Chou-Pong Pau

Quantitative Detection of Increasing HIV Type 1 Antibodies after Seroconversion: A Simple Assay for Detecting Recent HIV Infection and Estimating Incidence

We have devised a simple enzyme immunoassay (EIA) that detects increasing levels of anti-HIV IgG after seroconversion and can be used for detecting recent HIV-1 infection. Use of a branched peptide that included gp41 immunodominant sequences from HIV-1 subtypes B, E, and D allowed similar detection of HIV-specific antibodies among various subtypes. Because of the competitive nature of the capture EIA, a gradual increase in the proportion of HIV-1-specific IgG in total IgG was observed for 2 years after seroconversion. This was in contrast to results obtained with the conventional EIA using the same antigen in solid phase, which plateaus soon after seroconversion. The assay was used to test 622 longitudinal specimens from 139 incident infections in the United States (subtype B) and in Thailand (subtypes B and E). The assay was also performed with an additional 8 M urea incubation step to assess the contribution of high-avidity antibodies. Normalized optical density (OD-n) was calculated (ODspecimen/ODcalibrator), using a calibrator specimen. An incremental analysis indicated that a cutoff of 1.0 OD-n and a seroconversion period of 160 days offered the best combination of sensitivity and specificity for classifying incident or long-term infections. The urea step increased the seroconversion period to 180 days with similar sensitivity and specificity. Separate analysis of B and E subtype specimens yielded the same optimal OD-n threshold and similar seroconversion periods. The assay was further validated in African specimens (subtypes A, C, and D) where the observed incidence was within 10% of the expected incidence. This assay should be useful for detecting recent HIV-1 infection and for estimating incidence among diverse HIV-1 subtypes worldwide.

Alternative Algorithms for Human Immunodeficiency Virus Infection Diagnosis Using Tests That Are Licensed in the United States

ABSTRACT Serodiagnosis of human immunodeficiency virus (HIV) infection in the United States has traditionally relied on a sequential two-test algorithm: an initial screen with an enzyme immunoassay (EIA) and reflex testing of EIA-reactive specimens with a more specific supplemental test such as Western blotting or immunofluorescence. The supplemental tests are tedious, subjective, and expensive. In addition, there have been major improvements in the performance and accuracy of the EIA tests as well as the introduction of rapid serologic tests (RT) and HIV nucleic acid amplification tests (NAAT). Related to these improvements is the possibility that alternative algorithms using combinations of currently approved HIV tests may function as well as if not better than the current algorithm, with more flexibility, improved accuracy, and lower cost. To this end, we evaluated the performance of 12 currently licensed tests and 1 in-house HIV test (6 EIA, 4 RT, and 3 NAAT) on panels of plasma samples from HIV-infected (n = 621 HIV type 1 [HIV-1] and 34 HIV-2) and uninfected (n = 513) people and of sequential specimens from people early in seroconversion (183 specimens from 15 patients). Test combinations were analyzed in two dual-test (sensitivity-optimized and specificity-optimized) algorithms and in a three-test (tie-breaking) algorithm, and performance was compared to the conventional algorithm. The results indicate that alternative algorithm strategies with currently licensed tests compare favorably with the conventional algorithm in detecting and confirming established HIV infection. Furthermore, there was a lower frequency of discordant or indeterminate results that require follow-up testing, and there was improved detection of early infection.

Performance Characteristics of the Immunoglobulin G-Capture BED-Enzyme Immunoassay, an Assay To Detect Recent Human Immunodeficiency Virus Type 1 Seroconversion

ABSTRACT Recently, we developed an immunoglobulin G (IgG)-capture BED-enzyme immunoassay (BED-CEIA) to identify recent human immunodeficiency virus (HIV) type 1 (HIV-1) seroconversion for use in incidence estimates. We have established an algorithm for its use; developed quality control reagents to monitor the assay; and evaluated its performance for interrun, intrarun, and operator variability. Analysis of 144 individual plates, which involved multiple plate lots and several operators over more than a year, indicated that the coefficients of variation (CVs) were between 10 and 15% for raw optical density (OD) values in the dynamic range between 0.5 and 2.0 OD units; the CVs decreased to 5 to 10% when the OD was normalized (OD-n; OD-n = specimen OD/calibrator OD). The intrarun CVs were generally in the range of 5 to 10% for specimens with ODs <0.5 and less than 5% for specimens with ODs >0.5. The level of concordance between multiple plate lots (n = 6) and multiple operators (n = 7) was quite high (R2 > 0.9). Comparison of the results of the initial and the confirmatory tests with specimens with OD-n values ≤1.5 demonstrated a high degree of correlation (R2 = 0.92); 566 (92%) of 615 of specimens tested in the two modes retained the same classification (recent or long-term infection). The values for those specimens with changed classifications (n = 49) were close to the cutoff (OD-n = 1.0), as expected. The twofold difference in the HIV IgG contents between the controls and the calibrator reagents was exploited to monitor individual plate runs by using a control plot, which was incorporated into the spreadsheet for data entry and run monitoring. This information provides baseline data for the successful transfer of BED-CEIA to other laboratories and the use of BED-CEIA for the detection of recent HIV seroconversion and the calculation of incidence estimates worldwide.

Highly specific V3 peptide enzyme immunoassay for serotyping HIV-1 specimens from Thailand.

ObjectiveTo develop and evaluate a simple V3 peptide-based enzyme immunoassay (EIA) for large-scale serotyping of HIV-1 specimens from Thailand. DesignSerologic reactivities with synthetic peptides derived from the V3 loop of gp120 were used for typing HIV-1 specimens. MethodsSynthetic peptides PND-A and PND-B, derived from the consensus amino-acid sequences of the V3 loop of gp120 from two major genomic variants of HIV-1 in Thailand (A and B), were evaluated in an EIA on 61 Thai HIV-1 sera for which genotypes had been determined by polymerase chain reaction. The peptide EIA was then applied to sera from 188 HIV-1-infected patients, selected in non-random, convenience samples of known risk groups from four geographic regions of Thailand. ResultsThe sensitivities and specificities of PND-A and PND-B were 86% (30 out of 35) and 96% (25 out of 26) and 92% (24 out of 26) and 94% (33 out of 35), respectively, with 100% predictive values of a monoreactive positive test for both peptides. The assay classified 101 specimens as serotype A, 39 as serotype B, eight as serotype AB (dually reactive), and 40 as untypable (non-reactive). Excluding dual reactors and non-reactors, 92% (77 out of 84) of specimens from patients probably infected by sexual contact were serotype A; conversely, 76% (28 out of 37) of injecting drug users were serotype B. ConclusionThe serologic results corroborated previous findings, in a smaller subset of samples, of an apparent segregation of viral subtypes by mode of transmission, suggesting two separate HIV-1 epidemics in Thailand. This peptide EIA could be a valuable epidemiologic tool in determining the dynamics of the rapid spread of HIV-1 in Thailand.

Durable Protection from Vaginal Simian-Human Immunodeficiency Virus Infection in Macaques by Tenofovir Gel and Its Relationship to Drug Levels in Tissue

ABSTRACT A vaginal gel containing 1% tenofovir (TFV) was found to be safe and effective in reducing HIV infection in women when used pericoitally. Because of the long intracellular half-life of TFV and high drug exposure in vaginal tissues, we hypothesized that a vaginal gel containing TFV may provide long-lasting protection. Here, we performed delayed-challenge experiments and showed that vaginal 1% TFV gel protected 4/6 macaques against vaginal simian-human immunodeficiency virus (SHIV) exposures occurring 3 days after gel application, demonstrating long-lasting protection. Despite continued gel dosing postinfection, neither breakthrough infection had evidence of drug resistance by ultrasensitive testing of SHIV in plasma and vaginal lavage. Analysis of the active intracellular tenofovir diphosphate (TFV-DP) in vaginal lymphocytes collected 4 h to 3 days after gel dosing persistently showed high TFV-DP levels (median, 1,810 fmol/106 cells) between 4 and 24 h that exceed the 95% inhibitory concentration (IC95), reflecting rapid accumulation and long persistence. In contrast to those in peripheral blood mononuclear cells (PBMCs) following oral dosing, TFV-DP levels in vaginal lymphocytes decreased approximately 7-fold by 3 days, exhibiting a much higher rate of decay. We observed a strong correlation between intracellular TFV-DP in vaginal lymphocytes, in vitro antiviral activity, and in vivo protection, suggesting that TFV-DP above the in vitro IC95 in vaginal lymphocytes is a good predictor of high efficacy. Data from this model reveal an extended window of protection by TFV gel that supports coitus-independent use. The identification of protective TFV-DP concentrations in vaginal lymphocytes may facilitate the evaluation of improved delivery methods of topical TFV and inform clinical studies.

Prevalence of mutations associated with reduced antiretroviral drug susceptibility among human immunodeficiency virus type 1 seroconverters in the United States, 1993-1998

To assess the prevalence of mutations associated with decreased antiretroviral drug susceptibility, specimens were tested from persons infected with human immunodeficiency virus (HIV) during 1993-1998. Subjects were drug naive and were attending sexually transmitted disease clinics in 6 US cities. All were enrolled consecutively and had tested negative for HIV during the 2 years before enrollment. Plasma specimens from patients having >/=1 reverse transcriptase (RT) or primary protease mutation were tested phenotypically with a recombinant virus assay. Of 99 patients, 6 (6%) had mutations associated with zidovudine resistance, 2 (2%) had mutations associated with nonnucleoside RT inhibitor resistance, and 1 (1%) had a primary protease mutation. Overall, the prevalence of resistance-associated primary mutations was 5%, although high levels of decreased drug susceptibility (IC(50)s >/=10 times that of a reference virus) were observed in just 1%. These findings confirm the transmission of these mutations to drug-naive persons.

Detection of Human Immunodeficiency Virus Type 1 DNA in Dried Blood Spots by a Duplex Real-Time PCR Assay

ABSTRACT A dried blood spot (DBS) is a well-accepted means for the collection, transport, and storage of blood samples for various epidemiologic, serologic, and molecular assays for human immunodeficiency virus (HIV) studies. It is particularly important for mother-to-infant-transmission studies of affected individuals living in remote areas. We have developed a real-time PCR method to detect HIV type 1 (HIV-1) DNA in dried blood spots. A cellular gene, RNase P, was coamplified with the HIV-1 DNA in the same tube to monitor the DNA extraction efficiency and the overall assay performance. Our assay is a one-tube, single-step closed-system assay and uses a dUTP/uracil DNA glycosidase anti-PCR contamination control. The HIV-1 primers and probe were derived from a conserved region of the long terminal repeat. The detection of RNase P is attenuated by lowering the forward and reverse primer concentrations so that its amplification will not overwhelm the HIV-1 amplification and yet will provide a semiquantitative measurement of the quality of the isolated DBS DNA. We examined 103 HIV-1-seropositive and 56 seronegative U.S. adults and found that our assay has a sensitivity of 98.1% (95% confidence interval [CI], 95.5% to 100%) and specificity of 100% (95% CI, 99% to 100%). The positive and negative predictive values are 100% and 96.6%, respectively. This duplex PCR assay may be useful in identifying HIV-1-infected persons, particularly infants born to seropositive mothers in remote areas of the world.

Antiretroviral Resistance Mutations among Pregnant Human Immunodeficiency Virus Type 1–Infected Women and Their Newborns in the United States: Vertical Transmission and Clades

To assess the impact of antiretroviral resistance on perinatal transmission prevention efforts, human immunodeficiency virus type 1 (HIV-1) genotypic resistance testing was done for 220 HIV-1-infected, zidovudine (AZT)-exposed pregnant women and 24 of their infected infants. The women were prospectively enrolled in 4 US cities in 1991-1997. Phylogenetic and sequencing analyses revealed 5 women with non-clade B infections traced to western African origins. AZT-associated mutations were detected in 17.3% of pregnant women, whereas genotypic resistance to nonnucleoside reverse-transcriptase inhibitors and protease inhibitors was infrequent. No significant association was detected between perinatal transmission and the presence of either AZT or nucleoside reverse-transcriptase inhibitor resistance-associated mutations. AZT resistance mutations were detected in 2 (8.3%) neonatal samples, but the mutation pattern was not identical to the mothers. Although no effect of viral resistance on mother-infant transmission was demonstrated, the advent of more-potent drug classes and the potential for the rapid emergence of resistance warrant prospective surveillance.

Human Immunodeficiency Virus (HIV) Subtype Surveillance of African-Born Persons at Risk for Group O and Group N HIV Infections in the United States

A population-based surveillance registry was used to identify human immunodeficiency virus (HIV)-infected persons in the United States at increased risk for group O and group N infections (those born in or near African countries where group O infection has been reported). Of 155 eligible subjects, 37 gave samples. By phylogenetic and serologic analysis, 32 were infected with group M (16 with subtype A, 5 with B, 7 with C, and 1 each with subtypes D, F2, G, and recombinant A/J) and 2 with group O but none with group N virus. For 3, samples could not be typed by serology or amplified by polymerase chain reaction using group M-, O-, or N-specific primers. In the United States, group O HIV infection is uncommon; no case of group N infection was found. African-born persons may have HIV strains typical of their birth country. Ongoing subtype surveillance may allow early identification of novel or emerging HIV strains.

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.