Timothy C. Granade

Impact of HIV Type 1 Subtype Variation on Viral RNA Quantitation

We evaluated the performance of three HIV-1 RNA quantitation methods (Amplicor HIV-1 MONITOR-1.0, NASBA, and Quantiplex HIV RNA 2.0 [branched DNA (bDNA)]) using plasma specimens (N = 60) from individuals from Asia and Africa infected with one of three HIV-1 subtypes (A, Thai B [B] or E; N = 20 each). Our results demonstrate that of the 20 subtype A specimens, 19 were quantifiable by the bDNA assay compared with 15 by the MONITOR-1.0 and 13 by NASBA. Of those quantifiable, the mean log10 difference was 0.93 between bDNA and MONITOR-1.0 and 0.46 between bDNA and NASBA. For subtype B specimens, the correlation among methods was better with only 2 specimens missed by NASBA and 3 by the bDNA assay. However the missed specimens had viral burden near the lower limit (1000 copies/ml) for these assays. For the 20 subtype E specimens, MONITOR-1.0 and NASBA quantified RNA in 17 and 14 specimens, respectively, as compared with 19 specimens quantified by the bDNA assay. The correlation among different assays, especially between bDNA/NASBA and MONITOR-1.0/NASBA, was poor, although the mean log10 difference for subtype E specimens was 0.4 between bDNA and MONITOR-1.0 and only 0.08 between bDNA and NASBA. The addition of a new primer set, designed for non-B HIV-1 subtypes, to the existing MONITOR assay (MONITOR-1.0+) resulted in RNA detection in all 60 specimens and significantly improved the efficiency of quantitation for subtypes A and E. Our data indicate that HIV-1 subtype variation can have a major influence on viral load quantitation by different methods. Periodic evaluation and modification of these quantitative methods may be necessary to ensure reliable quantification of divergent viruses.

Maternal Virus Load and Perinatal Human Immunodeficiency Virus Type 1 Subtype E Transmission, Thailand

To determine the rate and risk factors for human immunodeficiency virus (HIV)-1 subtype E perinatal transmission, with focus on virus load, pregnant HIV-infected women and their formula-fed infants were followed prospectively in Bangkok. Of 281 infants with known outcome, 68 were infected (transmission rate, 24.2%; 95% confidence interval, 19.3%-29.6%). Transmitting mothers had a 4.3-fold higher median plasma HIV RNA level at delivery than did nontransmitters (P<.001). No transmission occurred at <2000 copies/mL. On multivariate analysis, prematurity (adjusted odds ratio [AOR], 4.5), vaginal delivery (AOR, 2.9), low NK cell percentage (AOR, 2.4), and maternal virus load were associated with transmission. As RNA quintiles increased, the AOR for transmission increased linearly from 4.5 to 24.8. Two-thirds of transmission was attributed to virus load>10,000 copies/mL. Although risk is multifactorial, high maternal virus load at delivery strongly predicts transmission. This may have important implications for interventions designed to reduce perinatal transmission.

Diagnosis of Human Immunodeficiency Virus Type 1 Infection with Different Subtypes Using Rapid Tests

ABSTRACT We evaluated six rapid tests for their sensitivity and specificity in diagnosing human immunodeficiency virus type 1 (HIV-1) infection using 241 specimens (172 HIV-1 positive, 69 HIV-1 negative) representing different HIV-1 subtypes (A [n = 40], B [n = 47], C [n = 28], E [n = 42], and F [n = 7]). HIVCHEK, Multispot, RTD and SeroStrip were 100% sensitive and specific. Capillus failed to identify two of eight subtype C specimens (overall sensitivity of 98.85%), while the SUDS test (the only test approved by the Food and Drug Administration) gave false-positive results for 5 of 69 seronegative specimens (specificity of 93.24%). Our results suggest that although rapid tests perform well in general, it may be prudent to evaluate a rapid test for sensitivity and specificity in a local population prior to its widespread use.

Rapid detection of HIV-1 p24 antigen using magnetic immuno-chromatography (MICT)

Detection of human immunodeficiency virus (HIV) infections has been enhanced by incorporating p24 antigen detection with current HIV antibody detection using enzyme immunoassays (EIAs). However, screening for HIV antibodies has increased through the use of rapid, lateral-flow HIV antibody detection assays that currently do not have the capability to detect HIV p24 antigen. In this report, a lateral-flow based assay using super-paramagnetic particles as the detection marker was developed for the detection of HIV-1 p24 antigen. This magnetic immuno-chromatographic test (MICT) uses an inexpensive, low-maintenance instrument that detects the magnetic moment of the super-paramagnetic particles in a magnetic field. MICT is simple to perform, provides a numerical output for easier determination of reactive results and can be completed in 40min. The lower limit of detection for HIV-1 p24 spiked into assay sample buffer and 50% plasma was 30pg/ml for both. Detection of HIV-1 p24 antigen at 50pg/ml was reproducible in both inter-run and intra-run assays with coefficients of variation of <13%. Furthermore, the MICT p24 assay was able to detect intact virus spiked into 50% plasma (lower detection limit of approximately 250,000 viral RNA copies/ml). MICT detection of increasing HIV-1 p24 levels in commercially available seroconversion panels by MICT was only slightly later than that detected by much more complex EIAs. MICT could provide a simple, low-cost, and portable method for rapid HIV-1 p24 detection in a variety of testing environments.

Modification of rapid human immunodeficiency virus (HIV) antibody assay protocols for detecting recent HIV seroconversion.

ABSTRACT Assay protocols of three rapid human immunodeficiency virus (HIV) assays, OraQuick-1/2, SeroStrip-1/2, and Determine-1/2, were modified to detect recent HIV seroconversion using a higher dilution of serum specimens. Optimal predilution of specimens resulted in negative test results during early periods of seroconversion (about 6 months), when antibody levels were low. A total of 269 seropositive specimens from routine HIV type 1 testing and from commercial sources (low-titer and seroconversion panels) were tested, and results were recorded as negative (score = 0) or positive using intensity scores from 0.5 (weak positive) to 4 (strongly positive). The same specimens were previously tested by a less sensitive (LS) enzyme immunoassay (EIA), Abbott 3A11-LS, and were classified as recent or long-term infections based on the standardized optical density (SOD) cutoff of 0.75. Overall concordance of >94% was observed between 3A11-LS and modified rapid tests (RT-LSs) for detecting and distinguishing recent HIV seroconversion from long-term HIV infection (kappa statistics = 0.894 to 0.901). Moreover, intensity scores on RT-LSs correlated well with median 3A11-LS SOD values (R2 > 0.98). Our results indicate that rapid HIV tests can be modified to detect recent seroconversion with results comparable to those from less sensitive EIA.

1970s and ‘Patient 0’ HIV-1 genomes illuminate early HIV/AIDS history in North America

The emergence of HIV-1 group M subtype B in North American men who have sex with men was a key turning point in the HIV/AIDS pandemic. Phylogenetic studies have suggested cryptic subtype B circulation in the United States (US) throughout the 1970s and an even older presence in the Caribbean. However, these temporal and geographical inferences, based upon partial HIV-1 genomes that postdate the recognition of AIDS in 1981, remain contentious and the earliest movements of the virus within the US are unknown. We serologically screened >2,000 1970s serum samples and developed a highly sensitive approach for recovering viral RNA from degraded archival samples. Here, we report eight coding-complete genomes from US serum samples from 1978–1979—eight of the nine oldest HIV-1 group M genomes to date. This early, full-genome ‘snapshot’ reveals that the US HIV-1 epidemic exhibited extensive genetic diversity in the 1970s but also provides strong evidence for its emergence from a pre-existing Caribbean epidemic. Bayesian phylogenetic analyses estimate the jump to the US at around 1970 and place the ancestral US virus in New York City with 0.99 posterior probability support, strongly suggesting this was the crucial hub of early US HIV/AIDS diversification. Logistic growth coalescent models reveal epidemic doubling times of 0.86 and 1.12 years for the US and Caribbean, respectively, suggesting rapid early expansion in each location. Comparisons with more recent data reveal many of these insights to be unattainable without archival, full-genome sequences. We also recovered the HIV-1 genome from the individual known as ‘Patient 0’ (ref. 5) and found neither biological nor historical evidence that he was the primary case in the US or for subtype B as a whole. We discuss the genesis and persistence of this belief in the light of these evolutionary insights.

Evaluation of Rapid Prenatal Human Immunodeficiency Virus Testing in Rural Cameroon

ABSTRACT Pregnant women (n = 859) in rural Cameroonian prenatal clinics were screened by two rapid human immunodeficiency virus (HIV) antibody tests (rapid tests [RT]) (Determine and Hema-Strip) using either whole blood or plasma. One additional RT (Capillus, HIV-CHEK, or Sero-Card) was used to resolve discordant results. RT results were compared with HIV-1 enzyme immunoassay (EIA) and Western blot (WB) results of matched dried blood spots (DBS) to assess the accuracy of HIV RTs. DBS EIA/WB identified 83 HIV antibody-reactive, 763 HIV antibody-nonreactive, and 13 indeterminate specimens. RT results were evaluated in serial (two consecutive tests) or parallel (two simultaneous tests) testing algorithms. A serial algorithm using Determine and Hema-Strip yielded sensitivity and specificity results of 97.6% and 99.7%, respectively, whereas a parallel RT algorithm using Determine plus a second RT produced a sensitivity and specificity of 100% and 99.7%, respectively. HIV RTs provide excellent alternatives for identifying HIV infection, and their field performance could be monitored using DBS testing strategies.

Comparison of alternative interpretive criteria for the HIV-1 Western blot and results of the Multispot HIV-1/HIV-2 Rapid Test for classifying HIV-1 and HIV-2 infections

BACKGROUNDnHIV-1 Western blot (WB) may be positive in specimens from persons with HIV-2 infection due to cross-reactive antibodies. HIV-1 and HIV-2 infections may be identified using assays designed to differentiate HIV-1 and HIV-2 antibody reactivity.nnnOBJECTIVESnTo evaluate the ability of the current CDC WB criteria, alternative more stringent HIV-1 WB criteria (2 env plus one gag or pol band) and the Multispot HIV-1/HIV-2 Rapid Test to accurately differentiate HIV-1 and HIV-2 infections.nnnSTUDY DESIGNnTwo panels were used to determine the ability of each method to properly classify HIV-1 and HIV-2 infections: an HIV-2 panel (n=114) determined to be HIV-2 antibody-positive by both Multispot and by a validated HIV-2 WB, and 2135 HIV-1/HIV-2 immunoassay repeatedly reactive (IA-RR) specimens from the New York State Department of Health Laboratory (NYS).nnnRESULTSnBy CDC WB criteria, 53 (46.5%) HIV-2 panel specimens were HIV-1 WB positive, 60 (52.6%) were indeterminate, and 1 (0.9%) was negative; the alternative WB criteria re-classified 75.5% of the positives as indeterminate. Among 2135 NYS IA-RR specimens, the alternative WB criteria increased the proportion of indeterminates by 0.8%. Only 6 (0.3%) of the NYS specimens were determined to be HIV-2 infections; all 6 were classified either as HIV-1 positive or indeterminate by both WB criteria, but were classified as HIV-2 (n=4) or HIV-1/2 undifferentiated (n=2) by Multispot.nnnCONCLUSIONSnThe alternative WB criteria classified most of the HIV-2 specimens that were HIV-1 positive by CDC criteria as indeterminate, but also slightly increased the proportion of HIV-1 specimens classified as indeterminate. The WB indeterminate specimens would require further testing or follow-up to resolve the infection status, whereas Multispot directly distinguished HIV-1 from HIV-2.

The use of simple, rapid tests to detect antibodies to human immunodeficiency virus types 1 and 2 in pooled serum specimens

BACKGROUNDnThe use of pooled specimens has been proposed as a means of expanding testing for human immunodeficiency virus (HIV) antibodies in population studies and in blood screening, while reducing laboratory costs.nnnOBJECTIVESnTo develop a strategic specimen pooling method to be used with rapid HIV antibody assays to detect positive specimens and to evaluate its performance in comparison with testing with commercial EIA and WB.nnnSTUDY DESIGNnTwo lateral flow rapid HIV antibody assays, Seroz*Strip HIV-1/2(1) and Determine HIV-1/2, were evaluated for their ability to detect HIV-1 antibodies in serum and/or plasma specimens pooled in sizes ranging from two to 20 following the respective manufacturers protocols. One thousand prospectively collected specimens and 55 seroconversion specimens were prepared in pools of five for evaluation by the two rapid HIV assays.nnnRESULTSnOptimal detection and discrimination of HIV-1 antibody-positive and HIV-1 antibody-negative specimens was observed in pool sizes of five to ten for both assays. The ability of the two rapid assays to detect HIV-1 antibody-positive samples from commercial HIV-1 seroconversion panels contained in the pools was equivalent to that of commercial enzyme immunoassays (EIAs) and Western blot (WB) to detect HIV-1 antibody in the non-pooled samples. Application of the pooling method in prospectively collected specimens yielded excellent concordance with EIA/WB results in both sensitivity (98.88% for Seroz*Strip HIV-1/2, 100% for Determine HIV-1/2) and specificity (99.56% for Seroz*Strip HIV-1/2, 99.45% for Determine HIV-1/2).nnnCONCLUSIONnUse of a pooling strategy with either assay reduced the number of tests required by almost 50% and could provide substantial cost reductions for HIV screening in settings where HIV-1 prevalence is less than 10%.

Rapid Detection and Differentiation of Antibodies to HIV-1 and HIV-2 Using Multivalent Antigens and Magnetic Immunochromatography Testing

ABSTRACT A simplified lateral-flow assay for the detection of antibodies to HIV using magnetic-bead conjugates and multibranched peptides from both HIV-1 and HIV-2 was developed. Magnetic immunochromatography testing (MICT) uses a standard lateral-flow platform that incorporates magnetic-bead conjugates for quantitative measurement of the magnetic field distortion associated with the bound magnetic conjugate (reported as adjusted relative magnetic units [MAR]). The results of the optimized MICT assay were compared to standard enzyme immunoassay (EIA) and Western blotting (WB) results using a blinded 649-member panel of specimens from the United States, Cameroon, and West Africa. The panel was comprised of samples from individuals infected with various HIV-1 subtypes (n = 234) or HIV-2 (n = 65) and HIV-seronegative specimens (n = 350). Additionally, 13 HIV-1 seroconversion panels (total specimens = 85), a worldwide panel containing seven of the major circulating HIV-1 subtypes (n = 18), an HIV-2 panel, an HIV-1/HIV-2 mixed panel, and 100 prospective specimens were tested with completely concordant results. Assay reproducibility (observed MAR) for both intra- and interrun testing was excellent, with coefficients of variation of <12%. MICT can provide a rapid, low-cost method of determining HIV antibody status requiring no subjective interpretations.