Preliminary study for the application of Raman spectroscopy for the identification of Leishmania infected dogs

Abstract

Raman spectroscopy is a rapid qualitative and quantitative technique that allows the simultaneous determination of several components in biological fluids. This methodology concerns an alternative technique to distinguish between non-healthy and healthy subjects. Leishmaniasis is a zoonosis of world interest, the most important agent is L. infantum. Dogs are the principal reservoirs affected by a broad spectrum of clinical features. During a clinical exam, blood samples were collected in tubes without anticoagulants, from twenty two dogs. One aliquot was used for serological test for Leishmaniasis, one aliquot was subjected to the Raman spectroscopic analysis. Animals were divided into two groups of equal subjects, Leishmania group (LG) constituted by infected dogs, and control group (CG) constituted by healthy dogs. The acquired spectra were different in the region 1200-1370 cm^-1, in which it is possible to distinguish the amide III vibration (~ 1300 cm^-1). In LG, an evident shift to the shortwave region is observed in spectral frequencies of the band centered at ~ 1250 cm^-1. Our results distinguished between LD group and CG. Further studies are necessary to exclude the effect of metabolic modification due to disease on the recorded spectra changes and to consolidate the achievability of Raman spectroscopy as rapid and less expensive diagnosis of Leishmaniasis.

Figure 1

Structural formula of leucine and isoleucine.

Figure 2

Average Raman spectrum of sera from control group (CG) subjects (n = 11) in the spectral range of 3100–400 cm⁻¹, after having performed a baseline correction of each of them in order to compensate eventual technical and/or sample variations, and having normalized them to the total integrated area. The spectrum put in evidence the main typical protein vibrational modes, which derive from the polypeptide backbone (amide bands) and from aromatic and non-aromatic amino acid residue side chains.

Figure 3

Mean (continuous line) and standard deviation (gradient color) of Raman spectra of LG (red line) and CG (blue line) subjects.

Figure 4

Acquired spectra, obtained from sera of LG (red line) and CG (blue line) subjects, in the region 900–1900 cm⁻¹. The attribution of amide III vibration is around the ~ 1300 cm⁻¹.

Figure 5

(a) ROC curve for the A1350/A1250 ratio (AUC = 1, 95% confidence interval). The diagonal joining the point (0.0) and (1.1) represents the line of equality. The black point corresponds to the 1-Specificity, Sensitivity calculated for the optimal cut-off for correctly identifying Leishmania (LG) or control (CG) subjects. (b) Trend of sensitivity (red line) and specificity (blue line) vs. A1350/A1250 ratios. The vertical dot line identifies the optimal cut-off (threshold value = 1.316) and the point of intersection of sensitivity and specificity curves corresponds to the black marker depicted in (a). (c) Scatter distribution of each group (CG and LG). The optimal cut-off is plotted as the horizontal dot line.