Sandra Mara Alessi Aristides

Photodynamic therapy for American cutaneous leishmaniasis: The efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

The aim of this study was to investigate the effectiveness of Photodynamic Therapy (PDT) using Methylene Blue (MB) as the photosensitizing compound and a Light-Emitting Diode (LED) in American cutaneous leishmaniasis (ACL). Hamsters were experimentally infected with Leishmania (Leishmania) amazonensis. After the development of the lesions in the footpad, the animals were treated with MB three times a week for 3 months. Ten minutes after each application of MB, the lesions were irradiated with LED for 1 h. The lesions were evaluated weekly by the measurement of the hamster footpad thickness. At the end of the treatment the parasitic load was quantified in the regional lymph node of the hamsters. The treatment promoted a decrease in the thickness of infected footpad (P=0.0001) and reduction in the parasitic load in the regional lymph node (P=0.0007) of the animals from group treated with MB+LED. PDT using MB+LED in ACL caused by L. amazonensis shows a strong photodynamic effect. This therapy is very promising, once it is an inexpensive system and the own patient can apply it in their wound and in their house without the need of technical assistance.

Leishmanicidal activity of synthetic chalcones in Leishmania (Viannia) braziliensis

The treatment of American cutaneous leishmaniasis (ACL) is based on a small group of compounds that were developed decades ago, all of which are highly toxic and have a high rate of treatment failure. The chalcones show leishmanicidal activity, yet few studies have evaluated this activity against Leishmania (Viannia) braziliensis, one of the most important species of Leishmania across Latin America. Four new synthetic chalcones (1-4) were evaluated for inhibitory activity in vitro against promastigotes and intracellular parasites 24h post infection of L. (V.) braziliensis, cytotoxicity for macrophages J774.A1 and red blood cells, and the ability to stimulate nitric oxide production. The results for the inhibitory concentration for 50% of the promastigotes (IC50) (1.38±1.09-6.36±2.04μM), cytotoxic concentration for 50% of the macrophages (CC50) (13.49±3.13-199.43±4.11μM), and selectivity index (SI) (3.76 to 33.94) indicate that all chalcones (1-4) showed an effect on promastigotes of L. (V.) braziliensis; chalcone 2 had the highest SI. The haemolytic assay with chalcones 1 (301.93μM), 2 (534.18μM), 3 (419.46μM) and 4 (381.11μM) showed 0.00%, 2.33%, 0.57% and 1.74% haemolysis, respectively. All chalcones significantly reduced the infection index of macrophages by parasites; for chalcones (1-3) this effect may be dependent on nitric-oxide production by macrophages. The chalcones tested exhibited inhibitory activity for promastigotes and intracellular parasites of L. (V.) braziliensis, with low toxicity for macrophages and red blood cells. The anti-Leishmania activity of chalcones (1-3) may depend on the stimulation of nitric-oxide production in the initial stage of infection. These results show an initially encouraging potential for the use of chalcones (1-4) to treat ACL.

Detection of DNA from Leishmania (Viannia): Accuracy of Polymerase Chain Reaction for the Diagnosis of Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) can occur in skin and mucosa, causing disfiguring lesions. The laboratory diagnosis of CL involves immunological methods and optical detection of the parasite, al of which have limitations. There is a need for more effective diagnostic methods for CL which wil allow treatment to be initiated more promptly in order to help prevent the development of severe forms of mucosal disease, and to estimate the prognosis of the infection. The polymerase chain reaction (PCR) has been widely used to diagnose CL, because of its higher sensitivity. This study estimated the accuracy and compared PCRs of samples from lesion scarification (PCR-L) and blood sample-enriched leukocytes (PCR-B) with three conventional diagnostic techniques: parasite direct search (DS), Montenegro skin test (MST), and indirect immunofluorescence reaction (IIF). The study included 276 patients under suspicion of CL. We conducted a cross-sectional study, in which patients were selected by convenience sampling. We used MP3H/MP1L primers to generate a Leishmania (Viannia) (minicircle kDNA) fragment of 70-bp. Of 106 patients with CL, 83.87%, 51.67%, 64.52%, 85.71%, or 96.10% tested positive by PCR-L, PCR-B, DS, IIF, or MST, respectively. Five patients tested positive only by PCR-L, and two other patients only by PCR-B. PCR-L is indicated for use in patients with chronic lesions or Leishmania reinfection, which may progress to mucosal lesion. PCR-B is indicated for use in patients with negative results in conventional tests or for patients with no apparent lesion. PCR is not only useful in diagnosing CL but also helps to identify the infecting species.

Safety and efficacy of current alternatives in the topical treatment of cutaneous leishmaniasis: a systematic review

Studies of topical treatments for leishmaniasis were systematically reviewed, to evaluate the therapeutic efficacy, safety and any adverse effects of these treatments. The papers identified in the databases PubMed and Web of Knowledge involved eight studies with a total of 1744 patients. The majority of trials was from Iran (4/8), covered a period of 8 years (2003-2011), and included patients 4-85 years of age. The most frequent Leishmania species in the studies were L. tropica (4/8) and L. major (2/8). The treatments administered were thermotherapy, paromomycin and combinations, CO2 laser, 5-aminolevulinic acid hydrochloride (10%) plus visible red light (633 nm) and cryotherapy. Six articles reported cure rates over 80·0%. Six studies reported on failure rates, three of them reporting rates lower than 10%. Four studies did not report relapses or recurrences, while the other studies reported low rates (1·8-6·3%). The most common adverse effects of the topical treatments were redness/erythema, pain, pruritus burning, oedema, vesicles and hyper- or hypopigmentation. The results provide strong evidence that the treatments topical evaluated showed high cure rates, safety and effectiveness, with low side-effects, relapse and recurrence rates, except for cryotherapy, which showed a moderate cure rate.

Evaluation of the reference value for the Montenegro skin test

INTRODUCTION The Montenegro skin test (MST) has good clinical applicability and low cost for the diagnosis of American tegumentary leishmaniasis (ATL). However, no studies have validated the reference value (5mm) typically used to discriminate positive and negative results. We investigated MST results and evaluated its performance using different cut-off points. METHODS The results of laboratory tests for 4,256 patients with suspected ATL were analyzed, and 1,182 individuals were found to fulfill the established criteria. Two groups were formed. The positive cutaneous leishmaniasis (PCL) group included patients with skin lesions and positive direct search for parasites (DS) results. The negative cutaneous leishmaniasis (NCL) group included patients with skin lesions with evolution up to 2 months, negative DS results, and negative indirect immunofluorescence assay results who were residents of urban areas that were reported to be probable sites of infection at domiciles and peridomiciles. RESULTS The PCL and NCL groups included 769 and 413 individuals, respectively. The mean ± standard deviation MST in the PCL group was 12.62 ± 5.91mm [95% confidence interval (CI): 12.20-13.04], and that in the NCL group was 1.43 ± 2.17mm (95% CI: 1.23-1.63). Receiver-operating characteristic curve analysis indicated 97.4% sensitivity and 93.9% specificity for a cut-off of 5mm and 95.8% sensitivity and 97.1% specificity for a cut-off of 6mm. CONCLUSIONS Either 5mm or 6mm could be used as the cut-off value for diagnosing ATL, as both values had high sensitivity and specificity.

Topical and Intradermal Efficacy of Photodynamic Therapy with Methylene Blue and Light-Emitting Diode in the Treatment of Cutaneous Leishmaniasis Caused by Leishmania braziliensis

INTRODUCTION The topical and intradermal photodynamic therapy (PDT) effect of methylene blue (MB) using light-emitting diode (LED) as light source (MB/LED-PDT) in the treatment of lesions of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis in hamsters were investigated. METHODS Hamsters were infected in the footpad with 4×10(7) promastigotes of L. braziliensis and divided in 4 groups: Control group was not treated, AmB group was treated with amphotericin B, MB-Id group received intradermal MB at the edge of the lesion and MB-Tp group received MB topic. After treatment with MB, the animals were illuminated using red LEDs at the 655 nm wavelength for 1 hour. The MB/LED-PDT was carried out three times a week for 12 weeks. RESULTS Animals of MB-Tp group presented lesion healing with significant diminution in extent of the lesion, and reduced parasite burden compared to control group; however, no significant difference was seen compared to the AmB group. MB-Tp group also showed reconstitution of the epithelium, the formation of collagen fibers, organization in the epidermis, a little disorganization and inflammation in the dermis. MB-Id was ineffective in all parameters evaluated, and it was comparable to the control group results. CONCLUSION These data show that PDT with the use of MB-Tp and LED may be an alternative for the treatment of ACL. However, additional studies are being conducted to assess the potential of MB/LED-PDT, alone or in combination with conventional therapy, for the treatment of ACL.

Antileishmanial and immunomodulatory effects of the essential oil from Tetradenia riparia (Hochstetter) Codd.

Cutaneous leishmaniasis usually presents therapeutic resistance to antimonials, and the existing therapies for leishmaniasis have many adverse effects and toxicity. Natural products may be regarded as possible candidates for alternative leishmaniasis treatment. The plant Tetradenia riparia has shown promise for the treatment of infectious diseases in folk medicine. We evaluated the antileishmanial activity of an essential oil from T. riparia (TrEO) and the modulatory effects of TrEO on cytokine modulation by peritoneal fluid cells that were infected with L. (L.) amazonensis. Peritoneal fluid cells were infected with Leishmania and incubated with TrEO (30 ng/mL) for 3, 6, and 24 h. Cytokines were screened using semi‐quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) and flow cytometry. Antileishmanial activity was evaluated at 24 h by microscopic counting and quantitative PCR (qPCR). TrEO treatment induced the death of 50% of Leishmania amastigotes (indicated by microscopic counting) and 91% of the parasite load (indicated by qPCR). TrEO inhibited some of the most critical cytokines for parasite growth and the establishment of infection, including granulocyte‐macrophage colony‐stimulating factor, interleukin‐4 (IL‐4), IL‐10, and tumour necrosis factor. The parasite inhibited interferon‐γ and IL‐12, and TrEO blocked this inhibition, indicating that these cytokines are critical for activating mechanisms associated with the death and elimination of the parasite. These results suggest that TrEO may be an alternative leishmaniasis therapy when considering its antileishmanial and immunomodulatory activity.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis, in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

Phototoxic effect of aluminium-chlorine and aluminium-hydroxide phthalocyanines on Leishmania (l.) amazonensis

This study investigated the activity of photosensitive phthalocyanines on promastigotes and amastigotes of Leishmania (L.) amazonensis. Aluminum phthalocyanine chloride (AlPcCl), Aluminum phthalocyanine hydroxide (AlPcOH) and zinc phthalocyanine (PcZn) were tested in the presence (matte red LED, potency of 2.5-2.3 μW for 30 min) and absence of light against L. amazonensis promastigotes and the parasite viability was evaluated after 24, 48 and 72 h. The amastigote forms were treated with AlPcCl and AlPcOH, following the same lighting protocols described for the promastigote forms, being evaluated after 24 h. Cytotoxicity to human erythrocytes and peritoneal macrophages was also evaluated. The results showed that AlPcCl and AlPcOH in the presence of light have antileishmania activity, with leishmanistatic effects on promastigotes and amastigotes of L. amazonensis, without causing cytotoxicity to peritoneal macrophages and erythrocytes. The concentrations that inhibited 50% of the promastigote forms after 24 h of light exposure were 0.21 ± 0.08 μM for AlPcCl and 0.23 ± 0.06 μM for AlPcOH. In 48 h and 72 h after the treatment, the IC50 of AlPcCl was 0.13 ± 0.02 and 0.12 ± 0.03 μM and for AlPcOH was 0.14 ± 0.01 μM and 0.11 ± 0.01 μM, respectively. PcZn showed no activity on promastigotes of L. amazonensis. This study showed a substantial photodynamic activity of the phthalocyanines AlPcCl and AlPcOH against intracellular amastigotes forms of L. amazonensis after irradiation, presenting IC50 values of 0.62 ± 0.06 μM and 0.92 ± 0.12 μM, respectively. These results support the possibility of using photodynamic therapy for the treatment of cutaneous leishmaniasis.

Cutaneous leishmaniasis with atypical clinical manifestations: Case report

This case report alerts to the existence of atypical forms of cutaneous leishmaniasis (CL). A woman with nodular cutaneous lesions over a neck with papules and pustules located deep in the hypodermis that formed plaques with subcutaneous induration and satellite papules was confirmed to have CL. After confirmation, the patient was treated with remission of the lesions, scarring and thickening of the skin.