Diagnostic Accuracy of LDBIO-Toxo II IgG and IgM Western Blot in Suspected Seroconversion in Pregnancy: A Multicentre Study.

The high sensitivity of the automated tests used for Toxoplasma gondii serology can yield false-positive IgM results due to aspecific reactions. On the other hand, specific therapy can delay IgG production and, therefore, the diagnosis of seroconversion. There is a need for confirmation tests to early detect seroconversions during pregnancy. We conducted a multicentre study to evaluate the diagnostic accuracy of the Toxo II IgG and a new, not yet commercialised Toxo II IgM western blot (WB) (LDBio diagnostics Lyon France) on 229 sera corresponding to 93 patients with seroconversions and 158 sera corresponding to 68 patients with nonspecific IgM. Sensitivity was 97.8% for IgM WB and 98.9% for IgG WB. Specificity was 89.7% and 100%, respectively. The concordance between IgM and IgG Toxo WB with the final diagnosis was very good, K = 0.89 and K = 0.99, respectively. In 5 cases (5.4%), the appearance of IgM, and in 55 cases (59.1%), the appearance of IgG was recorded by WB earlier than by traditional tests. In 10 cases (10.8%), IgM was detected after the traditional tests and in 2 cases (2.2%) for IgG. The association of IgG and IgM WB on the same sample not only detected all seroconversions but also correctly identified most of the false-positive results.

Effector Vγ9Vδ2 T cell response to congenital Toxoplasma gondii infection.

A major γδ T cell population in human adult blood are the Vγ9Vδ2 T cells that are activated and expanded in a TCR-dependent manner by microbe-derived and endogenously derived phosphorylated prenyl metabolites (phosphoantigens). Vγ9Vδ2 T cells are also abundant in human fetal peripheral blood, but compared with their adult counterparts they have a distinct developmental origin, are hyporesponsive toward in vitro phosphoantigen exposure, and do not possess a cytotoxic effector phenotype. In order to obtain insight into the role of Vγ9Vδ2 T cells in the human fetus, we investigated their response to in utero infection with the phosphoantigen-producing parasite Toxoplasma gondii (T. gondii). Vγ9Vδ2 T cells expanded strongly when faced with congenital T. gondii infection, which was associated with differentiation toward potent cytotoxic effector cells. The Vγ9Vδ2 T cell expansion in utero resulted in a fetal footprint with public germline-encoded clonotypes in the Vγ9Vδ2 TCR repertoire 2 months after birth. Overall, our data indicate that the human fetus, from early gestation onward, possesses public Vγ9Vδ2 T cells that acquire effector functions following parasite infections.

Adaptive Natural Killer Cell Functional Recovery in Hepatitis C Virus Cured Patients.

BACKGROUND AND AIMS: Current evidence suggests that dysfunctional natural killer (NK) cell responses during hepatitis C virus (HCV) infection can be restored after viral eradication with direct acting antivirals (DAAs). However, the fate of the recently described adaptive NK cell population, endowed with increased ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), during HCV infection is poorly defined, while no study has explored the effects of DAAs on this NK subset. APPROACH AND RESULTS: We performed multicolor flow cytometry to investigate CD57+ FcεRIγneg adaptive and FcεRIγpos conventional NK cell phenotype and function before and after DAA treatment in 59 patients chronically infected with HCV, 39 with advanced liver fibrosis, and 20 with mild-moderate liver fibrosis. Moreover, bulk NK cell phenotype and function were analyzed after cytokine activation following contact with K562 target cells. The proportion of FcεRIγneg NK cells in patients with HCV was associated with increased HCV load at baseline, and it was significantly reduced after treatment. Patients with an advanced fibrosis stage displayed increased NK cell activation and exhaustion markers that normalized after therapy. Of note, adaptive NK cells from patients with HCV were characterized by increased programmed death receptor 1 expression and reduced ADCC activity at baseline. DAA treatment restored ADCC ability and reduced programmed death receptor 1 expression. CONCLUSIONS: HCV profoundly affects the frequency, phenotype, and function of adaptive NK cells. DAA therapy restores a normal adaptive NK phenotype and enhances interferon-gamma production by this cell subset.

Comparison of the LIAISON®XL and ARCHITECT IgG, IgM, and IgG avidity assays for the diagnosis of Toxoplasma, cytomegalovirus, and rubella virus infections.

This study compared the performance of the LIAISON®XL system of immunoglobulin (Ig) G and IgM immunoassays for the diagnosis of Toxoplasma gondii, cytomegalovirus (CMV), and rubella virus infections with that of the ARCHITECT system. Patient serum samples, previously screened and clinically diagnosed with T. gondii, CMV or rubella, were used to compare LIAISON®XL and ARCHITECT IgG and IgM immunoassays. LIAISON®XL Toxo and CMV IgG avidity assays were also compared with equivalent ARCHITECT assays and reference methods. Overall agreement between the LIAISON®XL and ARCHITECT assays was 99% and 92% for the Toxo IgG and IgM assays, respectively, 98% and 96% for the CMV IgG and IgM assays, respectively, and 93% and 98% for the rubella virus IgG and IgM assays, respectively. LIAISON®XL IgG Toxo and CMV avidity assays showed high concordance with the VIDAS® Toxo IgG avidity assay and an in-house CMV avidity assay (reference methods), and faster IgG avidity maturation in a larger number of samples collected months after the primary infection compared with equivalent ARCHITECT assays. LIAISON®XL assays for detection of anti-T. gondii, CMV and rubella virus IgG and IgM are at least equal to the competitor assays on the ARCHITECT platform.

Role of microRNAs in host defense against Echinococcus granulosus infection: a preliminary assessment.

Cystic echinococcosis (CE) is a neglected helminthic zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus s.l. MicroRNAs (miRNAs) are regulators of gene expression that have been linked with the pathogenesis of several human diseases, but little exists in the available literature about miRNAs in CE. Here, we investigate the expression profiles of 84 microRNAs relevant to the function of lymphocytes and other immune cells during CE infection in the peripheral blood of patients with cysts in active and inactive stages. We applied the microRNA PCR array technology to blood samples from 20 patients with a single hepatic CE cyst in either the active (CE3b) or inactive (CE4-CE5) stage. Our results show a significant upregulation of eight miRNAs (let-7g-5p, let-7a-5p, miR- 26a-5p, miR- 26b-5p, miR- 195-5p, miR- 16-5p, miR- 30c-5p, and miR- 223-3p) in patients with active cysts compared to those with inactive cysts. The high expression of these miRNAs in patients with active cysts suggests their role in a specific host immune response against the infection. Further work in this direction may help shed light on the pathogenesis of human CE.

Correction to: Role of microRNAs in host defense against Echinococcus granulosus infection: a preliminary assessment.

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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.

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.

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.

Triagem para toxoplasmose na gestação: um ano de experiência em um laboratório de referência italiano / Screening for toxoplasmosis during pregnancy: one-year experience in an Italian reference laboratory

Pavia, IRCCS Foundation, San Matteo Polyclinic Pavia, a reference laboratory for diagnosis of toxoplasmosis, in the investigation of 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 (bioMérieux, Marcy l?Etoile, France), IgM ISAGA (bioMérieux, 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.

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 Kenya's 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.

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.

Surveillance of Toxoplasma gondii infection in recipients of thoracic solid organ transplants.

We evaluated the frequency of seroconversion for toxoplasmosis in seronegative recipients of thoracic solid organ transplants with seronegative or seropositive donors and the efficacy of chemoprophylaxis with pyrimethamine+sulfametopirazine. One hundred and sixty one patients seronegative for toxoplasmosis were followed-up at different intervals. Six patients out of 79 R-/D- and twelve out of 82 R-/D+ seroconverted after chemoprophylaxis interruption. There was no difference between matched and mismatched recipients as to the frequency of seroconversion which therefore could not be related to donor seropositivity. Seroconversions were almost asymptomatic. All positive recipients should be tested if symptoms of infection are present.

Evaluation of Elisa test for therapeutic monitoring of Nelfinavir in HIV-positive patients.

Therapeutic drug monitoring (TDM) is an important tool in the management of antiretroviral (ARV) therapy. The gold standard for measuring drugs plasma levels is High-Performance Liquid Chromatographic Assay (HPLC) however it is technically-demanding and time-consuming. We evaluated a new immunoenzymatic test (TDM-ELISA, Biostrands, Trieste, Italy) for nelfinavir and its active metabolite M8 in comparison with HPLC. A statistically significant difference in Ctrough between the two different tests was demonstrated but this difference was no longer significant when a value of 29% due to M8 aliquot was deleted. This faster TDM-ELISA may have an important role for TDM in HIV patients taking ARVs.