Antimicrobial Photodynamic Therapy (aPDT) for periodontal disease (PD) treatment has been studied for more than three decades, but there is no consensus among researchers about its role on PD control. PD is an oral infection linked with a variate of systemic diseases affecting mainly immunocompromised and elderly patients. The aim of this study is to evaluate aPDT for PD control and to analyze the periodontal microbiological profile regarding healthcare-associated infections (HAI). Six patients were enrolled in this study and the microbiological profile of 36 sites were evaluated in different moments: initial, after scaling and root planing, after aPDT and one-week post-treatment. aPDT was performed with 100μM water solution of methylene blue (MB), and a diode laser (100mW, 660nm, 90s per point, 9J). After periodontal therapy the pockets were irrigated with hydrogen peroxide (3%), washed with water, and MB was applied when no bleeding was detected. To collect the periodontal pocket content, a sterilized paper point was placed inside the pocket for 30s. The material was transferred to a vial with transport culture medium and sent to a microbiological facility to be analyzed blindly. The results showed the presence of pathogens linked with HAI as Acinetobacter baumannii, Candida albicans e Serratia marcescens immediately after therapy, but one week after treatment, none of them were detected and the periodontal index of all patients improved. This preliminary result point out that aPDT may not only improve periodontal clinical conditions, but also can diminish the systemic-health threat represented by this disease.
Computer vision technologies for diagnostic imaging applied to oral lesions, specifically, carious lesions of the teeth, are in their early years of development. The relevance of this public problem, dental caries, worries countries around the world, as it affects almost the entire population, at least once in the life of each individual. The present work demonstrates current techniques for obtaining information about lesions on teeth by segmentation laser speckle imagens (LSI). Laser speckle image results from laser light reflection on a rough surface, and it was considered a noise but has important features that carry information about the illuminated surface. Even though these are basic images, only a few works have analyzed it by application of computer vision methods. In this article, we present the latest results of our group, in which Computer vision techniques were adapted to segment laser speckle images for diagnostic purposes. These methods are applied to the segmentation of images between healthy and lesioned regions of the tooth. These methods have proven to be effective in the diagnosis of early-stage lesions, often imperceptible in traditional diagnostic methods in the clinical practice. The first method uses first-order statistical models, segmenting the image by comparing the mean and standard deviation of the intensity of the pixels. The second method is based on the distance of the chi-square (χ2 ) between the histograms of the image, bringing a significant improvement in the precision of the diagnosis, while a third method introduces the use of fractal geometry, exposing, through of the fractal dimension, more precisely the difference between lesioned areas and healthy areas of a tooth compared to other methods of segmentation. So far, we can observe efficiency in the segmentation of the carious regions. A software was developed for the execution and demonstration of the applicability of the models
Vulvovaginal candidiasis is a common cause of vaginal infections. This study investigates the efficiency of antimicrobial photodynamic therapy (aPDT) against yeast cells in mice. Methylene blue (MB), malachite green (MG), and a special designed protoporphirin (PpNetNI) were used as photosensitizers. Female BALB-c mice were infected with Candida albicans ATCC 90028. PDT was applied with two different light sources, intravaginal and transabdominal. Vaginal washes were performed and cultivated for microbial quantification. Antimicrobial PDT was able to decrease microbial content with MB and PpNetNI (p<0.05), it was not effective, however, with MG photosensitizer. The results of this study demonstrate that aPDT may be a viable alternative treatment for vaginal candidiasis.
Dental erosion is a non-carious lesion that causes progressive tooth wear of structure through chemical processes that do
not involve bacterial action. Its origin is related to eating habits or systemic diseases involving tooth contact with
substances that pose a very low pH. This work demonstrates a new methodology to quantify the erosion by coherent light
scattering of tooth surface. This technique shows a correlation between acid etch duration and laser speckle contrast map
(LASCA). The experimental groups presented a relative contrast between eroded and sound tissue of 17.8(45)%, 23.4
(68)% 39.2 (40)% and 44.3 (30)%, for 10 min, 20 min, 30 min and 40 min of acid etching, respectively.
Over the last few years, low-level light therapy (LLLT) has shown an incredible suitability for a wide range of applications for central nervous system (CNS) related diseases. In this therapeutic modality light dosimetry is extremely critical so the study of light propagation through the CNS organs is of great importance. To better understand how light intensity is delivered to the most relevant neural sites we evaluated optical transmission through slices of rat brain point by point. We experimented red (λ = 660 nm) and near infrared (λ = 808 nm) diode laser light analyzing the light penetration and distribution in the whole brain. A fresh Wistar rat (Rattus novergicus) brain was cut in sagittal slices and illuminated with a broad light beam. A high-resolution digital camera was employed to acquire data of transmitted light. Spatial profiles of the light transmitted through the sample were obtained from the images. Peaks and valleys in the profiles show sites where light was less or more attenuated. The peak intensities provide information about total attenuation and the peak widths are correlated to the scattering coefficient at that individual portion of the sample. The outcomes of this study provide remarkable information for LLLT dose-dependent studies involving CNS and highlight the importance of LLLT dosimetry in CNS organs for large range of applications in animal and human diseases.
Due to the great number of applications of Low-Level-Laser-Therapy (LLLT) in Central Nervous System
(CNS), the study of light penetration through skull and distribution in the brain becomes extremely
important. The aim is to analyze the possibility of precise illumination of deep regions of the rat brain,
measure the penetration and distribution of red (λ = 660 nm) and Near Infra-Red (NIR) (λ = 808 nm)
diode laser light and compare optical properties of brain structures. The head of the animal (Rattus
Novergicus) was epilated and divided by a sagittal cut, 2.3 mm away from mid plane. This section of rat's
head was illuminated with red and NIR lasers in points above three anatomical structures: hippocampus,
cerebellum and frontal cortex. A high resolution camera, perpendicularly positioned, was used to obtain
images of the brain structures. Profiles of scattered intensities in the laser direction were obtained from
the images. There is a peak in the scattered light profile corresponding to the skin layer. The bone layer
gives rise to a valley in the profile indicating low scattering coefficient, or frontal scattering. Another
peak in the region related to the brain is an indication of high scattering coefficient (μs) for this tissue.
This work corroborates the use of transcranial LLLT in studies with rats which are subjected to models of
CNS diseases. The outcomes of this study point to the possibility of transcranial LLLT in humans for a
large number of diseases.
Several studies indicate that low level laser therapy (LLLT) accelerates the healing process, however, for a determined
pathology, dosimetry remains difficult to be established. To understand the tissue optical properties under different
conditions is extremely relevant since the dose delivered to the target tissue is known to be critical. The skin
pigmentation influence on the laser attenuation is not yet well established on different mice lineages or human ethnical
groups, making the dose problematic. Along the same line, inflammatory processes may cause similar problems since the
tissues in this condition change their optical properties due to inflammatory cell accumulation. This work evaluated the
attenuation pattern of a HeNe laser (λ=632.8 nm) using ex vivo skin samples from Balb/C and C57BL/6 mice under
inflammatory stages induced in their paw by local carrageenan inoculation. The samples were placed between two
microscope slides, and a CCD camera was placed orthogonal to the beam path. The intensity distribution of the scattered
light was photographed in grayscale and analyzed by ImageJ software. Our findings suggest that even slight differences
of the epithelial pigmentation could result in a relevant dose loss delivered to the deeper tissues. The increase of the
inflammatory cell density in the connective tissue indicated a highly scattering area also resulting in a dose loss for the
deeper tissues when compared to control group.
Candida albicans is the most frequent human opportunistic pathogenic fungus and one of the most important causes of
nosocomial infections. In fact, diagnosis of invasive candidiasis presents unique problems. The aim of this work was to
evaluate, by fluorescence image analysis, cellular labeling of C. albicans with CdTe/CdS quantum dots conjugated or
not to concanavalin A (ConA). Yeast cells were incubated with CdTe/CdS quantum dots (QD) stabilized with
mercaptopropionic acid (MPA) (emission peak at 530 nm) for 1 hour. In the overall study we observed no morphological
alterations. The fluorescence microscopic analysis of the yeast cells showed that the non-functionalized QDs do not label
C. albicans cells, while for the QD conjugated to ConA the cells showed a fluorescence profile indicating that the
membrane was preferentially marked. This profile was expected since Concanavalin A is a protein that binds specifically
to terminal carbohydrate residues at the membrane cell surface. The results suggest that the QD-labeled Candida cells
represent a promising tool to open new possibilities for a precise evaluation of fungal infections in pathological
conditions.
Photodynamic antimicrobial chemotherapy (PACT) is based on the principal that microorganisms will be inactivated
using a light source combined to a photosensitizing agent in the presence of oxygen. Oxidative damage of cell
components occurs by the action of reactive oxygen species leading to cell death for microbial species. It has been
demonstrated that PACT is highly efficient in vitro against a wide range of pathogens, however, there is limited
information for its in vivo potential. In addition, it has been demonstrated that sublethal photodynamic inactivation may
alter the virulence determinants of microorganisms. In this study, we explored the effect of sublethal photodynamic
inactivation to the virulence factors of Candida albicans. Methylene Blue (MB) was used as photosensitizer for sublethal
photodynamic challenge on C. albicans associated with a diode laser irradiation (λ=660nm). The parameters of
irradiation were selected in causing no reduction of viable cells. The potential effects of PACT on virulence determinants
of C. albicans cells were investigated by analysis of germ tube formation and in vivo pathogenicity assays. Systemic
infection was induced in mice by the injection of fungal suspension in the lateral caudal vein. C. albicans exposed to
sublethal photodynamic inactivation formed significantly less germ tube than untreated cells. In addition, mice infected
with C. albicans submitted to sublethal PACT survived for a longer period of time than mice infected with untreated
cells. The oxidative damage promoted by sublethal photodynamic inactivation inhibited virulence determinants and
reduced in vivo pathogenicity of C. albicans.
Antimicrobial photodynamic inactivation (APDI) is based on the utilization of substances that can photosensitize
biological tissues and are capable of being activated in the presence of light. Cryptococcus neoformans is an yeast
surrounded by a capsule composed primarily of glucoronoxylomannan that plays an important role in its virulence. This
yeast causes infection on skin, lungs and brain that can be associated with neurological sequelae and neurosurgical
interventions, and its conventional treatment requires prolonged antifungal therapy, which presents important adverse
effects. The aim of this study was to evaluate the protective effect of Cryptococcus neoformans capsule against reactive
oxygen species generated by APDI. Cryptococcus neoformans KN99α, which is a strain able to produce capsule, and
CAP59 that does not present capsule production were submitted to APDI using methylene blue (MB), rose bengal (RB),
and pL-ce6 as photosensitizers (PS). Then microbial inactivation was evaluated by counting colony form units following
APDI and confocal laser scanning microscopy (CLSM) illustrated localization as well as the preferential accumulation of
PS into the fungal cells. C. neoformans KN99α was more resistant to APDI than CAP59 for all PSs tested. CLSM
showed incorporation of MB and RB into the cytoplasm and a preferential uptake in mitochondria. A nuclear
accumulation of MB was also observed. Contrarily, pL-ce6 appears accumulated in cell wall and cell membrane and
minimal florescence was observed inside the fungal cells. In conclusion, the ability of C. neoformans to form capsule
enhances survival following APDI.
The biofilm formed by Candida albicans is the mainly cause of infections associated to medical devices such as
catheters. Studies have shown that photodynamic antimicrobial therapy (PAT) has lethal effect on C. albicans, and it is
based on photosensitizer (PS) in the presence of low intensity light to generate reactive oxygen species in biological
systems. The aim of this study was to analyze in real time, by Optical Coherence Tomography (OCT), the alterations in
C. albicans biofilm in vitro during PAT using methylene blue (MB) as a PS and red light. An OCT system with working
at 930nm was used, sequential images of 2000×512 pixels were generated at the frame rate of 2.5frames/sec. The
dimension of the analyzed sample was 6000μm wide by 1170μm of depth corrected by refraction index of 1.35. We
recorded 1min. before and after the irradiation with LED for PAT, generating 8min. of video. For biofilm formation,
discs were made from elastomeric silicone catheters. The PS was dissolved in PBS solution, and a final concentration of
1mM MB was applied on biofilm, followed by a red LED irradiation (λ=630nm±20nm) during 6min. We performed a
curve of survival fraction versus time of irradiation and it was reduced by 100% following 6min. of irradiation. OCT was
performed for measurement of biofilm thickness of 110μm when biofilm was formed. During irradiation, the variation of
biofilm thickness was ~70μm. We conclude that OCT system is able to show real time optical changes provided by PAT
in yeasts organized in biofilm.
The reduction of pathogenic microorganisms in supragingival plaque is one of the principal factors in caries prevention
and control. A large number of microorganisms have been reported to be inactivated in vitro by photodynamic therapy
(PDT). The purpose of this study was to develop a rat model to investigate the effects of PDT on bacterial reduction in
induced dental caries. Twenty four rats were orally inoculated with Streptococcus mutans cells (ATCC 25175) for three
consecutive days. The animals were fed with a cariogenic diet and water with 10% of sucrose ad libitum, during all
experimental period. Caries lesion formation was confirmed by Optical Coherence Tomography (OCT) 5 days after the
beginning of the experiment. Then, the animals were randomly divided into two groups: Control Group: twelve animals
were untreated by either light or photosensitizer; and PDT Group: twelve animals were treated with 100μM of methylene
blue for 5min and irradiated by a Light Emitting Diode (LED) at λ = 640±30nm, fluence of 172J/cm2, output power of
240mW, and exposure time of 3min. Microbiological samples were collected before, immediately after, 3, 7 and 10 days
after treatment and the number of total microaerophiles was counted. OCT images showed areas of enamel
demineralization on rat molars. Microbiological analysis showed a significant bacterial reduction after PDT.
Furthermore, the number of total microaerophiles in PDT group remained lower than control group until 10 days posttreatment.
These findings suggest that PDT could be an alternative approach to reduce bacteria in dental caries.
Bloodstream infections are potentially life-threatening diseases. They can cause serious secondary infections, and may
result in endocarditis, severe sepsis or toxic-shock syndrome. Pseudomonas aeruginosa is an opportunistic pathogen and
one of the most important etiological factors responsible for nosocomial infections, mainly in immuno-compromissed
hosts, characteristic of patients with severe burns. Its multiresistance to antibiotics produces many therapeutic problems,
and for this reason, the development of an alternative method to antibiotic therapy is needed. Photodynamic inactivation
(PDI) may be an effective and alternative therapeutic option to prevent bloodstream infections in patients with severe
burns. In this study we report the use of PDI to prevent bloodstream infections in mice with third-degree burns. Burns
were produced on the back of the animals and they were infected with 109 cfu/mL of multi-resistant (MR) P. aeruginosa.
Fifteen animals were divided into 3 groups: control, PDT blue and PDT red. PDT was performed thirty minutes after
bacterial inoculation using 10μM HB:La+3 and a light-emitting diode (LED) emitting at λ=460nm±20nm and a LED
emitting at λ=645 nm±10nm for 120s. Blood of mice were colected at 7h, 10h, 15h, 18h and 22h pos-infection (p.i.) for
bacterial counting. Control group presented 1×104 cfu/mL in bloodstream at 7h p.i. increasing to 1×106 at 22h, while
mice PDT-treated did not present any bacteria at 7h; only at 22h p.i. they presented 1×104cfu/mL. These results suggest
that HB:La+3 associated to blue LED or red LED is effective to delay and diminish MR P.aeruginosa bloodstream
invasion in third-degree-burned mice.
In this work we described the potentiality of the Hypocrellin B (HB) modified with the presence of lanthanum (La3+)
ions, in eliminate Candida albicans in suspension. The results showed that the presence of lanthanum ions promotes a
red shift of the HB absorption band and an enhancement in singlet oxygen quantum yield in 32%. Also in this work we
obtained that the best molar ration between HB and La concentrations was 1:2. No photobleaching was observed in our
experimental conditions. Antimicrobial activity was studied exciting C. albicans suspension with a 460 nm LED and a
660 nm laser both with 330 mW/cm2 irradiance. Best irradiation time, PS concentration and ROS production profile
were determined showing that using 460 nm LED with 10 μM of PS, only 30 s of irradiation time was sufficient to
reduce 100 % C. albicans colonies.
Burns are frequently contamined by pathogenic microorganisms and the widespread occurrence of antibiotic resistant
strains of Pseudomonas aeruginosa in hospitals is a matter of growing concern. Hypocrellin B (HB) is a new generation
photosensitizer extracted from the fungus Hypocrella bambusae with absorption bands at 460, 546 and 584 nm.
Lanthanide ions change the HB molecular structure and a red shift in the absorption band is observed as well as an
increase in the singlet oxygen quantum yield. In this study, we report the use of HB:La+3 to kill resistant strain of P.
aeruginosa infected burns. Burns were produced on the back of mice and wounds were infected subcutaneously with
1x109 cfu/mL of P. aeruginosa. Three-hours after inoculation, the animals were divided into 4 groups: control, HB:La+3,
blue LED and HB:La+3+blue LED. PDT was performed using 10μM HB:La+3 and 500mW light-emitting diode (LED)
emitting at λ=470nm±20nm during 120s. The animals of all groups were killed and the infected skin was removed for
bacterial counting. Mice with photosensitizer alone, light alone or untreated infected wounds presented 1x108 cfu/g while
mice PDT-treated showed a reduction of 2 logs compared to untreated control. These results suggest that HB:La+3
associated to blue LED is effective in diminishing antibiotic resistant strain P. aeruginosa in infected burns.
KEYWORDS: Photodynamic therapy, Yeast, In vivo imaging, In vitro testing, Statistical analysis, Lasers, Tissues, Scanning electron microscopy, Skin, Nervous system
Cryptococcosis is an infection caused by the encapsulated yeast Cryptococcus neoformans and the most afflicted sites
are lung, skin and central nervous system. A range of studies had reported that photodynamic therapy (PDT) can
inactivate yeast cells; however, the in vivo experimental models of cryptococcosis photoinactivation are not commonly
reported. The aim of this study was to investigate the ability of methylene blue (MB) combined with a low-power red
laser to inactivate Cryptococcus neoformans in in vitro and in vivo experimental models. To perform the in vitro study,
suspension of Cryptococcus neoformans ATCC-90112 (106cfu/mL) was used. The light source was a laser (Photon Lase
III, DMC, São Carlos, Brazil) emitting at λ660nm with output power of 90mW for 6 and 9min of irradiation, resulting
fluences at 108 and 162J/cm². As photosensitizer, 100μM MB was used. For the in vivo study, 10 BALB/c mice had the
left paw inoculated with C. neoformans ATCC-90112 (107cfu). Twenty-four hours after inoculation, PDT was performed
using 150μM MB and 100mW red laser with fluence at 180J/cm2. PDT was efficient in vitro against C. neoformans in
both parameters used: 3 log reduction with 108J/cm² and 6 log reduction with 162J/cm². In the in vivo experiment, PDT
was also effective; however, its effect was less expressive than in the in vitro study (about 1 log reduction). In
conclusion, PDT seems to be a helpful alternative to treat dermal cryptococcosis; however, more effective parameters
must be found in in vivo studies.
The aim of this study was to determinate the efficiency of methylene blue (MB) to kill cultures of Aggregatibacter
actinomycetemcomitans under red light and to investigate MB photobleaching by optical absorption spectroscopy.
Bacteria were diluted in aqueous solution, putted in glass tubes and distributed in 5 groups: (L-MB-) control group;
(L+MB-) laser alone by 5min; (L-MB+) MB alone through 5min; (3L+MB+) MB+laser 3min; (5L+MB+) MB+laser
5min. Laser parameters were P=30mW, λ=660nm, E=9J in 5min and E=5.4J in 3min. The samples were diluted and
bacterial colonies were counted and converted into colony forming units (CFU). Absorption spectra of the MB-stained
bacterial suspension and photosensitized bacterial suspension were obtained. Groups L-MB-, L+MB-, and L-MB+ did
not show a decrease in CFU/mL. L+MB+ groups showed a significant decrease in CFU/mL but no statistically
significant differences were observed between 3min and 5min. Spectroscopy showed that MB is photodegraded after
irradiation and that dimer species are more notably consumed than monomeric species. These results suggest that MB is
a suitable photosensitizer to reduce A. actinomycetemcomitans, and that 3min of irradiation are enough to produce a
significant effect. Due to the spectral changes observed on MB solution after irradiation a type I mechanism may be
involved.
Severe burns cause extensive damage and are complicated by loss of body fluids, injury in the cutaneous vasculature and delayed wound healing. Low-intensity laser therapy (LILT) has been studied as an alternative method to accelerate wound healing. This study was carried out to evaluate LILT effects (λ= 660 nm) in rat burned skin with two different dose regimens. Thirty-six male adult Wistar rats with two burns created on their back using steam water were divided into 3 groups. In the fractioned dose laser group (FG), the lesions were irradiated with 1J/cm2 on days 1, 3, 8 and 10; in the single dose laser group (SG), the lesions were irradiated with 4J/cm2 on day 1. On control group (CG), lesions were not irradiated. Three animals per group were sacrificed on days 1, 3, 8, 10, 15 and 21 post-wounding and skin specimens were collected and processed to histomorphometry. At days 1, 3 and 8, statistical significant differences were not observed among groups. On the 10th day, mean values of the number of blood vessels for FG was significantly higher than CG. Irradiated groups showed a peak of new blood vessels formation at day 15 while for CG the peak was at day 21. The number of vessels in CG was significantly higher than FG and SG at day 21. These findings suggest that LILT may accelerate angiogenesis compared to control group, however, no significant differences were observed between laser groups with fractioned or single dose during all experiment.
It has been proposed that photodynamic therapy (PDT) can inactivate microbial cells. A range of photosensitizers and light sources were reported as well as different fluence parameters and dye concentrations. However, much more knowledge regarding to the role of fluences, irradiation time and irradiance are required for a better understanding of the photodynamic efficiency. The aims of this study were to investigate the role of light parameters on the photoinactivation of yeast cells, and compare cell survivors in different growing phases following PDT. To perform this study, a
suspension (106cfu/mL) of Candida albicans ATCC-90028 was used in log and stationary-phase. Three irradiances 100mW/cm2, 200mW/cm2 and 300mW/cm2 were compared under 3min, 6min and 9min of irradiation, resulting in fluences of 18, 36, 54, 72,108 and 162J/cm2. The light source used was a laser emitting at 660nm with output power of 30, 60 and 90mW. As photosensitizer, 100μΜ methylene blue was used. PDT was efficient against yeast cells (6 log reduction) in log and stationary-phase. Neither photosensitizer nor light alone presented any reduction of cell viability. The increase of irradiance and time of irradiation showed a clearly improvement of cell photoinactivation. Interestingly, the same fluences in different irradiances presented dissimilar effects on cell viability. The irradiance and time of irradiation are important in PDT efficiency. Fluence per se is not the best parameter to compare photoinativation effects on yeast cells. The growing-phases presented the same susceptibility under C. albicans photoinactivation.
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