An Unexpected Case of Opisthorchis felineus Infection Revealed during Liver Transplantation.

A man with hepatitis B infection was admitted to Pisa University Hospital for hepatological evaluation, which revealed multiple cystic lesions and suggested a cirrhotic evolution. Treatment with Entecavir 0.5 mg/day was started, resulting in rapid viral load suppression and alanine aminotransferase normalization. After 10 years, imaging documented a single nodule of hepatocellular carcinoma (HCC), and a robot-assisted nodule resection was performed. One year later, HCC recurrence prompted orthotopic liver transplantation, during which the patient died because of the sudden rupture of the donor's organ and rapid multiorgan deterioration before retransplantation. During post-mortem liver examination, adult worms were evidenced within large biliary ducts, suggesting infection with Opisthorchis or Clonorchis spp. flukes. Sequencing of the ITS2 locus, following PCR amplification of DNA extracted from liver tissue, revealed 100% identity with the reference sequence of O. felineus. Infection of the patient with O. felineus was confirmed by the presence of specific IgG detected by ELISA in the patient's sera. Two major alkaline phosphatase serum levels peaks observed during the first two years of antiviral therapy support the hypothesis that O. felineus infection worsened liver function. This case report highlights the importance of a very careful screening of parasitic infections in solid organ transplantation candidates.

Artesunate and dihydroartemisinin-piperaquine treatment failure in a severe Plasmodium falciparum malaria case imported from Republic of Côte d'Ivoire.

A 68-year-old man returning from Republic of Côte d'Ivoire (Ivory Coast) was diagnosed with severe Plasmodium falciparum malaria and treated with intravenous artesunate followed by oral dihydroartemisinin-piperaquine (DHA-PPQ). A month later the patient experienced a new P. falciparum episode; analysis of pfmsp-1 and pfmsp-2 revealed that the infection was caused by a genetic strain identical to the strain that caused the initial episode, indicating resurgence of the previous infection. No mutations in genes associated with resistance to artemisinin derivatives (pfk13) or piperaquine (pfexonuclease, pfplasmepsin 2/3) were detected, suggesting that treatment failure could have been caused by drug malabsorption or poor drug manufacturing practices. A second treatment with atovaquone-proguanil was successful in eliminating the infection, with no further relapses. To our knowledge, this is the first description of a treatment failure with both artesunate and DHA-PPQ in a traveler returning from a malaria-endemic region. Analysis of molecular markers of resistance to antimalarial drugs revealed mutations associated with resistance to sulfadoxine (pfdhps) and pyrimethamine (pfdhfr), highlighting the important contribution of surveillance of imported malaria cases to the monitoring of drug resistance globally.

Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility.

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

Molecular Identification of New Cases of Human Dirofilariosis (Dirofilaria repens) in Italy.

(1) Dirofilariosis is a vector-borne parasitic disease mainly in domestic and wild carnivores caused by Dirofilaria (Noctiella) repens, which is endemic in many countries of the Old World, and D. immitis, which has a worldwide distribution. In recent years, an increase in the number of human cases has been reported, suggesting that dirofilariosis is an emergent zoonosis. Here, we describe further cases (N = 8), observed in Central Italy during the years 2018-2019. (2) Molecular diagnosis was performed on: (i) live worms extracted from ocular conjunctiva, cheek, and calf muscle; (ii) histological sections of surgically removed nodules from parenchymal lung, coccyx, and breast. (3) Sequence analysis (650-bp) of the mitochondrial cytochrome oxidase subunit I gene (mtDNA cox1) showed a match of 100% with the sequences of D. repens previously deposited in GenBank. ELISA test to detect IgG against filarial antigens was performed on four patients' sera and resulted positive in two patients who showed ocular and subcutaneous dirofilariosis, respectively. Microfilariae have been never detected in the peripheral blood of the patients. (4) The occurrence of N = 8 new cases of human D. repens-infections observed in a two-year period suggests an increased circulation of the parasite in Italy. Therefore, dirofilariosis should be included in differential diagnosis in patients presenting subcutaneous and/or pulmonary nodules. Molecular diagnosis of the etiological agents is fundamental. Specific serological diagnosis needs to be improved in future research work.

Protein hydrolysates from anchovy waste: purification and chemical characterization.

The aim of this study is to prompt the recovery of industrial by-products through the production of new functional foods; it takes advantage from new throughput technologies with low environmental impact and high economic sustainability. In the field of fish processing, in order to recover the worthy protein-rich fish waste, residues from the production of Anchovies (Engraulis encrasicolus) have been converted into hydrolysate through enzymatic treatment. The obtained hydrolysate product showed a promising biological and nutritional content made by differently sized peptides and free amino acids endowed with assessed benefic effects. The study showed the possibility to produce a dry powder with an activity water (aw) of 0.3-0.5 and an essential amino acids (EAA) fraction of 42.0% over the total amino acids (TAAs). These results pave the way to the smart recovery of commercial products featured by high nutritional value, either as stand-alone items or as components of functional foods.

Screening of at-risk blood donors for Chagas disease in non-endemic countries: Lessons from a 2-year experience in Tuscany, Italy.

BACKGROUND: Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi and is transmitted by blood-sucking triatomine insects in endemic areas of Latin America. Transmission can also occur via blood transfusion and is a major cause of CD in non-endemic areas. OBJECTIVES: The aim of the study was to assess the prevalence of anti-T. cruzi antibodies in blood donors at risk of infection in Tuscany, Italy, following the introduction of blood safety Italian legislation. MATERIAL AND METHODS: Donors (N = 1985) were tested in 2016 to 2018 for anti-T. cruzi IgG using an immunochromatographic test (ICT). Chemiluminescent immunoassay (CLIA) was performed on ICT-positive donors to exclude CD, whereas enzyme-linked immunosorbent assay and western blot were performed in case of discordant results. All assays were performed on CD patients (N = 10) for validation. RESULTS: Ten blood donors had a positive ICT result, with a resulting T. cruzi seroprevalence of 0.5% but demonstrated negative results to CLIA, as well as to the other serological assays. The comparison of serological assays suggested a lower relative sensitivity of ICT. CONCLUSION: The results of this study confirm the significance of serological testing in the screening strategy for CD. However, they provide evidence for discontinuing the use of ICT as a screening test and suggest that a sensitive, specific and multi-sample format assay should be used at the national level for uniformity of results.

Antibody response to Schistosoma haematobium and other helminth species in malaria-exposed populations from Burkina Faso.

Infection with helminths in sub-Saharan Africa could modulate the immune response towards Plasmodium falciparum as well as susceptibility to malaria infection and disease. The aim of this study is to assess the antibody responses to helminths species in malaria-exposed populations from Burkina Faso. Plasma samples were collected in rural villages inhabited by Fulani, Mossi and Rimaibe communities, and IgG against parasitic helminths were measured by ELISA. The prevalence of IgG against antigens of Strongyloides stercoralis, Wuchereria bancrofti and Schistosoma haematobium (Soluble Egg Antigen, SEA) was 5%, 16% and 63% respectively, in line with estimates of infection prevalence in the region for the three parasites. Anti-SEA IgG prevalence was highest at 10-20 years of age, higher in males than females, and did not show differences between ethnic groups. However, the Fulani showed lower levels of anti-SEA IgG suggesting that lighter S. haematobium infections may occur in the ethnic group known for a marked lower susceptibility to P. falciparum. The present data support the use of serological methods for integrated surveillance of neglected tropical diseases such as soil-transmitted helminths, lymphatic filariasis and bilharzia. Furthermore, as helminth infections might promote downregulation of immune responses against intracellular pathogens, the observation of lower anti-SEA IgG levels in the malaria resistant Fulani population warrants further investigation into the immunological cross-talk between S. haematobium and P. falciparum in this geographical region.

Imported human Schistosoma japonicum: A report on two cases in Filipino migrants present in Italy and a systematic review of literature.

BACKGROUND: Schistosoma japonicum is endemic in the Philippines, China, and Indonesia, and is the third-most common schistosoma species. The infection can be asymptomatic for years but, if left untreated, can lead to irreversible complications. METHOD: We report the results of a systematic review of the literature on imported S. japonicum infection and describe two previously unpublished cases diagnosed in Filipino migrants in Italy. RESULTS: Twenty-five imported cases of S. japonicum schistosomiasis were retrieved. All patients but one were migrants. Most subjects acquired the infection in Philippines (n = 18, 72%). Median age at diagnosis was 46 years. Median period of residence in non-endemic countries before diagnosis was 14.5 years. Cases of prevalent hepatosplenic involvement were 10 (40%), those with prevalent intestinal involvement were 10 (40%), whereas five (20%) had overlapping manifestations. Ten patients suffered from cirrhosis; two underwent liver transplantation. Three patients presented with acute abdomen due to intestinal complications, leading to explorative laparotomy. In all cases, but one, the diagnosis was based on a histological examination of biopsy specimen, revealing S. japonicum ova. Seventeen patients were treated with praziquantel, and in three of them, possible treatment failures occurred. CONCLUSIONS: S. japonicum infection is uncommonly reported in non-endemic areas, but is probably underestimated because of a low threshold awareness of clinicians and unavailability of specific diagnostic tools. Viable S. japonicum adults may persist for decades, indicating that migrants or travellers previously exposed in areas with high-risk areas can harbour viable worms and deserve treatment.

Accumulation of PCBs, PAHs, plasticizers and inorganic elements in Hexanchus griseus from the strait of Messina (Central Mediterranean sea).

This study investigates the inorganic elements and the persistent organic pollutants (POPs) accumulated in liver of a sexually mature Hexanchus griseus living in the Mediterranean Sea. The casual finding of a specimen in the Strait of Messina (April 2018) allowed us to carefully analyse its liver which can be considered a very important biological indicator. The determination of inorganic elements was carried out by ICP-MS technique. Quantitative determination of polycyclic aromatic hydrocarbons (PAH) and poly-chloro-biphenyl compounds (PCB), distinguished in dioxine-like (DL) and not-dioxine-like (NDL) derivates, has been carried out by HRGC/MS and HRGC-MS/MS respectively. Worrying levels of PCB-DL (TEQ = Σ(PCB-DL)*TEF = 5.96 ng g-1), PCB-NDL (Σ(PCB-NDL) = 1390.4 ng g-1) together with the presence of pesticides and plasticizers were found in the shark liver oil. The levels of heavy metals were below the legal limits.

High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR) as quick and direct insight of almonds.

Almonds are the tasty seeds of Prunus dulcis plants globally appreciated for the pleasant palatability and remarkable nutritional value, therefore it is very spread as snack and as basic ingredient of the confectionery products. The HR-MAS-NMR is a simple spectroscopy able to directly and quickly explore the chemical composition of powdered seed samples dispersed in D2O. 1H spectra witness the remarkable presence of triglyceride fatty esters together with sucrose; other minor water soluble metabolites are also detectable. This very rough approach is effectively providing chemical profiles featuring almond samples. In this analysis we were able to statistically distinguish the "Avola" almonds from other marketed products submitted to the same analysis. This is just a first investigation based on the main compounds but it might pave the way toward the quantitative evaluation of many other compounds in the almond therefore implementing the HR-MAS-NMR knowledge of these precious seeds.

Risk of transfusion-transmitted malaria: evaluation of commercial ELISA kits for the detection of anti-Plasmodium antibodies in candidate blood donors.

BACKGROUND: Transfusion with Plasmodium-infected blood represents a risk for malaria transmission, a rare but severe event. Several non-endemic countries implement a strategy for the screening of candidate blood donors including questionnaire for the identification of at-risk subjects and laboratory testing of blood samples, often serology-based, with temporary deferral from donation for individuals with a positive result. In Italy, the most recent legislation, issued in November 2015, introduced the use of serological tests for the detection of anti-Plasmodium antibodies. METHODS: In the absence of a gold standard for malaria serology, the aim of this work was to evaluate five commercial ELISA kits, and to determine their accuracy (sensitivity and specificity) in comparison to immuno-fluorescence antibody test (IFAT), and their agreement (concordance of results). Serum samples from malaria patients or from subjects with malaria history (N = 64), malaria naïve patients with other parasitic infections (N = 15), malaria naïve blood donors (N = 8) and malaria exposed candidate blood donors (N = 36) were tested. RESULTS: The specificity of all ELISA kits was 100%, while sensitivity ranged between 53 and 64% when compared to IFAT on malaria patients samples. When tested on candidate blood donors' samples, ELISA kits showed highly variable agreement (42-94%) raising the possibility that the same individual could be included or excluded from donation depending on the test in use by the transfusion centre. CONCLUSIONS: These preliminary results indicate how the lack of a gold standard for malaria serology must be taken into account in the application and future revision of current legislation. There is need of developing more sensitive serological assays. Moreover, the adoption of a unique serological test at national level is recommended, as well as the development of screening algorithms based on multiple laboratory tests, including molecular assays.

Multiple analytical approaches for the organic and inorganic characterization of Origanum vulgare L. samples.

Origanum vulgare L. samples, marketed in different geographic locations, were characterized by their organic and inorganic chemical composition. A total of 35 commercial samples were collected from various sites and analyzed to determine the qualitative and quantitative profile of essential oils, phenolic compounds and some inorganic elements. The variation in the content and composition of the essential oil was assessed by GC and GC-MS analyses, the phenolic fraction was investigated by UPLC®/PDA, and the inorganic elements were determined by ICP-MS. The Principal Component Analysis (PCA) was applied with the aim to sort out the Origanum vulgare L. samples with different composition according to the different belonging origins. The results showed appreciable qualitative and quantitative differences among samples from different geographic origin.

Post-transplant liver graft schistosomiasis in a migrant from Sub-Saharan Africa.

We report a case of post-transplant liver graft infection with Schistosoma spp in a migrant from sub-Saharan Africa transplanted for HBV-related cirrhosis and with undiagnosed schistosomiasis pre-transplantation. The occurrence of tropical diseases in non-endemic areas warrants screening protocols for organ donors and recipients with a history of exposure in endemic areas.

Chemical characterization of a variety of cold-pressed gourmet oils available on the Brazilian market.

Different specialty extra virgin oils, produced by cold-pressing fruits/nuts (olive, pequi, palm, avocado, coconut, macadamia and Brazil nut) and seeds (grapeseed and canola), and retailed in the Brazilian region of Minas Gerais, were chemically characterized. Specifically, for each type of oil, the fatty acid composition was elucidated by GC-FID, the contents of selected polyphenols and squalene were determined respectively by UHPLC-MS and UHPLC-PDA, whereas minerals were explored by means of ICP-MS. Olive oil was confirmed to have the highest MUFA content due to a valuable level of oleic acid, while oils from grapeseed, Brazil nut and canola were marked by nutritionally important PUFA levels. The highest SFA content found in coconut oil was mainly due to the high levels of lauric acid, known for its advantageous HDL-raising effects. As for polyphenols, gourmet oils from palm, coconut and canola showed higher levels of phenolic acids (e.g. p-hydroxybenzoic, ferulic, syringic, acids) than olive oil, which was though characterized by peculiar antioxidants, such as tyrosol and hydroxytyrosol. Also, olive oil had the highest amount of squalene, followed by the oil from Brazil nut. Finally, all the investigated oils had very low levels (order of µg/kg) of pro-oxidant elements, such as Cu, Fe and Mn. Overall, these findings may fill the gaps still present in literature on certain compositional aspects of commercially available gourmet oils.

Toxic inorganic pollutants in foods from agricultural producing areas of Southern Italy: Level and risk assessment.

This study focused on the inorganic environmental contaminants in specific food products which form part of the food chain of the population residing in the area of Southern Italy (Gela) where there is a high mortality rate linked to tumor diseases and congenital malformations. Determination of toxic metals was performed by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The toxicological data for every toxic element has been applied to evaluate the risk for the consumer by calculating the amount of potentially toxic element that would ingest an average of 60kg weight individual adult. The analysis of the investigated samples shows a high level of metal contamination which can be linked both to the polluted water and air. The obtained results give us an idea about the agricultural products contamination and possible exposure of local people through the food chain.

Detection of Plasmodium falciparum male and female gametocytes and determination of parasite sex ratio in human endemic populations by novel, cheap and robust RTqPCR assays.

BACKGROUND: The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites. AIM AND OBJECTIVES: The aim of this work was to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on detection and amplification of gametocyte sex specific transcripts selected from the literature: the female-specific pfs25 and pf glycerol kinase (pfGK) and the male-specific pfs230p and pf13 transcripts. METHODS: RTqPCR assays were used to test the gametocyte- and sex-specific expression of the target genes using asexual stages of the gametocyteless parasite clone F12 and FACS purified male and female gametocytes of the PfDynGFP/P47mCherry line. Assays were performed on 50 blood samples collected during an epidemiological survey in the Soumousso village, Burkina Faso, West-Africa, and amplification of the human housekeeping gene 18S rRNA was employed to normalize RNA sample variability. RESULTS: SYBR Green assays were developed that showed higher sensitivity compared to Taqman assays at a reduced cost. RTqPCR results confirmed that expression of pfs25 and pfs230p are female and male-specific, respectively, and introduced two novel markers, the female-specific pfGK and the male-specific pf13. A formula was derived to calculate the ratio of male to female gametocytes based on the ratio of male to female transcript copy number. Use of these assays in the field samples showed, as expected, a higher sensitivity of RTqPCR compared to microscopy. Importantly, similar values of gametocyte sex-ratio were obtained in the field samples based on the four different target combinations. CONCLUSION: Novel, sensitive, cheap and robust molecular assays were developed for the detection and quantification of female and male P. falciparum gametocytes. In particular, the RTqPCR assays based on the female-specific pfs25 and the newly described male gametocyte-specific pf13 transcripts, including normalization by the human 18S, reliably assess presence and abundance of female and male gametocytes and enable to determine their sex-ratio in human subjects in endemic areas.

Resistance to malaria through structural variation of red blood cell invasion receptors.

The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against Plasmodium falciparum malaria, but the precise nature of the protective effecthas proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of P. falciparum infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual's level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of P. falciparum infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations.

Fast UPLC/PDA determination of squalene in Sicilian P.D.O. pistachio from Bronte: Optimization of oil extraction method and analytical characterization.

A fast reversed-phase UPLC method was developed for squalene determination in Sicilian pistachio samples that entry in the European register of the products with P.D.O. In the present study the SPE procedure was optimized for the squalene extraction prior to the UPLC/PDA analysis. The precision of the full analytical procedure was satisfactory and the mean recoveries were 92.8±0.3% and 96.6±0.1% for 25 and 50mgL-1 level of addition, respectively. Selected chromatographic conditions allowed a very fast squalene determination; in fact it was well separated in ∼0.54min with good resolution. Squalene was detected in all the pistachio samples analyzed and the levels ranged from 55.45-226.34mgkg-1. Comparing our results with those of other studies it emerges that squalene contents in P.D.O. Sicilian pistachio samples, generally, were higher than those measured for other samples of different geographic origins.