Discussion
We documented one gastric and two intestinal cases of invasive anisakiasis caused by the species
A. pegreffii. In Italy, previous cases of gastric [
12,
22‐
26] and intestinal [
24,
27,
28] anisakiasis were reported. However, in those cases, despite the histological findings and morphological features allowed disclosing the presence of a larval
Anisakis sp., the identification at the species level was not possible. By contrast, in the last decades, the application of molecular tools in this group of parasites has allowed the identification of larvae infecting humans, thus enlarging the knowledge about this fish-borne zoonosis [
9‐
16]. Furthermore, the implementation of the methodologies based on PCR, as those concerning DNA purification and extraction, have allowed to develop a method for the identification of
A. pegreffii even from the paraffin-embedded tissues of surgically removed granulomas caused by the parasite [
11]. This method was recently successfully applied also in an archival case in Croatia [
13].
In the present study, a more sensitive method for the accurate and rapid identification of the etiological agent of human anisakiasis, based on RT-PCR hydrolisis probe, was utilized. This method detected up to 0.0006 ng/μl of DNA of the parasite. The RT-PCR probe assay was assessed for the first time, on a human tissue, (eosinophilic granuloma) whose etiological agent would have been undetectable as inferred by direct DNA sequencing, due to the low quantity of DNA available. This situation could also occur when bioptic tissues are collected by endoscopy from ulcers caused by the parasite in the gastric and/or intestinal mucosa, without direct observation of the parasite itself. Indeed, since the symptoms of anisakiasias are not pathognomonic, the acute form could be misdiagnosed. As a consequence, the infection could become chronic, leading to granuloma formation. Therefore, the early removal and identification of the worm makes the best prevention of formation of eosinophilic granuloma caused by the allergic reaction to the degenerated larva in the chronic infection.
The possibility to use sensitive and specific methods for the molecular diagnosis of human anisakiasis, such as a RT-PCR, as here proposed, will allow to enlarge the knowledge about its occurrence in human populations that regularly consume raw or undercooked fish.
In this respect, in Italy the consumption of home-made raw marinated anchovies that were previously not exposed to rapid freezing (−20 °C for 24 h), as compulsory by European Community regulation (EC no 1276/2011), represents the main risk to contract this fish-borne zoonosis [
12]. Indeed, also in the cases, here described, the source of the infection was “home-made raw marinated anchovies”. On the other hand, anchovies
Engraulis encrasicolus, is one the most important fish resource in the Mediterranean countries; this species was found to be heavily infected by
Anisakis spp. larvae, even in its flesh (i.e. edible parts), in some fishing grounds of the Mediterranean Sea [
29,
30]. In addition, it was demonstrated that a migration of
A. pegreffii larvae from the viscera to the edible parts of the
E. encrasicolus occurs
post-mortem, with temperature- and time-dependent larval motility [
29]. It is therefore important to increase the control of the temperature of the fish storage after landing, up to the domestic level.
A. pegreffii is not the only species able to cause human anisakiasis. In Japan, Umehara et al. [
14] and Arai et al. [
15] recognized
A. simplex (s .s.) as the main etiological agent of anisakiasis in a large number of patients. The opposite situation was found in South Korea, where Lim et al. [
16] described the infection in several patients as caused by
A. pegreffii, but in one patient was due to
A. simplex (s. s.)
, However, those findings could be related both to the fishing ground of the source of the infection (i. e. the infected fish consumed), and to the geographical distribution of the two sibling species of
Anisakis. Indeed, while the Japanese waters are a sympatric area of the two species from South Pacific area, so far the species
A. simplex (s. s.) has not been recorded in fish hosts; by contrast, in the same geographical area,
A. pegreffii is present, also in co-infection with
A. berlandi, in several fish and cetacean hosts [
7,
8,
18].
Concerning the pathogenic potential of
A. pegreffii in comparison with
A. simplex (s. s.)
, according to the literature,
A. pegreffii has been identified at molecular level in several cases of Gastro Allergic Anisakiasis (GAA) in Italy, characterized by the occurrence of urticaria manifestation and/or edema of oral mucosae [
12]. Conversely, so far, no data have been recorded about the GAA due to
A. simplex (s. s.), identified in clinical cases by molecular means [
31]. Of course, this does not exclude the possibility that also
A. simplex (s. s.) is able to elicit that immune response in humans causing GAA, also in consideration that some authors are reporting a higher capacity of this species to survive the pH condition of human stomach [
32,
33] and to penetrate at higher percentage in the muscle of natural fish host [
34,
35], but also in its accidental host (humans) [
9‐
16]. However, in the clinical cases here described, in spite of the fact that their etiological agent was
A. pegreffii, we did not notice any allergic reaction; this finding is similar to that found by Lim et al. [
16].
In the present study, we have also performed Immunoblotting assay to study the
Anisakis-specific immune response in three different cases of anisakiasis due to
A. pegreffii. Interestingly, the serum from the patient suffering of the eosinophilic granuloma (CC3) showed immunoreactivity at the Immunoblotting assay, at both IgE and IgG against the three most frequent antigens indicative of the infection by
A. pegreffii (i.e.
Ani s 1-like,
Ani s 7-like and
Ani s 13-like). Whereas, the sera from patient CC1 and CC2 did not react with antigen around
Ani s 1-like
. Hypotheses to explain these last findings could be related to:
i) the serum available from the CC1 was taken just during patient’s hospitalization (i.e. few days after the infected fish intake); on the other hand, it has been reported that IgE reaction against
Ani s 1 increased after 1 month from the infection [
2,
36,
37];
ii) in both CC1 and CC2 cases, the parasite larva was found, at least, during the first 7 days after the infection
.
Interestingly, the only gastric case of anisakiasis (i.e. CC1), here studied, showed IgG
4 reactivity against
Ani s 7-like. Typical for a parasite-induced immunologic reaction, previous studies observed the concomitant production of both IgE and other immunoglobulin isotypes, such as IgG
4, which are mediated by the same Th-2 mechanism [
38‐
40]. Further, IgG
4 response to
Ani s 7 has been suggested as a marker of gastric allergic anisakiasis due to
A. simplex [
40]. Thus, the IgG4 seropositivity against
Ani s 7-like observed in patient CC1 seems to be in accordance with previous observation [
40].
Finally, a band of immunoreactivity having a relative mobility (Mr) around 37 kDa, likely corresponding to
Ani s 13-like [
21]
, was observed in all the sera tested in the present study; the same antigen was found in all the sera from patients in a large survey carried out on IgE sensitization by
A. pegreffii, in Italy [
21]. This suggests that
Ani s 13-like could be a good candidate in the diagnosis of human anisakiasis.