Prevalent subtypes and one-year outcomes of an HIV-cohort from an urban Philippine center.

ABSTRACT: Circulating HIV subtypes in the Philippines have increasingly diversified, potentially affecting treatment. We monitored outcomes of a treatment-naïve cohort and their virus subtype prevalence.Retrospective/prospective study cohort.HIV-I-REACT clinic patients co-enrolled in the Virology Quality Assurance Program (RUSH-VQA) from 7/2017-6/2019 were included. Relevant demographic and laboratory information were collected. The ViroSeq HIV-1 Genotyping System v.3 and HIV-1 Integrase Genotyping Kit identified protease-reverse transcriptase and integrase drug resistance mutations (DRM). Sequence subtyping followed using the Stanford University Drug Resistance Database and the REGA HIV-1 Subtyping Tool v.3. The jpHMM HIV-1 Tool and REGA HIV-1 Subtyping Tool provided additional subtype analysis of this cohort's 5'LTR-VIF regions after Sanger sequencing. One-year outcomes included virologic suppression, mortality, and follow-up.86/88 patients were males. Median age was 30 (range 19-65) years; 61/88 were MSM. 15/85 carried baseline DRM. ViroSeq-generated sequences included subtypes CRF01_AE (66/85), B (14/85), and newer recombinants (4/85). Extensive sequencing (n = 71) of the 5'-LTR-GAG-Pol genes showed CRF01_AE (n = 50), subtype B (n = 7), and other recombinants (n = 13). Bootstrap analysis identified 7 pairs of highly related strains. Discordant DRM appeared in 2/7 pairs, where 1/2 strains displayed DRM. After 1 year, 87 individuals were alive, with 19 lost to care. Viral load (VL) was repeated for only 31/77 (40.2%). Follow-up CD4 testing for 39/77 (50.6%) showed an increase to a median of 327 cells/mm3.Our cohort currently carries subtype CRF01_AE (∼68%-70%), followed by subtype B and CRF01_AE/B recombinants. Outcomes were favorable, regardless of subtype after 1 year on cART.

Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing.

The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) established a robust proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. While many of the lessons learned during the development of such programs may also apply to next generation sequencing (NGS)-based HIVDR assays, challenges remain for the ongoing evaluation of NGS-based testing. These challenges include a proper assessment of assay accuracy and the reproducibility of low abundance variant detection, intra- and inter-assay performance comparisons among laboratories using lab-defined tests, and different data analysis pipelines designed for NGS. In collaboration with the World Health Organization (WHO) Global HIVDR Laboratory Network and the Public Health Agency of Canada, the Rush VQA program distributed archived proficiency testing panels to ten laboratories to evaluate internally developed NGS assays. Consensus FASTA files were submitted using 5%, 10%, and 20% variant detection thresholds, and scored based on the same criteria used for SS. This small study showed that the SS External Quality Assurance (EQA) approach can be used as a transitional strategy for using NGS to generate SS-like data and for ongoing performance while using NGS data from the same quality control materials to further evaluate NGS assay performance.

The stability of HIV-1 nucleic acid in whole blood and improved detection of HIV-1 in alternative specimen types when compared to Dried Blood Spot (DBS) specimens.

BACKGROUND: Commercially-available kits for HIV-1 detection include instructions for detecting HIV-1 in plasma and DBS, but don't support other specimen types. OBJECTIVES: Show quantitative stability of HIV-1 total nucleic acid (TNA) in blood and improved HIV-1 detection in alternative specimen types. STUDY DESIGN: Whole blood and DBS specimens, tested as part of an external quality assurance program for qualitative HIV-1 detection, were used to evaluated error rates (false negative [FN], false positive [FP] and indeterminant [IND] results) across assays (internally developed [IH], Roche Amplicor [RA], and Roche TaqMan Qual [TQ]) and specimen types (frozen whole blood [BLD], DBS and cell pellets [PEL]). A modified Roche TaqMan HIV-1 assay was used to quantify HIV-1 TNA. RESULTS: Significantly higher error rates were noted in DBS across all of the assays (4% vs. 0% for DBS and PEL, IH, p = 0.005; 4% vs. 0.1% for DBS and PEL, RA, p < 0.001; 10% vs. 1% for DBS and PEL or BLD, TQ, p < 0.001). HIV TNA concentration is stable in BLD (day 1 vs. day 10, p = 0.39) and higher than DBS (p < 0.001). CONCLUSIONS: Transporting refrigerated whole blood for centralized processing into alternative specimen types will improve the sensitivitiy of HIV-1 detection in samples with low virus loads.

Use of External Quality Control Material for HIV-1 RNA Testing To Assess the Comparability of Data Generated in Separate Laboratories and the Stability of HIV-1 RNA in Samples after Prolonged Storage.

The National Institute of Allergy and Infectious Diseases (NIAID) AIDS Clinical Trials Group (ACTG) stores specimens from its clinical trials in a biorepository and permits the use of these specimens for nonprotocol exploratory studies, once the studies for the original protocol are concluded. We sought to assess the comparability of the data generated from real-time HIV-1 RNA testing during two clinical trials with the data generated from the retesting of different aliquots of the same samples after years of storage at -80°C. Overall, there was 92% agreement in the data generated for 1,570 paired samples (kappa statistic = 0.757; 95% confidence interval [CI], 0.716 to 0.797), where samples were tested in one laboratory using the microwell plate (MWP) version of the Roche HIV-1 Monitor test within 1 to 37 days of collection and retested in another laboratory using the Cobas version of the assay after a median of 6.7 years of storage (range, 5.7 to 8.6 years). Historical external quality control data submitted to the NIAID Virology Quality Assurance program (VQA) by client laboratories using the same two versions of the Monitor assay were used to differentiate between systematic differences in the assays to evaluate the stability of HIV-1 RNA in the stored samples. No significant loss of RNA was noted in samples containing either a low concentration (<50 copies/ml) or a high concentration (≥50 copies/ml) of HIV-1 RNA (P = 0.10 and P = 0.90, respectively) regardless of the time in storage. These data confirm the quality of the plasma samples in the ACTG biorepository following long-term storage.

Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms Confirms the Virologic Failure Endpoint of 200 Copies per Milliliter Despite Improved Assay Sensitivity.

Discrepancies between HIV-1 RNA results assayed by different FDA-approved platforms have been reported. Plasma samples collected from 332 randomly selected clinical trial participants during the second year of antiretroviral treatment were assayed with three FDA-approved platforms: UltraSensitive Roche Amplicor Monitor, v1.5 (Monitor), the Abbott RealTime HIV-1 test on the m2000 system (Abbott), and the Roche TaqMan HIV-1 test, v2.0 (TaqMan). Samples from 61 additional participants with confirmed HIV-1 RNA levels of >50 copies/ml during trial follow-up were also included. Endpoints were HIV-1 RNA quantification of ≤50 copies/ml versus >50 copies/ml at an individual-sample level (primary) and determination of confirmed virologic failure (VF) from longitudinal samples. A total of 389 participants had results obtained from all assays on at least one sample (median = 6). Proportions of results of >50 copies/ml were 19% (Monitor), 22% (TaqMan), and 25% (Abbott). Despite indication of strong agreement (Cohen's kappa, 0.76 to 0.82), Abbott was more likely to detect HIV-1 RNA levels of >50 copies/ml than Monitor (matched-pair odds ratio [mOR] = 4.2; modified Obuchowski P < 0.001) and TaqMan (mOR = 2.1; P < 0.001); TaqMan was more likely than Monitor (mOR = 2.6; P < 0.001). Despite strong agreement in classifying VF across assay comparisons (kappa, 0.75 to 0.92), at a 50-copies/ml threshold, differences in the probability of VF classification (in the same direction as primary) were apparent (all McNemar's P < 0.007). At a 200-copies/ml VF threshold, no differences between assays were apparent (all P > 0.13). Despite strong agreement among assays, significant differences were observed with respect to detecting HIV-1 RNA levels of >50 copies/ml and identifying VF at the 50-copies/ml threshold. This has important implications for the definition of VF in clinical trials and clinical practice.

Cross-platform analysis of HIV-1 RNA data generated by a multicenter assay validation study with wide geographic representation.

HIV-1 RNA quantitation continues to be extremely important for monitoring patients infected with HIV-1, and a number of assays have been utilized for this purpose. Differences in assay performance with respect to log(10) recovery and HIV-1 subtype specificity have been well documented for commercially available assays, although comparisons are usually limited to one or two assay platforms. Two new FDA-approved assays, the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test (RT) and the Abbott RealTime HIV-1 assay (AR), that utilize real-time PCR have replaced previous HIV-1 RNA platforms. Inadequate detection of some strains of HIV-1 resulted in the addition of a new primer/probe set and the introduction of a second version of the RT assay. In this study, comparisons of assay performance between the different FDA-approved HIV-1 RNA assay platforms (both new and existing) were performed by using validation data that included both well-characterized virus stock and locally collected clinical samples. Laboratories across diverse geographical regions performed the validation testing and submitted data to the Virology Quality Assurance program (VQA) for analysis. Correlation values for clinical sample testing varied across the assay platforms (r = 0.832 to 0.986), and average log(10) recoveries for HIV-1 RNA controls (compared to the nominal value) ranged from -0.215 to 0.181. These data demonstrate the need for use of one assay platform for longitudinal patient monitoring, but the data also reinforce the notion that no one assay is superior and that testing across platforms may be required for discordance reconciliation.

Feasibility of Repurposing the Polyanionic Microbicide, PPCM, for Prophylaxis against HIV Transmission during ART.

HIV-serodiscordant couples wishing to conceive often seek assisted reproduction, during which spermatozoa from infected men are washed to minimize the risk of HIV transmission to partner and fetus. We sought to improve this method by adding a microbicide, PPCM, as an HIV prophylactic. HIV-1 (BaL) inhibition by PPCM appears irreversible and independent of added Ca(2+). Without added Ca(2+), PPCM (≤10 mg/mL, ≤90 min), a stimulus of Ca(2+)-dependent acrosomal loss, has no effect on sperm motility, forward progression, or acrosomal status. PPCM-treated (10 mg/mL) sperm retain their ability to acrosome react when Ca(2+) is added. Sperm DNA integrity/function is unaffected by PPCM (≤10 mg/mL). Adding PPCM (5 mg/mL, 30 min) to washing media reduces infectivity (viral antigen p24 and RNA) of ex-vivo HIV-infected semen by 3-4 Logs compared with washing alone. Sperm washing with appropriate extracellular Ca(2+) levels and PPCM is significantly more effective than washing alone at reducing HIV infectivity.

Multilaboratory evaluation of real-time PCR tests for hepatitis B virus DNA quantification.

The performance characteristics of four different assays for hepatitis B virus (HBV) quantification were assessed: the Abbott RealTime HBV IUO, the Roche Cobas AmpliPrep/Cobas TaqMan HBV test, the Roche Cobas TaqMan HBV test with HighPure system, and the Qiagen artus HBV TM ASR. Limit of detection (LOD), linear range, reproducibility, and agreement were determined using a serially diluted plasma sample from a single chronically infected subject. Each assay was tested by at least three laboratories. The LOD of the RealTime and two TaqMan assays was approximately 1.0 log(10) IU/ml; for artus HBV (which used the lowest volume of extracted DNA), it was approximately 1.5 log(10) IU/ml. The linear range spanned 1.0 to at least 7.0 log(10) IU/ml for all assays. Median values were consistently lowest for artus HBV and highest for Cobas AmpliPrep/Cobas TaqMan HBV. Assays incorporating automated nucleic acid extraction were the most reproducible; however, the overall variability was minor since the standard deviations for the means of all tested concentrations were ≤0.32 log(10) IU/ml for all assays. False-positive results were observed with all assays; the highest rates occurred with tests using manual nucleic acid extraction. The performance characteristics of these assays suggest that they are useful for management and therapeutic monitoring of chronic HBV infection.

Complex mosaic composition of near full-length genomes of two NED (NIH-ENVA-DOD) subtype panel HIV type 1 strains, BCF-Dioum and BCF-Kita, originating from the Democratic Republic of Congo (DRC).

Sequence characterization of the near full-length genomes of HIV-1 isolates BCF-Dioum and BCF-Kita, originating from the Democratic Republic of Congo (DRC), was continued. These NED panel isolates, contributed by F. Brun-Vezinet (ENVA-France), were first identified as subtypes G and H, respectively. Our earlier analyses of portions of their pol genes showed that both were likely to be intersubtype recombinants of different composition. This study analyzed the remainder of each genome, confirming them to be complex recombinants. The BCF-Dioum genome resembles CRF06_cpx strains found in West Africa, composed of subtypes A/G/J/K. The BCF-Kita genome is a unique complex recombinant A-F-G-H-K-U strain. These data support previous observations of the complexity of strains originating from the DRC. BCF-Dioum may be a suitable strain for standards and reagents since it matches a defined circulating recombinant form. Studies and reagents made from BCF-Kita should take into account its complex genome.

Multisite comparison of anti-human immunodeficiency virus microbicide activity in explant assays using a novel endpoint analysis.

Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.

Rapid decline in the efficiency of HIV drug resistance genotyping from dried blood spots (DBS) and dried plasma spots (DPS) stored at 37 degrees C and high humidity.

OBJECTIVES: Dried blood spots (DBS) and dried plasma spots (DPS) are considered convenient alternatives to serum and plasma for HIV drug resistance testing in resource-limited settings. We sought to investigate how extreme conditions could affect the short-term ability to amplify and genotype HIV from DBS. METHODS: A panel of six matched DPS/DBS was generated using blood collected from HIV-infected donors. Replicate cards were prepared in 903 filter paper using 50 microL of blood and stored at either -20 degrees C or at 37 degrees C/100% humidity. Nucleic acids were extracted at baseline and after 1, 2, 8 and 16 weeks of storage and were amplified and sequenced using an in-house RT-nested PCR method or the ViroSeq assay. RESULTS: HIV-1 pol was successfully amplified in all DBS/DPS at baseline and in those stored for up to 16 weeks at -20 degrees C by the in-house assay. In contrast, amplification was rapidly lost during storage at 37 degrees C/100% humidity with only 6/6 and 4/6 DBS specimens amplifiable by the in-house assay at weeks 1 and 2, respectively. Similarly, only two DPS stored at 37 degrees C/100% humidity were amplified by the in-house assay at week 1. CONCLUSIONS: We show that resistance testing from DBS and DPS is severely compromised after 2 and 1 weeks of storage at 37 degrees C/100% humidity with desiccant, respectively. These findings underscore the importance of temperature and humidity for the efficient genotyping of HIV-1 from DBS and DPS, and reiterate the need to rapidly transport specimens from collection sites to locations that have appropriate storage conditions such as -20 degrees C.

Diminished human immunodeficiency virus type 1 DNA yield from dried blood spots after storage in a humid incubator at 37 degrees C compared to -20 degrees C.

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at -20 degrees C. DBS were created by spotting 50-microl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at -20 degrees C or at 37 degrees C with approximately 85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at -20 degrees C, and 398 stored at 37 degrees C. A chi-square test showed fewer positive reactions for DBS stored at 37 degrees C (55%) than for those stored at -20 degrees C (78%) (P < 0.0001). Samples stored at -20 degrees C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37 degrees C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37 degrees C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.

Human cytomegalovirus and human immunodeficiency virus type-1 co-infection in human cervical tissue.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type-1 (HIV-1) infect the female genital tract. A human cervical explant model was developed to study single and dual infection by these viruses in the genital compartment. An HCMV strain expressing green fluorescent protein, and two clinical HCMV strains produced peak viral DNA copies at 14 to 21 days post-infection. Peak levels of HIV-1(Ba-L) p24 antigen occurred at 7 days post-infection. HIV-1(Ba-L) appeared to enhance HCMV in co-infected tissues. Singly and dually infected explants produced increased levels of cytokines IL-6, IL-8, and GRO-alpha in culture supernatants. Immunohistochemical and flow cytometric analysis showed HCMV infection of leukocytes with the phenotype CD45+/CD1a+/CD14+/HLA-DR+ but not stromal or endothelial cells. Cells expressing both GFP and HIV-1 p24 antigen were detected in co-infected tissues. The cervical explants provide an ex vivo human model for examining mechanisms of virus-virus interaction and pathogenesis in clinically relevant tissue.

Evaluation of real-time PCR laboratory-developed tests using analyte-specific reagents for cytomegalovirus quantification.

Viral load testing for cytomegalovirus (CMV) has become the standard for the diagnosis of infection and monitoring of therapy at many transplant centers. However, no viral load test has been approved by the FDA. Therefore, many laboratories rely on laboratory-developed assays. This study evaluated the performance characteristics of two real-time PCR tests developed using the artus CMV analyte-specific reagents (ASRs). One version is distributed by Abbott Molecular and the other by QIAGEN. For plasma specimens, the Abbott test had a limit of detection of 2.3 log10 copies/ml and a linear range up to at least 6.0 log10 copies/ml. Comparison of plasma viral loads using the Abbott test and the Roche Amplicor Monitor test showed a mean difference of -0.012 log10 copies/ml. In addition, the Abbott test viral loads correlated with the Digene Hybrid Capture assay ratios. Viral loads obtained from plasma specimens tested by the Abbott and QIAGEN tests were in very close agreement (mean difference, 0.144 log10 copies/ml). When the QIAGEN test was evaluated with the QIAGEN, MagNA Pure, and easyMAG extraction methods, the viral loads for all three methods were within 0.370 log10 copies/ml. Thus, there is good agreement between viral loads obtained by the different tests using the same extraction method or by the same test using different extraction methods. The availability of real-time PCR ASRs provides additional reagents that can be used for CMV viral load testing.

Evaluation of dried blood spots for human immunodeficiency virus type 1 drug resistance testing.

Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at -20 degrees C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at -20 degrees C or at -70 degrees C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that -20 degrees C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.

Saliva can mediate HIV-1-specific antibody-dependent cell-mediated cytotoxicity.

HIV is not usually transmitted by saliva from HIV-1-infected individuals. Antiviral substances in saliva responsible for this may include HIV-1-specific antibody-dependent cell-mediated cytotoxicity (ADCC). We evaluated saliva ADCC titers of 62 HIV-1-infected women from the Women's Interagency HIV Study (WIHS) and 55 uninfected individuals. HIV-1-infected women were less likely to have ADCC activity in saliva than in serum or cervical lavage fluid (CVL). 24% of HIV-1-positive women and a similar percentage of uninfected women had HIV-1-specific saliva ADCC activity. A significant amount of saliva ADCC activity in infected women was HIV-gp120-specific. These studies demonstrate that HIV-specific ADCC activity can be present in saliva. This activity may contribute to host defence against initial infection with HIV.

Comparison of two human immunodeficiency virus (HIV) RNA surrogate assays to the standard HIV RNA assay.

Human immunodeficiency virus (HIV) RNA testing is the gold standard for monitoring antiretroviral therapy in HIV-infected patients. However, equipment and reagent costs preclude widespread use of the assay in resource-limited settings. The Perkin-Elmer Ultrasensitive p24 assay and the Cavidi Exavir Load assay both offer potentially simpler, less costly technologies for monitoring viral load. These assays were compared to the Roche Amplicor HIV-1 Monitor Test, v1.5, using panels of clinical samples (subtype B) from HIV-positive subjects and HIV-spiked samples (subtypes A, C, D, CRF_01AE, CRF_02AG, and F). The Ultrasensitive p24 assay detected 100% of the spiked samples with virus loads of >250,000 copies/ml and 61% of the clinical samples with virus loads of 219 to 288,850 copies/ml. Detection rates were improved substantially if an external lysis buffer was added to the procedure. The Cavidi assay detected 54 to 100% of spiked samples with virus loads >10,000 copies/ml and 68% of the clinical samples. These detection rates were also greatly improved with a newly implemented version of this kit. Coefficients of variation demonstrate good reproducibility for each of these kits. The results from the Cavidi v1.0, Cavidi v2.0, and Perkin-Elmer, and the Perkin-Elmer Plus external buffers all correlated well with the results from the Roche Monitor Test (r = 0.83 to 0.96, r = 0.84 to 0.99, r = 0.58 to 0.67, and r = 0.59 to 0.95, respectively). Thus, the use of these two assays for monitoring patients, together with less-frequent confirmation testing, offers a feasible alternative to frequent HIV RNA testing in resource-limited settings.

Model for assessment of proficiency of human immunodeficiency virus type 1 sequencing-based genotypic antiretroviral assays.

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of > or =98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.

Stability of human immunodeficiency virus type 1 proviral DNA in whole-blood samples.

The Roche Amplicor HIV-1 Test requires that whole blood be processed within four days of collection. However, this requirement may be too limiting for use in international settings. Thus, we demonstrate that blood may be processed up to 10 days after collection if maintained under ambient conditions (2 to 25 degrees C).

Women with cervicovaginal antibody-dependent cell-mediated cytotoxicity have lower genital HIV-1 RNA loads.

Antibodies that mediate human immunodeficiency virus (HIV)-specific antibody-dependent cell-mediated cytotoxicity (ADCC) are present in the cervical fluid of many HIV-positive women; however, the role that these antibodies play in host defense against HIV is not known. To understand the contribution of ADCC in cervical secretions as a protective mechanism against HIV, we evaluated ADCC titers in paired serum and cervical-lavage (CVL) samples from >300 HIV-1-positive women who participated in the multicenter Division of AIDS Treatment Research Initiative Study 009. The present study demonstrates that women with CVL ADCC activity had lower genital viral loads than did women with serum ADCC activity only. Women with CVL ADCC activity were likely to have HIV-1 gp120-specific immunoglobulin (Ig) G, but not IgA, in their cervical fluid. This finding suggests that specific IgG in cervical fluid can mediate ADCC activity that inversely correlates with genital viral load.