First molecular identification of the zoonotic parasite Anisakis pegreffii(Nematoda: Anisakidae) in a paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy
TL;DR: This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin, reinforcing the pathological significance of the species A. pegreffii to humans.
Abstract: Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus Anisakis. The detection and identification of human infections is difficult. This is due to: a) the low specificity of the clinical features and symptomatology related to human infections; b) the paucity of diagnostic features of larvae found in granulomatous lesions characteristic of "invasive anisakiasis"; and c) the lack morphological characters diagnostic at the specific level when larvae of Anisakis are detected. Thus, molecular-based diagnostic approaches are warranted. We have developed a PCR method that amplifies the DNA of Anisakis spp. in fixed paraffin-embedded tissues. This method was applied to a granuloma removed from a human case of intestinal anisakiasis in Italy. Specific primers of the mtDNA cox2 gene were used and sequence analysis was performed according to the procedures already established for species of Anisakis. The sequence obtained (629 bp) was compared with those of the other species of Anisakis which have so far been genetically characterized and with sequences obtained from larval stages of Anisakis collected from the Mediterranean fish Engraulis encrasicolus. This enabled the genetic identification of the larva in the human tissue as A. pegreffii. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin. The case of human anisakiasis presented reinforces the pathological significance of the species A. pegreffii to humans. The molecular/genetic methodological approach based on mtDNA cox2 sequence analysis, described here, can allow easy and rapid identification of Anisakis spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis.
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TL;DR: Human cases of gastric anisakiasis caused by the zoonotic parasite Anisakis pegreffii are increasing in Italy.
Abstract: Human cases of gastric anisakiasis caused by the zoonotic parasite Anisakis pegreffii are increasing in Italy. The disease is caused by ingestion of larval nematodes in lightly cooked or raw seafood. Because symptoms are vague and serodiagnosis is difficult, the disease is often misdiagnosed and cases are understimated.
181 citations
Cites background from "First molecular identification of t..."
...Previously, the most common haplotype associated with gastric and intestinal anisakiasis in humans in Italy was designated as H1 (7)....
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...pegreffii nematodes previously sequenced for the same gene (7,8,11) (Figure 1)....
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...pegreffii (7), mtDNA cox2 sequences from 5 specimens (from patients HuC1, HuC2, HuC3, HuC7, and HuC8) corresponded to the most frequent haplotype, designated as H1....
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...Since then, several cases of gastrointestinal anisakiasis have been reported there (7)....
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TL;DR: The morphological and morphometric analyses revealed the presence of morphological features that differed among the 3 biological species, and the tree topologies support the finding that A. simplex (s.s.), A. pegreffii, and A. berlandi n.
Abstract: Numerous specimens of the 3 sibling species of the Anisakis simplex species complex (A. pegreffii, A. simplex (senso stricto)), and A. simplex sp. C) recovered from cetacean species stranded within the known geographical ranges of these nematodes were studied morphologically and genetically. The genetic characterization was performed on diagnostic allozymes and sequences analysis of nuclear (internal transcribed spacer [ITS] of ribosomal [r]DNA) and mitochondrial (mitochondrial [mt]DNA cox2 and rrnS) genes. These markers showed (1) the occurrence of sympatry of the 2 sibling species A. pegreffii and A. simplex sp. C in the same individual host, the pilot whale, Globicephala melas Traill, from New Zealand waters; (2) the identification of specimens of A. pegreffii in the striped dolphin, Stenella coeruleoalba (Meyen), from the Mediterranean Sea; and (3) the presence of A. simplex (s.s.) in the pilot whale and the minke whale, Balaenoptera acutorostrata Lacepede, from the northeastern Atlantic wa...
166 citations
TL;DR: This review addresses the biodiversity, biology, distribution, ecology, epidemiology, and consumer health significance of the so far known species of Anisakis, both in their natural hosts and in human accidental host populations, worldwide.
Abstract: This review addresses the biodiversity, biology, distribution, ecology, epidemiology, and consumer health significance of the so far known species of Anisakis, both in their natural hosts and in human accidental host populations, worldwide. These key aspects of the Anisakis species' biology are highlighted, since we consider them as main driving forces behind which most of the research in this field has been carried out over the past decade. From a public health perspective, the human disease caused by Anisakis species (anisakiasis) appears to be considerably underreported and underestimated in many countries or regions around the globe. Indeed, when considering the importance of marine fish species as part of the everyday diet in many coastal communities around the globe, there still exist significant knowledge gaps as to local epidemiological and ecological drivers of the transmission of Anisakis spp. to humans. We further identify some key knowledge gaps related to Anisakis species epidemiology in both natural and accidental hosts, to be filled in light of new 'omic' technologies yet to be fully developed. Moreover, we suggest that future Anisakis research takes a 'holistic' approach by integrating genetic, ecological, immunobiological, and environmental factors, thus allowing proper assessment of the epidemiology of Anisakis spp. in their natural hosts, in human populations, and in the marine ecosystem, in both space and time.
158 citations
TL;DR: Allergic phenomena share common pathways with the immune response against helminth parasites, as well as putatively crossreacting antibodies, as are used in food allergy, depend on the clinical relevance of specific IgE and deserve careful interpretation in the various forms of A. simplex.
102 citations
TL;DR: Clinical symptoms associated with the infection, termed anisakidosis, vary from irritation of the oesophagus and stomach, via nausea, vomiting and diarrhoea to severe epigastric and abdominal pain.
99 citations
References
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TL;DR: The recently‐developed statistical method known as the “bootstrap” can be used to place confidence intervals on phylogenies and shows significant evidence for a group if it is defined by three or more characters.
Abstract: The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
40,349 citations
"First molecular identification of t..." refers methods in this paper
...The reliabilities of the phylogenetic relationships were evaluated using nonparametric bootstrap analysis [39] for the MP and NJ trees....
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TL;DR: ClUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W, providing an integrated system for performing multiple sequence and profile alignments and analysing the results.
Abstract: CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.
38,522 citations
"First molecular identification of t..." refers methods in this paper
...The cox2 sequences were aligned using Clustal X [37]....
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35,309 citations
TL;DR: Version 4 of MEGA software expands on the existing facilities for editing DNA sequence data from autosequencers, mining Web-databases, performing automatic and manual sequence alignment, analyzing sequence alignments to estimate evolutionary distances, inferring phylogenetic trees, and testing evolutionary hypotheses.
Abstract: We announce the release of the fourth version of MEGA software, which expands on the existing facilities for editing DNA sequence data from autosequencers, mining Web-databases, performing automatic and manual sequence alignment, analyzing sequence alignments to estimate evolutionary distances, inferring phylogenetic trees, and testing evolutionary hypotheses. Version 4 includes a unique facility to generate captions, written in figure legend format, in order to provide natural language descriptions of the models and methods used in the analyses. This facility aims to promote a better understanding of the underlying assumptions used in analyses, and of the results generated. Another new feature is the Maximum Composite Likelihood (MCL) method for estimating evolutionary distances between all pairs of sequences simultaneously, with and without incorporating rate variation among sites and substitution pattern heterogeneities among lineages. This MCL method also can be used to estimate transition/transversion bias and nucleotide substitution pattern without knowledge of the phylogenetic tree. This new version is a native 32-bit Windows application with multi-threading and multi-user supports, and it is also available to run in a Linux desktop environment (via the Wine compatibility layer) and on Intel-based Macintosh computers under the Parallels program. The current version of MEGA is available free of charge at (http://www.megasoftware.net).
29,021 citations
"First molecular identification of t..." refers methods in this paper
...0 [38], using Maximum Parsimony (MP) and Neighbour-Joining (NJ) based on p-distance values....
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TL;DR: This work uses computer simulations and a laboratory-generated phylogeny to test bootstrapping results of parsimony analyses, and indicates that any given bootstrap proportion provides an unbiased but highly imprecise measure of repeatability, unless the actual probability of replicating the relevant result is nearly one.
Abstract: Bootstrapping is a common method for assessing confidence in phylogenetic anal? yses. Although bootstrapping was first applied in phylogenetics to assess the repeatability of a given result, bootstrap results are commonly interpreted as a measure of the probability that a phylogenetic estimate represents the true phylogeny. Here we use computer simulations and a laboratory-generated phylogeny to test bootstrapping results of parsimony analyses, both as measures of repeatability (i.e., the probability of repeating a result given a new sample of characters) and accuracy (i.e., the probability that a result represents the true phylogeny). Our results indicate that any given bootstrap proportion provides an unbiased but highly imprecise measure of repeatability, unless the actual probability of replicating the relevant result is nearly one. The imprecision of the estimate is great enough to render the estimate virtually useless as a measure of repeatability. Under conditions thought to be typical of most phylogenetic analyses, however, bootstrap proportions in majority-rule consensus trees provide biased but highly con? servative estimates of the probability of correctly inferring the corresponding clades. Specifically, under conditions of equal rates of change, symmetric phylogenies, and internodal change of 70% usually correspond to a probability of >95% that the corresponding dade is real. However, under conditions of very high rates of internodal change (approaching randomization of the characters among taxa) or highly unequal rates of change among taxa, bootstrap proportions >50% are overestimates of accuracy. (Boot? strapping; accuracy; repeatability; phylogeny; parsimony; precision; statistical analyses; simu? lations.)
4,057 citations
"First molecular identification of t..." refers background in this paper
...Bootstrap values ≥70 were considered well supported [40]....
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