The burden of the variability introduced by the HEp-2 assay kit and the CAD system in ANA indirect immunofluorescence test.

According to the recent recommendations of the American College of Rheumatology, ANA Task Force, IIF technique should be considered the gold standard in antinuclear antibodies (ANAs) testing. To overcome the lack of standardization, biomedical industries have developed several computer-aided diagnosis (CAD) systems. Two hundred and sixty-one consecutive samples with suspected autoimmune diseases were tested for ANA by means of IIF on routinely HEp-2 assay kit (Euroimmun AG). Assignment of result was made if consensus for positive/negative was reached by at least 2 out of 3 expert physicians. ANA-IIF was also carried out using 3 CAD systems: Zenit G-Sight (n = 84), Helios (n = 85) and NOVA View (n = 92); human evaluation was repeated on the same substrate of each CAD system (Immco, Aesku and Inova HEp-2 cells, respectively). To anonymize the results, we randomly named these three systems as A, B and C. We ran a statistical analysis computing several measures of agreement between the ratings, and we also improved the evaluation by using the Wilcoxon's test for nonparametric data. Agreement between the human readings on routinely HEp-2 assay kit and human readings on CAD HEp-2 assay was substantial for A (k = 0.82) and B (k = 0.72), and almost perfect for C (k = 0.89). Such readings were statistically different only in case A. Comparing experts' readings with the readings of CAD systems, when the samples were prepared using CAD HEp-2 assay kits, we found almost perfect agreement for B and C (k = 0.86; k = 0.82) and substantial agreement for A (k = 0.73). Again, human and CAD readings were statistically different only in A. When we compared the readings of medical experts on routinely HEp-2 assay kit with the output of the CAD systems that worked using their own slides, we found substantial agreement for all the systems (A: k = 0.62; B: k = 0.65; C: k = 0.71). Such readings were not statistically different. The change of the assay kit and/or the introduction of a CAD system affect the laboratory reporting, with an evident impact on the autoimmune laboratory workflow. The CAD systems may represent one of the most important novel elements of harmonization in the autoimmunity field, reducing intra- and inter-laboratory variability in a new vision of the diagnostic autoimmune platform.

The clinical impact of Anti-DFS70 antibodies in undifferentiated connective tissue disease: case reports and a review of the literature.

Anti-nuclear antibody (ANA) positivity suggests CTD but can also lead to a diagnosis of UCTD when a patient does not fulfill the CTD diagnostic criteria. An anti-dense fine speckled (DFS) immunofluorescence (IIF) pattern can be observed when using an ANA test on HEp-2 cells and is due to the presence of antibodies to the nuclear DFS70 antigen that has rarely found in CTD. Serological testing for anti-DFS70 antibodies could therefore play a very interesting negative predictive role in stratifying patients on the basis of the evolution of UCTD to CTD. We described two patients ANA and anti-DFS70 positive in which the use of new method allowing the immunoadsorption of anti-DFS70 antibodies has permitted to exclude the incorrect diagnosis of CTD.

Natural history of human papillomavirus infection in non-vaccinated young males: low clearance probability in high-risk genotypes.

In this study, we aimed to investigate the clearance of type-specific genital human papillomavirus (HPV) infection in heterosexual, non-HPV-vaccinated males whose female partners were positive to HPV DNA tests. All consecutive men attending the same sexually transmitted diseases (STD) centre between January 2005 and December 2006 were considered for this study. All subjects (n = 1009) underwent a urologic visit and microbiological tests on first void, midstream urine and total ejaculate samples. One hundred and five patients were positive for HPV DNA (10.4 %; mean age: 34.8 ± 5.8 years) and consented to clinical examination and molecular diagnostic assays for HPV detection scheduled every 6 months (median surveillance period of 53.2 months). HPV genotypes were classified as high risk, probable high risk and low risk. HPV-positive samples which did not hybridise with any of the type-specific probes were referred to as positive non-genotypeable. At enrollment, the distribution of HPV genotypes was as follows: high-risk HPV (n = 37), probable high-risk HPV (n = 6), low-risk HPV (n = 23) and non-genotypeable HPV (n = 39). A high HPV genotype concordance between stable sexual partners emerged (kappa = 0.92; p < 0.001). At the end of the study, 71/105 (67.6 %) subjects were negative for HPV (mean virus clearance time: 24.3 months). With regard to the HPV genotype, virus clearance was observed in 14/37 (37.8 %) high-risk HPV cases, 6/6 (100 %) probable high-risk HPV cases, 20/23 (86.9 %) low-risk HPV cases and 31/39 (79.5 %) non-genotypeable cases. The high-risk HPV genotypes showed the lowest rate and probability of viral clearance (p < 0.001). In our series, high-risk HPV infections were more likely to persist over time when compared with other HPV genotypes.

Diagnostic accuracy of anti-gliadin antibodies in Non Celiac Gluten Sensitivity (NCGS) patients: A dual statistical approach.

BACKGROUND: Gluten is the target of several diseases such as wheat allergy (WA), celiac disease (CD) and non-celiac gluten sensitivity (NCGS). NCGS is a new clinical entity characterized by gastrointestinal and extraintestinal symptoms comparable to those of CD patients but to date still lacking of specific biomarkers so that NCGS diagnosis can be reached only by excluding CD and WA, and based on the direct association between gluten ingestion and symptoms onset. Previous studies showed that antigliadin antibodies (AGA) IgG are the most prevalent positive antibodies in NCGS population. AIM: The first aim of the study was to estimate AGA distribution and prevalence in a NCGS population. The second aim was to identify a serological pattern to help the diagnosis and/or to mark the NCGS disease. METHODS: Sera from 59 patients with suspected NCGS, 90 CD patients and 70 healthy individuals were assessed for AGA IgG/IgA, IgG/IgA deamidated gliadin peptide antibodies (DGP-AGA), tissue transglutaminase antibodies IgA (tTGA), endomysial antibodies IgA (EmA) and HLA typing (Eurospital, Trieste, Italy). RESULTS: We evaluated data by a dual statistical approach: logistic regression and receiver operating characteristic (ROC) analysis; therefore, we showed a poor diagnostic accuracy of AGA IgG in NCGS condition. CONCLUSION: Our preliminary data showed that AGA IgG didn't seem to be a strongly sensitive marker, even if it has been recently proposed as promising marker for NCGS condition, together with negativity for other celiac disease related antibodies. It can partially help the NCGS diagnosis, if it is integrated in the overall management of the patient. More in-depth clinical and laboratory researches are mandatory.

Polymyalgia rheumatica: inflammation suppression with low dose of methylprednisolone or modified-release prednisone.

OBJECTIVE: Polymyalgia rheumatica (PMR) is an inflammatory disease that affects people aged > 50 years, and is characterised by pain and morning stiffness in the shoulder and pelvic girdle with synovitis of the proximal joints and extra-articular synovial structures. It is currently mainly treated with glucocorticoids (GCs). The aim of the study was to evaluate changes in inflammatory markers and their correlations with cortisol levels after treatment with 6-methylprednisolone (6-MP) or modified-release prednisone (MR-P) in patients with "early" PMR. PATIENTS AND METHODS: The study involved 81 GC-naïve with "early" PMR diagnosed on the basis of the 2012 EULAR/ACR criteria: 38 treated with 6-MP at a starting dose of 12 mg at 8.00 a.m, gradually tapered to 8, 4 and 2 mg/day, and 43 treated with MR-P at a starting dose of 10 mg at 10 p.m, tapered to 7, 5, 3, 2 and 1 mg. The markers of inflammation (ESR mm/h, CRP mg/dL and fibrinogen mg/dL), the circulating serum levels of cytokines (TNFa and IL-6), and morning serum cortisol levels were evaluated at baseline and during GC treatment. RESULTS: There were significant differences between baseline and the end of treatment in the serum levels of IL-6 (5.3 ± 9.3 vs 2.8 ± 3.3 pg/mL; p < 0.05) and CRP (2.1 ± 3.3 vs 0.9 ± 1.7 mg/dL; p < 0.01) in the patients treated with MR-P, and in serum cortisol levels (15.8±6.4 vs 13.6+5.6 µg/dL; p < 0.01) in the patients treated with 6-MP. After the first month of treatment, 76.7% of the patients treated with MR-P had IL6 levels at or below the upper normal limit, whereas 52.6% of those treated with 6-MP had normal IL6 levels (p < 0.05). There was also a significant difference in the percentage of patients in whom the daily GC dose was tapered within eight months (6.7% in the MR-P group vs 25% in the 6-MP group; p < 0.001) and, by the end of the study, respectively 59.5% vs 35.1% patients were receiving a low GC dose or had discontinued treatment altogether (OR 2.7, 95% CI 1.0-6.77; p < 0.001). After six and 12 months, respectively 10.3% and 14.3% of the patients had discontinued MR-P, as against none of the patients treated with 6-MP (p < 0.05). CONCLUSIONS: In this prospective observational study of PMR patients receiving low-dose GCs, the changes in inflammatory markers were similar in those treated with 6-MP or MR-P, whereas morning cortisol levels remained unchanged only in the MR-P group. During the first month of treatment, MR-P chronotherapy given at bedtime significantly decreased IL-6 levels. The percentage of patients stopping GC treatment was higher in the MR-P group than in the 6-MP group.

Highlights on novel technologies for the detection of antibodies to Ro60, Ro52, and SS-B.

OBJECTIVE: We aimed to compare a chemiluminescent immunoassay (CIA, QUANTA Flash) on BIO-FLASH with a multiplex flow immunoassay (MFI) on BioPlex 2200 for the detection of antibodies to Ro60, Ro52, and SS-B. METHODS: The study included 241 samples, from patients suffering from systemic autoimmune diseases (n = 108) as well as disease controls (n = 133). All samples were tested for anti-Ro52, anti-Ro60, and anti-SS-B (La) antibodies on QUANTA Flash (INOVA Diagnostics, San Diego, USA) and BioPlex 2200 (Bio-Rad Laboratories Inc., Hercules, USA). Discrepant samples were tested by two independent methods: BlueDot/ANA and QUANTRIX Microarray (both D-tek, Belgium). RESULTS: The overall qualitative agreements were 95.4% (95% confidence interval, CI 92.0-97.7%) for anti-Ro52, 98.8% (95% CI 96.4-99.7%) for anti-Ro60, and 91.7% (95% CI 87.5-94.9%) for anti-SS-B antibodies. There were 34 discrepant samples among all assays (20 anti-SS-B, 11 anti-Ro52, 3 anti-Ro60). 30/33 of retested samples (by D-tek dot blot) agreed with the QUANTA Flash results. Similar findings were obtained with QUANTRIX Microarray kit. CONCLUSION: QUANTA Flash and BioPlex 2200 show good qualitative agreement. The clinical performances were similar for anti-Ro52 and anti-Ro60 autoantibodies while differences were observed for anti-SS-B (La) antibodies.

Role of radiotherapy boost in women with ductal carcinoma in situ: a single-center experience in a series of 389 patients.

BACKGROUND: The use of adjuvant radiotherapy in ductal carcinoma in situ is accepted by most radiation oncologists worldwide; the role of a boost on the tumor bed is however more controversial. MATERIALS AND METHODS: We reviewed our Institute experience in DCIS treatment, focusing on main prognostic factors and impact of radiation boost on local relapse. A total of 389 patients treated between 1990 and 2007 were retrospectively analyzed. All patients received adjuvant radiotherapy after breast-conserving surgery for a median dose of 50 Gy; 190 patients (48.8%) received and additional radiation boost on the tumor bed. RESULTS: At a mean follow up of 7.7 years, we recorded 26 local recurrence (6.7%). Concerning local relapse-free survival, at Cox regression univariate analyses <1 mm surgical margins (p < 0.0001) and young age (p = 0.01) emerged as significant unfavorable prognostic factors. At multivariate analysis Cox regression model, surgical margins (p < 0.001) and radiation boost (p = 0.014) resulted as the significant independent predictors of recurrence. CONCLUSIONS: Our experience showed the negative prognostic impact of surgical margins <1 mm and the protective role of radiation boost on LR rate. Anyway, results from ongoing prospective Phase III studies are strongly necessary to better identify high-risk DCIS patients.

Treatment of invasive male breast cancer: a 40-year single-institution experience.

PURPOSE: We conducted a retrospective analysis to evaluate the management and outcome of invasive male breast cancer treated in a single-institution over a period of 40 years. MATERIALS AND METHODS: We reviewed the clinical and pathological features of 60 male patients affected by breast carcinoma treated at our Radiotherapy Unit between 1971 and 2011. Tumours were classified according to histological type and the updated 2010 TNM classification of malignant tumours. RESULTS: At a median follow-up of 8.9 [range, 0.6-20; standard deviation (SD), 4.98] years, 32 patients (53.3%) were alive and 16 patients died (26.7%) due to disease progression and 12 (20%) due to other causes. At univariate analysis for overall survival, pathological tumour size (p=0.031), histological subtype (p=0.013) and nodal status (p=0.006) emerged as significant predictors of death. At multivariate analysis, independent death predictors were advanced pathological tumour size (p=0.016), positive nodal status (p=0.003) and invasive cribriform histological type (p=0.0003). CONCLUSIONS: In consideration of the rarity of the disease, many issues are still being debated, and future collaborative studies are required. However, our experience confirms the prognostic role of greater pathological tumour size and positive nodal status as unfavourable features for survival in male breast cancer.

Molecular detection of Leptospira interrogans in human tissues and environmental samples in a lethal case of leptospirosis.

A forensic case of suspected Leptospirosis with fatal course was resolved by the molecular detection of Leptospira interrogans in postmortem human tissues and in environmental samples. Polymerase chain reaction analysis and DNA sequencing confirmed the clinical diagnosis of Weil syndrome, and the death was considered to be an occupational accident with all the legal implications.

Detection of neurotropic viruses circulating in Tuscany: the incisive role of Toscana virus.

Acute meningitis is perhaps the most frequent among central nervous system infections. We report a study considering 277 cases of meningitis hospitalized in the southern Tuscany area (Italy) during the period from 1995 to 1998 investigated by tissue culture and polymerase chain reaction (PCR) methods. The cytochemical analysis of the cerebrospinal fluid samples suggested the diagnosis of aseptic meningitis, recognized as viral meningitis in 104 cases by detection of viral DNA or RNA. The results collected by tissue culture technique, available for 95 clinical samples, reported a positive isolation for only 12 cases. The viruses identified in the neurological infection were Toscana virus (81%), enterovirus (12%), mumps virus (3%), measles virus (1%), and herpes virus type 1 (3%). These data demonstrate the incisive role of the RNA viruses as the cause of meningitis, and overall the relevance of Toscana virus.

Human immunodeficiency virus (HIV)-specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV-seropositive persons.

The presence and activity of human immunodeficiency virus (HIV)-specific antibodies were analyzed in the sera of 15 sexually exposed seronegative persons who had systemic HIV-specific cell-mediated immunity and IgA-mediated mucosal immunity and in their HIV-infected partners. The HIV-positive subjects had HIV-specific serum IgG and IgA; the seronegative persons had HIV-specific serum IgA in the absence of IgG. Testing of the seronegative persons 1 year after the interruption of at-risk sex showed that no IgG seroconversion had occurred and that HIV-specific IgA serum concentrations had declined. Serum from the HIV-exposed seronegative persons was analyzed for the ability to neutralize primary HIV-1 isolates. Neutralizing activity was detected in 5 of 15 sera and in 2 cases was retained by serum-purified IgA. Thus, the immunologic picture for resistance to HIV infection should include HIV-specific cell-mediated immunity as well as HIV-specific IgA-mediated mucosal and systemic immunity.

A role for mucosal immunity in resistance to HIV infection.

In a recent, thought-provoking novel by Elizabeth McCracken (The Giant's House. Avon Books, New York, 1997), two characters discuss love and its impossibilities. One brashly claims to be "immune to love", explaining the concept to his perplexed interlocutor, "...people become immune to love like they become immune to any disease. Either they had it bad early in life, like chicken pox and that's that; or they keep getting exposed to it in little doses and build up an immunity; or somehow they just don't catch it, something in'em is born resistant. I'm the last type. I'm immune to love and poison ivy". (p. 275) (E. McCracken, The Giant's House. Avon Books, New York, 1997). Substitute the words 'HIV infection' for 'love' and this intriguing metaphor summarizes the state of the art working hypotheses for the phenomenon of resistance to HIV infection in HIV-exposed individuals who, against all odds, do not seroconvert. These hypotheses will be discussed hereafter and particular emphasis will be placed upon a possible role for mucosal immunity in this phenomenon.

HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals.

HIV-specific mucosal and cellular immunity was analyzed in heterosexual couples discordant for HIV status in serum and in HIV-unexposed controls. HIV-specific IgA but not IgG was present in urine and vaginal wash samples from HIV-exposed seronegative individuals (ESN), whereas both IgA and IgG were observed in their HIV-seropositive partners; antibodies were not detected in low-risk controls. Envelope protein (Env) peptide-stimulated interleukin-2 (IL-2) production by peripheral blood mononuclear cells (PBMCs) was detected in 9 out of 16 ESNs, 5 out of 16 HIV-infected patients and 1 out of 50 controls. Env peptide-stimulated PBMCs of ESNs produced more IL-2 and less IL-10 compared with those of HIV-infected individuals; no differences were observed in chemokine production or in CCR5 expression. These data demonstrate that a compartmentalized immune response to pathogens is possible in humans and raise the possibility of protective roles for cell-mediated immunity and mucosal IgA in HIV-seronegative individuals exposed to HIV.