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HIV testing was offered to 2,185 people receiving mpox (formerly monkeypox) vaccination, who reported not being HIV positive. Among them 390 were current PrEP users, and 131 had taken PrEP in the past. Of 958 individuals consenting testing, six were newly diagnosed with HIV. Two patients had symptomatic primary HIV infection. None of the six patients had ever taken PrEP. Mpox vaccination represents an important opportunity for HIV testing and counselling about risk reduction and PrEP.

Molecular investigation of primary HIV infections (PHI) is crucial to describe current dynamics of HIV transmission. Aim of the study was to investigate HIV transmission clusters (TC) in PHI referred during the years 2013&ndash, 2020 to the National Institute for Infectious Diseases in Rome (INMI), that is the Lazio regional AIDS reference centre, and factors possibly associated with inclusion in TC. These were identified by phylogenetic analysis, based on population sequencing of pol, a more in depth analysis was performed on TC of B subtype, using ultra-deep sequencing (UDS) of env. Of 270 patients diagnosed with PHI during the study period, 229 were enrolled (median follow-up 168 (IQR 96&ndash, 232) weeks). Median age: 39 (IQR 32&ndash, 48) years, 94.8% males, 86.5% Italians, 83.4% MSM, 56.8% carrying HIV-1 subtype B. Of them, 92.6% started early treatment within a median of 4 (IQR 2&ndash, 7) days after diagnosis, median time to sustained suppression was 20 (IQR 8&ndash, 32) weeks. Twenty TC (median size 3, range 2&ndash, 9 individuals), including 68 patients, were identified. A diagnosis prior to 2015 was the unique factor associated with inclusion in a TC. Added value of UDS was the identification of shared quasispecies components in transmission pairs within TC.

Background The 2013–2016 Ebola virus disease (EVD) outbreak showed a lack of diagnostic point-of-care methods. Currently, EBOV diagnosis relies on quantitative reverse-transcription-PCR (RT- qPCR), highly specific and sensitive, but requiring skilled personnel and well-equipped laboratories. In field settings, these factors and others, such as samples’ time of collection and transportation, determine a prolonged turnaround-time to final results. In outbreak scenarios, a rapid and transportable method could eliminate issues of cohorting suspected and actual EVD patients for lack of diagnostic certainty. The aim of this study was the field evaluation of the new fast, easy-to-use and reliable RT-qPCR assay and platform for EBOV detection, developed in the framework of the EbolaMoDRAD project by CLONIT S.r.l. and STMicroelectronics S.r.l. Study design We evaluated its performance during the outbreak and in further studies in the EVD laboratory at the Princess Christian Maternity Hospital (PCMH) in Freetown (Sierra Leone) run by Emergency NGO and the Italian National Institute for Infectious Diseases (INMI). The assay was tested on residual aliquots of clinical specimens from EBOV-positive or −negative patients (n = 116, EVD prevalence 37%). Results and conclusion Overall, the test was very easy-to-use and the instrument was robust and reliable in field-settings. The sensitivity of the assay was 100% and the specificity was 98.63% (95%CI: 96.34–100.92%). The positive and negative predictive values were 97.73 (95%CI:94.77–100.68%) and 100%, respectively. The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, especially in resource-limited settings.

Background Deep sequencing has a deep impact on the study of rapidly mutating RNA viruses, such as hepatitis C virus, proving to be an invaluable tool for analyzing virus diversity and evolution. Aim Genotype-independent high-throughput pyrosequencing was used to obtain near full length hepatitis C virus genome sequence reconstruction directly from clinical samples. Methods Samples from hepatitis C virus infected subjects harbouring different subtypes (1a, 1b, 2c) were analyzed (viral load range: 1.2–20.8 × 10 6 IU/ml). Data were generated with a modified sequence-independent single primer amplification method followed by 454 sequencing. Results the extent of reconstructed hepatitis C virus genome varied from 79.95% to 99.64%. No correlation between extent of genome reconstruction and either viral load ( r = 0.4857, p = 0.3556) or number of HCV reads ( r = 0.08571, p = 0.9194) was observed. Conclusion This study describes a protocol for obtaining whole genome sequences from different hepatitis C virus patients with different genotypes in a single sequencing run.

The advent of next generation sequencing platforms has greatly improved the direct assessment of human metagenomes permitting to obtain results in a time-frame compatible with diagnostic needs. The performances of two platforms, namely Roche 454 GS FLX+ and Illumina MiSeq, have been compared using a metagenomic approach aimed at the identification of the pathogenic agent, an RNA virus in this case, in a clinical sample. Both platforms resulted able to correctly identify the H1N1 virus in the sample, but also provided a similar overview of the microbial community. The detailed analysis of two very different but clinically relevant microorganisms that often co-infect patients, H1N1 and Streptococcus pneumoniae, showed differences in terms of depth of coverage and genome coverage, and showed that some genomic regions are more frequently represented in Illumina sequencing reads, while others in the 454 reads. These findings indicate that the platforms and the corresponding analysis procedures permit the high quality assembly of the H1N1 virus and the identification of the complex microbial community in the sample, supporting the usage of these approaches in a clincal setting.

Persistent residual viremia (RV) has been demonstrated in 70-90% of patients under successful cART. We analyzed the RV trend during the first year following cART-induced virological suppression (VS; HIVRNA50 copies/ml) to identify predictors of achievement and maintenance of ultra-deep RV suppression (URVS; HIV-RNA5 copies/ml) in 60 naïve patients. These patients were aligned at the time of reaching VS and were longitudinally tested with an ultrasensitive HIV-RNA assay. The influence of demographics, primary/chronic infection, pre-therapy HIV-RNA and CD4, cART regimen and time to reach VS on RV trends was evaluated. During the first year following VS, median RV levels steadily decreased. RV dropped below 5 copies/ml at least once in each patient, but URVS was maintained in 45% of patients. RV rebounded to levels fluctuating around 5-10 copies/ml while in the remaining 55% of patients. Predictors of early achievement and maintenance of stable URVS were fast (12 weeks) VS achievement after the start of therapy, better pre-treatment viro-immunological conditions (lower viremia and higher CD4 before cART), and treatment initiation during primary infection. These findings emphasize the importance of an early onset of potent antiretroviral regimens. RV trends should be further studied in detail in the following years of cART.

Background Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic. Methods RT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes. Results Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication. Conclusions Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development. Electronic supplementary material The online version of this article (doi:10.1186/s12953-017-0126-0) contains supplementary material, which is available to authorized users.

HCV quasispecies variability represents the background for the selection of mutations and for the development of drug resistance. Natural aminoacid changes in NS3, associated with reduced protease inhibitor susceptibility, have been observed in treatment-naïve patients. Massively parallel sequencing has been used to analyze NS3 quasispecies in patients infected with HCV genotype 1, naive to anti-HCV treatment, with/without HIV-coinfection, to establish the genetic heterogeneity and the presence of amino acid substitutions at positions responsible for drug resistance. Genomes carrying substitutions represented either predominant or minority components of viral quasispecies, and were observed in 85.7% of patients. Multiple substitutions, frequently associated on the same haplotype, were observed in 46.4% of patients. High resistance combinations were not detected, neither on the same genome, nor in the whole quasispecies. Heterogeneity of HCV NS3 was lower in HIV-coinfected as compared to HCV-monoinfected patients, but factors underlying this difference remain to be established. Although the relevance of naturally occurring mutations with respect of resistance development and probability of success of direct acting antivirals is questioned, UDPS may be beneficial to help understanding viral dynamics, providing high resolution view of viral diversity.

Background Entecavir is the drug of choice as first-line treatment for treatment-naive HBV patients. As a result of the high genetic barrier to resistance, treatment failure remains rare, but occurs within 3 years of initiation, suggesting that viral genetic characteristics may provide a fast lane to resistance. One of the main concerns is the long time to viral suppression observed in some (even treatment-naive) patients. The reasons for this phenomenon were investigated in a group of chronic hepatitis B treatment-naive patients. Methods Out of 23 treatment-naive patients starting entecavir, the 5 with the best and those with the worst viral load decay curves were selected for the study. Quasispecies analysis was performed for the reverse transcriptase/hepatitis B surface antigen (HBsAg) open reading frame (ORF) by ultra-deep pyrosequencing. For each patient, the analysis was performed at baseline (T0) and when viraemia reached between 15,000 and 200 IU/ml (T1). Results The few resistance mutations present at T0 were not selected by treatment; no other resistance mutations or suggestive mutational patterns were selected at T1. Selective pressure analysis indicated that both at T0 and T1 the HBsAg ORF was subjected to a significantly higher pressure in rapid responders, especially in a region rich in cytotoxic T-lymphocyte (CTL) epitopes. Conclusions The results did not provide evidence that a slower response to entecavir is due to the emergence of less sensitive variants. Rather, the lower selective pressure and variability in humoral and CTL epitopes in slow responders suggests that their immune response might be at odds in rapidly clearing infected cells from the liver.

Sir, ART effectively suppresses viral replication and controls infection for an undefined period of time; however, viral eradication is not achievable because of long-lived cellular HIV reservoirs. We previously showed that, in chronically infected subjects with undetectable plasma HIV-RNA, bone marrow CD34+ haematopoietic progenitor cells (HPCs) are apparently free of HIV replication, but are blunted in differentiation capability, and may harbour HIV-DNA even after a long period on successful ART. Moreover, in patients treated with successful ART for a very long time, a persistent impairment in the lymphopoietic capability of circulating CD34+ HPCs was found, and lymphopoiesis exhaustion resulted correlated to systemic immune activation, only partially reversed by prolonged ART. To date, the mechanisms of HIV-related lymphopoiesis dysfunction remain largely unexplained, and in particular, little information is available on the possibility of limiting the occurrence of irreversible damage by early ART introduction. We herein describe immune activation levels, T cell profile/response and circulating HPC kinetics in a patient with primary HIV infection receiving early treatment with ART. The patient was further followed for 12 months, and blood samples were analysed before (baseline) and after 2, 24 and 48 weeks of ART. A young adult male was recently diagnosed with HIV acute infection (Fiebig IV stage according to Fiebig et al.). Baseline plasma HIV-1 RNA was 1868262 copies/mL, and CD4+ T lymphocyte count was 389 cells/mm. Ritonavir-boosted darunavir, tenofovir+ emtricitabine and raltegravir were started on day 3 after diagnosis. After 12 weeks of ART, viral load dropped ,40 copies/mL and ART was simplified to rilpivirine+emtricitabine+tenofovir. Plasma HIV-RNA remained undetectable at all timepoints thereafter. The viro-immunological parameters are shown in Figure 1(a). CD4+ cell count steadily increased over time: 534, 1218 and 1072 cells/mL at weeks 2, 24 and 48, respectively. Proviral HIV-DNA, determined as described in Rozera et al., was 82479 copies/10 PBMC at baseline and 21534, 1752 and 6809 copies/10 PBMC at weeks 2, 24 and 48, respectively. CD8+ T cell activation, measured as CD38 expression by flow cytometry, paralleled plasma HIV-RNA viral load, reaching at week 24 the level found in healthy donors. On the other hand, the level of early CD8+ T cells, evaluated by CD127 expression, steadily increased from baseline to week 48 (Figure 1b). Peripheral blood CD4+ and CD8+ T cell differentiation was evaluated by CD45RA and CCR7 expression. As shown in Figure 1(c), the variation in CD4+ subsets included a decrease in effector memory (EM; CD45RA2/CCR72) and an increase in Research letters

In March/April 2009, a new pandemic influenza A virus (A(H1N1)pdm09) emerged and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century. Influenza virus may be present in the infected host as a mixture of variants, referred to as quasi-species, on which natural and immune-driven selection operates. Since hemagglutinin (HA) and non-structural 1 (NS1) proteins are relevant in respect of adaptive and innate immune responses, the present study was aimed at establishing the intra-host genetic heterogeneity of HA and NS1 genes, applying ultra-deep pyrosequencing (UDPS) to nasopharyngeal swabs (NPS) from patients with confirmed influenza A(H1N1)pdm09 infection. The intra-patient nucleotide diversity of HA was significantly higher than that of NS1 (median (IQR): 37.9 (32.8-42.3) X 10(-4) vs 30.6 (27.4-33.6) X 10(-4) substitutions/site, p = 0.024); no significant correlation for nucleotide diversity of NS1 and HA was observed (r = 0.319, p = 0.29). Furthermore, a strong inverse correlation between nucleotide diversity of NS1 and viral load was observed (r = - 0.74, p = 0.004). For both HA and NS1, the variants appeared scattered along the genes, thus indicating no privileged mutation site. Known polymorphisms, S203T (HA) and I123V (NS1), were observed as dominant variants (>98%) in almost all patients; three HA and two NS1 further variants were observed at frequency >40%; a number of additional variants were detected at frequency

This study aimed to define clinical and virological parameters associated with spontaneous control of HIV replication in patients having initiated HAART during primary HIV infection, who underwent structured therapy interruption by two protocols with either fixed or HIV viremia-guided scheme. At the end of the protocol all patients were changed to viremia-guided scheme and observed for 12 months (follow-up). Patients maintaining HIV viremia below the indications for resumption of HAART during the follow-up, were defined controllers, those who had to resume HAART were defined non-controllers. The following parameters were examined: pre-interruption therapy duration, CD4+, HIV RNA, proviral DNA, evolution of viral quasispecies. No specific advantage was conferred by either interruption of structured therapy in the proportion of controllers and non-controllers. Pre-HAART and zenith CD4+, pre-therapy interruption, HAART duration, but not pre-HAART HIV RNA, were significantly higher in controllers as compared to non-controllers. HIV RNA levels after the first interruption cycle of therapy were significantly lower in controllers than in non-controllers. Proviral DNA levels were also lower in controllers at this time point. HIV RNA and proviral DNA levels associated with the last interruption of therapy cycle were not different from those associated with the first cycle, and, in spite of multiple waves of virus rebound, very few gag quasispecies variants emerged in each patient. The data suggest that pre-treatment clinical parameters and virological events associated with the first viral rebound are crucial factors in determining the ability to control viral replication after multiple cycles of interruption of treatment. J. Med. Virol. 79: 1047–1054, 2007. © 2007 Wiley-Liss, Inc.

Although in decline after successful anti-HIV therapy, B-cell lymphomas are still elevated in HIV-1-seropositive (HIV+) persons, and the mechanisms are obscure. The HIV-1 matrix protein p17 persists in germinal centers long after HIV-1 drug suppression, and some p17 variants (vp17s) activate Akt signaling and promote growth of transformed B cells. Here we show that vp17s derived from four of five non-Hodgkin lymphoma (NHL) tissues from HIV+ subjects display potent B-cell growth-promoting activity. They are characterized by amino acid insertions at position 117-118 (Ala-Ala) or 125-126 (Gly-Asn or Gly-Gln-Ala-Asn-Gln-Asn) among some other mutations throughout the sequence. Identical dominant vp17s are found in both tumor and plasma. Three of seven plasma samples from an independent set of NHL cases manifested multiple Ala insertions at position 117-118, and one with the Ala-Ala profile also promoted B-cell growth and activated Akt signaling. Ultradeep pyrosequencing showed that vp17s with C-terminal insertions are more frequently detected in plasma of HIV+ subjects with than without NHL. Insertion of Ala-Ala at position 117-118 into reference p17 (refp17) was sufficient to confer B-cell growth-promoting activity. In contrast, refp17 bearing the Gly-Asn insertion at position 125-126 did not, suggesting that mutations not restricted to the C terminus can also account for this activity. Biophysical analysis revealed that the Ala-Ala insertion mutant is destabilized compared with refp17, whereas the Gly-Asn form is stabilized. This finding provides an avenue for further exploration of structure function relationships and new treatment strategies in combating HIV-1-related NHL.

Severe acute respiratory syndrome (SARS) is an emerging infection caused by a novel coronavirus known as SARS-CoV, characterized by an over-exuberant immune response with lung lymphomononuclear cells infiltration and proliferation that may account for tissue damage more than the direct effect of viral replication. This study is aimed at investigating the capability of SARS-CoV to activate IFN-alpha and -gamma expression in lymphomonocytes (PBMC) from healthy donors, evaluating whether viral replication is necessary for this activation.SARS-CoV virus is able to induce both IFN-alpha and -gamma mRNA accumulation and protein release in a dose-dependent manner, MOI 10 being the most effective. The time course curve indicated that IFN-alpha mRNA induction peaked at 24 h.p.i,. whereas IFN-gamma mRNA was still increasing at 48 h.p.i. Released IFN (both types) reached a plateau after 24-48 h.p.i. and remained rather stable over a 5-day period. A transient peak of negative strand viral RNA was detected after 1-2 days of infection, but neither infectious virus progeny yield nor newly produced viral genomic RNA could be evidenced in infected cultures, even after prolonged observation time (up to 13 days). Cocultivation of PBMC with fixed SARS-CoV-infected Vero cells was even more efficient than exposure to live virus in eliciting IFN-alpha and -gamma induction. A combination of IFN-alpha and -gamma strongly inhibited SARS-CoV replication in Vero cells, while the single cytokines were much less effective.This study provides evidence that SARS-CoV is able to induce in normal PBMC a coordinate induction of IFN-alpha and -gamma gene expression. Virus replication is not necessary for IFN induction since efficient IFN expression could be obtained also by the cocultivation of normal PBMC with fixed SARS-CoV-infected cells. Concomitant activation of IFN-alpha and -gamma gene expression by SARS-CoV in vivo may be relevant for the pathogenesis of the disease, both for the possible involvement in immunomediated damage of the tissues and for the strong inhibition of SARS-CoV replication as a result of combined cytokine action.

Summary. Vero cells transfected with either neo- or bcl-2-plasmid were infected with SARS-CoV at a high multiplicity of infection. Apoptosis appeared after the onset of CPE and completion of virus replication, and could be prevented by Bcl-2 expression. Apoptosis is likely mediated by the mitochondrial pathway, as demonstrated by its inhibition using Bcl-2, and by the activation of the caspase cascade, resulting in PARP cleavage. Prevention of apoptosis did not affect susceptibility to infection, kinetics and extent of viral replication and release, thus implying that apoptosis is not involved in facilitating release and/or dissemination of SARS-CoV in Vero cells.

Background Aim was to determine the dynamics of peripheral blood mononuclear cells (PBMC)- associated total HIV-1 DNA in successfully ART-treated HIV/HCV co-infected patients receiving DAA treatment and to explore possible virological hypotheses underlying the phenomenon. Methods Longitudinal, single-centre study measuring total HIV-1 DNA before the start of DAA, at the end of treatment (EOT), and 3 months after treatment. Univariable and multivariable analyses were used to assess factors associated with HIV-1 DNA increase ≥0.5 Log copies/million PBMC. Episomal 2-LTR forms, residual HIV-1 viremia and proviral DNA quasispecies evolution were also investigated. Results 119 successfully ART-treated HIV/HCV co-infected patients were included. Median baseline HIV-1 DNA was 3.84 Log copies/million PBMC (95%CI 3.49–4.05), and no significant variation with respect to baseline was found at EOT and after 3 months of DAA termination. In 17% of cases an increase ≥0.5 Log copies/million PBMC was observed at EOT compared to baseline. HIV-1 DNA increase was independently associated with lower baseline HIV-1 DNA, longer HIV suppression, raltegravir-based ART and previous exposure to interferon/ribavirin for HCV treatment. In none of the patients with HIV-1 DNA increase, 2-LTR forms were detected at baseline, while in 2 cases 2-LTR forms were found at EOT, without association with residual HIV-1 RNA viremia. No evidence of viral evolution was observed. Conclusions In successfully ART-treated HIV/HCV co-infected patients receiving DAA, PBMC-associated total HIV-1 DNA was quite stable over time, but some patients showed a considerable increase at EOT when compared to baseline. A significantly higher risk of HIV DNA increase was found, in presence of lower cellular HIV reservoir at baseline. Activation of replicative-competent virus generating new rounds of viral replication seems unlikely, while mobilization of cell-associated HIV from tissue reservoirs could be hypothesized.

ObjectiveThe false-positive rate (FPR) is a percentage-score provided by Geno2Pheno-algorithm indicating the likelihood that a V3-sequence is falsely predicted as CXCR4-using. We evaluated the correlation between FPR obtained by V3 population-sequencing and the burden of CXCR4-using variants detected by V3 ultra-deep sequencing (UDPS) and Enhanced-Sensitivity Trofile assay (ESTA).Methods54 HIV-1 B-subtype infected-patients (all maraviroc-naïve), with viremia >10,000copies/ml, were analyzed. HIV-tropism was assessed by V3 population-sequencing, UDPS (considering variants with >0.5% prevalence), and ESTA.ResultsBy UDPS, CCR5-using variants were detected in 53/54 patients, irrespective of FPR values, and their intra-patient prevalence progressively increased by increasing the FPR obtained by V3 population-sequencing (rho = 0.75, p = 5.0e-8). Conversely, the intra-patient prevalence of CXCR4-using variants in the 54 patients analyzed progressively decreased by increasing the FPR (rho = -0.61; p = 9.3e-6). Indeed, no CXCR4-using variants were detected in 13/13 patients with FPR>60. They were present in 7/18 (38.8%) patients with FPR 20-60 (intra-patient prevalence range: 2.1%-18.4%), in 5/7 (71.4%) with FPR 10-20, in 4/6 (66.7%) with FPR 5-10, and in 10/10(100%) with FPRConclusionsFPR by V3 population-sequencing can predict the burden of CXCR4-using variants. This information can be used to optimize the management of tropism determination in clinical practice. Due to its low cost and short turnaround time, V3 population-sequencing may represent the most feasible test for HIV-1 tropism determination. More sensitive methodologies (as UDPS) might be useful when V3 population-sequencing provides a FPR >20 (particularly in the range 20-60), allowing a more careful identification of patients harboring CXCR4-using variants.

The HIV’s envelope glycoprotein gp120 plays a major role in the entry of the virus into the host cell, through its successive interactions with the cell surface CD4 receptor and a co-receptor (CCR5 or CXCR4). The choice of a specific co-receptor by gp120 has an important consequence on HIV infection and pathogenesis. The third variable region within gp120, the V3 loop, is the principal determinant of the co-receptor usage by gp120. Here, we report the long time molecular dynamics simulations of four gp120 structures, having a V3 loop charge of +3 and +5, from both R5 and X4 specific strains of HIV. The results of the study highlight the properties of the V3 loop that can be critical for dictating the co-receptor recognition and selection in structural context. In detail, we observe that the structural orientation of the V3 loop in the 3D space is modulated by its net charge, whilst its co-receptor choice is likely dictated by a combined effect of both the electrostatics of the loop and its conformational variability at the level of its central crown region.

HCV genome variability is related to both disease progression and treatment response. De novo high-throughput pyrosequencing was used to obtain full length HCV genome characterization directly from clinical samples.

Viruses represent the most abundant biological components on earth.They can be found in every environment, from deep layers of oceans to animal bodies.Although several viruses have been isolated and sequenced, in each environment there are millions of different types of viruses that have not been identified yet.The advent of nextgeneration sequencing technologies with their high throughput capabilities make possible to study in a single experiment all the community of microorganisms present in a particular sample “microbioma”.They made more feasible the application of the metagenomic approach, by which it is also possible to discover and identify new pathogens, that may pose a threat to public health.This paper summarizes the most recent applications of nextgeneration sequencing to discover new viral pathogens during the occurrence of infection disease outbreaks.

Background Outbreaks of Acute Hepatitis A Virus (HAV) among men who have sex with men (MSM) have been reported in Europe and, recently, in Italy. From July 2008 through January 2010, 162 HAV infections were diagnosed at National Institute for Infectious Diseases, Rome, Italy, with high male-to-female ratio (M:F = 7.5). Objectives The aim of this study was to characterize viral strains involved in this outbreak. Study design The sequences of VP1-2A junction of HAV genome, obtained from 67/97 HAV-RNA-positive samples, were used for phylogenetic analysis. Results All but 1 of the HAV sequences were genotype 1A, 1 was genotype 1B. A monophyletic cluster, including 59/66 genotype IA sequences, was identified by phylogenetic analysis. This cluster included also 2 HAV strains isolated in Germany (2007) and France (2008) from MSM, that, in turn, were reported to be genetically correlated to HAV strains circulating in Tuscany in 2008. Among the males harboring an HAV strain belonging to the cluster, 62% reported to be MSM, and 25% were HIV-positive, 2 with acute HIV infection. Conclusion The outbreak occurred in Rome in 2008–2010, involving high proportion of HIV-infected MSM, is sustained by a monophyletic HAV strain, circulating around the same period also in other European countries. Possible factors favouring HAV spread among HIV-infected persons, such as high risk behavior and prolonged fecal excretion, need to be further elucidated. Timely identification of outbreaks with one or the same source of infection may be helpful to implement preventive measures addressing at risk populations.

Background Raltegravir is the first approved antiretroviral able to prevent HIV genome integration into the host chromosomes. The aim of the study is to test if raltegravir plasma concentrations can be associated with proviral DNA decline during raltegravir-based salvage therapy. Methods A total of 33 multidrug-resistant HIV-infected patients were enrolled in a longitudinal open-label pilot study and completed a 24-week follow-up. The CD4+ T-cell count, plasma viral load, proviral HIV DNA and two-long-terminal repeat (2-LTR) circular forms were assessed at baseline, day 14, 30, 60, 90 and 180. The raltegravir trough concentration (Ctrough) was measured by HPLC-ultraviolet and patients were divided into two groups according to the median raltegravir Ctrough. Results The mean ±sd values of baseline HIV RNA, CD4+ T-cell count and HIV DNA were 4.4 ±0.82 log copies/ml, 256 ±177 cells/mm3, and 2,668 ±4,721 copies/106 peripheral blood mononuclear cells, respectively. Despite a transient increase of total DNA at week 2, a marked proviral DNA decay ( P=0.01) with an increase of the 2-LTR unintegrated/total DNA ratio ( P=0.06) over time was observed. At univariate analysis, no correlation between raltegravir C and trough classical virological parameters was observed. Nevertheless, the decay of proviral HIV DNA was more pronounced in patients displaying Ctrough trough>158 ng/ml ( P=0.046). Conclusions Successful raltegravir-based therapy produces a significant decline in proviral DNA and is associated with an increase of the unintegrated/total DNA ratio. Further studies are necessary to define the possible role of pharmacokinetic raltegravir monitoring and the biological meaning of unintegrated proviral DNA.

This proof-of-concept study aimed to identify whether mutations considered not yet relevant for drug resistance (but located at key drug-resistance positions) can act as 'sentinels' of minority resistant variants in HIV-1 drug-naive patients.We focused our attention on three reverse transcriptase (RT) mutations (T69S, L210M and K103R) easily detected by standard population sequencing [i.e. the genotypic resistance test (GRT)]. Ultra-deep pyrosequencing (UDPS) of HIV-1 RT was performed using GS-FLX Roche, on plasma RNA from 40 drug-naive patients infected with HIV-1 subtype B without primary resistance detected by GRT. Only RT drug resistance mutations detected at0.1% in both forward and reverse directions were considered. Associations between GRT sentinel mutations and UDPS drug resistance were assessed using Fisher's exact test.UDPS detected drug resistance mutations in 18/40 drug-naive patients. Patients carrying HIV-1 strains with T69S and L210M by GRT showed a trend to greater infection by minority drug-resistant variants than control patients infected by HIV-1 without these mutations (5/10 and 7/10 versus 3/10; P = not significant). No association was found for K103R by GRT. Notably, T69S and L210M (but not K103R or control viruses) were associated with GRT minority drug-resistant variants with a prevalence1% (3/10 and 4/10 versus 0/20 in K103R and controls; P = 0.03 and P = 0.008, respectively). Moreover, the presence of L210M or T69S viruses by GRT significantly correlated with that of minority thymidine analogue mutations by UDPS (6/20 patients carrying HIV-1 strains with T69S/L210M versus 0/20 patients carrying HIV-1 having K103R or none of these mutations; P = 0.03).This proof-of-concept study suggests the existence of genetic markers, detectable by routine testing, potentially acting as sentinel mutations of minority drug resistance. Their identification may help in the selection of patients at high risk of resistance in reservoirs without the necessity of using UDPS.

De novo high-throughput pyrosequencing was used to detect and characterize 2009 pandemic influenza A (H1N1) virus directly in nasopharyngeal swabs in the context of the microbial community. Data were generated with a prior sequence independent amplification by 454 pyrosequencing on GS-FLX platform (Roche). Influenza A assembled reads allowed near full-length genome reconstruction with the simultaneous analysis of site-specific heterogeneity. The molecular approach applied proved to be a powerful tool to characterize the new pandemic H1N1 influenza virus in clinical samples. This approach could be of great value in identifying possibly new reassortants that may occur in the near future.

Objectives: HIV-1 V3 quasispecies was analyzed by ultra-deep pyrosequencing, in early HIV-infected patients, to assess possible correlations between quasispecies diversity, frequency of variants predicted to use CXCR4 and need for early antiretroviral treatment. Methods: Twenty patients were retrospectively enrolled: 10 patients (group A) required HAART within 6 months from seroconversion and 10 (group B) remained free of therapy during this period. V3 quasispecies was assessed on plasma viral RNA and in peripheral blood mononuclear cell-associated proviral DNA. Prediction of coreceptor usage was performed by position-specific score matrix analysis. Results: Variants predicted to use CXCR4 were detected (frequency >0.3%) in the plasma of 50% of early infected patients (60% from group A and 40% from group B). Intrapatient frequency of these variants was highly variable (0.3-56.3%). A positive correlation was observed between the proportion of X4 variants and intrapatient quasispecies diversity. Quasispecies diversity and absolute numbers of X4 variants were significantly higher in patients from group A. The analysis of proviral DNA quasispecies, performed in a subgroup of five patients, showed that X4 variants were not detected in patients with RNA frequency below 0.3%, and detected at 3.6% in the patient with 56.3% of X4 plasma variants. Conclusion: Our findings show that X4 variants may be frequently found, at variable intrapatient frequency, in early infected patients, and that quasispecies diversity and absolute numbers of X4 variants are significantly higher in patients undergoing early antiretroviral treatment. Further studies are mandatory to explore the clinical relevance of X4 variants present during early infection with respect to clinical progression and possible therapeutic implications.

In this work we evaluated plasmacytoid (pDC) and myeloid dendritic (mDC) cells activation before and during anti-HCV treatment in HCV+/HIV+ individuals.HCV+/HIV+ patients received Peg-IFN-α2b subcutaneously for 28. days, followed by oral weight-based ribavirin. DCs activation was evaluated by flow cytometry.Baseline pDC CD80 and CD86 expression was correlated with HIV, but not with HCV viral load. A transient decrease of HIV RNA was found not associated with DC activation. When patients were grouped according to early/sustained virological response (EVR/SVR) to anti-HCV treatment, baseline pDC CD80 and CD86 expression was higher in non-EVR and non-SVR compared to EVR and SVR. Moreover, in responder patients CD80 and CD86 were upregulated by IFN-α. Our data suggest a correlation between DCs activation and response to therapy. These findings could be helpful to better understand the mediators of IFN-α action in HCV+/HIV+ patients and to explore possible exploitation of this knowledge to improve therapeutic response. © 2010 Elsevier Inc.

Near full length genome characterization of a BF recombinant from a patient who died from multiorgan failure during HIV-1 seroconversion is reported. Massive parallel pyrosequencing was used with the shotgun approach. Intrahost genetic variability along the whole genome was calculated and coreceptor usage of viral quasispecies was predicted. A consensus sequence was established to perform subtype assignment, phylogenetic analysis, and recombination tests. The sequence clustered with two recently described BF unique recombinant forms from Brazil, consistent with the recombination pattern, yielding breakpoints located at the same positions, with the exception of the second env breakpoint. The actual prevalence of recombinant forms is probably underestimated if partial genomic regions are considered. Here the first full length BF recombinant from Italy is described, together with an evaluation of quasispecies heterogeneity. Our data provide evidence that next generation sequencing may provide a major contribution to HIV-1 molecular epidemiology and to the comprehension of intrapatient heterogeneity.

The use of HIV-1 DNA quantitation in cellular reservoirs to predict disease progression and treatment outcome in infected patients is hampered by the lack of standardization among the available methods. In the present study, real-time PCR methods used commonly for HIV-1 proviral DNA evaluation were compared, showing strong differences in the results, probably as a consequence of genome variability in the target regions. Standardization of HIV-1 proviral DNA quantitation assays is needed for use in clinical management of patients with HIV-1.