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Background: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) represent the most common agents of sexually transmitted pharyngeal infections. These conditions are frequently asymptomatic, acting as important reservoir for further transmission. The aim of this study was to assess the prevalence and predictors of pharyngeal infections due to CT and NG in a cohort of patients attending a STI Clinic in the North of Italy. Materials/methods: From January 2016 to September 2018, all the consecutive patients attending the STI Outpatients Clinic of St. Orsola-Malpighi Hospital (Bologna) and reporting unsafe oral intercourses were considered eligible for the study. After a clinical examination, a pharyngeal swab for the molecular detection of CT and NG (Versant CT/GC DNA 1.0 Assay; Siemens) was collected from each patient. Moreover, on the basis of patients’ sexual behavior and symptoms, urine samples and/or ano-rectal swabs for CT/NG detection were collected. In case of a CT positivity, a molecular genotyping based on omp1 gene PCR, was performed. Results: During the study period, 893 patients (119 females and 774 MSM) were enrolled. Globally, a total of 134 cases of gonorrhoea (15%) and 34 chlamydial infections (3.8%) were detected in the pharyngeal site, with no significant differences between males and females. More than 90% of the pharyngeal infections were completely asymptomatic and the presence of symptoms was not a predictor of a pharyngeal infection (P=0.7). On the contrary, a history of sexual contacts with a partner positive for CT and/or NG (P

Background There are no Italian data regarding the strategies for preventing neonatal group B streptococcal (GBS) infection. We conducted a national survey in order to explore obstetrical, neonatal and microbiological practices for the GBS prevention. Methods Three distinct questionnaires were sent to obstetricians, neonatologists and microbiologists. Questionnaires included data on prenatal GBS screening, maternal risk factors, intrapartum antibiotic prophylaxis, microbiological information concerning specimen processing and GBS antimicrobial susceptibility. Results All respondent obstetrical units used the culture-based screening approach to identify women who should receive intrapartum antibiotic prophylaxis, and more than half of the microbiological laboratories (58%) reported using specimen processing consistent with CDC guidelines. Most neonatal units (89 out of 107, 82%) reported using protocols for preventing GBS early-onset sepsis consistent with CDC guidelines. Conclusions The screening-based strategy is largely prevalent in Italy, and most protocols for preventing GBS early-onset sepsis are consistent with CDC guidelines. However, we found discrepancies in practices among centers that may reflect the lack of Italian guidelines issued by public health organizations.

OBJECTIVES:Detection of specific antibodies is crucial for routine diagnosis of Hepatitis C virus (HCV) infection. Nowadays, many commercial tests based on chemiluminescence technology are available as highsensitive screening methods. In case of weak reactivity on first-line assays, further investigations are required to discriminate true-positivity from biological false-positivity. For this purpose, immunoblot tests are used due to their highest specificity. Nonetheless, final interpretation problems and unsatisfactory cost-effectiveness strongly limit their usefulness in daily practice. In this study, we evaluated the performance of VIDAS® Anti-HCV (bioMérieux) as a second-line test for the detection of anti-HCV antibodies in order to limit the use of immunoblot in our laboratory setting. As preliminary data, we report on the concordance of VIDAS® Anti-HCV with RecomLine HCV IgG(Mikrogen) in a group of selected sera. METHODS: In our laboratory testing for anti-HCV antibodies was first performed on ARCHITECT Anti-HCV (Abbott), a full-automated, high throughput chemiluminescent assay. Sera were scored as positive or negative on the basis of signal/cut-off result (cut-off=1). In case of weak positivity and in certain clinical or epidemiological settings, further tests based on a different technology were performed to clarify doubtful results. Until June 2012, a second-line test based on enzyme-linked immunoassay (INNOTEST® HCV Ab IV, Innogenetics) was used. From July 2012, INNOTEST® HCV Ab IV was replaced by VIDAS Anti-HCV, a third-generation test combining a two-step enzyme immunoassay with a final fluorescent detection. Discordant results were arbitrated by RecomLine HCV IgG, a line immunoassay forqualitative antibodies detection against individual HCV antigens. To evaluate the performance of VIDAS® Anti-HCV, we compared the total amount of immunoblot assays performed during the year before and after the introduction of this new second-line test, in respect to the number of screening tests on ARCHITECT Anti-HCV. The concordance of VIDAS® Anti-HCV with RecomLine HCV IgG was calculated testing a panel of 127 sera with a s/co result ranging from 0.7 to 7.0 on ARCHITECT Anti-HCV assay. RESULTS: During the year before VIDAS® Anti-HCV introduction (July 2011-June 2012) a total of 634 immunoblot assays were performed. On the other hand, after its introduction (July 2012-June 2013) only 404 immunoblot tests were used as confirmatory method. Considering the total amount of anti-HCV screening tests, we noticed a significant decrease of cases requiring immunoblot from 7.5‰ (634/83802) to 4.8‰ (404/83769). Among selected sera, VIDAS® Anti-HCV showed a concordance with RecomLine HCV IgG of 73% (93/127). Border-line results of RecomLine HCV IgG were considered as positive. CONCLUSION: In our experience, VIDAS® Anti-HCV showed a good diagnostic performance as second-line test for anti-HCV antibodies detection, limiting the use of immunoblot tests.

INTRODUZIONE La ricerca di anticorpi specifici è di fondamentale importanza nella diagnosi di infezione da virus dell’epatite C (HCV). Attualmente la maggior parte dei test proposti come screening sono basati su metodiche di chemiluminescenza (CMIA), che garantiscono ottima sensibilità, facilità di esecuzione e costi contenuti. Tuttavia, considerata la loro limitata specificità, nei casi di debole reattività sono necessari ulteriori approfondimenti diagnostici allo scopo di discriminare i veri positivi dai falsi positivi. A tale scopo i test basati sulla metodica di immunoblot sono caratterizzati da alta specificità diagnostica e rappresentano il gold standard. Tuttavia, le difficoltà nella interpretazione finale e l’elevato costo, limitano fortemente il loro utilizzo nella routine diagnostica. In questo studio abbiamo valutato la performance del test VIDAS® Anti-HCV (bioMérieux) come esame di secondo livello per la ricerca di anticorpi anti-HCV, con l’obiettivo di ridurre il ricorso all’immunoblot. Come dati preliminari, riportiamo inoltre la concordanza tra il test VIDAS® Anti-HCV e l’immunoblot RecomLine HCV IgG (Mikrogen) in un gruppo di sieri selezionati. METODI Nel nostro laboratorio lo screening per la ricerca di anticorpi anti-HCV viene eseguito con il test ARCHITECT Anti-HCV (Abbott), basato su metodica CMIA. I sieri vengono considerati positivi o negativi in base al risultato di ‘signal/cutoff’ (positivo>=1). In caso di debole positività al test di screening, si procede a ulteriori accertamenti. Fino a Giugno 2012 era utilizzato come secondo livello un test immunoenzimatico (INNOTEST® HCV Ab IV, Innogenetics), successivamente sostituito dal test VIDAS Anti-HCV, basato su metodica immunoenzimatica abbinata a un sistema di rilevazione finale in fluorescenza. In caso di risultati discordanti si procedeva, quindi, ad eseguire un test di terzo livello rappresentato da un immunoblot (RecomLine HCV IgG) allestito con antigeni ricombinanti. Per valutare la performance del test VIDAS® Anti-HCV, abbiamo confrontato il numero di immunoblot eseguiti nel corso dell’anno precedente e dell’anno successivo al suo inserimento nella routine diagnostica, rapportando tale valore al numero totale dei test di screening. E’ stata inoltre calcolata la concordanza tra VIDAS® Anti-HCV e RecomLine HCV IgG su un gruppo di 127 sieri selezionaticon valori al test ARCHITECT Anti-HCV compresi tra 0.7 e 7.0 s/co. RISULTATI Nel corso dell’anno antecedente all’introduzione del test VIDAS® Anti-HCV (Luglio 2011-Giugno 2012) sono stati eseguiti 634 immunoblot. Nell’anno successivo (Luglio 2012-Giugno 2013) il numero è sceso a 404. Rapportando tale dato al numero totale di test di screening eseguiti, si rileva un calo significativo in termini percentuali del ricorso all’immunoblot dal 7.5‰ (634/83802) al 4.8‰ (404/83769). La concordanza tra VIDAS® Anti-HCV e RecomLine HCV IgG nel gruppo dei sieri selezionati è stata del 73% (93/127). I risultati border-line al RecomLine HCV IgG sono stati considerati come positivi. CONCLUSIONI Nel nostro contesto l’introduzione del test VIDAS® Anti-HCV, come secondo livello diagnostico per la ricerca di anticorpi anti-HCV, ha consentito una netta riduzione del numero di immunoblot

To investigate whether it is appropriate to assume comparability of hepatitis virus C (HCV)-RNA results across laboratories in multi-centre studies, nine laboratories of the European Paediatric HCV Network participated in an international proficiency study of HCV-RNA assays. A panel of 12 samples of different dilutions and genotypes was sent to each laboratory and tested with qualitative and/or quantitative HCV-RNA assays according to local procedures. Commercial assays were used in seven laboratories and in-house assays in two. All six laboratories in which a commercial qualitative assay was used were proficient, as were four of six runs (in five laboratories) in which a commercial quantitative assay was used. The proficiency of the laboratories where in-house assays were used could not be assessed according to the VQC definition because of differences in the methods used. Overall, there were several false-negative results, but only one false-positive result with a quantitative assay and none with a qualitative assay. The false-negative results may have implications for the diagnosis of infection, and highlight the need for an antibody test to be performed at 18 months to confirm the absence of infection. The results of qualitative assays were generally consistent across laboratories but it was difficult to evaluate and compare the results of quantitative assays. Multivariate analysis of data collected in multi-centre studies should therefore allow for centre and/or assay used.

The routine determination of drug resistance in newly HIV-1 infected individuals records a potential increase in transmissions of drug-resistant variants. Plasma samples from 38 individuals classified as newly infected (seroconversion time

To investigate whether it is appropriate to assume comparability of hepatitis virus C (HCV)-RNA results across laboratories in multi-centre studies, nine laboratories of the European Paediatric HCV Network participated in an international proficiency study of HCV-RNA assays. A panel of 12 samples of different dilutions and genotypes was sent to each laboratory and tested with qualitative and/or quantitative HCV-RNA assays according to local procedures. Commercial assays were used in seven laboratories and in-house assays in two. All six laboratories in which a commercial qualitative assay was used were proficient, as were four of six runs (in five laboratories) in which a commercial quantitative assay was used. The proficiency of the laboratories where in-house assays were used could not be assessed according to the VQC definition because of differences in the methods used. Overall, there were several false-negative results, but only one false-positive result with a quantitative assay and none with a qualitative assay. The false-negative results may have implications for the diagnosis of infection, and highlight the need for an antibody test to be performed at 18 months to confirm the absence of infection. The results of qualitative assays were generally consistent across laboratories but it was difficult to evaluate and compare the results of quantitative assays. Multivariate analysis of data collected in multi-centre studies should therefore allow for centre and/or assay used. © 2003 Wiley-Liss, Inc.

The possible relationships between the intensity of humoral response to full length Tat protein, the amount of proviral DNA reservoir in peripheral blood mononuclear cells and RNA viral load were analyzed in plasma samples obtained from a group of HIV-1 seropositive subjects, who never received any antiretroviral therapy. All HIV-1 patients showed detectable levels of serum IgG to full-length Tat by immunoenzymatic assay. We found a higher percentage of HIV-1 seropositive subjects with low levels of antibody in the presence of barely detectable proviral DNA copies (or =10 copies/1.5x10(5) PBMCs) and a high anti-Tat antibody response accompanied by variable (from10(1) toor =10(3) copies/1.5x10(5) PBMCs) levels of DNA load (p=0.011). Moreover, an inverse relationship between anti-Tat antibody titers and HIV-1 RNA viral load was demonstrated HIV-1 seropositive patients. In HIV-1-infected patients, a strong humoral immune response against HIV-1 transactivating Tat protein, able to down-modulate viral replication in peripheral blood, does not seem to inhibit the number of proviral DNA molecules in peripheral blood mononuclear cells. Even though our data strongly confirm the "positive" role of anti-Tat antibody on viral replication, the persistence of significant amount of DNA viral load in peripheral blood mononuclear cells, despite high level of anti Tat antibody, suggests a more cautious approach to HIV-1 Tat-containing vaccines, able to stimulate an immune specific response to transactivating Tat protein sufficient in inhibiting circulating virus, but not completely efficient in decreasing proviral DNA integration.

In order to compare HIV-1 p24 antigenemia and plasma HIV-1 RNA levels as markers of viral replication, 3,129 paired determinations of alkaline immunocomplex-dissociated serum HIV-1 p24 antigenemia (performed with an immunoenzymatic assay), and plasma HIV-1 RNA levels (carried out with a branched DNA method, a reverse transcriptase-coupled polymerase chain reaction, and a nucleic acid sequence-based assay) were assessed over a two-year-period. When excluding samples with undetectable plasma HIV-1 RNA levels (which tested negative or borderline positive at serum p24 antigen assay in 97.9% of cases), immunocomplex-dissociated p24 antigenemia proved significantly less sensitive than viral load at all considered HIV-1 RNA reference levels, although the profile of positive serum p24 antigen assays (values above 10 pg/ml) paralleled the trend of plasma HIV-1 viral load, especially at higher levels. However, serum HIV-1 p24 antigenemia (even after immunocomplex dissociation) can be longer suggested as a the sole virological tool in the laboratory management of HIV-1 infection, due to its significantly lower sensitivity levels compared with viral load assessment.

A branched DNA method for the quantification of human immunodeficiency virus type 1 (HIV-1) RNA levels (Quantiplex HIV RNA 2.0) was compared with a reverse transcriptase-coupled polymerase chain reaction method (Amplicor HIV-1 Monitor) and a nucleic acid sequence-based assay (Nuclisens HIV-1 QT) in plasma samples from a group of HIV-1 seropositive patients. We found a high correlation between Nuclisens and Quantiplex (r = 0.89; p0.001) and between Amplicor and Quantiplex (r = 0.94; p0.001), a shift of RNA viral load to higher Nuclisens and Amplicor values compared with the Quantiplex results and a significant positive correlation (rS = 0.60; p0.001) between the p24 antigen level and the RNA viral load determined with the Quantiplex assay. We also found higher sensitivities of the Nuclisens and the Amplicor procedures compared with the Quantiplex assay. The total sensivity of the Quantiplex assay in our study was 70% whereas that of the p24 antigen was only 29%.

In situ polymerase chain reaction represents an intriguing method to couple the sensitivity of PCR technology with the preservation of cell morphology. The target of this technique is the detection of specific DNA or retrotranscribed RNA inside the cell. In this brief report, we describe an in situ polymerase chain reaction technique revealed by flow cytometry in order to reveal the human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells. We also discuss the key steps and parameters of our assay in comparison with current opinion on in situ polymerase chain reaction.

The effects of the administration of recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) on HIV-1 replication were evaluated in 15 patients with advanced HIV-1 disease and severe leukopenia, by monitoring immunocomplex dissociated p24 antigenemia, during 21 overall courses of therapy with rHuGM-CSF (lasting 2 to 27 weeks), alone or associated with zidovudine. During most treatment courses with rHuGM-CSF (17 out of 21), no significant modifications of HIV-1 antigenemia were recognized. A remarkable increase in viral replication occurred in only two courses out of 13 performed with rHuGM-CSF alone, while a significant reduction of antigenemia was observed in two courses of rHuGM-CSF out of 8 administered with zidovudine, after 10 weeks of combined treatment. Our experience is discussed on the grounds of both experimental and clinical investigations dealing with interactions between rHuGM-CSF and zidovudine during HIV-1 disease, focusing on risks of increased viral burden during treatment with rHuGM-CSF alone, and the synergistic activity of the combination with zidovudine against HIV-1 replication.

Starting from our previous observations that the HIV-1-mediated engagement of CD4 induced apoptotic death of TF-1 hematopoietic progenitor cells, in this study we evaluated PKC activity and intracellular Ca2+ levels in TF-1 cells treated with viable and heat-inactivated HIV-1 (strain IIIB) or anti-CD4 Leu3a monoclonal antibody (mAb). Both viable and heat-inactivated HIV-1 or anti-CD4 mAb, but not anti-human cytomegalovirus (HCMV) 66kD protein or anti-CD8 mAb induced a rapid (5-10 min) increase in PKC activity under both serum-containing and serum-free conditions. The same treatment also induced both a transient and a long-lasting (48 hours) decrease (p0.05) in intracellular Ca2+ levels in serum-containing cultures. We propose that the observed changes in PKC activity and intracellular Ca2+ levels might be involved in the HIV-1 mediated apoptosis of hematopoietic progenitor cells.

In this study we evaluated the effect of human immunodeficiency virus type 1 (HIV-1) recombinant tat-protein on the production of interleukin-6 (IL-6), granulocyte/macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) by purified peripheral blood monocytes. Whereas no effects were observed on TNF-alpha and GM-CSF production, recombinant tat-protein was able to induce the production of IL-6 by peripheral blood monocytes in a dose-dependent fashion for concentrations ranging from 1 ng/ml to 1 micrograms/ml. Pre-exposure of tat-protein with a polyclonal neutralizing anti-tat antibody (dilution 1:100) completely abrogated the tat-dependent increase in IL-6 production. The ability of tat-protein to selectively stimulate the production of IL-6 by peripheral blood monocytes could help to explain the presence of elevated levels of IL-6 in the serum of HIV-1 seropositive individuals, especially in patients in advanced stages of the disease with an active viral replication.

The production of granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were evaluated in the supernatants of short-term cultures of purified CD4+ T-lymphocytes and enriched monocytes obtained from peripheral blood (PB) of 35 HIV-1 seropositive (+) asymptomatic individuals, stages I-II of the Walter Reed (WR) classification, 15 HIV (+) symptomatic patients (WR V-VI) and 40 HIV-1 seronegative normal blood donors. IL-1 beta and TNF-alpha production by either enriched monocytes or isolated CD4+ T-cells, was similar in HIV-1 (+) asymptomatic, symptomatic subjects and normal controls. GM-CSF level in enriched monocyte culture supernatants did not show any significant difference in the three groups of subjects under investigation. On the other hand, GM-CSF production by isolated CD4+ T-lymphocytes was two-fold decreased in HIV-1 (+) asymptomatic subjects and five-fold decreased in HIV-1 (+) symptomatic patients with respect to normal blood donors. The decline in GM-CSF production was clearly correlated with viral isolation from patient's PB light density mononuclear cells (r = -0.920, p less than 0.01). The selective and progressive decline in GM-CSF production by CD4+ T-lymphocytes, starting from early stages of HIV-1 infection, suggest a preferential lesion of a specific subset of CD4+ T-lymphocytes characterized by an intense production of GM-CSF and may contribute to explain the deranged inflammatory and immune responses which characterize the course of HIV-1 infection.