Evaluation of 16S rRNA Hypervariable Regions for Bioweapon Species Detection by Massively Parallel Sequencing.

Molecular detection and classification of the bacterial groups in a sample are relevant in several areas, including medical research and forensics. Sanger sequencing of the 16S rRNA gene is considered the gold standard for microbial phylogenetic analysis. However, the development of massively parallel sequencing (MPS) offers enhanced sensitivity and specificity for microbiological analyses. In addition, 16S rRNA target amplification followed by MPS facilitates the combined use of multiple markers/regions, better discrimination of sample background, and higher sample throughput. We designed a novel set of 16S rRNA gene primers for detection of bacterial species associated with clinical, bioweapon, and biohazards microorganisms via alignment of 364 sequences representing 19 bacterial species and strains relevant to medical and forensics applications. In silico results indicated that the hypervariable regions (V1V2), (V4V5), and (V6V7V8) support the resolution of a selected group of bacteria. Interspecies and intraspecies comparisons showed 74.23%-85.51% and 94.48%-99.98% sequencing variation among species and strains, respectively. Sequence reads from a simulated scenario of bacterial species mapped to each of the three hypervariable regions of the respective species with different affinities. The minimum limit of detection was achieved using two different MPS platforms. This protocol can be used to detect or monitor as low as 2,000 genome equivalents of bacterial species associated with clinical, bioweapon, and biohazard microorganisms and potentially can distinguish natural outbreaks of pathogenic microorganisms from those occurring by intentional release.

Planktonic microbial profiling in water samples from a Brazilian Amazonian reservoir.

Our comprehension of the dynamics and diversity of freshwater planktonic bacterial communities is far from complete concerning the Brazilian Amazonian region. Therefore, reference studies are urgently needed. We mapped bacterial communities present in the planktonic communities of a freshwater artificial reservoir located in the western Amazonian basin. Two samples were obtained from rainy and dry seasons, the periods during which water quality and plankton diversity undergo the most significant changes. Hypervariable 16S rRNA and shotgun sequencing were performed to describe the first reference of a microbial community in an Amazonian lentic system. Microbial composition consisted mainly of Betaproteobacteria, Cyanobacteria, Alphaproteobacteria, and Actinobacteria in the dry period. The bacteria distribution in the rainy period was notably absent of Cyanobacteria. Microcystis was observed in the dry period in which the gene cluster for cyanotoxins was found. Iron acquisition gene group was higher in the sample from the rainy season. This work mapped the first inventory of the planktonic microbial community of a large water reservoir in the Amazon, providing a reference for future functional studies and determining other communities and how they interact.

Cognitive performance of children living in endemic areas for Plasmodium vivax.

BACKGROUND: The role of repeated episodes of malaria on the cognitive development of children is a relevant issue in endemic areas since it can have a long-lasting impact on individual lifespan. The aim of the current paper was to investigate whether the history of malaria can impair the verbal and performance skills of children living in an endemic area with low transmission of Plasmodium vivax malaria. METHODS: A cross-sectional study was conducted with children living in an endemic area of P. vivax malaria in Brazilian Amazon basin. The history of episodes of malaria was used as criteria for inclusion of children in the groups. The cognitive performance was assessed by the Wechsler intelligence scale for children-III edition (WISC-III), which was applied to the participants of study by two trained psychologists. RESULTS: A total of 17 cases and 26 controls was included in the study. A significant low score of verbal quotient was found in the cases (p = 0.005), however, the performance IQ was similar in both groups (p = 0.304). The full-scale IQ was significantly lower in the cases when compared to the controls (p = 0.042). The factorials index showed significant difference only in the subtest of verbal comprehension with the lower values in the cases (p = 0.0382), compared to the controls. The perceptual organization (p = 0.363), freedom from distractability (p = 0.180) and processing speed (p = 0.132) were similar in both groups. CONCLUSIONS: Children with a history of vivax malaria has a significant impairment of verbal and full-scale quotients as well as a significant low index of verbal comprehension. These findings are likely due to the absenteeism caused by malaria and by the low parental education, which impairs an adequate response to the environmental stimulus.