Francesca Genco

Molecular evidence of the camel strain (G6 genotype) of Echinococcus granulosus in humans from Turkana, Kenya

Cystic echinococcosis (CE) is a zoonotic helminthic disease, which is widely distributed throughout the world. Although G1 is the Echinococcus granulosus genotype most commonly involved in CE in humans, the prevalence of infection with other genotypes, such as G6, may be higher than previously thought. We performed molecular analysis to identify which E. granulosus genotypes are the causative agents of CE in humans in Kenyas Turkana district. During a Hydatid Control Programme in 1993-1994, 71 cyst fluid isolates of E. granulosus were collected during PAIR (puncture, aspiration, injection, re-aspiration) sessions. DNA was amplified for two genes from 59 isolates. Of these, 49 isolates (83%) were identified as G1 and 10 (17%) as G6. This is the highest prevalence of G6 detected in humans of the Old World, and our results suggest that, in highly contaminated environments, G6 might be of greater public health significance than previously believed.

Factors influencing the serological response in hepatic echinococcus granulosus infection

Knowledge of variables influencing serology is crucial to evaluate serology results for the diagnosis and clinical management of cystic echinococcosis (CE). We analyzed retrospectively a cohort of patients with hepatic CE followed in our clinic in 2000-2012 to evaluate the influence of several variables on the results of commercial enzyme-linked immunosorbent assay (ELISA) and indirect hemagglutination (IHA) tests. Sera from 171 patients with ≥ 1 hepatic CE cyst, and 90 patients with nonparasitic cysts were analyzed. CE cysts were staged according to the WHO-IWGE classification and grouped by activity. A significant difference in ELISA optical density (OD) values and percentage of positivity was found among CE activity groups and with controls (P < 0.001). The serological response was also influenced by age (P < 0.001) and cyst number (P = 0.003). OD values and cyst size were positively correlated in active cysts (P = 0.001). IHA test showed comparable results. When we analyzed the results of 151 patients followed over time, we found that serology results were significantly influenced by cyst activity, size, number, and treatment ≤ 12 months before serum collection. In conclusion, serological responses as assessed by commercial tests depend on CE cyst activity, size and number, and time from treatment. Clinical studies and clinicians in their practice should take this into account.

Recombinant AgB8/1 ELISA test vs. commercially available IgG ELISA test in the diagnosis of cystic echinococcosis

The diagnosis and clinical management of cystic echinococcosis (CE) rely on imaging and serology, the latter still having a complementary role as its accuracy in assessing cyst viability is unsatisfactory. We used an experimental IgG ELISA test based on the recombinant antigen rEgAgB8/1 cloned from Echinococcus granulosus to differentiate active from inactive/cured CE infection, comparing its performance to that of a commercially available ELISA test used routinely in our hospital laboratory. Both tests were performed on sera from 88 patients with hepatic echinococcal cysts, grouped according to cyst stage based on ultrasonographical morphology, and on 17 patients surgically treated for echinococcosis and 18 patients with nonparasitic hepatic cysts included as controls. Tests’ performances did not differ significantly, but the overall concordance between tests drastically dropped when groups were analysed separately. Further longitudinal studies should evaluate whether these discrepancies reflect the different ability of either test to predict the evolution of cysts over time. Although the recombinant‐AgB8/1‐based ELISA test seems to have no clinical advantage over the commercially available ELISA test in the assessment of hepatic CE cyst viability, the easiness of production and reproducibility of high‐quality recombinant antigens makes rEgAgB8/1 a valid candidate for use in CE ELISA diagnostic tests.

Ex vivo assessment of serum cytokines in patients with cystic echinococcosis of the liver

To investigate the usefulness of serum cytokine levels in the diagnosis of active cystic echinococcosis, we evaluated the cytokine profile of patients with hepatic cystic echinococcosis in different cyst stages, CE 1‐2 (active), CE3a‐3b (transitional) and CE4‐5 (inactive). Ex vivo assessment of Th1 (IL12, TNFα) and Th2 (IL4, IL10) cytokines in sera was carried out using ELISA. Percentages of positive samples and median levels of IL12, TNFα and IL10 did not differ significantly between groups. However, patients with CE3b cysts, a stage clinically unresponsive to treatments, had statistically significantly higher median levels of IL4 and percentage of positive samples for IL4. We conclude that the analysis of serum cytokine dosage, at least in its present form, is not useful as a marker of cyst activity. However, our results support recent findings suggesting the chronic activity of CE3b cysts and suggest that this might be partly because of a skewed Th2 response.

Immunoblotting with Human Native Antigen Shows Stage-Related Sensitivity in the Serodiagnosis of Hepatic Cystic Echinococcosis

The diagnosis of hepatic cystic echinococcosis is based on ultrasonography and confirmed by serology. However, no biological marker of cyst viability is currently available implying years-long patient follow-up, which is not always feasible in endemic areas. We characterized the performance of an immunoblotting test based on human hydatid cyst fluid with particular regard to its ability to distinguish between cyst stages. Sera from patients with cysts in different stages showed distinctive band pattern recognition. Most importantly, the test discriminated in 80% of cases CE3a from CE3b transitional cysts, known to have different viability profiles. Interestingly, we observed a rapid change in band pattern recognition of sera from one patient at time points when his cyst passed from active to transitional to inactive stages. Further identification of different antigens expressed by different cyst stages will support the development of diagnostic tools that could early define cyst viability, to guide clinical decision making, and shorten patient follow-up.

Serum Cytokine Profile by ELISA in Patients with Echinococcal Cysts of the Liver: A Stage-Specific Approach to Assess Their Biological Activity

To investigate the usefulness of serum cytokine dosage in the clinical management of cystic echinococcosis (CE), we analyzed serum levels of Th1 and Th2 cytokines in patients with hepatic CE in different cyst stages, CE1-2 (active), CE3a-3b (transitional), and CE4-5 (inactive). Ex vivo assessment of Th1 (IFN-γ) and Th2 (IL-4, IL-13, and IL-10) cytokines in sera was carried out using ELISA. IL-10 was undetectable in all serum samples of patients and controls, while a few sera contained measurable amounts of IFN-γ, IL-4, and IL-13. No statistically significant difference was found between the percentages of positive samples for each cytokine and the different groups analyzed (patients/controls, stage, number, location, and size of the cyst, serology, and sex of patients), with the exception of the association of IL-4 and IL-13 with the cyst stage. Overall, this investigation showed many limits of serum cytokine dosage as a marker of biological activity of echinococcal cysts. Because of low sensitivity and lack of specificity of this test, we believe that other ways to evaluate ex vivo biological activity of the cysts should be explored.

Correlation of serum sHLA‐G levels with cyst stage in patients with cystic echinococcosis: is it an immune‐evasion strategy?

Patients with cystic echinococcosis (CE) can harbour cysts for years or even decades, apparently without effect of the immune system on the metacestode. Although several immune evasion mechanisms by echinococcal cysts have been described, it is unclear whether the human leucocyte antigen (HLA) system plays a role in the susceptibility or resistance to CE in humans. HLA‐G molecules are known to exert a suppressive action on dendritic cells maturation and on natural killer (NK) cells functions, therefore hampering T‐cell responses and NK cytolysis. HLA‐G plays an important role in immune tolerance, is involved in foetus and in allotransplant tolerance, and may be involved in tumoral and viral immune evasion. In this study, we assessed the presence and levels of soluble HLA‐G (sHLA‐G) in patients with CE using a commercial ELISA kit to determine whether hosts HLA‐G may have a role in the course of human CE.

Nelfinavir+M8 Plasma Levels Determined with an ELISA Test in HIV Infected Patients with or without HCV and/or HBV Coinfection: The VIRAKINETICS II Study

Virakinetics II was designed as an observational, multicenter cohort study conducted in HIV-positive patients treated with NFV-based combinations. Trough (pre-dose) concentrations of NFV+M8 in plasma were determined using a novel ELISA test (NFV TDM-ELISA) and analyzed using clinical and laboratory parameters. Drug levels were sorted as below, within or above a given interval (<0.8 microg/mL, 0.8-3.5 microg/mL and >3.5 microg/mL, respectively). Longitudinal analysis was performed in a subset of patients who underwent two or more determinations. Ninety patients on NFV-containing HAART were enrolled and 43 were coinfected with HCV and/or HBV. Among coinfected patients, 10 subjects had a clinical or histological diagnosis of cirrhosis. Compared to the HIV-monoinfected, the coinfected patients were significantly older, more treatment-experienced, with higher frequency of lipodystrophy and altered liver function test values (all p values: <0.05). Coinfected patients were also more likely to be on a reduced dose of NFV than monoinfected (p=0.03). No significant difference was observed between the two groups with regard to NFV+M8 trough values and concentration range distribution. Median NFV+M8 C(trough) concentrations were higher in coinfected patients, but without reaching statistical significance (p=0.2). This new ELISA test proved to be a rapid, convenient and reliable tool for assessing NFV+M8 plasma levels in HIV-positive patients. It could be suitable for use within the framework of routine clinical practice even in peripheral centers without specialized laboratories.

Screening for toxoplasmosis during pregnancy: One-year experience in an Italian reference laboratory

AIMS: To describe the experience of the Toxoplasmosis Laboratory of Infectious Disease Department University of Pavia, IRCCS Foundation, San Matteo Polyclinic Pavia, a reference laboratory for diagnosis of toxoplasmosis, in the investigation of pregnant women with suspected acute toxoplasmosis. METHODS: All sera were tested with LIAISON® Toxo IgM and IgG II, Toxo IgG Avidity II kits (DiaSorin, Saluggia, Italy), VIDAS Toxo IgG II and Toxo IgG Avidity (bioMerieux, Marcy l’Etoile, France ), IgM ISAGA (bioMerieux, Marcy l’Etoile, France) and ETI-TOXOK-A reverse PLUS (DiaSorin, Saluggia, Italy). When required (IgG negative/IgM positive women), IgG/IgM Western Blot II (LDBio, Lyon, France) was also performed. Prenatal diagnosis on amniotic fluid was done by nested PCR. All newborns were followed up to one year of age in order to exclude or confirm the diagnosis of congenital toxoplasmosis. All pregnant women with acute or undetermined stages of infection were treated. RESULTS: In the course of 2007, 236 women with suspected acute (IgM-positive) Toxoplasma infection were followed up. In the reference laboratory, 91 women had test results indicating acute toxoplasmosis, and 10 had undetermined status of infection. These 101 patients represented 42.8% of the 236 women referred. Acute toxoplasmosis could be excluded in the remaining 135 patients, of whom 53 were non-immune. Three infected newborns were observed, all from mothers tested for the first time during the third trimester of pregnancy. CONCLUSIONS: The role of a reference laboratory in suspected toxoplasmosis acquired during pregnancy is crucial to date the infection and discriminate between seroconversion and false positive anti- Toxoplasma IgM antibodies. This avoids unnecessary anxiety in immune women, provides correct counseling about primary prevention and periodic testing for seronegative ones, and allows early treatment and follow-up of pregnant women with acute infection and their newborns.

Toxoplasmosis in Transplant Recipients, Europe, 2010–2014

Transplantation activity is increasing, leading to a growing number of patients at risk for toxoplasmosis. We reviewed toxoplasmosis prevention practices, prevalence, and outcomes for hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT; heart, kidney, or liver) patients in Europe. We collected electronic data on the transplant population and prevention guidelines/regulations and clinical data on toxoplasmosis cases diagnosed during 2010–2014. Serologic pretransplant screening of allo-hematopoietic stem cell donors was performed in 80% of countries, screening of organ donors in 100%. SOT recipients were systematically screened in 6 countries. Targeted anti-Toxoplasma chemoprophylaxis was heterogeneous. A total of 87 toxoplasmosis cases were recorded (58 allo-HSCTs, 29 SOTs). The 6-month survival rate was lower among Toxoplasma-seropositive recipients and among allo-hematopoietic stem cell and liver recipients. Chemoprophylaxis improved outcomes for SOT recipients. Toxoplasmosis remains associated with high mortality rates among transplant recipients. Guidelines are urgently needed to standardize prophylactic regimens and optimize patient management.