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.

Accuracy of an experimental whole-blood test for detecting reactivation of echinococcal cysts.

BACKGROUND: Cystic echinococcosis (CE) is a complex disease for which clear understanding of clinical manifestations is needed to avoid misdiagnosis, inappropriate treatment, and severe complications. We evaluated the accuracy of a whole-blood stimulation test based on Interleukin (IL)-4 detection in response to Antigen B (AgB) of Echinococcus granulosus sensu lato to discriminate cyst viability and detect cyst reactivation in patients with hepatic CE. METHODOLOGY/PRINCIPAL FINDINGS: Thirty patients with CE3b cysts and 37 patients with spontaneously-inactivated CE4-CE5 cysts were recruited (T0). After enrollment, 5 patients with CE3b cysts received albendazole, resulting in cyst solidification (CE4) in 4/5. Within a two-year follow-up, the whole-blood test was repeated at two time-points, in ≥14 (T1) and in ≥4 (T2) patients per group. IL-4 and a panel of other soluble factors were measured in the stimulated plasma. Baseline IL-4 levels were significantly higher in patients with CE3b compared to those with CE4 cysts (p = 0.006). Test accuracy for CE3b diagnosis had a sensitivity of 33-60% and a specificity of 76-95%, depending on the cut-off applied. Overall, IL-4 levels did not change significantly over time in either group; however, patients within the CE3b group showed a significant decrease of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF at T1 compared to T0 (p≤0.042). CONCLUSIONS/SIGNIFICANCE: Whole-blood IL-4-response to AgB is significantly higher in patients with active compared to inactive CE but apparently not modulated over time after treatment. On the contrary, the levels of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF significantly decreased in active CE during follow-up. Additional studies are needed to understand whether these findings might have a clinical significance for patients' follow-up.

Collaborative Studies for the Detection of Taenia spp. Infections in Humans within CYSTINET, the European Network on Taeniosis/Cysticercosis.

Laboratory tools for diagnosing taeniosis/cysticercosis in non-endemic countries are available; however, there is little data on their performance. To provide information on the sensitivity, specificity, and reproducibility of these tools, inter-laboratory studies were organized within the EU COST-Action CYSTINET (TD1302). Two serological and one coprological Ring Trials (RTs) were organized to test a panel of human-derived sera and stool samples using assays routinely conducted by the participating laboratories to detect Taenia spp. infections. Four Western blots (WBs) and five ELISAs were used by nine laboratories for cysticercosis diagnosis. In the first serological RT, the overall sensitivity was 67.6% (95% CI, 59.1-75.4), whereas specificity was 97% (95% CI, 89.8-99.6). WBs recorded the best accuracy. A second serological RT was organized, to assess the three tests most frequently used during the first RT. Two out of six laboratories performed all the three tests. The overall sensitivity and specificity were 52.8% (95% CI, 42.8-62.7) and 98.1% (95% CI, 93.2-99.7), respectively. Laboratory performance strongly affected test results. Twelve laboratories participated in the coprological RT using conventional microscopy and six laboratories used molecular assays. Traditional diagnosis by microscopy yielded better results than molecular diagnosis. This may have been influenced by the lack of standardization of molecular tests across participating laboratories.

Differences in larval survival and IgG response patterns in long-lasting infections by Trichinella spiralis, Trichinella britovi and Trichinella pseudospiralis in pigs.

BACKGROUND: Domesticated and wild swine play an important role as reservoir hosts of Trichinella spp. and a source of infection for humans. Little is known about the survival of Trichinella larvae in muscles and the duration of anti-Trichinella antibodies in pigs with long-lasting infections. METHODS: Sixty pigs were divided into three groups of 20 animals and infected with 10,000 larvae of Trichinella spiralis, Trichinella britovi or Trichinella pseudospiralis. Four pigs from each group were sacrificed at 2, 6, 12, 18 and 24 months post-infection (p.i.) and the number of larvae per gram (LPG) of muscles was calculated. Serum samples were tested by ELISA and western blot using excretory/secretory (ES) and crude antigens. RESULTS: Trichinella spiralis showed the highest infectivity and immunogenicity in pigs and larvae survived in pig muscles for up to 2 years p.i. In these pigs, the IgG level significantly increased at 30 days p.i. and reached a peak at about 60 days p.i., remaining stable until the end of the experiment. In T. britovi-infected pigs, LPG was about 70 times lower than for T. spiralis at 2 months p.i. and only very few infecting larvae were detected at 6 months p.i., whereas no larvae were detected at 12, 18 and 24 months p.i. At 6 months p.i., degenerated/calcified larvae and cysts were detected in the muscles by trichinoscopy and histology. The IgG pattern showed by T. britovi-infected pigs was similar to that of T. spiralis-infected pigs, although seroconversion occurred some days later. The larval burden of T. pseudospiralis was slightly greater than for T. britovi at 2 months p.i., but no larvae were detected at 6 and 12 months p.i. In T. pseudospiralis-infected pigs, seroconversion occurred slowly, as in T. britovi-infected pigs. The IgG level showed a significant drop at 6 months p.i. and declining to the cut-off value at 12 months p.i. CONCLUSIONS: The longer survival of T. spiralis in pigs in comparison with the other two species highlights its exceptional dissemination potential. These results provide an explanation of the controversial data collected by parasitological and serological tools in the course of epidemiological investigations.

Delivery of SA35 and SA40 peptides in mice enhances humoral and cellular immune responses and confers protection against Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium parvum is a major cause of diarrhea in children and ruminants at the earliest stages of life. Maternal antibodies represent the main shield of neonate mammals for most of the infections. Two recombinant antigens (SA35 and SA40), portions of two C. parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate BALB/c mice born from females immunised by mucosal delivery of both peptides. METHODS: Adult BALB/c mice were intraperitoneally immunised with SA35 and SA40, separately or mixed, and their immune response was characterised. Furthermore, BALB/c pregnant mice were immunised by mucosal delivery with an SA35/40 mix, before and during pregnancy. Soon after birth, their offspring were infected with two doses (1 × 105 and 5 × 103) of C. parvum oocysts and the parasitic burden was determined at 5 and 9 days post-infection. RESULTS: Intraperitoneal immunisation with SA35 and SA40 induced specific IgG and IgG1 in serum, specific IgA in the intestinal mucosa, increase of CD3+/CD4+ and CD30+ cells in splenocytes, which produced IFN-γ. Neonates born from immunised mice and infected with 1 × 105 oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). A reduction of all parasitic forms (96%; P < 0.05) was observed when neonates were infected with 5 × 103 oocysts. CONCLUSIONS: SA35 and SA40 peptides induce specific humoral and cell-mediated immune responses to C. parvum in adult mice. Moreover, mucosal administration of the SA35/40 mix in pregnant mice reduces C. parvum burden in their litters.

Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts.

BACKGROUND: Cystic echinococcosis (CE) is a complex disease caused by Echinococcus granulosus (E.granulosus), and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS), the presence of CE specific immune signatures. METHODOLOGY/PRINCIPAL FINDINGS: We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25) and "inactive stages" (17). The ability of CD4+ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB) of E.granulosus. Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4+ T-cells producing IL-2+TNF-α+Th2+ or TNF-α+Th2+ were significantly increased in the "active stages" group compared to the "inactive stages" group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE. CONCLUSIONS: We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2+TNF-α+Th2+ triple-positive and TNF-α+Th2+ double-positive specific T-cells associate with cyst biological activity. These results contribute to increase knowledge of CE immunophatogenesis and the disease outcome in terms of control and persistence.

IL-4 specific-response in whole blood associates with human Cystic Echinococcosis and cyst activity.

OBJECTIVES: Human Cystic Echinococcosis (CE) is estimated in 2-3 million global cases. CE diagnosis and clinical management are based on imaging and serology, which lacks sensitivity and does not provide cyst stage information. This study aimed to evaluate tools for improving diagnosis by analysing the Interleukin (IL)-4-response to Antigen B (AgB) of Echinococcus granulosus. METHODS: Whole blood (WB) and peripheral blood mononuclear cells were stimulated with AgB. IL-4 levels were measured by enzyme-linked immunosorbent assay. RESULTS: WB 1-day stimulation resulted the best experimental condition for evaluating AgB IL-4-response. IL-4 levels were significantly higher in CE patients than healthy donors (p ≤ 0.0001). A ROC analysis showed significant area under the curve (AUC) results (AUC, 0.85; p = 0.0001) identifying an IL-4 level cut-off point ≥0.39 pg/mL which predicted CE with 71.4% sensitivity and 93.3% specificity. Moreover, we found that IL-4 levels were significantly increased in patients with active cysts compared to those with inactive cysts (p ≤ 0.0001). ROC analysis showed significant AUC results (0.94; p = 0.0001) with a cut-off point of 4.6 pg/mL which predicted active cysts with 84.6% sensitivity and 92% specificity. CONCLUSIONS: We found immunological correlates associated with CE and biological cyst activity.

Development of an ELISA to detect the humoral immune response to Trichinella zimbabwensis in Nile crocodiles (Crocodylus niloticus).

Crocodiles are known reservoir hosts of Trichinella papuae and Trichinella zimbabwensis, two zoonotic parasites that also infect mammals. Since commercial crocodile farming represents a key source of income in several countries, it is important to monitor this nematode infection in both farmed crocodiles and in breeding stocks which are frequently introduced from the wild. For this purpose, an indirect ELISA was developed to detect the anti-Trichinella immune response in crocodile sera. New Zealand rabbits were immunized with pooled sera from non-infected farmed crocodiles in the presence of Freund's complete adjuvant. The anti-crocodile serum was then conjugated with horseradish peroxidase. Serum samples from four Nile crocodiles (Crocodylus niloticus) experimentally infected with T. zimbabwensis and from four uninfected crocodiles were used to set up the ELISA. The larval burden per gram of muscle tissue was determined by muscle biopsy. The test was performed on serum samples from an additional 15 experimentally infected crocodiles as well as eight wild Nile crocodiles. Among the 19 experimentally infected crocodiles, seroconversion was observed in 11 animals. The highest antibody response was observed six weeks post infection (p.i.), but in most of these animals, antibodies were not detectable after six weeks p.i. even though live larvae were present in the muscles up to six months p.i.