On the morphology of Anisakis pegreffii: a comparative analysis of three microscopic techniques used to build a new parasite atlas

BACKGROUND: Human anisakidosis is a parasitic anthropozoonosis caused by larval nematodes of the family Anisakidae. Here, we report a detailed description of the morphology of Anisakis pegreffii third-stage larva performed using a conventional light and confocal microscopy, and scanning electron microscopy (SEM) that provide a basis for both phenotypic studies and genetic mutations. METHODS: The collected larvae from fish were morphologically identified as Anisakis larvae Type I, and they were characterized by PCR-RFLP to identify the Anisakis pegreffii specie. Using NC5/NC2 primers, ribosomal genomic regions ITS1, 5.8 SrRNA and ITS2 of DNA were amplified and PCR products were sequenced. Fifteen larvae belonging to Anisakis pegreffii were fixed, sectioned, and examined with a light and confocal microscope and by SEM. RESULTS: In our studies, have been acquired detailed ultrastructural images, which have been integrated with those derived from the dissection of the parasite, obtained with light and confocal microscopy. The structural and ultrastructural images concerning the third stage larvae of Anisakis pegreffii have been studied, analyzed and compared among them. The derived overall view has allowed detecting new interesting details of a well-known parasite and has been schematically showed. CONCLUSIONS: The aim of this study is to furnish an updated atlas of Anisakis pegreffii. Confocal microscopy, as well as the light and electron microscopy have played a pivotal role in the accumulation of new scientific data regarding the anatomical structures of this nematode. This work is the result of one year of engagement by the Authors and the outcome is a comprehensive atlas on Anisakis pegreffii microscopy

Anisakis sensitization in different population groups and public health impact. A systematic review

Anisakis simplex spp. sensitization rates have increased worldwide, with a significant impact on health-care systems. To date, no clear-cut diagnostic criteria and laboratory algorithm have been established, so anisakiasis still represents an under-reported health problem whose clinical manifestations, when present, mimic the much more common allergic and digestive disorders. Aim of the study was to systematically review the available literature on the prevalence of sensitization against Anisakis in the general population and in specific population groups, taking into account the impact of the different available diagnostic techniques on the epidemiological data. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, relevant papers reporting Anisakis sensitization epidemiological data were found covering a period ranging from 1996 to February 2017. Overall, 41 studies comprising 31,701 participants from eleven countries were included in the qualitative synthesis. General asymptomatic population resulted sensitized to Anisakis in 0.4 to 27.4% of cases detected by means of indirect ELISA or ImmunoCAP specific IgE detection, and between 6.6% and 19.6% of the samples by Skin prick test (SPT). Occupationally exposed workers (fishermen, fishmongers and workers of fish-processing industries) documented specific IgE between 11.7% and 50% of cases, whereas SPT positivity ranged between 8% and 46.4%. Symptomatic allergic patients to any kind of allergen were found to be positive to Anisakis specific IgE detection between 0.0% (in children with mastocytosis) to 81.3% (among adults with shellfish allergy). Results highlighted that hypersensitivity prevalence estimates varied widely according to geographical area, characteristics of the population studied, diagnostic criteria and laboratory assays. Further studies are needed to overcome the documented misdiagnosis by improving the diagnostic approach and, consequently, providing more affordable estimates in order to address public health interventions on populations at high risk of exposure to Anisakis and to tailor health services related to specific groups

Development of a rapid and eco-friendly UHPLC analytical method for the detection of histamine in fish products

We developed, validated, and confirmed with proficiency tests a fast ultra-high-performance liquid chromatography with diode array detector (UHPLC-DAD) method to determine histamine in fish and fishery products. The proposed method consists of two successive solid–liquid extractions: one with a dilute solution of perchloric acid (6%) and the second only with water. The instrumental analysis with UHPLC provides a very fast run time (only 6 min) with a retention time of approximately 4 min, a limit of quantification (LOQ) of 7.2 mg kg−1, a limit of detection (LOD) of 2.2 mg kg−1, a recovery around 100%, a relative standard deviation (RSD%) between 0.5 and 1.4, and an r2 of calibration curve equal to 0.9995. The method detected optimal values of the validation parameters and required a limited number of reagents in comparison to other methods reported in the literature. Furthermore, the method could detect histamine in a very short time compared with other methods. This method, in addition to being validated, precise, specific, and accurate, avoids wasting time, money, and resources, and limits the use of organic solvents

Trace elements in stomach oil of Scopoli's shearwater (Calonectris diomedea) from Linosa's colony

Calonectris diomedea is a colonial Procellariiform breeding on Mediterranean islands. The stomach oil produced during chick rearing is a peculiar trait of this species. The composition of the stomach oil is likely to reflect the composition of the prey ingested and might reveal the contaminants uptake with prey becoming a possible tool for the marine pollution monitoring. We examined the concentration of 15 trace elements by ICP-MS and direct mercury analyser. The principal component analysis revealed a heterogeneous pattern of metal concentration, showing a significant separation between samples collected 20 and 70 days after hatching. The data obtained in this work give preliminary information on the feeding habits and breeding ecology of Linosa's colony of Scopoli's shearwater. The trace metals variability found suggest that the stomach oil may have a role as trophic markers to understand predator-prey relationships and to have evidence on the accumulation of pollutants in the latter

An improved microtiter assay for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma

BACKGROUND: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. METHODS: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor(s), the tested sera or plasma was removed by a washout procedure after initial 3–6 h culture in the assay. RESULT: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. CONCLUSION: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma

Use of a comprehensive diagnostic algorithm for Anisakis allergy in a high seroprevalence Mediterranean setting

Background. Diagnosis of anisakis allergy (AA) is based on the skin prick test (SPT) and specific IgE (sIgE) determination. Anyway, false positivity cases are due to cross reactivity with numerous allergens. The aim of the study was to evaluate the reliability of a comprehensive diagnostic algorithm for the AA. Methods. An observational study was conducted on a sample of consecutive subjects accessing the allergology outpatient ambulatories of two hospitals located in Western Sicily. All the recruited outpatients were tested by Skin Prick Test performed using anisakis extracts by ALK-Abello (Madrid, Spain). Specific IgE dosage for anisakis extracts was then performed by using ImmunoCAP250 (Immunodiagnostics Uppsala, Sweden). Consequently, outpatients who tested positive to first line tests underwent sIgE testing for ascaris and tropomyosin. Lastly, outpatients positive to the first line were invited to be further tested by basophil activation test (BAT) by using Flow CAST kit and anisakis commercial extract (Buhlmann Laboratories AG, Schonenbuch, Switzerland), as confirmatory analysis. Results. One hundred and eleven outpatients with an anamnesis suggestive of sensitization to anisakis (AS) and 466 subjects with chronic urticaria (CU) were recruited in the study. Of these, 22 with AS and 41 with CU showed a sensitization to anisakis allergens. The diagnostic algorithm revealed that 8.8% of outpatients who tested positive to sIgE determination were affected by CU, while 82.5% of all the sIgE positivity was related to cross-reactivity. Overall, a genuine anisakis seroprevalence of 2.3% was documented. Within a sub-sample of 15 subjects with clinical symptoms related to AA, n. 8 showed a real positivity after BAT. A greater response to A. pegreffii allergens as compared to A. simplex was reported. Conclusions. Our preliminary findings support the high clinical specificity of BAT for AA diagnosis, suggesting implementing this method in a comprehensive diagnostic algorithm

Inducing Cross-Clade Neutralizing Antibodies against HIV-1 by Immunofocusing

Background: Although vaccines are important in preventing viral infections by inducing neutralizing antibodies (nAbs), HIV-1 has proven to be a difficult target and escapes humoral immunity through various mechanisms. We sought to test whether HIV-1 Env mimics may serve as immunogens. Methodology/Principal Findings: Using random peptide phage display libraries, we identified the epitopes recognized by polyclonal antibodies of a rhesus monkey that had developed high-titer, broadly reactive nAbs after infection with a simianhuman immunodeficiency virus (SHIV) encoding env of a recently transmitted HIV-1 clade C (HIV-C). Phage peptide inserts were analyzed for conformational and linear homology using computational analysis; some peptides mimicked various domains of the original HIV-C Env, such as conformational V3 loop epitopes and the conserved linear region of the gp120 C-terminus. Next, we devised a novel prime/boost strategy to test the immunogenicity of such phage-displayed peptides and primed mice only once with HIV-C gp160 DNA followed by boosting with mixtures of recombinant phages. Conclusions/Significance: This strategy, which was designed to focus the immune system on a few Env epitopes (immunofocusing), not only induced HIV-C gp160 binding antibodies and cross-clade nAbs, but also linked a conserved HIV Env region for the first time to the induction of nAbs: the C-terminus of gp120. The identification of conserved antige

The role of neutralizing antibodies in prevention of HIV-1 infection: what can we learn from the mother-to-child transmission context?

International audienceIn most viral infections, protection through existing vaccines is linked to the presence of vaccine-induced neutralizing antibodies (NAbs). However, more than 30 years after the identification of AIDS, the design of an immunogen able to induce antibodies that would neutralize the highly diverse HIV-1 variants remains one of the most puzzling challenges of the human microbiology. The role of antibodies in protection against HIV-1 can be studied in a natural situation that is the mother-to-child transmission (MTCT) context. Indeed, at least at the end of pregnancy, maternal antibodies of the IgG class are passively transferred to the fetus protecting the neonate from new infections during the first weeks or months of life. During the last few years, strong data, presented in this review, have suggested that some NAbs might confer protection toward neonatal HIV-1 infection. In cases of transmission, it has been shown that the viral population that is transmitted from the mother to the infant is usually homogeneous, genetically restricted and resistant to the maternal HIV-1-specific antibodies. Although the breath of neutralization was not associated with protection, it has not been excluded that NAbs toward specific HIV-1 strains might be associated with a lower rate of MTCT. A better identification of the antibody specificities that could mediate protection toward MTCT of HIV-1 would provide important insights into the antibody responses that would be useful for vaccine development. The most convincing data suggesting that NAbs migh confer protection against HIV-1 infection have been obtained by experiments of passive immunization of newborn macaques with the first generation of human monoclonal broadly neutralizing antibodies (HuMoNAbs). However, these studies, which included only a few selected subtype B challenge viruses, provide data limited to protection against a very restricted number of isolates and therefore have limitations in addressing the hypervariability of HIV-1. The recent identification of highly potent second-generation cross-clade HuMoNAbs provides a new opportunity to evaluate the efficacy of passive immunization to prevent MTCT of HIV-1