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We barcoded 25 in vitro isolates (representing 92 samples) of Giardia duodenalis from humans and other animals, which have been assembled by the Upcroft team at the Queensland Institute of Medical Research over a period of almost three decades. We used mutation scanning-coupled sequencing of loci in the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh) and beta-giardin (bg) genes, combined with phylogenetic analysis, to genetically characterise them. Specifically, the isolates (n = 14) of G. duodenalis from humans from Australia (AD113; BRIS/83/HEPU/106; BRIS/87/HEPU/713; BRIS/89/HEPU/1003; BRIS/91/HEPU/1279; BRIS/92/HEPU/1541; BRIS/92/HEPU/1590; BRIS/92/HEPU/2443; BRIS/93/HEPU/1706), Malaysia (KL/92/IMR/1106) and Afghanistan (WB), a cat from Australia (BAC2), a sheep from Canada (OAS1) and a sulphur-crested cockatoo from Australia (BRIS/95/HEPU/2041) represented assemblage A (sub-assemblage AI-1, AI-2 or AII-2); isolates (n = 10) from humans from Australia (BRIS/91/HEPU/1279; BRIS/92/HEPU/2342; BRIS/92/HEPU/2348; BRIS/93/HEPU/1638; BRIS/93/HEPU/1653; BRIS/93/HEPU/1705; BRIS/93/HEPU/1718; BRIS/93/HEPU/1727), Papua New Guinea (BRIS/92/HEPU/1487) and Canada (H7) represented assemblage B (sub-assemblage BIV); and an isolate from cattle from Australia (BRIS/92/HEPU/1709) had a match to assemblage E. Isolate BRIS/90/HEPU/1229 from a human from Australia was shown to represent a mixed population of assemblages A and B. These barcoded isolates (including stocks and derived lines) now allow direct comparisons of experimental data among laboratories and represent a massive resource for transcriptomic, proteomic, metabolic and functional genomic studies using advanced molecular technologies. (Nucleotide sequences reported in this paper are available in the GenBank database under accession nos. XXXXX-XXXXX).

Given the growing appreciation of serious health sequelae from widespread Trichomonas vaginalis infection, new tools are needed to study the parasite's genetic diversity. To this end we have identified and characterized a panel of 21 microsatellites and six single-copy genes from the T. vaginalis genome, using seven laboratory strains of diverse origin. We have (1) adapted our microsatellite typing method to incorporate affordable fluorescent labeling, (2) determined that the microsatellite loci remain stable in parasites continuously cultured for up to 17 months, and (3) evaluated microsatellite marker coverage of the six chromosomes that comprise the T. vaginalis genome, using fluorescent in situ hybridization (FISH). We have used the markers to show that T. vaginalis is a genetically diverse parasite in a population of commonly used laboratory strains. In addition, we have used phylogenetic methods to infer evolutionary relationships from our markers in order to validate their utility in future population analyses. Our panel is the first series of robust polymorphic genetic markers for T. vaginalis that can be used to classify and monitor lab strains, as well as provide a means to measure the genetic diversity and population structure of extant and future T. vaginalis isolates.

The gut protozoan parasite, Giardia duodenalis, is the best characterized example of the most ancient eukaryotes, which are anaerobic and appear to be primitively amitochondrial. Apart from its obvious medical importance, Giardia is fascinating in its own right. Its prokaryotic-like anaerobic metabolism renders it selectively sensitive to some bacterial drugs, especially the nitroimidazoles, which are activated to form toxic radicals. Other features, including an enzyme that reduces oxygen directly to water, cysteine as the keeper of redox balance, a plasmid, and toxin-like genes are also distinctly prokaryotic-like. But, unlike prokaryotes, Giardia has a sophisticated, highly developed cytoskeleton, bounded nuclei, linear chromosomes capped with telomeric repeats, and telomere positional regulation of gene expression.

The protozoan parasite, Giardia duodenalis, shares many metabolic and genetic attributes of the bacteria, including fermentative energy metabolism which relies heavily on pyrophosphate rather than adenosine triphosphate and as a result contains two typically bacterial glycolytic enzymes which are pyrophosphate dependent. Pyruvate decarboxylation and subsequent electron transport to as yet unidentified anaerobic electron acceptors relies on a eubacterial-like pyruvate:ferredoxin oxidoreductase and an archaebacterial/eubacterial-like ferredoxin. The presence of another 2-ketoacid oxidoreductase (with a preference for alpha-ketobutyrate) and multiple ferredoxins in Giardia is also a trait shared with the anaerobic bacteria. Giardia pyruvate:ferredoxin oxidoreductase is distinct from the pyruvate dehydrogenase multienzyme complex invariably found in mitochondria. This is consistent with a lack of mitochondria, citric acid cycle, oxidative phosphorylation and glutathione in Giardia. Giardia duodenalis actively consumes oxygen and yet lacks the conventional mechanisms of oxidative stress management, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. In their place Giardia contains a prokaryotic H2O-producing NADH oxidase, a membrane-associated NADH peroxidase, a broad-range prokaryotic thioredoxin reductase-like disulphide reductase and the low molecular weight thiols, cysteine, thioglycolate, sulphite and coenzyme A. NADH oxidase is a major component of the electron transport pathway of Giardia which, in conjunction with disulphide reductase, protects oxygen-labile proteins such as ferredoxin and pyruvate:ferredoxin oxidoreductase against oxidative stress by maintaining a reduced intracellular environment. As the terminal oxidase, NADH oxidase provides a means of removing excess H+, thereby enabling continued pyruvate decarboxylation and the resultant production of acetate and adenosine triphosphate. A further example of the bacterial-like metabolism of Giardia is the utilisation of the amino acid arginine as an energy source. Giardia contain the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, but not in other eukaryotes apart from trichomonads and Chlamydomonas reinhardtii. The pathway includes substrate level phosphorylation and is sufficiently active to make a major contribution to adenosine triphosphate production. Two enzymes of the pathway, arginine deiminase and carbamate kinase, are rare in eukaryotes and do not occur in higher animals. Arginine is transported into the trophozoite via a bacterial-like arginine:ornithine antiport. Together these metabolic pathways in Giardia provide a wide range of potential drug targets for future consideration.

International audience; Metronidazole (MTR) is frequently used for the treatment of Blastocystis infections, but with variable effectiveness, and often with treatment failures as a possible result of drug resistance. We have developed two Blastocystis MTR-resistant (MTRR) subtype 4 WR1 lines (WR1-M4 and WR1-M5), with variable susceptibility to a panel of anti-protozoal agents including various 5-nitroimidazoles, nitazoxanide and furazolidone. WR1-M4 and WR1-M5 were developed and assessed over an 18-month period and displayed persistent MTR resistance, being more than 2.5-fold less susceptible to MTR than the parent isolate. The MTRR lines grew with a similar g time to WR1, but were morphologically less consistent with a mixture of size. All Blastocystis isolates and the MTRR lines were most susceptible to the 5-nitroimidazole drug ronidazole. WR1-M5 was apparently cross-resistant to satranidazole and furazolidone, and WR1-M4 was cross-resistant to nitazoxanide. These MTRR lines now provide a valuable tool for the continued assessment of the efficacy and mechanism of action of new and established drugs against a range of Blastocystis sp. subtypes, in order to identify a universally effective drug and to facilitate understanding of the mechanisms of drug action and resistance in Blastocystis.

Giardiasis is a worldwide disease that can cause serious morbidity. Metronidozole is the current recommended drug for treatment, and is mostly still effective. However, Giardia duodenalis , the causative agent, is capable of developing resistance to high levels of metronidozole and other drugs, in vitro , via a number of mechanisms. Resistance, in vivo , has been reported and many cases of treatment failure have been variously attributed to a number of causes, including resistance. Here, Jacqueline and Peter Upcroft ask: is this the beginning of another chapter of drug resistance? or is the situation likely to remain as a ‘few refractory cases'? Should we wait to find out or can we act positivelyto avert the possibility of yet another valuable drug in our limited pharmacopoeia becoming obsolete?

Blastocystis is an emerging protistan parasite of controversial pathogenesis. Although metronidazole (Mz) is standard therapy for Blastocystis infections, there have been accumulating reports of treatment failure, suggesting the existence of drug-resistant isolates. Furthermore, very little is known about Blastocystis susceptibility to standard antimicrobials. In the present study, we established resazurin and XTT viability microassays for Blastocystis spp. belonging to subtypes 4 and 7, both of which have been suggested to represent pathogenic zoonotic subtypes. The optimized resazurin assay was used to screen a total of 19 compounds against both subtypes. Interestingly, subtype 7 parasites were resistant to Mz, a 1-position-substituted 5-nitroimidazole (5-NI), while subtype 4 parasites were sensitive. Some cross-resistance was observed to tinidazole, another 1-position 5-NI. Conversely, subtype 4 parasites were resistant to emetine, while subtype 7 parasites were sensitive. Position 2 5-NIs were effective against both subtypes, as were ornidazole, nitazoxanide, furazolidone, mefloquine, quinicrine, quinine, cotrimoxazole (trimethoprim-sulfamethoxazole), and iodoacetamide. Both subtypes were resistant to chloroquine, doxycycline, paromomycin, ampicillin, and pyrimethamine. This is the first study to report extensive variations in drug sensitivities among two clinically important subtypes. Our study highlights the need to reevaluate established treatment regimens for Blastocystis infections and offers clear new treatment options for Mz treatment failures.

The gut protozoan parasite, Giardia lamblia (Assemblage A), has 5 major chromosomes, 1 of which is 2 Mb, as determined from gel separations of whole chromosomes. We originally published a physical map of this chromosome and, now, using the sequence data from 46 chromosome-specific probes, have produced a sequence map of the 2 Mb chromosome. Comparison of the probe sequences with the Giardia genome database (http://GiardiaDB.org) has identified 4 scaffolds (CH991771, CH991780, CH991782, and CH991767) belonging to the 2 Mb, Assemblage A, chromosome. Because of the density of probe sequences, we have been able to predict the orientation of the scaffolds and have identified erroneous inclusions in scaffold CH991767. Exclusion of erroneously included sequences resulted in a 1.96 Mb chromosome sequence. This study brings together experimental data and the GiardiaDB data to compile the sequence of a whole chromosome.

Two genotypes, assemblages A and B, of the pathogenic gut protozoan parasite Giardia lamblia infect humans. Symptoms of infection range from asymptomatic to chronic diarrhea. Giardia chromosomes have long been characterized but not until the publication of the first Giardia genome sequence was chromosome mapping work, commenced nearly two decades ago, completed. Initial mapping studies identified and ordered Not I chromosome segments (summating to 1.8 Mb) of the estimated 2 Mb chromosome 3. The resulting map was confirmed with the release of the Giardia genome sequence and this revitalized mapping. The result is that 93% of the WB isolate genome sequence has now been assigned to one of five major chromosomes, and community access to these data has been made available through GiardiaDB, the database for Giardia genomes.

A 3000 bp cDNA insert (G6/1) from Giardia duodenalis, cloned into Escherichia coli is located on chromosome 3 of Giardia stocks which have 3 chromosomes detectable by field-inversion gel electrophoresis in the range 650–800 kb and on chromosome 3 and/or 4 of stocks with 4 chromosomes in this size range. The loss of this sequence from chromosome 4 but not chromosome 3 was associated with the induction of drug resistance in a previously sensitive laboratory stock. G6/1 appears to represent a single copy gene in Giardia as determined by hybridization of the probe to cleaved genomic DNA. Furthermore, the sequences flanking at least 12 kb of G6/1 are the same when G6/1 appears on both chromosomes 3 and 4. The cDNA encodes a protein associated with the nuclei of trophozoites during some stages of growth of the parasite. In a non-dividing culture, the antigen is associated with the nuclei of about 30% of trophozoites and fewer in a dividing culture. Three Giardia stocks with obvious chromosome rearrangements, which grow poorly and fail to divide normally, apparently lack the DNA sequence G6/1.

Analysis of 10 stocks of Blastocystis hominis isolated from human stools revealed two discrete groups of organisms. Proteins of the two groups were immunologically distinct and hybridization with random probes generated from the DNA of one stock showed that the DNA content of the two groups was different. Further studies are required to determine whether these should be classified as discrete species or whether these groups are epidemiologically significant.

Evidence for drug resistance in giardiasis is reviewed and biochemical studies undertaken to determine the basis for this resistance are discussed. Metronidazole and furazolidone, which produce toxic radicals within the cell, have different biochemical mechanisms of action. Resistance to metronidazole is negatively correlated with the intracellular concentration of pyruvateferredoxin oxidoreductase leading to a concomitant decrease in the uptake of free metronidazole into the cell, while resistance to furazolidone appears to be due to an increase in thiol cycling enzymes. At the molecular level resistance to metronidazole is associated with DNA changes. DNA probes which hybridize with specific chromosomes and repetitive sequences indicate that rearrangements both at the chromosome and repetitive DNA level occurred concurrently with the development of metronidazole resistance. The problems of cross-resistance and treatment failures that occur in the absence of resistance are additional difficulties which have important implications for the management of individual patients. New drugs such as azithromycin, while showing great variation in activity against different stocks may be useful in treating some refractory cases of giardiasis. In the community, it is important to recognize the occurrence and spread of drug resistant Giardia, and markers, such as DNA probes, provide methods to monitor potential epidemics and the spread of drug resistant Giardia.

Objectives: Two of the major diagnostic methods for Trichomonas vaginalis. wet mount and culture, rely on the continued viability of the organism. Methods to increase the viability of T. vaginalis in urine are needed. Goal: The goal of this study was to develop a method that increases the time of viability of T. vaginalis in urine. Study Design: Urine samples were inoculated with trichomonads, held at either room temperature or 37 degrees C, and processed through a column and frit, which was then placed in either a tube of culture medium containing antibiotics or a TV InPouch. Results: The column and polyethylene frit system was found to increase the duration of viability for T. vaginalis from urine specimens at least 6-fold. Conclusion: This novel method, which uses a column and frit system to increase the duration of viability of the organism, has the potential to increase the sensitivity of diagnostic tests.

This meeting, entitled `Future Chemotherapeutic Targets for the Anaerobic Protozoa', followed a very successful 10th ICOP, and was held in Brisbane Australia, 28–30 July 1997. The aim was to identify avenues of research that are likely to lead to new therapeutics to treat or prevent diseases caused by the anaerobic protozoa. We would like to thank QIMR for the venue and Quantum Scientific, BioRad, GeneSearch, New England Biologicals and Queensland Medical Laboratories for supporting this meeting.

We report on a new experimental technique suitable for measurement of light-activated processes, such as fluorophore transport. The usefulness of this technique is derived from its capacity to decouple the imaging and activation processes, allowing fluorescent imaging of fluorophore transport at a convenient activation wavelength. We demonstrate the efficiency of this new technique in determination of the action spectrum of the light mediated transport of rhodamine 123 into the parasitic protozoan Giardia duodenalis.

Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed.

Objective Trichomonas vaginalis, the causative agent of human trichomoniasis, is the most prevalent non-viral sexually transmitted infection and has been associated with increased risk of HIV transmission, making detection and treatment a global health priority. In this study, we evaluate the population genomics of globally distributed clinical isolates to characterise genetic diversity and identify population structure. Methods We use a panel of 21 microsatellite and three single copy gene markers to evaluate the population genomics of 18 clinical isolates collected from female patients attending New York City STD clinics in 2008, as well as 177 extant isolates collected from the USA, Mexico, Chile, Italy, Southern Africa, Papua New Guinea, and Australia. We use a panel of population genetic tools including Arlequin 3.11 and FSTAT to calculate expected heterozygosity (HE) and population differentiation (FST) statistics. To infer population structure, we use STRUCTURE 2.2, Network 4.516, and SeaView 4.2.4. We test for significance in clinical and demographic variables (χ2 and t-tests) using JMP Genomics 5.0. Results We detect significant genetic diversity within the species (HE=0.66) that is observed across global regions (range 0.52–0.67), and find that a two-phylotype population structure is maintained globally with few instances in population differentiation defined by geographical origin. This two-phylotype structure is further supported by minimum spanning networks, hierarchical clustering and phylogenies inferred from three single copy genes. These two phylotypes appear at nearly equal frequencies globally. Defining characteristics of the phylotypes include significantly different prevalence of T vaginalis virus infection (3% in phylotype 1 and 73% of phylotype 2 (p Conclusions We detect clear population structure and high levels of genetic diversity from global T vaginalis isolates. This preliminary data suggests that phylotypes differ phenotypically. An accurate survey of genetic diversity and population structure will facilitate responsible selection of drug and/or vaccine targets for future treatments, and will enable better understanding of virulence factors contributing to the wide range of severity of symptoms associated with trichomoniasis.

SUMMARYQuinacrine resistance has been induced in 3 Giardia duodenalis laboratory stocks and 4 lines resistant to other drugs. The quinacrine-resistant lines were maintained in normally lethal levels of 5 µM quinacrine and some lines are viable in 20 µM levels of the drug. Fluorescence studies indicated that quinacrine was taken up by sensitive cells but was actively excluded from resistant trophozoites. The nuclei were not a site of drug accumulation and no specific fluorescence in the trophozoite could be attributed to any structure. Blebs of concentrated drug appeared prior to disintegration of the membrane of drug-sensitive trophozoites exposed to drug overnight. Parasite lines already resistant to furazolidone adapted more readily to quinacrine exposure than drug-sensitive stocks. This multiple drug resistant phenotype was not as marked with metronidazole-resistant lines.

SUMMARYA gene,CRP136,from the intestinal protozoan parasiteGiardia duodenalis,expressed at a high level in a metronidazole-resistant line, encodes a 136 kDa protein with 23 copies of a 40 amino acid repeat. The protein is cysteine-rich and has the typical membrane-spanning region and CXXC amino acid motifs of a family ofGiardiacysteine-rich surface proteins (CRSPs). The repeat unit inCRP136, shares 57% homology with the gene encoding the precursor of the sarafotoxins, a group of snake toxins from the burrowing adder known to cause symptoms similar to those of humans acutely infected withGiardia.The sarafotoxins are low molecular weight sulphydryl cross-linked peptides which are proteolytically cleaved from a precursor polyprotein.CRP136has homology over the entire length of the sarafotoxin precursor, and the repeats are of the same length. ThusCRP136represents the first evidence for a potentialGiardiatoxin. The genomic copy number ofCRP136appears to be the same in both the parent and drug-resistant lines and expression of this gene, and at least one other, is associated with a conserved partial duplication, but not amplification, of one chromosome.

The nitroheterocyclic drugs have been available since the early 1960's for the treatment of anaerobic protozoa. The application of these drugs has widened since then and they are presently used to treat anaerobic pathogenic bacteria and protozoa. The activity of the nitroheterocyclic drugs depends on the all-important nitro group attached to the imidazole or furan ring. Although the nitro radicals, generated by reduction of the parent drugs, are similar for both families of nitroheterocyclics, the nitroimidazoles and the nitrofurans, the electron potential of each is different and thus the mechanism of action depends on different pathways. The nitroimidazoles depend on reduction by ferredoxin or flavodoxin. The nitrofurans require nitroreductase activity, but the natural substrate of these enzymes has not been identified. Increased use of nitroheterocyclic drugs, in response to drug resistance to other commonly used antibiotics, has in turn resulted in drug resistance to a number of nitroheterocyclic drugs. Bacteroides strains and other bacteria, including Helicobacter, have developed resistance. Among the protozoa, Trichomonas has developed resistance to metronidazole via a number of mechanisms, especially a decrease in drug reduction, as a result of alterations in the electron transport pathways. Resistance to both types of nitroheterocyclic drugs has been reported in Giardia. Although resistance to these drugs is not widespread, their increased use world-wide as a prophylaxis and in chemotherapy will inevitably result in increased resistance in organisms commonly found in asymptomatic infections, including Trichomonas, Giardia and Entamoeba. However, the variety of substitutions which can be attached to the ring structures has led to a great variety of drugs being synthesised, some of which are many-fold more active than the commonly prescribed nitroheterocyclics. With careful administration of currently available drugs and continued interest in synthesising more active compounds, we can optimistically expect to have useful nitroheterocyclic drugs available for some time.

Five Giardia stocks collected from animals and man in Alberta, Canada, were compared by DNA fingerprinting. Although many DNA bands were common to all stocks, differences in the DNA banding patterns were seen. These same stocks had previously been shown to be identical by restriction enzyme cleavage of genomic DNA.

Since the first description of Giardia by Antony van Leeuwenhoek in 1681, this parasite has proved to be enigmatic with a much more complicated epidemiology than originally envisaged. Even the nomenclature of this organism is confused. Despite numerous community prevalence studies, it was not until 1970 that the first proven report of waterborne transmission occurred. In 1981 the first common source outbreak due to contaminated food was recorded. It is also now recognized that venereal transmission occurs, especially among homosexual males. The faecal-oral route still remains the most important mode of infection despite the elucidation of these other methods of transmission. Recent developments in molecular biology especially DNA 'fingerprinting' and karyotyping which allow individual strain identification are being used to answer key epidemiological questions; for example are there truly pathogenic strains of Giardia, does drug resistance develop in vivo and is giardiasis a zoonotic disease? These and other molecular biological approaches will form the basis of research into the epidemiology of giardiasis and other intestinal protozoal infections until the end of the century and are certain to hold many surprises.

Three stocks of Blastocystis hominis were adapted to monophasic culture in minimal essential medium (MEM) and the chromosomes of these stocks separated by field inversion gel electrophoresis (FIGE). Ten-twelve chromosomes were distinguished in the electrophoretic karyotype of these three stocks over the range 200 kilobase pairs to > 1 megabase pairs. The karyotype of each stock was different. Three DNA probes, B10, B30 and B31, derived from the Netsky stock isolated in America were used as chromosome markers. Probe B10 hybridized to chromosomes of the same size in two of the stocks, one of which was isolated in the U.S.A. and the other in Queensland. B30 and B31 hybridized to a similar number of chromosomes of different sizes in these two stocks. The third stock, from Australia, did not hybridize at all with probes B10 and B30 and only weakly with probe B31.

A total of 37 Giardia stocks isolated from humans and 14 stocks derived from animal sources have been analysed for antigenic differences. Separation of the proteins of the stocks by polyacrylamide gel electrophoresis showed no major differences among the stocks. Immunoblotting of these antigens demonstrated some minor differences which were not correlated with geographic location, allozyme type, virulence or any other distinguishing characteristic of the stocks. Immunofluorescence tests using monoclonal antibodies revealed some differences between stocks but the monoclonal antibodies did not significantly inhibit growth in inhibition assays.