Evaluation of the potential of Delta-aminolevulinic acid for simultaneous detection of bioburden and anti-microbial photodynamic therapy of MRSA infected wounds in Swiss albino mice.

BACKGROUND: The dramatic increase of drug-resistant bacteria necessitates urgent development of platforms to simultaneously detect and inactivate bacteria causing wound infections, but are confronted with various challenges. Delta amino levulinic acid (ALA) induced protoporphyrin IX (PpIX) can be a promising modality for simultaneous bioburden diagnostics and therapeutics. Herein, we report utility of ALA induced protoporphyrin (PpIX) based simultaneous bioburden detection, photoinactivation and therapeutic outcome assessment in methicillin resistant Staphylococcus aureus (MRSA) infected wounds of mice. METHODS: MRSA infected wounds treated with 10% ALA were imaged with help of a blue LED (∼405 nm) based, USB powered, hand held device integrated with a modular graphic user interface (GUI). Effect of ALA application time, bacteria load, post bacteria application time points on wound fluorescence studied. PpIX fluorescence observed after excitation with blue LEDs was used to detect bioburden, start red light mediated antimicrobial photodynamic therapy (aPDT), determine aPDT effectiveness and assess selectivity of the approach. RESULTS: ALA-PpIX fluorescence of wound bed discriminates infected from uninfected wounds and detects clinically relevant load. While wound fluorescence pattern changes as a function of ALA incubation and post infection time, intra-wound inhomogeneity in fluorescence correlates with the Gram staining data on presence of biofilms foci. Lack of red fluorescence from wound granulation tissue treated with ALA suggests selectivity of the approach. Further, significant reduction (∼50%) in red fluorescence, quantified using the GUI, relates well with bacteria load reduction observed post topical aPDT. CONCLUSION: The potential of ALA induced PpIX for simultaneous detection of bioburden, photodynamic inactivation and "florescence-guided aPDT assessment" is demonstrated in MRSA infected wounds of mice.

In vitro photoinactivation effectiveness of a portable LED device aimed for intranasal photodisinfection and a photosensitizer formulation comprising methylene blue and potassium iodide against bacterial, fungal, and viral respiratory pathogens.

Antimicrobial photodynamic therapy (aPDT) can be a viable option for management of intranasal infections. However, there are light delivery, fluence, and photosensitizer-related challenges. We report in vitro effectiveness of an easily fabricated, low-cost, portable, LED device and a formulation comprising methylene blue (MB) and potassium iodide (KI) for photoinactivation of pathogens of the nasal cavity, namely, methicillin-resistant Staphylococcus aureus, antibiotic-resistant Klebsiella pneumoniae, multi-antibiotic-resistant Pseudomonas aeruginosa, Candida spp., and SARS-CoV-2.In a 96-well plate, microbial suspensions incubated with 0.005% MB alone or MB and KI formulation were exposed to different red light (~ 660 ± 25 nm) fluence using the LED device fitted to each well. Survival loss in bacteria and fungi was quantified using colony-forming unit assay, and SARS-CoV-2 photodamage was assessed by RT-PCR.The results suggest that KI addition to MB leads to KI concentration-dependent potentiation (up to ~ 5 log10) of photoinactivation in bacteria and fungi. aPDT in the presence of 25 or 50 mM KI shows the following photoinactivation trend; Gm + ve bacteria > Gm - ve bacteria > fungi > virus. aPDT in the presence of 100 mM KI, using 3- or 5-min red light exposure, results in complete eradication of bacteria or fungi, respectively. For SARS-CoV-2, aPDT using MB-KI leads to a ~ 6.5 increase in cycle threshold value.The results demonstrate the photoinactivation effectiveness of the device and MB-KI formulation, which may be helpful in designing of an optimized protocol for future intranasal photoinactivation studies in clinical settings.

Fluorescence photobleaching of urine for improved signal to noise ratio of the Raman signal - An exploratory study.

Urine analysis is an important clinical test routinely performed in pathology labs for disease diagnosis and prognosis. In recent years, near-infrared Raman spectroscopy has drawn considerable attention for urine analysis as it can provide rapid, reliable, and reagent-free analysis of urine samples. However, one important practical problem encountered in such Raman measurements is the orders of magnitude stronger spectral background preventing one to utilize the full dynamic range of the detector which is required for the measurement of Raman signal with good signal-to-noise ratio (SNR). We report here the results of an exploratory study carried out on human urine samples to show that the photobleaching, which is a major disadvantage during the fluorescence measurement, could be utilized for suppressing the measured background to improve the SNR of the Raman peaks. It was found that once the photobleaching reached its plateau, there were improvements by ~67% and ~47% in the SNR and the signal to background ratio (SBR), respectively, of the Raman signals as compared to the spectra measured at the start of acquisition. Further, the reduced background also allowed us to utilize the full dynamic range of the detector at increased integration time without saturating the detector indicating the possibility of obtaining an improved detection limit.

Fluorescence photo-bleaching of urine and its applicability in oral cancer diagnosis.

Photo-stability of urine is of crucial importance for the applicability of fluorescence spectroscopy of urine samples for diagnosis of cancer. We report the results of a detailed study on fluorescence photo-bleaching of human urine samples. We also present the results of a preliminary investigation on evaluation of the applicability of photo-bleaching characteristics of urine for discriminating patients with oral cancer from healthy volunteers. The time-lapse fluorescence induced by continuous shining of 405 nm radiation from a diode laser was recorded from the urine samples obtained from 18 patients with oral cancer as well as from 22 healthy volunteers with history of no known major illness in the past two months. The integrated fluorescence intensity (ΣI), calculated for each spectrum, was found to decrease with time till a point after which no further decrease was observed. Further, while significant differences were observed in the spectra of cancerous patients and healthy volunteers, these differences were found to be varying with time till the intensities of the observed fluorescence spectra corresponding to the two categories of urine samples became stable. The curve, generated by plotting ΣI vs. time, was found to be best fitted (R2 > 0.95) with a double-exponential decay function. The photo-bleaching constants, obtained from curve-fitting, were found to have statistically significant differences corresponding to the urine samples of cancerous patients and healthy volunteers. A classification algorithm developed based on nearest-mean classifier (NMC) and applied on the photo-bleaching constants in leave-one-subject-out cross-validation mode was found to provide a sensitivity and specificity of up to ∼ 86% in discriminating the two categories of urine samples.

Inverse spatially-offset Raman spectroscopy using optical fibers: An axicon lens-free approach.

Inverse spatially offset Raman spectroscopy (I-SORS) seeks to interrogate deep inside a Raman-active, layered, diffusely scattering sample. It makes a collimated laser beam incident onto the sample surface in the form of concentric illumination rings (of varying radii) from whose center the back-scattered Raman signal is collected for detection. Since formation of illumination rings of different sizes requires an axicon to be moved along the axis of the collimated laser beam and axicons below a certain minimum size (~1 inch) are not readily available, this classical configuration incorporating an axicon cannot be used for designing a compact I-SORS probe of narrower diameter. We report a novel scheme of implementing I-SORS which overcomes this limitation by implementing ring illumination and point collection using two multi-mode optical fibers. An important advantage of the proposed scheme is that unlike the previously reported inverse SORS configurations, it does not require physical movement of any of the optical components for generating spatial offsets needed for probing sub-surface depths. Another advantage is its fiber-optic configuration which is ideally suited for designing a compact and pencil-sized I-SORS probe, often desired in many practical situations for carrying out depth-sensitive Raman measurements in situ from a layered turbid sample.

Nanotrap-Enhanced Raman Spectroscopy: An Efficient Technique for Trace Detection of Bioanalytes.

Reliable diagnosis of disease using body fluids requires sensitive and accurate detection of disease-specific analytes present in the fluid. In recent years, there has been increasing interest in using surface-enhanced Raman spectroscopy (SERS) for this purpose. The demonstrable signal enhancement and sensitivity of SERS makes it ideally suited for detection of a trace quantity of any analyte. However, lack of reproducibility along with large spatial variability in the measured Raman intensities due to differential (and often random) distribution of surface "hot spots" limits its routine clinical use. We propose here a technique, nanotrap-enhanced Raman spectroscopy (NTERS), for overcoming these long-standing limitations and challenges of SERS. In this technique, hot spots are formed by drying up a microvolume drop of the liquid, containing the mixture of nanoparticles and analytes in the focal volume of the Raman excitation laser, and the Raman signal is detected from these spots containing the analytes localized within the nanoparticle aggregates. The performance of the technique was evaluated in detecting trace quantities of two Raman-active analytes, Rhodamine 6G (R6G) and urea. It was found that R6G and urea could be detected down to a concentration of 50 nM with signal-to-noise ratio (SNR) value of ∼75 and 4 mM with SNR value of ∼500, respectively. A comparison with SERS revealed that NTERS not only had significantly superior (around 2 orders of magnitude) signal enhancement but also had high reproducibility because of its intrinsic ability to form nanoparticle aggregates with high repetitiveness. Another advantage of NTERS is its simplicity and cost effectiveness as it does not require any specialized substrate.

Inverse SORS for detecting a low Raman-active turbid sample placed inside a highly Raman-active diffusely scattering matrix - A feasibility study.

The broad range of applications of spatially-offset Raman spectroscopy (SORS) were found to involve samples having only marginal differences in Raman cross-sections between the surface and subsurface targets. We report the results of a feasibility study to evaluate the potential of the approach to identify the presence of a very low Raman-active turbid sample placed inside a highly Raman-active diffusely scattering matrix. Paraffin sandwiched tissue blocks prepared by embedding slices of chicken muscle tissue into solid paraffin blocks were employed as representative samples for the study. It was found that in contrast to the several millimetres of probing depth reported in the earlier applications, the Raman signatures of tissue were best recovered when it was located beneath the surface of the paraffin block at a depth of around a millimetre, beyond which the quality of recovery was increasingly poorer. However, the probing depth could be further increased by increasing the thickness of the embedded tissue sections. The results clearly suggest that though the probing depth achievable under the current condition is less than that found in previous applications, nevertheless it is sufficient for various other applications that would not require probing as deep as was required earlier.

Depth-sensitive Raman spectroscopy combined with optical coherence tomography for layered tissue analysis.

Complete characterization of a layered tissue requires probing both the biochemical and the morphological information from its different layers at various depths. We report the development of a combined Raman spectroscopy (RS) and optical coherence tomography (OCT) system that is capable of measuring depth-sensitive Raman signal from the tissue layers imaged by the OCT. The sample arm of a real-time time-domain OCT system was modified to allow for co-alignment of the OCT with the Raman probe beam. The depth sensitivity of Raman was obtained by incorporating confocal Raman configuration that minimized out-of-focus Raman scattered light. The system was first validated using a layered phantom prepared by depositing a thin layer of paraffin over acetaminophen. A good correlation was observed between the OCT images and the Raman signal. The system was also used to record OCT and Raman images of a resected mucosal tissue sample. While OCT image showed the presence of epithelial and stromal layers, Raman spectra measured from these layers confirmed the biochemical difference between the two.

In vivo Raman spectroscopy for detection of oral neoplasia: a pilot clinical study.

We report a pilot study carried out to evaluate the applicability of in vivo Raman spectroscopy for differential diagnosis of malignant and potentially malignant lesions of human oral cavity in a clinical setting. The study involved 28 healthy volunteers and 171 patients having various lesions of oral cavity. The Raman spectra, measured from multiple sites of normal oral mucosa and of lesions belonging to three histopathological categories, viz. oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSMF) and leukoplakia (OLK), were subjected to a probability based multivariate statistical algorithm capable of direct multi-class classification. With respect to histology as the gold standard, the diagnostic algorithm was found to provide an accuracy of 85%, 89%, 85% and 82% in classifying the oral tissue spectra into the four tissue categories based on leave-one-subject-out cross validation. When employed for binary classification, the algorithm resulted in a sensitivity and specificity of 94% in discriminating normal from the rest of the abnormal spectra of OSCC, OSMF and OLK tissue sites pooled together.

Fluorescence spectroscopy for noninvasive early diagnosis of oral mucosal malignant and potentially malignant lesions.

BACKGROUND: We report the results of a clinical in vivo study to evaluate the potential of fluorescence spectroscopy for differential diagnosis of oral mucosal malignant and potentially malignant lesions. MATERIALS AND METHODS: The study involved 26 healthy volunteers and 144 patients enrolled for routine medical examination of the oral cavity at the outpatient department of the Tata Memorial Hospital, Mumbai. In vivo autofluorescence spectra were recorded using a N2 laser based portable fluorimeter developed in-house. The different tissue sites investigated belonged to either of the four histopathologic categories: 1) squamous cell carcinoma (SCC), 2) oral sub-mucous fibrosis (OSMF), 3) leukoplakia (LP) and 4) normal squamous tissue. A multivariate statistical algorithm capable of direct multi-class classification was used to predict pathological designations. RESULTS: With respect to histopathology as the "gold standard", the diagnostic algorithm was found to provide an accuracy of 82, 76, 81 and 85% based on leave-one-patient-out cross-validation in classifying the oral tissue spectra into four different pathology classes - SCC, OSMF, LP, and normal squamous tissue - respectively. When the algorithm was employed for delineating the normal oral tissues from all the abnormal oral tissues including SCC, OSMF and LP put together, a sensitivity of 98% and a specificity of 100% were obtained. CONCLUSION: The results suggest that it is possible to objectively classify the oral tissue into different pathology classes based on their in vivo autofluorescence spectra. Thus, the technique can potentially improve oral screening efforts in low resource settings where clinical expertise and resources are limited.