Invasive anisakiasis by the parasite Anisakis pegreffii (Nematoda: Anisakidae): diagnosis by real-time PCR hydrolysis probe system and immunoblotting assay

BACKGROUND: Anisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen. METHODS: Parasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. IgE, IgG4 and IgG immune response against antigens A. pegreffii by Immunoblotting assay was also studied. RESULTS: According to the mtDNA cox2 and the EF1 α - 1 nDNA sequence analysis, the larvae from stomach and intestine of two patients were assigned to A. pegreffii. The Real-time PCR primers/probe system, showed a fluorescent signal at 510 nm for A. pegreffii, in all the three cases. In Immunoblotting assay, patient CC1 showed IgE, IgG4 reactivity against Ani s 13-like and Ani s 7-like; patient CC2 revealed only IgG reactivity against Ani s 13-like and Ani s 7-like; while, the third patient showed IgE and IgG reactivity against Ani s 13-like, Ani s 7-like and Ani s 1-like. CONCLUSION: The Real-time PCR assay, a more sensitive method than direct DNA sequencing for the accurate and rapid identification of etiological agent of human anisakiasis, was successfully assessed for the first time. The study also highlights the importance to use both molecular and immunological tools in the diagnosis of human anisakiasis, in order to increase our knowledge about the pathological findings and immune response related to the infection by zoonotic species of the genus Anisakis

Rapid and persistent selection of the K103N mutation as a majority quasispecies in a HIV1-patient exposed to efavirenz for three weeks: a case report and review of the literature

<p>Abstract</p> <p>Introduction</p> <p>Selection of the K103N mutation is associated with moderately reduced in vitro fitness of HIV. Strains bearing K103N in vivo tend to persist, even in the absence of additional drug pressure, as minority quasispecies, often undetectable in genotyping resistance testing assays, performed at standard conditions. Here, we report on the rapid and long lasting selection of a K103N bearing strain as the dominant quasispecies after very short exposure to efavirenz in vivo.</p> <p>Case presentation</p> <p>A 55-year-old Caucasian man was switched to efavirenz, zidovudine and lamivudine in February 2003, while on viral suppression in his first-line highly active anti-retroviral treatment regimen. One month later, he reported inconsistent adherence and his viremia level was 5700 c/mL. He did not attend further checkups until September 2005, when his viral load was 181,000 c/mL. The patient reported interrupting his medications approximately three weeks after simplification. The genotyping resistance testing assay was performed both on HIV RNA and HIV DNA from plasma, yielding an identical pattern with the isolate presence of the K103N mutation in the prevalent strain.</p> <p>Conclusion</p> <p>Persistence of the K103N mutation as a majority quasispecies may ensue after a very short exposure to efavirenz. Our case would therefore suggest that the presence of the K103N mutation should always be ruled out by genotyping resistance testing assays, even after minimal exposures to efavirenz.</p

Anisakiasis and gastroallergic reactions associated with Anisakis pegreffii infection, Italy.

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

Has VZV epidemiology changed in Italy? Results of a seroprevalence study

The aim of the study was to evaluate if and how varicella prevalence has changed in Italy. In particular a seroprevalence study was performed, comparing it to similar surveys conducted in pre-immunization era. During 2013–2014, sera obtained from blood samples taken for diagnostic purposes or routine investigations were collected in collaboration with at least one laboratory/center for each region, following the approval of the Ethics Committee. Data were stratified by sex and age. All samples were processed in a national reference laboratory by an immunoassay with high sensitivity and specificity. Statutory notifications, national hospital discharge database and mortality data related to VZV infection were analyzed as well. A total of 3707 sera were collected and tested. In the studied period both incidence and hospitalization rates decreased and about 5 deaths per year have been registered. The seroprevalence decreased in the first year of life in subjects passively protected by their mother, followed by an increase in the following age classes. The overall antibody prevalence was 84%. The comparison with surveys conducted with the same methodology in 1996–1997 and 2003–2004 showed significant differences in age groups 1–19&nbsp;y. The study confirms that in Italy VZV infection typically occurs in children. The impact of varicella on Italian population is changing. The comparison between studies performed in different periods shows a significant increase of seropositivity in age class 1–4&nbsp;years, expression of vaccine interventions already adopted in some regions

Trichphyton violaceum and T. soudanese: re-emerging pathogens in Italy, 2005-2013

Dermatomycoses due to Trichophyton violaceum are described in Mediterranean Countries, North Africa and in the Horn of Africa where T. soudanense is present too, but it was rare until few years ago in Italy. Aim of the present study was to evaluate an Italian multicenter 9 year (2005-2013) experience concerning these re-emerging pathogens. Fifty three fungal strains were sent from clinical laboratories to the Medical Mycology Committee (CoSM) - Italian Association of Clinical Microbiology (AMCLI) for mycological confirmation. Strains were identified as T. violaceum (23) and T. soudanense (30) by phenotypic and genotypic methods. These dermatophytes present epidemiological (high rate of inter-human transmission, high risk among adopted children coming from countries of either the Horn of Africa or Sub-Saharan Africa also in outbreaks of tinea capitis) and clinical peculiarities (reduced alopecia, presence of exudative lesions) confirming the originality of these \u201cimported\u201d dermatophyte infection

Consequences of inaccurate hepatitis C virus genotyping on the costs of prescription of direct antiviral agents in an Italian district

Available commercial assays may yield inaccurate hepatitis C virus (HCV) genotype assignment in up to 10% of cases. We investigated the cost-effectiveness of re-evaluating HCV genotype by population sequencing, prior to choosing a direct acting antiviral (DAA) regimen. Between March and September 2015, HCV sequence analysis was performed in order to confirm commercial LiPA-HCV genotype (Versant\uae HCV Genotype 2.0) in patients eligible for treatment with DAAs. Out of 134 consecutive patients enrolled, sequencing yielded 21 (15.7%) cases of discordant results. For three cases of wrong genotype assignment, the putative reduction in efficacy was gauged between 15% and 40%. Among the eight cases for whom G1b was assigned by commercial assays instead of G1a, potentially suboptimal treatments would have been prescribed. Finally, for five patients with G1 and indeterminate subtype, the choice of regimens would have targeted the worst option, with a remarkable increase in costs, as in the case of the four mixed HCV infections for whom pan-genotypic regimens would have been mandatory. Precise assignment of HCV genotype and subtype by sequencing may, therefore, be more beneficial than expected, until more potent pan-genotypic regimens are available for all patients

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

<p>Abstract</p> <p>Background</p> <p>Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus <it>Anisakis</it>. 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 <it>Anisakis </it>are detected. Thus, molecular-based diagnostic approaches are warranted.</p> <p>Method</p> <p>We have developed a PCR method that amplifies the DNA of <it>Anisakis </it>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 <it>cox2 </it>gene were used and sequence analysis was performed according to the procedures already established for species of <it>Anisakis</it>.</p> <p>Results</p> <p>The sequence obtained (629 bp) was compared with those of the other species of <it>Anisakis </it>which have so far been genetically characterized and with sequences obtained from larval stages of <it>Anisakis </it>collected from the Mediterranean fish <it>Engraulis encrasicolus</it>. This enabled the genetic identification of the larva in the human tissue as <it>A. pegreffii</it>. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin.</p> <p>Conclusion</p> <p>The case of human anisakiasis presented reinforces the pathological significance of the species <it>A. pegreffii </it>to humans. The molecular/genetic methodological approach based on mtDNA <it>cox2 </it>sequence analysis, described here, can allow easy and rapid identification of <it>Anisakis </it>spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis.</p