Evaluation of DNA Recombinant Methodologies for the Diagnosis of Plasmodium falciparum and their Comparison with the Microscopy Assay

Since 1984, DNA tests based on the highly repeated subtelomeric sequences of Plasmodium falciparum (rep 20) have been frequently used in malaria diagnosis. Rep 20 is very specific for this parasite, and is made of 21 bp units, organized in repeated blocks with direct and inverted orientation. Based in this particular organization, we selected a unique consensus oligonucleotide (pf-21) to drive a PCR reaction coupled to hybridization to non-radioactive labeled probes. The pf-21 unique oligo PCR (pf-21-I) assay produced DNA amplification fingerprints when was applied on purified P. falciparum DNA samples (Brazil and Colombia), as well as in patient's blood samples from a large area of Venezuela. The performance of the Pf-21-I assay was compared against Giemsa stained thick blood smears from samples collected at a malaria endemic area of the Bolívar State, Venezuela, at the field station of Malariología in Tumeremo. Coupled to non-radioactive hybridization the pf-21-I performed better than the traditional microscopic method with a r=1.7:1. In the case of mixed infections the r value of P. falciparum detection increased to 2.5:1. The increased diagnostic sensitivity of the test produced with this homologous oligonucleotide could provide an alternative to the epidemiological diagnosis of P. falciparum being currently used in Venezuela endemic areas, where low parasitemia levels and asymptomatic malaria are frequent. In addition, the DNA fingerprint could be tested in molecular population studies

On a New Species of Hysterothylacium (Nematoda: Anisakidae) from Cauque mauleanum (Pisces: Atherinidae) by Brightfield and Scanning Electron Microscopy

Hysterothylacium geschei n. sp. (Nematoda, Anisakidae) is described from the intestine of Cauque mauleanum (Steindachner) (Pisces: Atherinidae) from Lake Panguipulli (39º43'S; 72º13'W), Chile. Eleven (78.6%) out of 14 fish were infected, with a mean intensity (range) of 14.4 (1-55) worms. The new species can be differentiated from the two previously described species of freshwater fishes from South America by the presence of lateral alae, the number of caudal papillae, and the length of the spicules, oesophagus, intestinal caecum, distance vulva-anterior extremity and the length ratio intestinal caecum: ventricular appendix. From the fishes examined in Lake Panguipulli, including the introduced salmonid species Oncorhynchus mykiss (Walbaum) and the authochthonous species Basilichthys australis Eigenmann (Atherinidae) and Percichthys trucha (Valenciennes) (Percichthyidae), only one specimen of P. trucha was found parasitized by a third-stage larva of this species.

Scolex Morphology of Proteocephalid Cestodes Parasites of Neotropical Freshwater Fishes

The morphology of the scolices and metascolices of 29 proteocephalid species, parasites of freshwater fishes from Brazil was compared by using scanning electron microscope, light and laser microscopy. The following species were evaluated: Proteocephalus vazzolerae, P. piramutab, Robertiella paranaensis, Travassiella avitellina, Monticellia loyolai, M. spinulifera, M. belavistensis, Houssayela sudobim, Zygobothrium megacephalum, Gibsoniela mandube, Cangatiella arandasi, Nomimoscolex sudobim, N. lopesi, N. admonticellia, N. piraeeba, N. pirarara, Harriscolex kaparari, Crepidobothrium eirasi, Spatulifer rugosa, Megathylacus brooksi, Choanoscolex abscisus, Amphoteromorphus peniculus, A. piraeeba, Paramonticellia itaipuensis, Peltidocotyle rugosa, Othinoscolex lenha, Rudolphiella rugata, R. piranabu, Jauella glandicephalus. Some features as overall shape of scolex, suckers, apical sucker, frontal glands and several kinds of metascolex were analyzed. The taxonomic importance of scolex and metascolex is discussed.

Human chronic chagasic cardiopathy: participation of parasite antigens, subsets of lymphocytes, cytokines and microvascular abnormalities

This article tries to demonstrate by new pathological findings (with the use of immunohistochemical technique and confocal laser microscopy) that chronic chagasic cardiomyopathy is a result of multiple factors involving myocarditis, immunodepression, severe fibrosis and microvessels dilatation and that all of these alterations are probably directly related with the presence of Trypanosoma cruzi parasites in the host associated with inadequate immunological response of the host.

Posterior spiracles of fourth instar larvae of four species of phlebotomine sand flies (Diptera: Psychodidae) under scanning electron microscopy

In the present study, posterior spiracles of laboratory-reared fourth instar larvae of Lutzomyia longipalpis, L. migonei, L. lenti, and L. whitmani (Diptera: Psychodidae) of the State of Ceará, Brazil, were examined under scanning electron microscopy. The number of papillae of spiracles examined varied according to the species examined, but no intraspecific differences were found. The importance of this structure to sand fly larva identification and phylogeny is commented.

Scanning and transmission electron microscopy of the tegument of Paranaella luquei Kohn, Baptista-Farias & Cohen, 2000 (Microcotylidae, Monogenea), parasite of a Brazilian catfish, Hypostomus regani

The surface topography and ultrastructure of the tegument of Paranaella luquei Kohn, Baptista-Farias & Cohen, 2000, a microcotylid monogenean parasite from the gills of Hypostomus regani (Ihering, 1905) (Loricariidae) was studied by scanning (SEM) and transmission electron microscopy (TEM). By SEM, it was observed that the tegument presents transversal ridges, forming folds in the ventral and dorsal surfaces and microvillous-like tegumental projections in the anterior and median regions of body. These projections were also observed by TEM. The tegument is made up of a syncytium delimited by apical and basal plasma membranes, containing inclusion bodies and mitochondria, connected to the nucleated region by means of cytoplasmatic processes. The tegumental cells present a well developed nucleus and cytoplasm containing inclusion bodies, similar to those found on the external layer, mitochondria, rough endoplasmatic reticulum and free ribossomes.

External morphology of sensory structures of fourth instar larvae of neotropical species of phlebotomine sand flies (Diptera: Psychodidae) under scanning electron microscopy

In the present study, some morphological structures of antennae, maxillary palps and caudal setae of fourth instar larvae of laboratory-reared phlebotomine sand flies (Lutzomyia longipalpis, L. migonei, L. evandroi, L. lenti, L. sericea, L. whitmani and L. intermedia) of the State of Ceará, Brazil, were examined under scanning electron microscopy. The antennal structures exhibited considerable variation in the morphology and position. A prominent digitiform distal segment has been observed only on the antenna of species of the subgenus Nyssomyia. The taxonomic relevance of this and other antennal structure is discussed. The papiliform structures found in the maxillae and the porous structures of the caudal setae of all species examined may have chemosensory function. Further studies with transmission electron microscopy are needed to better understand the physiological function of these external structures.

Ultrastructure of Proechinophthirus zumpti (Anoplura, Echinophthiriidae) by scanning electron microscopy

The ultrastructure of Proechinophthirus zumpti Werneck, 1955, mainly the external chorionic features of the egg, is described through electronic microscopy techniques. This species was first cited in Argentina, infesting Arctocephalus australis (Zimmermann, 1873). The morphological adaptations of adults and nymphs are described in both species of Proechinophthirus parasitic on Otariidae: P. fluctus (Ferris, 1916) and P. zumpti.

Intestinal fibrovascular nodules caused by Schistosoma mansoni infection in Calomys callosus Rengger, 1830 (Rodentia: Cricetidae): a model of concomitant fibrosis and angiogenesis

Human schistosomiasis develops extensive and dense fibrosis in portal space, together with congested new blood vessels. This study demonstrates that Calomys callosus infected with Schistosoma mansoni also develops fibrovascular lesions, which are found in intestinal subserosa. Animals were percutaneously infected with 70 cercariae and necropsied at 42, 45, 55, 80, 90 and 160 days after infection. Intestinal sections were stained for brightfield, polarization microscopy, confocal laser scanning, transmission and scanning electron microscopies. Immunohistological analysis was also performed and some nodules were aseptically collected for cell culture. Numerous intestinal nodules, appearing from 55 up to 160 days after infection, were localized at the interface between external muscular layer and intestinal serosa, consisting of fibrovascular tissue forming a shell about central granuloma(s). Intranodular new vessels were derived from the vasculature of the external vascular layer and were positive for laminin, chondroitin-sulfate, smooth muscle alpha-actin and FVIII-RA. Fibroblastic cells and extracellular matrix components (collagens I, III and VI, fibronectin and tenascin) comprised the stroma. Intermixed with the fibroblasts and vessels there were variable number of eosinophils, macrophages and haemorrhagic foci. In conclusion, the nodules constitute an excellent and accessible model to study fibrogenesis and angiogenesis, dependent on S. mansoni eggs. The fibrogenic activity is fibroblastic and not myofibroblastic-dependent. The angiogenesis is so prominent that causes haemorrhagic ascites.

Comparative study of the stridulatory sulcus, buccula and rostrum of the nymphs of Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998 by scanning electron microscopy (Hemiptera, Reduviidae)

The ultrastructural morphology of the ventral region of the head (rostrum and buccula) and proesternum (stridulatory sulcus) of nymphs from the 1st to 5th instars of Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998 was described. Morphological differences between the two species and of the five nymphal stages development of each species were observed. These structures showed systematic differential characteristics of the studied species and may be used to increase their taxonomic range.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Morphobiological aspects of Rhodnius brethesi Matta, 1919 (Hemiptera:Reduviidae) from the Upper and Middle Negro River, Amazon region of Brazil: I - scanning electron microscopy

The occurrence of autochthonous cases of Chagas disease in the Amazon region of Brazil over recent decades has motivated an intensification of studies in this area. Different species of triatomines have been identified, and ten of these have be proven to be carriers of the parasite Trypanosoma cruzi or " cruzi-like " parasites. Studies conducted in the municipalities of Santa Isabel do Rio Negro and Barcelos, located on the Upper and Middle of the Negro River, microregion of Negro River, state of Amazonas have confirmed not only that Rhodnius brethesi is present in the palm tree Leopoldinia piassaba, but also that this insect was recognized by palm fiber collectors. A morphological study of eyes, inter-ocular and inter-ocellar regions, antennae, buccula, labrum, rostrum, stridulatory sulcus and feet, including the apex of the tibia, spongy fossette and ctenidium was conducted by scanning electron microscopy. The buccula and the stridulatory sulcus presented notable differences in specimens of different genera and also of different species. These data make it possible to suggest that the details presented in these structures can be included as diagnostic characteristics to be used in new dichotomous keys, thereby contributing towards studies of taxonomy and systematics and furnishing backing for comparative analysis of specimens collected from different localities.

Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy

The blood flukes of mammals (Digenea: Schistosomatidae) are among trematodes unique whose adult worms have separeted sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59%) had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33% females had an incipient ovary (without cellular differentiation). At week four, 77.2% males presented testicular lobes with few germinative cells while 26% had developing tegumental tubercles. The immature ovary was observed in 69% females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold) more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.

A study of the scutellum in eight Chagas disease vector species from genus Triatoma (Hemiptera, Reduviidae) using optical and scanning electron microscopy

The aim of this study was to analyze the external morphology of the scutellum through optical microscopy and scanning electron microscopy (SEM) in male specimens of Triatoma costalimai, T. delpontei, T. eratyrusiformis, T. matogrossensis, T. infestans melanosoma, T. sherlocki, T. tibiamaculata, and T. vandae. A total of 30 photographs of the scutellum were made. Magnification varied from 50X to 750X. Regarding depth and forms of the central depression, the heart-shaped form was predominant, with some exceptions, so that this shape appears to be a common characteristic for species of genus Triatoma Laporte, 1832. In T. eratyrusiformis, a kind of sensillum with important taxonomic value was observed. The different sizes and shapes of the designs found on the posterior process of the scutellum were also of important taxonomic interest. The study of the scutellum based on SEM showed valuable characteristics, allowing the use of this structure to aid the diagnosis of triatomine species. Thus, more specimens in subsequent studies and analyses of morphometric parameters should contribute to agreement on phylogenetic aspects in this genus. A Key to eight species of Triatoma based on male scutellar morphology is presented.

Electron microscopy of trypanosomes: a historical view

Since the discovery of the electron microscope and the development of the initial techniques for the processing of biological samples for electron microscopy, the protozoan Trypanosoma cruzi has been the subject of intense investigation. This review analyzes the results obtained by observation of whole trypanosomes as well as thin sections and replicas using several microscopic approaches. Micrographs detailing the appearance of T. cruzi using several methods illustrate the evolution of electron microscopic techniques as well as its contribution to understanding the structural organization of the protozoan.