Application of qPCR method to hair and cerumen samples for the diagnosis of canine leishmaniosis in Araçatuba, Brazil.

Visceral leishmaniosis (VL) remains a serious public health problem in Brazil. Dogs are the main hosts of the parasite, developing canine leishmaniosis (CanL), hence the importance of an accurate diagnosis of the animals. Recently, the application of qPCR method to non-invasive samples obtained from dogs with CanL has shown high sensitivity. Thus, we analyzed by qPCR blood, hair (from healthy zones and cutaneous lesions) and cerumen of 16 dogs with confirmed leishmaniosis from Araçatuba, a Brazilian endemic area. Cerumen-qPCR showed the highest sensitivity (87.5%), followed by hair (lesions: 78.57%, healthy skin: 62.5%), and blood (68.75%). We also analyzed blood, hair and cerumen of 5 healthy dogs from a non-endemic area, obtaining 100% of specificity in all samples. The use of cerumen and hair for qPCR analysis provides high reliability, taking into account the sensitivity and total specificity of the method. The non-invasive sampling procedure without the need of specific conditions of storage and transport support the usefulness of hair and cerumen for the diagnosis of CanL.

First detection of Leishmania kDNA in canine cerumen samples by qPCR.

Nowadays, searching for alternative non-invasive methods for molecular diagnosis of canine visceral leishmaniosis is getting increasingly important. We previously described the presence of Leishmania kinetoplast DNA (kDNA) in canine hair; in this case we hypothesized whether foreign DNA might be present in cerumen of dogs with leishmaniosis, and be detected by Real time quantitative PCR (qPCR). A population of 38 dogs that lived in Leishmania endemic areas was divided in two groups: A (33 dogs with confirmed leishmaniosis by serological techniques) and B (5 healthy dogs). Blood, lymph node, bone marrow and cerumen samples from all animals were tested for the presence of parasite kDNA. Our method was 100% specific, and in dogs from group A, Leishmania infantum kDNA was detected and quantified in the 100% of lymph node samples, in 90.9% of cerumen samples, in 88.5% of the bone marrow samples and in 57.6% of the blood samples. The qPCR-cerumen is a new non-invasive method that shows a high potential for the diagnosis of zoonotic visceral leishmaniosis.

A new model for mapping the peptide backbone: predicting proton chemical shifts in proteins.

This paper describes a methodology that correlates experimental chemical shifts (at the alpha proton) of proteins with their geometrical data (both dihedral angles and distances) obtained from 13 representative proteins, which are taken from the Protein Data Bank (PDB) and the BioMagRes Data Bank (BMRB). To this end, the experimentally measured proton chemical shifts of simple amides have been correlated with DFT-based calculated structures, at the B3PW91/6-31G* level. This results in a series of mathematical relationships, which are extrapolated to the above-mentioned proteins giving rise to a modified equation for such skeleta. It is relevant to note that the equation is also supported by a clear comparison with NMR data of a protein beyond the chosen set, such as insulin, even with lower errors. The model also relates the dependence of chemical shifts on hydrophobic and anisotropic effects at the amino acid residues.