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A review of a large number of HIV-1 tropism test requests (n = 1148) performed at a London tertiary referral centre was carried out. The aim was to establish whether these were being performed in line with recommendations from published guidelines and whether this represented the most cost-effective use of these tests in informing prescribing decisions of the CCR5 antagonist drug, maraviroc. The cost of these assays within the UK was covered by commercial funding until April 2013 which has subsequently been withdrawn. Furthermore, all healthcare settings are under increasing cost constraints and hence establishing the real utility and appropriate use of these tests is of vital importance. [ABSTRACT FROM AUTHOR]

HIV co-receptor tropism determination is essential before prescribing the CCR5 antagonist maraviroc. British HIV Association guidelines suggest tropism testing may remain valid for only 90 days in antiretroviral-naïve patients. We aimed to determine the accuracy of this figure. Tropism was assessed in 26 antiretroviral-naïve patients with ongoing viral replication, sampled yearly from first clinic visit. The V3 region of HIV-1 was sequenced in triplicate, then tropism predicted using the Geno2Pheno system. Baseline tropism prediction remained valid for a median of 52 months (range 7-81). For 19/26 individuals baseline tropism remained unchanged throughout a median of 54 months follow-up; 18 R5 tropic and 1 X4 tropic. In seven patients (27%) baseline tropism switched at least once (range 1-4 switches) during follow-up; however, their baseline tropism prediction remained valid for a median of 45 months. Co-receptor tropism in treatment-naïve patients with ongoing viral replication appears highly stable over time, suggesting that baseline genotypic tropism prediction may be valid for a longer duration in patients delaying ART initiation. In this study, baseline tropism prediction remained valid for a median of 52 months, suggesting current guidelines recommending repeat testing after 90 days may be excessively conservative in their assessment of tropism stability., (© The Author(s) 2015.)

The current epidemic of Hepatitis C infection in HIV-positive men who have sex with men is associated with increasing use of recreational drugs. Multiple HCV infections have been reported in haemophiliacs and intravenous drug users. Using ultra-deep sequencing analysis, we present the case of an HIV-positive MSM with evidence of three sequential HCV infections, each occurring during the acute phase of the preceding infection, following risk exposures. We observed rapid replacement of the original strain by the incoming genotype at subsequent time points. The impact of HCV super-infection remains unclear and UDS may provide new insights., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Hepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options., Objective: To establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B., Study Design: Genotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA., Results: Of 100 samples from 50 patients, ≥ 2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively. LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available. Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be subtyped by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold., Conclusion: Genotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: Testing for HIV is a standard of care for people with active tuberculosis (TB). People investigated for TB in the UK often originate from areas with a high prevalence of HIV and other blood borne viruses (BBV). However, assessment for these infections is patchy. We determined the yield and costs of different testing strategies for BBV in a UK TB clinic., Methods: Since 2009, it has been routine to test all TB clinic attendees. Demographic, clinical and virological data were retrospectively extracted from patient notes and hospital databases., Results: Over 3 years, 1036 people were assessed in the TB service. 410 had a final diagnosis of active TB. HIV testing of the latter population diagnosed 27 new HIV cases at a cost of £3017. When BBV testing was offered to all clinic attendees, a further 6 (total 33) new HIV, 5 Hepatitis B (HBV) and 2 Hepatitis C (HCV) diagnoses were made at a total cost of £22,170., Conclusions: We have identified previously undiagnosed HIV, HBV and HCV in a TB clinic population. Our data suggest that despite increasing upfront expense, the associated yield argues strongly for BBV testing to be offered to all patients being investigated for possible TB, irrespective of their final diagnosis., (Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Hepatitis C virus (HCV) infection is a major health problem worldwide. The effects of chronic infection include cirrhosis, end-stage liver disease, and hepatocellular carcinoma. As a result of shared routes of transmission, co-infection with HIV is a substantial problem, and individuals infected with both viruses have poorer outcomes than do peers infected with one virus. No effective vaccine exists, although persistent HCV infection is potentially curable. The standard of care has been subcutaneous interferon alfa and oral ribavirin for 24-72 weeks. This treatment results in a sustained virological response in around 50% of individuals, and is complicated by clinically significant adverse events. In the past 10 years, advances in HCV cell culture have enabled an improved understanding of HCV virology, which has led to development of many new direct-acting antiviral drugs that target key components of virus replication. These direct-acting drugs allow for simplified and shortened treatments for HCV that can be given as oral regimens with increased tolerability and efficacy than interferon and ribavirin. Remaining obstacles include access to appropriate care and treatment, and development of a vaccine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Isolated HBV core antibody (anti-HBc) is defined as the presence of anti-HBc with a negative HBV surface antigen (HBsAg) and HBV surface antibody (anti-HBs <10 IU/l). In patients infected with HIV with isolated anti-HBc, the aim was to determine: The prevalence of isolated positive anti-HBc; The most effective method of identifying which patients have had previous Hepatitis B Virus (HBV) infection; The prevalence of false positive anti-HBc. HBV serology results were identified from 539 patients infected with HIV sampled between January 2010 and December 2012. In those with an isolated anti-HBc and negative anti-HBe, a second anti-HBc test was carried out using a different assay. Samples were also screened for HBV DNA. The anti-retroviral regimens at time of screening were documented. 101/539 had an isolated anti-HBc. Of these, 32 (32%) had a positive anti-HBe (including 1 equivocal) and 69(68%) were anti-HBe negative. Of those negative for anti-HBe, 32 were tested for both DNA and a second anti-HBc. Of these 26 (81%) were on cART at time of HBV testing, with 25 (78%) on ART with anti-HBV activity. The prevalence of isolated anti-HBc was 19%. Only 32% were also anti-HBe positive, whereas 97% of those anti-HBe negative were positive on a second anti-HBc assay suggesting lack of utility of anti-HBe in resolving serological quandaries. One subject (3%) had a false positive anti-HBc. There was no evidence of chronic HBV but 78% patients were on HBV-suppressive combination anti-retroviral therapy., (© 2014 Wiley Periodicals, Inc.)

As scientific techniques for the detection of cytomegalovirus (CMV) improve, we are able to detect small amounts of CMV in the mucosal wall. As clinicians, we are unsure how to interpret the results of this novel test. There is controversy in the literature as to the significance of the detection of CMV in the gut. Whilst the importance of CMV and reactivation of the virus is clear in those patients such as allograft recipients with established immune compromise, the role is less clear in patients with less damaged immune systems. We explore whether the detection of CMV in such cases influences outcome and how it should be optimally managed. We discuss the optimal management of such cases, according to current guidelines, with a review of the literature.

Background: The non-structural 5A (NS5A) protein of HCV is a multifunctional phosphoprotein involved in regulation of viral replication and virion assembly. NS5A inhibitors targeting domain I of NS5A protein have demonstrated high potency and pan-genotypic antiviral activity, however they possess a low genetic barrier to resistance. At present, only genotype 1, the most prevalent HCV genotype has been studied in detail for resistant variants., Methods: Utilizing a panel of genotypic-specific resistance assays, population sequencing was performed on plasma-derived viral RNA isolated from 138 patients infected with HCV genotypes 1-4 and not treated with direct-acting antiviral agents. Amino acid changes in HCV NS5A domain I at codon positions 28, 30, 31, 32 and 93, reported to confer reduced susceptibility to certain NS5A inhibitors were examined. Additionally, genotypic outcome based on NS5A sequences were compared with VERSANT HCV Genotype Assay (LiPA) 1.0 (Siemens Healthcare Diagnostics, Surrey, UK) and Abbott m2000 RealTime HCV genotype II assay (Abbott Molecular, Maidenhead, Berkshire, UK)., Results: Amino acid substitutions associated with moderate to high level resistance to NS5A inhibitors were detected in 2/42 (4.76%) HCV-1a, 3/23 (13.04%) HCV-1b, 4/26 (15.38%) HCV-2, 1/24 (4.17%) HCV-3 and 1/23 (4.35%) HCV-4 infected patients who had not been treated with NS5A inhibitors. Genotype prediction based on NS5A sequences were concordant with LiPA and/or Abbott RealTime for 97.10% of cases., Conclusions: Primary resistance mutations associated with resistance to first-generation NS5A inhibitors such as daclatasvir were observed in all genotypes, albeit at low frequencies. An excellent correlation based on NS5A genotyping and LiPA or Abbott RealTime was achieved.

Genetically engineered microbial biosensors have yet to realize commercial success in environmental applications due, in part, to difficulties associated with transducing and transmitting traditional bioluminescent information. Bioelectrochemical systems (BESs) output a direct electric signal that can be incorporated into devices for remote environmental monitoring. Here, we describe a BES-based biosensor with genetically encoded specificity for a toxic metal. By placing an essential component of the metal reduction (Mtr) pathway of Shewanella oneidensis under the control of an arsenic-sensitive promoter, we have genetically engineered a strain that produces increased current in response to arsenic when inoculated into a BES. Our BES-based biosensor has a detection limit of ~40 μM arsenite with a linear range up to 100 μM arsenite. Because our transcriptional circuit relies on the activation of a single promoter, similar sensing systems may be developed to detect other analytes by the swap of a single genetic part., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: HIV-1 tropism needs to be determined before the use of CCR5 antagonist drugs such as maraviroc (MVC), which are ineffective against CXCR4-using HIV-1. This study assessed how different computational methods for predicting tropism from HIV sequence data performed in a large clinical cohort. The value of adding clinical data to these algorithms was also investigated., Design and Methods: PCR amplification and sequence analysis of the HIV-1 gp120 V3 loop region was performed on triple replicates of plasma viral RNA or proviral DNA extracted from peripheral blood monocytes (PBMCs) in 242 patients. Coreceptor usage was predicted from V3 sequences using seven bioinformatics interpretation algorithms, combined with clinical data where appropriate. An intention-to-treat approach was employed for exploring outcomes and performance for different viral subtypes was examined., Results: The frequency of R5 predictions varied by 22.6%, with all seven algorithms agreeing for only 75.3% of tests. The identification of individuals likely to fail was poor for all algorithms. The addition of clinical data improved this, but at the expense of their ability to predict success. The clinical algorithms varied across subtypes, whereas other algorithms were more consistent. Furthermore, individuals with discordant clonal and clinical predictions were more likely to fail MVC treatment., Conclusion: Eligibility for MVC varied depending on the algorithm method used. The addition of clinical parameters alongside sequence data may help predict X4 emergence during treatment. It could be that V3 loop analysis in isolation may not be the best method for selecting individuals for MVC.

Coinfection with HIV adversely impacts every stage of hepatitis C (HCV) infection. Liver damage in HCV infection results from host antiviral responses rather than direct viral pathogenesis. Despite depressed cellular immunity, coinfected patients show accelerated hepatic fibrosis compared with HCV monoinfected patients. This paradox is poorly understood. T-regulatory (Treg) cells (CD4+ and FOXP3+) are hypothesized to limit hepatic damage in HCV. Our hypothesis was that reduced frequency of hepatic Treg in HIV/HCV coinfection compared with HCV monoinfection may explain poorer outcomes. We quantified FOXP3+, CD4+, CD8+ and CD20+ cells in liver biopsies of 35 male subjects matched by age and ISHAK fibrosis score, 12 HIV monoinfected, 11 HCV monoinfected and 12 HIV/HCV coinfected. Cell counts were performed using indirect immunohistochemical staining and light microscopy. HIV/HCV coinfected subjects had fewer hepatic FOXP3+ (P = 0.031) and CD4+ cells (P = 0.001) than HCV monoinfected subjects. Coinfected subjects had more hepatic CD8+ cells compared with HCV monoinfected (P = 0.023), and a lower ratio of FOXP3+ to CD8+ cells (0.08 vs 0.27, P < 0.001). Multivariate analysis showed number of CD4+ cells controlled for differences in number of FOXP3+ cells. Fewer hepatic FOXP3+ and CD4+ cells in HIV/HCV coinfection compared with HCV monoinfection suggests lower Treg activity, driven by an overall loss of CD4+ cells. Higher number of CD8+ cells in HIV/HCV coinfection suggests higher cytotoxic activity. This may explain poorer outcomes in HIV/HCV coinfected patients and suggests a potential mechanism by which highly active antiretroviral therapy may benefit these patients., (© 2013 John Wiley & Sons Ltd.)

Hepatitis E virus (HEV) infection is an important public health concern as a major cause of enterically-transmitted hepatitis worldwide. The detectable window of viraemia is narrow, and HEV IgM and IgG rise simultaneously in acute infection. Furthermore, previous investigators have shown HEV IgM false positive reactions occur against EBV, CMV and potentially hepatitis A. A retrospective analysis of HEV serology testing was performed at a London tertiary referral hospital over a 3-year period. A thousand four hundred and twenty three serum samples were tested for HEV serology, with 33 samples HEV IgM positive and 28 HEV IgM equivocal. One hundred and eleven samples were HEV IgG positive but IgM negative suggesting past infection. No patients with HEV IgM positivity had false positive reactions against hepatitis A. A high degree of EBV and CMV cross reactivity was noted, with 33.3% and 24.2% of HEV IgM positive samples also testing positive for EBV and CMV IgM, respectively. HEV RNA was detected in four HEV IgM positive samples, indicating true positivity, although three demonstrated cross reactivity against EBV. Only 13.3% of samples with positive HEV IgM were HEV PCR positive, highlighting a low positive predictive value of serology testing. Overall a high level of HEV, EBV and CMV IgM cross reactivity was demonstrated, indicating that serology is unreliable in the diagnosis of acute viral hepatitis. It is concluded that that the diagnosis of viral hepatitis should be based on clinical features, raised transaminases, serology, and confirmatory PCR testing., (© 2013 Wiley Periodicals, Inc.)

We report the case of a 28-year-old, HIV-positive woman presenting with painful vesicular and ulcerating lesions in the ano-genital region caused by varicella zoster virus that appeared similar to herpes simplex infection. The case highlights that herpes zoster needs to be considered in the differential diagnosis of genital lesions, particularly in HIV-positive individuals, and the importance of virological diagnosis by PCR to direct appropriate management.

Background: Studies have shown that low-frequency resistance mutations can influence treatment outcome. However, the lack of a standardized high-throughput assay has precluded their detection in clinical settings., Objective: To evaluate the performance of the Roche prototype 454 UDS HIV-1 drug resistance assay (UDS assay) in a routine diagnostic laboratory., Study Design: 50 plasma samples, previously characterized by population sequencing and that had shown ≥1 resistance associated mutation (RAM), were retrospectively tested by the UDS assay, including 18 B and 32 non-B subtypes; viral loads between 114-1,806,407 cp/ml; drug-naive (n=27) and drug-experienced (n=23) individuals., Results: The UDS assay was successful for 37/50 (74%) samples. It detected all RAMs found by population sequencing at frequencies above 20%. In addition, 39 low-frequency RAMs were exclusively detected by the UDS assay at frequencies below 20% in both drug-naïve (19/26, 73%) and drug-experienced (9/18, 50%) individuals. UDS results would lead to changes from susceptible to resistant to efavirenz (EFV) in one drug-naive individual with suboptimal response to an EFV-containing regimen and from susceptible to resistance to lamivudine (3TC) in one drug naïve subject who subsequently failed a 3TC-containing regimen and in a treatment experienced subject who had failed a 3TC-containing regimen., Conclusions: The UDS assay performed well across a wide range of subtypes and viral loads; it showed perfect agreement with population sequencing for all RAMs analyzed. In addition, the UDS assay detected additional mutations at frequencies below 20% which correlate with patients' treatment history and had in some cases important prognostic implications., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a serious reaction to drugs with a clinical presentation of rash, fever, lymph node enlargement and internal organ involvement. Reports have described the reactivation of human herpes virus 6 (HHV-6) and other HHVs in association with this syndrome. We report a 41-year-old woman who developed a rash, fever, liver dysfunction, eosinophilia and atypical monocytosis 21 days after initiation of the quadruple therapy for tuberculous cervical lymphadnitis. HHV-7 DNA was detected in blood by PCR suggesting infection with or more likely reactivation of HHV-7 as a contributing factor or consequence of this serious adverse drug reaction.

Controlled human malaria infection is used to measure efficacy of candidate malaria vaccines before field studies are undertaken. Mathematical modeling using data from quantitative polymerase chain reaction (qPCR) parasitemia monitoring can discriminate between vaccine effects on the parasite's liver and blood stages. Uncertainty regarding the most appropriate modeling method hinders interpretation of such trials. We used qPCR data from 267 Plasmodium falciparum infections to compare linear, sine-wave, and normal-cumulative-density-function models. We find that the parameters estimated by these models are closely correlated, and their predictive accuracy for omitted data points was similar. We propose that future studies include the linear model.

Acute colonic pseudo-obstruction syndrome, also known as Ogilvie's syndrome, is a rare condition associated with significant morbidity and mortality. We report a case that developed very rapidly after emergency caesarean section. A 20-year-old woman underwent an emergency caesarean section for failure to progress in the first stage of labour and a healthy male infant was delivered without incident. However, soon afterwards the patient developed significant abdominal distension and pain. Ogilvie's syndrome was diagnosed following an abdominal x-ray which revealed a gross large bowel obstruction without mechanical cause. To prevent caecal rupture, the patient underwent successful emergency colonic decompression.

Acute hepatitis C infection in the context of HIV is an emerging problem in men who have sex with men (MSM). We conducted a retrospective cohort study of MSM diagnosed with and treated for acute hepatitis C infection over 10 years. Genotype 1 was the commonest type representing 69% of cases; the spontaneous clearance rate was 20%. The overall sustained virological response (SVR) rate on an intention-to-treat basis was 83%; SVR and was 92% for those completing 48 weeks of treatment. The presence of detectable RNA at week 12 had a 100% negative predictive value for SVR. This is the largest single cohort treated with 48 weeks of interferon and ribavirin and the treatment SVR is one of the highest reported. We propose that a 48-week treatment regimen may be superior to shorter (24-week) regimens though we acknowledge the need for a randomized controlled trial.

Objectives: Infective endocarditis (IE) is a severe complication in Staphylococcus aureus bacteraemia (SAB) and recent guidelines from the BSAC recommend all patients undergo echocardiography. We assessed the use of echocardiography at a major tertiary referral centre and sought to identify those patients most likely to have positive findings., Methods: We retrospectively evaluated all cases of SAB at Oxford University Hospitals NHS Trust between September 2006 and August 2011., Results: Three-hundred-and-six out of 668 patients with SAB underwent cardiac imaging on average 9.8 ± 1.3 days from the first culture. Thirty-one patients (10.1%) had echocardiographic evidence of IE. Risk factors for observing evidence of IE on scanning included the presence of prosthetic heart valves (32% versus 4%, P < 0.001) or cardiac rhythm management (CRM) devices (16% versus 3%, P < 0.004). On excluding patients with prosthetic valves or CRM devices from the analysis, no patient with a line-related bacteraemia and only one patient (an intravenous drug user) with no/mild regurgitation on transthoracic echocardiography had echo evidence of IE., Conclusions: We propose that the use of scarce echocardiography resources could be prioritized. Patients with prosthetic heart valves or a CRM device should receive early cardiological input and transoesophageal echocardiography. In patients with a clearly defined line-related bacteraemia who do not have a prosthetic valve or CRM device or clinical features of IE, response to treatment could be closely monitored and imaging deferred. Patients without a line-related infection or prosthetic valve/device could receive a transthoracic echocardiogram as a screening tool.

We present two cases of non-capsulated Haemophilus influenzae hepatobiliary infection and review the literature. Such cases are rare, and prior to routine immunization against H. influenzae serotype b invasive Haemophilus disease was largely caused by capsulated strains. The epidemiology of invasive Haemophilus infections has changed and the number of cases of intra-abdominal and hepatobiliary infection may be underestimated due to current microbiological processing practices.

Objectives: The objectives of this study were: (i) to describe an outbreak of multidrug-resistant Klebsiella pneumoniae in our population; (ii) to identify the potential source of this outbreak by examining antibiotic resistance trends in urocultures; (iii) to evaluate the contribution of this outbreak to resistance patterns over time in the two commonest Gram-negative blood culture isolates, namely K. pneumoniae and Escherichia coli; and (iv) to assess risk factors for multidrug resistance and the impact of this resistance on mortality and length of stay., Methods: We searched Microbiology and Patient Administration Service databases retrospectively and describe resistance trends in E. coli and K. pneumoniae bloodstream infections (BSIs) in Oxfordshire, UK, over an 11 year period., Results: An outbreak of a multidrug-resistant, CTX-M-15 extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae clone was identified and shown by multilocus sequence typing to belong to a novel sequence type designated ST490. This was associated with a sporadic change in resistance rates in K. pneumoniae BSIs with rates of multidrug resistance (defined as resistance to three or more antibiotic classes) reaching 40%. A case-control study showed prior antibiotic exposure as a risk factor for infection with this organism. During the same time period, rates of ESBL-producing Klebsiella spp. isolated from urocultures increased from 0.5% to almost 6%. By contrast, the rate of multidrug resistance in E. coli rose more steadily from 0% in 2000 to 10% in 2010., Conclusions: Changes in resistance rates may be associated with outbreaks of resistant clones in K. pneumoniae. Changing resistance patterns may affect important health economic issues such as length of stay.

Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters--monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

We report what we believe is the first reported case of Streptococcus mutans endocarditis complicated by vertebral discitis. The case is particularly interesting and topical as it occurred in a patient with pre-existing cardiac valvular disease who had recently had a dental procedure without antibiotic prophylaxis following a dramatic shift in the UK guidelines.

The spread of dengue virus throughout the tropics represents a major, rapidly growing public health problem with an estimated 2.5 billion people at risk of dengue fever and the life-threatening disease, severe dengue. A safe and effective vaccine for dengue is urgently needed. The pathogenesis of severe dengue results from a complex interaction between the virus, the host, and, at least in part, immune-mediated mechanisms. Vaccine development has been slowed by fears that immunisation might predispose individuals to the severe form of dengue infection. A pipeline of candidate vaccines now exists, including live attenuated, inactivated, chimeric, DNA, and viral-vector vaccines, some of which are at the stage of clinical testing. In this Review, we present what is understood about dengue pathogenesis and its implications for vaccine design, the progress that is being made in the development of a vaccine, and the future challenges.

Hepatitis C virus (HCV) infection is a major and growing global health problem, affecting about 170 million people worldwide, and is a leading cause of liver cirrhosis and hepatocellular carcinoma. Currently, treatment is restricted to interferon alfa and ribavirin, which leads to a successful outcome in only about 50% of individuals. New effective treatments with tolerable side-effect profiles are needed urgently, but development has been hindered by an inability to culture HCV and a scarcity of animal models. Herein, we review progress in HCV biology, including cell culture and new animal models, and the contribution of this work to our understanding of the virus' life-cycle and pathogenesis and development of specifically targeted antiviral treatment. We also discuss changes in our understanding of HCV epidemiology, clinical manifestations, and diagnostics.

The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-gamma ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-gamma ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study.

Heterologous prime-boost immunisation with RTS,S/AS02A and the poxvirus MVA-CS was evaluated in 18 healthy malaria-naïve subjects in Oxford. Both priming with RTS,S and boosting MVA-CS, and the reverse, were found to be safe and well tolerated. T cell responses as measured by IFN-gamma ex vivo ELISPOT were induced, but the responses were low to moderate in both groups, with heterologous boosting yielding only small increments in T cell immunogenicity and no increased antibody response. Protection against 3D7 Plasmodium falciparum sporozoite challenge 4 weeks after the final vaccination was equal for both regimens at 33% (95% C.I. 4.3-77.7%), with one subject remaining fully protected on rechallenge at 5 months.

The ability to generate potent antigen-specific T cell responses by vaccination has been a major hurdle in vaccinology. Vaccinia virus and avipox viruses have been shown to be capable of expressing antigens in mammalian cells and can induce a protective immune response against several mammalian pathogens. We report on two such vaccine constructs, modified vaccinia virus Ankara and FP9 (an attenuated fowlpox virus) both expressing the pre-erythrocytic malaria antigen thrombospondin-related adhesion protein and a string of CD8+ epitopes (ME-TRAP). In prime-boost combinations in a mouse model MVA and FP9 are highly immunogenic and induce substantial protective efficacy. A series of human clinical trials using the recombinant MVA and FP9 malaria vaccines encoding ME-TRAP, both independently and in prime-boost combinations with or without the DNA vaccine DNA ME-TRAP, has shown them to be both immunogenic for CD8+ T cells and capable of inducing protective efficacy. We report here a detailed analysis of the safety profiles of these viral vectors and show that anti-vector antibody responses induced by the vectors are generally low to moderate. We conclude that these vectors are safe and show acceptable side effect profiles for prophylactic vaccination.

Immunological memory is a required component of protective antimalarial responses raised by T cell-inducing vaccines. The magnitude of ex vivo IFN-gamma T cell responses is widely used to identify immunogenic vaccines although this response usually wanes and may disappear within weeks. However, protection in the field is likely to depend on durable central memory T cells that are not detected by this assay. To identify longer-lived memory T cells, PBMC from malaria-naive vaccinated volunteers who had received prime boost vaccinations with a combination of DNA and/or viral vectors encoding the multiepitope string-thrombospondin-related adhesion protein Ag were cultured in vitro with Ag for 10 days before the ELISPOT assay. Ex vivo T cell responses peaked at 7 days after the final immunization and declined substantially over 6 mo, but responses identified after T cell culture increased over the 6-mo period after the final immunization. Moreover, individual cultured ELISPOT responses at the day of challenge time point correlated significantly with degree of protection against malaria sporozoite challenge, whereas ex vivo responses did not, despite a correlation between the peak ex vivo response and magnitude of memory responses 6 mo later. This cultured assay identifies long-lasting protective T cell responses and therefore offers an attractive option for assessments of vaccine immunogenicity.

Malaria is a major global health problem for which an effective vaccine is required urgently. Prime-boost vaccination regimes involving plasmid DNA and recombinant modified vaccinia virus Ankara-encoding liver-stage malaria antigens have been shown to be powerfully immunogenic for T cells and capable of inducing partial protection against experimental malaria challenge in humans, manifested as a delay in time to patent parasitemia. Here, we report that substitution of plasmid DNA as the priming vector with a specific attenuated recombinant fowlpox virus, FP9, vaccine in such prime-boost regimes can elicit complete sterile protection that can last for 20 months. Protection at 20 months was associated with persisting memory but not effector T cell responses. The protective efficacy of various immunization regimes correlated with the magnitude of induced immune responses, supporting the strategy of maximizing durable T cell immunogenicity to develop more effective liver-stage vaccines against Plasmodium falciparum malaria.

Heterologous prime-boost vaccination has been shown to be an efficient way of inducing T cell responses in animals and in humans. We have used three vaccine vectors, naked DNA, modified vaccinia virus Ankara (MVA), and attenuated fowlpox strain, FP9, for prime-boost vaccination approaches against Plasmodium falciparum malaria in humans. In this study, we characterize, using two types of ELISPOT assays and FACS analysis, cell-mediated immune responses induced by different prime-boost combinations where all vectors encode a multiepitope string fused to the pre-erythrocytic Ag thrombospondin-related adhesion protein. We show that these different vectors need to be used in a specific order for an optimal ex vivo IFN-gamma response. From the different combinations, DNA priming followed by MVA boosting and FP9 priming followed by MVA boosting were most immunogenic and in both cases the IFN-gamma response was of broad specificity and cross-reactive against two P. falciparum strains (3D7 and T9/96). Immunization with all three vectors showed no improvement over optimal two vector regimes. Strong ex vivo IFN-gamma responses peaked 1 wk after the booster dose, but cultured ELISPOT assays revealed longer-lasting T cell memory responses for at least 6 mo. In the DNA-primed vaccinees the IFN-gamma response was mainly due to CD4(+) T cells, whereas in the FP9-primed vaccinees it was mainly due to CD4-dependent CD8(+) T cells. This difference may be of importance for the protective efficacy of these vaccination approaches against various diseases.

A retrospective study was performed to evaluate the usefulness of various historical, clinical, and laboratory findings in differentiating acute appendicitis from pelvic inflammatory disease (PID) in women of childbearing age. The records of all female patients presenting to the emergency department with abdominal pain who were found to have histologically proven appendicitis (n = 80) or PID confirmed on endocervical culture (n = 71) were reviewed. Clinically useful indicators favoring appendicitis included the presence of anorexia and the onset of pain later than day 14 of the menstrual cycle. Indicators favoring PID included a history of vaginal discharge, urinary symptoms, prior PID, tenderness outside the right lower quadrant, cervical motion tenderness, vaginal discharge on pelvic examination, and positive urinalysis. Despite these indicators, differentiating acute appendicitis from PID remains difficult.