Detection of human bocavirus and human metapneumovirus by real-time PCR from patients with respiratory symptoms in Southern Brazil

The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5% of samples and hBoV in 13.2%. A unique causative viral agent was identified in 46.2% samples and the coinfection rate was 43.7%. For hBoV, 98.3% of all positive samples were from patients with mixed infections. Similarly, 84.8% of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

Molecular diagnosis of strongyloidiasis in tropical areas: a comparison of conventional and real-time polymerase chain reaction with parasitological methods

This study aimed to evaluate the use of conventional polymerase chain reaction (cPCR) and real-time quantitative PCR (qPCR) in the diagnosis of human strongyloidiasis from stool samples in tropical areas. Stool samples were collected from individuals and were determined to be positive for Strongyloides stercoralis (group I), negative for S. stercoralis (group II) and positive for other enteroparasite species (group III). DNA specific to S. stercoralis was found in 76.7% of group I samples by cPCR and in 90% of group I samples by qPCR. The results show that molecular methods can be used as alternative tools for detecting S. stercoralis in human stool samples in tropical areas.

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.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Relationship between the users' contact time in educational programs on diabetes mellitus and self-care skills and knowledge

Abstract OBJECTIVE Check the relationship between the users' contact time in educational programs and self-care and knowledge variables in diabetes mellitus. METHOD A longitudinal study with a quantitative approach with the participation, in the initial phase, of 263 users linked to Basic Health Units in Belo Horizonte, Brazil during the years 2012 and 2013. The data were collected with respect to the total contact time of the users' participation in the educational program as regards knowledge and self-care in acquired diabetes mellitus. The data were analyzed using the Student t-test for comparison of means, considering a 0.05 significance level. RESULTS The final sample included 151 users. The analysis showed that the improvement in self-care scores was statistically higher during an educational intervention of eight hours or more (p-value <0.05). In relation to the scores for knowledge, there was a statistically significant improvement at the end of the educational program. It was not possible to identify a value for the contact time from which there was an increase in mean scores for the ability of knowledge. CONCLUSION To improve the effectiveness of the promotion of skills related to knowledge and self-care in diabetes mellitus, it is necessary to consider the contact time as a relevant factor of the educational program.

Nymph developmental time and survivorship, adult longevity, reproduction and body weight of Dichelops melacanthus (Dallas) feeding on natural and artificial diets

The biology of nymphs and adults of the neotropical pentatomid, Dichelops melacanthus (Dallas), feeding on the natural foods, soybean, Glycine max (L.) Merrill immature pods, and corn, Zea mays L. immature seeds, and on an artificial dry diet, was studied in the laboratory. Nymph developmental time was shorter on the natural foods (ca. 21-22 days) than on the artificial diet (28 days), and most nymphs reached adulthood on the food plants (55% on soybean and 73% on corn) than on the artificial diet (40%). Fresh body weight at adult emergence was similar and higher for females raised as nymphs on the natural foods, compared to females from nymphs raised on the artificial diet; for males, weights were similar on all foods. Mean (female and male) survivorship up to day 20, decreased from 55% on soybean to 40% on corn, down to 0% on the artificial diet. Total longevity for females was higher on soybean, while for males was similar on all foods. About three times more females oviposited on soybean than on corn, but fecundity/female was similar on both foods. On the artificial diet, only one out of 30 females oviposited. Fresh body weight of adults increased significantly during the first week of adult life, and at the end of the 3rd week, weight gain was similar on all foods.

On the nesting biology of eumenine wasps yet again: Minixi brasilianum (de Saussure) is a builder and a renter... at the same time! (Hymenoptera, Vespidae, Eumeninae)

Our understanding of eumenine nesting biology is still elusive. The use of two nesting strategies, namely renting and building, are reported concomitantly for the first time for Minixi brasilianum (de Saussure, 1875). Ecological factors such as resource availability and protection against potential enemies may play an important role in eumenine nesting biology.

N2O emissions from a cultivated mollisol: optimal time of day for sampling and the role of soil temperature

The correct use of closed field chambers to determine N2O emissions requires defining the time of day that best represents the daily mean N2O flux. A short-term field experiment was carried out on a Mollisol soil, on which annual crops were grown under no-till management in the Pampa Ondulada of Argentina. The N2O emission rates were measured every 3 h for three consecutive days. Fluxes ranged from 62.58 to 145.99 ∝g N-N2O m-2 h-1 (average of five field chambers) and were negatively related (R² = 0.34, p < 0.01) to topsoil temperature (14 - 20 ºC). N2O emission rates measured between 9:00 and 12:00 am presented a high relationship to daily mean N2O flux (R² = 0.87, p < 0.01), showing that, in the study region, sampling in the mornings is preferable for GHG.

SWEET SORGHUM PERFORMANCE AFFECTED BY SOIL COMPACTION AND SOWING TIME AS A SECOND CROP IN THE BRAZILIAN CERRADO

ABSTRACT Increasing attention has recently been given to sweet sorghum as a renewable raw material for ethanol production, mainly because its cultivation can be fully mechanized. However, the intensive use of agricultural machinery causes soil structural degradation, especially when performed under inadequate conditions of soil moisture. The aims of this study were to evaluate the physical quality of aLatossolo Vermelho Distroférrico (Oxisol) under compaction and its components on sweet sorghum yield forsecond cropsowing in the Brazilian Cerrado (Brazilian tropical savanna). The experiment was conducted in a randomized block design, in a split plot arrangement, with four replications. Five levels of soil compaction were tested from the passing of a tractor at the following traffic intensities: 0 (absence of additional compaction), 1, 2, 7, and 15 passes over the same spot. The subplots consisted of three different sowing times of sweet sorghum during the off-season of 2013 (20/01, 17/02, and 16/03). Soil physical quality was measured through the least limiting water range (LLWR) and soil water limitation; crop yield and technological parameters were also measured. Monitoring of soil water contents indicated a reduction in the frequency of water content in the soil within the limits of the LLWR (Fwithin) as agricultural traffic increased (T0 = T1 = T2>T7>T15), and crop yield is directly associated with soil water content. The crop sown in January had higher industrial quality; however, there was stalk yield reduction when bulk density was greater than 1.26 Mg m-3, with a maximum yield of 50 Mg ha-1 in this sowing time. Cultivation of sweet sorghum as a second crop is a promising alternative, but care should be taken in cultivation under conditions of pronounced climatic risks, due to low stalk yield.

Basil conservation affected by cropping season, harvest time and storage period

Fresh basil (Ocimum basilicum L.) is used in food, phytotherapic industry, and in traditional therapeutic, due to its essential oil content and composition. Nevertheless basil can not be kept for long periods after harvest and its quality can be reduced. This work aimed to assess the influence of the season and harvest time in the postharvest conservation of basil stored for different periods. Basil was harvested at 8 am and 4 pm both in August/1999 and January/2000. Cuttings were conditioned in PVC packages and stored for 3, 6, and 9 days. During storage, chlorophyll content, essential oil content and composition were determined as well as microbiological analyses were carried out. Harvest season and the days of storage influenced the final content of essential oil. There was a linear decrease in the content of essential oil, in the chlorophyll content and in the number of mold and yeast colonies during storage. There was no effect of cropping season or harvest hour on essential oil composition, but the eugenol and linalool content increased during storage. Coliforms were under 0.3 MPN g-1 and the number of Staphylococcus aureus was under 1.0x10² UFC g-1.