In Silico Approaches for the Identification of Aptamer Binding Interactions to Leptospira spp. Cell Surface Proteins.

Aptamers are nucleic acids that can bind with high affinity and specificity to a range of target molecules. However, their functionality relies on their secondary and tertiary structures such that the combination of nucleotides determines their three-dimensional conformation. In this study, the binding mechanisms of candidate aptamers and their interactions with selected target proteins found in the cell surface of Leptospira were predicted to select high-affinity aptamers. Four aptamers were evaluated through molecular modeling and docking using available software and web-based tools, following the workflow previously designed for in silico evaluation of DNA aptamers. The most predominant and highly conserved surface-exposed proteins among pathogenic Leptospira species were used as aptamer targets. The highest number of interactions was seen in aptamers AP5 and AP1. Hydrogen bonds, along with a few hydrophobic interactions, occur in most aptamer-protein complexes. Further analysis revealed serine, threonine, glutamine, and lysine as main protein residues. H-bond interactions occur mostly with polar amino acids, as reflected in the predicted interaction profiles of aptamer-protein complexes. In silico strategies allowed the identification of key residues crucial in aptamer-target interaction during aptamer screening. Such information can be used in aptamer modification for improved binding affinity and accuracy for diagnostics application.

Application of simplified MLST scheme for direct typing of clinical samples from human leptospirosis cases in a tertiary hospital in the Philippines.

Despite the major threat of leptospirosis to public health in the Philippines, its epidemiologic data remain scarce. Multilocus sequence typing (MLST) is a method often used for identification of circulating Leptospira species and disease surveillance. Unfortunately, molecular typing of Leptospira isolates is not routinely done in most hospital settings. A simplified MLST scheme targeting three loci (adk, lipL41, mreA) was performed for rapid direct typing of Leptospira in clinical specimens. Blood samples from suspected or clinically diagnosed cases (n = 50) were initially screened via polymerase chain reaction (PCR) targeting 23S rRNA, 16S rRNA (rrs2), and lipL32 genes. From the nine positives, seven had interpretable data from MLST. Allelic profiles identified L. interrogans in all positive samples. Six were assigned to ST12 of serovar Manilae (serogroup Pyrogenes) while one sample cannot be clearly differentiated between two serovars/serogroups, Bataviae/Losbanos (serogroup Bataviae) or Australis (serogroup Australis), indicating possibility of a new ST. Phylogenetic analysis confirmed that the application of simplified MLST scheme produces consistent results with the seven-loci genetic profile of published Leptospira MLST schemes. Reduced scheme addressed the challenges often encountered in the amplification of full MLST genetic profile of Leptospira. The approach is a potential alternative to serological tests for rapid typing of clinical specimens and can also aid in investigations on disease epidemiology specifically to monitor occurrence, pathogen transmission, host specificity and susceptibility, and other factors that could lead to potential outbreaks.

Identification of Leptospira spp. from environmental sources in areas with high human leptospirosis incidence in the Philippines.

Leptospira is the causative agent of leptospirosis, which is considered an emerging major threat to public health due to its increasing frequency reported worldwide. In the Philippines, the prevalence of the disease continuously rises, particularly in urban areas. Because leptospirosis is commonly transmitted through contact with contaminated environment, water and soil samples were collected in regions in the Philippines where high incidence of human leptospirosis cases was reported recently. Of the 54 samples screened for the presence of Leptospira, 35% were found positive through 23S rRNA gene PCR-based detection. None were found positive when primers targeting lipL32, lipL41, and ompL1 genes were used. Most of these isolates were collected from rural areas. However, 16S rRNA gene sequencing identified all isolates to be L. yanagawae and L. meyeri, which are nonpathogenic. Despite the lack of evidence of the presence of pathogenic species in the environmental sources, the results still suggest that leptospires persist in these areas. These data are crucial for environmental monitoring and identification of contaminated areas where humans may be at risk of infection.