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Background and objectives Globally, blood safety interventions have been successful in mitigating risk of the major transfusion-transmitted (TT) viruses. However, strategies that address risk from parasites are comparatively limited. TT parasites are often regional in nature, posing unique challenges; we sought to understand their impact on blood safety. Materials and methods An electronic questionnaire was distributed to transfusion medicine leaders in 100 countries. The survey focused on specific questions pertaining to four parasitic diseases: babesiosis, Chagas, leishmaniasis and malaria. Respondents provided data on historical TT cases, local epidemiology, policies to mitigate risk and an assessment of public health perceptions for each aetiologic agent. Results Twenty-eight (28%) surveys were returned from countries in Europe (n = 13), the Americas (n = 6), Africa (n = 4), Asia (n = 3) and Oceana (n = 2). Historically, no cases of TT leishmaniasis were reported, TT babesiosis was exclusive to Canada and the USA, TT Chagas was limited to the Americas and Spain, while TT malaria was cosmopolitan. Mitigation efforts varied widely; malaria was the most frequently tested parasitic disease. The public's perception of risk for parasitic agents was low, while that of health authorities in endemic countries was higher. Conclusion The global impact of parasitic infections on blood safety and related mitigation efforts varied widely by parasite epidemiology, test availability, public health priorities and socioeconomic constraints. While parasites continue to pose a risk to blood safety, the successful mitigation of viral risk has elevated the prominence of TT parasites in many locations, thereby requiring consideration of mitigation efforts.

BACKGROUND Babesiosis is an emerging tick-borne infection in humans. The increasing numbers of reported cases of transfusion-associated babesiosis (TAB), primarily caused by Babesia microti, represents a concern for the safety of the US blood supply. STUDY DESIGN AND METHODS This study investigated kinetics of parasitemia and innate immune responses and dynamics of antibody responses during B. microti infection in rhesus macaques (RMs) using blood smears, quantitative polymerase chain reaction (qPCR), flow cytometry, and indirect fluorescent antibody testing. A total of six monkeys were transfused with either hamster or monkey-passaged B. microti–infected red blood cells (two and four monkeys, respectively) simulating TAB. RESULTS The prepatent period in monkeys inoculated with hamster-passaged B. microti was 35 days compared with 4 days in monkeys transfused with monkey-passaged B. microti; the latter monkeys also had markedly higher parasitemia levels. The duration of the window period from the first detected parasitemia by qPCR analysis to the first detected antibody response ranged from 10 to 17 days. Antibody responses fluctuated during the course of the infection. Innate responses assessed by the frequencies of monocytes and activated B cells correlated with the kinetics and magnitude of parasitemia. On Day 14, additional activation peaks were noted for CD14+CD16+ and CD14–CD16+ monocytes and for CD11c+ myeloid dendritic cells, but only in animals transfused with monkey-passaged B. microti. Parasitemia persisted in these immunocompetent animals, similar to human infection. CONCLUSION The results suggest that transfusion-associated transmission of B. microti leads to rapid onset of parasitemia (Day 4) in RMs, detectable antibody response 14 days later, and persistent parasitemia.

Infection with human T lymphotropic virus (HTLV), although asymptomatic in most cases, can lead to severe illnesses, such as adult T cell leukemia/lymphoma or myelopathy/tropical spastic paraparesis. HTLV can be transmitted by whole-blood (WB) transfusion. The prevalence of HTLV among blood donor populations has not been characterized in Vietnam, although the screening has been partially implemented on voluntary basis since 2016. To determine the seroprevalence of HTLV-1/2 among blood donors, a total of 14,819 healthy blood donors in northern, central, and southern Vietnam and 1,003 samples from hepatitis B surface antigen (HbsAg), anti-hepatitis C (anti-HCV), or HIV Ag/Ab reactive blood donors were screened for anti-HTLV-1/2 antibodies by a chemiluminescence immunoassay using the Abbott ARCHITECT rHTLV-I/II assay. The anti-HTLV-1/2 repeat reactive (RR) samples were further tested by immunoblot (IB) method using MP Biomedicals HTLV Blot 2.4 for confirmation and differentiation of HTLV-1/2 infection. Proviral HTLV subgenomic amplification of the gag and tax regions was performed on the available WB RR samples (N = 11) by polymerase chain reaction (PCR). Among 14,819 blood donors, 34 samples (0.23%) were RR for anti-HTLV-1/2 antibodies, but only 1 case was confirmed HTLV-2 positive (0.0067%) and 5 cases were classified as indeterminate (0.034%) by IB. The RR rate was 0.39% among HBsAg/anti-HCV/HIV reactive sample groups, but none of them was confirmed by IB. Subgenomic PCR failed to amplify proviral DNA from WB samples of 11 RR samples. HTLV-1/2 prevalence was found to be low among blood donors in the study. Continued vigilance remains essential to maintain a low transfusion-transmitted risk in Vietnam.

BACKGROUND: Xenotropic murine leukemia virus–related virus (XMRV) has been reported in patients with prostate cancer and chronic fatigue syndrome. Although results have been conflicting, the potential of XMRV as an infectious human retrovirus has raised concerns about transfusion safety. To address this issue, normal and retrovirus-infected blood donors were screened for evidence of XMRV infection. STUDY DESIGN AND METHODS: Plasma from 1000 US, 100 human immunodeficiency virus Type 1–infected Cameroonian, and 642 human T-lymphotropic virus Type I (HTLV-I)-infected or uninfected Japanese blood donors as well as 311 sexually transmitted disease diagnostic specimens were screened for antibodies to XMRV gp70 and p15E using chemiluminescent immunoassays (CMIAs). CMIA-reactive samples were evaluated by p30 CMIA, Western blot, and real-time reverse transcriptase polymerase chain reaction. RESULTS: XMRV seroreactivity was low (0%-0.6%) with the exception of the HTLV-I–infected donors (4.9%). Antibody was detected against only a single XMRV protein (p15E or gp70); none of the seroreactive samples had detectable XMRV pol or env sequences. The elevated seroreactivity in HTLV-I–infected donors was due to an increased p15E seroreactive rate (4.1%). Inspection of XMRV and HTLV sequences revealed a high level of conservation within the immunodominant region (IDR) of the transmembrane protein. In some cases, HTLV IDR peptide competitively reduced the XMRV p15E signal. CONCLUSIONS: Based on the low prevalence of seroreactivity, detection of antibody to only a single XMRV protein and the absence of XMRV sequences, this study finds no compelling evidence of XMRV in normal or retrovirus-infected blood donors. The increased p15E seroreactivity observed in HTLV infection is likely due to cross-reactive antibodies.

Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.

We report the second human immunodeficiency virus (HIV) belonging to the new HIV type 1 (HIV-1) group P lineage that is closely related to the simian immunodeficiency virus found in gorillas. This virus was identified in an HIV-seropositive male hospital patient in Cameroon, confirming that the group P virus is circulating in humans. Results from screening 1,736 HIV-seropositive specimens collected in Cameroon indicate that HIV-1 group P infections are rare, accounting for only 0.06% of HIV infections. Despite its rarity, group P shows evidence of adaptation to humans.

BACKGROUND: In a previous study of 66 human immunodeficiency virus (HIV)-infected US blood donors from 1999 to 2005, HIV-1 non-B and antiretroviral drug-resistant strains accounted for 4.7 and 6.5% of HIV infections, respectively. This study was expanded to include an additional 11 recently acquired infections and 197 established infections collected from January 2005 through December 2007. STUDY DESIGN AND METHODS: HIV-infected donors were detected using FDA-licensed assays. Drug resistance profiles for protease and reverse transcriptase (RT) genes were determined using a genotyping system (ViroSeq, Celera Diagnostics); genetic subtype was determined by phylogenetic analysis of these sequences. RESULTS: Drug resistance profiles were obtained for 203 of 208 specimens; 9.9% had mutations that confer drug resistance. Ten showed resistance to a single drug class: nine to nonnucleoside RT inhibitors (NNRTIs) and one to nucleoside RT inhibitors (NRTIs). Eight showed two drug class resistance: five NRTI plus NNRTI, two NRTI plus protease inhibitor (PI), and one NNRTI plus PI. Two showed three drug class resistance. Non-B strains were identified in 2.5% of donors and consisted of subtypes A1 and D, CRF02_AG, CRF43-02G, and URF_BF. CONCLUSIONS: Data from this and the previous study show that antiretroviral drug-resistant HIV-1 is present in 9.1% of HIV-infected donors from 1999 through 2007; 9.3% of established infections and 6.9% of recent infections. Diverse HIV-1 non-B strains presently account for 3.0% of HIV infections in US donors.

Although Cameroon, in west central Africa, has a relatively low HIV prevalence of 5–6%, all HIV-1 groups (M, N, O, and P), nearly all HIV-1 group M subtypes, and numerous intersubtype recombinant forms have been identified in Cameroon. In this report, we describe the near full-length sequence of 04CMU11421, an HIV-1 group M subtype J strain collected in Cameroon in 2004. Phylogenetic analysis of the genome sequence shows high bootstrap support with three subtype J reference sequences in the HIV Sequence database. Therefore, 04CMU11421 represents a fourth pure subtype J isolate and the first reported in Cameroon.

In comparison to current on-market assays, the ARCHITECT rHTLV-I/II assay is the first fully automated assay that simultaneously detects human T-cell lymphotropic virus type I (HTLV-I) and type II (HTLV-II) in human serum and plasma. Specificity was assessed on 5646 blood donors and 692 clinical specimens. For sensitivity determination, 301 HTLV-I-positive and 105 HTLV-II-positive specimens were tested. Precision was between 3.98% and 4.31% coefficient of variation (CV) for specimens with 1 to 6 sample to cutoff. Specificity was 99.95% and 99.86% on specimens from blood donors and hospitalized patients, respectively. Sensitivity evaluation showed 100% detection on 301 HTLV-I and 105 HTLV-II specimens. HTLV-I and HTLV-II viruses are still circulating among general populations even in the low prevalence areas. To control the further spread of these retroviruses, we need to know that it is important to continue screening of blood. The performance evaluation data from this study demonstrate that the high throughput and fully automated ARCHITECT rHTLV-I/II chemiluminescence immunoassay effectively serves this purpose.

We evaluated use of the ARCHITECT HIV Ag/Ab Combo assay (HIV Combo; Abbott Diagnostics; available for sale outside the United States only) for detection of acute HIV infection.Samples were obtained from a behavioral intervention study (EXPLORE). HIV-uninfected men who have sex with men were enrolled and tested for HIV infection every 6 months. Samples from seroconverters collected at their last seronegative visit (n = 217) were tested individually using 2 HIV RNA assays. Samples with detectable HIV RNA were classified as acute and were tested with HIV Combo. Samples from the enrollment visit (n = 83) and the time of HIV seroconversion (n = 219) were tested with HIV Combo as controls.Twenty-one samples (9.7%) from the last seronegative visit had detectable HIV RNA and were classified as acute. HIV Combo was positive for 13 of the acute samples (61.9%). Samples not detected by HIV Combo had viral loads of 724-15,130 copies per milliliter. Expected results were obtained for positive and negative controls tested with HIV Combo.HIV Combo detected nearly two thirds of acute HIV infections identified in this high-risk population by non-pooled HIV RNA assays. HIV Combo may be useful for high-throughput screening to identify individuals with acute HIV infection.

HIV-1 is characterized by an exceptional level of sequence diversity and a rapid rate of evolution. HIV diversity has implications for reliability of assays designed to detect and monitor infection, pathogenesis, disease progression, response to antiviral therapeutics, resistance pathways, and vaccine development. In the present study, HIV-1 strain diversity was assessed for a small clinical cohort (n = 15) from London, England at risk for infection with non-subtype B strains. Analysis of gag p24, pol IN, and env gp41 IDR revealed the presence of five subtypes (A, B, C, D, H), CRF02_AG, and four unique recombinant forms. Due to the paucity of complete subtype H genomes available, we performed near full-length genome sequence analysis on the candidate subtype H strain, designated as 00GB.AC4001. Phylogenetic analysis revealed that it formed a monophyletic cluster with the three available subtype H reference sequences. Bootscanning analysis confirmed that 00GB.AC4001 represents a new nonrecombinant subtype H genome.

Objective: The HIV epidemic in Cameroon is characterized by a high level of strain diversity despite a relatively low prevalence of infection. In this study, HIV strains infecting blood donors in Cameroon were characterized to determine the prevalence of subtypes and intersubtype recombinants and if strain prevalence was changing over time. Methods: From 1996 through 2004, 676 HIV-infected blood donations were collected at blood banks in Douala and Yaounde, Cameroon. A subset of the HIV-1 group M strains (n = 574) were classified based on phylogenetic analysis of viral sequences from the gag p24, pol integrase, and env gp41 regions. Results: HIV-1 group M accounted for 97.3% (n = 658) of infections, whereas group O was present in 2.2% (n = 15) and HIV-2 in 0.4% (n = 3). Within the group M infections, 14 subtypes and circulating recombinant forms (CRFs) and unique recombinant forms (URFs) were identified. Overall, CRF02_AG accounted for 58.2% of infections, URFs 14.8%, and levels of subtypes, A, B, C, D, F2, and G, and CRFs, 01, 06, 09, 11, 13, 22, and 37, varied from 0.2% to 6.1%. Evaluation of HIV strains present in the donor population over this 9-year period showed no substantial changes in the proportion of infections caused by each subtype and CRF, the percentage of intersubtype recombinants, or the strain composition of the URFs. Conclusions: HIV-1 strain diversity in Cameroon did not significantly change, suggesting a mature and relatively stable epidemic.

Recombinant forms of HIV-1 contribute significantly to the ongoing epidemic. In the present study, we characterized the near full-length genome of one candidate HIV-1 CRF25_cpx strain originating in Cameroon, 06CM-BA-040. Viral RNA was extracted from plasma, and the genome was obtained using RT-PCR amplification to generate 10 overlapping fragments. Bootscanning, recombination breakpoint analysis, and phylogenetic trees confirmed that 06CM-BA-040 had a genomic structure consistent with two available CRF25_cpx reference sequences. The CRF25_cpx mosaic composition consisted of nine segments derived from subtypes A and G as well as unclassified (U) regions. Subtype G and CRF25_cpx clusters diverged from each other with long branch lengths but were distinct from other known subtypes with high bootstrap support (94%). The epidemiological significance of CRF25_cpx strains is unknown; however, the availability of additional genomic sequences will improve our understanding of the overall genetic diversity within this recombinant form of HIV-1.

In the Brazilian HIV-1 epidemic subtypes B, C, and F1 are cocirculating in the high risk population groups, and there is a high prevalence of intersubtype recombinant forms. The dynamic nature of the HIV epidemic in Brazil led us to study HIV-1 subtypes present in HIV-infected blood donations collected from 2001 to 2003. Donations from 91 seropositive donors were evaluated. Genetic subtype was obtained for 88 specimens based on sequence analysis of gag p24, pol IN, and env gp41 IDR. HIV-1 subtype B was the predominant strain present in the donor population (73.9%). A significant prevalence of intersubtype recombinants of subtypes B and F1 was found (22.7%). Subtype C (1.1%) and F1 (2.3%) were rare. None of the B/F1 recombinants is CRF28_BF or CRF29_BF. The high level of unique B/F1 recombinant strains in this population demonstrates the dynamic and complex nature of the HIV epidemic in Brazil.

Genetically polymorphic targets present a significant challenge to the reliability of detection and quantification by nucleic acid-based assays. A probe system with enhanced mismatch tolerance would be advantageous for such applications. The present study introduces a novel class of DNA probes, designated as partially double-stranded linear probes, composed of a long target-specific strand 5' labeled with a fluorophore and a markedly shorter quencher strand, complementary to the 5' end of the target-specific strand, that is 3' end-labeled with a quencher moiety. The utility of this probe system for sensitive detection of amplification products was demonstrated in a real-time PCR format. Comparison of multiple partially double-stranded linear probe combinations revealed that increased asymmetry in strand length was associated with improved mismatch tolerance. Notably, for a 45-mer/11-mer combination, the difference in threshold cycle values obtained for a perfectly matched target and one containing six mismatches was

Early detection of HIV infection is critical for clinical diagnosis, prevention of transmission, and safety of blood products. Currently, several HIV antigen-antibody (Ag/Ab) combination assays that simultaneously detect HIV-1 p24 antigen, and antibodies to HIV-1 group M, group O, and HIV-2 are available only outside the USA, for diagnosis of viral infection. HIV Ag/Ab combination assays reduce the seroconversion window by 3-5 days relative to sensitive third-generation HIV antibody assays and approximately 14 days relative to first-generation antibody assays. HIV combination assays developed on several fully automated instruments provide cost-effective means for detection of early infections. The utility of HIV combination assays to identify primary HIV infections has been demonstrated in several settings outside the USA, including in low HIV prevalence countries.

Texto completo: acesso restrito. p. 237–243 Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-08-23T11:03:09Z No. of bitstreams: 1 1-s2.0-S0166093406000395-main.pdf: 212674 bytes, checksum: 411c7cb3b1da7567c1b1dc64cd693538 (MD5) Approved for entry into archive by Rodrigo Meirelles (rodrigomei@ufba.br) on 2013-11-16T13:11:50Z (GMT) No. of bitstreams: 1 1-s2.0-S0166093406000395-main.pdf: 212674 bytes, checksum: 411c7cb3b1da7567c1b1dc64cd693538 (MD5) Made available in DSpace on 2013-11-16T13:11:51Z (GMT). No. of bitstreams: 1 1-s2.0-S0166093406000395-main.pdf: 212674 bytes, checksum: 411c7cb3b1da7567c1b1dc64cd693538 (MD5) Previous issue date: 2006 The combination of automated sample preparation and real-time RT-PCR for measurement of HIV-1 viral load has the potential to significantly enhance throughput, reduce operator-associated error, and increase assay sensitivity and dynamic range. In this study, RNA was extracted from the plasma of 91 HIV-1 seropositive Brazilian blood donors using the Abbott m2000sp™ automated sample preparation system. Viral loads measured using the RealTime™ HIV-1 (RealTime HIV-1) assay and the Abbott m2000rt™ instrument were compared to values obtained in the LCx® HIV RNA quantitative assay. Subtype was determined for 89 of 91 specimens by sequence/phylogenetic analysis of three genomic regions: gag p24, pol integrase, and env gp41. The panel included 69 subtype B, 1 C, 2 F, and 17 recombinant strains. Eighty-seven specimens were quantified by both assays. Two specimens were quantified only in RealTime HIV-1. Two additional specimens below the detection limit of both assays were also negative on PCR amplification. Viral load results were highly correlated, and good agreement was observed between assays with 90% of values within 0.5 log10 copies/ml. The RealTime HIV-1 assay and m2000 system offer the advantages of automation while providing reliable quantification of diverse HIV strains.

An HIV-1 group N infection, 02CM-DJO0135, was identified among specimens collected in 2002 at the D'Joungolo Hospital, Yaoundé, Cameroon. Sequences were obtained from viral RNA extracted from plasma for regions of LTR-gag, pol-vif, and env. The virus amplified from the specimen is closely related to a previously reported group N virus, 02CM-DJO0131, that was also collected at this hospital in 2002. Although the viral sequences for the two isolates differ, their close relationship suggests that the two specimens are linked. No patient histories are available for 02CM-DJO0131 and 02CM-DJO0135; the specimens could have been drawn from a husband/wife, mother/child, or a single individual. However, differences in seroreactivity indicate that it is unlikely that the specimens were drawn from the same patient. This report documents the second case that suggests linkage between group N-infected individuals and indicates that there is ongoing transmission of HIV-1 group N in Cameroon.

HIV-1 is classified into three groups, M (major), N (non-M non-O), and O (outlier); each group arose from a separate transmission of SIVcpz into humans. HIV-1 group N was recently discovered and infections with this virus are rare with only eight documented cases. All group N infections have been found in Cameroon and there is no evidence of direct linkage between the infected patients. We report here the identification of HIV-1 group N infections in a husband and wife. The group N infection in the husband, 1131-03, was identified first based on seroreactivity in peptide EIAs and confirmed by PCR amplification of group N viral sequences. Subsequently the wife, 1015-04, was evaluated and confirmed to also be infected with a group N virus. Near full-length viral genomes were amplified and sequenced from each patient's specimen. The low level of diversity between the two viral sequences provides evidence of horizontal transmission of group N from one spouse to the other. Patient 1131-03 was receiving antiviral therapy consisting of reverse transcriptase inhibitors; the treatment appears effective for suppression of group N viral replication based on apparently low viral load in plasma specimens collected from the patient and the absence of drug resistance mutations in RT sequences amplified from 1131-03. This report brings to 10 the number of group N infections identified and to 5 the number of group N genomes sequenced. Although group N infections continue to be rare, group N is a pathogenic virus and its prevalence needs to be monitored.

Human immunodeficiency virus type 1 (HIV-1) evolution and changing strain distribution present a challenge to nucleic acid-based assays. Reliable patient monitoring of viral loads requires the detection and accurate quantification of genetically diverse HIV-1. A panel of 97 HIV-1-seropositive plasma samples collected from Cameroon, Brazil, and South Africa was used to compare the performance of four commercially available HIV RNA quantitative tests: Abbott LCx HIV RNA Quantitative assay (LCx), Bayer Versant HIV-1 RNA 3.0 (bDNA), Roche AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5), and bioMérieux NucliSens HIV-1 QT (NucliSens). The panel included group M, group O, and recombinant viruses based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified viral RNA in 97 (100%) of the samples. In comparison, bDNA, Monitor v1.5, and NucliSens quantified viral RNA in 96.9%, 94.8%, and 88.6% of the samples, respectively. The two group O specimens were quantified only by the LCx HIV assay. Analysis of nucleotide mismatches at the primer/probe binding sites for Monitor v1.5, NucliSens, and LCx assays revealed that performance characteristics reflected differences in the level of genetic conservation within the target regions.

In this study, we evaluated the performance of six HIV combined p24 antigen and antibody (Ag/Ab) assays versus two third-generation anti-HIV antibody assays. The assays were evaluated using p24 antigen panel of 31 HIV-1 subtypes (n = 124), 25 HIV-1 seroconversion panels (n = 176), HIV-1 antibody positive samples including group M subtypes and group O (n = 559), HIV-2 positive samples (n = 110), and unselected HIV negative samples from four French private laboratories (n = 1005). The results showed that overall HIV combined Ag/Ab assays present better performance, when compared to antibody-only assays. However, some differences were observed in the sensitivity of the six HIV combined Ag/Ab assays evaluated. The AxSYM and Murex Combo assays had the best sensitivity score in this study and reduced the window period by 2.0-2.35 days relative to antibody only assays and 1-2.17 days relative to the other combined Ag/Ab assays. Among combined HIV Ag/Ab assays, Genscreen Plus and AxSYM Combo presented the highest specificity, with 99.9% and 99.8%, respectively.

A panel of genetically diverse, well-characterized human immunodeficiency type 1 (HIV-1) virus isolates is a valuable tool for standardizing nucleic acid-based tests. Since HIV quantitative assays target different regions of the genome, thorough molecular characterization is useful for evaluating the effect of genetic variation on viral load assay performance. In this study, 39 virus isolates were examined. The representative HIV-1 group M subtypes A-G and group O strains included 30 members of the Walter Reed Army Institute of Research (WRAIR) clade panel. Though this panel is currently being used in laboratories around the world, only limited sequence information is available on most of these isolates. In this communication, gag p24 (gag), pol integrase (pol IN), and env gp41 immunodominant (IDR) region sequences were characterized to assess the level of genetic diversity and to verify group/subtype. The panel was composed of 37 group M and two group O strains.

HIV-1 group O strains have a level of genetic diversity similar to that of strains in group M; however, group O has not been readily classified into genetic subtypes. Phylogenetic classification of group O has been hindered by the limited sequence information available. To facilitate phylogenetic analysis, we sequenced the gag p24 (693 nt), pol p32 (864 nt), and env gp160 (approximately 2700 nt) genes from 39 group O-infected specimens. These specimens include 32 plasma samples collected in Cameroon between 1996 and 1999, 2 specimens collected in the United States, and 5 infections previously isolated in Equatorial Guinea. Phylogenetic analysis of HIV-1 group O sequences resulted in the identification of five clusters that are maintained across gag, pol, and env, generally supported by high bootstrap values, and approximately equidistant from each other. In addition to the group O clusters, several isolates branch independently and are equidistant from the other group O isolates. Cluster I comprises greater than 50% of the group O isolates and is a diverse set of isolates that is subdivided into subclusters. The average intra-, sub-, and intercluster distances for group O are similar to the corresponding distances for group M subtypes. The five group O clusters have characteristics similar to those of group M subtypes. Thus the data presented may form the basis for classification of group O into subtypes. However, full-length genomes representing each group O cluster will be required to formalize a group O subtype classification.

The performance of the LCx HIV RNA Quantitative (LCx HIV), AMPLICOR HIV-1 MONITOR version 1.5 (MONITOR v1.5), and Quantiplex HIV-1 RNA version 3.0 (bDNA v3.0) viral load assays was evaluated with 39 viral isolates (3 A, 7 B, 6 C, 4 D, 8 E, 4 F, 1 G, 4 mosaic, and 2 group O). Quantitation across the assay dynamic ranges was assessed using serial fivefold dilutions of the viruses. In addition, sequences of gag -encoded p24 ( gag p24), pol -encoded integrase, and env -encoded gp41 were analyzed to assign group and subtype and to assess nucleotide mismatches at primer and probe binding sites. For group M isolates, quantification was highly correlated among all three assays. In contrast, only the LCx HIV assay reliably quantified group O isolates. The bDNA v3.0 assay detected but consistently underquantified group O viruses, whereas the MONITOR v1.5 test failed to detect group O viruses. Analysis of target regions revealed fewer primer or probe mismatches in the LCx HIV assay than in the MONITOR v1.5 test. Consistent with the high level of nucleotide conservation is the ability of the LCx HIV assay to quantify efficiently human immunodeficiency virus type 1 group M and the genetically diverse group O.

Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus from sooty mangabey (SIVSM) form one of the six primate lentivirus lineages. The close phylogenetic relationship and ge...

A prototype assay was used to genotype integrase (IN) from 120 HIV-1- infected IN inhibitor-naive adults from Argentina, Brazil, Cameroon, South Africa, Thailand, and Uganda. Subtype designations based on analysis of pol IN sequences were A (14), B (15), C (12), D (11), F (12), G (7), H (1), CRF01_AE (9), CRF02_AG (34), CRF22_01A1 (4), and CRF37_cpx (1). Ten (8.3%) of 120 samples had mutations associated with reduced susceptibility to the IN inhibitors, raltegravir and elvitegravir. Two samples had E92Q (both subtype B) and eight had E157Q (2A, 1C, 1D, 1F, 3 CRF02_AG). Some samples had other mutations selected by these drugs including T97A, and some had amino acid polymorphisms at positions associated with raltegravir and elvitegravir resistance. Mutations associated with other investigational HIV IN inhibitors were also identified. This suggests that HIV strains may vary in their natural susceptibility to HIV IN inhibitors.

The prevalence of HIV infection in Brazil is one of the highest in the world. In addition, transfusion-transmitted HIV accounts for 2.3% of all AIDS cases in Brazil. The objective of this study was to evaluate genetic diversity and distribution of HIV-1 strains circulating in the blood-donor population. We characterized 43 seropositive blood units collected from volunteer blood donors residing throughout Rio de Janeiro, Brazil. Viral RNA was extracted from plasma, reverse transcribed, and amplified by nested polymerase chain reaction (PCR) using HIV group M degenerate primers. Genetic heterogeneity was evaluated by direct automated cycle sequencing of the following gene fragments: gag p24 (399 bp), env C2V3 (345 bp), and env gp41 (369 bp). Phylogenetic analysis reflected the complexity of the Brazilian HIV epidemic: the majority of specimens, 33 of 43 (76.7%) were subtype B, and 6 of 43 (14%) were subtype F. The remaining 4 samples (9.3%) involved potential mosaic viruses of subtypes B and F or B and D. This survey is the first to document HIV-1 genetic variation in the Brazilian blood-donor population.Brazil has the highest prevalence of HIV infection in Latin America and one of the highest such prevalences in the world. By 1996, 110,000 AIDS cases had been cumulatively reported by the Brazil National AIDS Program. HIV-1 subtypes B and F have previously been described in Brazil, accounting for 85% and 15% of infections, respectively. Findings are presented from a study conducted to evaluate the genetic diversity and distribution of HIV-1 strains circulating in the blood donor population. The authors characterized 43 HIV-seropositive blood units collected from volunteer blood donors living throughout Rio de Janeiro. Viral RNA was extracted from plasma, reverse transcribed, and amplified by nested polymerase chain reaction using HIV group M degenerate primers. Genetic heterogeneity was assessed through the direct automated cycle sequencing of gene fragments gag p24 (399 bp), env C2V3 (345 bp), and env gp41 (369 bp). 33 of the 43 (76.7%) specimens were of subtype B and 6 (14%) of subtype F, while the remaining 4 (9.3%) involved potential mosaic viruses of subtypes B and F or B and D.

Recombinant antigens and peptides were used to develop an HIV slot immunoblot assay to confirm and differentiate infection by HIV-1 group M, HIV-1 group O or HIV-2. Recombinant antigens from the gag , pol or env regions of HIV-1 and HIV-2, in addition to synthetic peptides from the immunodominant region (IDR) of transmembrane proteins gp41 (HIV-1) or gp36 (HIV-2), were blotted on nitrocellulose strips and used as a substitute for competitive Western blots. Evaluation of a large number of samples ( N =440) from various regions of the world, using the immunoblot, showed effective differentiation of HIV-1 group M, HIV-1 group O and HIV-2. The immunoblot identified correctly all (24/24) HIV-1 group O samples that were confirmed subsequently by PCR and sequence analysis. The immunoblot is a useful tool for identifying HIV-1 group O seropositive samples and has the potential to identify other serological HIV variants that may represent detection problems for HIV screening assays using HIV-1 group M subtype B reagents.

Multiple sclerosis (MS) patient samples were screened for known or novel retroviruses using an ultrasensitive technique, IMx PERT, that detects the presence of reverse transcriptase (RT). This procedure has 10(5)- to 10(7)-fold greater sensitivity than conventional RT assays and is capable of detecting 10 to 50 virions. Moreover, IMx PERT is at least as sensitive as polymerase chain reaction, and requires no previous knowledge of viral nucleotide sequence. The MS specimens analyzed in this study included 136 sera from 79 patients and 128 cerebrospinal fluid samples from 53 patients with relapsing or chronic progressive disease. In addition, peripheral blood mononuclear cells from 19 MS patients were cultured in an attempt to amplify or induce expression of low-copy number or cell-associated retrovirus. No evidence of retrovirus was found in any of the specimens obtained from MS patients.

This chapter explains the disease of acquired immune deficiency syndrome (AIDS), and the etiologic agents: human immunodeficiency virus (HIV) -1, and -2. The differences between HIV types 1 and 2 are described and there is a review of the subtypes of both viruses. The pathogenesis is also reviewed. Diagnosis and therapeutic monitoring of HIV-AIDS is explained, using the serology markers, HIV RNA, anti-HIV IgM, anti-HIV IgG, and HIV p24 Ag.

In this study, we evaluated the performance of two prototype human immunodeficiency virus (HIV) antigen-antibody (Ag-Ab) combination assays, one from Abbott Laboratories (AxSYM HIV Ag-Ab) and the other from bioMerieux (VIDAS HIV Duo Ultra), versus five combination assays commercially available in Europe. The assays were Enzygnost HIV Integral, Genscreen Plus HIV Ag-Ab, Murex HIV Ag-Ab Combination, VIDAS HIV Duo, and Vironostika HIV Uniform II Ag-Ab. All assays were evaluated for the ability to detect p24 antigen from HIV-1 groups M and O, antibody-positive plasma samples from HIV-1 groups M and O, HIV-2, and 19 HIV seroconversion panels. Results indicate that although all combination assays can detect antibodies to HIV-1, group M, subtypes A to G, circulating recombinant form (CRF) A/E, and HIV-1 group O, their sensitivity varied considerably when tested using diluted HIV-1 group O and HIV-2 antibody-positive samples. Among combination assays, the AxSYM, Murex, and VIDAS HIV Duo Ultra assays exhibited the best antigen sensitivity (at ∼25 pg of HIV Ag/ml) for detection of HIV-1 group M, subtypes A to G and CRF A/E, and HIV-1 group O isolates. However, the VIDAS HIV Duo Ultra assay had a lower sensitivity for HIV-1 group M and subtype C, and was unable to detect subtype C antigen even at 125 pg of HIV Ag/ml. The HIV antigen sensitivity of the VIDAS HIV Duo and Genscreen Plus combination assays was ∼125 pg of HIV Ag/ml for detection of all HIV-1 group M isolates except HIV-1 group O while the sensitivity of Vironostika HIV Uniform II Ag-Ab and Enzygnost HIV Integral Ag-Ab assays for all the group M subtypes was >125 pg of HIV Ag/ml. Among the combination assays, the AxSYM assay had the best performance for detection of early seroconversion samples, followed by the Murex and VIDAS HIV Duo Ultra assays.

Xenotropic murine leukemia virus-related virus (XMRV) has been reported in patients with prostate cancer and chronic fatigue syndrome. Although results have been conflicting, the potential of XMRV as an infectious human retrovirus has raised concerns about transfusion safety. To address this issue, normal and retrovirus-infected blood donors were screened for evidence of XMRV infection.Plasma from 1000 US, 100 human immunodeficiency virus Type 1-infected Cameroonian, and 642 human T-lymphotropic virus Type I (HTLV-I)-infected or uninfected Japanese blood donors as well as 311 sexually transmitted disease diagnostic specimens were screened for antibodies to XMRV gp70 and p15E using chemiluminescent immunoassays (CMIAs). CMIA-reactive samples were evaluated by p30 CMIA, Western blot, and real-time reverse transcriptase polymerase chain reaction.XMRV seroreactivity was low (0%-0.6%) with the exception of the HTLV-I-infected donors (4.9%). Antibody was detected against only a single XMRV protein (p15E or gp70); none of the seroreactive samples had detectable XMRV pol or env sequences. The elevated seroreactivity in HTLV-I-infected donors was due to an increased p15E seroreactive rate (4.1%). Inspection of XMRV and HTLV sequences revealed a high level of conservation within the immunodominant region (IDR) of the transmembrane protein. In some cases, HTLV IDR peptide competitively reduced the XMRV p15E signal.Based on the low prevalence of seroreactivity, detection of antibody to only a single XMRV protein and the absence of XMRV sequences, this study finds no compelling evidence of XMRV in normal or retrovirus-infected blood donors. The increased p15E seroreactivity observed in HTLV infection is likely due to cross-reactive antibodies.

Background PCR-based testing has been widely utilized to assess the prevalence of Xenotropic Murine Leukemia Virus-related Virus (XMRV) in prostate cancer and chronic fatigue syndrome patients. An alternative approach, screening for antibodies elicited by XMRV infection, represents a more desirable option for large-scale epidemiologic studies. In this study, blood donor and retrovirus-infected populations were screened for serologic evidence of XMRV infection.

Background Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a human gammaretrovirus recently identified in prostate cancer tissue and in lymphocytes of patients with chronic fatigue syndrome. To establish the etiologic role of XMRV infection in human disease requires large scale epidemiologic studies. Development of assays to detect XMRV-specific antibodies would greatly facilitate such studies. However, the nature and kinetics of the antibody response to XMRV infection have yet to be determined. Results Three rhesus macaques were infected with XMRV to determine the dynamics of the antibody responses elicited by infection with XMRV. All macaques developed antibodies to XMRV during the second week of infection, and the predominant responses were to the envelope protein gp70, transmembrane protein p15E, and capsid protein p30. In general, antibody responses to gp70 and p15E appeared early with higher titers than to p30, especially in the early period of seroconversion. Antibodies to gp70, p15E and p30 persisted to 158 days and were substantially boosted by re-infection, thus, were identified as useful serologic markers. Three high-throughput prototype assays were developed using recombinant proteins to detect antibodies to these viral proteins. Both gp70 and p15E prototype assays demonstrated 100% sensitivity by detecting all Western blot (WB) positive serial bleeds from the XMRV-infected macaques and good specificity (99.5-99.9%) with blood donors. Seroconversion sensitivity and specificity of the p30 prototype assay were 92% and 99.4% respectively. Conclusions This study provides the first demonstration of seroconversion patterns elicited by XMRV infection. The nature and kinetics of antibody responses to XMRV in primates were fully characterized. Moreover, key serologic markers useful for detection of XMRV infection were identified. Three prototype immunoassays were developed to detect XMRV-specific antibodies. These assays demonstrated good sensitivity and specificity; thus, they will facilitate large scale epidemiologic studies of XMRV infection in humans.

In a previous study of 66 human immunodeficiency virus (HIV)-infected US blood donors from 1999 to 2005, HIV-1 non-B and antiretroviral drug-resistant strains accounted for 4.7 and 6.5% of HIV infections, respectively. This study was expanded to include an additional 11 recently acquired infections and 197 established infections collected from January 2005 through December 2007.HIV-infected donors were detected using FDA-licensed assays. Drug resistance profiles for protease and reverse transcriptase (RT) genes were determined using a genotyping system (ViroSeq, Celera Diagnostics); genetic subtype was determined by phylogenetic analysis of these sequences.Drug resistance profiles were obtained for 203 of 208 specimens; 9.9% had mutations that confer drug resistance. Ten showed resistance to a single drug class: nine to nonnucleoside RT inhibitors (NNRTIs) and one to nucleoside RT inhibitors (NRTIs). Eight showed two drug class resistance: five NRTI plus NNRTI, two NRTI plus protease inhibitor (PI), and one NNRTI plus PI. Two showed three drug class resistance. Non-B strains were identified in 2.5% of donors and consisted of subtypes A1 and D, CRF02_AG, CRF43-02G, and URF_BF.Data from this and the previous study show that antiretroviral drug-resistant HIV-1 is present in 9.1% of HIV-infected donors from 1999 through 2007; 9.3% of established infections and 6.9% of recent infections. Diverse HIV-1 non-B strains presently account for 3.0% of HIV infections in US donors.

Analysis of 3555 HIV-seropositive specimens, collected in Cameroon from 2002 to 2006, led to the identification of four HIV-1 group N infections based on differential seroreactivity to HIV env-derived peptides and proteins and confirmation by nucleic acid amplification. Group N prevalence continues to be low accounting for only 0.1% of HIV infections in Cameroon. Near full-length genomic sequences were obtained from viral RNA or proviral DNA by PCR amplification of overlapping fragments for three isolates, 06CM-U14296, 06CM-U14842, and 02CM-SJGddd. Two genome segments, partial pol and env-nef, were obtained from viral RNA for the fourth isolate, 02CM-TIM0217. With the four group N isolates identified in this study and group N sequences previously reported, eight near full-length and five partial genome sequences are now available. Despite genetic divergence from HIV-1 group M and O, all of the group N infections evaluated by five commercial HIV immunoassays were detected.

Recommendations from the Centers for Disease Control and Prevention (CDC) in 2006 [1] aimed to identify the estimated 21% of HIV-infected persons who remain unaware of their human immunodeficiency virus (HIV) status and who may be responsible for up to 50% of transmissions [2]. The CDC specifically recommended that HIV antibody testing should be expanded from targeted, risk-based testing to universal screening of all adults in health care settings aged 13–64 years. Unfortunately, strategies limited to antibody testing may compete with goals of prevention programs in settings where highly infectious individuals in the antibody-negative “window period” [3] represent a significant proportion of HIV-infected testers, and false-negative test results could lead to critical missed opportunities to interrupt onward HIV transmission. The US Food and Drug Administration–approved HIV tests vary in their ability to detect recent infection. The Public Health–Seattle & King County (PHSKC) HIV testing program has used a first- or second-generation enzyme immunoassay (EIA) [4] performed on serum specimens and a rapid antibody test performed on oral fluid or finger-stick blood specimens. Since September 2003, we have tested serum samples obtained from antibody-negative men who have sex with men (MSM) using pooled HIV nucleic acid amplification testing (NAAT) to identify antibody-negative, NAAT-positive MSM with acute HIV infection in real time (Appendix). This analysis summarizes our experience from the first 5 years of the NAAT program. We will present estimates of the sensitivity of early-generation EIAs and a rapid antibody test in a population with a high prevalence (15%) and incidence (0.8 cases per 100 person-years) of HIV infection (Robert Wood, personal communication). We will also provide data on the ability of a fourth-generation antigen-antibody combination assay to detect HIV in frozen serum specimens obtained from persons with acute infection. These findings will illustrate that a one-size-fits-all, antibody test–focused approach may not be sufficient to detect HIV infection, particularly if rapid tests are used in high-incidence populations, and that a program to diagnose highly-infectious antibody-negative, NAAT-positive persons can be successfully integrated with rapid and anonymous HIV antibody testing.

Near full-length viral genome sequences were obtained for five putative subtype G candidates identified in HIV-infected Cameroonian blood donors, based on partial genome sequences for the gag, pol, and env regions. Phylogenetic analysis of the genome sequences shows that all five strains are pure subtype G with no indication of intersubtype recombination. The Cameroon subtype G sequences did not form a geographically based subcluster and were intermixed within the subtype G branch with isolates from several different countries. HIV-1 group M subtype G accounts for only 4.5% of HIV infections in Cameroon. However, genome segments of subtype G are present in 67% of all infections and 80% of infections due to intersubtype recombinant strains in Cameroon. The addition of five subtype G genome sequences to the HIV database may contribute to a better understanding of the origins and classification of HIV-1 subtypes and CRFs.

An enzyme immunoassay (EIA) which utilizes a solid phase coated with a recombinant antigen (c100-3) derived from the hepatitis C virus (HCV) genome was evaluated for efficacy in the detection of antibodies to HCV (anti-HCV). The sensitivity of the antibody test was demonstrated by the detection of anti-HCV in a well-characterized panel of human specimens known to contain the infectious agent of non-A, non-B hepatitis. The specificity of the anti-HCV test was evaluated by testing 6,118 serum specimens from volunteer blood donors considered to be at low risk for exposure to HCV. The specificity of the anti-HCV EIA was demonstrated to be 99.56%, since 6,069 of 6,096 specimens from this low-risk group were nonreactive. A total of 49 (0.80%) of the 6,118 specimens were repeatedly reactive in the test, and 22 (46.81%) of the 47 specimens available for additional testing were confirmed as positive for antibodies to HCV c100-3. Among commercial plasma donors, 390 (10.49%) of 3,718 specimens were repeatedly reactive in the EIA. A total of 375 (97.40%) of the 385 specimens available for further testing were confirmed as positive. These limited data indicate that the prevalence of antibodies to HCV is 0.36% (22 confirmed positives among 6,118 specimens) among volunteer blood donors and 10.08% (375 confirmed positives among 3,718 specimens) among commercial plasma donors. The importance of confirmatory testing is discussed.

BACKGROUND: In this study, human immunodeficiency virus type 1 (HIV-1)-infected blood donors were evaluated for genetic subtype and drug resistance to determine the prevalence of divergent HIV strains in the US donor population. STUDY DESIGN AND METHODS: Subtype was determined by phylogenetic analysis of viral sequences amplified by reverse transcription-polymerase chain reaction. The drug resistance profile of the protease and reverse transcriptase (RT) genes was determined using an HIV-1 genotyping system (ViroSeq). RESULTS: From 1999 through 2005, 26 recently infected donors, defined as HIV-1 RNA–positive, antibody-negative (RNA+/Ab−), were identified (yield, 1:1.61 million). Over the same period, the frequency of anti-HIV–positive donors was 1:34,700. Twenty RNA+/Ab− specimens were evaluated; all were infected with HIV-1 subtype B. Drug resistance profiles obtained for 18 donors identified one strain with protease mutation L90M that confers resistance to nelfinavir and one with RT mutation Y188H that confers resistance to nevirapine. Genetic subtype was determined for 44 of 46 HIV antibody–reactive and confirmed-positive (Ab+) specimens. Three infections (6.8%) were due to circulating recombinant forms: 2 CRF01_AE and 1 CRF02_AG. In the Ab+ group, one strain was resistant to all nucleoside RT inhibitors and one had mutations that confer resistance to protease inhibitors. CONCLUSION: The data show that antiretroviral drug–resistant HIV strains are being transmitted in the United States. Overall 6.5 percent (4 of 62) of HIV-1–infected donors harbored drug-resistant strains. HIV-1 non-B strains accounted for 4.7 percent (3 of 64) of the infections in donors. HIV-1 subtype B is still the predominant strain in the United States; however, non-B strains are increasing.

Recently, we reported a high level of HIV-1 strain diversity in patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Based on phylogenetic analysis of gag p24, pol integrase, and env gp41 sequences, subtypes A, B, C, D, and G, and CRF02_AG, as well as unique recombinant forms were identified. Subtype G accounted for 25% of the infections in the Saudi population and this high prevalence was unexpected. Although subtype G is found in west central Africa, pure subtype G strains are uncommon. To further characterize the subtype G infections in Saudi Arabia, six strains that appeared to be pure subtype G were selected for full genome sequencing. Near full-length genomes were obtained using RT-PCR amplification to generate overlapping fragments from viral RNA extracted from plasma. The six strains are not subtype G throughout their entire genome. Four isolates have a recombinant structure composed of CRF02_AG and subtype G and share three identical breakpoints. This recombinant form defines a new CRF designated CRF43_02G. The remaining two isolates are CRF25_cpx, a circulating recombinant form identified in Cameroon composed of subtypes A and G and unclassified segments. Reanalysis of the previously reported Saudi HIV-1 partial genome sequences revealed additional isolates classified as CRF43_02G and CRF25_cpx and one isolate was reclassified to CRF22_01A. Identification of CRF43_02G in Saudi Arabia could indicate a transmission network within the country. Alternatively, the new CRF could have been introduced from an external source where this CRF is not yet recognized.

A unique HIV-2 intergroup recombinant strain was identified in Cameroon. The virus, CM-03-510-03, was amplified from blood collected from a 47-year-old female patient in Douala, Cameroon in 2003 who was seroreactive for HIV-2. A near full-length genome 9089 nucleotides in length was amplified from proviral DNA. The genome for CM-03-510-03 is composed of segments of HIV-2 groups A and B with four recombination break-points and has open reading frames for all the structural and regulatory genes. A comparison of CM-03-510-03 to the only previously reported HIV-2 intergroup recombinant shows that the two strains share one recombination breakpoint but are otherwise distinct from each other. Similar to HIV-1, HIV-2 intergroup recombination is an indication that coinfection with more than one strain has occurred in individuals and is a mechanism that increases strain genetic diversity.

Screening blood donations for human T-lymphotropic virus types I and II (HTLV-I/II) continues to be important in protecting the safety of blood products and controlling the global spread of these retroviruses. We have developed a fully automated, third generation chemiluminescent immunoassay, ARCHITECT rHTLV-I/II, for detection of antibodies to HTLV-I/II. The assay utilizes recombinant proteins and synthetic peptides and is configured in a double antigen sandwich assay format. Specificity of the assay was 99.98% (9,254/9,256, 95% CI = 99.92–100%) with the negative specimens from the general population including blood donors, hospital patients and pregnant women from the US, Japan and Nicaragua. The assay demonstrated 100% sensitivity by detecting 498 specimens from individuals infected with HTLV-I (n = 385) and HTLV-II (n = 113). ARCHITECT rHTLV-I/II results were in complete agreement with the Murex HTLV-I/II reference assay and 99.7% agreement with the Genelabs HTLV Blot 2.4 confirmatory assay. Analytical sensitivity of the assay was equivalent to Murex HTLV-I/II assay based on end point dilutions. Furthermore, using a panel of 397 specimens from Japan, the ARCHITECT rHTLV-I/II assay exhibited distinct discrimination between the antibody negative (Delta Value = −7.6) and positive (Delta Value = 7.6) populations. Based on the excellent specificity and sensitivity, the new ARCHITECT rHTLV-I/II assay should be an effective test for the diagnosis of HTLV-I/II infection and also for blood donor screening. J. Med. Virol. 80:484–493, 2008. © 2008 Wiley-Liss, Inc.

BACKGROUND: Before the introduction of human immunodeficiency virus (HIV) combination assays, serologic diagnosis of HIV infection was performed with assays that detected either antibodies or p24 antigen. Owing to the capability to detect the early appearance of p24 antigen, combination assays that are designed for simultaneous detection of antibodies and antigen can significantly reduce the diagnostic window. STUDY DESIGN AND METHODS: Specificity and sensitivity of a commercially available HIV antigen-antibody combination assay (Abbott PRISM; assay is not licensed by the FDA for use in the United States) were evaluated in a multicenter study by testing volunteer blood donors, hospitalized patients, seroconversion panels, and p24 antigen and HIV antibody subtype panels. Performance data were compared to a commercially available HIV combination assay and the PRISM HIV O Plus assay. RESULTS: Apparent specificity of 99.95 percent was observed in the donor population for the PRISM HIV antigen-antibody combination assay, and better seroconversion sensitivity was demonstrated compared with another combination assay and the PRISM HIV O Plus assay. Analytical HIV antigen detection sensitivity averaged 33 pg per mL on the Agence Francaise de Securite Sanitaire des Produits de Sante (AFSSAPS) panel. Furthermore, comparable antigen sensitivity was demonstrated for 32 HIV-1 group M subtype and group O panels. The PRISM HIV combination assay detected all HIV-1 group M and O and HIV-2 antibody–positive specimens evaluated. CONCLUSIONS: The PRISM HIV antigen-antibody combination assay demonstrated a significant reduction of the window period for diagnosis of HIV infection. The assay demonstrated enhanced specificity and sensitivity along with broad subtype detection. The assay performance represents the “state-of-the art” technology for serologic blood screening of HIV infection.

HUMAN T-cell lymphotropic virus Type I (HTLV-I) is a retrovirus identified as the etiologic agent of adult T-cell leukemia and lymphoma.1 , 2 More recently, HTLV-I infection has been implicated in the causation of chronic progressive myelopathies, such as tropical spastic paraparesis3 , 4 and HTLV-I—associated myelopathy.5 HTLV-I—associated myelopathy affects patients from southern Japan, which has a temperate climate, whereas tropical spastic paraparesis affects persons from tropical areas. Despite these differences, the two conditions resemble each other,6 prompting some to suggest that they are different names for the same disease.7 HTLV-I has been implicated in the development of these conditions, because affected persons have . . .

Mouse monoclonal antibodies directed against the transmembrane proteins of HIV-1 or HIV-2 provided site-directed, unambiguous discrimination between HIV-1 and HIV-2 antibody-positive sera, when employed in immunoassays as competitive probes against serum antibodies. These monoclonal antibodies mapped to epitopes outside of the well-characterized immunodominant regions (IDR) of the transmembrane proteins. The monoclonal competitive immunoassay was a superior method for discrimination compared with immunoprecipitation of metabolically radiolabeled HIV envelope glycoproteins, Western blot against viral envelope glycoproteins, or noncompetitive enzyme immunoassays employing HIV recombinant transmembrane proteins or synthetic IDR peptides as serological targets. The monoclonal competitive assay was not affected by antigenic cross reactivity or nonspecific reactivity exhibited by selected serum samples toward envelope proteins or peptides, respectively. Results of the monoclonal competitive immunoassay were supported by results of a peptide inhibition assay employing free IDR peptides in competition with IDR peptides on a solid support for binding of serum antibody. IDR peptide inhibition clearly demonstrated non-cross-reactive antigenic specificity of sera toward either the HIV-1 IDR or the HIV-2 IDR. The monoclonal competitive assay also identified samples containing antibody to both HIV-1 and HIV-2 transmembrane proteins. Analysis of these samples by IDR peptide inhibition indicated they contained two distinct, non-cross-reactive populations of antibodies, one directed to the HIV-1 IDR and the other directed to the HIV-2 IDR.

The HIV fusion inhibitor enfuvirtide (ENF/Fuzeon) targets the env gp41 transmembrane domain. Mutations in gp41 are associated with ENF resistance. We developed a prototype assay to genotype a 676-bp region spanning the heptad repeat domains (HR1 and HR2) of HIV-1 gp41. Plasma samples were collected from 126 HIV-1-infected blood donors in Cameroon, Brazil, Uganda, South Africa, Thailand, and Argentina. Based on analysis of gag p24, pol integrase, and env gp41 genes, the panel was composed of subtypes A/A2 (18), B (11), C (14), D (10), F/F2 (9), G (7), CRF01_AE (9), CRF02_AG (33), and recombinant strains (15). Genotyping was successful for 119 of the 126 samples (94.4%). Although numerous amino acid polymorphisms were detected in some samples, none had primary mutations associated with ENF resistance. The gp41 HIV-1 research reagents developed by Celera are useful tools for genotyping analysis of the gp41 region in diverse HIV-1 strains.