Development of the 1st International Reference Panel for HIV-1 RNA genotypes for use in nucleic acid-based techniques.

Twenty-eight laboratories from 16 countries participated in a collaborative study to evaluate an HIV-1 RNA Genotype Reference Panel for use with nucleic acid-based tests (NAT). The Reference Panel consisted of 11 coded samples representing different HIV-1 genotypes (subtypes A-D, AE, F, G, AA-GH, groups N and O) as well as a negative diluent control. Each laboratory assayed the eleven panel members concurrently with the 1st International Standard for HIV-1 RNA (NIBSC Code 97/656) on at least three separate occasions and the data collated and analysed at NIBSC. Twenty-nine sets of data from NAT were received, 19 from quantitative and 10 from qualitative assays, with six different commercial assays and five "in-house" assays represented. The results showed that viruses from subtypes A-D and recombinant virus AE [CRF01_AE] were detected consistently, but that some assays had difficulty with the detection and quantification of viruses from subtypes F and G, a mixed recombinant virus AA-GH and a representative of group N. Furthermore, most assays failed to detect the group O representative. The study illustrated the limitations of some molecular assays particularly in detection of certain non-B genotypes which are important viruses in the global AIDS pandemic and illustrated the value of a well-characterised genotype panel. The panel has been established by the World Health Organisation's Expert Committee on Biological Standardisation as the 1st International Reference Panel HIV-1 RNA Genotypes (code 01/466).

Clostridium infections associated with musculoskeletal-tissue allografts.

BACKGROUND: Allografts are commonly used in orthopedic reconstructive surgery. In 2001, approximately 875,000 musculoskeletal allografts were distributed by U.S. tissue banks. After the death from Clostridium sordellii sepsis of a 23-year-old man who had received a contaminated allograft from a tissue bank (Tissue Bank A), the Centers for Disease Control and Prevention initiated an investigation, including enhanced case finding, of the methods used for the recovery, processing, and testing of tissue. METHODS: A case of allograft-associated clostridium infection was defined as a culture-proven infection of a surgical site within one year after allograft implantation, from January 1998 to March 2002. We traced tissues to tissue banks that recovered and processed these tissues. We also estimated the rates of and risk ratios for clostridium infections for tissues processed by the implicated tissue bank and reviewed processing and testing methods used by various tissue banks. RESULTS: Fourteen patients were identified, all of whom had received allografts processed by Tissue Bank A. The rates of clostridium infection were 0.12 percent among patients who received sports-medicine tissues (i.e., tendons, femoral condyles, menisci) from Tissue Bank A and 0.36 percent among those who received femoral condyles in particular. The risk-ratio estimates for clostridium infections from tissues processed by Tissue Bank A, as compared with those from other tissue banks, were infinite (P<0.001) for musculoskeletal allografts, sports-medicine tissues, or tendons. Because Tissue Bank A cultured tissues only after treating them with a nonsporicidal antimicrobial solution, some test results were probably false negatives. Tissues from implicated donors were released despite the isolation of clostridium or bowel flora from other anatomical sites or reports of infections in other recipients. CONCLUSIONS: Clostridium infections were traced to allograft implantation. We provide interim recommendations to enhance tissue-transplantation safety. Tissue banks should validate processes and culture methods. Sterilization methods that do not adversely affect the functioning of transplanted tissue are needed to prevent allograft-related infections.

The importance of standardisation of laboratory evaluations in HIV vaccine trials.

It is important to evaluate HIV-1-specific immunological responses elicited by therapeutic or prophylactic vaccines using precise, standardised assays, so that the immunogenicity and putative efficacy of candidate vaccines may be compared. Different well-validated assays must be used to quantitate specific responses, to determine which particular strategies may be efficacious.

Detection and quantitation of human immunodeficiency virus type-1 particles by confocal microscopy.

A method is described to visualise directly human immunodeficiency virus type-1 (HIV-1) particles. HIV-1 containing samples were adsorbed onto a plastic surface and doubly labeled with antibodies specific for viral proteins and sensitive nucleic acids dyes. Laser scanning confocal microscopy detected co-localization of viral proteins and nucleic acids, thus allowing specific identification of HIV. Using this technique, we have quantified eight different HIV-1 sub-types and three HIV-1 groups in tissue culture supernatants from infected peripheral blood mononuclear cells (PBMCs). Confocal counts correlated well with electron microscopy (EM) counts and HIV-1 RNA loads as determined by quantitative PCR. Confocal microscopy may prove to be a simple alternative to electron microscopy for virus identification and quantitation.

Calibration of HIV-1 working reagents for nucleic acid amplification techniques against the 1st international standard for HIV-1 RNA.

Many laboratories use working reagents/run controls to monitor the performance of their nucleic acid amplification techniques (NAT) for the measurement of HIV-1 RNA. A collaborative study was carried out in order to calibrate seven internationally available working reagents, QC105 (National Serology Reference Laboratory [NRL], Australia), B5 and B10 (Center for Biological Evaluation and Research [CBER], USA), Pelispy (Central Laboratory of the Netherlands Blood Transfusion Service [CLB], The Netherlands), PWS-1 and PWS-3 (National Institute for Biological Standards and Control [NIBSC], UK) and IRC (Virology Networks [VN], The Netherlands) against the 1st International Standard for HIV-1 RNA (code 97/656). Twenty-one laboratories from 12 different countries participated in the collaborative study and from the results it was determined that QC105 contained 4.0 log(10) International Units (IU)/ml, B5 2.2 log(10) IU/ml, B10 3.8 log(10) IU/ml, Pelispy 4.4 log(10) IU/ml, PWS-1 3.6 log(10) IU/ml, PWS-3 2.7 log(10) IU/ml and IRC 4.3 log(10) IU/ml. The seven working reagents calibrated in this international study may be used to validate and standardise the large number of qualitative and quantitative, commercial and in-house NAT assays that are currently being applied in the fields of blood safety and patient management. They will also help laboratories to comply with the sensitivity requirements that may be brought in by the regulatory authorities and may contribute to further harmonisation of guidelines on NAT published by organisations such as the European Medicines Evaluation Agency (EMEA), Paul-Ehrlich Institute and CBER, FDA.

A new antigen scanning strategy for monitoring HIV-1 specific T-cell immune responses.

Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensitive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast array of database information. This new system, termed Variable Overlapping Peptide Scanning Design (VOPSD), was used for preparing two peptide sets encompassing the candidate HIV-1 vaccine antigens Tat and Nef. Validation of the VOPSD strategy was obtained by direct comparison with 15mer or 20mer peptide sets in a trial involving six laboratories of the AVIP consortium. Cross-reactive background responses were measured in 80 HIV seronegative donors (HIV-), while sensitivity and magnitude of Tat and Nef-specific T-cell responses were assessed on 90 HIV+ individuals. In HIV-, VOPSD peptides generated background responses comparable with those of the standard sets. In HIV-1+ individuals the VOPSD pools showed a higher sensitivity in detecting individual responses (Tat VOPSD vs. Tat 15mers or 20mers: p≤0.01) as well as in generating stronger responses (Nef VOPSD vs. Nef 20mers: p<0.001) than standard sets, enhancing both CD4 and CD8 T-cell responses. Moreover, this peptide design allowed a marked reduction of the peptides number, representing a powerful tool for investigating novel HIV-1 candidate vaccine antigens in cohorts of HIV-seronegative and seropositive individuals.

Preparation and evaluation of the 1st international standard for the quantitation of HIV-2 RNA in plasma.

An international standard for the quantitation of HIV-2 RNA in plasma samples was developed. A collaborative study involving 29 laboratories from 15 countries was carried out in order to evaluate HIV-2 RNA candidate materials for use with nucleic acid-based tests (NATs). Candidate reference standards consisted of duplicate copies of two HIV-2 genotype A viruses, HIV-2 CAM2 and HIV-2 ROD and were coded S1-S4. Each laboratory assayed all four candidates on at least three separate occasions and data were collated and analysed at NIBSC. Of the data sets returned the majority were from qualitative assays. All assays detected both candidate standards with the exception of one commercial assay, the Nuclisens Easy Q, which was designed primarily for HIV-1 detection which did not detect HIV-2 CAM2 but showed good detection of HIV-2 ROD. This highlighted possible cross reactivity with HIV-2 ROD with some NAT primer/probe combinations; as a result the HIV-2 CAM2 material was established as the 1st international standard for HIV-2 RNA with an assigned unitage of 1000 International Units (IU) per ampoule and is available upon request from the National Institute for Biological Standardisation and Control (NIBSC) (www.nibsc.ac.uk).

Development of a World Health Organisation International Reference Panel for Anti-HIV.

In response to a recommendation made by the "World Health Organisation (WHO) Working Group on Reference Preparations for Testing HBsAg, Anti-HCV and Anti-HIV Diagnostic Kits", a reference panel for anti-HIV consisting of plasma samples representing the major groups and subtypes of HIV has been prepared. The panel consists of solvent-detergent treated anti-HIV-positive human plasma samples that have been diluted 1 in 40 in anti-HIV-negative human serum and freeze-dried and the anti-HIV-positive plasma samples were derived from individuals infected with HIV-1 group M subtypes A, B, C and CRF01_AE, HIV-1 group O and HIV-2. Fifteen laboratories from around the world took part in a collaborative study to evaluate the reference panel for anti-HIV and were requested to test the panel in as wide a range of assays as possible. Where appropriate, serial dilutions were performed and samples tested around their end-points to facilitate the comparison of analytical sensitivity between assays. For qualitative assays such as Western blots and rapid assays, the panel was tested undiluted. Results show that the HIV-negative serum sample was negative in all assays (except for a small number of Western blot assays) and that all HIV-positive samples were detected in all assays, with the exception of an anti-HIV-2 EIA that did not detect most HIV-1 samples and a small number of assays that failed to detect the group O sample. Considerable variability was seen in the end-point titres obtained with the various assays. A report on the study was submitted to the WHO Expert Committee on Biological Standardisation (ECBS) and the panel established as the 1st International Reference Panel for Anti-HIV (code 02/210); a unitage was not assigned to the panel members.

International network for comparison of HIV neutralization assays: the NeutNet report.

BACKGROUND: Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. METHODS: Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. FINDINGS: PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. CONCLUSIONS: The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation.

Mitochondrial DNA and retroviral RNA analyses of archival oral polio vaccine (OPV CHAT) materials: evidence of macaque nuclear sequences confirms substrate identity.

Inoculation of live experimental oral poliovirus vaccines (OPV CHAT) during the 1950s in central Africa has been proposed to account for the introduction of HIV into human populations. For this to have occurred, it would have been necessary for chimpanzee rather than macaque kidney epithelial cells to have been included in the preparation of early OPV materials. Theoretically, this could have led to contamination with a progenitor of HIV-1 derived from a related simian immunodeficiency virus of chimpanzees (SIVCPZ). In this article we present further detailed analyses of two samples of OPV, CHAT 10A-11 and CHAT 6039/Yugo, which were used in early human trials of poliovirus vaccination. Recovery of poliovirus by culture techniques confirmed the biological viability of the vaccines and sequence analysis of poliovirus RNA specifically identified the presence of the CHAT strain. Independent nested sets of oligonucleotide primers specific for HIV-1/SIVCPZ and HIV-2/SIVMAC/SIVSM phylogenetic lineages, respectively, indicated no evidence of HIV/SIV RNA in either vaccine preparation, at a sensitivity of 100 RNA equivalents/ml. Analysis of cellular substrate by the amplification of two distinct regions of mitochondrial DNA (D-loop control region and 12S ribosomal sequences) revealed no evidence of chimpanzee cellular sequences. However, this approach positively identified rhesus and cynomolgus macaque DNA for the CHAT 10A-11 and CHAT 6039/Yugo vaccine preparations, respectively. Analysis of multiple clones of mtDNA 12S rDNA indicated a relatively high number of nuclear mitochondrial DNA sequences (numts) in the CHAT 10A-11 material, but confirmed the macaque origin of cellular substrate used in vaccine preparation. These data reinforce earlier findings on this topic providing no evidence to support the contention that poliovirus vaccination was responsible for the introduction of HIV into humans and sparking the AIDS pandemic.

Surface sampling methods for Bacillus anthracis spore contamination.

During an investigation conducted December 17-20, 2001, we collected environmental samples from a U.S. postal facility in Washington, D.C., known to be extensively contaminated with Bacillus anthracis spores. Because methods for collecting and analyzing B. anthracis spores have not yet been validated, our objective was to compare the relative effectiveness of sampling methods used for collecting spores from contaminated surfaces. Comparison of wipe, wet and dry swab, and HEPA vacuum sock samples on nonporous surfaces indicated good agreement between results with HEPA vacuum and wipe samples. However, results from HEPA vacuum sock and wipe samples agreed poorly with the swab samples. Dry swabs failed to detect spores >75% of the time when they were detected by wipe and HEPA vacuum samples. Wipe samples collected after HEPA vacuum samples and HEPA vacuum samples collected after wipe samples indicated that neither method completely removed spores from the sampled surfaces.