Viability study of a multiplex diagnostic platform for Chagas disease

A new multiplex assay platform was evaluated to detect Trypanosoma cruzi infection using the recombinant antigens CRA, FRA, CRAFRA fusion and parasite lysate. The antigens presented different sensitivity and specificity in a singleplex test when compared to a serial dilution of two pools comprising 10 positive serum samples and one pool of 10 negative samples. The recombinant protein CRA presented lower sensitivity (55%) in contrast to the 100% specificity and sensitivity of FRA, CRAFRA and T. cruzi lysate. These antigens also showed good results in a duplex test and the duplex test with CRAFRA/T. cruzi lysate showed better performance with 100% specificity and sensitivity, as well as a lower cut-off value in comparison to the other duplex test, FRA/T. cruzi lysate. Hence, when the antigens were used in duplex format, both tests showed decreased cut-off values and no interference between different bead sets, resulting in increasing sensitivity and specificity. The results of these multiplex tests show that they could be an alternative to singleplex detection for Chagas disease, and also indicate the necessity of using multiplex diagnostic tools to increase the sensitivity and specificity for diagnostic tests. Emerging data from the T. cruzi genome and from its ORFeome project will also allow the identification of new antigens for this disease detection application.

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.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Is the capture success of orchid bees (Hymenoptera, Apoidea) influenced by different baited trap designs? A case study from southern Brazil

Orchid bees are increasingly applied on Neotropical biomonitoring and bioindication studies due to the relative easiness of sampling and identification when compared to other bee groups. A considerable number of orchid bee community studies have been adopting baited traps as a sampling method, especially for replication purposes. However, the trap attributes are variable, and hitherto no evaluation of different designs was carried out. Here, five attributes of baited traps were tested: trap volume, number of entrance holes, presence of landing platform, kind of landing platform, and fixation content. We use Mann-Whitney tests to access differences in richness and abundance capture rates for each trap design. We found that volume, number of entrance holes, and fixation content do not influence orchid bees capture. However, the design without landing platforms had a significantly higher capture rate for richness when compared with sanded landing platforms. On the other hand, analyzing the kind of landing platform, we detected a significantly higher richness and abundance for the trap with landing platforms glued with sand. Despite the fact that the effects of different designs tested here were very punctual, we consider that results from samples taken with different baited trap designs are comparable. Some adjustments on trap design can be done according to the particularities of future studies.

Modeling temporal variations of Gracilaria Greville and Hypnea J.V. Lamouroux (Rhodophyta) assemblages on a midlittoral reef platform at Piedade Beach, Pernambuco State, Brazil

The diversity of algal banks composed of species out the genera Gracilaria Greville and Hypnea J.V. Lamouroux have been impacted by commercial exploitation and coastal eutrophication. The present study sought to construct dynamic models based on algal physiology to simulate seasonal variations in the biomasses of Gracilaria and Hypnea an intertidal reef at Piedade Beach in Jaboatão dos Guararapes, Pernambuco State, Brazil. Five 20 × 20 cm plots in a reef pool on a midlittoral reef platform were randomly sampled during April, June, August, October, and December/2009 and in January and March/2010. Water temperature, pH, irradiance, oxygen and salinity levels as well as the concentrations of ammonia, nitrate and phosphate were measured at the sampling site. Forcing functions were employed in the model to represent abiotic factors, and algal decay was simulated with a dispersal function. Algal growth was modeled using a logistic function and was found to be sensitive to temperature and salinity. Maximum absorption rates of ammonia and phosphate were higher in Hypnea than in Gracilaria, indicating that the former takes up nutrients more efficiently at higher concentrations. Gracilaria biomass peaked at approximately 120 g (dry weight m-2) in March/2010 and was significantly lower in August/2009; Hypnea biomasses, on the other hand, did not show any significant variations among the different months, indicating that resource competition may influence the productivity of these algae.

Neuroethologic differences in sleep deprivation induced by the single- and multiple-platform methods

It has been proposed that the multiple-platform method (MP) for desynchronized sleep (DS) deprivation eliminates the stress induced by social isolation and by the restriction of locomotion in the single-platform (SP) method. MP, however, induces a higher increase in plasma corticosterone and ACTH levels than SP. Since deprivation is of heuristic value to identify the functional role of this state of sleep, the objective of the present study was to determine the behavioral differences exhibited by rats during sleep deprivation induced by these two methods. All behavioral patterns exhibited by a group of 7 albino male Wistar rats submitted to 4 days of sleep deprivation by the MP method (15 platforms, spaced 150 mm apart) and by 7 other rats submitted to sleep deprivation by the SP method were recorded in order to elaborate an ethogram. The behavioral patterns were quantitated in 10 replications by naive observers using other groups of 7 rats each submitted to the same deprivation schedule. Each quantification session lasted 35 min and the behavioral patterns presented by each rat over a period of 5 min were counted. The results obtained were: a) rats submitted to the MP method changed platforms at a mean rate of 2.62 ± 1.17 platforms h-1 animal-1; b) the number of episodes of noninteractive waking patterns for the MP animals was significantly higher than that for SP animals (1077 vs 768); c) additional episodes of waking patterns (26.9 ± 18.9 episodes/session) were promoted by social interaction in MP animals; d) the cumulative number of sleep episodes observed in the MP test (311) was significantly lower (chi-square test, 1 d.f., P<0.05) than that observed in the SP test (534); e) rats submitted to the MP test did not show the well-known increase in ambulatory activity observed after the end of the SP test; f) comparison of 6 MP and 6 SP rats showed a significantly shorter latency to the onset of DS in MP rats (7.8 ± 4.3 and 29.0 ± 25.0 min, respectively; Student t-test, P<0.05). We conclude that the social interaction occurring in the MP test generates additional stress since it increases the time of forced wakefulness and reduces the time of rest promoted by synchronized sleep.