Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Is there an efficient trap or collection method for sampling Anopheles darlingi and other malaria vectors that can describe the essential parameters affecting transmission dynamics as effectively as human landing catches? - A Review

Distribution, abundance, feeding behaviour, host preference, parity status and human-biting and infection rates are among the medical entomological parameters evaluated when determining the vector capacity of mosquito species. To evaluate these parameters, mosquitoes must be collected using an appropriate method. Malaria is primarily transmitted by anthropophilic and synanthropic anophelines. Thus, collection methods must result in the identification of the anthropophilic species and efficiently evaluate the parameters involved in malaria transmission dynamics. Consequently, human landing catches would be the most appropriate method if not for their inherent risk. The choice of alternative anopheline collection methods, such as traps, must consider their effectiveness in reproducing the efficiency of human attraction. Collection methods lure mosquitoes by using a mixture of olfactory, visual and thermal cues. Here, we reviewed, classified and compared the efficiency of anopheline collection methods, with an emphasis on Neotropical anthropophilic species, especially Anopheles darlingi, in distinct malaria epidemiological conditions in Brazil.

Using a single tablet daily to treat latent tuberculosis infection in Brazil: bioequivalence of two different isoniazid formulations (300 mg and 100 mg) demonstrated by a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry method in a randomised, crossover study

The recommended treatment for latent tuberculosis (TB) infection in adults is a daily dose of isoniazid (INH) 300 mg for six months. In Brazil, INH was formulated as 100 mg tablets. The treatment duration and the high pill burden compromised patient adherence to the treatment. The Brazilian National Programme for Tuberculosis requested a new 300 mg INH formulation. The aim of our study was to compare the bioavailability of the new INH 300 mg formulation and three 100 mg tablets of the reference formulation. We conducted a randomised, single dose, open label, two-phase crossover bioequivalence study in 28 healthy human volunteers. The 90% confidence interval for the INH maximum concentration of drug observed in plasma and area under the plasma concentration vs. time curve from time zero to the last measurable concentration “time t” was 89.61-115.92 and 94.82-119.44, respectively. The main limitation of our study was that neither adherence nor the safety profile of multiple doses was evaluated. To determine the level of INH in human plasma, we developed and validated a sensitive, simple and rapid high-performance liquid chromatography-tandem mass spectrometry method. Our results showed that the new formulation was bioequivalent to the 100 mg reference product. This finding supports the use of a single 300 mg tablet daily strategy to treat latent TB. This new formulation may increase patients’ adherence to the treatment and quality of life.

A sensitive, specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivaxandPlasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivaxand Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed withPlasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochromeb-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.

Adaptação para a língua portuguesa e validação do Lunney Scoring Method for Rating Accuracy of Nursing Diagnoses

O Lunney Scoring Method for Rating Accuracy of Nursing Diagnoses (LSM) é uma escala de diferencial semântico que foi desenvolvida por Lunney para estimar a acurácia dos diagnósticos de enfermagem. O objetivo deste estudo foi adaptar o LSM para a língua portuguesa e avaliar as sua propriedades psicométricas. A escala original foi traduzida para o português, revertida para o inglês e as duas versões em inglês foram comparadas para ajustar a versão em português que passou a ser denominada Escala de Acurácia de Diagnóstico de Enfermagem de Lunney - EADE. Quatro enfermeiras foram orientadas sobre a EADE e a aplicaram em 159 diagnósticos formulados para 26 pacientes de três estudos primários com base nos registros de entrevista e exame físico de cada paciente. Os índices Kappa de Cohen mostraram ausência de concordância entre as avaliadoras, o que indica que o instrumento adaptado não tem confiabilidade satisfatória. Em virtude desse resultado, não foi realizada estimativa de validade.

Rearing method of Oryzaephilus surinamensis (L.) (Coleoptera, Silvanidae) on various wheat grain granulometry

Oryzaephilus surinamensis is one of most common insect pest of grains and a variety of stored products, and has been found in high numbers in almost all storage facilities. However, laboratory mass rearing of this insect for bioassays is not a simple task, mainly because of its feeding behavior, small size, and high mobility. Thus, the aim of this work was to develop a simple and efficient laboratory rearing method for O. surinamensis, using wheat kernels milled into different granulometry to obtain large number and standardized population at different life stages for bioassays. The adults were collected from storage grain facilities in the southern region of Brazil and 100 specimens were placed inside glass jars with wheat kernels milled at different grades and kept at 25±0.5ºC and 65±5% relative humidity. The insects were allowed to copulate and lay eggs for 10 days and then removed. The number of eggs, larvae, and pupae was counted at five-day intervals; longevity of the second generation adults was evaluated. The kernels milled at grade 20 were the best medium for offspring production: 89% of eggs by the 5th day; 30.5% larvae by the 10th day; 43% pupae by the 30th day and 63.4% adults at the 46th day. The adults survived up to 450 days. Culturing O. surinamensis under the described conditions, transferring the parental adults by the 10th day after infestation and replacing the media when population builds up will produce enough insects of each stage for various laboratory bioassays.

Evaluation of oviposition traps as an entomological surveillance method for Aedes aegypti (Diptera, Culicidae)

This study aimed to describe the behavior of oviposition traps for Aedes aegypti over time, to compare it with the larval survey and to investigate the association with climatic variables. It was conducted in São José do Rio Preto city, São Paulo. Daily climatic data and fortnightly measurements for oviposition traps and larval infestation were collected from October 2003 to September 2004. Three different periods were identified in the behavior of oviposition traps' positivity and mean number of eggs: increase, plateau and decrease in values. These measurements followed the variation of climatic data from the first and third periods. High correlation was obtained between the positivity and the mean number of eggs. The oviposition traps showed higher capacity to detect the vector than did larval survey. It was observed that the first (October to December) and third (May to September) periods were considered to be the most suitable to use oviposition traps than larval surveys.

Spatial evaluation of larvae of Culicidae (Diptera) from different breeding sites: application of a geospatial method and implications for vector control

Spatial evaluation of Culicidae (Diptera) larvae from different breeding sites: application of a geospatial method and implications for vector control. This study investigates the spatial distribution of urban Culicidae and informs entomological monitoring of species that use artificial containers as larval habitats. Collections of mosquito larvae were conducted in the São Paulo State municipality of Santa Bárbara d' Oeste between 2004 and 2006 during house-to-house visits. A total of 1,891 samples and nine different species were sampled. Species distribution was assessed using the kriging statistical method by extrapolating municipal administrative divisions. The sampling method followed the norms of the municipal health services of the Ministry of Health and can thus be adopted by public health authorities in disease control and delimitation of risk areas. Moreover, this type of survey and analysis can be employed for entomological surveillance of urban vectors that use artificial containers as larval habitat.

New method for rearing Spodoptera frugiperda in laboratory shows that larval cannibalism is not obligatory

New method for rearing Spodoptera frugiperda in laboratory shows that larval cannibalism is not obligatory. Here we show, for the first time, that larvae of the fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera, Noctuidae), can be successfully reared in a cohort-based manner with virtually no cannibalism. FAW larvae were reared since the second instar to pupation in rectangular plastic containers containing 40 individuals with a surprisingly ca. 90% larval survivorship. Adult females from the cohort-based method showed fecundity similar to that already reported on literature for larvae reared individually, and fertility higher than 99%, with the advantage of combining economy of time, space and material resources. These findings suggest that the factors affecting cannibalism of FAW larvae in laboratory rearings need to be reevaluated, whilst the new technique also show potential to increase the efficiency of both small and mass FAW rearings.

Soil spectral library and its use in soil classification

Soil science has sought to develop better techniques for the classification of soils, one of which is the use of remote sensing applications. The use of ground sensors to obtain soil spectral data has enabled the characterization of these data and the advancement of techniques for the quantification of soil attributes. In order to do this, the creation of a soil spectral library is necessary. A spectral library should be representative of the variability of the soils in a region. The objective of this study was to create a spectral library of distinct soils from several agricultural regions of Brazil. Spectral data were collected (using a Fieldspec sensor, 350-2,500 nm) for the horizons of 223 soil profiles from the regions of Matão, Paraguaçu Paulista, Andradina, Ipaussu, Mirandópolis, Piracicaba, São Carlos, Araraquara, Guararapes, Valparaíso (SP); Naviraí, Maracajú, Rio Brilhante, Três Lagoas (MS); Goianésia (GO); and Uberaba and Lagoa da Prata (MG). A Principal Component Analysis (PCA) of the data was then performed and a graphic representation of the spectral curve was created for each profile. The reflectance intensity of the curves was principally influenced by the levels of Fe2O3, clay, organic matter and the presence of opaque minerals. There was no change in the spectral curves in the horizons of the Latossolos, Nitossolos, and Neossolos Quartzarênicos. Argissolos had superficial horizon curves with the greatest intensity of reflection above 2,200 nm. Cambissolos and Neossolos Litólicos had curves with greater reflectance intensity in poorly developed horizons. Gleisols showed a convex curve in the region of 350-400 nm. The PCA was able to separate different data collection areas according to the region of source material. Principal component one (PC1) was correlated with the intensity of reflectance samples and PC2 with the slope between the visible and infrared samples. The use of the Spectral Library as an indicator of possible soil classes proved to be an important tool in profile classification.

Method to estimate soil macroporosity and microporosity based on sand content and bulk density

Macroporosity is often used in the determination of soil compaction. Reduced macroporosity can lead to poor drainage, low root aeration and soil degradation. The aim of this study was to develop and test different models to estimate macro and microporosity efficiently, using multiple regression. Ten soils were selected within a large range of textures: sand (Sa) 0.07-0.84; silt 0.03-0.24; clay 0.13-0.78 kg kg-1 and subjected to three compaction levels (three bulk densities, BD). Two models with similar accuracy were selected, with a mean error of about 0.02 m³ m-3 (2 %). The model y = a + b.BD + c.Sa, named model 2, was selected for its simplicity to estimate Macro (Ma), Micro (Mi) or total porosity (TP): Ma = 0.693 - 0.465 BD + 0.212 Sa; Mi = 0.337 + 0.120 BD - 0.294 Sa; TP = 1.030 - 0.345 BD 0.082 Sa; porosity values were expressed in m³ m-3; BD in kg dm-3; and Sa in kg kg-1. The model was tested with 76 datum set of several other authors. An error of about 0.04 m³ m-3 (4 %) was observed. Simulations of variations in BD as a function of Sa are presented for Ma = 0 and Ma = 0.10 (10 %). The macroporosity equation was remodeled to obtain other compaction indexes: a) to simulate maximum bulk density (MBD) as a function of Sa (Equation 11), in agreement with literature data; b) to simulate relative bulk density (RBD) as a function of BD and Sa (Equation 13); c) another model to simulate RBD as a function of Ma and Sa (Equation 16), confirming the independence of this variable in relation to Sa for a fixed value of macroporosity and, also, proving the hypothesis of Hakansson & Lipiec that RBD = 0.87 corresponds approximately to 10 % macroporosity (Ma = 0.10 m³ m-3).

An improved method for measuring soil microbial activity by gas phase flow injection analysis

The rate of carbon dioxide production is commonly used as a measure of microbial activity in the soil. The traditional method of CO2 determination involves trapping CO2 in an alkali solution and then determining CO2 concentration indirectly by titration of the remaining alkali in the solution. This method is still commonly employed in laboratories throughout the world due to its relative simplicity and the fact that it does not require expensive, specific equipment. However, there are several drawbacks: the method is time-consuming, requires large amounts of chemicals and the consistency of results depends on the operator's skills. With this in mind, an improved method was developed to analyze CO2 captured in alkali traps, which is cheap and relatively simple, with a substantially shorter sample handling time and reproducibility equivalent to the traditional titration method. A comparison of the concentration values determined by gas phase flow injection analysis (GPFIA) and titration showed no significant difference (p > 0.05), but GPFIA has the advantage that only a tenth of the sample volume of the titration method is required. The GPFIA system does not require the purchase of new, costly equipment but the device was constructed from items commonly found in laboratories, with suggestions for alternative configurations for other detection units. Furthermore, GPFIA for CO2 analysis can be equally applied to samples obtained from either the headspace of microcosms or from a sampling chamber that allows CO2 to be released from alkali trapping solutions. The optimised GPFIA method was applied to analyse CO2 released from degrading hydrocarbons from a site contaminated by diesel spillage.

Soil CO2 flux: a method comparison of closed static chambers in a sugarcane field

A large variety of techniques have been used to measure soil CO2 released from the soil surface, and much of the variability observed between locations must be attributed to the different methods used by the investigators. Therefore, a minimum protocol of measurement procedures should be established. The objectives of this study were (a) to compare different absorption areas, concentrations and volumes of the alkali trapping solution used in closed static chambers (CSC), and (b) to compare both, the optimized alkali trapping solution and the soda-lime trapping using CSC to measure soil respiration in sugarcane areas. Three CO2 absorption areas were evaluated (7; 15 and 20 % of the soil emission area or chamber); two volumes of NaOH (40 and 80 mL) at three concentrations (0.1, 0.25 and 0.5 mol L-1). Three different types of alkaline traps were tested: (a), 80 mL of 0.5 mol L-1 NaOH in glass containers, absorption area 15 % (V0.5); (b) 40 mL of 2 mol L-1 NaOH retained in a sponge, absorption area 80 % (S2) and (c) 40 g soda lime, absorption area 15 % (SL). NaOH concentrations of 0.5 mol L-1 or lower underestimated the soil CO2-C flux or CO2 flux. The lower limit of the alkali trap absorption area should be a minimum of 20 % of the area covered by the chamber. The 2 mol L-1 NaOH solution trap (S2) was the most efficient (highest accuracy and highest CO2 fluxes) in measuring soil respiration.