New diagnostic tools that can detect malaria parasites in conjunction with other diagnostic parameters are urgently required. In this study, Attenuated Total Reflection Fourier transform infrared (ATR-FTIR) spectroscopy in combination with Partial Least Square Discriminant Analysis (PLS-DA) and Partial Least Square Regression (PLS-R) have been applied as a point-of-care test for identifying malaria parasites, blood glucose, and urea levels in whole blood samples from thick blood films on glass slides. The specificity for the PLS-DA was found to be 98% for parasitemia levels >0.5%, but a rather low sensitivity of 70% was achieved because of the small number of negative samples in the model. In PLS-R the Root Mean Square Error of Cross Validation (RMSECV) for parasite concentration (0-5%) was 0.58%. Similarly, for glucose (0-400 mg/dL) and urea (0-250 mg/dL) spiked samples, relative RMSECVs were 16% and 17%, respectively. The method reported here is the first example of multianalyte/disease diagnosis using ATR-FTIR spectroscopy, which in this case, enabled the simultaneous quantification of glucose and urea analytes along with malaria parasitemia quantification using one spectrum obtained from a single drop of blood on a glass microscope slide.

Atomic force microscopy-infrared (AFM-IR) spectroscopy is a powerful new technique that can be applied to study molecular composition of cells and tissues at the nanoscale. AFM-IR maps are acquired using a single wavenumber value: they show either the absorbance plotted against a single wavenumber value or a ratio of two absorbance values. Here, we implement multivariate image analysis to generate multivariate AFM-IR maps and use this approach to resolve subcellular structural information in red blood cells infected with Plasmodium falciparum at different stages of development. This was achieved by converting the discrete spectral points into a multispectral line spectrum prior to multivariate image reconstruction. The approach was used to generate compositional maps of subcellular structures in the parasites, including the food vacuole, lipid inclusions, and the nucleus, on the basis of the intensity of hemozoin, hemoglobin, lipid, and DNA IR marker bands, respectively. Confocal Raman spectroscopy was used to validate the presence of hemozoin in the regions identified by the AFM-IR technique. The high spatial resolution of AFM-IR combined with hyperspectral modeling enables the direct detection of subcellular components, without the need for cell sectioning or immunological/biochemical staining. Multispectral-AFM-IR thus has the capacity to probe the phenotype of the malaria parasite during its intraerythrocytic development. This enables novel approaches to studying the mode of action of antimalarial drugs and the phenotypes of drug-resistant parasites, thus contributing to the development of diagnostic and control measures.

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A new experimental platform for probing nanoscale molecular changes in living bacteria using atomic force microscopy-infrared (AFM-IR) spectroscopy is demonstrated. This near-field technique is eminently suited to the study of single bacterial cells. Here, we report its application to monitor dynamical changes occurring in the cell wall during cell division in using AFM to demonstrate the division of the cell and AFM-IR to record spectra showing the thickening of the septum This work was followed by an investigation into single cells, with particular emphasis on cell-wall signatures, in several bacterial species. Specifically, mainly cell wall components from and containing complex carbohydrate and phosphodiester groups, including peptidoglycans and teichoic acid, could be identified and mapped at nanometre spatial resolution. Principal component analysis of AFM-IR spectra of six living bacterial species enabled the discrimination of Gram-positive from Gram-negative bacteria based on spectral bands originating mainly from the cell wall components. The ability to monitor molecular changes during cellular processes in bacteria at the nanoscale opens a new platform to study environmental influences and other factors that affect bacterial chemistry.