Evaluation of an Antigen-Antibody “Combination” Enzyme Linked Immunosorbent Assay for Diagnosis of Hepatitis C Virus Infections

Background

Development of “combination” assays detecting in parallel, within a single test, Hepatitis C Virus (HCV) antigens and antibodies, not only reduces the window period in HCV-infection but also costs. Reduction of costs is important for developing countries where money and personal resources are limited.

Methods

We compared the Monolisa® HCV Antigen-Antibody Ultra (Bio-Rad Laboratories Limited, Marnes La Coquette, France) with the AXSYM HCV version 3.0 (Abbot Diagnostics, Germany)-the latter assay detecting only antibodies to HCV. Seventy three HCV-PCR positive and negative samples were tested.

Results

Although the two assays showed comparable results, two samples from a bone marrow transplant (BMT) patient of viral loads 7.8 × 10₅ and 8.9 × 10₆ IU/mL could not be detected by the Monolisa® HCV Antigen-Antibody Ultra assay. Failure to detect the two samples with viral loads considered above threshold of detection for antigen proteins suggested a lack of sensitivity by this assay to discover viral capsid protein in patient samples. Genotyping of these samples revealed genotype 1b, a HCV-subtype which is widespread and should thus be easily detected.

Conclusion

We conclude that although this assay depicts high sensitivity and specificity in detecting antibodies to HCV, it seems not to add further benefit in our study population to detect HCV infections by enhanced sensitivity due the potential contingency to trace viral capsid antigens.     

Surgical repair of the incompetent femoral vein valve.

Incompetent femoral vein valves are identified by reflux femoral phlebography in selected patients with postphlebitic syndrome. In addition to conventional stripping of varices and subfascial interruption of communicating veins, 14 patients with 17 involved extremities have undergone surgical repair of an incompetent valve in the superficial femoral veins. Pathologic condition of the valve consisted of elongation of the cusp edge. Repair was achieved by a series of tucking sutures shortening the cusp and restoring competence to the valve. Healing of ulcers and relief of swelling occurred in 90% of the patients. This rate was maintained up to seven years. These cases demonstrate the feasibility of surgery on the femoral vein valve. The relative importance of conventional stripping, subfascial communicating vein interruption, and femoral valve repair will have to be established by way of further clinical experience.     

West Nile virus and the safety of plasma derivatives: verification of high safety margins,and the validity of predictions based on model virus data

BACKGROUND: During the 2002 West Nile virus (WNV) epidemic in the US, virus transmission through solid organ transplantation and transfusion of blood components was observed. This raised concerns about the safety of plasma derivatives. To verify the safety margins of these products, which were initially shown with a panel of model viruses including some very similar to WNV, the effectiveness of the virus inactivation procedures incorporated into their manufacturing processes was reinvestigated. STUDY DESIGN AND METHODS: An infectivity assay for 1999 New York isolate of WNV was established to investigate virus inactivation steps commonly used during the manufacture of plasma derivatives, such as pasteurization for human albumin, S/D treatment for IVIG and FVIII, vapor heating for FVIII inhibitor-bypassing activity, and incubation at low pH for IVIG. RESULTS: The results show that WNV behaves exactly as had been predicted based on available data for similar model viruses; that is, it is readily inactivated by all the commonly used virus inactivation procedures tested. CONCLUSION: Our investigation verifies the safety margins of plasma derivatives against a potential transmission of WNV and that the model virus concept is valid for predicting the behavior of closely related viruses.     

Bupivacaine-induced cellular entry of QX-314 and its contribution to differential nerve block

Background and Purpose: Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314.Experimental Approach: We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed.Key Results: Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine''s nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine''s blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314.Conclusions and Implications: Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful.     

Venous severity scoring: An adjunct to venous outcome assessment

Some measure of disease severity is needed to properly compare the outcomes of the various approaches to the treatment of chronic venous insufficiency. Comparing the outcomes of two or more different treatments in a clinical trial, or the same treatment in two or more reports from the literature cannot be done with confidence unless the relative severity of the venous disease in each treatment group is known. The CEAP (Clinical-Etiology-Anatomic-Pathophysiologic) system is an excellent classification scheme, but it cannot serve the purpose of venous severity scoring because many of its components are relatively static and others use detailed alphabetical designations. A disease severity scoring scheme needs to be quantifiable, with gradable elements that can change in response to treatment. However, an American Venous Forum committee on venous outcomes assessment has developed a venous severity scoring system based on the best usable elements of the CEAP system. Two scores are proposed. The first is a Venous Clinical Severity Score: nine clinical characteristics of chronic venous disease are graded from 0 to 3 (absent, mild, moderate, severe) with specific criteria to avoid overlap or arbitrary scoring. Zero to three points are added for differences in background conservative therapy (compression and elevation) to produce a 30 point-maximum flat scale. The second is a Venous Segmental Disease Score, which combines the Anatomic and Pathophysiologic components of CEAP. Major venous segments are graded according to presence of reflux and/or obstruction. It is entirely based on venous imaging, primarily duplex scan but also phlebographic findings. This scoring scheme weights 11 venous segments for their relative importance when involved with reflux and/or obstruction, with a maximum score of 10. A third score is simply a modification of the existing CEAP disability score that eliminates reference to work and an 8-hour working day, substituting instead the patient's prior normal activities. These new scoring schemes are intended to complement the current CEAP system.     

A cell culture-derived whole-virus H9N2 vaccine induces high titer antibodies against hemagglutinin and neuraminidase and protects mice from severe lung pathology and weight loss after challenge with a highly virulent H9N2 isolate

Background

Influenza viruses of subtype A/H9N2 are enzootic in poultry across Asia and the Middle East and are considered to have pandemic potential. The development of new vaccine manufacturing technologies is a cornerstone of influenza pandemic preparedness.

Methods

A non-adjuvanted whole-virus H9N2 vaccine was developed using Vero cell culture manufacturing technology. The induction of hemagglutination inhibition (HI) and virus-neutralizing antibodies was assessed in CD1 mice and guinea pigs. A highly sensitive enzyme-linked lectin assay was used to investigate the induction of antibodies capable of inhibiting the enzymatic activity of the H9N2 neuraminidase. Protective efficacy against virus replication in the lung after challenge with the homologous virus was evaluated in BALB/c mice by a TCID₅₀ assay, and prevention of virus replication in the lung and associated pathology were evaluated by histology and immunohistochemistry. To investigate the ability of the vaccine to prevent severe disease, BALB/c mice were challenged with a highly virulent mouse-adapted H9N2 isolate which was generated by multiple lung-to-lung passage of wild-type virus.

Results

The vaccine elicited high titers of functional H9N2-specific HA antibodies in both mice and guinea pigs, as determined by HI and virus neutralization assays. High titer H9N2-specific neuraminidase inhibiting (NAi) antibodies were also induced in both species. Vaccinated mice were protected from lung virus replication in a dose-dependent manner after challenge with the homologous H9N2 virus. Immunohistochemical analyses confirmed the lack of virus replication in the lung and an associated substantial reduction in lung pathology. Dose-dependent protection from severe weight loss was also provided after challenge with the highly virulent mouse-adapted H9N2 virus.

Conclusions

The induction of high titers of H9N2-specific HI, virus-neutralizing and NAi antibodies and dose-dependent protection from virus replication and severe disease in animal models suggest that the Vero cell culture-derived whole-virus vaccine will provide an effective intervention in the event of a H9N2 pandemic situation.     

A non-adjuvanted whole-virus H1N1 pandemic vaccine is well tolerated and highly immunogenic in children and adolescents and induces substantial immunological memory

This phase 1/2 open-label, randomized clinical study investigated the safety and immunogenicity of a non-adjuvanted, whole virus, Vero cell-derived H1N1 pandemic influenza vaccine (A/H1N1/California/07/2009) in children and adolescents (6 months to 17 years). Subjects were stratified by age (6–11 months, 12–35 months, 3–8 years, 9–17 years) to receive two vaccinations 21 days apart of either the 3.75 μg or 7.5 μg dose. A booster with a licensed trivalent seasonal (2010/2011) influenza vaccine was administered one year after the first vaccination to a subgroup that had previously received the 7.5 μg dose.     

A cell culture-derived whole-virus H5N1 vaccine induces long-lasting cross-clade protective immunity in mice which is augmented by a homologous or heterologous booster vaccination

Background

Preparation for an H5N1 influenza pandemic in humans could include priming the population in the pre-pandemic period with a vaccine produced from an existing H5N1 vaccine strain, with the possibility of boosting with a pandemic virus vaccine when it becomes available. We investigated the longevity of the immune response after one or two priming immunizations with a whole-virus H5N1 vaccine and the extent to which this can be boosted by later immunization with either a homologous or heterologous vaccine.

Methods

Mice received one or two priming immunizations with a Vero cell culture-derived, whole-virus clade 1 H5N1 vaccine formulated to contain either 750 ng or 30 ng hemagglutinin. Six months after the first priming immunization, mice received either a booster immunization with the same clade 1 vaccine or a heterologous clade 2.1 vaccine, or buffer. Humoral and cellular immune responses were evaluated before and at regular intervals after immunizations. Three weeks after booster immunization, mice were challenged with a lethal dose of wild-type H5N1 virus from clades 1, 2.1 or 2.2 and survival was monitored for 14 days.

Results

One or two priming immunizations with the 750 ng or 30 ng HA formulations, respectively, induced H5N1-neutralizing antibody titers which were maintained for ≥6 months and provided long-term cross-clade protection against wild-type virus challenge. Both humoral and cellular immune responses were substantially increased by a booster immunization after 6 months. The broadest protective immunity was provided by an immunization regimen consisting of one or two priming immunizations with a clade 1 vaccine and a boosting immunization with a clade 2.1 vaccine.

Conclusions

These data support the concept that pre-pandemic vaccination can provide robust and long-lasting H5N1 immunity which could be effectively boosted by immunization either with another pre-pandemic vaccine or with the pandemic strain vaccine.