Reliability of CE-marked NATs for HIV-1 subtype C detection and quantitation.

We compared seven CE-marked HIV-1 RNA nucleic acid amplification technology (NAT) based assays for their detection efficiency and quantitation concordance in regard to HIV-1 subtype C. We used 398 plasma samples from South African repeat blood donors identified as HIV positive at occasion of routine screening NAT performed mainly during the years 2010-2013, with most plasma samples reflecting recent HIV-1 infections. All HIV-1 subtype C specimens were detected, independent of mono- or dual-target assay design. In the same time period new variants of HIV-1 subtype B had been identified which were missed by some mono-target assays, a finding which was not corroborated for subtype C in our study. A high level of concordance of HIV-1 subtype C quantitation was determined for the HIV-1 NATs, showing successful standardization in this diagnostic field.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains.

BACKGROUND AND OBJECTIVES: Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the 'repository'), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. MATERIALS AND METHODS: Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10-25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. RESULTS: Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. CONCLUSION: The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

Minor allergen patterns in birch pollen allergen products-A question of pollen?

BACKGROUND: Contrary to the scientific differentiation between major and minor allergens, the regulatory framework controlling allergen products in the EU distinguishes relevant and non-relevant allergens. Given the lack of knowledge on their clinical relevance, minor allergens are usually not controlled by allergen product specifications. Especially, in birch pollen (BP) allergen products, minor allergens are commonly disregarded. OBJECTIVES: To quantify three minor allergens in BP allergen products from different manufacturers and to assess the influence of the utilized BP on minor allergen patterns. METHODS: Apart from common quality parameters such as Bet v 1 content, Bet v 4, Bet v 6 and Bet v 7 were quantified in 70 BP allergen product batches from six manufacturers, using ELISA systems developed in-house. Batch-to-batch variability was checked for agreement with a variability margin of 50%-200% from mean of the given batches for individual allergen content. Subsequently, minor allergen patterns were generated via multidimensional scaling and related to information on the pollen lots used in production of the respective product batches. RESULTS: Like the already established Bet v 4 ELISA, the ELISA systems for quantification of Bet v 6 and Bet v 7 were successfully validated. Differences in minor allergen content between products and batch-to-batch consistency were observed. Correlations between minor and major allergen content were low to moderate. About 20% of batches exceeded the variability margin for at least one minor allergen. Interestingly, these fluctuations could not in all cases be linked to the use of certain BP lots. CONCLUSIONS AND CLINICAL RELEVANCE: The impact of the observed minor allergen variability on safety and efficacy of BP allergen products can currently not be estimated. As the described differences could only in few cases be related to the used pollen lots, it is evident that additional factors influence minor allergens in BP allergen products.

Collaborative study for the establishment of the Ph. Eur. Hepatitis E virus RNA for NAT testing biological reference preparation batch 1.

A new European Pharmacopoeia (Ph. Eur.) biological reference preparation (BRP) had to be established further to the decision to include nucleic acid testing (NAT) for the detection of hepatitis E virus (HEV) RNA in the monograph Human plasma (pooled and treated for virus inactivation) (1646). To this purpose, an international collaborative study was launched in the framework of the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the Commission of the European Union (EU). The study was run in conjunction with the establishment of the 1st World Health Organization (WHO) international reference panel (IRP) for hepatitis E virus RNA genotypes (8578/13). Twenty-three laboratories used in-house developed and commercially available assays to calibrate a lyophilised candidate BRP prepared from a HEV 3f strain positive human plasma against the 1st WHO International Standard (IS) for HEV RNA (6329/10). Results from quantitative and qualitative assays were in good agreement and were combined to calculate an assigned potency. Real-time stability studies indicated that the candidate BRP is very stable at lower temperatures and is thus suitable for long-term use. Based on these results, in February 2016, the Ph. Eur. Commission adopted the candidate material as the hepatitis E virus RNA for NAT testing BRP batch 1, with an assigned unitage of 2.1 × 104 IU/vial (4.32 log10 IU/vial).

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

[Overweight in primary school-age children. Prevalence and risk factors]. / Gewichtsentwicklung im frühen Grundschulalter. Prävalenz, Inzidenz und Risikofaktoren für Übergewicht und Adipositas.

BACKGROUND: Various studies show that pre-school age is a sensitive period for the development of overweight and obesity. During a longitudinal study between 2010 and 2013, the municipal health authority (city of Frankfurt) in cooperation with the university children's hospital investigated the development of weight in children aged 5 to 8. MATERIALS AND METHODS: The weight and height of a collective of 5720 children were measured (2010/11). In addition, nutritional and exercise habits, as well as media consumption was documented for 4758 children through a questionnaire during the school enrolment procedure. The weight and height of 3481 children were measured again in the second grade (2012/13). RESULTS: Over a period of 24 months, the percentage of overweight (not obese) children increased from 7.5 to 9.4 % and that of obese children from 4.5 to 5.0 %. 164 of 2818 children with a normal initial weight (5.8 %) changed to percentile class overweight or obese. 79 of 260 children who were initially overweight, not obese (30 %), changed to the group of normal weight, but only 4 out of 156 obese children (3 %). Increased TV consumption (> 1 h per day), availability of their own television, lack of physical activity, and consumption of high-calorie drinks were risk factors for the development of overweight during the primary school age. 72 % of parents of overweight children and 22 % of obese children falsely classified their children as normal weight. CONCLUSIONS: Targeted education about the risk of obesity in the primary school age and offers for early intervention should be established in the healthcare services concerned.

IgE recognition patterns in peanut allergy are age dependent: perspectives of the EuroPrevall study.

BACKGROUND: We tested the hypothesis that specific molecular sensitization patterns correlate with the clinical data/manifestation in a European peanut-allergic population characterized under a common protocol. METHODS: Sixty-eight peanut-allergic subjects and 82 tolerant controls from 11 European countries were included. Allergy to peanut and lowest symptom-eliciting dose was established by double-blind placebo-controlled food challenge in all but anaphylactic subjects. Information of early or late (before or after 14 years of age) onset of peanut allergy was obtained from standardized questionnaires. IgE to peanut allergens rAra h 1-3, 6, 8-9, profilin and CCD was determined using ImmunoCAP. RESULTS: Seventy-eight percent of peanut allergics were sensitized to peanut extract and 90% to at least one peanut component. rAra h 2 was the sole major allergen for the peanut-allergic population. Geographical differences were observed for rAra h 8 and rAra h 9, which were major allergens for central/western and southern Europeans, respectively. Sensitization to rAra h 1 and 2 was exclusively observed in early-onset peanut allergy. Peanut-tolerant subjects were frequently sensitized to rAra h 8 or 9 but not to storage proteins. Sensitization to Ara h 2 ≥ 1.0 kUA /l conferred a 97% probability for a systemic reaction (P = 0.0002). Logistic regression revealed a significant influence of peanut extract sensitization and region on the occurrence of systemic reactions (P = 0.0185 and P = 0.0436, respectively). CONCLUSION: Sensitization to Ara h 1, 2 and 3 is usually acquired in childhood. IgE to Ara h 2 ≥ 1.0 kUA /l is significantly associated with the development of systemic reactions to peanut.

Performance of hepatitis B surface antigen tests with the first WHO international hepatitis B virus genotype reference panel.

BACKGROUND: Standardization of hepatitis B surface antigen (HBsAg) tests is indispensable for consistent quality and comparability. Ideally, the assays should detect all known hepatitis B virus (HBV) genotypes equally well. OBJECTIVE: Development of an HBV genotype reference panel for HBsAg assays representing the most prevalent HBV subgenotypes to address commutability and traceability of the heat-inactivated 2nd WHO International Standard (IS) for HBsAg in relation to native HBsAg and to HBV genotypes. STUDY DESIGN: An HBV panel of 15 non-inactivated lyophilized specimens representing the subgenotypes A1, A2, B1, B2, C2, D1-D3, E, F2, and H was evaluated in parallel to the IS by 15 laboratories using 19 different HBsAg tests and tree unitages. The virus content of the samples was reduced by ultracentrifugation and dilution to <2×10(4) IU HBV DNA/mL. RESULTS: Twenty-two qualitative and 6 quantitative data sets were evaluated. Overall, the results demonstrated consistent detection of HBV genotypes by the majority of tests with a mean potency variability relative to the IS of 36%. Some assays showed significant genotype-dependent differences in analytical sensitivity. Some tests were more sensitive with the IS, others less. On average, one IU HBsAg corresponded to 0.88±0.20 ng HBsAg protein. CONCLUSIONS: The panel was accepted by the WHO as the "1st International Reference Panel for HBV genotypes for HBsAg-based assays". The panel is a helpful complementation to the IS to validate HBV genotype specific analytical test sensitivities.

Thromboembolic events associated with immunoglobulin treatment.

OBJECTIVE: Due to an increasing number of reported thromboembolic events (TEE) after the administration of one intravenous immunoglobulin (IVIG) and one subcutaneous immunoglobulin (SCIG), pharmacovigilance and laboratory data were collected to analyse the root cause and assess the reporting frequency of TEEs for various IG products. METHODS: Paul-Ehrlich-Institut retrospectively analysed 228 reports of TEEs associated with six different IG products and estimated annual TEE-reporting rates based on worldwide sale figures over a period of 6 years (2006-2011). In addition, non-activated partial thromboplastin time (NAPTT) testing was performed to capture pro-coagulant potential of six IG products (four IVIG and two SCIG). RESULTS: For three IVIGs, the drug-related TEE-reporting rates remained stable from 2006 to 2011 (0-0·83 cases per 1000 kg IVIG distributed). In contrast, the TEE rate of one IVIG increased significantly from 0·33 cases in 2006 to nearly nine cases in 2010 (P < 0·001). The NAPTT testing of IG products with a low TEE rate revealed a NAPTT time >200 s and a NAPTT ratio >0·8, whereas TEE-associated batches of IG products with an increased TEE rate had a NAPTT ratio <0·8. After modifications of manufacturing processes, a normalization of NAPTT results and a decrease in TEE rates could be demonstrated.

First WHO International Reference Panel containing hepatitis B virus genotypes A-G for assays of the viral DNA.

BACKGROUND: WHO International Standards (IS) are provided for the calibration and validation of diagnostic and screening assays, e.g. for hepatitis B virus (HBV). HBV forms numerous subgenotypes and the current IS for HBV DNA reflects subgenotype A2. OBJECTIVE: A reference panel with the most prevalent subgenotypes should facilitate evaluation of genotype-specific detection efficiencies. STUDY DESIGN: 215 HBV positive plasma samples collected worldwide were characterized for HBV markers and sequenced. Fifteen subgenotype A1, A2, B2, B4, C2, D1, D3, E, F2 and G samples were selected for the panel. The lyophilized samples were tested in parallel with the IS in an international collaborative study with 16 laboratories using 13 different nucleic acid amplification techniques (NATs). RESULTS: Eight of 13 NAT had a HBV DNA detection efficiency which was independent of the genotype and consistent with the IS, while with five assays, certain deviations were noted, particularly with genotype F which was under quantitated or even missed by three assays. The panel was accepted by the WHO as the "1st WHO International Reference Panel for HBV Genotypes for HBV NAT-Based Assays". CONCLUSIONS: The evaluation of HBV DNA assays should include many different genotypes. The WHO Reference Panel is universally available for manufacturers of HBV DNA assays, diagnostic laboratories and control authorities to facilitate standardized validation of HBV genotype specific detection efficiency of both diagnostic (quantitative and qualitative) and screening NAT assays.

Component-resolved in vitro diagnosis of carrot allergy in three different regions of Europe.

BACKGROUND: Carrot is a frequent cause of food allergy in Europe. The objective of this study was to evaluate a panel of carrot allergens for diagnosis of carrot allergy in Spain, Switzerland and Denmark. METHODS: Forty-nine carrot allergic patients, 71 pollen allergic but carrot-tolerant patients and 63 nonatopic controls were included. Serum IgE to carrot extract, recombinant carrot allergens (rDau c 1.0104; rDau c 1.0201; rDau c 4; the isoflavone reductase-like proteins rDau c IFR 1, rDau c IFR 2; the carrot cyclophilin rDau c Cyc) were analyzed by ImmunoCAP. RESULTS: The sensitivity of the carrot extract-based test was 82%. Use of the recombinant allergens increased the sensitivity to 90%. The Dau c 1 isoforms were major allergens for Swiss and Danish carrot allergic patients, the profilin rDau c 4 for the Spanish patients. The rDau c IFR 1 and rDau c IFR 2 were recognized by 6% and 20% of the carrot allergics, but did not contribute to a further increase of sensitivity. Among pollen allergic controls, 34% had IgE to carrot extract, 18% to each of rDau c 1.0104, rDau c 1.0201 and rDau c 4, 8% to rDau c IFR 1 and 7% to rDau c IFR 2. Sensitization to rDau c Cyc occurred in one carrot allergic patient and one nonatopic control. CONCLUSION: Component-resolved in vitro analyses revealed a significant difference in IgE sensitization pattern between geographical regions and in the prevalence of sensitization to carrot components between carrot allergic and carrot-tolerant but pollen sensitized patients.

Benefit of transfusion-related acute lung injury risk-minimization measures--German haemovigilance data (2006-2010).

OBJECTIVE: Based on the frequency of immune-mediated and non-immune-mediated transfusion-related acute lung injury (TRALI), the effect of risk-minimization measures was evaluated during a period of 5 years (2006-2010). Risk-minimization measures were implemented in 2008/2009, consisting of exclusion of female donors with a history of pregnancy or exclusion of female donors with human leucocyte antigen (HLA)/human neutrophil alloantigen (HNA) antibodies. METHODS: TRALI was confirmed according to the criteria of the International Haemovigilance Network. Based upon the results of donor testing of white-blood-cell antibodies (WBC-Ab) against HLA or HNAs, confirmed cases were classified as immune- or non-immune-mediated TRALI. Reporting rates were calculated on the basis of the annually transfused blood components, and pre- and post-implementation periods were compared. RESULTS: In total, 60 immune-mediated (75%) and 20 non-immune-mediated (25%) TRALI reactions were confirmed. A total of 68 (64 women and four men) donors were involved: seven red-blood-cell concentrates donors (13%), six platelet concentrate donors (10%), and 48 fresh frozen plasma (FFP) donors (77%). The reporting rate of immune-mediated TRALI caused by FFP decreased continuously; from 12·71 per million units in 2006/2007 to 6·81 per million units in 2008/2009 and no case in 2010. CONCLUSION: The comparison of the pre- and the post-implementation period demonstrated a significantly reduced risk of TRALI events comparing 2006/2007 with 2010 (P-value: <0·01). Furthermore, no case of TRALI-induced fatality occurred after the implementation of risk-minimization measures.

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. MATERIAL AND METHODS: Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. RESULTS: Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. CONCLUSION: The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

The rapid fluorescent focus inhibition test is a suitable method for batch potency testing of inactivated rabies vaccines.

The European Pharmacopoeia proposes two methods for potency determination of inactivated rabies vaccines for veterinary use: The first one is a classical mouse challenge test, which is imprecise, time-consuming, and causes severe distress to the test animals. Alternatively, the potency may be determined serologically by measuring the neutralizing antibody titers induced after vaccination of mice by using a rapid fluorescent focus inhibition test (RFFIT). Although this method is faster and less painful for the animals, it is not widely used yet, and only little data exist concerning the comparability of both methods. We have therefore performed a comparative study, in which we demonstrated a good correlation between the challenge test results and the mean titers determined by RFFIT. Furthermore, all vaccine batches failing the challenge test were also recognized as insufficient in the serological assay. This publication further describes the influence of different vaccine administration routes on the resulting antibody titers, and it proposes various modifications to the serological assay protocol which could improve its overall practicability. Finally, we recommend that the serological assay be used for the potency testing of inactivated rabies vaccines.

Disease progression in HIV-1 infected children and adolescents--results of a German-Austrian cohort study.

OBJECTIVE: The Paediatric Working Group AIDS (PAAD) initiated a prospective cohort study in order to investigate disease progression in HIV- infected children and adolescents and the effect of antiretroviral treatment regimes. PATIENTS AND METHODS: Between 1998 and 2003, paediatric centres documented HIV-infected patients under clinical care using a questionnaire for basic data and annual follow up. Main outcome measures were: use of antiretroviral therapy, adverse events, disease progression and change of therapeutic regimes. RESULTS: 174 HIV- infected paediatric patients were followed up in 12 centres in Germany and Austria between 1998 and 2003. Initially 54 (31%) patients had no antiretroviral therapy, 35 (20%) received a two-drug regimen (ART) and 85 patients (49%) a highly active antiretroviral therapy (HAART>or=3 drugs). After an observation period of 5 years, 8 patients (4%) had no therapy, 17 (10%) were on ART and 134 patients on HAART (77%). The number of patients with salvage therapy (>or=4 drugs) increased from 5 (3%) to 15 patients (9%). 72 of 166 treated patients (43%) had no change of their drug regimes, 68 patients (41%) had one change and 26 patients (16%)>or=2 changes. Main reasons for changes were increased viral load (49%), immunologic deterioration (21%) and adverse events (14%). During the follow up period no patient died. According to the CDC classification, disease progression was seen in 48 of 174 patients (28%), of whom 20 had deteriorations of clinical categories (A, B, C) and 28 of immunologic categories. Using Kaplan-Meier curves, the mean time from study onset until change of clinical categories was 61 months for patients on HAART, 26 months for patients on ART and 14 months for patients without ART. CONCLUSION: In paediatric patients with HIV infection, disease progression has declined substantially by introduction of HAART. Superiority of HAART compared with ART was demonstrated. Non-adherence as well as other reasons for treatment failure have to be studied more carefully.

Antitumour effects in mycosis fungoides of the immunomodulatory, tellurium-based compound, AS101.

BACKGROUND: The immunomodulator AS101 [ammonium trichloro (dioxoethylene-O,O') tellurate], a nontoxic tellurium (IV) compound, has antitumoral effects which were demonstrated in several preclinical and clinical studies. OBJECTIVES: To investigate the antitumour activity of AS101 on cutaneous T-cell lymphoma (CTCL), of which mycosis fungoides (MF) is the most frequent disease variant. METHODS: We used a newly established mouse xenograft model for MF to test the effect of AS101 in vivo and analysed apoptosis induction in vitro. RESULTS: When injected intratumorally, AS101 delayed tumour growth in a dose-dependent manner. In vitro, AS101 induced a dose-dependent G2/M arrest in the CTCL cell lines Hut78 and MyLa. Moreover, higher concentrations of AS101 induced apoptosis in MyLa cells. Programmed cell death was associated with the loss of mitochondrial transmembrane potential and activation of caspase 9 and caspase 3. AS101 also elevated intracellular reactive oxygen species (ROS) production; the antioxidant, Mn superoxide dismutase, significantly reduced the degree of apoptosis, suggesting that ROS play a key role in apoptosis induction. CONCLUSIONS: These findings indicate that AS101 may be a promising antitumour drug for CTCL.