Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Methylene blue vital staining for Trypanosoma cruzi trypomastigotes and epimastigotes

The morphological identification of Trypanosoma cruzi is currently considered to have a high specificity, but its sensitivity, which depends on the volume of the sample examined, is rather low. Trypanosome developmental stages suspended in blood, reduviid feces, and culture media are routinely searched for by means of fresh film examination (about 2 µL). High speed centrifugation of blood samples separates the buffy coat, where most trypomastigotes concentrate. As the parasites are transparent and colorless, their detection is mostly dependent on their motility. The fluorescent vital stain acridine orange has been used to enhance image contrast, as exemplified by the QBC (Quantitative Buffy Coat) technique. Staining blood, buffy coat, reduviid feces, and culture media samples with methylene blue (also a vital dye) is a means of producing sharp, well contrasted images of motile or non-motile T. cruzi developmental stages, only standard laboratory microscopes being required. Slides previously coated with a thin layer of methylene blue are used to stain fresh blood films. Photomicrographs exemplify the results of methylene blue staining applied to living and fixed parasites.

Intestinal parasitoses and environmental factors in a rural population of Argentina, 2002-2003

We evaluate the prevalence of intestinal parasites in 504 people and the degree of association between environmental variables and parasites found in population, soil and water in a rural area of Argentina during 2002-2003. A structured survey was used to evaluate the environmental variables and fecal-human, soil and water samples were analyzed. The prevalence of parasites was 45.4%. Most prevalent protozoa were Blastocystis hominis (27.2%) and Giardia lamblia (6.9%), while the most prevalent helminth was Ascaris lumbricoides (3.8%). The analyzed environmental variables showing association (p < 0.05) with presence of parasites in population were: cardboard-tin or wooden house, dirt floor, home or communal water pump, faucet outside the house or public faucet and cesspool or latrine. Parasite forms were found in 82.3% of the soil samples and in 84.2% of the water samples. In both samples we found parasites that were also found in people. In this study we have found deficient sanitary conditions associated with presence of parasites in population and we have evidenced that contaminated soil and water were the source of these parasites.

American tegumentary leishmaniasis: langerhans cells in montenegro skin test

This work analyzed the histopathology and epidermal Langerhans cells (LC) of Montenegro skin test (MST) in patients with American tegumentary leishmaniasis (ATL) in order to in situ characterize and compare the immunological reaction of the two major clinical forms of ATL, localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). MST histopathology of both LCL and MCL showed superficial and deep perivascular inflammatory infiltrate composed mainly of lymphocytes and histiocytes. Epidermal LC population was higher in MST biopsies taken from LCL patients when compared to MCL group, at 48 and 72 hours after antigen inoculation. Increased number of epidermal LC displayed in MST biopsies of LCL patients represents specific cellular immunity against parasites. The decrease of LC in MST biopsies of MCL patients does not necessarily indicate a worse specific cellular immunity in this clinical form of leishmaniasis.

Enteroparasitosis and their ethnographic relationship to food handlers in a tourist and economic center in Paraná, Southern Brazil

This study reports on the occurrence of enteroparasites based on data from an ethnographic study of food handlers in the city of Cascavel, Paraná, Brazil. Fecal material from 343 food handlers of both sexes, between 14 and 75 years of age, was analyzed using Lutz, modified Ritchie and Ziehl-Neelsen techniques. Ethnographic relationships were investigated by means of specific questionnaires. Positive fecal samples were found for 131 (38.2%) handlers. Endolimax nana (67.9%) was the predominant species, followed by Entamoeba coli (35.9%), Blastocystis hominis (28.2%), Entamoeba histolytica/dispar (10.1%) and Giardia duodenalis (8.4%). Protozoan infections were more common than helminth infections (p = 0.00). The positive results for some parasites were associated with the male sex, professional category, and the performance of other activities (p < 0.05). The high overall occurrence of enteroparasites found indicates improper hygiene and sanitation conditions. Effective educational measures should be implemented to prevent the transfer of pathogenic organisms to food via handling.

Immunoactivation and immunopathogeny during active visceral leishmaniasis

Visceral leishmaniasis is caused by protozoan parasites of the Leishmania donovani complex. During active disease in humans, high levels of IFN-γ and TNF-α detected in blood serum, and high expression of IFN-γ mRNA in samples of the lymphoid organs suggest that the immune system is highly activated. However, studies using peripheral blood mononuclear cells have found immunosuppression specific to Leishmania antigens; this poor immune response probably results from Leishmania antigen-engaged lymphocytes being trapped in the lymphoid organs. To allow the parasites to multiply, deactivating cytokines IL-10 and TGF-β may be acting on macrophages as well as anti-Leishmania antibodies that opsonize amastigotes and induce IL-10 production in macrophages. These high activation and deactivation processes are likely to occur mainly in the spleen and liver and can be confirmed through the examination of organ samples. However, an analysis of sequential data from studies of visceral leishmaniasis in hamsters suggests that factors outside of the immune system are responsible for the early inactivation of inducible nitric oxide synthase, which occurs before the expression of deactivating cytokines. In active visceral leishmaniasis, the immune system actively participates in non-lymphoid organ lesioning. While current views only consider immunocomplex deposition, macrophages, T cells, cytokines, and immunoglobulins by diverse mechanism also play important roles in the pathogenesis.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.

In vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants

Terrestrial plants have been demonstrated to be sources of antimalarial compounds. In Cuba, little is known about antimalarial potentials of plant species used as medicinals. For that reason, we evaluated the antimalarial activity of 14 plant species used in Cuba as antimalarial, antipyretic and/or antiparasitic. Hydroalcoholic extracts were prepared and tested in vitro for the antimalarial activity against Plasmodium falciparum Ghana strain and over human cell line MRC-5 to determine cytotoxicity. Parasite multiplication was determined microscopically by the direct count of Giemsa stained parasites. A colorimetric assay was used to quantify cytotoxicity. Nine extracts showed IC50 values lower than 100 µg/mL against P. falciparum, four extracts were classified as marginally active (SI < 4), one as partially active (Parthenium hysterophorus) exhibiting SI equal to 6.2 and two extracts as active (Bambusa vulgaris and Punica granatum), showing SI > 10. B. vulgaris showed the most potent and specific antiplasmodial action (IC50 = 4.7 µg/mL, SI = 28.9). Phytochemical characterization of active extracts confirmed the presence of triterpenoids in B. vulgaris and polar compounds with phenol free groups and fluorescent metabolites in both extracts as major phytocompounds, by thin layer chromatography. In conclusion, antimalarial use of B. vulgaris and P. hysterophorus was validated. B. vulgaris and P. granatum extracts were selected for follow-up because of their strong antimalarial activity.

Melanoides tuberculata (Mollusca: Thiaridae) as an intermediate host of Centrocestus formosanus (Trematoda: Heterophyidae) in Brazil

Pleurolophocercous cercariae emerged from naturally infected Melanoides tuberculata from Minas Gerais State, Brazil, were used to perform experimental infection of laboratory-reared Poecilia reticulata. Mature metacercariae were obtained from the gills of fishes and force-fed to Mus musculus. The adult parasites which recovered from small intestines of mice were identified as Centrocestus formosanus. This is the first report of M. tuberculata as intermediate host of this heterophyid in Brazil.

Melanoides tuberculata as intermediate host of Philophthalmus gralli in Brazil

Melanoides tuberculata that naturally harbored trematode larvae were collected at the Pampulha dam, Belo Horizonte (Minas Gerais, Brazil), during malacological surveys conducted from 2006 to 2010. From 7,164 specimens of M. tuberculata collected, 25 (0.35%) were infected by cercariae, which have been morphologically characterized as belonging to the Megalurous group, genus Philophthalmus. Excysted metacercariae were used for successful experimental infection of Gallus gallus domesticus, and adult parasites recovered from the nictitating membranes of chickens were identified as Philophthalmus gralli. This is the first report of P. gralli in M. tuberculata in Brazil.

Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ß-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ß-hematin synthesis (IßHS) and hemoglobin proteolysis (IHbP) in vitro. The ß-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5%, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 µM and 14.4 ± 1.4 µM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0% and 55.3 ± 3.6%, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia (%P) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0% compared to 26.6 ± 3.7%, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.

Microbiological quality of drinking rainwater in the inland region of Pajeú, Pernambuco, Northeast Brazil

Despite all efforts to store and reduce its consumption, water is becoming less inexhaustible and its quality is falling faster. Considering that water is essential to animal life, it is necessary to adopt measures to ensure its sanitary conditions in order to be fit for consumption. The aim of this study was to analyze the microbiological quality of drinking rainwater used by rural communities of Tuparetama, a small town located in Northeast Brazil. The study covered seven rural communities, totaling 66 households. In each household two samples were collected, one from a tank and the other from a clay pot located inside the home, resulting in 132 samples (tank plus clay pot). Approximately 90% of samples were below the standard recommended by the current legislation, being considered unfit for human consumption. Part of this high microbiological contamination of drinking rainwater could be related to the lack of sanitary education and of an adequate sewerage sanitation system.

Occurrence of Ancylostoma in dogs, cats and public places from Andradina city, São Paulo state, Brazil

The aim of this study was to determine the frequency and intensity of Ancylostoma spp. in 33 dogs and 52 cats by means of coproparasitological examinations and parasitological necropsy, and assess the presence of contaminated feces with eggs of that parasite in public places of Andradina Municipality, São Paulo State, Brazil. Willis-Mollay and Sedimentation methods indicated Ancylostoma spp. eggs in 87.8% (29/33) dogs and 94.2% (49/52) cats. The species A. caninum and A. braziliense were found in 63.6% (21/33) and 30.3% (10/33) of dogs, respectively. Considering cats, 67.3% (35/52) were parasitized by A. braziliense, 21.1% (11/52) by A. caninum, and 9.6% (5/52) by A. tubaeforme. Forty-two canine fecal samples were collected from public environments, including 23 squares/gardens and 19 streets/sidewalks. Positive samples for Ancylostoma spp. accounted for 64.3% (27/42); squares/gardens had 60.9% (14/23) positive samples, and streets and sidewalks, 68.4% (13/19). No association was observed between the number of Ancylostoma spp parasites and age, sex and breed of the animals and also the ratio of EPG counts and the parasitic intensity observed at necropsy (p > 0.05). Based on the high occurrence of hookworm in dogs and cats in this study, the treatment with anti helminthics are needed even in those animals with negative stool tests, besides adopting control of the number of animals in public places, in order to decrease the likelihood of environmental contamination, since this parasite represents a potential hazard to human and animal health.