Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.

The antifungal drug therapy often employed to treat paracoccidiodomycosis (PCM), an important neglected fungal systemic infection, leads to offensive adverse effects, besides being very long-lasting. In addition, PCM compromises the oral health of patients by leading to oral lesions that are very painful and disabling. In that way, photodynamic therapy (PDT) arises as a new promising adjuvant treatment for inactivating Paracoccidioides brasiliensis (Pb), the responsible fungus for PCM, and also for helping the patients to deal with such debilitating oral lesions. PDT has been linked to an improved microbial killing, also presenting the advantage of not inducing immediate microbial resistance such as drugs. For the present study, we investigated the generation of reactive oxygen species (ROS) by using the fluorescent probes hydroxyphenyl fluorescein (HPF) and aminophenyl fluorescein (APF) after toluidine blue (TBO-37.5 mg/L)-mediated PDT (660 nm, 40 mW, and 0.04 cm spot area) and the action of TBO-PDT upon Pb cultures grown for 7 or 15 days in semisolid Fava Netto's culture medium; we also targeted oral PCM manifestations by reporting the first clinical cases (three patients) to receive topic PDT for such purpose. We were able to show a significant generation of hydroxyl radicals and hypochlorite after TBO-PDT with doses around 90 J/cm; such ROS generation was particularly useful to attack and inactivate Pb colonies at 7 and 15 days. All three patients reported herein related an immediate relief when it came to pain, mouth opening, and also the ability to chew and swallow. As extracted from our clinical results, which are in fact based on in vitro outcomes, TBO-PDT is a very safe, inexpensive, and promising therapy for the oral manifestations of PCM.

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