A protein microarray immunoassay for the serological evaluation of the antibody response in vertically transmitted infections.

The detection of specific serum antibodies is mainly achieved by enzyme-linked immunosorbent assay (ELISA). Here, we describe the setting up of a microarray-based serological assay to screen for IgG and IgM against vertically transmitted pathogens (Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus types 1 and 2, varicella zoster virus, Chlamydia trachomatis). The test, accommodated onto a restricted area of a microscope slide, consists of: (a) the immobilization of antigens and human IgG and IgM antibody dilution curves, laid down in an orderly manner; (b) addition of serum samples; (c) detection of antigen-serum antibodies complexes by indirect immunofluorescence. The IgG and IgM curves provide an internal calibration system for the interpolation of the signals from the single antigens. The test was optimized in terms of spotting conditions and processing protocol. The detection limit was 400 fg for the IgG assay and 40 fg for the IgM assay; the analytical specificity was >98%. The clinical sensitivity returned an average value of 78%, the clinical specificity was >96%, the predictive values were >73%, and the efficiency was >88%. The results obtained make this test a promising tool, suitable for introduction in the clinical diagnostic routine of vertically transmitted infections, in parallel (and in future as an alternative) to ELISA.

Interferon-gamma-release assays detect recent tuberculosis re-infection in elderly contacts.

The tuberculin skin test (TST) does not discriminate between recent and remote latent tuberculosis infection (LTBI). This study was carried out to test two interferon-gamma-based blood assays in recent contacts with high prevalence of remote LTBI. We performed a contact tracing investigation in a nursing home for the elderly, where elderly patients were exposed to a case of pulmonary tuberculosis. TST, QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB (TS.TB) were performed 8 weeks after the end of potential exposure. IFN-gamma measurements were recorded and correlation with exposure was evaluated. Twenty-seven (37.5%), 32 (44.4%) and 16 (22.2%) subjects were TST, TS.TB and QFT-G positive, respectively; agreement between TS.TB and QFT-G was good among exposed subjects only (K=0.915, 0.218 in unexposed, p<0.001). When amounts of IFN-gamma were corrected for the number of producing T cells, specific IFN-gamma production was significantly different between exposed and unexposed individuals (16.75+/-5.40 vs 2.33+/-0.71 IFN-gamma IU/1000 SFC, p=0.0001). QFT-G and TS.TB provided discordant results among elderly contacts. Unlike TST, the specific IFN-gamma response might discriminate between recent and long-lasting tuberculosis infection.

[Screening of latent tuberculosis infection in health care workers by QuantiFERON-TB and tuberculin skin test]. / Screening dell'infezione tubercolare latente in operatori sanitari con QuantiFERON-TB e test cutaneo tubercolinico.

UNLABELLED: Recent guidelines (MMWR 2005) recommend the use of QuantiFERON-TB (QFT-TB) as an alternative to the tuberculin skin test (TST) for the screening of latent tuberculosis infection (LTBI) in health care workers (HCWs). MATERIALS AND METHODS: 590 HCWs were screened for LTBI by TST and QFT-TB. Results were compared with risk factors for LTBI, determined by questionnaires. RESULTS: Both tests were significantly associated with non-Italian nationality [TCT (OR = 9.17), QFT-TB (OR = 3.65)], age > or = 45 years old [TCT (OR = 1.81), QFT-TB (OR = 2.36)], history of household contacts [TCT (OR = 2.65), QFT-TB (OR = 3.37], occupational exposure to tuberculosis (TB) patients [TCT (OR = 2.14), QFT-TB (OR = 1.93)]. 55 cases were discordant (28 QFT-TB-negatives/TCT-positives; 27 QFT-TB-positives/TCT-negatives). Both tests were not associated with workplace risk factors or TB risk level assessed in different hospital units. CONCLUSIONS: In HCWs employed in a low TB incidence area both QFT-TB and TCT were more associated with non occupational risk factors (nationality, age, household contacts) than with main determinants of workplace risk for LTBI.

Usutu virus infections in humans: a retrospective analysis in the municipality of Modena, Italy.

OBJECTIVE: To monitor the spread and to evaluate the role for public health of Usutu virus (USUV) in an endemic area of Italy. METHODS: The survey was retrospectively conducted by detecting USUV RNA and USUV antibodies in cerebrospinal fluid and serum samples collected between 2008 and 2011 from 915 patients with or without neurologic impairments in the area of the municipality of Modena, Italy. Organs of birds and pools of mosquitoes were also tested for USUV RNA. Positive samples were partially sequenced and used for phylogenetic analysis. RESULTS: The presence of USUV RNA (1.1%; 95% confidence interval (CI) 0.6-2.0) was significantly (p <0.05) higher than that of West Nile virus (0%; 95% CI 0-0.33). USUV antibody level was 6.57% (95% CI 4.87-8.82), and it was significantly higher (p <0.05) compared to that of West Nile virus (p 2.96, 95% CI 1.89-4.62). Partial genome sequencing of USUV strains detected in humans, birds and mosquitoes revealed high nucleotide sequence identity within them and with the USUV strains isolated in Central Europe. CONCLUSIONS: USUV infection in humans is not a sporadic event in the studied area, and USUV neuroinvasiveness has been confirmed.

Posttransplant Mycobacterium tuberculosis disease following liver transplantation and the need for cautious evaluation of Quantiferon TB GOLD results in the transplant setting: a case report.

This report describes a case of pulmonary tuberculosis in a liver transplant patient without a history of previous exposure to Mycobacterium tuberculosis (MTB) complex. Prior to transplantation, the tuberculin skin test was negative and the QuantiFERON-TB Gold (QFT Gold), an interferon gamma-based blood test, was negative before and after transplant including a period beginning on postoperative day 55 when the patient developed a febrile illness with an interstitial infiltrate and pleural effusion that was unresponsive to broad-spectrum antibiotic therapy. Empiric treatment with isoniazid, ethambutol, and levofloxacin resulted in resolution of the clinical symptoms. A sputum culture grew MTB on postoperative day 87. This case illustrates the need for caution when QFT Gold is used as diagnostic tool for latent tuberculosis during the pretransplant assessment. Further studies evaluating the usefulness of QFT Gold and other interferon gamma tests in posttransplantation active infection are warranted.

Clinical and Microbiological Characteristics of Visceral Leishmaniasis Outbreak in a Northern Italian Nonendemic Area: A Retrospective Observational Study.

Background. Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean area. In the last decades a northward spread of the parasite has been observed in Italy. This paper describes a VL outbreak in Modena province (Emilia-Romagna, Northern Italy) between 2012 and 2015. Methods. Retrospective, observational study to evaluate epidemiological, microbiological characteristics, and clinical management of VL in patients referring to Policlinico Modena Hospital. Results. Sixteen cases of VL occurred in the study period. An immunosuppressive condition was present in 81.3%. Clinical presentation included anemia, fever, leukopenia, thrombocytopenia, and hepatosplenomegaly. Serology was positive in 73.3% of cases, peripheral blood PCR in 92.3%, and bone marrow blood PCR in 100%. Culture was positive in 3/6 cases (50%) and all the isolates were identified as L. infantum by ITS1/ITS2 sequencing. The median time between symptom onset and diagnosis was 22 days (range 6-131 days). All patients were treated with liposomal amphotericin b. 18.8% had a VL recurrence and were treated with miltefosine. Attributable mortality was 6.3%. Conclusions. VL due to L. infantum could determine periodical outbreaks, as the one described; thus it is important to include VL in the differential diagnosis of fever of unknown origin, even in low-endemic areas.

Multidrug-resistant tuberculosis outbreak in an Italian prison: tolerance of pyrazinamide plus levofloxacin prophylaxis and serial interferon gamma release assays.

The optimal treatment for latent tuberculosis infection (LTBI) in subjects exposed to multidrug-resistant (MDR) tuberculosis (TB) remains unclear, and the change in response of the QuantiFERON-TB Gold In-Tube (QTB-IT) test during and after treatment is unknown. Between May 2010 and August 2010, 39 prisoners at the 'Casa Circondariale' of Modena, Italy, were exposed to a patient with active pulmonary MDR TB. All contacts were tested with the tuberculin skin test and QTB-IT. Upon exclusion of active TB, subjects positive to both tests were offered 6 months' treatment with pyrazinamide (PZA) and levofloxacin (LVX). QTB-IT testing was repeated at 3 and 6 months after initial testing in all subjects who were offered LTBI treatment. Seventeen (43.5%) of 39 subjects tested positive to both tuberculin skin test and QTB-IT test, and 12 (70.5%) agreed to receive therapy with PZA and LVX at standard doses. Only five (41.6%) of 12 subjects completed 6 months' treatment. Reasons for discontinuation were asymptomatic hepatitis, gastritis and diarrhoea. The QTB-IT values decreased in all subjects who completed the treatment, in two (33%) of six of those who received treatment for less than 3 months and in one (50%) of two patients who discontinued therapy after 3 months. The QTB-IT test results never turned negative. Despite the small number of subjects, the study confirmed that PZA plus LVX is a poorly tolerated option for MDR LTBI treatment. We observed a large degree of variation in the results of the QTB-IT test results among participants. The study confirmed that the interferon gamma release assay is not a reliable tool for monitoring the treatment of MDR LTBI in clinical practice.

Phenotypic characteristics and tendency to apoptosis of peripheral blood mononuclear cells from HIV+ long term non progressors.

The aim of this study was to analyze (i) phenotype, (ii) in vitro spontaneous and induced apoptosis, (iii) glutathione (GSH) intracellular content and (iv) inhibitors of apoptosis of potential therapeutical use in peripheral blood mononuclear cells (PBMC) from HIV+ long term non progressors (LTNP), in comparison with progressors (HIV+P) and seronegative controls (HIV-). Three groups of subjects were studied: 15 HIV+P (patients losing >150 CD4+/year), 9 LTNP (subjects infected by HIV for at least 7 years without clinical and immunological signs of progression, with a mean of 898 CD4+/microL) and 18 HIV-. All subjects were living in a large community for former drug addicts, and were matched for age and sex. We used flow cytometry for analyzing PBMC phenotype and apoptosis; high performance liquid chromatography for measuring intracellular GSH content. PBMC phenotype of LTNP shared characteristics with those of both HIV- and HIV+P. Indeed, LTNP showed a normal number CD4+ cells (an inclusion criteria), but significantly increased numbers of CD8+ lymphocytes, activated T cells, CD19+, CD5+ B lymphocytes and CD57+ cells, as well as a decrease in CD19+, CD5- B lymphocytes and CD16+ cells. In LTNP, spontaneous apoptosis was similar to that of HIV- and significantly lower than that of HIV+P. Adding interleukin-2 (IL-2) or nicotinamide (NAM) significantly decreased spontaneous apoptosis in LTNP and HIV+P. Pokeweed mitogen-induced apoptosis was also similar in LTNP and HIV-, but significantly lower than that of HIV+P. In HIV+P, but also in LTNP, spontaneous apoptosis was inversely correlated to the absolute number and percentage of CD4+ cells and directly correlated to the number and percentage of activated T cells present in peripheral blood. GSH intracellular content was greatly decreased in PBMC from HIV+P and slightly, but significantly, reduced in LTNP. Adding 2-deoxy-D-ribose, an agent provoking apoptosis through GSH depletion, to quiescent PBMC resulted in similar levels of massive cell death in the three groups. This phenomenon was equally prevented in the three groups by N-acetyl-cysteine but not by IL-2. A complex immunological situation seems to occur in LTNP. Indeed, PBMC from LTNP are characterized by a normal in vitro tendency to undergo apoptosis despite the presence of a strong activation of their immune system, unexpectedly similar to that of HIV+P. Our data suggest that NAM and IL-2 are possible candidates for reducing spontaneous apoptosis in HIV infection.

Treatment of advanced non-small cell lung cancer (NSCLC): the "Umbria" cooperative study.

One hundred fifty-six patients with metastatic or locally advanced non-small cell lung cancer (NSCLC) were randomized to 3-week cycles of treatment with either: (A) cisplatin (120 mg/m2 on day 1); (B) cisplatin (120 mg/m2 on day 1) plus etoposide (VP-16) (100 mg/m2 on days 1-3; and (C) the cisplatin plus etoposide (VP-16) regimen plus mitomycin C (10 mg/m2 on days 1, 21, and 42; then every 6 weeks for a maximum dose of 100 mg). The overall objective response rates for the combination regimens (30% with two drugs and 26% with three drugs) were superior to that obtained with one drug (4%). Likewise, the median duration of survival with the combination therapy arms (8 to 9 months) was superior to that obtained with the single agent (5 months). Both performance status and limited disease were correlated with response in all groups, and with survival in the combined chemotherapy arms. The dose-limiting toxicity was myelosuppression, especially for the group receiving the three-drug regimen. In summary, combination chemotherapy using cisplatin and etoposide (VP-16) appears to be the most active and safest regimen in NSCLC.

Growth of human herpesvirus 6 in HEPG2 cells.

HepG2 cells, a well differentiated liver cell line, were shown to be permissive for both human herpesvirus 6 (HHV-6) A and B strains by three independent methods of analysis: detection of viral antigens, viral DNA sequences and infectious virus. HepG2 cell infection with HHV-6 resulted in functional damage as shown by the increased release in the culture medium of some hepatocyte markers. Cells surviving the acute infection were serially passaged without showing cytopathic effect, but, some months later, HHV-6 DNA was still present in the cells and virus induction with a phorbol ester was successful. A possible pathogenetic role of HHV-6 in liver diseases is discussed. Experiments of HepG2 infection with human herpesvirus 7 (HHV-7) were also carried out. The lack of an efficient virus replication suggested a difficulty for HHV-7 to infect hepatic cells.

Quantitation of HIV-1 p24 antibody expressed as relative binding capacity p24 antigen (p24 RBC) in infants born to HIV-1 seropositive mothers: correlation of this serological marker with the HIV-1 maternal antibody course and p24 antigenemia detection.

The antibody content to HIV-1 p24 Ag expressed as relative binding capacity to the target antigen (p24 RBC) was retrospectively quantified in serum samples from 20 HIV-1-uninfected infants born to HIV-1 seropositive mothers. p24 RBC values quantified at birth were included either in a low (0-20%) or high (80-100%) range of values, classified as group A (11 infants) and group B (9 infants), respectively. The course of maternal antibodies to HIV-1 antigens p17, p24, p31, gp41, p51, p66, gp120, and gp160 was studied in each group. A substantial difference in the amount and subsequently in the decline of maternal antibodies to gag proteins p17, p24, and p55 and to pol proteins p51 and p66 was observed in the two infant groups in contrast with a similar content and decline of the remaining antibodies. In 7 HIV-1-infected infants of whom 4 resembled infant group A and 3 infant group B for p24 RBC values, a relationship appeared between p24 antibody decline and p24 antigenemia detection.

Chemotherapy with cisplatin and teniposide for cerebral metastases in non-small cell lung cancer.

Twenty-three patients with brain metastases from non-small cell lung cancer (NSCLC) (median age 62 years, Karnofsky PS 50-100) were treated with cisplatin (100 mg/m2, day 1) and teniposide (80 mg/m2, days 1, 3 and 5) every 3 weeks. Response was evaluated by contrast-enhanced brain CT every two to three cycles of treatment. The objective response rate of brain metastases was 35% (8/23); three patients achieved complete response (CR) and five partial response (PR). The median response duration was 24 weeks for CR patients and 32 weeks for PR patients. The median survival was 21 weeks overall and 45 weeks for responding patients. Grade 3/4 leukocytopenia and thrombocytopenia were seen in 28 and 9%, respectively. Two patients died from infections while in neutropenia. Cisplatin and teniposide seems an active regimen against brain metastases in NSCLC. These data may indicate the need for reconsideration of the role of chemotherapy for brain metastases of NSCLC.

Herpesvirus DNA is frequently detected in liver tissue from hepatitis C patients.

BACKGROUND AND AIMS: Herpesviruses infect the liver and cause minor hepatitis. Our aim is to verify the presence of herpesviruses in the liver from hepatitis C patients and the possible influence of these viruses in the liver disease. METHODS: We searched for herpesvirus DNA in liver biopsies from patients with hepatitis C and from a control group without hepatitis by means of nested polymerase chain reaction. Serological investigations were carried out as well. RESULTS: Thirty-four liver specimens from hepatitis C patients were examined, 12 of which (35.3%) were positive for at least one herpesvirus DNA, whereas among the 19 control specimens only two were positive (10.5%; P = 0.049). Liver biopsies from seven patients, three with acute hepatitis of unknown origin, three with non-alcoholic steatohepatitis and one with autoimmune hepatitis were also investigated and three positive samples were found. CONCLUSIONS: The prevalence of herpesvirus DNA was found higher in patients with hepatitis C than in individuals without hepatitis. The influence of herpesviruses on the clinical course of hepatitis C is considered.

A randomized trial fo three cisplatin-containing regimens in advanced non-small-cell lung cancer (NSCLC): a study of the Umbrian Lung Cancer Group.

Survival in patients with locally advanced (stage III Mo) and metastatic (Ml) non-small-cell lung cancer (NSCLC) is short. Phase II studies have reported objective responses ranging from 20% to 60% using cisplatin-based chemotherapeutic regimens, yet few have shown improvement in median survival. In our phase II pilot studies with cisplatin (CDDP) and etoposide (VP-16), we observed a 26% response rate; with CDDP, VP-16, and mitomycin-C, a 38% response rate was obtained in advanced NSCLC patients. A total of 156 consecutive patients with locally advanced and metastatic NSCLC were randomized to one of three treatment arms to determine whether the chemotherapy protocols had any effect on response rate and median survival in a large, randomized study. Arm 1 consisted of CDDP (120 mg/m2 x 3 weeks); arm 2, of CDDP (120 mg/m2) and VP-16 (100 mg/m2 given i.v. on days 1-3), repeated every 3 weeks; and arm 3, of CDDP (120 mg/m2) and VP-16 (100 mg/m2 on days 1-3) given every 3 weeks, plus mitomycin C (10 mg/m2 on days 1, 21, and 42, then every 6 weeks, for a maximal dose of 100 mg). After 71 patients had been enrolled in the study, we stopped accrual in the CDDP arm due to a lack of response [1 complete response (CR) in 24 patients; 4%] and continued enrollment in the two combination-chemotherapy arms. In the CDDP/VP-16 arm a 30% response rate [1 CR, 18 partial responses (PRs)] was obtained, and in the CDDP/VP-16 mitomycin C arm a 26% response rate (4 CRs, 11 PRs) was seen among a total of 150 evaluable patients. Responses were observed in 31% of patients with favorable performance status (PS) (ECOG 0-1) vs 14% in patients with a poor PS (ECOG 2-3). Of patients with locally advanced disease (III Mo), 17 (33%) obtained an objective response, compared with 20 patients (20%) with metastatic disease. Median survival was 18 weeks in the CDDP arm, 35 weeks in the CDDP/VP-16 arm, and 37 weeks in the CDDP/VP-16/mitomycin C arm. The median survival in the multimodal chemotherapy arms was significantly greater than that obtained with CDDP alone. Toxicity was predominantly myelosuppression in the mitomycin C-containing arm (27%, wtto grade 3-4). Our study shows that combination chemotherapy using CDDP/VP-16 is active and safe in the treatment of advanced NSCLC patients with a good performance status. The addition of mitomycin C did not improve the therapeutic response.

Italian clinical experience from the major co-operative groups in small cell lung cancer.

The clinical experience of Italian co-operative groups started in the early 1980s with the first randomised studies on alternating non-cross resistant combination chemotherapy carried out by the Italian Oncology Group for Clinical Research (G.O.I.R.C.) and by the Umbria Lung Cancer Study Group. Later, most of the investigators moved their interest to exploring the clinical relevance of increased dose intensity in the treatment of small cell lung cancer (SCLC). Several trials have been completed and published from Padova, Perugia, Genova and the Oncology Co-operative Group South Italy (G.O.C.S.I.). At the same time, another issue which arose in clinical research of SCLC was the late intensification of treatment after induction chemotherapy. Two ongoing phase II trials were designed at the Milan National Cancer Institute and at the Department of Medical Oncology, in Perugia and Rome. Finally, G.O.C.S.I. and a large Italian co-operative group are evaluating the integration of different modalities of treatment, mainly exploring the role of surgery in the management of limited SCLC.

Do Italian small cell lung cancer (SCLC) specialists share a common language? An analysis based on 549 questionnaires.

Different specialists are involved in the treatment of SCLC: medical oncologists, pneumologists, radiotherapists, and thoracic surgeons; only in large institutions the therapeutic policy is the result of a multidisciplinary approach. In order to investigate the opinions of the Italian physicians about the state of the art in the diagnosis and treatment of SCLC, 2369 questionnaires have been sent to an equal number of specialists. Each questionnaire contained 16 topics addressing what we consider major open questions. The analysis is based on 549 interpretable questionnaires received back (23.1%). The general attitude of responding physicians is quite pessimistic on the present state of the art; the large majority considering insufficient the current knowledge of both clinical and basic research. Some differences have been registered, among different specialists, regarding the role of surgery and radiation therapy in prolonging the expected survival; while a nearly unanimous consensus has been reached on the role of radiation therapy for local control. Optimism merges about the possibilities of ameliorating the survival in the next decades: 48% have confidence in new drugs, 45% in the development of integrated modalities, and 41% in the application of basic research.

SupT-1: a cell system suitable for an efficient propagation of both HHV-7 and HHV-6 variants A and B.

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.

Detection of Epstein-Barr virus DNA in cerebrospinal fluid from immunocompetent individuals with brain disorders.

Fifty four cerebrospinal fluid samples obtained from as many immunocomponent patients with disorders of the central nervous system were investigated for the presence of herpesvirus DNA by nested polymerase chain reaction in order to determine an etiological diagnosis. Four of these samples proved positive for the presence of Epstein-Barr virus DNA (7.4%). The result of this diagnostic study is reported to draw insiders' attention to the possible presence of EBV in cerebrospinal fluid from patients with central nervous system diseases.

Primary infection by HHV-6 variant B associated with a fatal case of hemophagocytic syndrome.

The association of a human herpesvirus 6 primary infection with a fatal case of hemophagocytic syndrome in a 3 month old baby is described. The trigger mechanism of the virus for the clinical syndrome is discussed.

Epstein-barr virus DNA in the cerebrospinal fluid of patients with human immunodeficiency virus infection and central nervous system disorders.

Routine search for herpesvirus types 1-5 by nested polymerase chain reaction revealed Epstein-Barr virus (EBV) DNA in the cerebrospinal fluid (CSF) of ten out of seventy-nine patients with human immunodeficiency virus (HIV) infection and central nervous system (CNS) disorders not associated with the presence of primary CNS lymphomas. One out of the ten CSF samples was positive for EBV DNA only, six were also positive for microbial agents of recognised neurological pathogenicity while the remaining three samples had a high content of HIV p24 Ag. When six available CSF samples out of the ten EBV DNA positive specimens were investigated for an intrathecal EBV antibody response, all six samples proved EBV antibody-free. The concurrent detection of neurotropic infectious agents and the absence of EBV antibodies in the CSF contribute to the uncertainty on the role of EBV in the neurological illness of the patients studied. One hypothesis considered is that the presence of EBV DNA in the CSF of a large fraction of the ten patients under study is an incidental event associated with EBV reactivation in the host's peripheral blood monocytes, but not related to the genesis of neurological disorders.