Mobile phone-based UV fluorescence microscopy for the identification of fungal pathogens.

BACKGROUND AND OBJECTIVE: Dermatophytes are fungi that cause infections in hair, skin, and nails. Potassium Hydroxide (KOH) microscopy is the most frequently used method for identifying dermatophytes. KOH helps in the visualization of the hyphae as it clears the debris present in the specimen but needs a trained eye for final diagnosis of the infection. Fluorescence microscopy using staining agents, such as calcofluor white (CFW) or blankophor, is a better method for identification of dermatophytes but is not used in clinics due to the cost and complexity of fluorescence microscopes. The objective of the present work is to develop a simple low-cost mobile phone-based device for the identification of fungal pathogens in skin samples. MATERIALS AND METHODS: A fluorescence spectrometer was used to establish the excitation/emission peaks of fluorescence intensity of CFW and KOH and Methyl Cellulose, a surrogate of fungi used for system development. A transillumination microscopy prototype was fabricated using off-the-shelf components, 3D printing and a mobile phone. The system was optically characterized using contrast resolution targets and verified using fungi isolate samples. An isolate of Trichophyton (T) rubrum was grown for 10-14 days for formation of fungal colonies. The surface of a single colony was gently scraped with a sterile loop and transferred to a glass slide. CFW with KOH was added to the T. rubrum and covered with cover slip for microscopic examination. The images of T. rubrum obtained with the prototype device were compared to those obtained using a commercial microscope. RESULTS: The excitation/emission wavelength pair for CFW was found to be 370/430 nm. The proposed device design is a transillumination microscopy setup using a mobile phone. It consists of a 365 nm LED as the excitation source, a 3V battery to power the LED, a slide to hold the sample, a lens for magnification and a phone to capture and store the images of the sample. The fabricated prototype has a resolution of 70 to 99 µm, a 2% to 30% distortion, and 60% contrast value for well illuminated samples. Images of T. rubrum samples obtained under brightfield illumination clearly show the long septate hyphae of the dermatophyte. As expected, images of the same samples with CFW and KOH show blue fluorescence, which results from the binding of the CFW to the chitin and cellulose in the fungal hyphae. These images are similar to those obtained with a commercial microscope. SUMMARY AND CONCLUSIONS: The concept and design of a mobile phone-based fluorescence microscope to identify dermatophytes has been demonstrated in a prototype and laboratory samples. The concept and design offer a simple, low-cost, compact but robust method for identification of fungal pathogens. This method is shown to be feasible for detecting fluorescence accurately and imaging the fungal structure at a resolution of 100 µm or better. Lasers Surg. Med. 51:201-207, 2019. © 2018 Wiley Periodicals, Inc.

Point-of-care detection of neutrophils in live skin microsamples using chemiluminescence.

Many skin diseases are defined by the presence of neutrophils, which are among the first cells to respond to infection and inflammation. Currently, neutrophil identification in the skin is costly and slow. The objectives of the present work are to investigate the feasibility of detecting the presence of neutrophils in live skin microsamples using chemiluminescence and develop a device and procedures that will enable preclinical and clinical investigations. Our approach consists of collecting skin microsamples and exposing them to reagents that activate neutrophils and amplify the light emission produced by chemiluminescence. Experiments using live pig skin with and without inflammation show that it is feasible to detect the presence of neutrophils in the skin. The proposed method is minimally invasive, simple, fast, and does not require user specialization. The developed system is compact in size with a small footprint, which makes it portable and suitable for point-of-care diagnostics.