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.

Detection and dynamics of porcine circovirus 2 shedding in semen using conventional and real-time PCR

The dynamics of porcine circovirus type 2 (PCV2) shedding in semen of naturally infected boars was studied. Semen was collected serially each 15 or 20 days during 62 days from 5 boars from a herd and from 11 boars from an artificial insemination center. All boars were positive for PCV2 DNA by nested polymerase chain reaction of raw semen in at least two sampling dates, and most of them had detectable shedding in all sampling dates. Real-time quantitative PCR was performed in 23 samples. All samples showed low amounts of PCV2 DNA, ranging from 98 to 652 PCV2 copies/mL. No differences between the frequencies of PCV2 DNA shed in semen were found considering herds and age of boars. PCV2 shedding in the semen can occur continuously or intermittently up to 60 days in naturally infected boars at 12 to 42 months old in absence of PCV2 clinical signs. These results demonstrate sporadic and long-term shedding patterns of low amounts of PCV2 DNA in semen from naturally infected boars.

Bioeffects of albumin-encapsulated microbubbles and real-time myocardial contrast echocardiography in an experimental canine model

Myocardial contrast echocardiography has been used for assessing myocardial perfusion. Some concerns regarding its safety still remain, mainly regarding the induction of microvascular alterations. We sought to determine the bioeffects of microbubbles and real-time myocardial contrast echocardiography (RTMCE) in a closed-chest canine model. Eighteen mongrel dogs were randomly assigned to two groups. Nine were submitted to continuous intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) plus continuous imaging using power pulse inversion RTMCE for 180 min, associated with manually deflagrated high-mechanical index impulses. The control group consisted of 3 dogs submitted to continuous imaging using RTMCE without PESDA, 3 dogs received PESDA alone, and 3 dogs were sham-operated. Hemodynamics and cardiac rhythm were monitored continuously. Histological analysis was performed on cardiac and pulmonary tissues. No hemodynamic changes or cardiac arrhythmias were observed in any group. Normal left ventricular ejection fraction and myocardial perfusion were maintained throughout the protocol. Frequency of mild and focal microhemorrhage areas in myocardial and pulmonary tissue was similar in PESDA plus RTMCE and control groups. The percentages of positive microscopical fields in the myocardium were 0.4 and 0.7% (P = NS) in the PESDA plus RTMCE and control groups, respectively, and in the lungs they were 2.1 and 1.1%, respectively (P = NS). In this canine model, myocardial perfusion imaging obtained with PESDA and RTMCE was safe, with no alteration in cardiac rhythm or left ventricular function. Mild and focal myocardial and pulmonary microhemorrhages were observed in both groups, and may be attributed to surgical tissue manipulation.

Detection and quantification of Roundup Ready® soybean residues in sausage samples by conventional and real-time PCR

The increasing presence of products derived from genetically modified (GM) plants in human and animal diets has led to the development of detection methods to distinguish biotechnology-derived foods from conventional ones. The conventional and real-time PCR have been used, respectively, to detect and quantify GM residues in highly processed foods. DNA extraction is a critical step during the analysis process. Some factors such as DNA degradation, matrix effects, and the presence of PCR inhibitors imply that a detection or quantification limit, established for a given method, is restricted to a matrix used during validation and cannot be projected to any other matrix outside the scope of the method. In Brazil, sausage samples were the main class of processed products in which Roundup Ready® (RR) soybean residues were detected. Thus, the validation of methodologies for the detection and quantification of those residues is absolutely necessary. Sausage samples were submitted to two different methods of DNA extraction: modified Wizard and the CTAB method. The yield and quality were compared for both methods. DNA samples were analyzed by conventional and real-time PCR for the detection and quantification of Roundup Ready® soybean in the samples. At least 200 ng of total sausage DNA was necessary for a reliable quantification. Reactions containing DNA amounts below this value led to large variations on the expected GM percentage value. In conventional PCR, the detection limit varied from 1.0 to 500 ng, depending on the GM soybean content in the sample. The precision, performance, and linearity were relatively high indicating that the method used for analysis was satisfactory.

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.

Brazilian avian metapneumovirus subtypes A and B: experimental infection of broilers and evaluation of vaccine efficacy

Avian metapneumovirus (aMPV) is a respiratory pathogen associated with the swollen head syndrome (SHS) in chickens. In Brazil, live aMPV vaccines are currently used, but subtypes A and, mainly subtype B (aMPV/A and aMPV/B) are still circulating. This study was conducted to characterize two Brazilian aMPV isolates (A and B subtypes) of chicken origin. A challenge trial to explore the replication ability of the Brazilian subtypes A and B in chickens was performed. Subsequently, virological protection provided from an aMPV/B vaccine against the same isolates was analyzed. Upon challenge experiment, it was shown by virus isolation and real time PCR that aMPV/B could be detected longer and in higher amounts than aMPV/A. For the protection study, 18 one-day-old chicks were vaccinated and challenged at 21 days of age. Using virus isolation and real time PCR, no aMPV/A was detected in the vaccinated chickens, whereas one vaccinated chicken challenged with the aMPV/B isolate was positive. The results showed that aMPV/B vaccine provided a complete heterologous virological protection, although homologous protection was not complete in one chicken. Although only one aMPV/B positive chicken was detected after homologous vaccination, replication in vaccinated animals might allow the emergence of escape mutants.

No-till corn performance in response to P and fertilization modes

No-tillage systems provide soil changes that affect nutrient dynamics, hence, changing rates and forms of fertilizer application. This study aimed to evaluate the effect of phosphorus (P) and modes of nitrogen (N) and P application in corn under long-term no-tillage in a clayey Oxisol. Two experiments were carried out in the same experimental area and in the same year, in a randomized blocks design with four replications. In experiment I, the treatments consisted of five doses of phosphorus (0, 40, 80, 120 and 160 kg ha-1 of P2O5) applied in the sowing furrow. In experiment II, the treatments consisted of the N and P application modes (topdressing, in the sowing furrow and control - without N and P). Experiment I evaluated the root length, P uptake and grain yield and, the Experiment II, the firing height and yield. The P rates provided linear increases in root length in the 0-10 cm layer, P uptake and grain production. The different modes of application provided differences in the firing height and corn yield. The control treatment (0 kg ha-1 of N and P) provided the highest firing height, superior than those of topdressing and application in the furrow, which were not significantly different. The topdress application of N and P provided an increase in corn yield that exceeded 16 and 42% of the application in the furrow and the control, respectively. Thus, the results confirmed that increasing rates of P2O5, in soil with high initial P content, influence positively corn production factors, but with little significant responses, and the topdress application of N and P on soil with high P content, without water restriction, provided increased grain yield in relation to the application in the furrow.

Notes on health care planning in Latin America and the Caribbean

Attention is called to the fact that the efforts to improve health of populations in Latin America have generally failed. The inequality in the distribution of ill-health is great. The authors accept the fact that the lack of resources available to the health sector may be a restriction towards the improvement of the situation, but they argue that a much more important issue is the misuse of such resources and their maldistribution within the health sector. The lack of integration and coordination between the health services, the conflict of public and private health systems, the under-utilization of existing services and the gap between planning and real implementation are discussed.

Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

BACKGROUND: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31%) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9%). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16%) followed by gB3 (35.48%). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017), and almost all fatal cases (80%) had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

Enterococcus faecium AND Enterococcus faecalis IN BLOOD OF NEWBORNS WITH SUSPECTED NOSOCOMIAL INFECTION

Enterococci are Gram-positive cocci saprophyte of the human gastrointestinal tract, diners who act as opportunistic pathogens. They can cause infections in patients hospitalized for a long time or who have received multiple antibiotic therapy. Enterococcus faecalis and Enterococcus faecium are the most common species in human infections. To evaluate the possibility of rapid detection of these species and their occurrence in the blood of newborns with suspected nosocomial infection, blood samples were collected from 50 newborns with late infections, admitted to the Neonatal Care Unit of the University Hospital Federal de Mato Grosso do Sul (UFMS-HU), from September 2010 to January 2011. The samples were subjected to conventional PCR and real time PCR (qPCR) to search for Enterococcus faecium and Enterococcus faecalis, respectively. The PCR results were compared with respective blood cultures from 40 patients. No blood cultures were positive for Enterococci, however, eight blood samples were identified as genomic DNA of Enterococcus faecium by qPCR and 22 blood samples were detected as genomic DNA of Enterococcus faecalis by conventional PCR. These findings are important because of the clinical severity of the evaluated patients who were found positive by conventional PCR and not through routine microbiological methods.

EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

LABORATORY EVALUATION OF THE DEVELOPMENT OF Aedes aegypti IN TWO SEASONS: INFLUENCE OF DIFFERENT PLACES AND DIFFERENT DENSITIES

Aedes aegypti is an important vector in Brazil being the main vector of the dengue-fever. This paper employs survival curves to describe the time in days from larvae to adult forms of Aedes aegypti raised, individually and collectively, and compares it during winter and spring when positioned inside and outside a laboratory. The study was conducted in São Vicente, a coastal city in Southeastern Brazil. The lowest water temperature in winter and in spring was 20 °C and the highest was 26 °C in spring. Higher and more stable temperatures were measured in the intra compared to the peri in both seasons. Consequently, larvae positioned in the intra resulted in the lowest median time to develop in the individual and collective experiment (nine and ten days, respectively). At least 25% of the larvae positioned in the intra in the individual experiment in the spring took only seven days to reach adulthood. Sex ratios and the median time development by sex did not show significant differences. These results indicate that efforts to control Aedes aegypti must be continuous and directed mainly to prevent the intra-domiciliary sites that can be infested in a week in order to reduce the human-vector contact.

SEROLOGICAL AND MOLECULAR SURVEY OF Leptospira spp. AMONG CART HORSES FROM AN ENDEMIC AREA OF HUMAN LEPTOSPIROSIS IN CURITIBA, SOUTHERN BRAZIL

Introduction: Cart horses are a re-emerging population employed to carry recyclable material in cities. Methods: Sixty-two horses were sampled in an endemic area of human leptospirosis. The microscopic agglutination test (MAT) and real-time polymerase chain reaction (qPCR) were performed. Results: A seropositivity of 75.8% with serovar Icterohaemorrhagiae in 80.8% of the horses was observed. Blood and urine were qPCR negative. MAT showed positive correlations with rainfall (p = 0.02) and flooding (p = 0.03). Conclusions: Although horses may be constantly exposed to Leptospira spp. in the environment mostly because of rainfall and flooding, no leptospiremia or leptospiruria were observed in this study.

High prevalence of HTLV-1 and 2 viruses in pregnant women in São Luis, state of Maranhão, Brazil

INTRODUCTION: Human T cell lymphotropic virus type 1 (HTLV-1) is endemic in the Caribbean, Japan, South America and regions of Africa. HTLV-2 is present in Native American populations and associated with IV drug use in Europe and North America. In Brazil, it is estimated that 1.5 million people are infected with HTLV-1/2. The study objective was to determine HTLV-1/2 prevalence in pregnant women in the prenatal care from three public services in São Luis, State of Maranhão, Brazil, and to counsel seropositive women to reduce viral transmission. METHODS: A cross-sectional study was conducted from February to December 2008; women with age of 18 to 45 years, with low risk for sexually transmitted disease (STD) were invited to participate. Blood samples were collected in filter paper, and HTLV-1/2 immunoenzymatic test (ELISA) was performed as a screening test. Women with reactive results were submitted to peripheral venous blood collection for ELISA repetition, followed by Western blot (WB) and real-time PCR to confirm and discriminate the infection between virus types 1 and 2. RESULTS: Of the 2,044 women tested, seven (0.3%) were ELISA reactive and confirmed positive (four were HTLV-1, and three were HTLV-2). All positive women were oriented not to breastfeed their newborns. CONCLUSIONS: This study showed that the virus is present in high prevalence in that population. Further studies covering other segments of the population are necessary to better characterize the presence of HTLV-1/2 in Maranhão and to elicit measures to prevent its spread.

Time-variations of equivalent water heights'from Grace Mission and in-situ river stages in the Amazon basin

Gravity Recovery and Climate Experiment (GRACE) mission is dedicated to measuring temporal variations of the Earth's gravity field. In this study, the Stokes coefficients made available by Groupe de Recherche en Géodésie Spatiale (GRGS) at a 10-day interval were converted into equivalent water height (EWH) for a ~4-year period in the Amazon basin (from July-2002 to May-2006). The seasonal amplitudes of EWH signal are the largest on the surface of Earth and reach ~ 1250mm at that basin's center. Error budget represents ~130 mm of EWH, including formal errors on Stokes coefficient, leakage errors (12 ~ 21 mm) and spectrum truncation (10 ~ 15 mm). Comparison between in situ river level time series measured at 233 ground-based hydrometric stations (HS) in the Amazon basin and vertically-integrated EWH derived from GRACE is carried out in this paper. Although EWH and HS measure different water bodies, in most of the cases a high correlation (up to ~80%) is detected between the HS series and EWH series at the same site. This correlation allows adjusting linear relationships between in situ and GRACE-based series for the major tributaries of the Amazon river. The regression coefficients decrease from up to down stream along the rivers reaching the theoretical value 1 at the Amazon's mouth in the Atlantic Ocean. The variation of the regression coefficients versus the distance from estuary is analysed for the largest rivers in the basin. In a second step, a classification of the proportionality between in situ and GRACE time-series is proposed.