An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum.

We propose a taxonomic revision of the dixenous trypanosomatids currently classified as Endotrypanum and Leishmania, including parasites that do not fall within the subgenera L. (Leishmania) and L. (Viannia) related to human leishmaniasis or L. (Sauroleishmania) formed by leishmanias of lizards: L. colombiensis, L. equatorensis, L. herreri, L. hertigi, L. deanei, L. enriettii and L. martiniquensis. The comparison of these species with newly characterized isolates from sloths, porcupines and phlebotomines from central and South America unveiled new genera and subgenera supported by past (RNA PolII gene) and present (V7V8 SSU rRNA, Hsp70 and gGAPDH) phylogenetic analyses of the organisms. The genus Endotrypanum is restricted to Central and South America, comprising isolates from sloths and transmitted by phlebotomines that sporadically infect humans. This genus is the closest to the new genus Porcisia proposed to accommodate the Neotropical porcupine parasites originally described as L. hertigi and L. deanei. A new subgenus Leishmania (Mundinia) is created for the L. enriettii complex that includes L. martiniquensis. The new genus Zelonia harbours trypanosomatids from Neotropical hemipterans placed at the edge of the Leishmania-Endotrypanum-Porcisia clade. Finally, attention is drawn to the status of L. siamensis and L. australiensis as nomem nudums.

Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates.

Trypanosomatids are widespread in several plant families and although most isolates have been classified as Phytomonas, other trypanosomatid genera can also infect plants. In order to assess the natural occurrence of non-Phytomonas trypanosomatids in plants we characterized 21 new trypanosomatid cultures, 18 from fruits and three from seeds of 17 plant species. The trypanosomatids from fruit and seeds were compared in terms of morphological, growth, biochemical and molecular features. The high diversity among the isolates permitted the classification of the new flagellates into the genera Crithidia and Leptomonas as well as Phytomonas. The data showed that natural fruit infection with non-Phytomonas trypanosomatids is more common than usually thought, being detected in 43% of the fruit isolates.

Genetic variability of trypanosomatids isolated from phytophagous hemiptera defined by morphological, biochemical, and molecular taxonomic markers.

In the present study, we investigated the genetic variability among 49 new isolates of trypanosomatids from phytophagous Hemiptera by means of morphological characters, growth features, and biochemical (enzymes of ornithine-arginine cycle) and molecular markers (based on spliced-leader, and ribosomal genes). From 402 phytophagous insects dissected and examined for the presence of trypanosomatids, 228 species belonging to Pyrrhocoridae, Coreidae, Lygaeidae, and Pentatomidae families harbored trypanosomatids in their salivary glands, or digestive tubes. Among these insects, 211 carried promastigotes and only 17 had choanomastigote forms. The results show a strong association among morphology, growth features, and biochemical and molecular markers and reveal the genetic diversity of the isolates, which were assigned to Crithidia, Phytomonas, and Leptomonas; we found genetic polymorphism within all these genera, thus indicating high genetic variability among trypanosomatids from phytophagous insects.

Early clinical experience using custom excimer laser ablations to treat irregular astigmatism.

PURPOSE: To assess the viability of custom excimer laser ablations for treating irregular astigmatism. SETTING: Single-center prospective study of a new custom-ablation technique. METHODS: Twelve patients received 15 custom ablations for irregular astigmatism resulting from keratoconus, penetrating keratoplasty for keratoconus, prior decentered laser in situ keratomileusis, or incisional refractive surgery. Follow-up ranged from 6 weeks to 14 months. Initially, the laser beam was manually decentered; later, the Contoured Ablation Patterns (CAP) method (VISX, Inc.) was used to automatically decenter the ablation over the corneal elevation. RESULTS: Results are presented in a case-by-case fashion. In the manual decentration group, the uncorrected visual acuity (UCVA) was 20/50 or better in 9 of 11 eyes (81.8%) and 20/40 or better in 7 eyes (63.6%). Surgery resolved or decreased visual symptoms when present. The best corrected visual acuity (BCVA) was maintained or improved in all eyes. Persistent

New data from old Trypanosomatid preparations.

Assessing the diversity of the Trypanosomatidae is difficult because of the relatively small number of species that can be cultured. This same problem thwarts efforts to identify the hosts and insect vectors of Phytomonas, a genus of parasites of plants that includes species responsible for devastating epiphytotics of economically important plantations. Here, Myrna Serrano, Marta Teixeira and Erney Camargo review the studies that have led to the development of a PCR-based technique for processing insect and plant juices fixed on glass slides. The method overcomes the need for cultivation, facilitates field collections and also permits the molecular examination of archival smears of Phytomonas. In principle, the method can be adapted to any trypanosomatid as well as to any fastidious parasitic or free-living organism.

Phytomonas: analysis of polymorphism and genetic relatedness between isolates from plants and phytophagous insects from different geographic regions by RAPD fingerprints and synapomorphic markers.

The random amplification of polymorphic DNA was used for easy, quick and sensitive assessment of genetic polymorphism within Phytomonas to discriminate isolates and determine genetic relationships within the genus. We examined 48 Phytomonas spp., 31 isolates from plants and 17 from insects, from different geographic regions. Topology of the dendrogram based on randomly amplified polymorphic DNA fingerprints segregated the Phytomonas spp. into 5 main clusters, despite the high genetic variability within this genus. Similar clustering could also be obtained by both visual and cross-hybridization analysis of randomly amplified synapomorphic DNA fragments. There was some concordance between the genetic relationship of isolates and their plant tissue tropism. Moreover, Phytomonas spp. from plants and insects were grouped according to geographic origin, thus revealing a complex structure of this taxon comprising several clusters of very closely related organisms.

PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears.

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.

Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene.

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes.

Trypanosomatidae: a spliced-leader-derived probe specific for the genus Phytomonas.

We probed DNA from all trypanosomatid genera by slot blot hybridization with an oligonucleotide (SL3') complementary to a sequence of the Phytomonas spliced-leader or mini-exon RNA. The 19-nucleotide probe target site was previously shown to be highly conserved among a limited number of Phytomonas isolates, but diverges in other kinetoplastid genera. Our examination of 84 isolates of various genera of trypanosomatids showed hybridization of this probe exclusively with isolates from plants or insects which could, by morphological, biochemical, and molecular criteria, be considered to belong to the genus Phytomonas. In contrast, no hybridization was observed with flagellates of the genera Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leptomonas, Leishmania, and Trypanosoma. The method detected DNA quantities as low as 50 ng using either radioactive or nonradioactive probes, and was effective with as few as 10(4) intact flagellates. Together, these results suggest that this probe will serve as a convenient marker for taxonomic and epidemiological studies requiring reliable identification of Phytomonas spp. in plants or in putative insect vectors.