dIn this study, we introduce a multilocus sequence typing (MLST) scheme, comprised of seven single-copy housekeeping genes, to genetically characterize Trichomonas vaginalis. Sixty-eight historical and recent isolates of T. vaginalis were sampled from the American Type Culture Collection and female patients at area health care facilities, respectively, to assess the usefulness of this typing method. Forty-three polymorphic nucleotide sites, 51 different alleles, and 60 sequence types were distinguished among the 68 isolates, revealing a diverse T. vaginalis population. Moreover, this discriminatory MLST scheme retains the ability to identify epidemiologically linked isolates such as those collected from sexual partners. Population genetic and phylogenetic analyses determined that T. vaginalis population structure is strongly influenced by recombination and is composed of two separate populations that may be nonclonal. MLST is useful for investigating the epidemiology, genetic diversity, and population structure of T. vaginalis.

Artemisinin compounds inhibit in vitro growth of cultured Trypanosoma cruzi and Trypanosoma brucei rhodesiense at concentrations in the low micromolar range. Artemisinin also inhibits calcium-dependent ATPase activity in T. cruzi membranes, suggesting a mode of action via membrane pumps. Artemisinins merit further investigation as chemotherapeutic options for these pathogens.Diseases caused by insect-borne trypanosomatid parasites are a significant and neglected public health problem worldwide. Chagas' disease, caused by infection with Trypanosoma cruzi, is a major agent of disease in Latin America, with 16 to 18 million infected individuals and an annual death toll of 50,000. It is also an emerging problem in the United States, where an estimated 100,000 infected individuals reside (16). Trypanosoma brucei subspp., the causative agents of human African trypanosomiasis or "sleeping sickness," infect 50,000 annually. Approximately 300,000 to 500,000 people have trypanosomiasis and will die if not treated (18). Current therapeutic options for Trypanosoma infections, benznidazole and nifurtimox for Chagas' disease treatment and suramin, pentamidine, melarsoprol, and eflornithine for treatment of sleeping sickness, are far from ideal (8,17). These drugs all suffer from one or more disadvantages-high cost, parenteral administration, long treatment courses (months), high clinical failure rates, or parasite drug resistance-and they elicit multiple, serious, and potentially fatal toxic side effects. New therapeutic alternatives are obviously desirable for treatment of these lifethreatening infections.Artemisinin is a sesquiterpene lactone isolated from Artemisia annua, an annual herb that has been used in traditional Chinese medicine for over 2,000 years (21). Artemisinin is hydrophobic, passes biological membranes easily, and is a potent antimalarial with effective 50% inhibitory concentrations (IC 50 values) ranging from 4.2 to 16.2 nM for different derivatives. Oral, parenteral, or rectal dosages achieve micromolar plasma concentrations (22). Artemisinin derivatives have been used to treat malaria cases around the world, and their extensive usage has not been associated with any significant toxicity (19). Artemisinin generates bioreactive radicals capable of intracellular damage, depolarizes mitochondrial membrane potential in yeast, and inhibits the Plasmodium falciparum endoplasmic reticulum calcium pump (SERCA), and artemisininresistant P. falciparum contains SERCA mutations (9,13,14,(20)(21)(22).Artemisinin compounds also show efficacy against Leishmania spp. of trypanosomatid parasites, achieving 50% killing at 750 nM for Leishmania major promastigotes, at 3 to 30 M for intracellular amastigote stages in macrophages, and at 1.4 to 382.9 M against Leishmania infantum promastigotes (1, 23). Artemether treatment (50 mg/kg of body weight/day) of footpad lesions in mice, by oral, intralesional, intramuscular, or intravenous administration, significantly reduces lesion size and L. major parasite numbers (23). Oral dih...

Amastigotes (tissue forms) of Leishmania donovani isolated from infected hamster spleens carried out several physiological activities (respiration, catabolism of energy substrates, and incorporation of precursors into macromolecules) optimally at pH 4.0 to 5.5. All metabolic activities that were examined decreased sharply above the optimal pH. Promastigotes (culture forms), on the other hand, carried out the same metabolic activities optimally at or near neutral pH. This adaptation to an acid environment may account in part for the unusual ability of amastigotes to survive and multiply within the acidic environment of the phagolysosomes in vivo.

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Leishmania amastigotes do not appear to alter the phagolysosomal pH (27), but rather possess membrane transport systems that are designed to exploit the acidic environment provided by the lysosome. Adaptation of amastigotes to high extracellular proton concentrations is strikingly evidenced by their metabolic behavior. Amastigotes optimally respire, metabolize glucose, and incorporate thymidine, uridine, and proline when bathed in medium between pH 4.0 and 5.5 (22). pH levels comparable to those within phagolysosomes. The effect of extracellular protons on the metabolic activity of amastigotes is probably due to increased nutrient transport through proton-coupled symporters, such as the glucose transporter (37,38). Proton influx is countered in both amastigotes and promastigotes by a plasmatemma proton pump, which is capable of generating a proton gradient across the plasma membrane (37; T. Glaser and A. Mukkada, personal communication). While the possible existence of other cation pumps has not been rigorously excluded, available evidence suggests that L. donotiani may rely solely on a proton pump to drive the uptake of nutrients and to regulate ion flow (37

Recent advances in genetic characterisation of Trichomonas vaginalis isolates show that the extensive clinical variability in trichomoniasis and its disease sequelae are matched by significant genetic diversity in the organism itself, suggesting a connection between the genetic identity of isolates and their clinical manifestations. Indeed, a high degree of genetic heterogeneity in T vaginalis isolates has been observed using multiple genotyping techniques. A unique two-type population structure that is both local and global in distribution has been identified, and there is evidence of recombination within each group, although sexual recombination between the groups appears to be constrained. There is conflicting evidence in these studies for correlations between T vaginalis genetic identity and clinical presentation, metronidazole susceptibility, and the presence of T vaginalis virus, underscoring the need for adoption of a common standard for genotyping the parasite. Moving forward, microsatellite genotyping and multilocus sequence typing are the most robust techniques for future investigations of T vaginalis genotype-phenotype associations.

Abstract. Subtyping isolates of Trichomonas vaginalis is an essential tool for understanding the epidemiology of this common sexually-transmitted disease. Restriction fragment length polymorphism (RFLP) analysis employing a probe from the heat-inducible cytoplasmic HSP70 gene family hybridized with EcoR I-digested genomic DNA was used in the molecular typing of Trichomonas isolates. Analysis of five American Type Culture Collection (ATCC) reference strains and 31 Jackson, Mississippi, isolates from six male and 21 female patients, revealed 10 distinct RFLP pattern subtypes of Trichomonas. The subtypes were temporally stable and cosmopolitan. The RFLP profiles seen in Maryland, Ohio, Massachusetts, and New York ATCC strains were identical to those of some Mississippi isolates, even though the samples were isolated 10-35 years apart. There was no correlation between metronidazole resistance and RFLP subtype with resistant isolates from eight patients distributed among six different subtypes.

Restriction fragment length polymorphism (RFLP) analysis using a multilocus heat-inducible cytoplasmic heat-shock protein 70 (Hsp70) hybridization probe with EcoRI-digested genomic DNA was used in molecular typing of 129 Trichomonas vaginalis isolates. Results indicate that Trichomonas organisms exhibit considerable polymorphism in their Hsp70 RFLP patterns. Analysis of seven American Type Culture Collection reference strains and 122 clinical isolates, including 84 isolates from Jackson, Mississippi, 18 isolates from Atlanta, Georgia, and 20 isolates from throughout the United States, showed 105 distinct Hsp70 RFLP pattern subtypes for Trichomonas. Phylogenetic analysis of the Hsp70 RFLP data showed that the T. vaginalis isolates were organized into two clonal lineages. These results illustrate the substantial genomic diversity present in T. vaginalis and indicate that a large number of genetically distinct Trichomonas isolates may be responsible for human trichomoniasis in the United States.