Rapid diagnosis of parasitic diseases: current scenario and future needs.

BACKGROUND: Parasitic diseases are one of the world's most devastating and prevalent infections, causing millions of morbidities and mortalities annually. In the past, many of these infections have been linked predominantly to tropical or subtropical areas. Nowadays, however, climatic and vector ecology changes, a significant increase in international travel, armed conflicts, and migration of humans and animals have influenced the transmission of some parasitic diseases from 'book pages' to reality in developed countries. It has also been noted that many patients who have never travelled to endemic areas suffer from blood-borne infections caused by protozoa. In the light of existing knowledge, this new trend can be explained by the fact that in the process of migration a large number of asymptomatic carriers become a part of the blood bank donor and transplant donor populations. Accurate and rapid diagnosis represents the crucial weapon in the fight against parasitic infections. AIMS: To review old and new approaches for rapid diagnosis of parasitic infections. SOURCES: Data for this review were obtained through searches of PubMed using combinations of the following terms: parasitological diagnostics, microscopy, lateral flow assays, immunochromatographic assays, multiplex-PCR, and transplantation. CONTENT: In this review, we provide a brief account of the advantages and limitations of rapid methods for diagnosis of parasitic diseases and focus our attention on current and future research in this area. The approximate costs associated with the use of different techniques and their applicability in endemic and non-endemic areas are also discussed. IMPLICATIONS: Microscopy remains the cornerstone of parasitological diagnostics, especially in the field and low-resource settings, and provides epidemiological assessment of parasite burden. However, increased use and availability of point-of-care tests and molecular assays in modern era allow more rapid and accurate diagnoses and increased sensitivity in the identification of parasitic infections.

Helminth Microbiomes - A Hidden Treasure Trove?

There is increasing attention on the complex interactions occurring between gastrointestinal parasitic helminths and the microbial flora (microbiota) inhabiting the host gut. However, little is known about the occurrence, structure, and function of microbial populations residing within parasite organs and tissues. In this article, we argue that an in-depth understanding of the interplay between parasites and their microbiomes may significantly enhance current knowledge of parasite biology and physiology, and may lead to the discovery of entirely novel, anthelmintic-independent interventions against parasites and parasitic diseases.

The relationships between faecal egg counts and gut microbial composition in UK Thoroughbreds infected by cyathostomins.

A growing body of evidence, particularly in humans and rodents, supports the existence of a complex network of interactions occurring between gastrointestinal (GI) helminth parasites and the gut commensal bacteria, with substantial effects on both host immunity and metabolic potential. However, little is known of the fundamental biology of such interactions in other animal species; nonetheless, given the considerable economic losses associated with GI parasites, particularly in livestock and equines, as well as the global threat of emerging anthelmintic resistance, further explorations of the complexities of host-helminth-microbiota interactions in these species are needed. This study characterises the composition of the equine gut commensal flora associated with the presence, in faecal samples, of low (Clow) and high (Chigh) numbers of eggs of an important group of GI parasites (i.e. the cyathostomins), prior to and following anthelmintic treatment. High-throughput sequencing of bacterial 16S rRNA amplicons and associated bioinformatics and statistical analyses of sequence data revealed strong clustering according to faecal egg counts (P = 0.003). A trend towards increased populations of Methanomicrobia (class) and Dehalobacterium (genus) was observed in Clow in comparison with Chigh. Anthelmintic treatment in Chigh was associated with a significant reduction of the bacterial Phylum TM7 14 days post-ivermectin administration, as well as a transient expansion of Adlercreutzia spp. at 2 days post-treatment. This study provides a first known insight into the discovery of the intimate mechanisms governing host-parasite-microbiota interactions in equines, and sets a basis for the development of novel, biology-based intervention strategies against equine GI helminths based on the manipulation of the commensal gut flora.

Deep sequencing approach for investigating infectious agents causing fever.

Acute undifferentiated fever (AUF) poses a diagnostic challenge due to the variety of possible aetiologies. While the majority of AUFs resolve spontaneously, some cases become prolonged and cause significant morbidity and mortality, necessitating improved diagnostic methods. This study evaluated the utility of deep sequencing in fever investigation. DNA and RNA were isolated from plasma/sera of AUF cases being investigated at Cairns Hospital in northern Australia, including eight control samples from patients with a confirmed diagnosis. Following isolation, DNA and RNA were bulk amplified and RNA was reverse transcribed to cDNA. The resulting DNA and cDNA amplicons were subjected to deep sequencing on an Illumina HiSeq 2000 platform. Bioinformatics analysis was performed using the program Kraken and the CLC assembly-alignment pipeline. The results were compared with the outcomes of clinical tests. We generated between 4 and 20 million reads per sample. The results of Kraken and CLC analyses concurred with diagnoses obtained by other means in 87.5 % (7/8) and 25 % (2/8) of control samples, respectively. Some plausible causes of fever were identified in ten patients who remained undiagnosed following routine hospital investigations, including Escherichia coli bacteraemia and scrub typhus that eluded conventional tests. Achromobacter xylosoxidans, Alteromonas macleodii and Enterobacteria phage were prevalent in all samples. A deep sequencing approach of patient plasma/serum samples led to the identification of aetiological agents putatively implicated in AUFs and enabled the study of microbial diversity in human blood. The application of this approach in hospital practice is currently limited by sequencing input requirements and complicated data analysis.

Molecular characterization and phylogenetic inferences of Dermanyssus gallinae isolates in Italy within an European framework.

In order to investigate the genetic relationships between Dermanyssus gallinae (Metastigmata: Dermanyssidae) (de Geer) isolates from poultry farms in Italy and other European countries, phylogenetic analysis was performed using a portion of the cytochrome c oxidase subunit 1 (cox1) gene of the mitochondrial DNA and the internal transcribed spacers (ITS1+5.8S+ITS2) of the ribosomal DNA. A total of 360 cox1 sequences and 360 ITS+ sequences were obtained from mites collected on 24 different poultry farms in 10 different regions of Northern and Southern Italy. Phylogenetic analysis of the cox1 sequences resulted in the clustering of two groups (A and B), whereas phylogenetic analysis of the ITS+ resulted in largely unresolved clusters. Knowledge of the genetic make-up of mite populations within countries, together with comparative analyses of D. gallinae isolates from different countries, will provide better understanding of the population dynamics of D. gallinae. This will also allow the identification of genetic markers of emerging acaricide resistance and the development of alternative strategies for the prevention and treatment of infestations.

Bioinformatics meets parasitology.

The advent and integration of high-throughput '-omics' technologies (e.g. genomics, transcriptomics, proteomics, metabolomics, glycomics and lipidomics) are revolutionizing the way biology is done, allowing the systems biology of organisms to be explored. These technologies are now providing unique opportunities for global, molecular investigations of parasites. For example, studies of a transcriptome (all transcripts in an organism, tissue or cell) have become instrumental in providing insights into aspects of gene expression, regulation and function in a parasite, which is a major step to understanding its biology. The purpose of this article was to review recent applications of next-generation sequencing technologies and bioinformatic tools to large-scale investigations of the transcriptomes of parasitic nematodes of socio-economic significance (particularly key species of the order Strongylida) and to indicate the prospects and implications of these explorations for developing novel methods of parasite intervention.

Bioinformatic analysis of abundant, gender-enriched transcripts of adult Ascaris suum (Nematoda) using a semi-automated workflow platform.

Expressed sequence tag (EST) data representing transcripts with a high level of differential hybridization in suppressive-subtractive hybridization (SSH)-based microarray analysis between adult female and male Ascaris suum were subjected to detailed bioinformatic analysis. A total of 361 ESTs clustered into 209 sequences, of which 52 and 157 represented transcripts that were enriched in female and male A. suum, respectively. Thirty (57.7%) of the 'female' subset of 52 sequences had orthologues/homologues in other parasitic nematodes and/or Caenorhabditis elegans, 13 (25%) exclusively in other parasitic nematodes and nine (17.3%) had no match in any other organism for which sequence data are currently available; the C. elegans orthologues encoded molecules involved in reproduction as well as embryonic and gamete development, such as vitellogenins and chitin-binding proteins. Of the 'male' subset of 157 sequences, 73 (46.5%) had orthologues/homologues in other parasitic nematodes and/or C. elegans, 57 (37.5%) in other parasitic nematodes only, and 22 (14.5%) had no significant similarity match in any other organism; the C. elegans orthologues encoded predominantly major sperm proteins (MSPs), kinases and phosphatases, actins, myosins and an Ancylostoma secreted protein-like molecule. The findings of the present study should support further genomic investigations of A. suum.

A portrait of the "SCP/TAPS" proteins of eukaryotes--developing a framework for fundamental research and biotechnological outcomes.

A wide range of proteins belonging to the SCP/TAPS "family" has been described for various eukaryotic organisms, including plants and animals (vertebrates and invertebrates, such as helminths). Although SCP/TAPS proteins have been proposed to play key roles in a number of fundamental biological processes, such as host-pathogen interactions and defence mechanisms, there is a paucity of information on their genetic relationships, structures and functions, and there is no standardised nomenclature for these proteins. A detailed analysis of the relationships of members of the SCP/TAPS family of proteins, based on key protein signatures, could provide a foundation for investigating these areas. In this article, we review the current state of knowledge of key SCP/TAPS proteins of eukaryotes, with an emphasis on those from parasitic helminths, and undertake a comprehensive, systematic phylogenetic analysis of currently available full-length protein sequence data (considering characteristic protein signatures or motifs) to infer relationships and provide a framework (based on statistical support) for the naming of these proteins. This framework is intended to guide genomic and molecular biological explorations of key SCP/TAPS molecules associated with infectious diseases of plants and animals. In particular, fundamental investigations of these molecules in parasites and the integration of structural and functional data could lead to new and innovative approaches for the control of parasitic diseases, with important biotechnological outcomes.

Exploring transcriptional conservation between Ancylostoma caninum and Haemonchus contortus by oligonucleotide microarray and bioinformatic analyses.

In this study, we identified, using an established oligonucleotide microarray platform for the parasitic nematode Haemonchus contortus, transcripts that are 'conserved' between serum-activated and non-activated L3s of Ancylostoma caninum (aL3 and L3, respectively) and H. contortus by cross-species hybridization (CSH) at high stringency and conducted extensive bioinformatic analyses of the cross-hybridizing expressed sequence tags (ESTs). The microarray analysis revealed significant differential hybridization between aL3 and L3 for 32 molecules from A. caninum, of which 29 were shown to have homologues/orthologues in the free-living nematode Caenorhabditis elegans and/or A. caninum and the other three molecules had no homologues in current gene databases. 'Non-wildtype' RNAi phenotypes were recorded for 13 of the C. elegans homologues. A subset of 16 C. elegans homologues/orthologues (i.e. genes abce-1, act-2, C08H9.2, C55F2.1, calu-1, col-181, cpr-6, elo-2, asp-1, K07E3.4, rpn-2, sel-9, T28C12.4, hsb-1, Y57G11C.15 and ZK593.1) were predicted to interact genetically with a total of 156 (range 1-88) other genes. Gene ontology (GO) analysis of the interacting genes revealed that the most common subcategories were signal transduction (7%), intracellular protein transport and glycolysis (6.2%) within 'biological process'; nuclear (25.7%) and intracellular (19.8%) within 'cellular component'; and ATP-binding (14.4%) and protein-binding (8.4%) within 'molecular function'. The potential roles of key molecules in the two blood-feeding parasitic nematodes are discussed in relation to the known roles of their homologues/orthologues in C. elegans. The CSH approach used may provide a tool for the screening of genes conserved across a range of different taxa of parasites for which DNA microarray platforms are not available.

Molecular characterization and phylogenesis of Steganinae (Diptera, Drosophilidae) inferred by the mitochondrial cytochrome c oxidase subunit 1.

The subfamily Steganinae (Diptera, Drosophilidae) includes flies which display zoophilic feeding behaviour in the larval and/or adult stages, some of which act as vectors of Spirurida eyeworms, which infect both carnivores and humans. To date, the taxonomy and phylogeny of the subfamily Steganinae has been studied only superficially and many aspects of their systematics remain unresolved. Thus, the present study aimed to provide a molecular dataset to facilitate the identification and phylogenetic analysis of Steganinae species based on partial ( approximately 700 basepairs) mitochondrial cytochrome c oxidase subunit 1 (cox1) sequences. A total of 134 flies belonging to 13 species and eight genera of Steganinae were subjected to molecular and phylogenetic analyses. The mean nucleotide variation within the Steganinae subfamily was 8.1%, with a variation within genera for which more than one species was examined ranging from 1.6% (in Phortica spp.) to 21.8% (in Amiota spp.). Interspecific pairwise divergence ranged from 1.6% (Phortica variegata vs. Phortica semivirgo) to 24.8% (Cacoxenus indagator vs. Amiota alboguttata) and intraspecific variation ranged from 0% to 1%. Seventy of the 233 amino acids were variable, including 26 parsimony informative sites and 44 singleton sites, with some highly conserved residues identified within the genera Stegana and Amiota. Parsimony and maximum likelihood-based phylogenetic analyses provided strong support for the genus Phortica, phylogenetically distinct from the genus Amiota. Gitona distigma was placed in an unresolved position adjacent to the outgroup taxa, Drosophila yakuba and Drosophila melanogaster. The molecular data reported here represent the first molecular dataset based on cox1 of Steganinae flies and provide a base for further investigations into the evolutionary relationships among this little-studied subfamily.

DNA technological progress toward advanced diagnostic tools to support human hookworm control.

Blood-feeding hookworms are parasitic nematodes of major human health importance. Currently, it is estimated that 740 million people are infected worldwide, and more than 80 million of them are severely affected clinically by hookworm disease. In spite of the health problems caused and the advances toward the development of vaccines against some hookworms, limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of hookworms is central to their effective control. While traditional diagnostic methods have considerable limitations, there has been some progress toward the development of molecular-diagnostic tools. The present article provides a brief background on hookworm disease of humans, reviews the main methods that have been used for diagnosis and describes progress in establishing polymerase chain reaction (PCR)-based methods for the specific diagnosis of hookworm infection and the genetic characterisation of the causative agents. This progress provides a foundation for the rapid development of practical, highly sensitive and specific diagnostic and analytical tools to be used in improved hookworm prevention and control programmes.

First reports of autochthonous eyeworm infection by Thelazia callipaeda (Spirurida, Thelaziidae) in dogs and cat from France.

Thelazia callipaeda (Spirurida, Thelaziidae) is a small nematode living in the conjunctival sac of domestic and wild carnivores, rabbits and humans causing lacrimation, epiphora, conjunctivitis, keratitis and even corneal ulcers. The first autochthonous cases of thelaziosis affecting four dogs and one cat living in South Western France (Dordogne area) are reported and described. Nematodes recovered from the animals were morphologically identified as T. callipaeda and a partial region of the cytochrome oxidase c subunit 1 gene (cox1) was amplified by PCR from nematode specimens (from two dogs and the cat). In each case, this was shown to have an identical sequence to the haplotype 1 (h1) of T. callipaeda. So far, the arthropod acting as intermediate host of T. callipaeda eyeworms has not been identified in France although it might be Phortica variegata (Steganinae, Drosophilidae) as recently described in Italy.

Phortica variegata as an intermediate host of Thelazia callipaeda under natural conditions: evidence for pathogen transmission by a male arthropod vector.

Knowledge about Phortica variegata (Drosophilidae, Steganinae), the intermediate host of the eyeworm Thelazia callipaeda (Spirurida, Thelaziidae), is confined to experimental studies. To investigate the role P. variegata plays in the transmission of T. callipaeda under natural conditions, the population dynamics of these flies in the natural environment and their feeding preferences (on vegetables and/or animal lachrymal secretions) were examined. From April to November 2005, a total number of 969 (557 males and 412 females) P. variegata flies were collected weekly in a region of southern Italy with a history of canine thelaziosis. The flies were identified and dissected or subjected to a PCR assay specific for a region within the ribosomal ITS-1 DNA of T. callipaeda. The zoophilic preferences of P. variegata were assessed by collecting flies around the eyes of a person or around a fruit bait. Seven hundred and twenty flies (398 males and 322 females) were dissected under a stereomicroscope; 249 flies (158 males and 91 females) that died prior to the dissection were subjected to molecular investigation. Only P. variegata males were infected with larval T. callipaeda both at dissection (six, 0.83%) and with the specific PCR (seven, 2.81%), representing a total percentage of 1.34% flies infected. Interestingly, only males were collected around the eyes, compared with a male/female ratio of 1:4 around the fruit. This survey indicated that P. variegata males act as intermediate hosts of T. callipaeda under natural conditions in Europe. Both the zoophilic behaviour of P. variegata males on lachrymal secretions and their role as vector of T. callipaeda have been discussed as they represent a peculiarity in medical and veterinary entomology. The synchrony between the fly population dynamics and the biology of the nematode in the definitive host provides an interesting model for exploring the co-evolution of Thelazia spp. with their hosts.

The zoophilic fruitfly Phortica variegata: morphology, ecology and biological niche.

Flies belonging to the subfamily Steganinae (Drosophilidae) display unusual zoophilic feeding habits at the adult and/or larval stage. Phortica variegata (Fallén) feeds on tears or eye liquid around the eyes of humans and carnivores. When feeding it is a potential vector of Thelazia callipaeda (Railliet and Henry) eyeworms. Adult and larval stages of this fly may be easily confused with other species belonging to the same genus, and little is known on the biology and ecology of P. variegata. In April-November 2005, a total of 969 P. variegata were collected in an area with a high prevalence of canine thelaziosis. The number of flies collected weekly was then related to climatic and environmental parameters (e.g. temperature, relative humidity and total rainfall) recorded daily at the collection site. The highest number of Phortica were collected during July-August. The sex ratio (number of males : females) rose from approximately 0.5 during May-July, to approximately 3.0 in August and 181 during September-October. Distributional data, representing 242 sites at which P. variegata has been collected in Europe, were analysed using a desktop implementation of the genetic algorithm for rule-set prediction (GARP) to model ecological requirements across Europe, as well as in Italy. P. variegata is shown to be mainly active at 20-25 degrees C and 50-75% RH. The ecological niche model suggests with a high degree of confidence that large areas of Europe are likely to represent suitable habitat for this species, mostly concentrated in central Europe. The results reported here contribute basic knowledge on the ecology and geographical distribution of P. variegata flies, which will be fundamental to gaining a better understanding of their role as vectors of human and animal pathogens.

Nematode biology and larval development of Thelazia callipaeda (Spirurida, Thelaziidae) in the drosophilid intermediate host in Europe and China.

Thelazia callipaeda, commonly known as the 'oriental eyeworm', has been recently reported in Italy and other European countries. The insect/s that act as intermediate hosts and details of larval development inside the vector remain unclear. In order to (1) demonstrate the species of fly that may act as vector/s for T. callipaeda in southern Italy (Site A) and China (Site B) and (2) describe the larval development of the nematode in the body of flies, 847 Phortica (Drosophilidae) flies were collected from the above two sites, each with a history of human and/or canine thelaziosis. Flies were identified as Phortica variegata (245 - site A) and Phortica okadai (602 - site B), experimentally infected by 1st-stage larvae (L1), kept at different temperatures and dissected daily until day 180 post-infection (p.i.). Dead flies from site A were subjected to specific polymerase chain reaction (PCR) assay to detect T. callipaeda. To demonstrate the role of Phortica as vectors of T. callipaeda, 3rd-stage larvae (L3) recovered from the proboscis of flies were deposited onto the cornea of the eyes of dogs and rabbits. Following dissection, 3 (2.9%) of P. variegata in site A were found to be infected by L3 in the proboscis on days +14, +21 and +53 p.i., compared with 26 (18.4%) of Phortica flies recorded as being positive by PCR. Sequences from positive PCR products were 99% identical to sequences of the corresponding species available in GenBank (AY207464). At site B, 106 (17.6%) of 602 dissected P. okadai were found to be infected by T. callipaeda larvae (different stages) and in total 62 L3 were recovered from the proboscis of 34 (5.6%) flies. The shortest time in which L3 were found was at day +14, +17, +19, and +50 p.i. respectively, depending on the environmental temperatures. Of 30 flies overwintered for 6 months, 6 L3 were detected at day +180 p.i. in 3 flies (10%). The biology of larval development was reconstructed on the basis of the dissection of 602 P. okadai-infected flies and the morphology of larval stages in the insect body described. The present work provides evidence that P. variegata and P. okadai act as vectors for T. callipaeda in southern Europe and in China, respectively. The phenomenon of overwintering is described here for the first time for T. callipaeda and discussed. Finally, the relationship between T. callipaeda and its fly vector is considered in light of disease prophylaxis and to model its dissemination into habitats and environments favourable to Phortica flies.