Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

Evaluation of neuromotor development by means of the Harris Infant Neuromotor Test

Objective: To investigate the neuromotor development of at-risk children between three and 12 months of life, administering the Brazilian version of the Harris Infant Neuromotor Test (HINT).Method: A longitudinal study, with 78 children and 76 parents/guardians discharged from a neonatal intensive care unit in Fortaleza-CE/Brazil. Two instruments were administered: HINT and a socioeconomic questionnaire, between July/2009 to August/2010. Data from 55 preterm and 23 term children were analyzed. Results: The final mean scores ranged from 14.6 to 25.2 and from 11.2 to 24.7, for preterm and term, respectively, showing that 91% of children demonstrated good neuromotor performance; seven premature infants showed alterations which led to the referral of three children to a specialized clinic for examination and diagnostics.Conclusion: The test allowed nurses to assess infant development, identify deviations early, and plan interventions.

Pedotransfer functions to estimate proctor test parameters under different tillage systems

The Proctor test is time-consuming and requires sampling of several kilograms of soil. Proctor test parameters were predicted in Mollisols, Entisols and Vertisols of the Pampean region of Argentina under different management systems. They were estimated from a minimum number of readily available soil properties (soil texture, total organic C) and management (training data set; n = 73). The results were used to generate a soil compaction susceptibility model, which was subsequently validated using a second group of independent data (test data set; n = 24). Soil maximum bulk density was estimated as follows: Maximum bulk density (Mg m-3) = 1.4756 - 0.00599 total organic C (g kg-1) + 0.0000275 sand (g kg-1) + 0.0539 management. Management was equal to 0 for uncropped and untilled soils and 1 for conventionally tilled soils. The established models predicted the Proctor test parameters reasonably well, based on readily available soil properties. Tillage systems induced changes in the maximum bulk density regardless of total organic matter content or soil texture. The lower maximum apparent bulk density values under no-tillage require a revision of the relative compaction thresholds for different no-tillage crops.

Portable penetrometer for agricultural soil: sensitivity test to identify critical compaction depth

To express the negative effects of soil compaction, some researchers use critical values for soil mechanical strength that severely impair plant growth. The aim of this study was to identify this critical compaction depth, to test the functionality of a new, portable penetrometer developed from a spring dynamometer, and compare it to an electronic penetrometer traditionally used in compaction studies of agricultural soils. Three soils with distinct texture were conventionally tilled using a disk plow, and cultivated with different plant species. The critical soil resistance defined to establish critical compaction depth was equal to 1.5 MPa. The results of the new equipment were similar to the electronic penetrometer, indicating its viability as a tool for assessing the soil physical conditions for plant growth.

Conductivity test in seeds of different passion flower species

The objective of this work was to evaluate the use of the conductivity test as a means of predicting seed viability in seven Passiflora species: P. alata, P. cincinnata, P. edulis f. edulis, P. edulis f. flavicarpa, P. morifolia, P. mucronata, and P. nitida. Conductivity of non-desiccated (control), desiccated, and non-desiccated cryopreserved seeds was determined and related to their germination percentage. The obtained results suggest that the electrical conductivity test has potential as a germination predictor for P. edulis f. flavicarpa seed lots, but not for the other tested species.

Whole-body MRI: comprehensive evaluation on a 48-channel 3T MRI system in less than 40 minutes. Preliminary results

OBJECTIVE: To evaluate a comprehensive MRI protocol that investigates for cancer, vascular disease, and degenerative/inflammatory disease from the head to the pelvis in less than 40 minutes on a new generation 48-channel 3T system. MATERIALS AND METHODS: All MR studies were performed on a 48-channel 3T MR scanner. A 20-channel head/neck coil, two 18-channel body arrays, and a 32-channel spine array were employed. A total of 4 healthy individuals were studied. The designed protocol included a combination of single-shot T2-weighted sequences, T1-weighted 3D gradient-echo pre- and post-gadolinium. All images were retrospectively evaluated by two radiologists independently for overall image quality. RESULTS: The image quality for cancer was rated as excellent in the liver, pancreas, kidneys, lungs, pelvic organs, and brain, and rated as fair in the colon and breast. For vascular diseases ratings were excellent in the aorta, major branch vessel origins, inferior vena cava, portal and hepatic veins, rated as good in pulmonary arteries, and as poor in the coronary arteries. For degenerative/inflammatory diseases ratings were excellent in the brain, liver and pancreas. The inter-observer agreement was excellent. CONCLUSION: A comprehensive and time efficient screening for important categories of disease processes may be achieved with high quality imaging in a new generation 48-channel 3T system.

Evaluación de la estructura porosa de los materiales MCM-22, MCM-36 e ITQ-2 empleando el test catalitico del n-decano

The void structure of zeolites MCM-22, MCM-36 and ITQ-2 were discussed on the bases of catalytic reaction tests. The hydromerization of n-decane on bifunctional Pt/Zeolite Catalysts have been used as model reactions. Beta and ZSM-5 zeolites were used for comparison. It is concluded that all materials show features of 10MR zeolites and have also pores bigger than 12MR in this order MCM-226

Losartan potassium dissolution test for drug release evaluation in pharmaceutical capsules using HPLC and UV spectrophotometry

This work describes the development and validation of a dissolution test for 50 mg losartan potassium capsules using HPLC and UV spectrophotometry. A 2(4) full factorial design was carried out to optimize dissolution conditions and potassium phosphate buffer, pH 6.8 as dissolution medium, basket as apparatus at the stirring speed of 50 rpm and time of 30 min were considered adequate. Both dissolution procedure and analytical methods were validated and a statistical analysis showed that there are no significant differences between HPLC and spectrophotometry. Since there is no official monograph, this dissolution test could be applied for quality control routine.

Terbinafine: optimization of a LC method for quantitative analysis in pharmaceutical formulations and its application for a tablet dissolution test

A simple liquid chromatographic method was optimized for the quantitative determination of terbinafine in pharmaceutical hydroalcoholic solutions and tablets, and was also employed for a tablet dissolution test. The analysis was carried out using a RP-C18 (250 mm × 4.6 mm, 5 _6;m) Vertical® column, UV-Vis detection at 254 nm, and a methanol-water (95:5, v/v) mobile phase at a flow-rate of 1.2 mL min-1. Method validation investigated parameters such as linearity, precision, accuracy, robustness and specificity, which gave results within the acceptable range. The tablets dissolution was quite fast: 80% of the drug was dissolved within 15 min.

Development and validation of a dissolution test for diltiazem hydrochloride in immediate release capsules

This work describes the development and validation of a dissolution test for 60 mg of diltiazem hydrochloride in immediate release capsules. The best dissolution in vitro profile was achieved using potassium phosphate buffer at pH 6.8 as the dissolution medium and paddle as the apparatus at 50 rpm. The drug concentrations in the dissolution media were determined by UV spectrophotometry and HPLC and a statistical analysis revealed that there were significant differences between HPLC and spectrophotometry. This study illustrates the importance of an official method for the dissolution test, since there is no official monograph for diltiazem hydrochloride in capsules.

Spot-test identification and rapid quantitative sequential analysis of dipyrone

A qualitative spot-test and tandem quantitative analysis of dipyrone in the bulk drug and in pharmaceutical preparations is proposed. The formation of a reddish-violet color indicates a positive result. In sequence a quantitative procedure can be performed in the same flask. The quantitative results obtained were statistically compared with those obtained with the method indicated by the Brazilian Pharmacopoeia, using the Student's t and the F tests. Considering the concentration in a 100 µL aliquot, the qualitative visual limit of detection is about 5×10-6 g; instrumental LOD ≅ 1.4×10-4 mol L-1 ; LOQ ≅ 4.5×10-4 mol L-1.

Sensibilidade e especificidade do "California Mastitis Test" como recurso diagnóstico da mastite subclínica em relação à contagem de células somáticas

O "California Mastitis Test" (CMT) estima o conteúdo de células somáticas no leite e é interpretado subjetivamente, estabelecendo-se escores que, na maioria dos casos, variam de 1 a 5. O escore 1 indica uma reação completamente negativa e os de 2-5 indicam graus crescentes de resposta inflamatória do úbere, sendo normalmente considerados como indicativos de mastite subclínica. Dependendo da interpretação dos escores, o CMT pode produzir resultados falso-positivos ou falso-negativos. Esse trabalho teve o objetivo de avaliar a sensibilidade e a especificidade do CMT em relação à contagem de células somáticas (CCS). Foram utilizadas 3.012 amostras de leite provenientes de 760 vacas Holandesas ou mestiças Holandês-Zebu, pertencentes a 15 rebanhos. Todas as amostras foram submetidas ao CMT e processadas para CCS em equipamento Fossomatic 90. Os valores médios de CCS (x 1.000 células/ml) obtidos para os escores de CMT foram 1 (79,9), 2 (333,5), 3 (670,3), 4 (1.354,0) e 5 (4.455,6). Três opções de interpretação (doente/não-doente) para o CMT foram testadas, em relação aos valores de CCS, iniciando com 100.000 células/ml: (a) 1 versus 2, 3, 4, e 5; (b) 1 e 2 versus 3, 4 e 5; (c) 1, 2, 3 versus 4 e 5. As sensibilidades do CMT em identificar corretamente quartos mamários acima de 200.000 células/ml foram 79%, 61% e 34%, para as opções a, b e c, respectivamente. Para identificar corretamente contagens acima de 500.000 células/ml, as sensibilidades do CMT, para as opções a, b e c, foram, respectivamente: 93%, 82% e 54%. A sensibilidade do CMT em identificar quartos mamários com mastite subclínica foi adequada (acima de 80%) quando a interpretação do teste foi mais rigorosa (opções a e b). A interpretação da reação 3 como negativa (opção c) só alcançou sensibilidade de 80% para contagens entre 1.200.000 e 1.400.000 células/ml. As especificidades do CMT, para CCS de 200.000 e 500.000 foram, respectivamente, 90% e 80% (opção a), 97% e 90% (opção b) e 99% e 97% (opção c).

A conglutination test for rapid detection of antibodies against Babesia bigemina

A rapid conglutination test (RCT) with performance comparable to the indirect fluorescent antibody technique (IFAT) was developed to detect antibodies against Babesia bigemina (B. bigemina-RCT). The B. bigemina-RCT is a sensitive, specific, economical, and rapidly performed serological test suitable for field application or minimally equipped laboratories. This test had a sensitivity of 90.9%, and specificity of 97.6%, compared to IFAT, which showed for the same parameters respectively, 98.3% and 99.7%. The early detection of anti- B. bigemina immunoglobulins by RCT in experimental infections was nearly parallel to that of IFAT. Cross reactions were observed with sera from calves experimentally infected with Babesia bovis (1.8%) and with Anaplasma marginale (1.2%). RCT antigen prepared with non parasitized erythrocytes (negative antigen) showed 1.5%, 3.5% and 2.2% of positive reactions with sera from animals experimentally infected with B. bigemina, B. bovis and A. marginale. However, none of the sera from animals of endemic areas for babesia infection resulted in positive reactions with the negative antigen. Considering these results and shelf life over six months, the B. bigemina-RCT could be used for epidemiological surveys and evaluation of control measures against this species of Babesia.

Evaluation of resistance in a selected field strain of Haemonchus contortus to ivermectin and moxidectin using the Larval Migration on Agar Test

Haemonchus contortus is one of the most common and economically significant causes of disease in small ruminants worldwide, and the control programs of parasitic nematodes - including H. contortus - rely mostly on the use of anthelmintic drugs. The consequence of the use of this, as the sole sanitary strategy to avoid parasite infections, was the reduction of the efficacy of all chemotherapeutic products with a heavy selection for resistance. The widespread of anthelmintic resistance and the difficulty of its early diagnosis has been a major concern for the sustainable parasite management on farms. The objective of this research was to determine and compare the ivermectin (IVM) and moxidectin (MOX) effect in a selected field strain of H. contortus with a known resistance status, using the in vitro larval migration on agar test (LMAT). Third stage larvae of the selected isolate were obtained from faecal cultures of experimentally infected sheep and incubated in eleven increasing diluted concentrations of IVM and MOX (6, 12, 24, 48, 96, 192, 384, 768, 1536, 3072 and 6144µg/mL). The dose-response sigmoidal curves were obtained using the R² value of >0.90 and the lethal concentration (LC50) dose for the tested anthelmintic drugs using a four-parameter logistic model. The LC50 value for MOX was significantly lower than IVM (1.253µg/mL and 91.06µg/mL), identifying the H. contortus isolate as considerably less susceptible to IVM compared to MOX. Furthermore, the LMAT showed a high consistency (p<0.0001) and provided to be a useful diagnostic tool for monitoring the resistance status of IVM and MOX in H. contortus field isolate, as well as it may be used for official routine drug monitoring programs under the Ministry of Agriculture (MAPA) guidance.

Development and application of polymerase chain reaction test for detection of Conidiobolus lamprauges

Conidiobolomycosis is a granulomatous disease caused by the fungus Conidiobolus spp. in humans and animals. Traditional technique for diagnosis of the disease is isolation of the agent associated with the presence of typical clinical signs and pathological conditions. The aim of this study was to describe the development of a specific polymerase chain reaction (PCR) test for Conidiobolus lamprauges to detect the fungus in clinical samples. Samples from suspected animals were collected and submitted to isolation, histopathological analysis and amplification by PCR. DNA from tissues was subjected to PCR with fungi universal primers 18S rDNA gene, and specific primers were designed based on the same gene in C. lamprauges that generated products of about 540 bp and 222 bp respectively. The culture was positive in 26.6% of clinical samples. The PCR technique for C. lamprauges showed amplification of DNA from fresh tissues (80%) and paraffin sections (44.4%). In conclusion, the PCR technique described here demonstrated a high sensitivity and specificity for detection of fungal DNA in tissue samples, providing a tool for the rapid diagnosis of C. lamprauges.