A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.

Magnetic resonance imaging and BMB score in the evaluation of bone involvement in Gaucher’s disease patients

AbstractObjective:To evaluate by magnetic resonance imaging changes in bone marrow of patients undergoing treatment for type I Gaucher’s disease.Materials and Methods:Descriptive, cross-sectional study of Gaucher’s disease patients submitted to 3 T magnetic resonance imaging of femurs and lumbar spine. The images were blindly reviewed and the findings were classified according to the semiquantitative bone marrow burden (BMB) scoring system.Results:All of the seven evaluated patients (three men and four women) presented signs of bone marrow infiltration. Osteonecrosis of the femoral head was found in three patients, Erlenmeyer flask deformity in five, and no patient had vertebral body collapse. The mean BMB score was 11, ranging from 9 to 14.Conclusion:Magnetic resonance imaging is currently the method of choice for assessing bone involvement in Gaucher’s disease in adults due to its high sensitivity to detect both focal and diffuse bone marrow changes, and the BMB score is a simplified method for semiquantitative analysis, without depending on advanced sequences or sophisticated hardware, allowing for the classification of the disease extent and assisting in the treatment monitoring.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Estimation of the potential evapotranspiration by a simplified penman method

The numerous methods for calculating the potential or reference evapotranspiration (ETo or ETP) almost always do for a 24-hour period, including values of climatic parameters throughout the nocturnal period (daily averages). These results have a nil effect on transpiration, constituting the main evaporative demand process in cases of localized irrigation. The aim of the current manuscript was to come up with a model rather simplified for the calculation of diurnal daily ETo. It deals with an alternative approach based on the theoretical background of the Penman method without having to consider values of aerodynamic conductance of latent and sensible heat fluxes, as well as data of wind speed and relative humidity of the air. The comparison between the diurnal values of ETo measured in weighing lysimeters with elevated precision and estimated by either the Penman-Monteith method or the Simplified-Penman approach in study also points out a fairly consistent agreement among the potential demand calculation criteria. The Simplified-Penman approach was a feasible alternative to estimate ETo under the local meteorological conditions of two field trials. With the availability of the input data required, such a method could be employed in other climatic regions for scheduling irrigation.

SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

Detection of Dientamoeba fragilis in patients with HIV/AIDS by using a simplified iron hematoxylin technique

INTRODUCTION: Studies strongly indicate Dientamoeba fragilis as one of the causes of diarrhea in human immunodeficiency virus (HIV) patients. METHODS: The objective of the present study was to evaluate the prevalence of D. fragilis associated with the causes of diarrhea in 82 HIV/ AIDS patients hospitalized at the Instituto de Infectologia Emílio Ribas from September 2006 to November 2008. RESULTS: In total, 105 samples were collected from 82 patients. Unprotected sex was the most frequent cause of HIV infection (46.3%), followed by the use of injectable or non-injectable drugs (14.6%). Patients presented with viral loads of 49-750,000 copies/ mL (average: 73,849 ± 124,850 copies/mL) and CD4 counts ranging of 2-1,306 cells/mm³ (average: 159 ± 250 cells/mm³). On an average, the odds of obtaining a positive result by using the other techniques (Hoffman, Pons and Janer or Lutz; Ritchie) were 2.7 times higher than the chance of obtaining a positive result by using the simplified iron hematoxylin method. Significant differences were found between the methods (p = 0.003). CONCLUSIONS: The other techniques can detect a significantly greater amount of parasites than the simplified iron hematoxylin method, especially with respect to Isospora belli, Cryptosporidium sp., Schistosoma mansoni, and Strongyloides stercoralis, which were not detected using hematoxylin. Endolimax nana and D. fragilis were detected more frequently on using hematoxylin, and the only parasite not found by the other methods was D. fragilis.

A simplified procedure for determination of clofentezine residues in fruits by liquid chromatography with UV detection

A rapid and sensitive method is described for the determination of clofentezine residues in apple, papaya, mango and orange. The procedure is based on the extraction of the sample with a hexane:ethyl acetate mixture (1:1, v/v) and liquid chromatographic analysis using UV detection. Mean recoveries from 4 replicates of fortified fruit samples ranged from 81% to 96%, with coefficients of variation from 8.9% to 12.5%. The detection and quantification limits of the method were of 0.05 and 0.1 mg kg-1, respectively.

Multiple centroid method to evaluate the adaptability of alfalfa genotypes

This study aimed to evaluate the efficiency of multiple centroids to study the adaptability of alfalfa genotypes (Medicago sativa L.). In this method, the genotypes are compared with ideotypes defined by the bissegmented regression model, according to the researcher's interest. Thus, genotype classification is carried out as determined by the objective of the researcher and the proposed recommendation strategy. Despite the great potential of the method, it needs to be evaluated under the biological context (with real data). In this context, we used data on the evaluation of dry matter production of 92 alfalfa cultivars, with 20 cuttings, from an experiment in randomized blocks with two repetitions carried out from November 2004 to June 2006. The multiple centroid method proved efficient for classifying alfalfa genotypes. Moreover, it showed no unambiguous indications and provided that ideotypes were defined according to the researcher's interest, facilitating data interpretation.

An appraisal of the health planning method proposed by the Pan-American Health Organization for Latin America

A brief description of the main features of the health planning technique developed by the "Centro de Estudios del Desarrollo" (CENDES) in Venezuela, and proposed by the Pan-American Health Organization for use in Latin America, is presented. This presentation is followed by an appraisal of the planning method which includes comments both upon its positive aspects and upon its negative points. Comments are also made referring to other recent publications of the WHO/PAHO on health planning. In conclusion, the CENDES technique is considered a health planning method of great potential for use especially in underdeveloped areas, the success of its application depending upon the hability of the health planners to introduce the necessary modifications to adapt to the local circunstamces.

The use of wide-mesh gauze impregnated with lambda-cyhalothrin covering wall openings in huts as a vector control method in Suriname

An alternative vector control method, using lambda-cyhalothrin impregnated wide-mesh gauze covering openings in the walls of the houses was developed in an area in the Eastern part of the interior of Suriname. Experimental hut observations showed that Anopheles darlingi greatly reduced their biting activity (99-100%) during the first 5 months after impregnation. A model assay showed high mortality both of mosquitoes repelled by the gauze as well as of those that succeeded in getting through it. A field application test in 270 huts showed good acceptance by the population and good durability of the applied gauze. After introducing the method in the entire working area, replacing DDT residual housespraying, the malaria prevalence, of 25-37% before application dropped and stabilized at between 5 and 10% within one year. The operational costs were less than those of the previously used DDT housespraying program, due to a 50% reduction in the cost of materials used. The method using widemesh gauze impregnated with lambdacyhalothrin strongly affects the behavior of An. darlingi. It is important to examine the effect of the method on malaria transmission further, since data indirectly obtained suggest substantial positive results.

Ill-defined causes of death in Brazil: a redistribution method based on the investigation of such causes

OBJECTIVE To propose a method of redistributing ill-defined causes of death (IDCD) based on the investigation of such causes.METHODS In 2010, an evaluation of the results of investigating the causes of death classified as IDCD in accordance with chapter 18 of the International Classification of Diseases (ICD-10) by the Mortality Information System was performed. The redistribution coefficients were calculated according to the proportional distribution of ill-defined causes reclassified after investigation in any chapter of the ICD-10, except for chapter 18, and used to redistribute the ill-defined causes not investigated and remaining by sex and age. The IDCD redistribution coefficient was compared with two usual methods of redistribution: a) Total redistribution coefficient, based on the proportional distribution of all the defined causes originally notified and b) Non-external redistribution coefficient, similar to the previous, but excluding external causes.RESULTS Of the 97,314 deaths by ill-defined causes reported in 2010, 30.3% were investigated, and 65.5% of those were reclassified as defined causes after the investigation. Endocrine diseases, mental disorders, and maternal causes had a higher representation among the reclassified ill-defined causes, contrary to infectious diseases, neoplasms, and genitourinary diseases, with higher proportions among the defined causes reported. External causes represented 9.3% of the ill-defined causes reclassified. The correction of mortality rates by the total redistribution coefficient and non-external redistribution coefficient increased the magnitude of the rates by a relatively similar factor for most causes, contrary to the IDCD redistribution coefficient that corrected the different causes of death with differentiated weights.CONCLUSIONS The proportional distribution of causes among the ill-defined causes reclassified after investigation was not similar to the original distribution of defined causes. Therefore, the redistribution of the remaining ill-defined causes based on the investigation allows for more appropriate estimates of the mortality risk due to specific causes.