Synthesis of calcium phosphate nanostructures by combustion in solution as a potential encapsulant system of drugs with photodynamic properties for the treatment of cutaneous leishmaniasis.

The traditional drugs used in the treatment of cutaneous leishmanisis (CL) have multiple disadvantages, such as high toxicity, high costs, and more recently the appearance of parasites resistant to those drugs. For this reason, some research has focused on the development of new drugs or treatment therapies. Photodynamic therapy (PDT) that involves the use of a photosensitive or photosensitizing compound capable of producing reactive oxygen species to which Leishmania parasites are sensitive, has emerged as an alternative for the treatment of CL. However, some of these sensitizing compounds exhibit some toxicity (cytotoxicity, allergic reaction, etc), low selectivity, and some of them are insoluble in aqueous media limiting their use. Therefore, the PDT can be improved using encapsulation systems which protect drugs, prevent their degradation, help them overcome physical barriers and increase their selectivity. In this study, we propose the use of calcium phosphate as a potential encapsulant or photodynamic support for photoactive drugs, using hypericin (HY) as a photosensitizer agent. The self-combustion route was used to synthesize the CP nanostructures. The structure and morphology of CP nanoparticles were evaluated via X-ray diffraction (XRD), Raman and field-emission scanning electron microscopy (FE-SEM). Phases rich in hydroxyapatite and CP ß phase, with granular morphology and an average grain size of 42.9 nm were obtained. The encapsulation uptake and the interactions between HY and the encapsulated system were evaluated by fluorescence spectroscopy and Fourier-transform infrared spectroscopy (FTIR), respectively. Approximately 13% of HY was enapsulated per 1 µg of nanoparticles of calcium phosphate. Composites were submitted to in vitro assays of cytotoxicity and anti-leishmanial activity. The CP nanoparticles did not affect the photodynamic activity of HY. On the contrary they showed antileishmanial response.

Diagnosis of cutaneous leishmaniasis in Colombia: the sampling site within lesions influences the sensitivity of parasitologic diagnosis.

Parasitologic confirmation of cutaneous leishmaniasis is obligatory before chemotherapy can be considered. Direct microscopic examination of scrapings taken from indurated borders of ulcers has been routinely used as primary method of diagnosis. In this report we compared the sensitivity of examination of dermal scrapings taken from the bottoms of ulcers (BDS) with that of dermal scrapings taken from indurated active margins of lesions (MDS) in a total of 115 patients. The sensitivities of the microscopic examination were 90.4 and 78.3% for BDS and MDS samples, respectively. When the PCR method was used with a group of 40 patients, we also observed a higher sensitivity when BDS samples were examined (80.8% in BDS samples versus 57.7% in MDS samples). The improvement of the diagnostic sensitivity in the BDS samples appears to be related to the higher parasite load and more easily detectable morphology of amastigotes in the centers of the ulcers. Other parasitologic diagnostic methods, such as culture and histopathologic examination of biopsies, are less sensitive (67.5 and 64.3%, respectively). Aspirate culture, however, was shown to be the most sensitive method for the diagnosis of patients with chronic ulcers. When microscopic examinations of both MDS and BDS samples are combined, the sensitivity of diagnosis may rise up to 94%. We therefore recommend this method as a primary routine procedure for diagnosis of cutaneous leishmaniasis.

Enzymatic polymorphism and phylogenetic relationships in Leishmania Ross, 1903 (Sarcomastigophora: Kinetoplastida): a case study in Colombia.

Leishmaniasis is widespread in Colombia and is found in 30 of 32 Departments. More than 200 infection zones have been reported from different regions, which vary from sea-level to an altitude of 2,300 m along the Atlantic Coast, Pacific coast, Amazon basin, Cauca and Magdalena valleys. We report 76 Leishmania stocks isolated from humans, dogs and phlebotomine hosts. Isoenzyme electrophoresis revealed 16 zymodemes, which could be divided into four phylogenetic complexes, i.e., L. braziliensis, L. amazonensis, L. guyanensis/panamensis and L. infantum. Three zymodemes became integrated into the subgenus Leishmania and the other zymodemes into the subgenus Viannia. Cutaneous infections were due to the L. braziliensis (9.2%) and L. guyanensis/panamensis (85.54%) complexes. Mucous secondary involvement was due to the L. braziliensis and L. guyanensis/panamensis complexes. In this work the specific status of L. (V.) guyanensis and L. (V.) panamensis is discussed.

Human antibody response to a 56-kDa purified excretory/secretory product of Dirofilaria immitis.

Canine dirofilariasis is widespread in urban areas of central and northern Colombia. Previously we detected specific antibodies against complex antigens from Dirofilaria immitis adult worms in individuals from an isolated Tikuna Indian community in the Colombian Amazon. In this study a 56 kDa polypeptide from the adult D. immitis excretory/secretory (E/S) products is identified by Western blot, isolated by elution from polyacrilamide gels and applied in an ELISA-based test for the detection of specific IgG. Eleven of 74 serum samples analysed were positive by ELISADi56. Positive individuals came from five different areas of Colombia. The highest number of positives was found in the Amazon (4), followed by Bogotà (3). The physicians of the area must be alerted regarding the existence of human D. immitis infections and include dirofilariasis in the differential diagnosis of pulmonary nodules.

The humoral immune response to the kinetoplastid membrane protein-11 in patients with American leishmaniasis and Chagas disease: prevalence of IgG subclasses and mapping of epitopes.

The kinetoplastid membrane protein-11 (KMP-11) is a major target of the humoral immune response during Leishmania-infections. The majority of sera from visceral leishmaniasis, mucocutaneous leishmaniasis and even some cutaneous leishmaniasis patients contain detectable IgG antibodies against KMP-11. We also provide evidence that this protein may act as a potent antigen in T. cruzi infections, since most Chagas sera show immunological cross-reactivity. Therefore, KMP-11 cannot be used as a specific diagnostical tool for the serodiagnosis of leishmaniasis in those regions where both, Leishmania and T. cruzi infections overlap geographically. When analyzing the subclass specificity of the antibody response to KMP-11 we observed the following order of reactivity: IgG1 > > IgG3 > IgG2 > IgG4, which is similiar to that seen in crude parasite extract. The mapping of antigenic determinants by using synthetic 20-mer peptides revealed the existence of predominantly conformational epitopes in leishmaniasis, while 50% of sera from Chagas patients reacted with a particular KMP-11 peptide. These results therefore suggest the presence of disease-specific B-cell epitopes.

10-Oximeguanacone, the first nitrogenated acetogenin derivative found to be a potent inhibitor of mitochondrial complex I.

A new 10-keto bis-tetrahydrofuran acetogenin, guanacone (1), has been isolated from a cytotoxic extract of Annona aff. spraguei seeds. The 10-oximeguanacone derivative 1f is the first bioactive nitrogenated acetogenin found to be a very potent inhibitor of complex I. In addition, a SAR study of guanacone analogues is reported based on the titration of the NADH oxidase and NADH:ubiquinone oxidoreductase activities.

Visceral leishmaniasis in HIV-1-infected individuals: a common opportunistic infection in Spain?

OBJECTIVE: To investigate the epidemiological, clinical and biological features of visceral leishmaniasis (VL) in patients with HIV-1 infection. DESIGN: Retrospective study. SETTING: Three university hospitals in southern Spain. PATIENTS: Forty-seven adult patients with VL and HIV-1 infection diagnosed between January 1986 and November 1991. RESULTS: Forty-five out of the 47 (96%) cases were diagnosed in the last 2 years. Fever (87%), hepatomegaly (74%), splenomegaly (72%) and pancytopenia (77%) were the most common presenting features. Most patients (79%) were strongly immunocompromised when VL was diagnosed, and were in stage IV of the Centers for Disease Control classification; 87% had a CD4 lymphocyte count < 200 x 10(6)/l. However, VL was the first severe infection diagnosed in 10 cases. Significant titres (> 1:40) of antileishmanial antibodies were detected by indirect immunofluorescence in five out of 16 (31%) cases only. Clinical response to the therapy was difficult to assess. Microbiological response was achieved in only 38% of the patients evaluated. CONCLUSIONS: Leishmaniasis is a relatively common infection in HIV-1-infected individuals in southern Spain. Its clinical picture is quite uniform and it can be the first opportunistic infection in individuals with HIV-1. In endemic areas, a high index of clinical suspicion should be maintained in order to avoid underdiagnosis of leishmaniasis.

PIP: Physicians examined the records of 47 adults with visceral leishmaniasis (VL) and HIV-1 infection who were patients at 3 urban teaching hospitals in the Andalucia region in southern Spain between January 1986 and November 1991. They wanted to identify the clinical, biological, and epidemiological features of VL in HIV-1 positive patients. 96% of the cases were diagnosed with both infections during the last 2 years of the study period and 79% between January and November 1991. All the patients had risk factors for HIV infection (65.9% IV drug use, 21.3% sexual contact, and 12.8% blood transfusion). 70% exhibited the classic symptoms of VL (fever, enlarged liver and spleen, and depressed counts of blood cells). Most patients were already very immunocompromised when VL was diagnosed. 87% had a total lymphocyte count of less than 1000 x 1 million/1 and a CD4 lymphocyte count of less than 200 x 1 million/1. In fact, 66% had full blown AIDS prior to diagnosis of VL. VL was the first severe infection in 10 cases. 68% also suffered from opportunistic infections, especially candidiasis, extrapulmonary tuberculosis, and Pneumocystis carinii pneumonia. Microscopic examination of Leishmania amastiogotes in tissue samples led to a diagnosis in 94% of cases, isolation of motile amastigotes in culture of bone marrow aspirate in 2%, and microscopic and culture in 4%. Just 46% completed a full course of treatment (pentavalent antimony, allopurinol, and/or pentamidine). Only 38% had a microbiological response. Immunofluorescence detected sizeable titers (1:40) of antileishmanial antibodies in just 31% of cases. 17% experienced clear clinical improvement. Physicians in endemic areas should consider VL in every HIV-1 infected patient with fever, hepatosplenomegaly, or hematological abnormalities to avoid underdiagnosis of leishmaniasis.