An improved HIV antigen/antibody prototype assay for earlier detection of acute HIV infection.

BACKGROUND: Early detection of acute HIV infection by HIV antigen/antibody assays depends on antigen sensitivity. Maintaining consistently high sensitivity across diverse HIV strains is critical to ensure equal detection. OBJECTIVES: The performance of an improved HIV antigen/antibody prototype, HIV Combo Next, was evaluated for detection of genetically-diverse HIV strains and seroconversion samples. STUDY DESIGN: Antigen sensitivity of the prototype was evaluated and compared to five FDA-approved HIV antigen/antibody assays using World Health Organization (WHO) HIV p24 antigen standard and reference panels, 17 virus isolates and 9 seroconversion panels. Antibody sensitivity and assay specificity of the prototype were also assessed with 1062 disease-staged and genotyped samples, and samples from 3000 blood donors and 955 individuals with low-risk for HIV infection. RESULTS: Compared with other assays evaluated, the prototype demonstrated the best analytical sensitivity for WHO antigen standard, reference panels including 12 HIV-1 variants (0.04 - 0.25 IU/ml) and one HIV-2 variant, and 17 HIV virus isolates including HIV-1 group M, N, P and O and HIV-2 (0.3 -16 pg/ml). The enhanced sensitivity was also observed for seroconversion samples, detecting more PCR-positive samples with detection up to 7 days earlier than the other assays. Improvement in antigen sensitivity did not compromise antibody sensitivity or assay specificity, detecting all HIV disease-staged and genotyped samples, with assay specificity of 99.97% for blood donors and 99.68% for the low-risk population. CONCLUSIONS: These data indicate that the new prototype HIV Combo Next assay will be of diagnostic value, providing improved early detection for acute HIV infection from divergent HIV strains.

Comparative evaluation of three FDA-approved HIV Ag/Ab combination tests using a genetically diverse HIV panel and diagnostic specimens.

BACKGROUND: HIV Ag/Ab combination assays are recommended by CDC for routine screening and several HIV Ag/Ab combination tests are now FDA-approved. Maintaining high specificity and consistent sensitivity across diverse HIV strains is critical for these assays to accurately detect HIV infection and expedite delivery of patient results. OBJECTIVES: To evaluate performance of three FDA-approved HIV tests: ARCHITECT HIV Combo (Abbott), ADVIA Centaur HIV Combo (Siemens) and BioPlex HIV Ag-Ab (Bio-Rad). STUDY DESIGN: Sensitivity and specificity were evaluated using an extensive panel of 28 HIV infected human specimens and 17 cultured virus isolates representing multiple genotypes, 6 seroconversion panels, 4 human samples with acute infection, WHO p24 standard and 4020 clinical specimens. RESULTS: The p24 limit of detection (LOD) for the WHO standard was 0.19IU/ml, 0.70IU/ml, and 1.77IU/ml in BioPlex, ARCHITECT, and Centaur respectively. The distribution of LODs across 15 HIV-1 isolates was substantially narrower in ARCHITECT (5-33pg/ml) than in BioPlex (11-198pg/ml) and Centaur (6-384pg/ml). All assays detected antibodies to the majority of HIV-1 and HIV-2 variants. However, reduced sensitivity was observed for Centaur in detection of antibodies to HIV-1 group M (CRF02_AG), O and N variants. BioPlex and ARCHITECT showed better seroconversion sensitivity than Centaur, detecting one bleed (3-7 days) earlier in 4 (BioPlex) and 3 (ARCHITECT) of 6 seroconversion panels. ARCHITECT demonstrated the highest specificity (99.90-100%) compared to BioPlex (99.80%) and Centaur (99.42%). CONCLUSIONS: The overall performance of ARCHITECT and BioPlex was superior to Centaur, especially for detection of acute HIV infection.

Genetic Characterization of a Panel of Diverse HIV-1 Isolates at Seven International Sites.

HIV-1 subtypes and drug resistance are routinely tested by many international surveillance groups. However, results from different sites often vary. A systematic comparison of results from multiple sites is needed to determine whether a standardized protocol is required for consistent and accurate data analysis. A panel of well-characterized HIV-1 isolates (N = 50) from the External Quality Assurance Program Oversight Laboratory (EQAPOL) was assembled for evaluation at seven international sites. This virus panel included seven subtypes, six circulating recombinant forms (CRFs), nine unique recombinant forms (URFs) and three group O viruses. Seven viruses contained 10 major drug resistance mutations (DRMs). HIV-1 isolates were prepared at a concentration of 107 copies/ml and compiled into blinded panels. Subtypes and DRMs were determined with partial or full pol gene sequences by conventional Sanger sequencing and/or Next Generation Sequencing (NGS). Subtype and DRM results were reported and decoded for comparison with full-length genome sequences generated by EQAPOL. The partial pol gene was amplified by RT-PCR and sequenced for 89.4%-100% of group M viruses at six sites. Subtyping results of majority of the viruses (83%-97.9%) were correctly determined for the partial pol sequences. All 10 major DRMs in seven isolates were detected at these six sites. The complete pol gene sequence was also obtained by NGS at one site. However, this method missed six group M viruses and sequences contained host chromosome fragments. Three group O viruses were only characterized with additional group O-specific RT-PCR primers employed by one site. These results indicate that PCR protocols and subtyping tools should be standardized to efficiently amplify diverse viruses and more consistently assign virus genotypes, which is critical for accurate global subtype and drug resistance surveillance. Targeted NGS analysis of partial pol sequences can serve as an alternative approach, especially for detection of low-abundance DRMs.

Failure to identify HIV-infected individuals in a clinical trial using a single HIV rapid test for screening.

BACKGROUND: In the HIV Prevention Trials Network (HPTN) 061 study, 8 (2.3%) of 348 HIV-infected participants identified as HIV uninfected at study enrollment using a single HIV rapid test for screening were found to be HIV infected after additional testing. OBJECTIVES: To evaluate the performance of different HIV assays for detection of HIV infection in HPTN 061 participants with missed infection and individuals with viral suppression. METHODS: Plasma samples from 8 HPTN 061 participants, 17 elite controllers, and 101 individuals on antiretroviral treatment (ART) were tested for HIV with 3 rapid tests, 2 laboratory-based immunoassays, and a Western blot assay. The HPTN 061 samples were also tested with 2 HIV RNA assays and an antiretroviral drug assay. RESULTS: Of the 8 HPTN 061 participants with missed infection, 1 was an elite controller, 1 was taking ART, 2 were missed because of testing or clerical errors, 1 had recent HIV infection (identified using a multi-assay algorithm), and 3 had acute HIV infection. Two (1.7%) of 118 individuals with viral suppression (both taking ART) had at least 1 false-negative test. CONCLUSIONS: In clinical trials, HIV infections can be missed for a variety of reasons. Using more than one assay to screen for HIV infection may reduce the number of missed infections.

Clinical implications of elevated HIV-1 viral load results obtained from samples stored frozen in vacutainer plasma preparation tubes.

Studies have demonstrated that plasma samples collected and stored frozen using vacutainer plasma preparation tubes (PPT) may result in falsely elevated viral load (VL) values with the Roche COBAS TaqMan HIV-1 v1.0 test. At the University of Connecticut Health Center, a total of 349 samples from HIV-1-infected patients on HAART were collected and stored frozen in PPT. Viral load (VL) results were obtained using the Roche COBAS TaqMan HIV-1 v2.0 test (CTM v2.0) and Abbott RealTime HIV-1 assay (RealTime HIV-1). Of the 349 samples, 260 (74.5%) had VL values that differed by >0.5log10copies/mL; 64 of these were quantified by both assays. The remaining 196 samples were detected by CTM v2.0 but not detected in RealTime HIV-1: 62 of the most discordant samples in this category (CTM v2.0 detected/RealTime HIV-1 not detected) were further analyzed using two nested RT-PCR assays targeting pol integrase: full-length (864nt) and a highly conserved subregion (134nt). No HIV-1 RNA was detected in the discordant samples, confirming RealTime HIV-1 results. The increase in VL reactivity with the CTM v2.0 assay was presumably due to proviral DNA captured by the CTM total nucleic acid extraction chemistry but not the RNA-specific extraction procedure used in RealTime HIV-1. These results suggest that using CTM v2.0 with samples frozen in PPT could have significant clinical implications for HIV-1 patient management.

Estimation of HIV incidence in a large, community-based, randomized clinical trial: NIMH project accept (HIV Prevention Trials Network 043).

BACKGROUND: National Institute of Mental Health Project Accept (HIV Prevention Trials Network [HPTN] 043) is a large, Phase III, community-randomized, HIV prevention trial conducted in 48 matched communities in Africa and Thailand. The study intervention included enhanced community-based voluntary counseling and testing. The primary endpoint was HIV incidence, assessed in a single, cross-sectional, post-intervention survey of >50,000 participants. METHODS: HIV rapid tests were performed in-country. HIV status was confirmed at a central laboratory in the United States. HIV incidence was estimated using a multi-assay algorithm (MAA) that included the BED capture immunoassay, an avidity assay, CD4 cell count, and HIV viral load. RESULTS: Data from Thailand was not used in the endpoint analysis because HIV prevalence was low. Overall, 7,361 HIV infections were identified (4 acute, 3 early, and 7,354 established infections). Samples from established infections were analyzed using the MAA; 467 MAA positive samples were identified; 29 of those samples were excluded because they contained antiretroviral drugs. HIV prevalence was 16.5% (range at study sites: 5.93% to 30.8%). HIV incidence was 1.60% (range at study sites: 0.78% to 3.90%). CONCLUSIONS: In this community-randomized trial, a MAA was used to estimate HIV incidence in a single, cross-sectional post-intervention survey. Results from this analysis were subsequently used to compare HIV incidence in the control and intervention communities. TRIAL REGISTRATION: ClinicalTrials.gov NCT00203749.

ARCHITECT® HIV Ag/Ab Combo assay: correlation of HIV-1 p24 antigen sensitivity and RNA viral load using genetically diverse virus isolates.

BACKGROUND: HIV antigen/antibody (Ag/Ab) combination assays represent a significant advancement in assays used for diagnosing HIV infection based on their ability to detect acute and chronic infections. During acute HIV infection (AHI), detection depends on assay sensitivity for p24 Ag. OBJECTIVE: To directly compare the Ag sensitivity of the ARCHITECT(®) HIV Ag/Ab Combo assay to RNA viral load using cell culture supernatants of virus isolates. HIV-1 isolates allow correlation in the total absence of an antibody response to infection and across genetically diverse HIV-1 group M strains. METHODS: Thirty-five HIV-1 isolates comprising subtypes A-D, F and G, CRF01_AE, CRF02_AG, and unique recombinant forms were evaluated. Cell-free culture supernatant for each isolate was diluted to four levels and tested in the HIV Combo assay to determine a signal to cutoff ratio and the RealTime(®) HIV-1 assay to quantify RNA. The RNA copies/mL at the HIV Combo assay cutoff was determined. RESULTS: The median RNA copies/mL at the HIV Combo assay cutoff was 57,900 for individual virus isolates (range 26,440-102,400). A single plot of all the data gave a value of 58,500RNA copies/mL. An analysis of data published for acute HIV infection in human subjects gave a similar result; HIV Combo detected 97% of AHIs with RNA copies/mL > 30,700. CONCLUSIONS: Based on analysis of virus isolates, the ARCHITECT HIV Combo assay can detect p24 Ag when RNA is above approximately 58,000copies/mL. The correlation of viral load and Ag sensitivity was consistent across genetically diverse HIV-1 group M strains.

HIV genotypes and primary drug resistance among HIV-seropositive blood donors in Brazil: role of infected blood donors as sentinel populations for molecular surveillance of HIV.

BACKGROUND: There are few surveillance studies analyzing genotypes or primary (transmitted) drug resistance in HIV-infected blood donors in Brazil. The aim of this study was to characterize patterns of HIV genotypes and primary resistance among HIV-seropositive donors identified at 4 geographically dispersed blood centers in Brazil. METHODS: All HIV-infected donors who returned for counseling at the 4 REDS-II Hemocenters in Brazil from January 2007 to March 2011 were invited to participate in a case-control study involving a questionnaire on risk factors. Viral sequencing was also offered to positive cases to assign genotypes and to detect and characterize primary resistance to reverse transcriptase and protease inhibitors according to World Health Organization guidelines. RESULTS: Of the 341 HIV-seropositive donors who consented to participate in the risk factor and genetics study, pol sequences were obtained for 331 (97%). Clade B was predominant (76%) followed by F (15%) and C (5%). Primary resistance was present in 36 [12.2%, 95% confidence interval (CI) 8.2 to 15.5] of the 303 individuals not exposed to antiretroviral therapy, varying from 8.2% (95% CI: 2.7 to 13.6) in Recife to 19.4% in São Paulo (95% CI: 9.5 to 29.2); there were no significant correlations with other demographics or risk factors. CONCLUSIONS: Although subtype B remains the most prevalent genotype in all 4 areas, increasing rates of subtype C in Sao Paulo and F in Recife were documented relative to earlier reports. Transmitted drug resistance was relatively frequent, particularly in the city of Sao Paulo which showed an increase compared with previous HIV-seropositive donor data from 10 years ago.

Xenotropic murine leukemia virus-related virus does not pose a risk to blood recipient safety.

BACKGROUND: When xenotropic murine leukemia virus-related virus (XMRV) was first reported in association with chronic fatigue syndrome, it was suggested that it might offer a risk to blood safety. Thus, the prevalence of the virus among blood donors and, if present, its transmissibility by transfusion need to be defined. STUDY DESIGN AND METHODS: Two populations of routine blood donor samples (1435 and 13,399) were obtained for prevalence evaluations; samples from a linked donor-recipient repository were also evaluated. Samples were tested for the presence of antibodies to XMRV-related recombinant antigens and/or for XMRV RNA, using validated, high-throughput systems. RESULTS: The presence of antibodies to XMRV could not be confirmed among a total of 17,249 blood donors or recipients (0%; 95% confidence interval [CI], 0%-0.017%); 1763 tested samples were nonreactive for XMRV RNA (0%; 95% CI, 0%-0.17%). Evidence of infection was absent from 109 recipients and 830 evaluable blood samples tested after transfusion of a total of 3741 blood components. CONCLUSIONS: XMRV and related murine leukemia virus (MLV) markers are not present among a large population of blood donors and evidence of transfusion transmission could not be detected. Thus, these viruses do not currently pose a threat to blood recipient safety and further actions relating to XMRV and MLV are not justified.

Seroprevalence of xenotropic murine leukemia virus-related virus in normal and retrovirus-infected blood donors.

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.

Prevalence of xenotropic murine leukemia virus-related virus infection in different risk populations in Spain.

Human infection with the xenotropic murine leukemia virus-related virus (XMRV) has been associated controversially with prostate cancer and chronic fatigue syndrome. Information is lacking about the mechanisms of transmission and potential risk groups for XMRV infection. Plasma and peripheral blood mononuclear cells (PBMCs) from individuals with retroviral infections, chronic viral hepatitis, autoimmune diseases, prostate cancer, chronic fatigue syndrome, and blood donors were tested for XMRV markers. Antibodies to XMRV proteins p15E and gp70 were examined using research assays. DNA extracted from PBMCs was tested for the presence of XMRV gag and env sequences. A total of 1103 specimens belonging to individuals with chronic fatigue syndrome and/or fibromyalgia (437), prostate cancer (69), HIV-1 (149), HTLV-1/2 (31), chronic hepatitis B (81), chronic hepatitis C (72), autoimmune diseases (18), and blood donors (246) were examined. Overall, three samples (0.3%) were p15E seroreactive (two HTLV-1 and one HCV patient). Another 15 (1.4%) were gp70 seroreactive (six chronic fatigue syndrome-fibromyalgia, four blood donors, two HIV-1, one prostate cancer, one HBV, and one HCV). Four specimens were initially positive for XMRV gag sequences, but none could be confirmed by repeated testing. In summary, no evidence of XMRV infection was found in populations with retroviral and viral hepatitis infections in Spain. Likewise, XMRV was not recognized in patients with autoimmune diseases, chronic fatigue syndrome-fibromyalgia, prostate cancer, or healthy blood donors.

Performance of the Abbott RealTime HIV-1 viral load assay is not impacted by integrase inhibitor resistance-associated mutations.

The Abbott RealTime HIV-1 viral load assay uses primers and probes targeted to integrase, which is also the target of integrase inhibitors such as raltegravir. Viral loads of 42 raltegravir-susceptible and 40 raltegravir-resistant specimens were determined using RealTime HIV-1 and Roche Monitor (v1.5). The differences in viral load measurements between assays were comparable in the two groups, demonstrating that the RealTime HIV-1 assay can tolerate raltegravir-selected mutations.

Characterization of antibodies elicited by XMRV infection and development of immunoassays useful for epidemiologic studies.

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.

Analysis of pol integrase sequences in diverse HIV type 1 strains using a prototype genotyping assay.

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.

Identification of new CRF43_02G and CRF25_cpx in Saudi Arabia based on full genome sequence analysis of six HIV type 1 isolates.

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.

The prevalence of diverse HIV-1 strains was stable in Cameroonian blood donors from 1996 to 2004.

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 Yaoundé, 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, CRFO2_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.

Near full-length genome characterization of an HIV type 1 CRF25_cpx strain from Cameroon.

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.

Evaluation of a new, fully automated immunoassay for detection of HTLV-I and HTLV-II antibodies.

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.

Analysis of HIV type 1 gp41 sequences in diverse HIV type 1 strains.

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.

Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR.

Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.