Evaluation of contingency management as a strategy to improve HCV linkage to care and treatment in persons attending needle and syringe programs: A pilot study.

BACKGROUND: A greater proportion of HCV-infected people who inject drugs (PWID) need to be linked to care for HCV antiviral treatment. This study sets out to evaluate the efficacy of contingency management (CM) for improving HCV linkage to care, treatment initiation, adherence, and cure for PWID recruited from a needle and syringe program. METHODS: Between March 2015 and April 2016, 20 participants were enrolled into the CM arm, and then subsequently enrolled 20 participants in the enhanced standard of care (eSOC) arm. Participants in the eSOC arm received an expedited appointment and a round-trip transit card. Participants enrolled in the CM arm received eSOC plus $25 for up to ten HCV clinical visits and $10 for each returned weekly medication blister pack. Adherence was measured via electronic blister packs. RESULTS: Overall the median age was 47 years; most were men (67%) and Hispanic (69%). There were no significant differences in demographic characteristics between participants in the study arms. In the CM arm 74% were linked to HCV care, compared to 30% in the eSOC arm (p = 0.01). In the CM arm, 75% (9/12) of treatment eligible participants initiated treatment, compared to 100%(4/4) in the eSOC arm (p = 0.53). All patients (9/9) achieved cure in the CM arm, as compared to 75% (3/4) of patients in the eSOC arm. There were no differences in adherence between study arms. CONCLUSIONS: In this pilot study, contingency management led to higher rates of HCV linkage to care for PWID, as compared to standard of care. CM should be considered as a possible intervention to improve the HCV treatment cascade for PWID.

Retention in buprenorphine treatment is associated with improved HCV care outcomes.

Persons who inject drugs, most of whom are opioid dependent, comprise the majority of the HCV infected in the United States. As the national opioid epidemic unfolds, increasing numbers of people are entering the medical system to access treatment for opioid use disorder, specifically with buprenorphine. Yet little is known about HCV care in patients accessing buprenorphine-based opioid treatment. We sought to determine the HCV prevalence, cascade of care, and the association between patient characteristics and completion of HCV cascade of care milestones for patients initiating buprenorphine treatment. We reviewed electronic health records of all patients who initiated buprenorphine treatment at a primary-care clinic in the Bronx, NY between January 2009 and January 2014. Of the 390 patients who initiated buprenorphine treatment, 123 were confirmed to have chronic HCV infection. The only patient characteristic associated with achieving HCV care milestones was retention in opioid treatment. Patients retained (vs. not retained) in buprenorphine treatment were more likely to be referred for HCV specialty care (63.1% vs. 34.0%, p<0.01), achieve an HCV-specific evaluation (40.8% vs. 21.3%, p<0.05), be offered HCV treatment (22.4% vs. 8.5%, p<0.05), and initiate HCV treatment (9.2% vs. 6.4%, p=0.6). Given the current opioid epidemic in the US and the growing number of people receiving buprenorphine treatment, there is an unprecedented opportunity to access and treat persons with HCV, reducing HCV transmission, morbidity and mortality. Retention in opioid treatment may improve linkage and retention in HCV care; innovative models of care that integrate opioid drug treatment with HCV treatment are essential.

The Hepatitis C virus treatment cascade at an urban postincarceration transitions clinic.

Transitions clinics, which provide medical care to individuals who have been released from incarceration, reach a population at high risk for hepatitis C Virus (HCV) infection. We used the HCV treatment cascade to describe HCV care at an urban postincarceration transitions clinic, identifying gaps in care and determining reasons for lapses in care. In this retrospective cohort study, we reviewed electronic health records for all formerly incarcerated individuals receiving care at the Bronx Transitions Clinic. HCV treatment cascade measures included the following: detection of HCV antibodies, confirmation of chronic infection, specialist referral, specialist evaluation, initiation of treatment, completion of treatment and achievement of SVR. We recorded reasons for lapses in care. Of 451 patients accessing care, 317 (70%) were screened for HCV antibodies, and 106 (33%) tested positive. Of the 106 antibody-positive patients, 93 (88%) were evaluated for HCV viremia and 84 (79%) were confirmed to have chronic HCV infection; 19% of the total sample had chronic HCV infection. Of these 84 with chronic HCV, 48 (57%) received specialist referral, 30 (36%) were evaluated, 8 (10%) initiated treatment, and 5 (6%) completed treatment and achieved SVR. Some treatment lapses occurred because patients were deemed unstable for treatment (12%) or were re-incarcerated (5%). Chronic HCV infection was common among transitions clinic patients. Few were treated and cured. Patients lost contact with providers before consideration for antiviral therapy. Referral to specialty providers was a gap in care. Increasing HCV treatment in this population will likely require intensive delivery models.

HIV status, trust in health care providers, and distrust in the health care system among Bronx women.

Trust in health care providers and the health care system are essential. This study examined factors associated with trust in providers and distrust in the health care system among minority HIV-positive and -negative women. Interviews were conducted and laboratory tests performed with 102 women from the Women's Interagency HIV Study Bronx site. Interviews collected information about trust in providers, distrust in the system, substance use, mental health symptoms and medications, and sociodemographic characteristics. Many reported distrust of the health care system related to HIV, and most reported trust in their providers. On linear regression analyses, characteristics associated with distrust in the health care system included depressive symptoms (beta=0.48, p<0.05). Characteristics associated with trust in providers included HIV-positive status (beta=0.35, p<0.05), taking mental health medications (beta=0.39, p<0.05), and having a white provider (beta=0.36, p<0.05). Despite distrust in the health care system related to HIV, most reported high trust in their providers, with HIV-positive women trusting their providers more than HIV-negative women. Studies are needed to understand how trust in providers and the health care system is achieved and maintained, and how trust is correlated with HIV-related health outcomes.

Development, application and validation of a Taqman real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar).

Infectious salmon anaemia (ISA) is a disease of cultured Atlantic salmon (Salmo salar) which was successfully eradicated from Scotland following its emergence in 1998. The rapid deployment of sensitive diagnostic methods for the detection of ISA virus (ISAV) was fundamental to the swift eradication of ISA disease in Scotland and continues to be of crucial importance to surveillance of the aquaculture industry. This study reports the development, validation, application and interpretation of two independent, highly sensitive and specific semi-quantitative Taqman real-time RT-PCR (qRT-PCR) methods for the detection of ISAV. Such technology offers considerable advantages over conventional RT-PCR methods in current routine use for ISAV surveillance. These include an increased sensitivity, enhanced specificity, semi-quantification using endogenous controls, a lack of subjectivity in results interpretation, speed of processing and improved contamination control.

Moving molecular diagnostics from laboratory to clinical application: a case study using infectious pancreatic necrosis virus serotype A.

AIMS: Using an RT-PCR method for detection of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon as a model, this study examined the optimization and validation required to provide a method suitable for IPNV detection from fish tissue. METHODS AND RESULTS: IPNV-positive Atlantic salmon kidney samples that had been titred or kidney spiked with IPNV were used. The amount of RNA in the reverse transcription (RT), RT denaturation temperature and incubation time, PCR annealing temperature and number of cycles were optimized. The optimized RT-PCR was able to detect IPNV in Atlantic salmon kidney calculated to have a titre of ten infectious units. CONCLUSIONS: Extensive optimization is required to produce a PCR for detection of fish pathogens from methods designed in the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated some of the many variables that should be optimized before a fully validated assay can be claimed and illustrates the extensive validation required to fulfil requirements of the OIE and is of relevance to laboratories carrying out clinical testing.

A molecular phylogeny of the Dactylogyridae sensu Kritsky & Boeger (1989) (Monogenea) based on the D1-D3 domains of large subunit rDNA.

Phylogenetic analyses based on the partial large subunit rDNA (LSU) sequences of polyonchoinean monogeneans belonging to the Dactylogyridea and Monocotylidea were generated to investigate relationships among various subfamilies of the Dactylogyridae sensu Kritsky & Boeger, 1989. Monophyly of the Dactylogyridae was supported by all analyses performed. Status of the Ancyrocephalidae sensu Bychowsky & Nagibina, 1978 and Ancyrocephalinae sensu Kritsky & Boeger, 1989 was revised based on the present data. All phylogenetic analyses indicated polyphyletic origins of the Ancyrocephalidae and Ancyrocephalinae. Freshwater species of Ancyrocephalinae (Actinocleidus, Ancyrocephalus, Cleidodiscus and Urocleidus) and Ancylodiscoidinae (Thaparocleidus) collected from the fish in European waters were positioned at the base of the Dactylogyridae. The Dactylogyrinae formed a monophyletic group, sister to a clade including the Pseudodactylogyrinae and the tropical and subtropical Ancyrocephalinae. Analyses including only data set on Dactylogyridea were focused on relationships between representatives of the Asian and European Dactylogyrus species. Dactylogyrus species formed a monophyletic group, and the parasite colonization appeared to follow the dispersal history of the Cyprinidae from Asia to Europe. Three lineages of Dactylogyrus species were recognized: the first including species specific to hosts of Asian origin, the second by Dactylogyrus species from Chinese fish hosts, and the third included Dactylogyrus species from European cyprinids and one species from a percid host. The position of D. cryptomeres from Gobio gobio seems to be unresolved.

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates.

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).

Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV).

The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N- and subsequent G-gene based analyses. The range of Genotype I included subgroups of isolates associated with rainbow trout aquaculture (Genotype Ia) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also confirmed. Genotype III included marine isolates from around the British Isles in addition to those associated with turbot mariculture, highlighting a continued risk to the development of this industry. Genotype IV consisted of isolates from the marine environment in North America. Taken together, these findings suggest a marine origin of VHSV in rainbow trout aquaculture. The implications of these findings with respect to the future control of VHSV are discussed. The capacity for molecular phylogenetic analysis to resolve complex epidemiological problems is also demonstrated and its likely future importance to disease management issues highlighted.

Identification of European diplozoids (Monogenea, Diplozoinae) by restriction digestion of the ribosomal RNA internal transcribed spacer.

The second internal transcribed spacer (ITS2) of the ribosomal RNA genes of Diplozoon paradoxum and Paradiplozoon nagibinae were amplified and sequenced. The polymerase chain reaction product of D. paradoxum was bigger (840 bp) than that of P. nagibinae (820 bp). There was no intraspecific variability recorded in sequences from either species. Sequence comparisons and ITS2 restriction fragment length polymorphism (RFLP) pattern of 8 European diplozoid species aimed to resolve their identification and amend the previous studies. RFLP was used to distinguish the 2 species from each other and from P. bliccae, P. homoion, P. megan, P. pavlovskii, P. sapae, and Eudiplozoon nipponicum, using restriction enzymes AluI, HaeIII, HinfI, RsaI, and SphI. The criteria for morphological identification of 8 European diplozoids are also included, with the main morphological characters of clamps, trapeze spur, and anterior joining sclerites of 8 diplozoid species being illustrated. Combination of the shape and comparison of length of the trapeze spur and anterior joining sclerites could lead to accurate identification of diplozoid species.

The first complete monogenean ribosomal RNA gene operon: sequence and secondary structure of the Gyrodactylus salaris Malmberg, 1957, large subunit ribosomal RNA gene.

The sequence of the Gyrodactylus salaris Malmberg, 1957, large subunit, or 28S, ribosomal RNA (rRNA) gene has been determined. This gene is the final portion of the Gyrodactylus rRNA gene operon to be sequenced and results in the first complete sequence of all rRNA genes and spacers from a monogenean. The nucleotide sequence was used to predict the secondary structure of the large subunit rRNA, and regions of conserved and variable sequence and structure were identified. The site where the 5' terminus of the 5.8S rRNA binds to a region within the large subunit rRNA was predicted and complements the anticipated interaction of the 3' terminus of the 5.8S with the 5' terminus of the large subunit rRNA. The large subunit gene may be useful in phylogenetic analysis of the Monogenea or Platyhelminthes and comparisons with other eukaryotes. The variable domains C and H may be most suitable for this purpose.

Characterisation of a beta-tubulin gene from the monogenean parasite, Gyrodactylus salaris Malmberg, 1957.

The cDNA and two partial genomic sequences of beta-tubulin genes have been isolated from the monogenean parasite Gyrodactylus salaris. The cDNA sequence is not represented by either of the genomic sequences, implying that at least three isotypes of the gene exist in G. salaris. The sequences show regions of high homology with other helminth beta-tubulin genes. This represents the first isolation of a beta-tubulin gene from a monogenean and contributes to the overall characterisation of these genes within the helminths. This is an important area, as anthelmintic resistance is increasing against benzimidazole drugs that target the beta-tubulin gene. Benzimidazole drugs have been tested successfully against Gyrodactylus parasites, but their use is not widespread. Should it increase, analysis of the beta-tubulin gene may provide a tool for monitoring resistance development and improving management practises. Use of the beta-tubulin gene in the identification of Gyrodactylus species may prove complex due to the presence of different isotypes.

Characterization of a Gyrodactylus salaris variant: infection biology, morphology and molecular genetics.

A laboratory population of a Danish Gyrodactylus salaris variant founded by 1 single specimen was established and infection studies performed. Rainbow trout as well as Atlantic salmon of 3 different stocks were infected both in cohabitation systems and as single-parasite infections on isolated hosts. Both infection systems revealed that this particular morphotype exhibits low virulence towards Atlantic salmon. Thus, in isolated hosts, the parasites could either not establish or only reproduce to a limited degree on salmon. Rainbow trout, in contrast, proved to be rather susceptible to infection with this G. salaris variant and abundances on this host species always attained significantly higher values in cohabitation systems compared to salmon. Detailed morphological examination confirmed the very high resemblance to G. salaris (sensu stricto), as the range of variation in sclerite characters of the morphotype is almost fully covered by the total range of variation reported for reference G. salaris. Morphological similarities to the closely related congeneric species G. bohemicus were noted. Molecular studies, however, showed that the morphotype most likely represents a G. salaris variant, as it differed only slightly from G. salaris sensu Malmberg, 1957, which is also known to inhabit Danish watercourses. The genomic target region investigated does not allow us to rule out the possibility that it represents a variant form of G. thymalli. Sequences of the ribosomal RNA internal transcribed spacer (ITS) revealed that single individuals contained 2 different ITS sequences, one identical to reference sequence of G. salaris while the other differed at 3 positions. This can be interpreted as an example of a hybrid or, more likely, as intra-individual variation of ITS within single individuals. As one of the nucleotide changes in the variant ITS affects an Hae III restriction site, the current G. salaris variant can be distinguished from G. salaris sensu Malmberg by RFLP diagnosis.

A molecular study of Eubothrium rugosum (Batsch, 1786) (Cestoda: Pseudophyllidea) using ITS rDNA sequences, with notes on the distribution and intraspecific sequence variation of Eubothrium crassum (Bloch, 1779).

The internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal DNA (rDNA) of fish cestodes of the genus Eubothrium were sequenced. The ITS sequences of Eubothrium rugosum were determined and compared with previously analysed congeneric species, Eubothrium crassum and Eubothrium salvelini. The ITS-1 sequences of E. rugosum and E. crassum were 535 bp long, the length of E. salvelini ITS-1 was 536 bp. The ITS-2 region was found to be 403 bp in E. rugosum and E. crassum, and 401-402 bp in E. salvelini. The ITS-1 region of E. rugosum was closer to E. salvelini (identity 98.9-99.1%) than to E. crassum(97.9-98.1%), while the sequence similarity within the ITS-2 region was almost identical (97.5-98.0% for E. crassum; 97.7-98.3% for E. salvelini). Several restriction enzymes were found to be suitable for the differentiation of the three Eubothrium species by PCR-RFLP. The intraspecific sequence variation of E. crassum from different fish hosts (freshwater and marine) and European regions was very low, 0.2% for ITS-1 and 0.5% for ITS-2. Analysis of the ITS sequences of specimens from rainbow trout from three localities in Scotland revealed that both E. crassum and E. salvelini are present in this fish host.

Isolation and characterisation of segment 1 of the infectious salmon anaemia virus genome.

The isolation and characterisation of the largest genomic segment of infectious salmon anaemia virus (ISAV) is reported. Following identification of ISAV-specific clones from a cDNA library, a rapid amplification of cDNA ends-PCR strategy was designed to obtain the sequence of the full length mRNA transcript. The full length open reading frame (ORF) of this gene was shown to be 2169 nucleotides in length, encoding a putative protein of 722 aa. This sequence was demonstrated by RT-PCR to be specific to ISAV-infected cell cultures. The start codon of this ORF was preceded by the ISAV consensus sequence 5' GCTAAGA 3' indicating the full 5' end of the gene to have been obtained. Based on protein size and amino acid composition, this protein was shown to be similar to the PB2 protein of other orthomyxoviruses. Furthermore, a bipartite nuclear localisation signal was identified in the C-terminus of the protein as is found on all of the influenza virus P proteins. Expression of the putative PB2 as a green fluorescent marker protein-fusion protein confirmed that this protein exhibited nuclear localisation in a fish cell line. Sequences of the ISAV segment 1 gene were obtained from Scottish, Norwegian and Canadian ISAV isolates. Analyses confirmed the close genetic relationship between Norwegian and Scottish ISAV and indicated that this segment was among the most conserved of the ISAV genes identified to date. Thus, this evidence strongly suggests that the genomic segment 1 of ISAV encodes a polymerase protein which is thought to be analagous in function to the PB2 protein of influenza viruses.

Analysis of ribosomal RNA intergenic spacer (IGS) sequences in species and populations of Gyrodactylus (Platyhelminthes: Monogenea) from salmonid fish in Northern Europe.

The intergenic spacer (IGS) region of ribosomal RNA genes was amplified and sequenced from a variety of Gyrodactylus specimens collected from wild and farmed Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, and grayling Thymallus thymallus, from various locations in Northern Europe. Phylogenetic analysis of the sequences confirmed the distinction between G. salaris Malmberg, 1957 and G. thymalli Zitnan, 1960, supporting their validity as separate species. G. salaris adapted to rainbow trout are also distinct from the parasites found on Atlantic salmon, supporting the existence of a rainbow-trout form that was initially identified on the basis of morphological differences. Analysis of the IGS did not provide good resolution of different populations of G. salaris sensu stricto, but was consistent with epidemiological evidence which indicates that introduction of the parasite to Norway was recent and limited. The IGS may be helpful in distinguishing forms of G. salaris that are pathogenic to Atlantic salmon from those that are not.

Molecular phylogenetic analysis of the genus Gyrodactylus (Platyhelminthes: Monogenea) inferred from rDNA ITS region: subgenera versus species groups.

Analyses of small subunit ribosomal RNA gene sequences of representatives of major taxa of Monopisthocotylea were performed to identify the sister group of Gyrodactylus. Nuclear ribosomal DNA sequences from the complete internal transcribed spacer (ITS) region were used to infer phylogeny of 37 Gyrodactylus species and Gyrodactyloides bychowskii, Macrogyrodactylus polypteri and Gyrdicotylus gallieni, using maximum likelihood, parsimony and Bayesian inference. The genus Gyrodactylus appeared to be a monophyletic group in all analyses, based on the present data set. Within the genus, there were 3 major groups recognized by high bootstrap values and posterior probabilities. None of the 6 subgenera appeared to be monophyletic, and the most basal subgenus G. (Gyrodactylus) was paraphyletic. Characteristics of the excretory system of Gyrodactylus do not seem to be conservative enough to reveal subgenera within Gyrodactylus and we suggest abandoning existing subgenera as indicators of phylogeny. The grouping of species based on the morphology of the ventral bar and marginal hooks seems to have sufficient power to infer relationships between the Gyrodactylus species.

Paradiplozoon homoion Bychowsky & Nagibina, 1959 versus P. gracile Reichenbach-Klinke, 1961 (Monogenea): two species or phenotypic plasticity?

Specimens of the Paradiplozoon homoion-complex were collected from ten species of cyprinid fish in the Czech Republic. A combined molecular and morphometric approach was performed to distinguish Paradiplozoon homoion and P. gracile. The second internal transcribed spacer (ITS2) of the ribosomal RNA genes was amplified and sequences were analysed. No variability in the analysed sequences was detected. Measurements of clamps and the central hooks obtained from specimens from different host species were compared. Great variability was found in the length and width of the third pair of clamps. No significant differences were detected in the measurements of the central hook sickle. A positive relationship was found between host size and each of the following measurements of the third pair clamps: length and width of the whole clamp; and length of the median plate of the third pair of clamps. The length of the median plate of the attachment clamps may be a useful character for species identification of diplozoids. Further molecular and morphometric studies are required to resolve this taxonomic problem and, henceforth, we suggest considering P. gracile as a species inquirenda.

The use of host specificity, pathogenicity, and molecular markers to differentiate between Gyrodactylus salaris Malmberg, 1957 and G. thymalli Zitnan, 1960 (Monogenea: Gyrodactylidae).

The validity of Gyrodactylus thymalli has been questioned, based on its morphological and genetic resemblance to G. salaris. This taxonomic problem has practical implications regarding correct diagnosis of G. salaris, which has proved to be highly pathogenic to stocks of wild Norwegian Atlantic salmon. The host specificity and pathogenicity of G. salaris and G. thymalli were experimentally tested on salmon and grayling. Both parasite species were able to infect, live and reproduce on both fish species. G. salaris was highly pathogenic for the experimental stock of salmon, while grayling mounted an effective response against this parasite. Both fish species responded to an infection with G. thymalli. The results did not support the hypothesis of conspecificity between G. thymalli and G. salaris. The ribosomal RNA gene intergenic spacer of both species was sequenced. Variation in sequence was lower than expected for different species. Variation in the sequences of tandemly repeated elements was found and may prove useful in distinguishing G. salaris and G. thymalli.

Genetic characterization of six species of diplozoids (Monogenea; Diplozoidae).

The second internal transcribed spacer (ITS2) of the ribosomal RNA gene array of 6 species of diplozoids; Eudiplozoon nipponicum, Paradiplozoon bliccae, P. homoion, P. megan, P. pavlovskii and P. sapae, was amplified by PCR and sequenced. These sequences clearly demonstrate discrimination at the species level and confirm the validity of species determined by morphological identification. No intraspecific variation was found in the ITS2 sequences. There were no differences in the ITS2 sequences of P. homoion from populations parasitizing different host species. The length of the PCR product allowed discrimination of E. nipponicum from the Paradiplozoon species. Digestion of the amplified ITS2 fragment with enzymes AluI, HaeIII and HinfI provided useful genetic markers for species identification. The genetic relationships between diplozoids again demonstrated that E. nipponicum was the most genetically distinct species, whereas P. bliccae and P. sapae were the species most closely related. This represents the first molecular taxonomic study of these interesting parasites and demonstrates the utility of these methods for addressing questions of systematics.