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Background Elite controllers are able to control viral replication without antiretroviral therapy. Exceptional elite controllers do not show disease progression for more than 25 years. Different mechanisms have been proposed and several elements of both innate and adaptive immunity are implicated. Vaccines are immune stimulating agents that can promote HIV-RNA transcription; transient plasma HIV-RNA detectability has been described within 7-14 days after different vaccinations. The most reliable mechanism involved in virosuppressed people living with HIV is a generalized inflammatory response that activates bystander cells harboring latent HIV. So far no data about viral load increase in elite controllers after SARS-CoV-2 vaccination are reported in literature. Case Presentation We report the case of a 65-year-old woman of European ancestry, diagnosed with HIV-1/HCV co-infection more than 25 years ago. Since then, HIV-RNA remained undetectable and she never received ARV therapy. In 2021 she was vaccinated with mRNA-BNT162b2 vaccine (Pfizer-BioNTech®). She was administered with three doses in June, July and October 2021, respectively. The last available viral load was undetectable in March 2021. We observed an increase of VL at 32 cp/ml and 124 cp/mL, two and seven months after the first vaccine dose, respectively. During monthly follow-up, HIV-RNA gradually and spontaneously dropped becoming undetectable without ARV intervention. COVID-19 serology was positive with IgG 535 BAU/mL, showing response to vaccination. We measured total HIV-DNA at different time-points and we found it detectable both at the time of the higher plasma HIV-RNA (30 cp/10^6 PBMCs) and when it was undetectable (13 cp/10^6 PBMCs), in reduction. Conclusions This case is the first report, to our knowledge, describing a rebound of plasma HIV-RNA in an elite controller after three doses of mRNA-BNT162b2 vaccine for SARS-CoV-2. Concomitantly with a spontaneous reduction of plasma HIV-RNA ten months after the third dose of mRNA-BNT162b2 vaccine (Pfizer-BioNTech®) without antiretroviral therapy intervention, we observed a reduction of total HIV-DNA in peripheral mononuclear cells. The potential role of vaccinations in altering HIV reservoir, even in elite controllers population when plasma HIV-RNA is undetectable, could be a valuable aspect to take into account for the future HIV eradication interventions.

Background & Aims There are limited data on patients with chronic HCV infection in whom combination voxilaprevir (VOX), velpatasvir (VEL), sofosbuvir (SOF) retreatment fails. Thus, we aimed to assess treatment failure and rescue treatment options in these patients. Methods Samples from 40 patients with HCV genotypes (GT) 1-4 in whom VOX/VEL/SOF retreatment failed were collected within the European Resistance Study Group. Population-based resistance analyses were conducted and clinical parameters and retreatment efficacies were evaluated retrospectively in 22 patients. Results Most VOX/VEL/SOF failure patients were infected with HCV GT3a (n = 18, 45%) or GT1a (n = 11, 28%) and had cirrhosis (n = 28, 70%). Previous treatments included an NS3-inhibitor (30%), an NS5A-inhibitor (100%) and SOF (85%). Baseline RAS data from a subgroup of patients before VOX/VEL/SOF retreatment (78%) showed few NS3 RASs apart from Q80K in GT1a (40%), typical NS5A RAS patterns in most patients (74%) and no S282T in NS5B. Sequencing after VOX/VEL/SOF failure was available in 98% of patients and showed only minor changes for NS3 and NS5A RASs. In 22 patients, rescue treatment was initiated with glecaprevir, pibrentasvir alone (n = 2) or with SOF±ribavirin (n = 15), VOX/VEL/SOF±ribavirin (n = 4) or VEL/SOF and ribavirin (n = 1) for 12 to 24 weeks. Sustained virologic response was achieved in 17/21 (81%) patients with a final treatment outcome. Of these, 2 GT3a-infected patients had virologic failure after rescue treatment with VEL/SOF or glecaprevir/pibrentasvir+SOF+ribavirin, and 2 patients with cirrhosis died during treatment or before reaching SVR12. Conclusions VOX/VEL/SOF failure was mainly observed in HCV GT3- and GT1a-infected patients with cirrhosis and was not associated with specific RAS patterns within NS3, NS5A or NS5B target regions. Rescue treatment with multiple targeted therapies was effective in most patients. Lay summary The advent of direct-acting antivirals has enabled the effective cure of chronic hepatitis C in most patients. However, treatment failure occurs in some patients, who are often retreated with a combination regimen called VOX/VEL/SOF, which is associated with very high rates of cure. However, VOX/VEL/SOF retreatment also fails in some patients. Herein, we analysed samples from patients in whom VOX/VEL/SOF retreatment failed and we assessed the efficacy of different rescue therapies, showing that rescue treatment is effective in most patients (81%).

Objective HIV infection chronically affects the central nervous system (CNS). Olfactory mucosa is a unique site in the respiratory tract that is directly connected to the CNS; thus we wanted to evaluate olfactory mucosa as a surrogate of CNS sampling. Design We conducted a preliminary study examining HIV populations and susceptible cells in the olfactory mucosa. Methods Olfactory mucosa was sampled by minimally invasive brushing. Cerebrospinal fluid (CSF) analyses were performed as per routine clinical procedures. Olfactory marker protein, CD4+, CD8+, and trans-activator of transcription (TAT) expressions were assessed by immunohistochemistry. Plasma, CSF, and olfactory mucosa HIV-RNA were quantified using the Cobas AmpliPrep/Cobas TaqMan assay, whereas HIV proviral DNA was evaluated on peripheral blood mononuclear cell and olfactory mucosa. HIV-1 env deep sequencing was performed for phylogenetic analysis. Results Among ART-naive participants, 88.2% (15/17), and among ART-treated participants, 21.4% (6/28) had detectable HIV-RNA in samples from their olfactory mucosa; CSF escape was more common in patients with olfactory mucosa escape (50 vs. 7.9%; P = 0.010). Olfactory mucosa samples contained few cells positive for CD4, CD8, or HIV-DNA, and no HIV TAT-positive cells, indicating that this approach efficiently samples virions in the olfactory mucosa, but not HIV-infected cells. Yet, using a deep sequencing approach to phylogenetically compare partial HIV env genes in five untreated participants, we identified distinct viral lineages in the OM. Conclusions The results of this study suggest that nasal brushing is a well tolerated and useful technique for sampling the olfactory mucosa. HIV-RNA was detected in most naive and in some treated patients, warranting larger longitudinal studies.

At the time of writing, FIND has listed four CE-marked SARSCoV-2 antigen tests. We evaluated the recently CE-approved rapid POCT SD-Biosensor for SARS-CoV-2 nucleoprotein detection in nasopharyngeal secretions from 330 patients admitted to the Emergency Room for a suspect of COVID-19 and travelers returning home from high risk countries. Sensitivity, specificity, accuracy, negative and predictive values were consistent with the use of the test to mass-screening for SARS-CoV-2 surveillance.

In Emergency Room, Point-of-care antigen testing for SARS-CoV-2 antigen can expedite clinical strategies for patient management. We tested 1,232 consecutive patients during Italian second wave peak using the recent LumiraDx microfluidic assay. This assay showed high concordance (96.9 %), sensitivity and specificity compared to molecular testing, being highly valuable.

Background Zika virus (ZIKV) remains a public health concern due to its association with fetal malformation and neurologic disease. Objective To report a reference centre experience on ZIKA virus (ZIKV) infection in travelers from epidemic countries from January 1 to September, 30, 2016 in Italy North-West (a geographic area covering 4.424 million inhabitants, corresponding to almost 73% of Italy North-West area). Study design One hundred and twelve febrile travelers were studied to rule out a tropical fever [e.g. malaria, dengue (DENV), chikungunya (CHIKV), West Nile (WNV) and ZIKV]. Molecular tests for detecting ZIKV RNA were applied on serum or urine as well as IgG and IgM specific serology. Results ZIKV was the most frequent “tropical infection (11.6%) with 12 infected travelers and one sexual partner of an infected traveler. At the time of the diagnosis, ZIKV RNA was detected in the blood from 9 patients (69%) within 7 days from symptom onset; afterwards, the virus was detected only in urine (5 patients) and ZIKV IgM was reactive in 9 patients (69%). Travelers with ZIKV infection tested negative for DENV, CHIKV, WNV and malaria and completely recovered. Other infections identified in travelers were DENV (5 patients, 4.5%), CHIKV (1, 0.9%), malaria (Plasmodium vivax, 1, 0.9%), measles (1, 0.9%) and tuberculosis (1, 0.9%). Conclusions The etiologic diagnosis of a febrile illness in travelers where ZIKV is endemic is highly desirable as they are sentinel of a challenging epidemiology including the risk of autochthonous transmission in non endemic countries where the competent or carrier vector is present.

Background More and more countries test for HIV infection using combination assays that simultaneously detect p24 antigen and HIV antibodies. Objective To assess the performance of a new HIV combo assay: LIAISON ® XL murex HIV Ab/Ag HT. Study design The assays were examined with a total of 3090 samples that included 769 selected HIV antibody-negative samples, 1849 unselected HIV samples, 15 HIV-1 p24 Ag reference samples, 90 primary HIV-1 infection (PHI) samples, 167 HIV-1 antibody-positive samples (well characterized of groups M and O), 95 HIV-1 antibody-positive samples and 105 HIV-2 antibody-positive samples. Results The specificity of the LIAISON ® XL murex HIV Ab/Ag HT was 99.7%. The analytical sensitivity of Ag p24 of the LIAISON ® XL murex HIV Ab/Ag HT was 0.58 IU/mL and 9.93 pg/mL when using WHO and French national standards, respectively. All screened HIV subtypes was identified by this assay. Also, 90 PHI specimens were detected by this screening assay. Conclusion The sensitivity and specificity of the LIAISON ® XL murex HIV Ab/Ag HT assay are high. Hence the assay offers automated high-throughput screening with ability to detect primary infection.

Background Cerebrospinal fluid (CSF) HIV RNA is commonly used as a marker of compartmental antiviral activity in HIV-positive patients. Undetectable CSF HIV RNA levels have been associated with low CSF neopterin levels and better neurocognitive performances. The aim of this study was to analyse the prevalence and predictors of non-detectable CSF HIV RNA using a commercial assay. Methods In adult HIV-positive HAART-treated patients with confirmed plasma HIV RNA Results 112 adult patients were included. Plasma and CSF HIV RNA were non-detectable (target not detected [TND]) in 29 (25.9%) and 36 (32.1%) patients, respectively. CSF TND was observed more frequently in patients with plasma TND ( P=0.005, OR=3.87). CSF neopterin levels were associated with age (rho =0.333, P=0.002) and current (rho= -0.272, P=0.015) and nadir (rho =-0.240, P=0.038) CD4+ T-lymphocytes; the lowest CSF neopterin concentration was observed in patients with CSF TND versus other viral load strata (0.62 mg/dl versus 0.78 mg/dl; P=0.048). Conclusions Efficaciously treated HIV-positive patients with detectable plasma HIV RNA might imperfectly control CSF viral replication. Prospective studies addressing the management and neurocognitive consequences of CSF low-level viraemia are warranted.

Background In patients with A(H1N1)pdm09 infection, severe lung involvement requiring admission to intensive care units (ICU) has been reported. Mutations at the hemagglutinin (HA) receptor binding site (RBS) have been associated with increased virulence and disease severity, representing a potential marker of critical illness. Objectives To assess the contribution of HA-RBS variability in critically ill patients, A(H1N1)pdm09 virus from adult patients with severe infection admitted to ICU for extracorporeal membrane oxygenation support (ECMO) during influenza season 2009–2011 in Piemonte (4·2 million inhabitants), northwestern Italy, was studied. Patients and methods We retrospectively analyzed HA-RBS polymorphisms in ICU patients and compared with those from randomly selected inpatients with mild A(H1N1)pdm09 disease and outpatients with influenza from the local surveillance program. Results By HA-RBS direct sequencing of respiratory specimens, D222G and D222N viral variants were identified in a higher proportion in ICU patients (n = 8/24, 33·3%) than in patients with mild disease (n = 2/34, 6%) or in outpatients (n = 0/44) (Fisher's exact test P

Objectives: Valganciclovir is a well established drug for the management of cytomegalovirus (CMV) infection in haematopoietic stem cell transplantation (HSCT). Data concerning its safety regarding the development of drug resistance are required. The aim of the present study was to retrospectively investigate CMV drug resistance in a group of HSCT patients experiencing relapses of CMV infection after a first-line pre-emptive antiviral therapy. Methods: Thirteen adult HSCT patients out of 26 with asymptomatic CMV infection, experiencing relapsing infections 45‐155 days after either intravenous (iv) ganciclovir (2 patients) or valganciclovir (11 patients), were studied. Genotypic assays for mutations in the viral phosphotransferase (UL97) and DNA-polymerase (UL54) genes were directly applied on patient specimens. Baseline CMV sequences were compared with those at the time of relapses to identify drug-resistant strains. Results: UL97 mutations A594V and M460V known to confer drug resistance developed in one relapsing patient who received iv ganciclovir as first-line therapy, corresponding to a rate of 7.7% of relapses due to drug-resistant strains and an overall 3.8% rate of infections due to CMV drug-resistant strains. UL54 drug resistance mutations were absent. No evidence of drug resistance was found in patients on valganciclovir either as first-line therapy or as treatment for relapses. Conclusions: The safety profile of valganciclovir as anti-CMV pre-emptive therapy was confirmed, as well as that monitoring CMV drug resistance with genotypic tests on sequential isolates over the timecourse of therapy offers guidance to tailor antiviral treatment in a clinically relevant time frame.

Antiviral compounds including ganciclovir, foscarnet, and cidofovir are routinely used in the treatment of cytomegalovirus (CMV) infection and disease; however, these agents have a poor oral bioavailability and have the inconvenience and expense of intravenous administration. Aim of the study. To evaluate the safety and efficacy of oral valganciclovir (VGCV) for preemptive treatment of CMV reactivation in the setting of allogeneic hematopoietic stem cell transplantation (HSCT). Patients and methods. We treated 15 patients receiving allogeneic HSCT from related (n=9) or unrelated (n=6) donors. In all patients, either the donor, host, or both were CMV Ig G positive pretransplant. Indication for therapy was preemptive treatment of CMV infection defined as one or two consecutive positive tests of pp65 antigenemia assay or CMV-polymerase chain reaction (PCR). VGCV was administered orally in a dosage of 900 mg b.i.d. for 2 weeks, followed by 450 mg b.i.d. for 2 additional weeks. Results. Patients developed a positive CMV-PCR after a median of 52 days (range 37–427) post HSCT and a positive pp65 antigenemia after a median time of 74 days (range 37–427) post HSCT. Preemptive treatment with VGCV was started a median time of 56 days (range 37–429) after transplant. In all, 11 patients (73%) completed the 28 days of therapy with VGCV. All patients showed a complete clearance of the virus. The median time to achieve a negative CMV-PCR was 6 days (range 4–18). A relapse of CMV infection after VGCV preemptive therapy occurred in 6 patients (40%). No patient developed early or late CMV disease. Six patients (40%) presented hematological toxicity including neutropenia and/or thrombocytopenia that required drug discontinuation in 4 cases. Conclusion. VGCV administered as preemptive therapy for CMV infection in patients receiving an allogeneic HSCT showed promise for treating this frequent complication. Prospective randomized studies in this setting are mandatory to yield more definitive results.

Diagnosis and monitoring of HCV infection relies on sensitive and accurate HCV RNA detection and quantitation. The performance of the COBAS AmpliPrep™/COBAS TaqMan™ 48 (CAP/CTM) (Roche, Branchburg, NJ), a fully automated, real-time PCR HCV RNA quantitative test was assessed and compared with the branched-DNA (bDNA) assay. Clinical evaluation on 576 specimens obtained from patients with chronic hepatitis C showed a good correlation (r = 0.893) between the two test, but the CAP/CTM scored higher HCV RNA titers than the bDNA across all viral genotypes. The mean bDNA versus CAP/CTM log10 IU/ml differences were −0.49, −0.4, −0.54, −0.26 for genotype 1a, 1b, 2a/2c, 3a, and 4, respectively. These differences reached statistical significance for genotypes 1b, 2a/c, and 3a. The ability of the CAP/CTM to monitor patients undergoing antiviral therapy and correctly identify the weeks 4 and 12 rapid and early virological responses was confirmed. The broader dynamic range of the CAP/CTM compared with the bDNA allowed for a better definition of viral kinetics. In conclusion, the CAP/CTM appears as a reliable and user-friendly assay to monitor HCV viremia during treatment of patients with chronic hepatitis. Its high sensitivity and wide dynamic range may help a better definition of viral load changes during antiviral therapy. J. Med. Virol. 80:254–260, 2008. © 2007 Wiley-Liss, Inc.

Background Since the HLA-B*57:01 allele is strongly associated with abacavir hypersensitivity reaction, testing for the presence of HLA-B*57:01 is mandatory before administration of abacavir. While HLA-B*57:01 testing is usually provided by pharmacogenetics, genetics or blood transfusion services, clinical virology laboratories can be an optimal opportunity for HLA-B*57:01 testing since they receive blood samples for routine HIV monitoring and have the expertise for convenient and less expensive PCR-based point mutation assays. Objectives The Italian HLA-B*57:01 Network gathers accredited clinical virology laboratories offering HLA-B*57:01 testing in Italy with the aim to share protocols, test new methods, develop and maintain external quality assurance (EQA) programs. Study design A panel of 9HLA-B*57:01-positive and 16HLA-B*57:01-negative frozen blood samples were blindly distributed to 10 units including 9 clinical virology laboratories and one reference pharmacology laboratory. Each laboratory was free to use its own routine method for DNA extraction and HLA-B*57:01 testing. Results DNA was extracted by automated workstations in 6 units and by manual spin columns in 4. Eight units used the Duplicα Real Time HLA-B*57:01 kit by Euroclone and two units used two different PCR homemade protocols. All the 10 units correctly identified all the 25 samples. Conclusions The first HLA-B*57:01 EQA program run in Italy showed that clinical virology units are equipped and proficient for providing HLA-B*57:01 testing by inexpensive assays easy to integrate into their routine.

In the era of direct-acting antiviral agents (DAAs), hepatitis C virus (HCV) genotyping tests at baseline are controversial. The HCV NS3-Q80K polymorphism is associated with resistance to the recently approved NS3 inhibitor simeprevir (SMV) when combined with PEG-interferon and ribavirin (PEG-IFN/RBV) and alternative therapy should be considered for patients with baseline Q80K. The aim of this study was to provide an estimate of Q80K prevalence at baseline in a study group of 205 DAA-naïve patients (21% of them with HIV coinfection) using NS3 full-population direct sequencing to detect resistance-associated amino acid variants (RAVs). NS3 RAVs were identified in 56 patients (27.3%). Q80K was the most frequently reported one (41%), in both HIV/HCV-coinfected and HCV-monoinfected patients, but it was only detectable in cases of HCV-subtype 1a infection. Therefore, in clinical practice, an NS3-Q80K genotyping test prior to simeprevir plus PEG-IFN/RBV treatment is highly recommended.

We evaluated performances of the molecular test Septi Fast (SF) for the detection of agents of bloodstream infection (BSI) in patients with suspected sepsis, the majority of them under antibiotic treatment and at high prevalence of HIV-1 infection (10.5%). Matched SF and blood culture (BC) samples (n=1186) from 1024 patients were studied. Two hundred fifty-one episodes of BSI out of 1144 were identified with the combined methods (22%). SF identified more episodes of BSI than BC: 206 versus 176 (χ 2 =7.008, P =0.0081) and a significantly higher number of Gram-negative bacteria than BC (77 versus 53, χ 2 =9.12; P =0.0025), as well as of polymicrobial infections (χ 2 =4.50, P =0.0339). In conclusion, SF combined with BC improved the diagnosis of sepsis, especially in immunocompromised patients.

Enteroviruses (EVs) are common human viral pathogens, causing a variety of diseases, including aseptic meningitis. Recently, EV aseptic meningitis outbreaks have been reported across Europe, but, in Italy, knowledge of recent EV molecular epidemiology is very limited.We report an outbreak of EV aseptic meningitis in 10 adults in North-Western Italy, from October to November 2012. Patients were parents or close relatives of children5 years old attending the same class of a nursery school, suffering from a mild febrile upper respiratory disease. Phylogenetic relationship with other European circulating strains was analyzed updating E30 circulation in Italy in recent years.EVs were detected from cerebrospinal fluid (CSF) specimens with a real-time reverse transcription polymerase chain reaction and virus isolation was achieved from rectal and pharyngeal swabs. For cluster definition and phylogenetic studies, viral VP1 region was directly amplified and sequenced from CSF.EVs were identified in CSF from all patients and from rectal and pharyngeal swabs in 7 of them. Direct sequencing of CSF revealed the presence of the same Echovirus 30 (E30) in all patients and phylogenetic analysis identified it as a diverging clade within E30 genotype VII, the most recent strain circulating in UK, Finland and Denmark since 2006.Molecular techniques allowed the rapid identification and typing of E30 from CSF. Phylogenetic analysis revealed that the cluster might be due to a new E30 variant within the genotype VII currently circulating in Europe, thus updating the epidemiology of EV circulation in Italy.

A/H1N1/09 influenza is associated with a high risk of complications in patients with chronic diseases, but data on morbidity and mortality in patients with cirrhosis are limited. A cluster of A/H1N1/09 infection in 48 patients admitted to a Gastro-Hepatology Unit is reported. Nosocomial spread, clinical outcome, and viral characteristics of A/H1N1/09 strains from a study group of 48 inpatients (21 and 27 with and without cirrhosis, respectively) were compared with those from a control group of 44 outpatients with mild influenza-like illness and without cirrhosis. A/H1N1/09 infection was confirmed in 8/48 (17%) inpatients. A/H1N1/09 infection rate did not differ in patients with and without cirrhosis (4/21, 19%; 4/27, 15%), but three patients with cirrhosis died of pneumonia and acute respiratory distress syndrome, with fungal or bacterial superinfection in two cases, despite antiviral treatment. None of patients without cirrhosis died. Viral sequences showed the presence of hemagglutinin mutation D222G in two out of three fatal cases and S183P in seven out of eight infected patients. These mutants were not detected in the outpatients group. Even if A/H1N1/09 infection rate in hospitalized patients with and without cirrhosis was not significantly different, cirrhosis and D222G/S183P substitutions were significantly associated with severe disease and poor outcome, also suggesting fungal or bacterial superinfection and portal hypertension as risk factors for A/H1N1/09 disease severity in patients with cirrhosis. Vaccination, preventive and early treatment and a strict control of nosocomial spread should be activated carefully in patients with cirrhosis during epidemics influenza.

Background Domestic outbreaks of Dengue (DENV) fever from imported cases have to be considered a possible risk in non-endemic countries where Dengue vectors are present, such as in Italy. Objective To review imported acute/recent DENV infections in a one-year survey in a North West Italy region where the presence of Aedes albopictus is documented. Study design We retrospectively reviewed laboratory and clinical records of Italian febrile travelers from Dengue endemic areas referring to the local reference Centre for Infectious Disease, covering a population of about 4 million people. Results Acute/recent DENV infection was identified in 15 out of 91 travelers from endemic areas (16.5%) including 12 primary and 3 secondary infections; in 6 patients the virus was detectable in blood according to molecular real-time Polymerase Chain Reaction-based assays: in 9 patients the diagnosis of DENV infection was accomplished by the combination of specific IgM reactivity, high IgG titers, IgG seroconversion from negative to positive and increasing (four-fold) IgG titers in paired serum samples. Two cases of DENV infections were imported from South Egypt in patients travelling together, confirming the importance of returning travelers as sentinels of a rapidly changing epidemiology in specific geographic areas. Conclusions Our findings outline the high rate of imported Dengue infection in North West Italy and emphasize the need for a continued Dengue surveillance in non-endemic countries as well as a careful evaluation and follow-up of febrile patients returning from Dengue endemic countries.

Multicentric Castleman’s disease (MCD) is a rare lymphoproliferative disorder with systemic symptoms and poor prognosis and is characterized by an abnormal proliferation of polyclonal plasmablasts in the mantle zone of B-cell follicles. The disease is found primarily in chronic HIV carriers and is usually strictly associated with human herpes virus type 8 (HHV-8) coinfection, which is believed to play a key role in the pathogenesis of MCD. The disease is also diagnosed in HIV-negative patients, who are usually elderly or immunosuppressed; however, in about half of these cases, no evidence of HHV8 infection is found. The anti-CD20 monoclonal antibody rituximab is now the preferred treatment for HIV-positive MCD. However, it is not clear whether rituximab is effective in HIV-negative patients with MCD, particularly in the HHV8-positive subset. We report here the clinical and biologic courses of two HIV-negative, HHV8-positive patients with MCD who were treated with rituximab. In both cases, a significant clinical improvement was observed after the first two infusions, which was shortly followed by a drop in HHV8 viremia to undetectable levels. Both patients underwent complete clinical remission, which persisted without relapse at 30 and 9 months of follow-up, respectively. No reactivation of the Kaposi sarcoma found in a lymph node of one of the patients was observed. Our report, along with additional data present in the literature, suggests that rituximab may be an appropriate and safe first-line therapy for HIV-negative, HHV8-positive MCD.

In high risk patients such as in eldery, newborns and immunosuppressed subjects, a timely diagnosis of influenza is required for the most appropriate antiviral strategy in order to avoid severe secondary respiratory complications and viral spreading. Influenza is preventable by vaccination and chemoprophylaxis and is treatable by specific antiviral indications. The need for a timely diagnosis has led to the introduction of numerous rapid diagnostic tests.These are mostly antigen detection test giving results within 30 minutes, a clinically relevant time-frame to complement with the use of antiviral medications or chemoprophylaxis strategy. When evaluating performances of rapid test for influenza viruses, it is important to consider the type and quality of specimen to be tested, as well as sensitivity and specificity of the assays. Nasal/nasopharyngeal swabs are the most frequently submitted specimens, but nasal/nasopharingeal aspirates and washs can improve the diagnostic sensitivity of the test. Only some rapid assays can be successful used with broncoalveolar washings. In this review,we evaluated the sensitivity, specificity, reproducibility and feasibility of the most currently licensed rapid tests for influenza virus A and B. A flow-chart for the laboratory diagnosis of influenza with rapid test in combination with confirmatory test is proposed.

Performances of the new automatic system COBAS AmpliPrep/COBAS TaqMan 48 (CAP/CTM) (Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared with the endpoint PCR COBAS AMPLICOR HBV Monitor (CAHBM, Roche). Analytical evaluation with proficiency panels from UK National External Quality Assessment Scheme (UK NEQAS) over a 1-year period of distribution showed that CAP/CTM correctly measured HBV DNA levels with a close correlation between expected and observed values (r=0.995). Clinical evaluation as tested with samples from 11 HBsAg-positive patients undergoing antiviral therapy (71 serial specimens of plasma), demonstrated excellent correlation with CAHBM (r=0.958, mean difference in quantitation: 0.14 log, IU/ml), but CAP/CTM detected longer period of residual viremia. HBV DNA reduction was much higher in the combination schedule (Lamivudine+Adefovir), than in Adefovir monotherapy (5.1 vs. 3.5 logs). In conclusion, CAP/CTM allows for an accurate and standardized quantification of HBV DNA in high through-put laboratories. Due to it high sensitivity, it may further improve the detection of emerging drug resistance strains and the assessment of antiviral therapy.

Success of antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for Hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding and reliable PCR results depend it. Performances of the fully automatic system COBAS AmpliPrep™/COBAS TaqMan™ 48 (CAP/CTM) (Roche, Branchburg, NJ) for HBV DNA extraction and real -time PCR quantification were assessed and compared with the end-point PCR COBAS AMPLICOR HBV Monitor (CAHBM, Roche). Analytical evaluation with a proficiency panel showed that CAP/CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (r=0.976, interassay variability below 5%). Clinical evaluation as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r=0.966, mean difference in quantitation: 0.36 log10 IU/ml). CAP/CTM detected 10% more viremic patients and longer period of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 vs. 3.5 logs) suggesting a benefit in continuing LAM. In conclusion,CAP/CTM can improve the management of HBV infection, the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of drug resistance.

Successful pre-emptive anti-cytomegalovirus (CMV) therapy relies on sensitive, specific and reproducible tests for CMV detection. Real-time polymerase chain reaction (PCR) for CMV-DNA provides a superior reproducibility and sensitivity than pp65-antigenemia. Evaluation of a novel commercial real-time PCR for CMV-DNA associated with a fully automated DNA extraction from whole blood (WB) was performed, studying the correlation with pp65-antigenemia in guiding pre-emptive therapy. Analytical evaluation showed that PCR correctly quantitated CMV from 500 to 500,000copies/ml with a close correlation with expected values (r=0.999). Clinical evaluation on 375 consecutive WB samples from 48 infected patients (18 pre-emptively treated for pp65 values >/=50 positive cells) showed that according to pp65-antigenemia of 0, 1-10, 11-49 and >/=50 positive cells, median DNA levels were 3.7, 3.9, 4.6 and 5.6 log(10)copies/ml, respectively. According to existing pre-emptive treatment criteria based on pp65-antigenemia, receiver-operating curve analysis indicated 5.3log/ml (200,000genomes/ml) as the best CMV-DNA level to discriminate between patients requiring pre-emptive therapy and those who did not (positive predictive value: 91%; negative predictive value: 74%; sensitivity and specificity: 68 and 93%). In conclusion, real-time PCR provides reliable results for monitoring the developing of CMV infection, allowing for the definition of CMV-DNA thresholds associated with infection progress.

Success in antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding, and reliable PCR results depend on it. The performances of the fully automatic system COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared to the endpoint PCR COBAS AMPLICOR HBV monitor (CAHBM; Roche). Analytical evaluation with a proficiency panel showed that CAP-CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values ( r = 0.976, interassay variability below 5%). Clinical evaluation, as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM ( r = 0.966, mean difference in quantitation = 0.36 log 10 IU/ml). CAP-CTM detected 10% more viremic patients and longer periods of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, the reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 logs versus 3.5 logs), suggesting a benefit in continuing LAM. CAP-CTM detected HBV DNA in liver biopsy samples from 15% of HBsAg-negative, anti-HBcAg-positive graft donors with no HBV DNA in plasma. The amount of intrahepatic HBV DNA was significantly lower in occult HBV infection than in overt disease. CAP-CTM can improve the management of HBV infection and the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of occult HBV infection.

The polymerase chain reaction (PCR) for cytomegalovirus (CMV) DNA quantitation provides sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy. A recently introduced real-time PCR assay for CMV DNA quantitation was applied on 158 peripheral blood leukocytes (PBLs) from 32 liver-transplanted patients with CMV asymptomatic infection and correlated with a commercial quantitative end-point PCR (COBAS AMPLICOR CMV Monitor) and CMV pp65 antigenemia. A good correlation was found between real-time PCR and pp65 antigen test (r2 = 0.691) and the two PCR assays (r2 = 0.761). Real-time PCR data were higher in pre-emptive treated patients (>20 pp65 + positive cells, median CMV DNA value: 3.8 log10 copies/500,000 PBLs) than in not-treated ones (2.9 logs). According to pp65 levels of 0, 1–10, 11–20, 21–50, 51–100, and >100 positive cells/200,000 PBLs, median CMV DNA by real-time PCR was 2.6, 3.0, 3.6, 4.0, 4.2, and 4.8 logs, respectively, (CMV DNA levels by COBAS AMPLICOR: 2.8, 2.9, 3.8, 3.7, 3.9, and 4.0 logs). For samples with >20 pp65 + cells, real-time PCR gave significantly higher values than in groups with

Quantitave Polymerase Chain Reaction (PCR) for Cytomegalovirus (CMV) DNA provides highly sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy.To determine the clinical application of a recently introduced real-time (RT) PCR assay for CMV DNA quantitation in peripheral blood leukocytes (PBLs) and defining its correlation with the commercial quantitative end-point (EP) PCR method COBAS AMPLICOR CMV Monitor and pp65 antigen test. Sequential PBL samples (n=158) from 32 liver transplanted patients with CMV asymptomatic infection and positive for CMV DNA by EP-PCR were retrospectively analysed with RT-PCR and studied according to pp65 antigen levels. A good correlation was found between RT-PCR and pp65 antigen test (r=0.691) and between the two PCR assays (r=0.761). RT-PCR data were significantly higher in pre-emptive treated patients (those with >20 pp65+positive cells, median value: 3.8 log10 copies/500,000 PBLs) than in not-treated ones (2.9 logs).According to pp65 levels of 0, 1-10, 11-20, 21-50, 51-100 and >100 positive cells/200,000 PBLs, median CMV DNA load by RT-PCR was 2.6, 3.0, 3.6, 4.0. 4.2 and 4.8, log10 copies/ 500,000 PBLs, respectively (EP-PCR CMV DNA levels: 2. 8, 2.9, 3.8, 3.7, 3.9 and 4.0 logs). For samples with >20 pp65+cells, that is above the level at which pre-emptive therapy was started, RT-PCR values were significantly higher than in groups with less than 20 pp65+cells, whereas EP-PCR values did not significantly differ and showed a slower progression rate. Dilutions of DNA from CMV AD169 strain were used to probe RT-PCR reproducibility (between and intra-assay variability < 2%) and sensitivity (100% detection rate at 10 copies/reaction, 28.5% with EP-PCR). A significant improvement is coming from the introduction of RT-PCR to the study of CMV DNA dynamics in differently CMV infected patients due to a more reliable quantitation of CMV DNA for moderate and high DNA level compared to EP-PCR with better sensitivity and specificity. RTPCR gives more precise informations on viral load kinetics for evaluating the infection progress and assessing antiviral response, significantly simplifying and accelerating the process of producing a reliable quantification of CMV DNA for clinical purposes.

Summary Objectives To characterize by molecular techniques Burkholderia strains responsible for respiratory tract infections in cystic fibrosis (CF) patients (children and adults), to assign the Burkholderia cepacia complex (Bcc) isolates to a genomovar and to assess the presence of cblA and esmR genes in bacteria. Unique or sequential Burkholderia isolates ( n =48) that had been collected from eight CF children and 17 adults over several (4–6) years were investigated; moreover 11 reference strains were analyzed. Methods The microorganisms were identified by using biochemical methods, genotyped by pulse field gel electrophoresis (PFGE) and random-amplified polymorphic DNA fingerprinting–PCR (RAPD–PCR), and assessed by PCR assays for the genomovar and cblA and esmR genes of Bcc. Results Among isolates 70.8% were identified as Bcc genomovar III-A; one child was infected by Burkholderia ambifaria and four adults were colonized with Burkholderia gladioli . The cblA gene was not detected in any of the isolates, while the esmR gene was detected in the 52.1% of the strains, all belonging to genomovar III-A. Conclusion Molecular analysis of strains revealed in CF patients a colonization with a persistent Burkholderia flora with strains of one genotype. The prevalence of Bcc of genomovar III-A in the two categories of patients and of B. gladioli in four adults demonstrated that transmission may have occurred between subjects. Moreover the B. ambifaria infection demonstrated in a child may be environmentally derived.

Streptococcus pyogenes (group A Streptococcus [GAS]) is an important pathogen whose virulence is related to the production of exotoxins and the presence of particular surface components. One hundred eighty-two GAS strains were collected in northwestern Italy between 1994 and 2002 and analyzed for phenotypic characteristics (opacity factor, proteolyic activity, and antimicrobial susceptibility) and by polymerase chain reaction for the presence of genes responsible for the production of exotoxins implicated in pathogenesis speA and speF and of prtF(1) (encoding fibronectin-binding protein F1). All strains were speF positive and 19.2% were speA positive and prtF(1) negative, whereas the prtF(1) gene was identified in 39.5% of the other strains. Of these, approximately half revealed the same pulse-field gel electrophoresis (PFGE) pattern but differed in both speA gene and macrolide resistance.

The proteasomes of some protozoa are possible targets for chemotherapy. Leishmaniasis is a major health problem in human immunodeficiency virus (HIV) co-infected subjects. Two HIV protease inhibitors (PI), indinavir and saquinavir, have been shown to block proteasome functions; we therefore investigated their effects on the growth of two Leishmania spp. ( Leishmania major and Leishmania infantum ). After 24 h of treatment, both drugs exhibited a dose-dependent antileishmanial activity, with 50% lethal dose (LD 50 ) values of, respectively, 8.3 μM and 7 μM on L. major ; minor activity was observed on L. infantum . These results add new in vitro insights into the wide-spectrum efficacy of PI and suggest studying their action on amastigote forms of leishmania within macrophages in order to validate their potential contribution against opportunistic infections in treated seropositive patients.

Strains of the marine ciliate protist Euplotes crassus produce exclusive terpenoids called euplotins that play an ecological role. Among these derivatives, euplotin C is the main of four secondary metabolites isolated from cultures of this protozoon and represents the sesquiterpene taxonomic marker from E. crassus . Because different terpenoid metabolites of plant origin showed a certain antimicrobial activity, we assessed the compound euplotin C, purified by high-pressure liquid chromatography and solubilized in two solubility enhancers, against the protozoa Leishmania major and Leishmani infantum , the fungus Candida albicans , and nine strains of gram-positive and gram-negative microorganisms. An activity of euplotin C against Leishmania promastigotes was demonstrated (50% lethal doses were 4.6 or 8.1 μg/ml depending on the agent used to solubilize the compound), while the effect was less evident on Candida and nearly absent on bacteria. A nonsignificant cytotoxicity (50% lethal dose, >200 μg/ml) against the J774 cell line was observed. A leishmanicidal activity was also shown by the living, euplotin-producing cells of E. crassus cultured together with promastigotes; this activity increased with time from 10 min to 6 h of incubation. This study provides an initial rationale for the evaluation of euplotin C and other similar natural products as alternative or possibly synergistic compounds for current antiprotozoon chemotherapeutics.