Use of Monoclonal Antibodies for the Identification of Leishmania spp. Isolated from Humans and Wild Rodents in the State of Campeche, Mexico

The genus Leishmania includes 30 described species which infect a wide variety of mammalian hosts. The precise identification of leishmanial parasites at the species level is very important in order to determine whether an organism, causing the disease in a given area, is of the same biotype as that found in suspected mammalian reservoirs. The objectives of the present study were (1) to identify leishmanial parasites isolated from humans and wild rodents from the State of Campeche, an endemic focus of localized cutaneous leishmaniasis (LCL) in southern Mexico, using an indirect immunofluorescent assay (IFA) with monoclonal antibodies (Mabs); and (2) to determine if the parasites of the two types of hosts were of the same biotype. All the wild rodents (six Ototylomys phyllotis, eight Oryzomys melanotis, five Peromyscus yucatanicus and two Sigmodon hispidus) and 96% (24/25) of the human isolates were identified as Leishmania (L.) mexicana confirming that this specific LCL focus is a wild zoonosis. The presence of one human isolate of L. (Viannia) braziliensis in the State of Campeche, confirmed the importance of an accurate taxonomic identification at species level.

In vitro activity of Brazilian strains of the predatory fungi Arthrobotrys spp. on free-living nematodes and infective larvae of Haemonchus placei

In vitro tests were carried out to assess the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Haemonchus placei, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to H. placei, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.

Simulium spp. control program in Rio Grande do Sul, Brazil

Insects of the Simuliidae family have been the object of control in Rio Grande do Sul since the 70s. Their constant attacks became a social-economical problem as well as a problem of Public Health, with serious consequences to men and to the economy of the areas in which the insects develop. At first, the control was done with a chemical larvicide Themephos ABATE 500 E, but an imperfect measuring of outflow to determine the quantity of the product made Simulium spp. resistant to it. From 1983 on, following a study of a new method for the outflow measuring, we started to use a biological larvicide Bacillus thuringiensis serovar israelensis based. The biological control uses the new method in 36.4% of the state area, assisting about 3,500,000 inhabitants.

Anesthesia of Biomphalaria spp. (Mollusca, Gastropoda): sodium pentobarbital is the drug of choice

The anesthetic effect of some water-soluble anesthesic or narcotic drugs currently used in mice was tested in molluscs of the Biomphalaria genus. Sodium thiopental was very toxic to the snails resulting in high rates of mortality in all the treatment schedules tested. Cetamine base, at concentration of 0.25 mg/ml of water, resulted in partial snail anesthesia (40% of snails were anesthetized) only after 20 h of exposition. The association of Cetamine base with Tiazine chloridrate did not improve the anesthesic effect, and higher concentrations of these drugs were toxic to the snails. Sodium pentobarbital at 0.4 mg/ml in water for 8 h was the best treatment schedule to anesthetize Biomphalaria snails. In this schedule, the snails were anesthetized without any toxic effect. The procedure provides a powerful tool for in vivo studies that demande a complete state of snail anesthesia.

In vitro interaction of Brazilian strains of the Nematode-trapping fungi Arthrobotrys spp. on Panagrellus sp. and Cooperia punctata

In vitro tests were carried out to verify the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Cooperia punctata, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to C. punctata, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.

The distribution of Biomphalaria spp. in different habitats in relation to physical, biological, water contact and cognitive factors in a rural area in Minas Gerais, Brazil

A total of 256 sites in 11 habitats were surveyed for Biomphalaria in Melquiades rural area (State of Minas Gerais) in August and November 1999 and in March 2000. Of the 1,780 Biomphalaria collected, 1,721 (96.7%) were B. glabrata and 59 (3.3%) B. straminea. Snails were found in all habitats except in wells, with the largest mean numbers in tanks, seepage ponds and canals, and the smallest numbers in springs, rice fields and fishponds. People's knowledge of the occurrence of Biomphalaria at the collection sites and the presence of Biomphalaria ova were strongly correlated with the occurrence of snails, and distance between houses and collection sites, as well as water velocity were inversely correlated with Biomphalaria occurrence (p < 0.001). The strongest predictor o f Biomphalaria occurrence was the presence of tilapia fish in fishponds. Fourteen Biomphalaria (0.8% of all snails) found at 6 sites were infected with Schistosoma mansoni. Suggestions are made for the utilization of local people's knowledge in snail surveys and further studies are recommended on the possible use of tilapia for biological control of Biomphalaria in fishponds, as well as modeling of S. mansoni transmission and reinfection.

Cotton Rats (Sigmodon hispidus) and Black Rats (Rattus rattus) as Possible Reservoirs of Leishmania spp. in Lara State, Venezuela

A total of 519 wild animals belonging to eleven species were collected during a two year study in a cutaneous leishmaniasis endemic area in Venezuela (La Matica, Lara State). The animals were captured in home-made Tomahawk-like traps baited with maize, bananas or other available local fruits, and parasites were isolated from 27 specimens. Two different species were found naturally infected with flagellates, i.e., cotton rats (Sigmodon hispidus) and black rats (Rattus rattus). Characterization of the parasites using PCR, kDNA restriction pattern and hybridization with species-specific probes revealed the presence of Leishmania (L.) mexicana in three of the black rats and Leishmania (V.) braziliensis in two others. The latter species was also identified in the single positive specimen of S. hispidus. The results suggested both species of animals as possible reservoirs of Leishmania sp.

Morphologic and morphometric analysis of Hepatozoon spp. (Apicomplexa, Hepatozoidae) of snakes

Hepatozoon species are the most abundant hemoparasites of snakes. Its identification has been based mainly on the morphologic characterization of the gamonts in the peripheral blood of the vertebrate host and also of the cysts found in the internal organs of the vertebrate and invertebrate hosts. Using a computerized image analysis system, we studied five species of Hepatozoon from recently captured snakes in Botucatu, State of São Paulo, Brazil, to evaluate the importance of the morphology and morphometry of the gamonts for the characterization of Hepatozoon species and to analyze the morphologic changes induced in the erythrocytes by the parasite. The studied species were H. terzii of Boa constrictor amarali, Hepatozoon sp. of Crotalus durissusterrificus, H. philodryasi of Philodryas patagoniensis, and H. migonei and H. cyclagrasi of Hydrodynastes gigas. We observed three different groups, one of them including the species H. terzii, H. philodryasi and Hepatozoon sp. of C. durissus terrificus; and the other two consisting of H. migonei and H. cyclagrasi. Degree of alterations in the erythrocytes was variable and it may be useful for characterization of Hepatozoon species.

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Use of polymerase chain reaction and enzymatic cleavage in the identification of Helicobacter spp. in gastric mucosa of human beings from North Paraná, Brazil

Helicobacter pylori is the most common gastric bacteria of human beings. Animal-borne helicobacter have been associated with gastritis, ulceration, and gastric mucosa-associated lymphoid-tissue lymphoma in people. We attempted to identify the species of Helicobacter spp. that infect human beings in north Paraná, Brazil. Samples of gastric mucosa from 38 dyspeptic patients were analyzed by optic microscopy on silver stained slides, polimerase chain reaction (PCR), and enzymatic cleavage. Genus and species-specific primers to H. pylori, H. heilmannii, H. felis, and consensual primers to H. bizzozeronii or H. salomonis were used. The PCR products were submitted to enzymatic cleavage by VspI (Helicobacter spp. product) and HinfI (species products) enzymes. Thirty-two out of 38 patients evaluated had 3.2 to 5 µm long bacteria that resembled H. pylori in Warthin-Starry stained slides and were positive to the genus Helicobacter by PCR. In 30 of these patients the bacteria were identified as H. pylori. Two samples positive by silver stain were negative to all species tested by PCR. None of the 38 samples was positive to animal-origin helicobacter species. These results show that PCR and enzymatic restriction are practical methods to identify the species of helicobacters present in gastric mucosa of human beings. People in north Paraná appear to be infected mostly with H. pylori.

Species distribution and antifungal susceptibility profile of Candida spp. bloodstream isolates from Latin American hospitals

From March 1999 to March 2000, we conducted a prospective multicenter study of candidemia involving five tertiary care hospitals from four countries in Latin America. Yeast isolates were identified by classical methods and the antifungal susceptibility profile was determined according to the National Committee for Clinical Laboratory Standards microbroth assay method. During a 12 month-period we were able to collect a total of 103 bloodstream isolates of Candida spp. C. albicans was the most frequently isolated species accounting for 42% of all isolates. Non-albicans Candida species strains accounted for 58% of all episodes of candidemia and were mostly represented by C. tropicalis (24.2%) and C. parapsilosis (21.3%). It is noteworthy that we were able to identify two cases of C. lusitaniae from different institutions. In our casuistic, non-albicans Candida species isolates related to candidemic episodes were susceptible to fluconazole. Continuously surveillance programs are needed in order to identify possible changes in the species distribution and antifungal susceptibility patterns of yeasts that may occurs after increasing the use of azoles in Latin American hospitals.

Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in São Paulo city, Brazil

Species of Clostridium are widely distributed in the environment, inhabiting both human and animal gastrointestinal tracts. Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders, such as diarrhea. In this study, the prevalence of Clostridium spp. and C. difficile, from hospitalized children with acute diarrhea, was examined. These children were admitted to 3 different hospitals for over 12 months. Eighteen (20%) and 19 (21%) stool specimens from children with (90) and without (91) diarrhea respectively, were positive to clostridia. Only 10 C. difficile strains were detected in 5.5% of the stool samples of children with diarrhea. None healthy children (without diarrhea) harbored C. difficile. From these 10 C. difficile, 9 were considered as toxigenic and genotyped as tcdA+/tcdB+ or tcdA-/tcdB+, and 1 strain as nontoxigenic (tcdA-/tdcB-). They were detected by the citotoxicity on VERO cells and by the multiplex-polymerase chain reaction. Thirty clinical fecal extracts produced minor alterations on VERO cells. The presence of C. difficile as a probable agent of acute diarrhea is suggested in several countries, but in this study, the presence of these organisms was not significant. More studies will be necessary to evaluate the role of clostridia or C. difficile in diarrhoeal processes in children.

Infection rate of Babesia spp. sporokinetes in engorged Boophilus microplus from an area of enzootic stability in the State of Minas Gerais, Brazil

The infection rates of Babesia sporokinetes in engorged Boophilus microplus were evaluated during a 2-year period in a dairy farm located in an area of enzootic stability. Every 14 days engorged females were collected from calves and from adult animals. Ticks were incubated at 27 ± 0.5ºC and 80-90% relative humidity and Babesia infection rates were determined by microscopic examination of Giemsa-stained hemolymph smears. After 52 collections, 2105 ticks were obtained, from which 982 were collected from calves and 1123 from cows. The total Babesia infection rate was 10%, however the incidence was higher (p < 0.05) in ticks collected from calves (17.5%) than in those collected from cows (3.6%). Females collected from cows showed the highest infection rates in January, March, and August, and absence of infection in April and May. Ticks feeding on calves were infected throughout the experimental period. The infection rates of engorged females collected from naturally infected calves that were artificially infested with Babesia-free-larvae of B. microplus gradually decreased until the calves were four months old. No differences were observed among infection rates of ticks collected from calves maintained under natural conditions.

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.