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Chikungunya fever is caused by Chikungunya virus (CHIKV) and is generally considered a self-limiting disease. However, severe clinical presentations with a high mortality rate have been reported in association with underlying medical conditions. This study reports the molecular characterization of the virus and an abnormal pattern of circulating cytokines in a unique lethal CHIKV case during the 2017 outbreak in Italy, which involved an elderly patient with underlying cardiac disease. Analysis of inflammatory cytokines revealed a strong increase of interferon (IFN)-α and IFN-β, as well as interleukin-6, suggesting a possible role of type-I IFN in the cytokine storm, which may be correlated with unfavorable prognosis of CHIKV infection.

Acute hepatitis B infection (AHB) is still a common viral acute hepatitis worldwide. As vaccination, antiviral treatment, and immigration are bound to affect the epidemiological landscape of HBV infections, and some of its aspects need to be investigated: (1) the circulation of vaccine escape mutants and of primary drug resistant strains, (2) the change in HBV genotype prevalence, and (3) the clinical implications of AHB and the probability of chronification. The serological, virological, and clinical parameters of 75 patients, acutely infected by HBV, were gathered for a retrospective study. Long-term follow up, either to complete seroconversion or for up to five years, was possible for 44 patients. Sequence analysis of the reverse transcriptase/HBsAg and precore regions was performed to investigate the molecular epidemiology and pathogenesis of recent infections by HBV. Genotype distribution in AHB in Italian patients was radically different from that of chronic infections, with a dramatic increase of extra-European genotypes (A1, F), suggesting that a proportion of AHBs are currently related to imported strains. None of the documented infections occurred in vaccinated individuals, while HBsAg variants (potentially vaccine escape variants) were rare and less prevalent than in chronic infections. No drug resistant strains were observed. Spontaneous viral clearance occurred in all but three cases. Time to viral clearance was inversely proportional to liver damage, but HBsAg titer on day 28 and, better still, HBsAg decay from day 0 to day 28 after admission, were the best predictors of chronification. They are, thus, potentially useful to guide antiviral treatment to prevent chronic evolution.

This study reports the presence of dengue virus RNA in longitudinally collected semen samples of a previously healthy Caucasian man, returning to Italy from Thailand with primary dengue fever, up to 37 days post-symptom onset, when viraemia and viruria were undetectable. This finding, coupled with the evidence of dengue virus negative-strand RNA, an indirect marker of ongoing viral replication, in the cellular fraction of semen, indicates a need to further investigate possible sexual transmission.

The present paper aims to examine the prospects for sustainability transitions in Arab-Islamic countries. Starting from the analysis of the activities performed by the various constellations of social movements for sustainability in Egypt, it will be demonstrated that: (a) the formation of ‘eco-citizens’ occurs through a daily-based and active grassroots participation in political and social struggles related to sustainability issues; (b) the so-called Value-based Social Movements (VSMs) are acting as new social forces capable of participating in decision-making processes and suggesting sound policy solutions to environmental degradation, unsustainable modes of production and consumption, socio-economic injustices and inequalities.

Objectives Efficient interruption of Ebola virus disease (EVD) transmission chains critically depends on reliable and fast laboratory diagnosis. We evaluated the performance of the EBOLA Virus Antigen Detection K -SeT (EBOLA Ag K -SeT), a new rapid diagnostic antigen test in field settings. Methods The study was conducted in a field laboratory located in Freetown (Sierra Leone) by the Italian National Institute for Infectious Diseases ‘L. Spallanzani' and the EMERGENCY Onlus NGO. The EBOLA Ag K -SeT was tested on 210 residual plasma samples (EVD prevalence 50%) from patients hospitalized at the EMERGENCY Ebola treatment center in Goderich (Freetown), comparing the results with quantitative real-time PCR. Results Overall, the sensitivity of EBOLA Ag K -SeT was 88.6% (95% confidence interval (CI), 82.5–94.7), and the corresponding specificity was 98.1% (95% CI, 95.5–100.7). The positive and negative predictive values were 97.9% (95% CI, 95.0–100.8) and 89.6% (95% CI, 84–95.2), respectively. The sensitivity strongly increased up to 98.7% (95% CI, 96.1–101.2) for those samples with high virus load (≥6.2 log RNA copies/mL). Conclusions Our results suggest that EBOLA Ag K -SeT could represent a new effective diagnostic tool for EVD, meeting a need for resource-poor settings and rapid diagnosis for individuals with suspected EVD.

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.

Antiviral therapy has revolutionized treatment of chronic HBV infections. First generation compounds, lamivudine and adefovir, displayed a high rate of treatment failures, and have been replaced by more potent compounds with high genetic barrier to resistance. However, the evolution of the virus towards resistance due the use of first generation compounds may still provide useful information for a better management of current antivirals. A single center sequence database including 705 HBV reverse transcriptase sequences from patients failing antiviral treatments (2002-2012) has been statistically analyzed to highlight viral evolution in relationship to the use of antiviral compounds and to their associations/sequencing in those years. The influence of viral genotypes and polymorphisms on resistance-related mutational patterns was also investigated. This study documents how, after the first years of antiviral therapy, the use of adefovir as an add-on strategy allowed a consistent reduction treatment failures. It also documents the effects of the initial misuse of entecavir in lamivudine experienced patients. In the latest years, the correct use of entecavir and the introduction of tenofovir allowed further curbing of resistance-related treatment failures, which virtually disappeared. Furthermore, the study allows a better understanding of how viral genotype (A vs D) conditions specific mutational pathways to resistance against lamivudine and entecavir, and demonstrates that the use of adefovir in lamivudine experienced patients is associated to peculiar mutational patterns, in particular A181V + F/Y221L. Despite some concern may arise for patients previously treated with lamivudine/adefovir, in sequence or combination, where the virus may have developed a lower genetic barrier against resistance to tenofovir, the outlook of antiviral treatment of HBV infection should be quite optimistic.

Since December 2013, the world has experienced the worst ever epidemic of Ebola virus disease (EVD), which has caused thousands of deaths in several West African countries. When the epidemic began, the European Union (EU) was not unprepared, thanks to the 10-year-long commitment of the European Commission (EC) to fund several networks in the area of highly infectious diseases. The European Network of Biosafety-Level 4 (BSL-4) laboratories (Euronet-P4, later called ENP4-Lab) was one of them; it has been operating since 2004, bringing together the facilities where RiskGroup 4 (RG-4) pathogens such as Ebola virus can be safely handled. In 2010, with the aim of increasing European preparedness in the fight against highly infectious trans-border threats, a new Joint Action was launched, resulting from the union of the networks that had previously worked on the diagnostics of highly infectious viruses and bacteria: ENP4-Lab and EQADeBa (Establishment of Quality Assurance for Detection of Highly Pathogenic Bacteria of Potential Bioterrorism Risk). (...) The authors are grateful to the European Commission and CHAFEA for financially and technically supporting the following Networks: EURONET-P4 2003214, ENP4Lab 2006208, QUANDHIP 2010-21-02, and EMLab (European Mobile Laboratory Project). Part of this work was also supported by the Italian Ministry of Health, ‘Ricerca Corrente’ grants awarded to the ‘Lazzaro Spallanzani’ National Institute for Infectious Diseases-IRCCS, Rome, Italy. info:eu-repo/semantics/acceptedVersion

Drug resistance is a crucial problem emerging frequently during treatment of hepatitis B, resulting in treatment failure and progression of liver damage. A direct sequencing method based on a nested PCR was established to detect mutations in samples with low viral load. Primers were designed to obtain an amplicon encompassing the A, B, C, D and E functional domains of HBV polymerase. Fifty-five samples were tested, containing HBV DNA ranging from 19 to 1700 IU/mL. Sixteen samples were also tested by the commercially available assay INNO-LiPA HBV DR v2. Sequencing was successful for all samples, and mutations were detected in 24/55 (43.6%). When used in parallel with DR v2, concordant results were found in 8/16 samples. In the eight discordant cases, four were resolved by sequencing and not by DR v2, and four had differences in the mutation patterns. Direct sequencing was able to show pol mutations not revealed by DR v2, such as rtV214A, rtQ215H/S, and rtM250V. Genotype and env variations were also established. This highly sensitive sequencing protocol, providing valuable sequencing data from samples with a low viral load, is suitable for detection of mutations at the very early signs of failure of treatment, thereby allowing to maximize the success of early treatment changes.

A retrospective review was performed comparing lamivudine-resistance mutation patterns between patients infected with hepatitis B virus (HBV) with or without human immunodeficiency virus (HIV) co-infection. Medical records that included a genotypic test of patients infected with HBV and treated with lamivudine as the only anti-HBV drug were reviewed. Pol gene mutations were assessed by direct sequencing of the reverse transcriptase fragment 125–213 aa. Eighty-nine patients infected with HBV (29 co-infected with HIV) with rtM204V or rtM204I mutations were included. Multiple mutations associated with the YMDD motif were observed in 33 (55%) of 60 patients infected with HBV only and in 28 (96.6%) of patients co-infected with HIV/HBV. In this latter group, the prevalence of the rtV173L + rtL180M + rtM204V triple mutation was 31% versus a prevalence of 3.4% observed among patients infected with HBV only. All patients with the triple mutational pattern showed sE164D + sI195M changes in the envelope gene. Multivariate analysis demonstrated that HIV co-infection (adjusted OR 11.2, 95% CI 2.0–61.0) and HBV genotype A (adjusted OR 7.2, 95% CI 1.5–34.8) were the only independent variables associated with the chance of harboring rtM204V. Patients with HBV genotype A or HIV co-infection were more likely to harbor the rtM204V mutation. Patients co-infected with HIV showed multiple mutations more frequently, including the triple mutation that may elicit a vaccine escape phenotype. Among patients co-infected with HIV/HBV, strict HBV DNA monitoring is essential to detect treatment failure and adapt therapy to avoid limitations of future therapeutic options or the emergence of a public health threat. J. Med. Virol. 81:1151–1156, 2009. © 2009 Wiley-Liss, Inc.

Background: A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients. Objectives: The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples. Patients and Methods: Clinical samples from a cohort of CHB patients, including liver biopsies in some, were collected for the analysis of intracellular HBV molecular markers using novel molecular assays. Results: A plasmid construct, including sequences from the HBV genome and from the human gene hTERT, was generated as an isomolar multi-standard for HBV quantitation and normalization to the cellular contents. The specificity of the real-time assay for the cccDNA was assessed using Dane particles isolated on a density gradient. A comparison of liver tissue from 6 untreated and 6 treated patients showed that the treatment deeply reduced the replicative capacity (total DNA/cccDNA), but had limited impact on the parenchymal colonization. The peripheral blood mononuclear cells (PBMCs) and granulocytes from the treated and untreated patients were also analyzed. Conclusions: A straightforward method for the quantification of intracellular HBV molecular parameters in clinical samples was developed and validated. The widespread use of such versatile assays could better define the prognosis of CHB, and allow a more rational approach to time-limited tailored treatment strategies.

Summary Tuberculosis is the most frequent co-infection in human immunodeficiency virus (HIV)-infected individuals, and which still presents diagnostic difficulties. Recently we set up an assay based on interferon (IFN)-γ response to region of difference 1 (RD1) peptides selected by computational analysis which is associated with active Mycobacterium tuberculosis replication. The objective of this study was to investigate the response to RD1 selected peptides in HIV-1-infected individuals in a clinical setting. The mechanisms of this immune response and comparison with other immune assays were also investigated. A total of 111 HIV-infected individuals with symptoms and signs consistent with active tuberculosis were enrolled prospectively. Interferon (IFN)-γ responses to RD1 selected peptides and recall antigens were evaluated by enzyme-linked immunospot assay. Results were correlated with CD4+ T cell counts, individuals' characteristics, tuberculin skin test, QuantiFERON-TB Gold and T-SPOT.TB. Results from 21 (19%) individuals were indeterminate due to in vitro cell anergy. Among ‘non-anergic’ individuals, sensitivity for active tuberculosis of the assay based on RD1 selected peptides was 67% (24 of 36), specificity was 94% (three of 54). The assay also resulted positive in cases of extra-pulmonary and smear-negative pulmonary active tuberculosis. The response was mediated by CD4+ effector/memory T cells and correlated with CD4+ T cell counts, but not with plasma HIV-RNA load. Moreover, the RD1 selected peptides assay had the highest diagnostic odds ratio for active tuberculosis compared to tuberculin skin test (TST), QuantiFERON-TB Gold and T-SPOT.TB. RD1 selected peptides assay is associated with M. tuberculosis replication in HIV-infected individuals, although T cell anergy remains an important obstacle to be overcome before the test can be proposed as a diagnostic tool.

A previous case-control study reported that an in-vitro interferon (IFN)-gamma response to early secreted antigenic target (ESAT)-6 selected peptides was associated with active tuberculosis (A-TB). The objective of the present pilot study was to evaluate the diagnostic accuracy of this assay for TB disease in a clinical setting. An IFN-gamma ELISPOT assay was performed on samples from patients with suspected A-TB using two peptides selected from ESAT-6 protein and three peptides selected from culture filtrate 10 (CFP-10) proteins. The results were compared with those obtained by two commercially available assays approved for diagnosis of TB infection (T SPOT-TB and QuantiFERON-TB Gold) which use ESAT-6/CFP-10 (RD1) overlapping peptides. Sensitivity to the RD1 selected peptides was 70% (positive for 16 of 23 patients with microbiologically diagnosed A-TB) and specificity was 91% (positive for three of 32 controls). In contrast, the sensitivity and specificity were 91% and 59%, respectively, for T SPOT-TB, and were 83% and 59%, respectively, for QuantiFERON-TB Gold. The RD1 selected peptides assay had the highest diagnostic odds ratio for A-TB. Thus, the results suggest that an assay based on RD1 selected peptides has a higher diagnostic accuracy for A-TB in a clinical setting compared with commercially available assays based on RD1 overlapping peptides.

We recently set up a gamma interferon (IFN-γ) enzyme-linked immunospot assay (ELISPOT), using selected early secreted antigenic target 6 (ESAT-6) peptides, that appears specific for active tuberculosis (A-TB). However, ELISPOT is difficult to automate. Thus, the objective of this study was to determine if the same selected peptides may be used in a technique more suitable for routine work in clinical laboratories, such as whole-blood enzyme-linked immunosorbent assay (WBE). For this purpose, 27 patients with A-TB and 41 control patients were enrolled. Our WBE, using the already described selected peptides from ESAT-6 plus three new ones from culture filtrate protein 10, was performed, and data were compared with those obtained by ELISPOT. Using our selected peptides, IFN-γ production, evaluated by both WBE and ELISPOT, was significantly higher in patients with A-TB than in controls ( P < 0.0001). Statistical analysis showed a good correlation between the results obtained by WBE and ELISPOT ( r = 0.80, P < 0.001). To substantiate our data, we compared our WBE results with those obtained by QuantiFERON-TB Gold, a whole-blood assay based on region of difference 1 (RD1) overlapping peptides approved for TB infection diagnosis. We observed a slightly higher sensitivity with QuantiFERON-TB Gold than with our WBE (89% versus 81%); however, our test provided a better specificity result (90% versus 68%). In conclusion, results obtained by WBE based on selected RD1 peptides significantly correlate with those generated by ELISPOT. Moreover, our assay appears more specific for A-TB diagnosis than QuantiFERON-TB Gold, and thus it may represent a complementary tool for A-TB diagnosis for routine use in clinical laboratories.

To the Editor: The biologic role of amino acid variants at position 222 of the hemagglutinin (HA) gene of pandemic (H1N1) 2009 virus in severe infections has been extensively discussed. A recent series of studies (1–3) confirm the initial suggestions that G or N in this position might confer greater pathogenic potential to the virus than to the wild type. In contrast, their data suggest that no particular pathogenicity is associated with the 222E variant because it occurs at the same frequency in severe and mild infections. Most authors also seem to agree that D222G or N appears sporadically in phylogenetically distant viruses, with limited transmissibility. However, Puzelli et al. (4) reported transmission of a 222G mutant from son to father (with the appearance of an additional G155E mutation). In Italy, the pattern of D222 variants has been peculiar, with extremely rare appearances of 222G and high diffusion of 222E isolates. At the National Institute for Infectious Diseases in Rome, 82 isolates (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"CY063455-CY063469","start_term":"CY063455","end_term":"CY063469","start_term_id":"296238920","end_term_id":"296238948"}}CY063455-CY063469 for new sequences in this study) were monitored for D222 variants. No 222G or N variants were detected, even in 24 severe infections, nor was the G155E mutation detected. This finding was not surprising, given the worldwide low frequency of this mutation, even in severe infections. Conversely, D222E was detected in 12 of the 82 cases, peaking in September 2009, when it was present in most of the infections, with no overrepresentation in severe cases. Subsequently, it was substituted by different 222D viruses during the autumn–winter outbreak. The analysis of publicly available sequences from other centers in Italy confirmed the trend: 222E was the dominant variant during the summer, 222G was detected only in 4 cases, and 222N was never detected. As we previously reported (5), phylogenetic analysis of 222E variants allowed identification of them as an authentic circulating subclade of clade 7 (6) or cluster 2 (7), rather than as sporadically occurring variants. To further investigate the origin and the evolution of 222 variants, we have extended the phylogenetic analysis (neighbor-joining) to 2,492 complete HA sequences from the Global Initiative on Sharing All Influenza Data database (expanded Figure online, www.cdc.gov/EID/content/17/4/749-F.htm), confirming the clustering of all the worldwide 222E variants in a well-defined subclade (Figure; expanded Figure online). The same analysis showed that D222G variants could be reconciled with 2 different phylogenetic patterns. The first less frequent pattern includes sequences appearing sparsely throughout the tree, confirming the mentioned hypothesis of sporadic mutation. In contrast, the second pattern (the majority) relates to small groups of sequences appearing in monophyletic microclusters. Among these microclusters, 2 are particularly interesting because they include only 222G sequences, isolated in different parts of the world (Figure). This finding is still compatible with sporadic mutation; bootstrap values are low because of the low general variability of these sequences. However, the possibility that D222G variants are transmissible and might sustain small epidemics of their own or that they might arise more easily from specific, phylogenetically related backgrounds, is intriguing. In a few countries, such as Italy, Norway, or Sweden, where the 222E virus has been circulating as a substantial proportion of the total virus, the 222G variants appeared more frequently in the genetic context of the 222E virus (1,4), as demonstrated by phylogenetic analysis and confirmed by the analysis of codon 239 (the codon determining the 222 residue specificity): GAA to GGA (E to G), instead of GAT to GGT (D to G). In these cases, the correct definition of 222G variants would therefore be E222G rather than D222G. From this point of view, the 222N variant would have a higher genetic barrier to change from E because it would require 2 mutations (GAA to AAT) instead of 1 (GAT to AAT), and indeed none of the 16 available (worldwide) 222N full-length variants clustered with the 222E virus. On the basis of these findings, 2 different amino acids, D and E, might be considered polymorphic variants at position 222, and the potentially more pathogenic mutants or circulating variants would be G or N. Figure Phylogenetic relationships among 2,492 complete hemagglutinin (HA) genes of pandemic (H1N1) 2009. At the center, the whole neighbor-joining tree. On the left, enlargement of 2 regions of the tree including pure monophyletic D222G clusters, indicated in ...

The transmission of hepatitis B virus by donors with occult HBV infection (OBI) is a threat for blood transfusion and organ/tissue transplantation. The risk of carrying HBV DNA is currently not predictable by simple serologic markers, while HBV DNA testing is not universally deployed. This study evaluated an integrated serologic approach for assessing this risk. Anti-HBc positive subjects (461 HIV-negative, 262 HIV-positive) were selected for the study. Serology was analyzed by a commercial CMIA technique. HBV DNA was analyzed by both commercial and home-brew real-time amplification assays. A penalized maximum likelihood logistic approach was used to analyze the data. In HBsAg-negative subjects (HIV-negative), anti-HBc signal/cut off values, the presence of anti-HBc IgM, the absence of anti-HBsAg, and the absence of anti-HCV were correlated to the probability of finding circulating HBV DNA. A model for predicting HBV DNA presence by 4 serological parameters is therefore proposed. The predictive value of the logistic model based on simple serologic markers may represent a reasonable tool for the assessment of HBV transmission risk by transfusion or organ/tissue donation in the context of limited resources and where nucleic acid testing is not performed. In addition, it may be helpful for assessing the risk of reactivation in immunosuppressed OBI patients.

Primary structure analysis of the four river buffalo alpha-globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two alpha-globin chains, Ialpha1 and IIalpha3, which differ at positions 129 and 131: Ialpha1 has 64 Ala, 129 Phe, 131 Asn; IIalpha3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two alpha-globin chains, Ialpha2 and IIalpha4, which differ at positions 10 and 11: Ialpha2 has 10 I1e, 11 Gln, 64 Asn; IIalpha4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic alpha-globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo alpha-globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.

Background Some reports have documented the coexistence of Hepatitis B surfage Antigen (HBsAg) and anti-HBsAg antibodies (HBsAb) in patients with chronic hepatitis B (CHB), often in the absence of amino acid substitutions in the HBsAg sequences of the Hepatitis B Virus (HBV) genome able to explain an immunological escape variant. HBV genome has a very compact coding organization, with four partially overlapping open reading frames (ORFs). Because the reverse transcriptase region (rt) of HBV polymerase overlaps the HBsAg ORF, it is possible that some mutations in the HBsAg region correspond to mutations in the rt ORF, conferring resistance to current antiviral therapies. This unique case explores the response to antiviral therapies of a CHB with concurrent HBsAg and HBsAb positivity, and analyse the clinical implications of possible mutations in rt and HBsAg ORFs. Case presentation Here we describe the case of a 59 year-old Italian man suffering from Hepatitis B envelope Antigen (HBeAg) positive CHB with concurrent HBsAb positivity. By ultra-deep pyro-sequencing (UDPS) technique, mutations conferring immunological escape or resistance to antiviral therapies were found neither in HBsAg nor in HBV rt ORFs, respectively. The patient was unsuccessfully treated with interferon, adefovir monotherapy and adefovir plus entecavir combination. Surprisingly, during entecavir plus tenofovir combination, anti-HBe seroconversion and HBsAg loss were observed, while the titer of HBsAb persisted. Conclusions Concurrent HBsAg/HBsAb positivity in active CHB is a clinical and virological dilemma. In this setting, there are not consistent data about the response to conventional therapies and the immunological balance between host and virus remains so far unexplained. This is, to our knowledge, the first case described of a CHB with HBsAg/HBsAb positivity, wild type for clinically relevant mutations in HBsAg and rt ORFs, successfully treated with a combination of nucleot(s)ide analogues (NAs).

Monitoring the efficacy of antituberculosis therapy is crucial both for the individual patient and for better control of the spread of tuberculosis. We studied 18 patients with microbiologically confirmed tuberculosis, both at the time of diagnosis and 3 months after they started therapy, using an in vitro assay that detects T cell-mediated interferon- gamma response to selected peptides of Mycobacterium tuberculosis-specific early secretory antigenic target 6 (ESAT-6) protein. All patients had positive results at diagnosis; however, 3 months later, the response to ESAT-6 peptides was still detectable only in the 5 patients with microbiological isolation and/or absence of clinical improvement after treatment. On the basis of these data, we conclude that our assay is a useful tool in monitoring the efficacy of antituberculosis therapy.

A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct population sequencing and reverse hybridization (line probe assay [LiPA])-based methods are the most common methods for detecting hepatitis B virus (HBV) drug resistance mutations, although only mutations present in viral quasispecies with a prevalence of ≥20% can be detected by sequencing, and only known mutations are detected by LiPA. Massively parallel ultradeep pyrosequencing (UDPS; GS FLX platform) was used to analyze HBV quasispecies in reverse transcriptase (RT) and hepatitis B S antigen (HBsAg) from five drug-naive patients and eight drug-resistant patients. Eight primer pairs were used to obtain partially overlapping amplicons, covering the RT gene from codons 1 to 288 and the complete overlapping HBsAg sequence. A 1% mutation frequency was selected as the cutoff based on an error rate estimated on plasmid DNA. This technology enabled simultaneous analysis of between 2,852 and 18,016 clonally amplified fragments from each patient. The results indicate that UDPS has a relative sensitivity much higher than both direct sequencing and LiPA. In addition, the UDPS results are quantitative, allowing establishment of the relative frequency of both known mutations and novel substitutions. Some of the detected RT substitutions led to changes also in HBsAg. On the whole, genotype D presented a higher heterogeneity than genotype A. Considering the high quantity of information that can be provided by a single test from one patient, the short turnaround time, the information on substitution frequency, and the detection of rare variants, there are strong advantages conferred by UDPS, and the new method could play a relevant role in the clinical management of HBV infection and therapy.

On 398 river buffalo samples, randomly collected in distinct breeding areas of the Campania region, high-resolution analytical systems were used to identify both qualitative and quantitative variations of the Hb phenotype. Polyacrylamide gel isoelectric focusing and HPLC were used to determine the ratio between HBA1 and HBA2 globin chains; restriction endonuclease analysis was performed to assess whether quantitative variations in Hb bands were related to an unusual number of a-globin genes. In the two buffalo subpopulations, allele frequencies of the alpha and beta globin systems were calculated, and F statistics (FIS, FIT and FST) were estimated as parameters of genetic diversity. The results suggest that: i) as shown by RFLP analysis, only a couple of associated a globin genes account for the quantitative variations recorded at the phenotypic level; ii) as expected, in the a globin gene system (HBA), the frequency of haplotype B (HBA-B) largely exceeded that of haplotype A (HBA-A) (95.1% vs 4.9%); iii) the frequency of the usual allele at the beta locus is 0.6, as opposed to 0.4 of the slow variant; iiii) the most significant component of variation of the genetic system of hemoglobin is between individuals within the same location.

Controversial results have been obtained in studies of the effect of Mycobacterium tuberculosis on human immunodeficiency virus type 1 (HIV-1) replication in cells of the macrophage lineage. In the present study, monocyte-derived macrophages (MDMs), previously incubated for 2 days with heat-inactivated M. tuberculosis, were infected with HIV-1. M. tuberculosis consistently inhibited viral replication, and a similar result also was observed in the presence of supernatants from M. tuberculosis-stimulated MDMs, which indicates that this effect was mediated by soluble factors. Although CCR5-binding chemokines were induced by M. tuberculosis stimulation, the results of neutralization experiments indicated that it is unlikely that they were responsible for viral suppression. Inhibition occurred mainly after viral entry (demonstrated by use of a vesicular stomatitis virus G-pseudotyped HIV-1 and by analysis of HIV-1 early and late reverse-transcription products). Therefore, M. tuberculosis-induced factors may inhibit in vitro HIV-1 replication in macrophages by affecting an early postentry step in the HIV-1 cycle.

T cells expressing Vgamma9Vdelta2 display lytic and proliferative responses against human immunodeficiency virus (HIV)-infected cells and release antiviral soluble factors. Chronic HIV-positive patients have deep changes in the composition and function of the circulating gammadelta T cell pool that are restored by highly active antiretroviral therapy (HAART). gammadelta T cells were analyzed during the rapid plasma HIV RNA rebound in HIV-infected patients undergoing structured treatment interruption (STI). A loss in circulating Vgamma9Vdelta2 T cells was observed, indicating that acute HIV replication may influence Vgamma9Vdelta2 homeostasis. These cells were lost among CD45RA(-)CD27(-) Vgamma9Vdelta2 T cell effectors, and a significant unresponsiveness, measured as antigen-driven interferon-gamma production, was observed during STI. After HAART resumption and consequent inhibition of HIV replication, Vgamma9Vdelta2 T cell reactivity was restored both quantitatively and functionally. These observations indicate that Vgamma9Vdelta2 T cells are activated early after active HIV replication but are rapidly lost when viremia is not controlled.

Background Next-generation sequencing (NGS) offers a unique opportunity for high-throughput genomics and has potential to replace Sanger sequencing in many fields, including de-novo sequencing, re-sequencing, meta-genomics, and characterisation of infectious pathogens, such as viral quasispecies. Although methodologies and software for whole genome assembly and genome variation analysis have been developed and refined for NGS data, reconstructing a viral quasispecies using NGS data remains a challenge. This application would be useful for analysing intra-host evolutionary pathways in relation to immune responses and antiretroviral therapy exposures. Here we introduce a set of formulae for the combinatorial analysis of a quasispecies, given a NGS re-sequencing experiment and an algorithm for quasispecies reconstruction. We require that sequenced fragments are aligned against a reference genome, and that the reference genome is partitioned into a set of sliding windows (amplicons). The reconstruction algorithm is based on combinations of multinomial distributions and is designed to minimise the reconstruction of false variants, called in-silico recombinants. Results The reconstruction algorithm was applied to error-free simulated data and reconstructed a high percentage of true variants, even at a low genetic diversity, where the chance to obtain in-silico recombinants is high. Results on empirical NGS data from patients infected with hepatitis B virus, confirmed its ability to characterise different viral variants from distinct patients. Conclusions The combinatorial analysis provided a description of the difficulty to reconstruct a quasispecies, given a determined amplicon partition and a measure of population diversity. The reconstruction algorithm showed good performance both considering simulated data and real data, even in presence of sequencing errors.

Immunity to M.tuberculosis (MTB) infection consists of interactions between various T-cell subsets that control the infection and prevent further reactivation. We analysed the effector/memory T-cell dynamics and cytokines production in the peripheral blood of patients with pulmonary tuberculosis (TB). We observed that the frequency of CD4+ T-cell effectors was significantly increased during active TB, confirming a major role of this T-cell subset in TB immunity. Pre-terminally differentiated CD8+ T-lymphocytes were increased in the peripheral blood as well. In contrast, we observed a reduced number of effector mycobacteria-reactive γδ+ T-lymphocytes with a specific defects in reacting to mycobacterial nonpeptidic antigens, suggesting that this innate response is rapidly lost during TB infection. Nevertheless, the frequency of γδ+ T-cells effectors in TB patients was higher than the αβ+ T-cell response to peptide from MTB-ESAT-6 protein and quantitatively similar to PPD reactivity. Thus, αβ+and γδ+ T-cell differentiation and function are differently triggered by active TB infection.

The evaluation of diagnostic accuracy of new in vitro diagnostic assays for tuberculosis infection has been hampered by the lack of a standard reference test. The aim of this study was to compare sensitivity and specificity of interferon gamma assays for latent tuberculosis infection by assessing the association of test results with tuberculosis occupational exposure and by using latent class analysis. We analysed data from 115 healthcare workers on whom tuberculin skin test (TST) and the following in vitro tests were performed: in-house ELISPOT for RD1 proteins, T.SPOT-TB and Quantiferon-TB Gold. Results of all tests were associated with increased occupational risk of exposure to Mycobacterium tuberculosis, but only TST was associated with Bacillus Calmette-Guerin (BCG) vaccination. Sensitivity/specificity (95% confidence intervals) estimated by a latent class model were: 99.9%/64.2% (53.0-74.1) for TST, 95.3% (61.8-99.6)/87.5% (78.0-93.2) for in-house ELISPOT, 96.7% (69.3-99.7)/85.6% (75.3-92.0) for T.SPOT-TB, and 76.3% (55.9-89.1)/93.6% (85.4-97.3) for Quantiferon. The estimated specificity of in vitro assays was higher than that of TST also among individuals who were not BCG-vaccinated. In conclusion, when used in healthcare workers, in vitro assays may provide a significant increase of specificity for tuberculosis infection compared to TST, even among non vaccinated individuals, at the cost of some sensitivity. .

The early secretory antigenic target (ESAT)-6 purified protein and peptides from Mycobacterium tuberculosis were evaluated as antigens for the immunodiagnosis of tuberculosis (TB). Because the control of TB requires improved diagnostic procedures, efforts have increased to identify Mycobacterium tuberculosis-specific epitopes for the immunodiagnosis of active TB and to discriminate between active and latent states of infection. Two multiepitopic peptides from ESAT-6 protein were selected by computational analysis. Patients with active TB (7 HIV(+) and 20 HIV(-)) and control patients (17 HIV(+) and 28 HIV(-)) were enrolled. Enzyme-linked immunospot assay analysis for interferon-g expression by peripheral blood mononuclear cells was quantified after stimulation with selected ESAT-6 peptides, purified protein derivative, or the intact ESAT-6 protein. During active TB, 20 of 27 patients responded in vitro to ESAT-6 peptides and 23 of 27 patients to purified protein derivative. None of the controls without active TB, including individuals with latent TB infection, recognized ESAT-6 peptides. By contrast, latently infected individuals did respond in vitro to both intact ESAT-6 protein and purified protein derivative. Thus, high T-cell response frequencies to ESAT-6 peptides are present only during active TB and can be used to discriminate between active and latent forms of infection.

The effect of malondialdehyde on structural features of bovine alpha-crystallin has been investigated by absorption and fluorescence spectroscopy as well as by far-UV circular dichroism. Experimental evidence suggests the occurrence of intermolecular cross-linking induced by malondialdehyde. This cross-linking does not seem to affect the tryptophan environment, as suggested by intrinsic protein fluorescence. On the contrary, the time dependence of far-UV dichroic activity indicates that the cross-linking is accompanied by a secondary structure change. The formation of high molecular mass aggregates, evidence by electrophoresis in denaturing conditions, leads to irreversible alpha-crystallin aggregation due to extensive intermolecular cross-linking. Since malondialdehyde is produced in vivo as a breakdown product of lipid peroxidation the possible involvement of this molecule in the pathological mechanism of cataract formation has been briefly discussed.