Sherwin Lee

Evaluation of indeterminate c22-3 reactivity in volunteer blood donors

Background: Approximately 25 percent of blood donor sera that are repeatably reactive for hepatitis C virus (HCV) on second‐generation enzyme immunoassay (EIA 2.0) are indeterminate on second‐generation recombinant immunoblot assay (RIBA 2.0), and over 76 percent of these results are due to single reactivity to the HCV recombinant antigen c22‐ 3.

A pattern of 5-1-1 and c100-3 only on hepatitis C virus (HCV) : recombinant immunoblot assay does not reflect HCV infection in blood donors

Current criteria for a reactive (positive) interpretation on hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) require > or = 1+ reactivity to at least two of the four HCV antigens present in the assay. Given that 5‐1‐1 is a subcomponent of c100‐3, there is concern that donor samples reacting only with these two antigens (and not with c22‐3 or c33c) could be incorrectly classified as positive on the basis of limited reactivity to only one HCV gene product. It is determined that 0.23 to 0.44 percent of HCV enzyme immunoassay‐repeatably reactive donor sera demonstrate a pattern of 5‐1‐1 and c100‐3 only on RIBA. Evaluation of six such donor sera using peptide enzyme immunoassays spanning the c100‐3 antigen showed highly restricted reactivity to the 5‐1‐1 N‐terminal region of c100‐3, in contrast to broad 5‐1‐1 and c100‐ 3 C‐terminal peptide reactivity observed in the majority of donor sera with other positive RIBA patterns. HCV polymerase chain reaction and follow‐up serologic evaluations of four of these donors indicated the absence of viremia or evolving seroconversion in all cases. It is concluded that, in the blood donor setting, a pattern of only 5‐1‐1 and c100‐3 reactivity is typically not indicative of HCV infection. To avoid overinterpretation, it is recommended that RIBA grading criteria be revised to require reactivity to two or more HCV‐encoded gene products.

Novel approach for differential diagnosis of HIV infections in the face of vaccine-generated antibodies : Utility for detection of diverse HIV-1 subtypes

Summary:Because increasing numbers of HIV vaccine candidates are being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Most of the currently tested vaccines contain multiple viral components. As a result, many vaccine recipients give positive results in FDA-licensed HIV serodetection tests. We have identified conserved sequences in Env-gp41 and Gag-p6, which are recognized soon after infection but are not included in most HIV vaccine candidates. A new HIV serodetection assay, the HIV-SELECTEST, was established that distinguishes between vaccine-induced antibodies and seroconversion due to true HIV infections. It is important to make this assay globally relevant, because many clinical trials are conducted around the world where most HIV infections are due to non-B subtype HIV-1. Therefore, the current study examined the reactivity of plasma samples from >3000 infections with diverse HIV subtypes worldwide. The HIV-SELECTEST performed at >99% specificity and sensitivity. Both recent and established infections with clades A, B, C, D, E, F, G, J, and CRFs were detected. Antibodies elicited by other vaccinations or infections endemic to the clinical trial sites did not react in this assay. Therefore, HIV-SELECTEST could be an important differential diagnostic tool for HIV vaccine trials, blood banks, and population screening worldwide.

Performance of second- and third-generationRIBAs for confirmation of third-generationHCV EIA-reactive blood donations

BACKGROUND: Licensure of an enhanced HCV screening assay (HCV 3.0 EIA) without concurrent licensure of a complementary supplemental assay (i.e., RIBA HCV 3.0 strip immunoblot assay [RIBA‐3]) decoupled screening and supplemental testing. In March 1998, the FDA Center for Biologics Evaluation and Research (CBER) recommended the use of RIBA‐3 on RIBA HCV 2.0 strip immunoblot assay (RIBA‐2)‐indeterminate units screened with HCV EIA 3.0.

Comparative analysis of cell culture and prediction algorithms for phenotyping of genetically diverse HIV-1 strains from Cameroon.

BackgroundWith the advent of entry inhibitors, monitoring of viral tropism in the clinical setting is important. Conventional methods are cell-based and lengthy, therefore V3 sequence based prediction algorithms are becoming increasingly attractive as monitoring tools. Here we report a comparative analysis of viral tropism of strains circulating in Cameroon where diverse and emerging variant strains are prevalent.MethodsViruses were isolated from 17 HIV positive individuals from three cities in Cameroon. Ghost cell lines expressing either CCR5 or CXCR4 with CD4 or CD4 alone (NIH AIDS Reagent Program) were used to determine co-receptor usage. HIV replication was determined by measuring p24 antigen levels. Plasma viral load (VL) was determined using the Versant bDNA assay. Nucleotide sequencing was performed on the V3 region and sequences were edited, aligned and translated into amino acids as described in the algorithm. Bio-informatics tools based on the 11/25 and charge rule were used to predict co-receptor usage.ResultsThe majority of patient isolates in our study were CRF02_AG or CRF02_AG containing recombinants. Tropism of these complex viruses based on the cell culture assay was determined to be R5 in 15/17 (88.2%) patients. However, two patient isolates were dual tropic R5X4 and had drug-specific mutations. Of these two patients, one was on antiretroviral treatment with a VL of 20,899 copies/ml and the other was drug-naïve with 141,198 copies/ml. Genotype based prediction was overall in good agreement with phenotype for R5 viruses, where 93% (14/15) of results were comparable, dual tropic viruses being reported as X4 viruses by prediction.ConclusionOur results indicate that most HIV strains in Cameroon were R5 tropic and some harbored drug-resistant mutations. V3 sequence based prediction compared well with cell based assays for R5 strains and may be useful even in settings where highly diverse strains are prevalent.

XMRV: usage of receptors and potential co-receptors.

BackgroundXMRV is a gammaretrovirus first identified in prostate tissues of Prostate Cancer (PC) patients and later in the blood cells of patients with Chronic Fatigue Syndrome (CFS). Although XMRV is thought to use XPR1 for cell entry, it infects A549 cells that do not express XPR1, suggesting usage of other receptors or co-receptors.MethodsTo study the usage of different receptors and co- receptors that could play a role in XMRV infection of lymphoid cells and GHOST (GFP- Human osteosarcoma) cells expressing CD4 along with different chemokine receptors including CCR1, CCR2, etc., were infected with XMRV. Culture supernatants and cells were tested for XMRV replication using real time quantitative PCR.ResultsInfection and replication of XMRV was seen in a variety of GHOST cells, LNCaP, DU145, A549 and Caski cell lines. The levels of XMRV replication varied in different cell lines showing differential replication in different cell lines. However, replication in A549 which lacks XPR1 expression was relatively higher than DU145 but lower than, LNCaP. XMRV replication varied in GHOST cell lines expressing CD4 and each of the co- receptors CCR1-CCR8 and bob. There was significant replication of XMRV in CCR3 and Bonzo although it is much lower when compared to DU145, A549 and LNCaP.ConclusionXMRV replication was observed in GHOST cells that express CD4 and each of the chemokine receptors ranging from CCR1- CCR8 and BOB suggesting that infectivity in hematopoietic cells could be mediated by use of these receptors.

Performance of hepatitis C virus (HCV) second‐generation enzyme immunoassay (EIA) on blood samples that reacted in the first‐generation HCV EIA

To the Editor: The recent report by Linden et a1.l in TRANSFUSION provides an invaluable lesson in the care required in dealing with the ABO blood group system and blood transfusion. The report demonstrated the continued need to take all possible precautions to prevent ABO-incompatible transfusions. The authors, and the editors of TRANSFUSION, inadvertently also demonstrate how simple it is to make an ABO error, even after detailed review. In their report, the authors calculated the possibility of random ABO compatibility by using frequencies specified in the American Association of Blood Banks Technical Manual.2 A serious error appeared in the article, although the erroneous number was not used in the overall calculation of ABO compatibility (0.64). The authors included the coincidence frequency of A x B (0.044) in their list of red cellcompatible groups. This is an error that, in a clinical setting, can be considered analogous to issuing blood of an incorrect blood group. The need to check carefully, and most critically, the ABO blood group is intrinsic to the work of every blood transfusion laboratory. Errors will continue, however, and we must all remain vigilant. Failure to complete one check may prove deadly.

Performance of serological assays used to test blood from recent smallpox vaccinees

Vaccination against influenza has been reported to interfere with the performance of assays used to test donated blood, giving false-positive results for various disease markers. Current regulations require blood donors to be deferred indefinitely if their donated blood tests reactive for the presence of markers for human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B virus (HBV), or hepatitis C virus (HCV). More than 30 years ago, smallpox vaccination was required in the United States and was usually given as a condition of entry into public schools. Thus, smallpox vaccinations were administered many years before recipients became eligible blood donors and before current donor screening assays were implemented. As a result, no information is available on the incidence of false-positive reactions for infectious disease markers after vaccination with live vaccinia virus. In the following report, we describe a study of the potential interference of smallpox vaccination on the results of testing donated blood for infectious disease markers using currently licensed serologic assays. We collected blood from 75 volunteers who participated in two ongoing clinical trials for smallpox vaccines. All subjects had received a licensed live vaccinia vaccine (Dryvax, Wyeth, Collegeville, PA). Details of vaccinee demographics and vaccine strain derivations were not provided to us. We collected ethylenediaminetetraacetateor citrate-anticoagulated venous blood on Days 0 (before vaccination), 3, 7, 14, 28, and 56 after vaccination. Blood samples were shipped to our laboratory on wet ice. Plasma was separated using standard separation methods and stored at −70°C until tested. We collected a total of 356 plasma samples from participants in the two clinical studies. There were 216 samples from 45 individuals from Study A and 140 samples from 30 individuals from Study B. Study A samples were tested for HIV and HTLV markers with Abbott HIV-1 antigen (enzyme immunoassay [EIA]), Abbott HIV-1 and -2 (EIA), Abbott HTLV-I and -II (EIA), Bio-Rad HIV-1 (EIA), Bio-Rad HIV-2 (EIA), BioRad HIV-1 and -2 (EIA), bioMérieux HIV-1 (EIA), bioMérieux HTLV-I and -II (EIA), and the Coulter p24 antigen (EIA). Assays used for testing HBV markers were Abbott AUSZYME (EIA), Abbott CORZYME (EIA), Abbott AUSAB (EIA), Ortho HBc (EIA), Ortho HBsAg (EIA), and Bio-Rad HBsAg (EIA). Assays for HCV markers were Ortho third-generation HCV (EIA) and Abbott second-generation HCV (EIA). Study B samples were tested for HIV and HTLV markers only, because of insufficient sample. No false-positive reactions for hepatitis B or C markers were observed with postsmallpox vaccination samples from either study. Of 45 prevaccination samples from Study A, 27 were repeatedly reactive for the presence of anti-HBs by the Abbott AUSAB assay. These individuals had been vaccinated recently against hepatitis B. These samples did not react when tested by all other hepatitis assays listed above. Samples from three vaccinees gave false-positive results when tested by certain HIV and HTLV assays. Plasma samples collected from two different vaccinees in Study A on Days 28 and 56 demonstrated cross-reactivity with the Bio-Rad HIV-1 and -2 antibody assay. Additional testing by Western blot (Bio-Rad Laboratories Blood Virus Division, Redmond, WA) yielded negative results. Also, plasma from one subject in Study B showed reactivity on Days 3, 8, 15, and 28 with the Bio-Rad HIV-2 antibody test kit. These samples were tested using the Bio-Rad HIV-1 Western blot and gave negative results. Confirmation of HIV-2 antibodies was not feasible because licensed HIV-2 supplemental tests are currently not available. This study involved a limited number of vaccinees because of concerns regarding the safety of live vaccinia virus as an immunogen at the time the studies were conducted. We do not have information about the effectiveness of the vaccine preparations used. We are not aware of serologic tests for vaccinia antibody, and questions have been raised as to the degree of correlation between the presence of antibodies and protection. Safety concerns have prompted the development of new vaccinia strains and alternative smallpox immunogens not involving live virus. Extrapolating from the incidence of cross-reactivity observed with these HIV, HTLV, HBV, and HCV marker assays, we estimate a false-positivity rate that could potentially defer as many as 2 percent of vaccinated donors if their blood is tested by the Bio-Rad HIV-1 and -2 or HIV-2 assays. Of 356 samples, 6 showed consistent low-level reactivity with the two licensed HIV antibody assays, both assays containing HIV-2 antigens. The reason for this low level of cross-reactivity observed with the 6 samples is unknown and warrants further investigation. The question of cross-reactivity caused by smallpox immunization will need to be revisited, however, as new and different virus preparations become available. The number of samples in this study is not sufficient for an accurate estimate of the number of donors who would be deferred, should mass vaccination with live Dryvaxderived vaccinia become necessary. Our results indicate, however, that the likelihood of an acute shortage of transfusable blood and blood products due to false-positive reactions as a result of smallpox vaccination would be low.

Genetic diversity and high proportion of intersubtype recombinants of HIV-1 in urban Cameroon.

Results All the samples were infected with HIV-1 group M. No group N and O viruses were identified in the samples studied although they are endemic to Cameroon. HIV-1 infections were dominated by circulating recombinant form CRF02_AG (59%) accompanied by additional group M subtypes or CRFs: A, D, F2, G, CRF01_AE, CRF13_cpx and CRF22_01A1. A high percentage (26%) of the samples had discordant subtype designations between gag and env, and appear to be unique intersubtype recombinant forms (URF) with the majority (91%) involving recombination with either CRF01_AE or/and CRF02_AG. URFs containing CRF19 or CRF22 as well Fgag-Genv and Cgag-Aenv are reported. Some samples were not consistently detected by current HIV-1 testing assays. Conclusion Our study confirmed the existence of high number of cocirculating HIV-1 subtypes, the high intrasubtype diversity, and the high numbers of possible new recombinant viruses in urban Cameroon. These results highlight the epidemiologic importance of HIV-1 CRFs, especially CRF02_AG. from 2006 International Meeting of The Institute of Human Virology Baltimore, USA. 17–21 November, 2006