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Opportunistic parasitic infections are an important cause of morbidity and mortality in people infected with HIV. Since the introduction of highly active antiretroviral therapy (HAART), there has been a marked reduction in the occurrence and clinical course of these parasitic infections. Although these changes have been attributed to the restoration of cell-mediated immunity induced by either non-nucleoside reverse transcriptase inhibitors or HIV protease inhibitors, in combination with at least two nucleoside reverse transcriptase inhibitors included in HAART, there is evidence that HIV protease inhibitors have a direct inhibitory effect on the proteases of parasites. The results of studies on opportunistic parasitic infections conducted both before and during the HAART era indicate the need to develop clinical trials on the efficacy of HIV protease inhibitors in controlling parasitic infections in individuals with HIV or other immunocompromised individuals and laboratory investigations on aspartyl proteases of parasites as an important target for the development of new drugs.

The metabolism of the naturally occurring polyamines-putrescine, spermidine and spermine-is a highly integrated system involving biosynthesis, uptake, degradation and interconversion. Metabolic differences in polyamine metabolism have long been considered to be a potential target to arrest proliferative processes ranging from cancer to microbial and parasitic diseases. Despite the early success of polyamine inhibitors such as alpha-difluoromethylornithine (DFMO) in treating the latter stages of African sleeping sickness, in which the central nervous system is affected, they proved to be ineffective in checking other major diseases caused by parasitic protozoa, such as Chagas' disease, leishmaniasis or malaria. In the use and design of new polyamine-based inhibitors, account must be taken of the presence of up-regulated polyamine transporters in the plasma membrane of the infectious agent that are able to circumvent the effect of the drug by providing the parasite with polyamines from the host. This review contains information on the polyamine requirements and molecular, biochemical and genetic characterization of different transport mechanisms in the parasitic agents responsible for a number of the deadly diseases that afflict underdeveloped and developing countries.

Parasitic apicomplexans are responsible for some of the most severe worldwide health problems, including malaria, toxoplasmosis and cryptosporidiosis. These parasites are characterized by a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), which has a crucial role in pyrimidine biosynthesis. Inhibitors of DHFR have been successful in the treatment of toxoplasmosis and malaria. However, there is currently no effective therapy for cryptosporidiosis, and despite early successes against malaria, resistance to DHFR inhibitors in malaria parasites has now become a global problem. Novel DHFR inhibitors, designed using the recently revealed crystal structures of the enzymes from two parasitic protozoa, are in development.

Unlabelled: OBJECTIVE We sought to determine the etiologies, manifestations, and risk factors for persistent (> or =7 days) diarrhea in human immunodeficiency virus type 1 (HIV-1)-infected persons in Peru., Design: The present study is a case-control study of 147 HIV-1-infected case subjects with persistent diarrhea and 147 HIV-1-infected control subjects without diarrhea., Methods: We obtained clinical, demographic, and exposure data, CD4 lymphocyte counts, and stool samples for detection of enteric parasitic and bacterial pathogens and rotavirus., Results: One or more enteric pathogen was identified in 55% of case subjects and 21% of control subjects (odds ratio adjusted for CD4 lymphocyte count, 3.8; 95% confidence interval, 2.2-6.5). The median CD4 lymphocyte count was highest with pathogen-free diarrhea and lowest with Cryptosporidium infection. Cryptosporidium species (the most frequent pathogen), Giardia lamblia, Aeromonas species, Campylobacter species, and rotavirus were all significantly associated with diarrhea. Bacterial pathogens were significantly associated with G. lamblia and rotavirus infection. Of the bacterial pathogens (Aeromonas, Campylobacter, Salmonella, and Vibrio species and enterotoxigenic Escherichia coli), only 24% were susceptible to cotrimoxazole, whereas 90% were susceptible to ciprofloxacin. In no case did the sensitivity or positive predictive value of specific clinical and laboratory findings for curable enteric infections exceed 50%., Conclusions: Several enteric pathogens were associated with diarrhea in HIV-1-infected case subjects in Peru, especially among those who were heterosexual. Clinical findings were poor predictors of detectable microbial etiology. The guidelines for initial management of chronic diarrhea with sulfamethoxazole-trimethoprim in HIV-1-infected persons require revision, at least in settings where prophylaxis with this agent is common.

The aim of the present study was to investigate the impact of a successful anti-myxosporean medication on the innate immune system of fish intensively cultured in the Mediterranean basin. For this purpose, juvenile and adult gilthead seabream (S. aurata L.) naturally infected with Polysporoplasma sparis in the kidney were used in a small-scale field trial. The infected fish were treated orally with the combination of salinomycin and amprolium, two drugs well known for their anti-coccidial effect in other animals. Drug efficacy and safety was evaluated in terms of changes observed in histopathology, mortality and P. sparis intensity and prevalence rate. Phagocytic functions of head-kidney leucocytes were also investigated at the end as well as one month post the medication. Salinomycin with amprolium exhibited a significant reduction in intensity and prevalence rate in both juvenile and adult fish, and no histopathological evidence for toxic side effects was observed. In addition, the successful treatment was closely correlated with a complete restoration of the diminished phagocytic ability and capacity as well as NO, and lysozyme secretion in a time dependent manner. This data suggests that salilomycin with amprolium can be an alternative treatment for myxosporean infections in tropical fish, possibly exhibiting their action through the enhancement of host innate functions.

Apicomplexa are unicellular, obligate intracellular parasites of great medical importance. They include human pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease (toxoplasmosis). They alone affect several hundred million people worldwide so that new drugs, especially for plasmodial infections, are urgently needed. This review will focus on a recently emerged, potential drug target, a plant-type redox system consisting of ferredoxin-NADP(+) reductase (FNR) and its redox partner, ferredoxin (Fd). Both reside in an unique organelle of these parasites, named apicoplast, which is of algal origin. The apicoplast has been shown to be required for pathogen survival. In addition to other pathways already identified in this compartment, the FNR/Fd redox system represents a promising drug target because homologous proteins are not present in host organisms. Furthermore, a wealth of structural information exists on the closely related plant proteins, which can be exploited for structure-function studies of the apicomplexan protein pair. T. gondii and P. falciparum FNRs have been cloned, and the T. gondii enzyme was shown to be a flavoprotein active as a NADPH-dependent oxidoreductase. Both phylogenetic and biochemical analyses indicate that T. gondii FNR is similar in function to the isoform present in non-photosynthetic plastids whereby electron flow is from NADPH to oxidized Fd. The resulting reduced Fd is then presumably used as a reductant for various target enzymes whose nature is just starting to emerge. Among the likely candidates is the iron-sulfur cluster biosynthesis pathway, which is also located in the apicoplast and dependent on reducing power. Furthermore, lipoic acid synthase and enzymes of the isoprenoid biosynthetic pathway may be other conceivable targets. Since all these metabolic steps are vital for the parasite, blocking electron flow from FNR to Fd by inhibition of either FNR activity or its molecular interaction with Fd should also interfere with these pathways, ultimately killing the parasite. Although the three-dimensional structure of FNR from T. gondii is not yet known, experimental and computational evidence shows that apicomplexan and plant enzymes are very similar in structure. Furthermore, single amino acid changes can have profound effects on the enzyme activity and affinity for Fd. This knowledge may be exploited for the design of inhibitors of protein-protein interaction. On the other hand, specifically tailored NAD(P) analogues or mimetics based on previously described substances might be useful lead compounds for apicomplexan FNR inhibitors.

Objective: To determine the long-term outcome of cats infected with Tritrichomonas foetus and identify treatment and management strategies influencing resolution of infection or associated diarrhea., Design: Prospective study., Sample Population: 26 cats with T. foetus-associated diarrhea at least 22 months prior to the study., Procedure: A standardized survey regarding clinical course and management was administered to owners of cats with T. foetus infection and associated diarrhea. Fecal samples were obtained from each cat; the presence of T. foetus was assessed via microscopic examination of smears, culture in commercial media, and polymerase chain reaction amplification of T. foetus rDNA involving species-specific primers., Results: Survey responses were obtained from owners of all 26 cats. Twenty-three cats had complete resolution of diarrhea a median of 9 months after onset. Analysis of fecal samples obtained from 22 cats revealed persistent T. foetus infection in 12, with a median of 39 months after resolution of diarrhea. History of implementation of a dietary change, treatment with paromomycin, or higher numbers of cats in the household was associated with significantly longer duration of time to resolution of diarrhea., Conclusions and Clinical Relevance: Results suggested chronic T. foetus-associated diarrhea in most cats is likely to resolve spontaneously within 2 years of onset. Chronic infection with T. foetus (without clinical signs) after resolution of diarrhea appears to be common. Although often temporarily effective in decreasing severity of diarrhea, attempts to treat cats with T. foetus infection may result in prolongation of time to resolution of diarrhea.

Current biomedical research has its focus on the search for newer intervention strategies to control public health impact of parasitic diseases. The dramatic advances of molecular and cellular biology in recent times have provided opportunities for discovering and evaluating molecular targets for drug designing, which now form a rational basis for the development of improved anti parasitic therapy. DNA topoisomerases, the "cellular magicians" involved in nearly all biological processes governing DNA, have emerged as one such biological target. Over the last two decades, interest in topoisomerases has expanded beyond the realm of the basic science laboratory into the clinical arena. This review aims at providing a comprehensive insight into the biology of DNA topoisomerases and also focus on its evolution as a drug target in the unicellular kinetoplastids.

Toll-like receptor (TLR) agonists are being developed for use as vaccine adjuvants and as stand-alone immunomodulators because of their ability to stimulate innate and adaptive immune responses. Among the most thoroughly studied TLR agonists are the lipid A molecules that target the TLR4 complex. One promising candidate, monophosphoryl lipid A, which is a derivative of lipid A from Salmonella minnesota, has proven to be safe and effective as a vaccine adjuvant in > 120,000 human doses. A new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs), have been engineered specifically to target human TLR4 and are showing promise as vaccine adjuvants and as monotherapeutic agents capable of eliciting nonspecific protection against a wide range of infectious pathogens. In this review, the authors provide an update of the preclinical and clinical experiences with the TLR4 agonists, MPL (Corixa Corporation) adjuvant and the AGPs.

Constitutive nitric oxide (NO) is generated by constitutively expressed types of NO-synthase enzymes (NOS-I and -III), being involved in physiological processes such as nervous transmission and vasodilatation. Inducible NO, synthesized by the NO-synthase isoform NOS-II, is an anti-pathogen and tumoricidal agent. However, inducible NO production requires a tight control because of cytotoxic and immune-modulation activity. NO produced by human and canine macrophages has long been demonstrated to be involved in the intracellular killing of Leishmania. Mechanisms of parasite survival and persistence in the host have been throughly investigated, and include suppression of NOS-II and the parasite entry into NOS-II negative cells. Both intracellular and extracellular morphotypes of Trypanosoma cruzi are killed by NO in vitro and in vivo, although a role of NO in the pathogenesis of heart disease has been reported. Killing of extracellular protozoa such as Trichomonas vaginalis and Naegleria fowleri by activated macrophages is also mediated by NO. The main control of Plasmodium spp infection in human and murine hepatocytes, and in human monocytes is achieved by NO-mediated mechanisms. Protection from severe malaria in African children has been found associated with polymorphisms of the NOS-II promoter; however, a pathogenic role of endogenous NO has been documented in cerebral malaria. Although several macromolecules are putative NO targets, recent experimental work has shown that NO-releasing compounds inhibit cysteine proteases (CP) of P. falciparum, T. cruzi and L. infantum in a dose-dependent manner. CPs are present in a wide range of parasitic protozoa and appear to be relevant in several aspects of the life cycle and of the parasite-host relationships. Comparative analysis of 3-D amino acid sequence models of CPs from a broad range of living organisms, from viruses to mammals, suggests that the Sy atom of the Cys catalytic residue undergoes NO-dependent chemical modification (S-nytrosilation and disulfide bridge formation), with the concomitant loss of enzyme activity. The NO-donor S-nitroso-N-acetilpenicillamine (SNAP) was shown to kill T. cruzi epimastigotes and L. infantum promastigotes in culture, while a combination of nitrite plus acid organic salts was highly effective against L. major amastigotes in mouse macrophages. A parasitostatic effect--with both encystation and excystation inhibition--of S-nitrosoglutathione and spermine-NONOate was documented in trophozoite cultures of Giardia duodenalis. Recently, a novel formulation of metronidazole bearing a NO-releasing group was found to enhance significantly the in vitro killing of Entamoeba histolytica trophozoites, compared to metronidazole. So far, only two clinical studies were performed on human patients, suffering from cutaneous leishmaniasis. In one study, 16 Ecuadorean patients were treated with a SNAP cream administered on lesions for 10 days. All lesions were parasitologically cured and clinically healed by day 30. In the second study, a different NO-producing cream (basically nitrite in acidic environment) was employed to treat 40 Syrian patients. Only 28% of them showed improvement and 12% were cured by day 60. In conclusion, despite the wide evidence that NO can be regarded as a natural anti-protozoal weapon, little efforts have been made to develop and test NO-based drugs in human medicine. This is mainly due to the difficulty in designing suitable chemical carriers able to release the right amount of NO, in the right place and in the right time, to avoid toxic effects against non-target host cells.

Whereas parasitic diseases are always a heavy burden for humanity, few are the new antiparasitic molecules marketed during the last 25 years. Thus on the 1393 new molecules marketed between 1975 and 1999, only 7 have antiprotozoan properties. This talk will detail the progress made in the treatment of the intestinal protozoa, malaria, visceral leishmaniasis and toxoplasmosis, problems with which are especially confronted the European parasitologists. The treatment of Giardia and intestinal amoebas is based on 5-nitro-imidazoles derivatives. Single-dose treatments can be used with tinidazole or secnidazole. Resistance to these compounds of Giardia were described and in these cases, treatment by quinacrine or nitazoxanide are possible alternatives. Nitazoxanide is marketed in the United States and in Australia. It seems to be a well tolerated antiparasitic agent with a broad spectrum because it is active on a lot of intestinal protozoa and helminths. It acts on the same metabolic way as the 5-nitro-imidazoles (inhibition of the ferredoxine reductase) but without synthesis of free radicals and DNA deterioration of the target cell. It is thus neither teratogenic nor mutagenic. Artemisinin derivatives allowed considerable progress in the treatment of malaria. They have short half-lifes, allowing a fast parasitic clearance and these derivatives do no provoke resistance. They are first line drugs for the treatment of malaria in areas of drug resistance. The arthemeter-lumefantrine association (Riamet, Coartem) ensures a rapid disappearance of the circulating parasites and is well tolerated. Atovaquone-proguanil (Malarone) is usable in the treatment of acute malaria but also in disease prevention with the advantage of continuing drug intake for only 7 days after having left the infected area. The treatment of leishmaniasis is always delicate and is characterized by the worrying development of antimony resistances, probably related in the European zones to the treatment of dogs. Liposomal amphotricin is an alternative of choice but remains very expensive. The heating of amphotericin to 70 degrees C during 20 minutes gives it experimental properties and efficacies comparable with that of liposomal amphotericin, but at a less cost. Miltefosine, an alkyl-phospholipide antimetabolite, is very active on visceral leishmaniasis resistant to antimonial treatment. However, its long half-life could induce the emergence of resistances. Miltefosine induces much less side effects than conventional amphotericin B. The commonly used anti-toxoplasmic drugs (sulphadiazine and pyrimethamine) were marketed some 50 years ago and are only active on the rapid forms in multiplication. No drug is really efficient on the cysts although preliminary tests with atovaquone are encouraging to treat ophthalmologic forms in immunocompetent patients. To conclude, it is important to continue to search for new antiprotozoan molecules because, for some parasites, drug resistance is an important problem. Moreover, the treatment of the pregnant women, particularly during the first trimester, is often impossible and there is a lack of galenic forms easily usable in children. A better knowledge of the metabolic pathways of protozoa (particularly the apicoplast of Apicomplexa parasites) would certainly open the posssibility to identify new drugs. To reduce and delay the appearance of resistances, mass-treatments of mass should be avoided and targeted treatments prefered as well as the use of associations of molecules having different modes of action.

Remarkable progress has been made in antiretroviral therapeutics, as well as in the prophylaxis and treatment of opportunistic infections, since the beginning of the AIDS epidemic. The patient's life expectancy and quality of life have consequently improved, thanks to better management of opportunistic diseases. The introduction of protease inhibitors-containing regimen (i.e. Highly Active Antiretroviral Therapy or HAART), since 1996, has drastically reduced the incidence of opportunistic infections by restoring immunity. The large panel of antiretroviral drugs responsible for frequent sustained viral and immune responses has thus allowed a new definition of guidelines for the prophylaxis and treatment of opportunistic infections. A better use of prophylactic drugs should help reduce the risk of drug-related toxicity and pharmaceutical interactions. It should also decrease the cost of HIV management and eventually increase compliance to treatment and quality of life.

Nine drugs with known or suspected antiprotozoal activity were tested in vitro, and in vivo for activity against Histomonas meleagridis. The nitroimidazoles dimetridazole, metronidazole, ornidazole, and tinidazole suppressed growth of H. meleagridis in vitro at 10 microg/ml or higher. Paromomycin sulfate, and carbadox were weakly effective at high levels. Quinolinol, mebendazole, diloxanide furoate, and albendazole had no demonstrable efficacy in vitro. Drugs showing some activity in vitro were tested in young chickens inoculated intracloacally with 2 x 10(5) H. meleagridis/bird. Dimetridazole, metronidazole, ornidazole, and tinidazole were highly effective at 200 ppm in feed. Paromomycin sulfate, and carbadox were ineffective in vivo, with no improvement in liver or cecal lesion scores compared to that of infected controls. Thus, the only new entities with efficacy against blackhead disease in vivo were nitroimidazoles, related to the positive control dimetridazole.

Four commercial producers of discus (Symphysodon aequifasciatus) were found to have fish infested with the flagellate Cryptobia iubilans. Affected fish had granulomatous gastritis, and many also had granulomatous disease of other organs. The parasite had to be differentiated from the related flagellates Spironucleus spp, which induce different lesions. Transmission electron microscopy was found to be useful in detecting and identifying the parasite. Morbidity and mortality rates in the various fish populations appeared to be linked to a number of variables, including water quality, presence of other parasites and bacteria, diet, species, size, and age of the fish, and optimization of husbandry appeared to be important in alleviating the severity of disease. Metronidazole was not effective for treatment of C iubilans, but bath treatments with dimetridazole (80 mg/L for 24 hours, repeated daily for 3 days) or 2-amino-5-nitrothiazol (10 mg/L for 24 hours, repeated daily for 3 days) may be useful in decreasing the prevalence of infestation.

Lincomycin and clindamycin are lincosamide antibiotics used in clinical practice. Both antibiotics are bacteriostatic and inhibit protein synthesis in sensitive bacteria. They may even be bactericidal at the higher concentrations that can be reached in vivo. Clindamycin is usually more active than lincomycin in the treatment of bacterial infections, in particular those caused by anaerobic species; and it can also be used for the treatment of important protozoal diseases, e.g. malaria, most effectively in combination with primaquine. Resistance to lincomycin and clindamycin may be caused by methylation of 23S ribosomal RNA, modification of the antibiotics by specific enzymes or active efflux from the periplasmic space., (Copyright 2004 Springer-Verlag)

Nitazoxanide (Alinia, Romark Laboratories) was synthesized based on the structure of niclosamide. In vitro studies have demonstrated activity against a broad range of parasites as well as some bacteria. Three controlled trials demonstrated efficacy in cryptosporidiosis, however, the efficacy in advanced AIDS patients (CD4 cell counts = 50) at approved doses was limited. Trials have also demonstrated efficacy comparable to metronidazole (Flagyl, GD Searle and Co.) in giardiasis with fewer side effects. Nitazoxanide is also effective versus intestinal helminths and tapeworms as well as in chronic fascioliasis. Side effects in clinical trials have been similar to placebo. Nitazoxanide is the first agent proven to be effective in cryptosporidiosis. It has also proven efficacy in giardiasis. Nitazoxanide is efficacious again intestinal helminths. Additional indications may be developed in the future.

Protozoal infections of the gastrointestinal tract occur worldwide and have substantial morbidity and mortality. Prevalence is higher in the economically deprived regions of the world, especially the developing countries. Infections like amoebiasis and giardiasis have a worldwide distribution, being endemic in India. Apart from producing GI symptoms, growth and development of children is also impaired. It is seen that protozoa multiply rapidly in their hosts and as there is a lack of effective vaccines, chemotherapy has been the only practiced way to treat individuals and reduce transmission. The current treatment modalities for protozoal diarrhoea include 5-nitrosoimidazoles, iodoquinol, diloxanide furoate, paromomycin, chloroquine, and trimethoprim-sulphamethoxazole.

We report the synthesis of substituted quinolines and their in vitro biological evaluation against the causal agents of cutaneous leishmaniasis, visceral leishmaniasis, African trypanosomiasis and Chagas' disease. Furthermore, several quinolines have also been tested for their anti-retroviral activity in HIV-1 infected cells. The structure-activity relationships of these new synthetic compounds are discussed and emphasis was placed on the treatment of leishmania/HIV co-infections.

Salmonid cryptobiosis is caused by Cryptobia (Trypanoplasma) salmositica. The haemoflagellate has been reported from all species of Pacific Oncorhynchus spp. on the west coast of North America. It is normally transmitted by the freshwater leech, Piscicola salmositica, in streams and rivers, and sculpins, Cottus spp., are considered important reservoir hosts. The pathogen can also survive on the body surface of fish because it has a contractile vacuole to osmoregulate when the fish is in fresh water. This allows for direct transmission between fish, especially in aquaculture facilities. The parasite divides rapidly by binary fission in the blood to cause disease, the severity of which is directly related to parasitaemia. Cryptobia salmositica has a mitochondrium and it normally undergoes aerobic respiration; however, if its mitochondrium is damaged it will switch to glycolysis. Its glycolytic enzymes and catalase are contained in glycosomes. Cysteine protease is a metabolic enzyme, and its neutralization inhibits oxygen consumption and multiplication of the parasite. An important virulent factor in cryptobiosis is a secretory metalloprotease. The protective mechanism involves production of complement fixing antibodies, phagocytosis by macrophages, and cell-mediated cytotoxicity. Recovered fish are protected, probably for life as the immunity is non-sterile. Clinical signs of the disease include anaemia, anorexia, splenomegaly, general oedema and abdominal distension with ascites. The metabolism and swimming performance of infected fish are significantly reduced and the bioenergetic cost of the disease is very considerable. Fish are susceptible to hypoxia and their immune system is depressed during acute cryptobiosis. Severity of the disease and mortality rates vary significantly between species and stocks of salmon. Protective strategies include selective breeding of Cryptobia-resistant fish. This is innate resistance to infection and it is controlled by a dominant Mendelian locus. In these fish the parasite is lysed via the alternative pathway of complement activation. In Cryptobia-tolerant fish (infected with the pathogen but which do not suffer from disease) the metalloprotease secreted by the parasite is neutralized by alpha2 macroglobulin. Hence, the production of a transgenic Cryptobia-tolerant salmon is an option. This strategy has the advantage in that human intervention (e.g. vaccination, chemotherapy) is not required once the transgenic fish is produced. Acquired immunity is another option; a single dose of the attenuated live vaccine protects fish for at least 2 years. The protective mechanism in vaccinated fish is similar to that in recovered fish. The trypanocidal drug, isometamidium chloride, is an effective therapeutic and prophylactic agent. It accumulates in the mitochondrium of the parasite and significantly disrupts aerobic respiration by causing lesions in the organelle. Efficacy of the drug is significantly increased after its conjugation to antibodies. This immuno-chemotherapeutic strategy has the advantage in that it will lower the drug dosage and hence side-effects of chemotherapy. It will probably reduce the accumulation of the drug in fish, an important consideration in food fish.

Protozoa infections are an important cause of chronic diarrhea in patients infected with HIV. The introduction of highly active antiretroviral treatment to the management of HIV in the mid 1990s has led to a dramatic reduction in the incidence of these opportunistic infections in Europe and America. In contrast, in the developing world where such treatments are not readily affordable, protozoa-related diarrhea remains a major cause of morbidity and mortality in HIV-infected individuals. In this review, the optimum investigations required to diagnose these pathogens in HIV-related diarrhea, as well as current treatment options, will be discussed.

Diseases caused by parasitic protozoa present a health problem of immense magnitude, and there is an urgent need for safe and effective new therapies. DNA topoisomerases are clinically relevant targets for anti-cancer and anti-bacterial agents. Inhibitor studies on parasite topoisomerases have revealed that these enzymes have great promise as molecular targets for anti-parasitics, and have helped to dissect the basic biology of DNA topoisomerases in these organisms. This review provides a brief introduction to DNA topoisomerases and anti-topoisomerase drugs, and an overview of studies on protozoal DNA topoisomerases and their inhibitors.

New chemotherapies for human and animal apicomplexan infections are needed as a component of future strategies to deal with these diseases. An extensive search for new treatments exploring the unique developmental physiology, metabolism and molecular structures of Apicomplexa is under way. The description of the full complement of about 5,300 Plasmodium falciparum genes and fast growing sequence databases for other Apicomplexa allow reconstruction of metabolic pathways of these parasites and thus accelerate identification and biochemical analysis of potential targets. The apicoplast de novo fatty acid biosynthetic pathway shows great potential as a target for small-molecule inhibitors in a stand-alone or combination chemotherapy. Three enzymatic activities, acetyl-CoA carboxylase, beta-ketoacyl-ACP synthase and enoyl-ACP reductase, respond to inhibitors previously identified for bacteria and plants, and deserve to be explored in depth. In this connection, screening systems have been established to seek more potent and specific antiparasitic compounds that are harmless to the host. To this end the interconnections of fatty acid biosynthesis in Apicomplexa with other metabolic and cellular processes must be investigated.

Protozoan parasites are causing some of the most devastating diseases world-wide. It has now been recognised that a major effort is needed to be able to control or eliminate these diseases. Genome projects for the most important protozoan parasites have been initiated in the hope that the read-out of these projects will help to understand the biology of the parasites and identify new targets for urgently needed drugs. Here, I will review the current status of protozoan parasite genome projects, present findings obtained as a result of the availability of genomic data and discuss the potential impact of genome information on disease control.

A 59-year-old woman suffering from chronic lymphocytic leukemia developed pulmonary lesions; bronchoalveolar lavage was performed for possible systemic fungal infection. However, direct microscopic analysis revealed ciliated protozoa identified as Balantidium coli. B. coli is the only known pathogenic ciliate, and is usually associated with intestinal infection in areas associated with pig rearing. On very rare occasions the organisms may invade extra-intestinal organs, in this case the lungs of an immunocompromised patient. This case is unusual as balantidiasis is rare in Europe, the patient had no obvious contact with pigs, and there was no history of diarrhea prior to pulmonary colonization. Metronidazole was rapidly administered, and the condition improved after 24-48 hr., (Copyright 2003 Wiley-Liss, Inc.)

Apicomplexan parasites are a large phylum of unicellular and obligate intracellular organisms of great medical importance. They include the human pathogens Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease, together affecting several hundred million people worldwide. The search for new and effective drugs against these pathogens has been boosted during the last years by an unexpected finding. Through molecular and cell biological analysis it was realized that probably most members of this phylum harbor a plastid-like organelle, called the apicoplast, which probably is derived from the engulfment of a red alga in ancient times. Although the apicoplast itself contains a small circular genome, most of the proteome of this organelle is encoded in the nuclear genome, and the proteins are subsequently transported to the apicoplast. It is assumed to contain a number of unique metabolic pathways not found in the vertebrate host, making it an ideal "playground" for those interested in drug targets. Recent reports have shown that the rationale of this approach is valid and that new drugs which are urgently needed especially for plasmodial infections, might be developed in the near future based on these targets. Amongst them are three enzymes of the plant-like fatty acid synthesis machinery and enzymes of the non-mevalonat isoprenoid biosynthesis pathway. From their presence in the apicoplast it can be concluded that fatty acid and lipid biosynthesis seems to be a major function of the apicoplast. Another recently described apicoplast enzyme, ferredoxin-NADP(+)-reductase and its redox partner, ferredoxin, points to another interesting organelle-specific biosynthetic pathway, namely [Fe-S] cluster biosynthesis. In the present review, the fundamental aspects of the apicoplast as drug target will be described, together with the specific pathways and their currently known inhibitors. Furthermore, based on the recent findings potentially new targets will be discussed. A short overview of the presently available high-throughput methods for Apicomplexa to evaluate the potency of new inhibitory substances will also be given.

The group of biologically active nitroheterocyclic compounds includes various 5- and 2-nitroimidazoles and 5-nitrofurans, which can be used as therapeutic agents against a variety of protozoan and bacterial (anaerobic) infections of humans and animals. The current status in the the treatment of giardiasis, trichomoniasis, balantidiasis, histomoniasis, and amebiasis (including infections due to opportunistic amebas) is presented. The most relevant drugs (benznidazole, furazolidone, metronidazole, misonidazole, nifurtimox, nimorazole, nitazoxanide, ornidazole, secnidazole, and tinidazole) are characterized with regard to their chemical, chemotherapeutic, toxicological, pharmacokinetic, and pharmacological properties, including the mechanism of action and resistance in certain parasitic protozoa.

The purpose of this study was to evaluate the efficacy and the tolerance of nitazoxanide in children as a single broad-spectrum antiparasitic agent in the treatment of mixed parasite infections with both intestinal protozoa and helminths. Two hundred seventy-two children (age range = 2-14 years) participated in this study. We systematically surveyed every household head using questionnaires designed to obtain information about household socioeconomic status and hygiene. Parasitic infections were confirmed by three stool examinations using direct smear, Ferreira concentration, and cold acid-fast Kinyoun staining methods. One hundred twenty-one (44%) children tested positive for protozoa such as Giardia lamblia (18%), Entamoeba histolytica/E. dispar (10%), Blastocystis hominis (7%), Cryptosporidium parvum (4%), and Cyclospora cayetanensis (3%), and helminths such as Hymenolepis nana (10%), Trichuris trichiura (6%), and Ascaris lumbricoides (6%). There were also two cases of infection with Enterobius vermicularis. After a complete physical examination was performed, 121 patients received treatment with nitazoxanide. Overall, 84% of the protozoa and 95% of the helminths were completely eliminated from the patients. Nitazoxanide was very well tolerated, with no serious adverse effects reported.

Objectives: To examine the presence of intestinal protozoan and helminth infections and their association with clinical signs and symptoms in children in Trujillo, Venezuela., Methods: Conventional microscopic methods (thick-smear, saline and iodine solutions) were used to identify parasites in stool samples of 301 children attending day care centres. A subgroup of 45 children was evaluated clinically and parasitologically five times during a 1-month period using conventional methods and the Kinyoun acid-fast stain for Cryptosporidium identification., Results: The point prevalence of protozoan infections was 21% for Giardia duodenalis, 1.0% for Entamoeba histolytica/dispar, 4% for Entamoeba coli, 16% for Blastocystis hominis, and 89% for Cryptosporidium parvum. Prevalence of helminth infection was 11% for Ascaris lumbricoides, 10% for Trichuris trichiura, 0.3% for Strongyloides stercoralis, and 1.3% for Hymenolepis nana. Over a 1-month time frame, new infections were observed at a rate of 11% for G. duodenalis, 4% for E. histolytica/dispar, 7% for A. lumbricoides, 11% for T. trichiura, 0% for S. stercoralis, and 2% for H. nana. Intestinal symptoms (diarrhoea, vomiting, gas, stomach pain, and loss of appetite) were associated with presence of one or more of C. parvum or B. hominis organisms in stool samples., Conclusions: Intestinal parasitic infections contribute significantly to the enteric disease burden experienced by this group of children. The organisms most strongly implicated by this study are common and difficult-to-treat protozoan pathogens.

Molecular, biochemical and genetic characterization of ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase establishes that these polyamine-biosynthetic enzymes are essential for growth and survival of the agents that cause African sleeping sickness, Chagas' disease, leishmaniasis and malaria. These enzymes exhibit features that differ significantly between the parasites and the human host. Therefore it is conceivable that exploitation of such differences can lead to the design of new inhibitors that will selectively kill the parasites while exerting minimal, or at least tolerable, effects on the parasite-infected patient.

Diarrhea is only one of the many manifestations of intestinal parasites. Environmental influences are inescapable, regardless of an individual's state of health: in a highly endemic region, intestinal parasitic colonization is almost the rule. The clinical expression of the parasitoses, however, is largely determined by host defenses; and when they are weakened, parasitic diarrhea is frequent and severe. Protein-energy malnutrition is by far the most important cause of immune deficiency in developing countries. Diarrhea caused by Strongyloides or Giardia is common and severe in malnourished children, while well-nourished children remain healthy carriers. These parasites require specific treatment in the malnourished; and the well-nourished should have preventive treatment when they are to receive corticosteroids or immunosuppressive agents. Diarrhea caused by Cryptosporidium spp. may be severe in malnourished or immunodeficient children, and recovery is achieved only after renutrition or treatment of the immunodeficiency.

Apicomplexan parasites cause severe diseases such as malaria, toxoplasmosis, and coccidiosis (caused by Plasmodium spp., Toxoplasma, and Eimeria, respectively). These parasites contain a relict plastid-termed "apicoplast"--that originated from the engulfment of an organism of the red algal lineage. The apicoplast is indispensable but its exact role in parasites is unknown. The apicoplast has its own genome and expresses a small number of genes, but the vast majority of the apicoplast proteome is encoded in the nuclear genome. The products of these nuclear genes are posttranslationally targeted to the organelle via the secretory pathway courtesy of a bipartite N-terminal leader sequence. Apicoplasts are nonphotosynthetic but retain other typical plastid functions such as fatty acid, isoprenoid and heme synthesis, and products of these pathways might be exported from the apicoplast for use by the parasite. Apicoplast pathways are essentially prokaryotic and therefore excellent drug targets. Some antibiotics inhibiting these molecular processes are already in chemotherapeutic use, whereas many new drugs will hopefully spring from our growing understanding of this intriguing organelle.

There is an urgent need to develop new drugs against eukaryotic parasitic protozoa such as Plasmodium, Trypanosoma and Leishmania, which cause the diseases malaria, trypanosomiasis and the leishmaniases respectively. The biology of these organisms has many unusual facets that might be exploited for drug design, and the recent availability of parasite genome sequence data has facilitated the search for novel drug targets. Here we review current understanding of the cell cycle in these parasites and show that important structural and functional differences exist between parasite and mammalian cell cycle control machineries and signal transduction pathways, which might be utilised for rational drug design. Potential targets include protein kinases from the cyclin-dependent kinase, cAMP-dependent kinase and mitogen activated protein kinase families.

Polyamines are essential cell constituents for all organisms. The present review highlights important differences in the synthesis, degradation, and interconversion of polyamines between the protozoan parasites (Trypanosoma brucei, Trypanosoma cruzi, Cryptosporidium parvum and Trichomonas vaginalis) and their mammalian hosts. Approaches include development of mono- and di-substituted polyamine analogs targeting polyamine interconversion, as well as more traditional targeting of synthetic enzymes and related pathways.

Trypanosoma cruzi and Leishmania parasites have a strict requirement for specific endogenous sterols (ergosterol and analogs) for survival and growth and cannot use the abundant supply of cholesterol present in their mammalian hosts. Squalene synthase (SQS, E.C. 2.5.1.21) catalyzes the first committed step in sterol biosynthesis and is currently under intense study as a possible target for cholesterol-lowering agents in humans, but it has not been investigated as a target for anti-parasitic chemotherapy. SQS is a membrane-bound enzyme in both T. cruzi epimastigotes and Leishmania mexicana promastigotes with a dual subcellular localization, being almost evenly distributed between glycosomes and mitochondrial/microsomal vesicles. Kinetic studies showed that the parasite enzymes display normal Michaelis-Menten kinetics and the values of the kinetic constants are comparable to those of the mammalian enzyme. We synthesized and purified 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH), a potent and specific inhibitor of mammalian SQS and found that it is also a powerful non-competitive inhibitor of T. cruzi and L. mexicana SQS, with K(i)'s in the range of 12-62 nM. BPQ-OH induced a dose-dependent reduction of proliferation the extracellular stages of these parasites with minimal growth inhibitory concentrations (MIC) of 10-30 microM. Growth inhibition and cell lysis induced by BPQ-OH in both parasites was associated with complete depletion of endogenous squalene and sterols, consistent with a blockade of de novo sterol synthesis at the level of SQS. BPQ-OH was able to eradicate intracellular T. cruzi amastigotes from Vero cells cultured at 37 degrees C, with a MIC of 30 microM with no deleterious effects on host cells. Taken together, these results support the notion that SQS inhibitors could be developed as selective anti-trypanosomatid agents.

Tropical malabsorption remains an important clinical problem for both the indigenous population of tropical countries and for short-term visitors and longer-term residents from the industrialized world. In young children, persistent diarrhea and malabsorption can result in severe retardation of growth and development. The most common cause is an intestinal infection notably the small intestinal protozoa including Giardia intestinalis, Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis, and the microsporidia. Tropical sprue still remains an important diagnostic option but is less common than it was 20 to 30 years ago. It is important to attempt to make a specific microbiological diagnosis as this will influence the choice of antibiotic. However, if laboratory facilities are not available, it is possible to offer empirical therapy although this may involve a trial of more than one antibiotic.

Histomoniasis or blackhead is a life-threatening disease of turkeys that is caused by a flagellated protozoan, Histomonas meleagridis. The development of an assay to measure the sensitivity of drugs traditionally used against this parasite, as reputed to be effective against other protozoan parasites, is described. The in vitro minimum lethal concentrations (MLC), time for drug efficacy, and parasite viability after removal of residual drugs were determined. Three of the 10 tested drugs, fenbendazole, albendazole, and sulfadiazine, were found to be ineffective against H. meleagridis. Nifursol, the only compound still authorized as a feed additive in Europe, is an inhibiting agent but is not lethal in vitro. Roxarsone, an arsenical derivate similar to nitarsone (the only authorized drug in United States), is effective at high concentration (200 microg/mL) after a long exposure (48 h). The lethal activity of dimetridazole, metronidazole, ronidazole, tinidazole, and furazolidone in vitro was demonstrated. Dimetridazole (MLC = 25 microg/mL after 6 h of exposure), metronidazole (MLC = 50 microg/mL after 24 h), and furazolidone (MLC = 50 microg/mL after 24 h) are rapidly effective at low concentrations. These results confirm the effectiveness of dimetridazole, a drug that has been used in the treatment and prevention of blackhead. In May 2002 this compound was removed as feed additive in Europe.

Single species infections with schistosomes, geohelminths, and intestinal protozoans are common over large parts of sub-Saharan Africa, and it is expected that polyparasitism affects a considerable proportion of the population, hence posing a great toll on public health. However, few investigations have been carried out to quantify the extent of polyparasitism. Here, a detailed assessment is reported for the epidemiology of Schistosoma mansoni, geohelminths, and intestinal protozoan infections, with particular emphasis on polyparasitism among 260 community members in rural Cĵte d'Ivoire. Schistosoma mansoni, Entamoeba coli, and hookworm were the predominant species with prevalences of 71.5, 64.6, and 51.9%, respectively. Only 8 individuals displayed no infection, whereas two-thirds of the population harbored 3 or more parasites concurrently. There were a series of significant pairwise parasite co-occurrences, e.g., between S. mansoni and hookworms and between S. mansoni and E. coli. It is concluded that polyparasitism in the population studied here was very common, which is probably the case also in other areas of rural Cĵte d'Ivoire and elsewhere in sub-Saharan Africa. These findings call for integrated approaches to effectively control multiple parasitic and protozoan infections.

The greatest challenge to human health of recent times is the pandemic caused by the retroviruses known as HIV1 and HIV2. These have unleashed a plethora of opportunistic infections with organisms ranging from other viruses to helminths, some of which are newly recognised taxa or organisms never before identified as serious pathogens of man. Animal and bird migration as well as human travel and international trade have extended, to an alarming degree, the dissemination of other viral infections. Malaria is an increasing global challenge to human health, a reversal of the downward trend that was achieved in the latter half of the 20th century. Moreover, evidence is currently accumulating for the existence of a fifth species of Plasmodium in man. In the bacterial and protozoal fields, other novel organisms are being described. Powerful new tools of molecular biology are being deployed to probe parasite genetics, for example to reveal the genome of Plasmodium falciparum. These and molecular immunological techniques are also helping to resolve puzzling taxonomic questions, as well as aiding epidemiological studies that are revealing the unforeseen scale of human infection with a number of parasitic helminths in both tropic and temperate climatic areas. This paper presents a brief review of the major, as well as some of the relatively minor changes and advances of recent decades.

Protozoa are among the most important pathogens that can cause infections in immunocompromised hosts. These microorganisms particularly infect individuals with impaired cellular immunity, such as those with hematological neoplasias, renal or heart transplant patients, patients using high doses of corticosteroids, and patients with acquired immunodeficiency syndrome. The protozoa that most frequently cause disease in immunocompromised patients are Toxoplasma gondii, Trypanosoma cruzi, different Leishmania species, and Cryptosporidium parvum; the first two species cause severe acute meningoencephalitis and acute myocarditis, Leishmania sp. causes mucocutaneous or visceral disease, and Cryptosporidium can lead to chronic diarrhea with hepatobiliary involvement. Various serological, parasitological, histological and molecular methods for the diagnosis of these infections are currently available and early institution of specific therapy for each of these organisms is a basic measure to reduce the morbidity and mortality associated with these infections.

The present study was carried out to evaluate the effectiveness of nitazoxanide compared with that of quinfamide, mebendazole, or both in the treatment of intestinal protozoa and helminthic infections. A total of 677 stool specimens from children aged 2-12 years living in 3 communities of Colima, México, were analyzed in order to detect the presence of cysts, trophozoites, eggs, or larvae of intestinal protozoa or helminths. A total of 275 infected children were enlisted in a double-blind controlled study and randomly assigned to one of 2 treatment groups: Group A, nitazoxanide (200 mg for 3 days) and Group B, quinfamide (100 mg for 1 day), mebendazole (200 mg for 3 days), or both. A posttreatment fecal examination was conducted on Day 14 from treatment initiation. In Group A (n = 143), the parasitosis eradication rate was superior to that of Group B (n = 132). However, there was no significant difference between the 2 groups (P > 0.05).