Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice

AbstractMycobacterium avium subspecies paratuberculosis (MAP) can infect ruminants and remain subclinical for long periods within herds. The identification of organs that are more susceptible to infection and the evaluation of cytokine expression at the site of infection are important to understand the pathogenesis of MAP. In this study, the probability of detection of MAP-DNA and the expression of cytokines in organs of C57BL/6 mice infected intraperitoneally for 120 days were evaluated. Among the evaluated organs, the spleen (85%), colon (75%) and liver (60%) had the highest frequency of positivity. When compared these frequencies between organs, it has been found that the spleen had 1.54 times as likely to be positive in relation to the ileum, and 2.0 times more likely in relation to the Peyer's patches. In addition, at 60 days post-infection, the spleen and the liver were responsible for upregulation of IFN-γ , and the ileum by TNF-α and IL-4. The results indicate that the spleen is the best organ for evaluating an experimental infection by MAP, especially in the initial stages of the infection. Moreover, it showed that the spleen, liver and ileum have a direct role in the inflammatory response in experimental models.

Domestic Rhodnius ecuadoriensis (Hemiptera, Reduviidae) infestation in Northern Peru: a comparative trial of detection methods during a six-month follow-up

Two passive methods in the assessment of intradomiciliary infestation by Rhodnius ecuadoriensis were tested: (i) the Gomes Nuñez sensor box (GN), (ii) sheets of white typing paper and (iii) one active timed manual method. The study was carried out in the Alto Chicama River Valley, Province of Gran Chimú, Department of La Libertad. The study design consisted of an initial searching of triatomines inside of the domestic environment by the manual capture active procedure (man/hour) covering all the studied houses. Then, matched pairs of GN boxes and paper sheets were simultaneously installed in the bedrooms of 207 households distributed in 19 localities. A comparative prospective trial of these passive detection devices were monitored at 2, 4 and, finally 6 months follow-up. Parasitological Trypanosoma rangeli and/or T. cruzi infections were investigated in two houses with high level of infestation by R. ecuadoriensis. 16.9% of the 207 households investigated by an initial active manual method were infested with R. ecuadoriensis. The proportion of infested houses fluctuated from 6.2 to 55.5% amongst the 19 localities investigated. T. rangeli natural infection was detected in R. ecuadoriensis specimens collected in two households. Parasite rates in the bugs ranged from 16.6 to 21.7% respectively. The most striking fact was an average rate of salivary gland infection ranging from 7.4 to 8.3%. At the end of the sixth month period, a cumulative incidence of 31.4% of positive GN boxes against 15.9% for paper sheets was recorded. All three methods combined detected domestic infestation in 129 (62.3%) of the 207 houses studied in the 19 localities. The range of houses infested varies from 6.7% to 92.9%. In areas with low bug density infestation rates, the methodology experienced in our studies, seems to be the best choice for investigations on domestic R. ecuadoriensis populations.

Theoretical estimates of consumable food and probability of acquiring food in larvae of Chrysomya putoria (Diptera: Calliphoridae)

An indirect estimate of consumable food and probability of acquiring food in a blowfly species, Chrysomya putoria, is presented. This alternative procedure combines three distinct models to estimate consumable food in the context of the exploitative competition experienced by immature individuals in blowfly populations. The relevant parameters are derived from data for pupal weight and survival and estimates of density-independent larval mortality in twenty different larval densities. As part of this procedure, the probability of acquiring food per unit of time and the time taken to exhaust the food supply are also calculated. The procedure employed here may be valuable for estimations in insects whose immature stages develop inside the food substrate, where it is difficult to partial out confounding effects such as separation of faeces. This procedure also has the advantage of taking into account the population dynamics of immatures living under crowded conditions, which are particularly characteristic of blowflies and other insects as well.

The modulation of simple reaction time by the spatial probability of a visual stimulus

Simple reaction time (SRT) in response to visual stimuli can be influenced by many stimulus features. The speed and accuracy with which observers respond to a visual stimulus may be improved by prior knowledge about the stimulus location, which can be obtained by manipulating the spatial probability of the stimulus. However, when higher spatial probability is achieved by holding constant the stimulus location throughout successive trials, the resulting improvement in performance can also be due to local sensory facilitation caused by the recurrent spatial location of a visual target (position priming). The main objective of the present investigation was to quantitatively evaluate the modulation of SRT by the spatial probability structure of a visual stimulus. In two experiments the volunteers had to respond as quickly as possible to the visual target presented on a computer screen by pressing an optic key with the index finger of the dominant hand. Experiment 1 (N = 14) investigated how SRT changed as a function of both the different levels of spatial probability and the subject's explicit knowledge about the precise probability structure of visual stimulation. We found a gradual decrease in SRT with increasing spatial probability of a visual target regardless of the observer's previous knowledge concerning the spatial probability of the stimulus. Error rates, below 2%, were independent of the spatial probability structure of the visual stimulus, suggesting the absence of a speed-accuracy trade-off. Experiment 2 (N = 12) examined whether changes in SRT in response to a spatially recurrent visual target might be accounted for simply by sensory and temporally local facilitation. The findings indicated that the decrease in SRT brought about by a spatially recurrent target was associated with its spatial predictability, and could not be accounted for solely in terms of sensory priming.

Detection of enteroviruses in cases of neurological disorders in the State of Pará, Brazil

Eighty-one cerebrospinal fluid (CSF) samples mainly from cases of aseptic meningitis and motor deficiency syndrome were sent to the Virology Section of Evandro Chagas Institute, Belém Pará, in the period of January 1995 to January 1996 in order to isolate viruses. All samples were inoculated onto HEp-2 cell culture and newborn mice, with negative results. The probability of isolating viruses by these methods is reduced because of the low concentration of viral particles in these specimens. In order to obtain more information about the etiology of these cases, a group of 23 samples were selected to be tested by a more sensitive technique than the virus isolation - the reverse transcription polymerase chain reaction (RT-PCR). Specific primers directed to conserved regions in the enterovirus genome were used, considering that this group of viruses is frequently associated with these neurological disorder. The age of the patients ranged from 1 to 55 years and nearly all of them lived in Belém, State of Pará, North of Brazil. Of 15 samples analyzed by RT PCR nine (60%) were positive; of these, 6 (66.6%) had motor deficiency and 3 (33.3%) developed aseptic meningitis. These results show that it is important to investigate enterovirus as cause of these syndromes.

Genetic associations in the detection of QTLs for wheat spike-related traits

The objective of this work was to assess the genetic diversity and population structure of wheat genotypes, to detect significant and stable genetic associations, as well as to evaluate the efficiency of statistical models to identify chromosome regions responsible for the expression of spike-related traits. Eight important spike characteristics were measured during five growing seasons in Serbia. A set of 30 microsatellite markers positioned near important agronomic loci was used to evaluate genetic diversity, resulting in a total of 349 alleles. The marker-trait associations were analyzed using the general linear and mixed linear models. The results obtained for number of allelic variants per locus (11.5), average polymorphic information content value (0.68), and average gene diversity (0.722) showed that the exceptional level of polymorphism in the genotypes is the main requirement for association studies. The population structure estimated by model-based clustering distributed the genotypes into six subpopulations according to log probability of data. Significant and stable associations were detected on chromosomes 1B, 2A, 2B, 2D, and 6D, which explained from 4.7 to 40.7% of total phenotypic variations. The general linear model identified a significantly larger number of marker-trait associations (192) than the mixed linear model (76). The mixed linear model identified nine markers associated to six traits.

Comparison of AutoSetä and polysomnography for the detection of apnea-hypopnea events

The use of the flow vs time relationship obtained with the nasal prongs of the AutoSetä (AS) system (diagnosis mode) has been proposed to detect apneas and hypopneas in patients with reasonable nasal patency. Our aim was to compare the accuracy of AS to that of a computerized polysomnographic (PSG) system. The study was conducted on 56 individuals (45 men) with clinical characteristics of obstructive sleep apnea (OSA). Their mean (± SD) age was 44.6 ± 12 years and their body mass index was 31.3 ± 7 kg/m2. Data were submitted to parametric analysis to determine the agreement between methods and the intraclass correlation coefficient was calculated. The Student t-test and Bland and Altman plots were also used. Twelve patients had an apnea-hypopnea index (AHI) <10 in bed and 20 had values >40. The mean (± SD) AHI PSG index of 37.6 (28.8) was significantly lower (P = 0.0003) than AHI AS (41.8 (25.3)), but there was a high intraclass correlation coefficient (0.93), with 0.016 variance. For a threshold of AHI of 20, AS showed 73.0% accuracy, 97% sensitivity and 60% specificity, with positive and negative predictive values of 78% and 93%, respectively. Sensitivity, specificity and negative predictive values increased in parallel to the increase in AHI threshold for detecting OSA. However, when the differences of AHI PSG-AS were plotted against their means, the limits of agreement between the methods (95% of the differences) were +13 and -22, showing the discrepancy between the AHI values obtained with PSG and AS. Finally, cubic regression analysis was used to better predict the result of AHI PSG as a function of the method proposed, i.e., AHI AS. We conclude that, despite these differences, AHI measured by AutoSetä can be useful for the assessment of patients with high pre-test clinical probability of OSA, for whom standard PSG is not possible as an initial step in diagnosis.

Polymerase chain reaction (PCR) for the detection of Salmonella in artificially inoculated chicken meat

The aim of this study was to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in artificially contaminated chicken meat. Tests were performed with different dilutions of Salmonella Typhimurium or Salmonella Enteritidis cells (10-7, 10-8 or 10-9 CFU/mL) inoculated in chicken meat samples, in order to establish the limits of detection, incubation times (0, 6, 8 and 24 hours of pre-enrichment in PBW 1%) and three DNA extraction protocols (phenol-chloroform, thermal treatment and thermal treatment and Sephaglass). The assay was able to detect until 10-9 CFU/mL of initial dilution of Salmonella cells inoculated in chicken meat, which allows detection of Salmonella within 48 hours, including 24 hours of pre-enrichment and using the phenol-chloroform DNA extraction protocol. As the results are obtained in a shorter time period than that of microbiological culture, this procedure will be useful in the methodology for detection of Salmonella in chicken.

Evaluation of dengue NS1 antigen detection for diagnosis in public health laboratories, São Paulo State, 2009

The present work evaluated the diagnostic accuracy of detection of Dengue NS1 antigen employing two NS1 assays, an immunochromatographic assay and ELISA, in the diagnostic routine of Public Health laboratories. The results obtained with NS1 assay were compared with virus isolation and, in a subpopulation of cases, they were compared with the IgM-ELISA results obtained with convalescent samples. A total of 2,321 sera samples were analyzed by one of two NS1 techniques from March to October 2009. The samples were divided into five groups: groups I, II and III included samples tested by NS1 and virus isolation, and groups IV and V included patients with a first sample tested by NS1 and a second sample tested by IgM-ELISA. Sensitivity, specificity, positive and negative predictive values, Kappa Index and Kappa Concordance were calculated. The results showed that NS1 testing in groups I, II and III had high sensitivity (98.0%, 99.5% and 99.3%), and predictive values and Kappa index between 0.9 - 1.0. Groups IV and V only had Kappa Concordance calculated, since the samples were analyzed according to the presence of NS1 antigen or IgM antibody. Concordance of 92.1% was observed when comparing the results of NS1-negative samples with IgM-ELISA. Based on the findings, it is possible to suggest that the tests for NS1 detection may be important tools for monitoring the introduction and spread of Dengue serotypes.

The use of saliva as a practical and feasible alternative to urine in large-scale screening for congenital cytomegalovirus infection increasesinclusion and detection rates

INTRODUCTION: Although urine is considered the gold-standard material for the detection of congenital cytomegalovirus (CMV) infection, it can be difficult to obtain in newborns. The aim of this study was to compare the efficiency of detection of congenital CMV infection in saliva and urine samples. METHODS: One thousand newborns were included in the study. Congenital cytomegalovirus deoxyribonucleic acid (DNA) was detected by polymerase chain reaction (PCR). RESULTS: Saliva samples were obtained from all the newborns, whereas urine collection was successful in only 333 cases. There was no statistically significant difference between the use of saliva alone or saliva and urine collected simultaneously for the detection of CMV infection. CONCLUSIONS: Saliva samples can be used in large-scale neonatal screening for CMV infection.

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g-1)] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g-1, while the threshold for the ST was greater than 0.1.10³ CFU-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.

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.

Trypanosoma vivax infection dynamics in a cattle herd maintained in a transition area between Pantanal lowlands and highlands of Mato Grosso do Sul, Brazil

Trypanosoma vivax outbreaks in beef cattle in the Pantanal region of Mato Grosso do Sul state, Brazil, causes relevant economical impact due to weight loss, abortion and mortality. Cattle moved from the Pantanal to adjacent areas of this ecosystem for breeding and fattening is a common feature. Therefore an epidemiological study on breeding cows in the transition area between Pantanal lowland and adjacent highlands of Mato Grosso do Sul was performed to determine the T. vivax infection dynamics and outbreak risk. Three experimental groups were formed: Group 1 consisted of cows parasitologically negative by the Woo test and in the enzyme-linked immunosorbent assay for T. vivax antibody detection (Tv-ELISA-Ab); Group 2 parasitologically negative and positive in the Tv-ELISA-Ab; and in Group 3 cows were parasitologically positive and with positive reactions in the Tv-ELISA-Ab. During 24 months, the cows' dislodgment between the above established groups was monitored by Woo test and Tv-ELISA-Ab exams. The tabanid population was also monitored and the highest number occurred during the rainy season. Although parasitemias were detected only in the first four samplings of the experimental period, the cows could be considered as trypanotolerant, because no clinical signs were observed. Despite the higher T. vivax incidence during the dry season, no disease symptoms were seen. Even though T. vivax epidemiological situation in the herd was characterized as endemic with seasonal variation, the probability of outbreaks was null within the conditions of the study.

Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Chance of psychiatric morbidity amongst recently diagnosed cancer outpatients attending a chemotherapy unit

The prevalent rate of psychiatry morbidity amongst patients with cancer reported in various studies ranges from 5 to 50%, a variation that can be attributed to differences in sample size, the disease itself and treatment factors. The objectives of the present study were to determine the frequency of psychiatric morbidity amongst recently diagnosed cancer outpatients and try to identify which factors might be related to further psychological distress. Two hundred and eleven (70.9%) female patients and 87 (29.1%) male patients from the chemotherapy unit of the Cancer Hospital A.C. Camargo (São Paulo) completed a questionnaire that featured data on demographic, medical and treatment details. The Self Reporting Questionnaire (SRQ-20) was administered to the patients to determine their personal psychiatric morbidity. Seventy-two patients (25.8%) scored > or = 8 in the SRQ-20, the cut-off point for a patient to be considered a psychiatric case. When the low and high scoring groups were compared no differences were detected regarding age, marital status, tumor site, sex, or previous treatment. Nonetheless, patients in the lowest social class and those who were bedridden less than 50% of the time had a significantly higher probability of being a psychiatric case. Regarding help-seeking behavior in situations in which they had doubts or were frightened, about 64% of the total sample did not seek any type of support and did not talk to anyone. This frequency of psychiatric morbidity agrees with data from the cancer literature. According to many investigators, the early detection of a comorbid psychiatric disorder is crucial to relieve a patient's suffering.