Ready-to-eat salads and berry fruits purchased in Italy contaminated by Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica.

Ready-to-eat (RTE) salads and berries are increasingly consumed in industrialized countries. These products can be contaminated by pathogenic parasites that have been responsible for foodborne outbreaks worldwide. In Italy, there are few data on contamination of RTE salads and berries with parasite transmission stages and this requires more-in-depth investigations. To estimate the prevalence of contamination with Cryptosporidium spp. and Giardia duodenalis in these fresh products, a total of 324 packages of local RTE mixed salads - belonging to three different industrial brands - and 324 packages of berries - blueberries from Peru, blackberries from Mexico, raspberries from Italy - were bought from supermarkets located in the Provinces of Bari and Foggia, Apulia, Italy. A pool size of nine packages was chosen and a total of 72 pools were processed in the whole year. After washing, the pellets were examined by microscopy (FLOTAC) and tested using conventional simplex PCR, targeting Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp., and sequencing. Several Cryptosporidium species and Giardia duodenalis assemblages, some of which are of potential zoonotic relevance, as well as Entamoeba spp., were identified in both matrices. By microscopy, Giardia-like cysts in local raspberries and Entamoeba-like cysts in imported blueberries were detected. Giardia duodenalis (Assemblages A, B and E) and Entamoeba histolytica were molecularly confirmed with overall prevalences of 4.6% (95% C.I. 3.0-6.8) and 1% (95% C.I. 0.3-2.1), respectively. Molecular methods identified Cryptosporidium ryanae, Cryptosporidium bovis, Cryptosporidium xiaoi, and Cryptosporidium ubiquitum in both matrices, with a prevalence of 5.1% (95% C.I. 3.3-7.3). A distinct seasonality in prevalence was observed for G. duodenalis, with most positives occurring in spring, whereas Cryptosporidium showed no significant seasonal variations. These results highlight that inadequate management of fresh produce, both locally produced and imported, along the food chain may have the potential for consequences on human health.

A validated method to identify Echinococcus granulosus sensu lato at species level.

The zoonotic tapeworm Echinococcus granulosus sensu lato (s.l.) represents a species complex encompassing multiple causative agents of cystic echinococcosis, a neglected tropical disease affecting more than one million people in the world. At least eight genotypes, grouped in five species, are currently recognized within this species complex, and they differ in terms of relative public health impact. Here we present a molecular method that first identifies the common E. granulosus sensu stricto (s.s.) (genotypes G1 and G3) based on a PCR-RFLP assay, and can further identify the remaining species based on a multiplex PCR assay. We demonstrate the applicability of the method to DNA extracted from parasitic cyst material of human and animal origin, preserved in ethanol or frozen. The method has been developed and validated at the European Union Reference Laboratory for Parasites (EURLP), according to the ISO/IE 17025.

Occurrence and Molecular Typing of Giardia psittaci in Parakeets in Germany-A Case Study.

A grey-hooded parakeet (Psilopsiagon aymara) and two budgerigars (Melopsittacus undulatus) from different owners presented with decreased activity, vomitus, and diarrhea. A microscopic examination of feces showed trophozoites of the protozoan flagellate Giardia. A commercial immunochromatographic dipstick test for Giardia sp. antigens confirmed the infection. These findings were assured by PCR of the small subunit ribosomal RNA (SSU rRNA) gene and coproantigen ELISA. Sequencing of PCR products of the SSU rRNA (292 bp) and ß-giardin genes (511 bp) identified Giardia psittaci as the species involved. Therefore, our results show that a GSA 65-based coproantigen ELISA, which was established for diagnosis of Giardia duodenalis is applicable for the detection of G. psittaci. A treatment with ronidazole was started. Additionally, fecal examination and dissection of the dead birds revealed coinfection with the fungal pathogen Macrorhabdus ornithogaster. One budgerigar survived and repeatedly tested negative after treatment with ronidazole. The described cases indicate that a single infection with G. psittaci has a good prognosis, whereas the prognosis is poor when coinfections occur, especially with M. ornithogaster.

Reporte de caso- Presentación y tipificación molecular de Giardia psittaci en periquitos en Alemania: Un estudio de caso. Un periquito catita aimará (Psilopsiagon aymara) y dos periquitos australianos (Melopsittacus undulatus) de diferentes propietarios presentaron actividad disminuida, vómito y diarrea. El examen microscópico de las heces mostró trofozoitos del protozoo flagelado Giardia. Una prueba de tira reactiva inmunocromatográfica comercial para antígenos de Giardia sp. confirmó la infección. Estos resultados fueron confirmados por PCR para el gene de ARN de la subunidad pequeña ribosomal (SSU rRNA) y por ELISA de coproantígeno. La secuenciación de los productos de PCR del ARNr de SSU (292 pb) y los genes de ß-giardina (511 pb) identificaron a Giardia psittaci como la especie involucrada. Por lo tanto, estos resultados muestran que el método de ELISA de coproantígeno basado en GSA 65, que se estableció para el diagnóstico de Giardia duodenalis, es aplicable para la detección de G. psittaci. Se inició un tratamiento con ronidazol. Además, el examen fecal y la disección de las aves muertas revelaron coinfección con el patógeno fúngico Macrorhabdus ornithogaster. Un periquito australiano sobrevivió y dio negativo repetidamente después del tratamiento con ronidazol. Los casos descritos indican que la infección única con G. psittaci tiene un buen pronóstico, mientras que el pronóstico es malo cuando ocurren coinfecciones, especialmente con M. ornithogaster. Abbreviations: GSA = Giardia-specific antigen; OD = optical density; rRNA = ribosomal ribonucleic acid; SSU = small subunit.

Human cryptosporidiosis in Europe.

Cryptosporidium has emerged as a significant cause of diarrhoeal disease worldwide, with severe health consequences for very young, malnourished children living in endemic areas and for individuals with highly impaired T-cell functions. In Europe, as elsewhere, the burden of disease has been difficult to measure as a result of the lack of appropriate, standardized surveillance and monitoring systems. The recent occurrence of large water- and foodborne outbreaks in several EU countries, as well as the results of many surveys of human and animal cryptosporidiosis, indicate that this parasite is widespread. Specific subtypes of the zoonotic Cryptosporidium parvum and the anthroponotic C. hominis are responsible for the majority of human cases in Europe. No treatment is currently available to clear the infection, but recent progress in genetic engineering of the parasite, coupled with advances in genomics, have opened important avenues for future research. Here we explore the possible reasons for underascertainment of cryptosporidiosis and the importance of accurate diagnosis in clinical management, the epidemiology of human cryptosporidiosis and key messages from recent outbreaks to highlight important interventions and emerging public health issues.

A comparison of sequence and length polymorphism for genotyping Cryptosporidium isolates.

Simple sequence repeat markers have played an important role in elucidating the epidemiology of human and animal cryptosporidiosis. The drawback of sequence length polymorphisms is that nucleotide substitutions remain undetected. As some laboratories have opted for using length polymorphisms, while others have relied on sequencing, there is a need to compare both methods. We used a diversified set of unique length polymorphisms and matching nucleotide sequences to assess the ability of each genotyping protocol to discern clusters of related Cryptosporidium parvum isolates. We found a weak correlation between the two distance measures for individual markers. This analysis was extended to four-locus genotypes based on sequence length data or concatenated sequences from the same loci. We interrogated these data to assess whether one would reach the same conclusions regardless of the genotyping method. Clusters of isolates generated with the concatenated sequences were not observed with amplicon length, indicating that inferences on the structure of a Cryptosporidium population depend on the genotyping method. Moreover, isolate clusters derived from concatenated sequences were dependent on the algorithm used to calculate distances. These results emphasize the need for harmonizing genotyping tools, not only by selecting informative markers, but also by standardizing the entire genotyping method.

Cryptosporidium parvum Caused a Large Outbreak Linked to Frisée Salad in Finland, 2012.

Over 250 individuals fell ill in five outbreaks caused by Cryptosporidium parvum in Finland, October-November 2012. The cases were connected by lunch meals at restaurants in four different cities. In two outbreaks, the same C. parvumIIdA17G1 subtype was found in patients' stool samples which supports a single source of infection. Frisée salad was the only common food item served at the restaurants, and consumption of lunch salad containing the frisée salad was associated with the illness. Lunch customers who responded that they had eaten lunch salad were three times more likely to have become ill than those who had not answered whether they had eaten the salad or not (RR 2.66; 95% Cl 1.02-6.9, P-value <0.01). Cryptosporidiosis should be considered as a causal agent in long-lasting watery diarrhoea combined with abdominal cramps, and clinical samples should be tested for Cryptosporidium at the same time bacteria and viruses are tested. Measures to prevent contamination of 'ready-to-eat vegetables' with Cryptosporidium oocysts and methods to test frozen food samples should be developed.

Detection and molecular characterization of Giardia and Cryptosporidium in common dolphins (Delphinus delphis) stranded along the Galician coast (Northwest Spain).

The ubiquitous protozoan parasites Giardia and Cryptosporidium have been detected from many species of captive and free-living wildlife, representing most mammalian orders. Twenty species of marine mammals have been reported to inhabit Galician waters and the region has one of the highest rates of stranding in Europe. Evidence from stranding, reported by-catches and sightings, suggests that the common dolphin (Delphinus delphis) is the most abundant cetacean on the Galician coast (Northwest Spain). The objective of this study was to detect and molecularly characterize isolates of Giardia and Cryptosporidium obtained from common dolphins stranded in this area. Between 2005 and 2012, sections of large intestine from 133 common dolphins stranded along the Galician coast were collected by the personnel of the Galician Stranding Network (Coordinadora para o Estudo dos Mamíferos Mariños, CEMMA). Using direct immunofluorescence antibody test (IFAT) and PCR amplification and sequencing of the SSU-rDNA, ß-giardin genes and the ITS1-5.8S-ITS2 region, Giardia and Cryptosporidium were detected in 8 (6.0%) and 12 samples (9.0%), respectively. In two samples, co-infection by both parasites was observed. The molecular characterization revealed the presence of Giardia duodenalis assemblages A (genotypes A1 and A2) and B and Cryptosporidium parvum in these samples. This constitutes the first study in which the presence of Giardia and Cryptosporidium has been investigated in common dolphins on the European Atlantic coast, and it is also the first report of C. parvum in this host. Our findings indicate that these animals could act as reservoir of these waterborne parasites or could be victims of the contamination originated by anthropogenic activities.

Occurrence and molecular typing of Giardia isolates in pet rabbits, chinchillas, guinea pigs and ferrets collected in Europe during 2006-2012.

A total of 1180 faecal samples (528 from rabbits, 531 from chinchillas and 121 from guinea pigs) collected during 2006-2012 by veterinarians in Germany and in other European countries were submitted to a diagnostic laboratory for Giardia testing by means of coproantigen ELISA. Of these samples, 40 rabbits (7.6 per cent), 326 chinchillas (61.4 per cent) and five guinea pigs (4.1 per cent ) were found to be positive. To gain insights into the genetic identity of Giardia in small mammals, ELISA-positive samples from 23 chinchillas, five ferrets, a rabbit, and a Desmarest's hutia were investigated by PCR and sequencing of fragments of the small subunit ribosomal DNA (ssu), the triose phosphate isomerase (tpi) and the ß-giardin (bg) genes. At the ssu locus, assemblage B was identified in 28 of 30 isolates, whereas assemblage A and D were each detected in one sample. The majority of isolates from chinchillas and those from ferrets had Giardia duodenalis sequences identical to sub-assemblages AI or BIV, based on either a single locus (tpi or bg) or multiple loci (tpi and bg). As sub-assemblages AI or BIV are associated with human infection, these results indicate that small mammals can act as reservoirs of cysts potentially infectious to humans.

Molecular typing of Giardia duodenalis isolates from German travellers.

Giardia duodenalis isolates from German travellers returning from tropical areas were characterised by PCR amplification and sequencing of fragments of the beta-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. Assignment of isolates to specific G. duodenalis assemblages was found to differ according to the marker used. Indeed, at the bg locus, assemblages A and B were identified, with a higher prevalence of the former over the latter, whereas at the tpi and gdh loci, most samples were classified as assemblage B. In agreement with previous studies, sequence analysis showed that assemblage B isolates have a higher genetic polymorphism than assemblage A isolates, and novel variants were described. The degree of polymorphism was shown in a graphical representation of the polymorphic sites generating a novel sequence, the heterogeneous positions common to assemblages A and B (double peaks), that may represent mixed assemblage infection and the heterogeneous positions detected at random sites. Notably, assemblage D, which is considered to be adapted to dogs, was found at the gdh locus in two samples originating from southern Asia, as novel genotypes. By comparing the geographical origin of the infected cases and the number of German travellers visiting the areas considered, India and west Africa appeared to be the areas associated to the highest risk of acquiring Giardia infection. The analysis of the geographical distribution of the genotypes did not suggest any particular geographical clustering pattern, but it may be useful to evaluate these results with a higher number of isolates. Most of the samples typed at the three markers could not be assigned unequivocally to either assemblage A or B, and this was confirmed also by a real-time PCR assay, using a set of assemblage-specific primers. The results of this study reinforce the notion that genetic exchanges and allelic sequence heterogeneity represent major obstacles towards understanding the epidemiology of giardiasis and that exposure to Giardia parasites in endemic areas often results in mixed infections in returning travellers.

Cases of cryptosporidiosis co-infections in AIDS patients: a correlation between clinical presentation and GP60 subgenotype lineages from aged formalin-fixed stool samples.

Nine cases of cryptosporidiosis co-infections in AIDS patients were clinically categorised into severe (patients 1, 3, 8 and 9), moderate (patients 4 and 5) and mild (patients 2, 6 and 7). Formalin-fixed faecal specimens from these patients were treated to obtain high quality DNA competent for amplification and sequencing of the 60-kDa glycoprotein (GP60) gene. Sequence analysis revealed that one patient was infected with Cryptosporidium hominis whereas the remaining eight patients were infected with C. parvum. Interestingly, the patients showing severe cryptosporidiosis harboured two subtypes within the C. parvum allelic family IIc (IIcA5G3 and IIcA5G3R2), whereas patients with moderate or mild infections showed various subtypes of the C. parvum allelic family IIa (IIaA14G2R1, IIaA15G2R1, IIaA17G3R1 and IIaA18G3R1). DNA extraction and genotyping of Cryptosporidium spp. is a challenging task on formalin-fixed stool samples, whose diagnostic outcome is age-dependent. The method herein reported represents a step forward routine diagnosis and improves epidemiology of HIV-related clinical cases. Due to the need to elucidate genetic richness of Cryptosporidium human isolates, this approach represents a useful tool to correlate individual differences in symptoms to subgenotyping lineages.

Multilocus genotyping of Cryptosporidium and Giardia in non-outbreak related cases of diarrhoea in human patients in Belgium.

Stool samples from Belgian patients suffering from abdominal pain and/or diarrhoea were examined for Cryptosporidium and Giardia. Cryptosporidium-positive samples were genotyped using the 70 kDa heat shock protein and the 60 kDa glycoprotein (GP60) genes: C. hominis was identified in 54.2% and C. parvum in 45.8% of the samples. Sequencing at the GP60 locus indicated that subgenotype IbA10G2 of C. hominis and subgenotype IIaA15G2R1 of C. parvum were the most prevalent, although several other subgenotypes were identified. For Giardia, sequencing at the beta-giardin, triose phosphate isomerase (TPI) and glutamate dehydrogenase (GDH) genes revealed assemblage B as the most prevalent (74.4%) in human patients. A high degree of heterogeneity was found, especially on the beta-giardin gene, and to a lesser extent on the GDH gene. Furthermore, using a novel species-specific PCR based on the TPI gene, mixed infections with both assemblage A and B were detected in a large number (32.4%) of human patients, which might have important epidemiological implications.

Risk factors associated with Cryptosporidium parvum infection in cattle.

A 2-year, cross-sectional study was conducted to identify risk factors for Cryptosporidium sp. infection in bovine farms in central Italy. Faecal samples were collected on 248 farms, from 2024 calves and analysed using ELISA and immunofluorescent assay (IFA) commercial kits. In all 101 samples confirmed to be positive with IFA, the aetiological agent was identified as Cryptosporidium parvumand a large genetic variability was detected by subtype analysis. The prevalence of farm infection ranged from 3.4% to 35.6%. Univariate analysis showed a number of putative risk factors, including the type of farm, stalling of calves, late supply of colostrum, number of heads and contact between calves and adults. However, multivariate analysis confirmed that the higher risk for calves was associated with housing calves separately from their dams, a characteristic practice of dairy herd, whereas calves being nursed by their dams, a characteristic of cow-calf herd resulted as a protective factor.

Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B.

Giardia duodenalis is a widespread parasite of mammalian species, including humans. Due to its invariant morphology, investigations of aspects such as host specificity and transmission patterns require the direct genetic characterisation of parasites from faecal samples. We performed a sequence analysis of four genes (ssrRNA, ß-giardin, glutamate dehydrogenase and triose phosphate isomerase) of 61 human isolates and 29 animal isolates. The results showed that multilocus genotypes (MLGs) can be readily defined for G. duodenalis isolates of assemblage A but not for assemblage B. Indeed, for assemblage A isolates, there was no evidence of intra-isolate sequence heterogeneity, and congruent genotyping results were obtained at the four genetic loci investigated. Sequence comparison and phylogenetic analysis showed that human-derived and animal-derived MLGs are different, and further indicated the presence of a new sub-assemblage (referred to as "AIII"), which was found exclusively in wild hoofed animals. On the other hand, there were variable levels of intra-isolate sequence heterogeneity (i.e., the presence of two overlapping nucleotide peaks at specific positions in the chromatograms, or "heterogeneous templates") in assemblage B isolates from humans and animals, and this prevented the unambiguous identification of MLGs. Furthermore, in five human isolates and one non-human primate isolate, the assignment to assemblage B was problematic, given that one of the four markers supported an assignment to assemblage A. These findings raise concerns about the interpretation of genotyping data based on single markers, and indicate the need to understand the mechanisms that are responsible for the differences between G. duodenalis assemblages A and B.

Distribution of Cryptosporidium parvum subtypes in calves in Germany.

Cryptosporidium DNA was extracted from 134 faecal specimens from pre-weaned calves from different German Federal States (age range, 3-15 days old), which tested positive for oocysts by microscopic analysis. The 18S rDNA gene and the oocyst wall protein gene (COWP) were used as targets for PCR and RFLP techniques. Cryptosporidium species were identified by using SspI, MboII and RsaI endonucleases for the digestion of 18S rDNA and COWP amplified fragments, respectively. In all samples, restriction patterns corresponding to Cryptosporidium parvum were identified, which is in agreement with abundant literature data indicating C. parvum as the most common species in pre-weaned calves. In order to estimate the genetic heterogeneity among C. parvum calf isolates, 53 samples chosen to represent different German Federal States were successfully subtyped by sequence analysis of the highly polymorphic 60-kDa glycoprotein gene. All isolates belonged to the allele IIa (with seven subtypes), with the exception of one isolate that belonged to the allele IId. Moreover, three novel subtypes of the allele family IIa have been found. This study confirms the utility of genotyping and subtyping tools in characterizing the transmission of Cryptosporidium spp. This is the first molecular epidemiological report about subtyping of Cryptosporidium bovine isolates in Germany.

Cryptosporidium and Giardia as foodborne zoonoses.

Cryptosporidium and Giardia are major causes of diarrhoeal disease in humans, worldwide and are major causes of protozoan waterborne diseases. Both Cryptosporidium and Giardia have life cycles which are suited to waterborne and foodborne transmission. There are 16 'valid'Cryptosporidium species and a further 33+ genotypes described. Parasites which infect humans belong to the Giardia duodenalis "type", and at least seven G. duodenalis assemblages are recognised. Cryptosporidium parvum is the major zoonotic Cryptosporidium species, while G. duodenalis assemblages A and B have been found in humans and most mammalian orders. In depth studies to determine the role of non-human hosts in the transmission of Cryptosporidium and Giardia to humans are required. The use of harmonised methodology and standardised and validated molecular markers, together with sampling strategies that provide sufficient information about all contributors to the environmental (oo)cyst pool that cause contamination of food and water, are recommended. Standardised methods for detecting (oo)cysts in water are available, as are optimised, validated methods for detecting Cryptosporidium in soft fruit and salad vegetables. These provide valuable data on (oo)cyst occurrence, and can be used for species and subspecies typing using appropriate molecular tools. Given the zoonotic potential of these organisms, epidemiological, source and disease tracking investigations involve multidisciplinary teams. Here, the role of the veterinarian is paramount, particularly in understanding the requirement for adopting comprehensive sampling strategies for analysing both sporadic and outbreak samples from all potential non-human contributors. Comprehensive sampling strategies increase our understanding of parasite population biology and structure and this knowledge can be used to determine what level of discrimination is required between isolates. Genetic exchange is frequent in C. parvum populations, leading to recombination between alleles at different loci, the generation of a very large number of different genotypes and a high level of resolution between isolates. In contrast, genetic exchange appears rare in Cryptosporidium hominis and populations are essentially clonal with far fewer combinations of alleles at different loci, resulting in a much lower resolution between isolates with many being of the same genotype. Clearly, more markers provide more resolution and high throughput sequencing of a variety of genes, as in multilocus sequence typing, is a way forward. Sub-genotyping tools offer increased discrimination, specificity and sensitivity, which can be exploited for investigating the epidemiology of disease, the role of asymptomatic carriers and contaminated fomites and for source and disease tracking for food and water contaminated with small numbers of (oo)cysts.

A novel Giardia duodenalis assemblage A subtype in fallow deer.

The molecular identification of species and genotypes of Giardia spp. infecting wild mammals represents the most reliable tool to understand the role played by these animals as reservoirs of cysts infectious for human and other animals. Of 139 fecal samples collected from fallow deer (Dama dama L.) hunted in a Natural Reserve of northern Italy, the prevalence of Giardia sp. was 11.5% (16 of 139 animals), and it was higher in fawns than in older animals. Fragments of the betagiardin and triose phosphate isomerase (tpi) genes were successfully polymerase chain reaction amplified and sequenced from 8 isolates. No sequence variation was observed between isolates at the 2 genetic loci. Sequence and phylogenetic analyses identified a Giardia duodenalis subtype that clusters with assemblage A isolates and that shows homologies of 98 and 97% at the beta-giardin and tpi loci, respectively, compared with the A1 subtype. Because the G. duodenalis subtype found in fecal samples of fallow deer has never been detected previously, its role as a pathogen for humans and domestic animals is unknown, but, considering its genetic distinctiveness, it is likely to be low.

Molecular epidemiology of human cryptosporidiosis.

Species within the genus Cryptosporidium are protozoan parasites that infect a wide range of vertebrates, and represent a significant cause of morbidity and mortality in those animals. In humans, cryptosporidiosis is a common cause of diarrhoeal disease with a global distribution. Unravelling the epidemiology of human infection has proven to be difficult, due to the existence of multiple transmission routes (person-to-person, animal-to-person, waterborne, foodborne and airborne transmission), and to the difficulties in identifying the different species using conventional criteria, such as oocyst morphology. The advent of molecular techniques has had a remarkable impact on the way the epidemiology of cryptosporidiosis can be studied. Molecular investigations have shown that the vast majority of human cases are caused by C. hominis and C. parvum. Interestingly, differences in geographical and temporal distribution, disease presentations and risk factors for infection have been identified for both C. hominis and C. parvum. Further, molecular analyses have revealed that other species, including C. meleagridis, C. felis, C. canis, C. suis, C. muris and two Cryptosporidium genotypes, can infect humans and may be linked to clinical disease, not only in immunocompromised but also in immunocompetent individuals.

Dientamoeba fragilis is more prevalent than Giardia duodenalis in children and adults attending a day care centre in Central Italy.

Giardia duodenalis is a well recognised enteropathogen, while Dientamoeba fragilis is rarely detected and consequently it is not recognised as an important human pathogen. In 2002-2003, a survey has been carried out on enteroparasites in faecal samples of outpatients attending a day care centre in the town of Perugia (Central Italy). To improve the detection level, at least three samples from each patient were collected at different days and within two hours from defecation. The coproparasitological examination has been carried out by direct microscopic examination, faecal concentration, and Giemsa and modified Ziehl-Nielsen stainings of faecal smears. The genotypes of Giardia duodenalis isolates were determined by PCR of the beta-giardin gene. Of 1,989 enrolled people (966 children, 1,023 adults), 165 persons (8.3%; 153 adults, 15.0%; 12 children, 1.2%), were positive for parasites, but only 1 12 adults (73.2% of those infected) and eight children (66.7% of those infected) harboured D. fragilis and G. duodenalis. Both the Assemblages A and B were detected in 18 G. duodenalis isolates examined at the beta-giardin gene. The higher prevalence of D. fragilis infections than that of G. duodenalis is probably related to the method used, a procedure, which is rarely followed in laboratories for the diagnosis of enteric parasites. These epidemiological data suggest that when faecal samples are examined after a period of time and without Giemsa staining, most D. fragilis infections goes undetected.

[New methods for the diagnosis of Cryptosporidium and Giardia]. / Nuovi metodi per la diagnosi di Cryptosporidium e Giardia.

The accurate identification of a parasite at the species and/or genotype level has major implications for various aspects of human and veterinary parasitology, including the diagnosis, the taxonomy, the treatment and the control. The advent of molecular techniques, in particular those based on the in vitro amplification of nucleic acids, has dramatically improved our ability to detect infections caused by parasites. To illustrate the progress in molecular diagnostics, Cryptosporidium and Giardia are used here as examples of parasites for which both the diagnosis and the taxonomy have traditionally been problematic. These protozoan parasites, while very different for many aspects of their biology, shares a complex series of transmission routes, including anthroponotic and zoonotic transmission, as well as waterborne and foodborne transmission. The resistant stages produced by Cryptosporidium and Giardia (oocysts and cysts, respectively) are remarkably stable, and can survive for weeks to months in the environment. Further, the infective dose is low, and infectious dose studies and models suggest that even a single oocyst or cyst carries some probability of causing an infection. Finally, most faeces that contain (oo)cysts end up in the environment and can be spread to foods by irrigation or by direct contact, and can persist in the water, as routine treatments eliminate only a fraction of these stages. This situation explains the growing interest towards the development of methods that allows such stages to be detected with the highest sensitivity and specificity. A variety of Polymerase Chain Reaction (PCR) assays have been described for both Cryptosporidium and Giardia. The choice of a particular assay mainly depends on the amount of information carried by the genetic locus under analysis. Indeed, some assays can be used to identify the different species within a genus, while others allowed to distinguish between isolates of the same species (genotypes), and some can even be used for both purposes. Post-PCR analyses are usually based on the direct sequencing of the amplification products, or on the digestion with endonucleases followed by gel electrophoresis of the restriction fragments. In the last few years, the molecular characterization of a large number of isolates, collected from infected hosts and from the environment, has considerably changed our view of the epidemiology of cryptosporidiosis and giardiasis. Indeed, several species/genotypes have been established as human pathogens, and the nature of the parasites present in the water and in food have been investigated, allowing a better understanding of the complex circulation of the parasites in the environment, that may eventually led to implemented control measures. Finally, phylogenetic analysis of several nuclear genes is having a major impact in the revision of the taxonomy of Cryptosporidium and Giardia. The main limitation of PCR is that it doesn't provide information on the viability and infectivity of the pathogen. To obtain additional information on these important aspects, indirect methods, such as inclusion/exclusion assays using vital dyes or the Reverse-Transcriptase PCR (RT-PCR), can be used. Since RT-PCR relies on the integrity of mRNA, which usually has very short half-life (seconds), its use is thought to provide a more closely correlated indication of viability status compared to DNA-based methods. RT-PCR assays usually target the heat shock protein (hsp) 70 gene. The rationale behind this choice is that hsps are known to be synthesized with a high level of efficiency in stressed organisms; therefore, when (oo)cysts are exposed to a thermal shock, the induction of heat shock response provides both a level of amplification to increase detection sensitivity and an index of viability. Moreover, with the recent introduction of real-time PCR, that allows the continuous monitoring of amplicon formation throughout the reaction, quantitative aspect of the infection could be studied with exquisite sensitivity. This will, for example, allow (1) to detect carrier states, (2) to determine the number of oocysts/cysts present in a sample, (3) to study quantitative aspects of gene expression during the various phases of the infection.