Best practices in dengue surveillance: a report from the Asia-Pacific and Americas Dengue Prevention Boards

Background

Dengue fever is a virus infection that is spread by the Aedes aegypti mosquito and can cause severe disease especially in children. Dengue fever is a major problem in tropical and sub-tropical regions of the world.

Methodology/Principal Findings

We invited dengue experts from around the world to attend meetings to discuss dengue surveillance. We reviewed literature, heard detailed reports on surveillance programs, and shared expert opinions.

Results

Presentations by 22 countries were heard during the 2.5 day meetings. We describe the best methods of surveillance in general, the stakeholders in dengue surveillance, and the steps from mosquito bite to reporting of a dengue case to explore how best to carry out dengue surveillance. We also provide details and a comparison of the dengue surveillance programs by the presenting countries.

Conclusions/Significance

The experts provided recommendations for achieving the best possible data from dengue surveillance accepting the realities of the real world (e.g., limited funding and staff). Their recommendations included: (1) Every dengue endemic country should make reporting of dengue cases to the government mandatory; (2) electronic reporting systems should be developed and used; (3) at minimum dengue surveillance data should include incidence, hospitalization rates, deaths by age group; (4) additional studies should be completed to check the sensitivity of the system; (5) laboratories should share expertise and data; (6) tests that identify dengue virus should be used in patients with fever for four days or less and antibody tests should be used after day 4 to diagnose dengue; and (7) early detection and prediction of dengue outbreaks should be goals for national surveillance systems.     

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD₅₀ values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.     

“Vision for Action” in Young Children Aligning Multi-Featured Objects: Development and Comparison with Nonhuman Primates

Effective vision for action and effective management of concurrent spatial relations underlie skillful manipulation of objects, including hand tools, in humans. Children’s performance in object insertion tasks (fitting tasks) provides one index of the striking changes in the development of vision for action in early life. Fitting tasks also tap children’s ability to work with more than one feature of an object concurrently. We examine young children’s performance on fitting tasks in two and three dimensions and compare their performance with the previously reported performance of adult individuals of two species of nonhuman primates on similar tasks. Two, three, and four year-old children routinely aligned a bar-shaped stick and a cross-shaped stick but had difficulty aligning a tomahawk-shaped stick to a matching cut-out. Two year-olds were especially challenged by the tomahawk. Three and four year-olds occasionally held the stick several inches above the surface, comparing the stick to the surface visually, while trying to align it. The findings suggest asynchronous development in the ability to use vision to achieve alignment and to work with two and three spatial features concurrently. Using vision to align objects precisely to other objects and managing more than one spatial relation between an object and a surface are already more elaborated in two year-old humans than in other primates. The human advantage in using hand tools derives in part from this fundamental difference in the relation between vision and action between humans and other primates.     

Extending glacial refugia for a European tree: genetic markers show that Iberian populations of white elm are native relicts and not introductions

Conservation policies usually focus on in situ protection of native populations, a priority that requires accurate assessment of population status. Distinction between native and introduced status can be particularly difficult (and at the same time, is most important) for species whose natural habitat has become both rare and highly fragmented. Here, we address the status of the white elm (Ulmus laevis Pallas), a European riparian tree species whose populations have been fragmented by human activity and is protected wherever it is considered native. Small populations of this species are located in Iberia, where they are unprotected because they are considered introductions due to their rarity. However, Iberia and neighbouring regions in southwestern France have been shown to support discrete glacial refuge populations of many European trees, and the possibility remains that Iberian white elms are native relicts. We used chloroplast RFLPs and nuclear microsatellites to establish the relationship between populations in Iberia and the Central European core distribution. Bayesian approaches revealed significant spatial structure across populations. Those in Iberia and southwestern France shared alleles absent from Central Europe, and showed spatial population structure within Iberia common in recognized native taxa. Iberian populations show a demographic signature of ancient population bottlenecks, while those in Central European show a signature of recent population bottlenecks. These patterns are not consistent with historical introduction of white elm to Iberia, and instead strongly support native status, arguing for immediate implementation of conservation measures for white elm populations in Spain and contiguous areas of southern France.     

The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate

The active sites of eukaryotic arginase enzymes are strictly conserved, especially the first- and second-shell ligands that coordinate the two divalent metal cations that generate a hydroxide molecule for nucleophilic attack on the guanidinium carbon of l-arginine and the subsequent production of urea and l-ornithine. Here by using comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I, we demonstrate that several metal binding ligands are actually quite tolerant to amino acid substitutions. Of >2800 double mutants of first- and second-shell residues analyzed, we found more than 80 unique amino acid substitutions, of which four were in first-shell residues. Remarkably, certain second-shell mutations could modulate the binding of both the nucleophilic water/hydroxide molecule and substrate or product ligands, resulting in activity greater than that of the wild-type enzyme. The data presented here constitute the first comprehensive saturation mutagenesis analysis of a metallohydrolase active site and reveal that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution.     

Recovery of intracellular recombinant proteins from the yeast Pichia pastoris by cell permeabilization

A cell permeabilization method for the release of intracellular proteins from microbial cells was developed. The method was applied to the recovery of recombinant botulinum neurotoxin fragments, expressed intracellularly in the yeast Pichia pastoris, by suspending the cells in an aqueous solution containing N,N-dimethyltetradecylamine. For the botulinum neurotoxin serotype B C-terminal heavy chain fragment, 1.8 mg g(-1) biomass were recovered. For the botulinum neurotoxin serotype A C-terminal heavy chain fragment, 3.7 mg g(-1) biomass were recovered. The concentration of recombinant protein in the cell extracts remained stable for up to 48 and 24 h for the serotype B and serotype A fragments, respectively. The permeabilization method was compared with high-pressure homogenization; the permeabilization method proved to be both more selective and more efficient.     

Rapid recent radiation of S-RNase lineages in Witheringia solanacea (Solanaceae)

Strong frequency-dependent selection as found in the self-incompatibility loci of flowering plants maintains allelic lineages for extremely long time scales, such that allelic genealogies can shed insight into long-term demographic patterns of species. Effective mutation rate, as well as demographic change such as population bottlenecks, can influence genealogical structure. In addition, loss of functionality at the self-incompatibility locus is likely to affect radiation rates. Partial sequences for 21 S-RNase alleles of the mid-elevation tropical species Witheringia solanacea were obtained in order to compare their substitution rates and genealogy with those of Witheringia maculata and two species in the closely related genus Physalis. Sequences for W. solanacea fell into the three clades within the Solanaceae already identified for the genus. Terminal branch lengths for W. solanacea, scaled to the total depth of its phylogeny, were intermediate between the unusually short terminal branches of W. maculata and those of the two Physalis species. In contrast to the Physalis species, where interspecific dN/dS for closely related alleles exceeded 1.0 to the same degree as did intraspecific dN/dS, in Witheringia only intraspecific comparisons showed an excess of nonsynonymous substitutions, suggesting postspeciation radiation of alleles. Alleles associated with lowered S-RNase production and self-compatibility showed extremely short terminal branches. In summary, it appears that rapid recent diversification of alleles characterizes the Witheringia lineages. In some cases, this rapid diversification can be attributed to relaxed constraints due to breakdown of self-incompatibility.     

Agmatine transport into spinal nerve terminals is modulated by polyamine analogs

Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that Snca deletion had on whole brain lipid composition. We analysed masses of individual phospholipid (PL) classes and neutral lipid mass as well as PL acyl chain composition in brains from wild-type and Snca-/- mice. Although total brain PL mass was not altered, cardiolipin and phosphatidylglycerol mass decreased 16% and 27%, respectively, in Snca-/- mice. In addition, no changes were observed in plasmalogen or polyphosphoinositide mass. In ethanolamine glycerophospholipids and phosphatidylserine, docosahexaenoic acid (22 : 6n-3) was decreased 7%, while 16 : 0 was increased 1.1-fold and 1.4-fold, respectively. Surprisingly, brain cholesterol, cholesteryl ester, and triacylglycerol mass were increased 1.1-fold, 1.6-fold, and 1.4-fold, respectively in Snca-/- mice. In isolated myelin, cholesterol mass was also increased 1.3-fold, but because there was also a net increase in myelin PL mass, the cholesterol to PL ratio was unaltered. No changes in the expression of cholesterogenic enzymes were observed, suggesting these did not account for the observed changes in cholesterol. These data extend our previous results in astrocytes and kinetic studies in vivo demonstrating a role for Snca in brain lipid metabolism and demonstrate a clear impact on brain neutral lipid metabolism.     

Age and seasonal effects on predator-sensitive foraging in squirrel monkeys (Saimiri sciureus): a field experiment

A field experiment was conducted to examine the effect of perceived predation risk on the use of foraging areas by juvenile and adult primates under different conditions of local food abundance. Wild squirrel monkeys, Saimiri sciureus, were observed in an experiment conducted during the dry and the wet seasons at a site in Eastern Amazonia, Brazil. Animals were presented with feeding platforms that differed in food quantity and exposure to aerial predators through varying vegetative cover. In the dry season, juveniles and adults chose platforms based solely on food quantity. However, in the wet season, juveniles foraged preferentially on high-reward platforms only if cover level also was high (i.e., potentially offered greater concealment from predators). In contrast, adults showed the same pattern of platform use regardless of season. These results indicate that age and local resource availability based on seasonality affect whether primates forage in a predator-sensitive manner. Juveniles may be more sensitive to predation risk when foraging, and individuals may take fewer risks when resource abundance is high in their environment.