0.68; 95% CI=0.64-0.72). The coefficient of intraclass correlation estimated by the intraobserver agreement in regard to age at the time of the diagnosis of hypertension was 0.74. A perfect agreement between the 2 answers (k=1.00) was observed for 22 interviewees who reported prior prescription of antihypertensive medication. CONCLUSION: In the population studied, estimates of the reliability of the history of medical diagnosis of hypertension and its treatment ranged from substantial to almost perfect reliability.

Validation study of an automated wrist monitor, omron model HEM-608, compared with the standard methods for blood pressure measurement

OBJECTIVE - The aim of our study was to assess the profile of a wrist monitor, the Omron Model HEM-608, compared with the indirect method for blood pressure measurement. METHODS - Our study population consisted of 100 subjects, 29 being normotensive and 71 being hypertensive. Participants had their blood pressure checked 8 times with alternate techniques, 4 by the indirect method and 4 with the Omron wrist monitor. The validation criteria used to test this device were based on the internationally recognized protocols. RESULTS - Our data showed that the Omron HEM-608 reached a classification B for systolic and A for diastolic blood pressure, according to the one protocol. The mean differences between blood pressure values obtained with each of the methods were -2.3 +7.9mmHg for systolic and 0.97+5.5mmHg for diastolic blood pressure. Therefore, we considered this type of device approved according to the criteria selected. CONCLUSION - Our study leads us to conclude that this wrist monitor is not only easy to use, but also produces results very similar to those obtained by the standard indirect method.

Electrocardiographic Alterations in Patients Hospitalized with Leptospirosis in the Brazilian City of Salvador

OBJECTIVE: To report the frequency and types of electrocardiographic alterations in patients with leptospirosis in the first 24 hours of hospitalization. METHODS: We analyzed the electrocardiograms of 157 patients admitted to the Hospital Couto Maia in the city of Salvador, in the State of Bahia, Brazil, from March 1998 to June 1999. The electrocardiograms were performed in the first 24 hours after hospital admission, independent of the clinical manifestations of the patients. RESULTS: The mean ± SD for patients' age was 35.5± 13.7 (median = 32) years, and jaundice was present in 95.5% of them. Alterations in the electrocardiogram were detected in 68.2% (107/157) of the patients (95% confidence interval = 60.6% - 75.1%). Atrial fibrillation was the most frequent arrhythmia, occurring in 10.8% (17/157) of the patients. Other frequent findings were alterations in ventricular repolarization detected in 38.9% (61/157) of patients and first-degree atrioventricular block in 10.2% (16/157). The patients with atrial fibrillation were older and had higher levels of creatinine and aminotransferases. CONCLUSION: In this sample, approximately 2/3 of the patients had electrocardiographic alterations after hospital admission. Of the major arrhythmias, atrial fibrillation was the most frequent, and the patients with this arrhythmia had evidence of more severe disease. The relation between the presence and type of electrocardiographic alteration and the prognosis of leptospirosis is yet to be assessed.

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.

Characterization of the in vivo cardiac electrophysiologic effects of high-dose cocaine in closed-chest, anesthetized dogs with normal hearts

OBJECTIVE: To characterize the cardiac electrophysiologic effects of cocaine. METHODS: In 8 dogs (9-13 kg), electrophysiologic parameters and programmed stimulation were undertaken using transvenous catheters at baseline, and after cocaine intravenous infusion (12 mg/kg bolus followed by 0.22 mg/kg/min for 25 minutes). RESULTS: Cocaine plasma levels (n=5) rose to 6.73± 0.56 mg/mL. Cocaine did not affect sinus cycle length and arterial pressure. Cocaine prolonged P wave duration (54±6 vs 73±4 ms, P<0.001), PR interval (115±17 vs 164±15 ms, P<0.001), QRS duration (62±10 vs 88±14 ms, P<0.001), and QTc interval (344±28 vs 403±62 ms, P=0.03) but not JT interval (193±35 vs 226±53 ms, NS). Cocaine prolonged PA (9±6 vs 23±8 ms, P<0.001), AH (73±16 vs 92±15 ms; P=0.03), and HV (35±5 vs 45±3ms; P<0.001) intervals and Wenckebach point (247±26 vs 280±28 ms, P=0.04). An increase occurred in atrial (138±8 vs 184± 20 ms; P<0.001) and ventricular (160±15 vs 187±25 ms; P=0.03) refractoriness at a cycle length of 300 ms. Atrial arrhythmias were not induced in any dog. Ventricular fibrillation (VF) was induced in 2/8 dogs at baseline and 4/8 dogs after cocaine. CONCLUSION: High doses of cocaine exert significant class I effects and seem to enhance inducibility of VF but not of atrial arrhythmias.

Performance of Surgical Risk Scores to Predict Mortality after Transcatheter Aortic Valve Implantation

AbstractBackground:Predicting mortality in patients undergoing transcatheter aortic valve implantation (TAVI) remains a challenge.Objectives:To evaluate the performance of 5 risk scores for cardiac surgery in predicting the 30-day mortality among patients of the Brazilian Registry of TAVI.Methods:The Brazilian Multicenter Registry prospectively enrolled 418 patients undergoing TAVI in 18 centers between 2008 and 2013. The 30-day mortality risk was calculated using the following surgical scores: the logistic EuroSCORE I (ESI), EuroSCORE II (ESII), Society of Thoracic Surgeons (STS) score, Ambler score (AS) and Guaragna score (GS). The performance of the risk scores was evaluated in terms of their calibration (Hosmer–Lemeshow test) and discrimination [area under the receiver–operating characteristic curve (AUC)].Results:The mean age was 81.5 ± 7.7 years. The CoreValve (Medtronic) was used in 86.1% of the cohort, and the transfemoral approach was used in 96.2%. The observed 30-day mortality was 9.1%. The 30-day mortality predicted by the scores was as follows: ESI, 20.2 ± 13.8%; ESII, 6.5 ± 13.8%; STS score, 14.7 ± 4.4%; AS, 7.0 ± 3.8%; GS, 17.3 ± 10.8%. Using AUC, none of the tested scores could accurately predict the 30-day mortality. AUC for the scores was as follows: 0.58 [95% confidence interval (CI): 0.49 to 0.68, p = 0.09] for ESI; 0.54 (95% CI: 0.44 to 0.64, p = 0.42) for ESII; 0.57 (95% CI: 0.47 to 0.67, p = 0.16) for AS; 0.48 (95% IC: 0.38 to 0.57, p = 0.68) for STS score; and 0.52 (95% CI: 0.42 to 0.62, p = 0.64) for GS. The Hosmer–Lemeshow test indicated acceptable calibration for all scores (p > 0.05).Conclusions:In this real world Brazilian registry, the surgical risk scores were inaccurate in predicting mortality after TAVI. Risk models specifically developed for TAVI are required.

Methods for field detection of resistance to temephos in simuliids. Larval esterase level and topical application of the insecticide to adults

Two practical field methods for indirect detection of simuliid populations resistant to temephos are proposed. The first is based on high esterase activity in resistant larvae and involves adaptations of a filter paper test in which faintly stained spots indicate susceptible populations and strongly stained ones reveal populations resistant to temephos. The second is based on the resistance to the larvicide when adults are topically exposed, and involves the use of diagnostic doses obtained by the comparison between the LD50 for susceptible and resistant populations. The relevance of such methods is discussed in order to help resistance detection in Simulium pertinax Kollar control programmes.

Diagnosis of Streptococcus pneumoniae meningitis by polymerase chain reaction amplification of the gene for pneumolysin

Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear, is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established. Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96% (95% confidence interval, CI, 90-99%) compared to a sensitivity of 59% for culture (95% CI 49-69%), 66% for Gram stain (95% CI 56-74%), and 78% for latex agglutination test (95% CI 69-86%); PCR specificity was 100% (95% CI 83-100%). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.

The efficiency of different estimation methods of hydro-physical limits

The soil water available to crops is defined by specific values of water potential limits. Underlying the estimation of hydro-physical limits, identified as permanent wilting point (PWP) and field capacity (FC), is the selection of a suitable method based on a multi-criteria analysis that is not always clear and defined. In this kind of analysis, the time required for measurements must be taken into consideration as well as other external measurement factors, e.g., the reliability and suitability of the study area, measurement uncertainty, cost, effort and labour invested. In this paper, the efficiency of different methods for determining hydro-physical limits is evaluated by using indices that allow for the calculation of efficiency in terms of effort and cost. The analysis evaluates both direct determination methods (pressure plate - PP and water activity meter - WAM) and indirect estimation methods (pedotransfer functions - PTFs). The PTFs must be validated for the area of interest before use, but the time and cost associated with this validation are not included in the cost of analysis. Compared to the other methods, the combined use of PP and WAM to determine hydro-physical limits differs significantly in time and cost required and quality of information. For direct methods, increasing sample size significantly reduces cost and time. This paper assesses the effectiveness of combining a general analysis based on efficiency indices and more specific analyses based on the different influencing factors, which were considered separately so as not to mask potential benefits or drawbacks that are not evidenced in efficiency estimation.

Highly sensitive spectrophotometric methods for the determination of olanzapine

Highly sensitive and selective spectrophotometric methods (A and B) were developed for the determination of micro amounts of olanzapine (OLZ). Method A (direct method) is based on the oxidation of olanzapine with a known excess of iodine monochloride (ICl) in an acidic medium. Under the same condition, thymol blue was iodinated by unreacted ICl, and the absorbance of uniodinated thymol blue was measured at 536 nm. The decrease in ICl concentration is a measure of drug concentration. In method B (indirect method), oxidation of OLZ by a known excess of Ce(IV) in sulfuric acid medium followed by the reaction of unreacted Ce(IV) with leuco crystal violet (LCV) to crystal violet (CV), which is measured in an acetate buffer medium ( pH 4.9) at 580 nm. These methods obey the Beer's law in the concentration range of 0.2-1.6 µg mL-1 (method A) and 0.1-1.4 µg mL-1 (method B). The developed procedures have been successfully applied to the determination of OLZ in pure and in dosage forms. The results exhibit no interference from the presence of excipients. The reliability of the methods was established by parallel determination of OLZ against the reference method.

Rainfall erosivity for the State of Rio de Janeiro estimated by artificial neural network

The Artificial Neural Networks (ANNs) are mathematical models method capable of estimating non-linear response plans. The advantage of these models is to present different responses of the statistical models. Thus, the objective of this study was to develop and to test ANNs for estimating rainfall erosivity index (EI30) as a function of the geographical location for the state of Rio de Janeiro, Brazil and generating a thematic visualization map. The characteristics of latitude, longitude e altitude using ANNs were acceptable to estimating EI30 and allowing visualization of the space variability of EI30. Thus, ANN is a potential option for the estimate of climatic variables in substitution to the traditional methods of interpolation.

Estimation methods of reference evapotranspiration (ETo) for Uberlândia -MG

One approach to verify the adequacy of estimation methods of reference evapotranspiration is the comparison with the Penman-Monteith method, recommended by the United Nations of Food and Agriculture Organization - FAO, as the standard method for estimating ET0. This study aimed to compare methods for estimating ET0, Makkink (MK), Hargreaves (HG) and Solar Radiation (RS), with Penman-Monteith (PM). For this purpose, we used daily data of global solar radiation, air temperature, relative humidity and wind speed for the year 2010, obtained through the automatic meteorological station, with latitude 18° 91' 66" S, longitude 48° 25' 05" W and altitude of 869m, at the National Institute of Meteorology situated in the Campus of Federal University of Uberlandia - MG, Brazil. Analysis of results for the period were carried out in daily basis, using regression analysis and considering the linear model y = ax, where the dependent variable was the method of Penman-Monteith and the independent, the estimation of ET0 by evaluated methods. Methodology was used to check the influence of standard deviation of daily ET0 in comparison of methods. The evaluation indicated that methods of Solar Radiation and Penman-Monteith cannot be compared, yet the method of Hargreaves indicates the most efficient adjustment to estimate ETo.