Priscilla Swanson

Impact of Human Immunodeficiency Virus Type 1 (HIV-1) Genetic Diversity on Performance of Four Commercial Viral Load Assays: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR v1.5, VERSANT HIV-1 RNA 3.0, and NucliSens HIV-1 QT

ABSTRACT 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.

Performance of the Celera Diagnostics ViroSeq HIV-1 Genotyping System for Sequence-Based Analysis of Diverse Human Immunodeficiency Virus Type 1 Strains

ABSTRACT The Celera Diagnostics ViroSeq HIV-1 Genotyping System is a Food and Drug Administration-cleared, integrated system for sequence-based analysis of drug resistance mutations in subtype B human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT). We evaluated the performance of this system for the analysis of diverse HIV-1 strains. Plasma samples were obtained from 126 individuals from Uganda, Cameroon, South Africa, Argentina, Brazil, and Thailand with viral loads ranging from 2.92 to >6.0 log10 copies/ml. HIV-1 genotyping was performed with the ViroSeq system. HIV-1 subtyping was performed by using phylogenetic methods. PCR products suitable for sequencing were obtained for 125 (99%) of the 126 samples. Genotypes including protease (amino acids 1 to 99) and RT (amino acids 1 to 321) were obtained for 124 (98%) of the samples. Full bidirectional sequence data were obtained for 95 of those samples. The sequences were categorized into the following subtypes: A1/A2 (16 samples), B (12 samples), C (13 samples), D (11 samples), CRF01_AE (9 samples), F/F2 (9 samples), G (7 samples), CRF02_AG (32 samples), H (1 sample), and intersubtype recombinant (14 samples). The performances of the individual sequencing primers were examined. Genotyping of duplicate samples in a second laboratory was successful for 124 of the 126 samples. The identity level for the sequence data from two laboratories ranged from 98 to 100% (median, 99.8%). The ViroSeq system performs well for the analysis of plasma samples with diverse non-B subtypes. The availability of this genotyping system should facilitate studies of HIV-1 drug resistance in non-subtype B strains of HIV-1.

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, 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.

HIV-1 subtypes among blood donors from Rio de Janeiro, Brazil.

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.

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.

Molecular characterization of 39 HIV-1 isolates representing group M (subtypes A-G) and group O: Sequence analysis of gag p24, pol integrase, and env gp41

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.

Comparative Performance of Three Viral Load Assays on Human Immunodeficiency Virus Type 1 (HIV-1) Isolates Representing Group M (Subtypes A to G) and Group O: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR Version 1.5, and Quantiplex HIV-1 RNA Version 3.0

ABSTRACT 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 ofgag-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.

Quantification of HIV-1 group M (subtypes A-G) and group O by the LCx HIV RNA quantitative assay.

Human immunodeficiency virus type 1 (HIV-1) genetic diversity presents a challenge to nucleic acid-based assays with regard to sensitivity of detection and accuracy of quantification. The Abbott LCx HIV RNA Quantitative assay (LCx(R) HIV assay), a competitive RT-PCR targeting the pol integrase region, was evaluated using a panel of 297 HIV-1 seropositive plasma samples from Cameroon, Uganda, Brazil, Thailand, Spain, Argentina and South Africa. The panel included group M subtypes A-G, mosaics, and group O based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified 290 (97.6%) of the samples, including all the group O samples tested. In comparison, the Roche AMPLICOR HIV-1 MONITOR test versions 1.0 and 1.5 quantified 67.3 and 94.6% of the samples, respectively. No group O specimens were quantified by either version of AMPLICOR HIV-1 MONITOR. Seven specimens were below the detectable limits of all the three assays. The LCx HIV assay had fewer nucleotide mismatches at primer/probe binding sites as compared with both AMPLICOR HIV-1 MONITOR tests. The high degree of nucleotide conservation within the pol target region enables the LCx HIV assay to efficiently quantify the HIV-1 subtypes A-G and the most genetically diverse HIV-1, group O.

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

BackgroundXenotropic 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.ResultsThree 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.ConclusionsThis 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.

Evaluation of HIV type 1 group O isolates: identification of five phylogenetic clusters.

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.