Effect of Shipment, Storage, Anticoagulant, and Cell Separation on Lymphocyte Proliferation Assays for Human Immunodeficiency Virus-Infected Patients

Lymphocyte proliferation assays (LPA), which can provide important information regarding the immune reconstitution of human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral therapy, frequently involve shipment of specimens to central laboratories. In this study, we examine the effect of stimulant, anticoagulant, cell separation, storage, and transportation on LPA results. LPA responses of whole blood and separated peripheral blood mononuclear cells (PBMC) to different stimulants (cytomegalovirus, varicella-zoster virus, candida and tetanus toxoid antigens, and phytohemagglutinin) were measured using fresh specimens shipped overnight and frozen specimens collected in heparin, acid citrate dextrose (ACD), and citrate cell preparation tubes (CPT) from 12 HIV-infected patients and uninfected controls. Odds ratios for positive LPA responses were significantly higher in separated PBMC than in whole blood from ACD- and heparin-anticoagulated samples obtained from HIV-infected patients and from ACD-anticoagulated samples from uninfected controls. On separated PBMC, positive responses were significantly more frequent in fresh samples compared with overnight transportation for all antigens and compared with cryopreservation for the candida and tetanus antigens. In addition, viral antigen LPA responses were better preserved in frozen PBMC compared with specimens shipped overnight. CPT tubes yielded significantly more positive LPA results for all antigens, irrespective of the HIV patient status compared with ACD, but only for the candida and tetanus antigens and only in HIV-negative controls compared with heparin. Although HIV-infected patients had a significantly lower number of positive antigen-driven LPA responses compared with uninfected controls, most of the specimen processing variables had similar effects on HIV-positive and -negative samples. We conclude that LPA should be performed on site, whenever feasible, by using separated PBMC from fresh blood samples collected in either heparin or ACD. However, if on-site testing is not available, optimal transportation conditions should be established for specific antigens.     

Assessing Human Immunodeficiency Virus Type 1 Tropism: Comparison of Assays Using Replication-Competent Virus versus Plasma-Derived Pseudotyped Virions

Detection of CXCR4-using human immunodeficiency virus by the Trofile assay was compared to that by assays using virus isolates or replication-competent recombinants. Concordance with the Trofile assay was good, but assays using replicating viruses did not increase substantially the ability to detect the presence of CXCR4-using virus.Human immunodeficiency virus type 1 (HIV-1) can be assigned to one of three classes based on its ability to utilize the CCR5 and CXCR4 coreceptors: viruses that use CCR5 but not CXCR4 (R5 virus), those that use CXCR4 but not CCR5 (X4 virus), and those that can use either coreceptor (dualtropic virus). HIV-1 also can be classified according to its ability to replicate and induce syncytia in MT-2 cells (8, 12). The use of CXCR4 is a defining feature of syncytium-inducing (SI) viruses in MT-2 cells; most but not all non-SI (NSI) viruses are R5 (1, 13, 16).Testing to determine coreceptor usage of HIV-1 isolates is essential to identify patients who are suitable candidates for treatment with CCR5 antagonists. The tropism assay (Trofile; Monogram BioSciences, South San Francisco, CA) used in clinical trials of CCR5 antagonists to date is a validated single-cycle assay performed in a Clinical Laboratory Improvement Amendments/College of American Pathologists-certified laboratory; the assay is based on pseudotyped virus and sensitively detects the presence of CXCR4-using virus (14). However, up to 10% of subjects identified as having exclusively R5 virus at screening had evidence of dualtropic or mixed-tropic (D/M) virus at the time treatment with maraviroc or vicriviroc (VCV) was begun (3, 4). To test the hypothesis that the sensitivity of tropism testing could be improved by use of replicating viruses instead of pseudotyped viruses, we compared the results of tropism testing with the Trofile assay to those of assays using replication-competent viruses using clinical samples from AIDS Clinical Trials Group (ACTG) protocol A5211, a phase 2b trial of the investigational CCR5 antagonist VCV (SCH-D; SCH417690; Schering-Plough, Kenilworth, NJ) (4).(This work was presented in part at the 14th Conference on Retroviruses and Opportunistic Infections, Los Angeles, CA, 25-28 February 2007 [5a].)     

Reduction of Diagnostic Window by New Fourth-Generation Human Immunodeficiency Virus Screening Assays

In order to reduce the diagnostic window between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new screening enzyme-linked immunosorbent assays (ELISAs) which permit the simultaneous detection of HIV antigen and antibody have been developed. Two fourth-generation assays, HIV DUO (Biomérieux) and HIV Combi (Boehringer Mannheim), for the combined detection of HIV antigen and antibody, were compared with a third-generation assay (HIV-1/HIV-2 3rd Generation Plus enzyme immunoassay [EIA]; Abbott) and a p24 antigen test (HIV-1 Ag monoclonal; Abbott). A total of 17 seroconversion panels, 15 cell culture supernatants infected with different HIV type 1 (HIV-1) subtypes, and 255 potentially cross-reactive serum samples were tested. Ten seroconversions were detected an average of 8.1 days earlier with HIV DUO and 7.5 days earlier with HIV Combi than with the third-generation ELISA. Overall, in the 17 seroconversion panels tested, HIV DUO detected HIV-1 infection an average of 4.8 days and HIV Combi detected infection an average of 4.4 days earlier than HIV-1/HIV-2 3rd Generation Plus EIA. HIV antigen was detected with HIV DUO and HIV Combi in all of the 15 cell culture supernatants infected with different HIV-1 subtypes, including subtype O. With fourth-generation assays, considerably fewer false-positive results (n = 4 to 6) were obtained, in comparison with the third-generation EIA (n = 18). Fourth-generation assays permit an earlier diagnosis of HIV infection than third-generation antibody screening assays through the detection of p24 antigen, which may be present in serum samples from individuals with recent HIV infection prior to seroconversion.     

Comparison of NucliSens and Roche Monitor Assays for Quantitation of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma

We compared the performance of Organon Teknika’s NucliSens and Roche Diagnostic Systems’ Monitor quantitative human immunodeficiency type 1 RNA assays. Both had similar linearity and sensitivity over most of the dynamic range of the assays, although the Monitor assay was superior at the low range of RNA values while the NucliSens assay was more consistent at higher RNA values. NucliSens generally showed less interassay variability.     

Early Detection of Human Immunodeficiency Virus Infection Using Third- and Fourth-Generation Screening Assays

 Early detection of infection with human immunodeficiency virus (HIV) is critical for clinical diagnosis and treatment of patients, as well as for ensuring the safety of blood transfusion products. Recently, a number of fourth-generation HIV screening assays have been developed that offer increased sensitivity over earlier tests by combining detection of anti-HIV antibodies with detection of the p24 viral antigen. Previously, six different HIV assays were compared against a broad range of 30 seroconversion panels. In the present study, three of the newer fourth-generation assays were tested together with three of the third-generation HIV antibody-only assays. This extensive analysis highlights (i) the importance of p24 antigen detection for early diagnosis, (ii) the improved sensitivity of fourth-generation assays over antibody-only tests, and (iii) the superior performance of the Vidas Duo assay, which allows reduction of the diagnostic window by up to 2 weeks. Finally, the results emphasize the detection limitations of the different assays and suggest improvements for future HIV screening assays.     

Human T Lymphocyte Differentiation Antigens as Target for Immunotoxins or Complement-Mediated Cytotoxicity

Graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) is initiated by immunocompetent T cells present in the graft. Selective elimination of distinct T-cell subsets or a sufficient, but not complete T-cell depletion, might abolish severe GVHD without graft rejection and loss of the anti-tumour potential. In this study we analysed the efficacy of different monoclonal antibodies (MoAb) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7), and WT82 (CD8) with respect to their cytotoxicity to T cells either as immunotoxin (IT) or in combination with complement. The cytotoxic potential was assessed by protein synthesis inhibition and clonogenic assays. The ricin A conjugated MoAb exerted only a minor effect on blood or bone marrow T cells, although they were highly inhibitory to T-cell lines. However, in the presence of 20 mM ammonium chloride, IT directed against CD3, CD5, and CD7 were highly cytotoxic. IT directed against CD4 and CD8 were less effective, due to a low internalization. The complement-mediated cytotoxicity was efficient for all antigens used. The natural killer (NK) activity, as measured by cytotoxicity to K562, was hardly depressed by anti-CD3, anti-CD4, anti-CD5, and anti-CD8, but was eliminated by anti-CD7. All procedures used had only a minimal effect on haematopoietic progenitors as measured by CFU-GM and BFU-E assays. We concluded that, although the T-cell population can be eliminated with the combination of anti-CD3, anti-CD5, and anti-CD7 antibodies plus complement, IT with 20 mM NH4Cl appear to kill higher amounts of T cells. Selective elimination of CD4- and CD8-positive cells is effectively obtained by MoAb with complement.     

New Automated Chemiluminescence Immunoassay for Simultaneous but Separate Detection of Human Immunodeficiency Virus Antigens and Antibodies

The recently launched Liaison XL Murex HIV Ab/Ag assay (DiaSorin S.p.A) uses chemiluminescence immunoassay technology for the combined qualitative determination of p24 antigen of HIV-1 and specific antibodies to both HIV-1 and HIV-2. We studied 571 serum samples from those submitted to our laboratory for HIV screening. The samples were divided into 3 subsets: subset A, 365 samples collected prospectively during 1 week; subset B, 158 samples from confirmed HIV-positive patients; and subset C, 48 samples with a positive screening result but a negative or indeterminate confirmatory test result. Our standard screening/confirmatory algorithm was used as a reference. In subset A (prospective), 5 samples were positive and 360 negative by the standard procedure. Liaison XL Murex HIV Ab/Ag correctly identified all 5 positive samples (100%) and 357 negative samples (99.2%). In subset B (confirmed positive), all 158 positive samples were in total agreement in both procedures. In subset C (screen positive only), Liaison XL Murex HIV Ab/Ag yielded accurate results in 42 out of 48 samples (87.5%). Global sensitivity and specificity for Liaison XL Murex HIV Ab/Ag (all subsets included) were 98.3% and 98.5%, respectively. Considering only nonselected prospective samples and confirmed positive samples (subsets A and B), the corresponding sensitivity and specificity values were 100% and 99.2%, respectively. The new fully automated HIV screening test showed high sensitivity and specificity compared to our standard algorithm. Its added advantage of being able to detect HIV-1 and HIV-2 antibodies and p24 antigen separately could prove useful in the diagnosis of early infections.     

Localization of Human Immunodeficiency Virus Antigens in Infected Cells by Scanning/Transmission-Immunogold Techniques

An application of high resolution scanning/transmission electron microscopy (STEM) and gold-labeling techniques for the rapid detection of human immunodeficiency virus (HIV) in infected cells has been developed. Experimental in vitro studies for detecting two HIV structural proteins, gp41 and p17, were performed following an indirect labeling procedure that uses monoclonal anti-p17 and anti-gp41 antibodies as primary antibodies and 40 nm gold-linked goat antimouse IgG as secondary antibodies. The cells were then studied by STEM in the scanning mode. Unambiguous localization of the viral antigens was possible by combining the three-dimensional image provided by the secondary electron image and the atomic number-dependent backscattered electron image for the identification of the gold marker. This technique combines both the morphological information and the rapid procedures of scanning electron microscopy with the precise and sensitive antigen detection provided by the use of STEM and immunological methods. The preliminary results of its application to the study of peripheral blood mononuclear cells from four anti-HIV-seropositive patients showing the presence of specific labeling in all of them suggest that it might prove useful for early detection of HIV infection before seroconversion, as well as for quantitative studies.     

Localization of Human Immunodeficiency Virus Antigens in Infected Cells by Scanning/Transmission-Immunogold Techniques

An application of high resolution scanning/transmission electron microscopy (STEM) and gold-labeling techniques for the rapid detection of human immunodeficiency virus (HIV) in infected cells has been developed. Experimental in vitro studies for detecting two HIV structural proteins, gp41 and p17, were performed following an indirect labeling procedure that uses monoclonal anti-p17 and anti-gp41 antibodies as primary antibodies and 40 nm gold-linked goat antimouse IgG as secondary antibodies. The cells were then studied by STEM in the scanning mode. Unambiguous localization of the viral antigens was possible by combining the three-dimensional image provided by the secondary electron image and the atomic number-dependent backscattered electron image for the identification of the gold marker. This technique combines both the morphological information and the rapid procedures of scanning electron microscopy with the precise and sensitive antigen detection provided by the use of STEM and immunological methods. The preliminary results of its application to the study of peripheral blood mononuclear cells from four anti-HIV-seropositive patients showing the presence of specific labeling in all of them suggest that it might prove useful for early detection of HIV infection before seroconversion, as well as for quantitative studies.     

Intra-Assay Performance Characteristics of Five Assays for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma

Three kits (Roche AMPLICOR human immunodeficiency virus type 1 [HIV-1] Monitor, Chiron enhanced-sensitivity bDNA, and Organon Teknika NASBA HIV-1 QT) and two in-house assays (from National Genetics Institute and Baylor College of Medicine) were compared with a blinded panel. The results were evaluated as to intra-assay sensitivity, precision, and ability to detect differences in a dilution series.     

Neurotropism of Human Immunodeficiency Virus

Three major characteristics of human immunodeficiency virus (HIV) infection define HIV as neurotropic. 1) Clinically, distinct neurological syndromes are associated with HIV infection and 2) presence of the virus as well as 3) pathological changes can be demonstrated in the central nervous system. Spread of HIV to the brain seems to be the general rule. Virus expression appears to be restricted during the asymptomatic period but increases with severity of HIV infection. Whether this reflects the emergence of virus variants with increased replicative capacity in brain cells has yet to be elucidated.     

Comparison of Costs of Strategies for Measuring Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma by Using Amplicor and Ultra Direct Assays

The costs of four algorithms for monitoring plasma human immunodeficiency virus type 1 RNA were compared. For patients with strong virologic responses, the use of Ultra Direct exclusively was the cheapest strategy. For patients with weak virologic responses, small savings could be obtained by using Amplicor and retesting only samples with values below 500 copies/ml with Ultra Direct.     

Quantification of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma by Using Small-Volume-Format Branched-DNA Assays

We have developed small-volume (50 or 250 μl)-format branched-DNA assays for human immunodeficiency virus type 1 (HIV-1) RNA for use with specimens in which the volume is limited and/or a high viral load is anticipated. These formats exhibited good correlation with the standard 1-ml format; high specificity, reproducibility, and linearity; and no significant difference in the quantification of HIV-1 subtypes.     

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.Studies using human tissues cultured ex vivo (i.e., tissue explants) are performed for the preclinical evaluation of topical microbicides, compounds that can be applied vaginally or rectally to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1) (19). Various tissue explant models and the procedures used for evaluating test compounds for anti-HIV activity have been described (1, 2, 4, 7-9, 13, 14). At present, there is no standard methodology, including calculation of an endpoint, for comparing results between ex vivo experiments.Although cell-based assays can be standardized (5, 20), tissue explants may represent a more relevant method of testing, since this is where HIV-1 infection occurs in vivo. Since most parameters of cell-based assays (i.e., size of the virus inoculum, number of target cells, and use of a single endpoint measure) can be standardized, the data are often less variable and more easily reproduced than those obtained in assays with tissue explants. For example, the number of target cells in cell-based assays can be controlled, whereas the concentration and distribution of target cells in explant tissues are highly variable. In addition, explant methods vary across laboratories, and as a consequence, a number of parameters may affect the reliability of the results, including (i) tissue type, (ii) HIV-1 strain or isolate, (iii) culture medium formulation, (iv) size of the virus inoculum, (v) length of virus incubation, (vi) frequency of medium change, (vii) concentration of test compound, (viii) drug treatment period prior to or after viral exposure, (ix) use of controls, and (x) endpoint viral growth measurements.For the reasons described above, a group of microbicide investigators joined a mutual effort to identify an approach for improving the reliability and reproducibility of data from explant studies. To this end, results obtained from different explant models were analyzed using a range of endpoint methods. In general, due to the interdependency of repeated measurements, standard statistical methods (e.g., t test, analysis of variance [ANOVA], Mann-Whitney test, and Kruskal-Wallis test) do not apply in comparing virus growth in the presence of inhibitory compounds. For example, a high level of virus growth on one day in one explant sample is likely to continue to be followed by high-level virus growth throughout the assay period, while a low level of virus growth in a different sample will be followed by low-level growth. In some cases, the relationships between consecutive data points follow certain assumptions, thus allowing statistical modeling techniques to be applied (e.g., repeated-measures ANOVA and generalized linear and mixed models [26]). However, this type of statistical modeling may be outside the expertise of many microbiological research teams, and the inherent assumptions of normal distributions and homogeneity of variance are rarely met for virus growth data. The soft endpoint (SOFT) is presented as a single, summary measure of virus growth to enable direct comparisons between explant experiments.There is currently no consensus on a method to determine a summary measure of virus growth (25). A number of methods, including the area under the virus growth curve (AUC) (22), the slope of the virus growth curve (33), and virus growth at specific time points (1, 7, 8, 15), have been used. The first aim of this study was to compare a selection of alternative, single measures of virus growth, including SOFT, using data from multisite explant studies, as part of a wider effort to improve the quality of explant studies by standardizing the method used to objectively measure HIV-1 replication in tissues (J. E. Cummins, N. Richardson-Harman, J. Bremer, P. Anton, C. Dezzutti, P. Gupta, N. Lurain, L. Margolis, R. Shattock, and P. Reichelderfer, presented at the 2006 Conference on Retroviruses and Opportunistic Infections, Denver, CO).The second aim of this study was to apply SOFT to compare treatment conditions relevant to microbicide research, using the explant methods developed by the participating microbiological laboratories. It should be noted that the goal of this work was to identify those key parameters that would improve the overall reliability and reproducibility of explant studies, as opposed to having the investigators standardize every aspect of their protocols. Seven laboratories used their preferred human tissue type and explant method to infect samples with an in-house or commonly sourced HIV-1 stock in the presence or absence of the same commonly sourced drug. The null hypothesis of no effect of the various treatment conditions on virus growth was tested. When the null hypothesis was rejected, similarities in statistically significant treatment effects across explant methods provided evidence of interlaboratory and/or intermethod reproducibility in explant experimental results. Conversely, differences in treatment effects across explant methods indicated a need for either standardization of explant methodology or thorough characterization of the observed differences. A multisite study design was used to evaluate the conditions that could affect virus growth in explants and subsequent interpretation of the efficacy of a candidate microbicide (PRO 2000). These variables included (i) source/type of assay reagents (virus stocks and medium), (ii) tissue type (cervical, rectal, or tonsil tissue), (iii) how the tissue was cultured, (iv) HIV isolate (HIV-1_Ba-L and clinical isolates of differing clades), and (v) PRO 2000 concentration.     

Performance of Antigens Used in Detecting Delayed-Type Hypersensitivity in Adolescents Infected with the Human Immunodeficiency Virus

We examined the performance of delayed-type hypersensitivity (DTH) antigens employing a new Candida albicans product in a human immunodeficiency virus (HIV)-infected and nonanergic adolescent population. Diameters of induration (in millimeters) for three intradermally applied antigens (C. albicans, tetanus toxoid, and mumps) were compared in a population of HIV-infected 12 to 18 year olds at study entry in a national multicenter study of HIV disease progression. CD4⁺ T-cell counts were measured in quality-controlled laboratories. The influence of past immunization, gender, and clinical status on antigen reactivity was evaluated with contingency table comparisons and relative risk estimation. Nearly one-half of the 123 eligible subjects were untreated, and almost three-quarters were early in HIV disease by clinical indicators. There was no statistically significant difference in reactivity by past immunization status. Candida antigen (CASTA; Greer Laboratories) evoked DTH response in a significantly higher number of males and females at every level of induration (largest P value, 0.049 for male comparisons; all P values, <0.001 for females) and in subjects with early and intermediate HIV disease at every level of induration (all P values, <0.0001) than either tetanus or mumps antigens. No two-antigen combination was as useful as all three antigens across either gender or clinical categories, although candida and tetanus was the most useful two-antigen combination at indurations of <3 mm. The superior performance of a new C. albicans antigen may extend the utility of DTH assessment in monitoring immune function.     

Neuropathology of Human Immunodeficiency Virus Infection

Neuropathology has defined novel HIV-specific diseases at tissue level: HIV encephalitis and HIV leukoencephalopathy. Both occur usually in the later stages of the AIDS infection and consistently demonstrate large amounts of HIV products. In contrast to this HIV-specific neuropathology, HIV-associated neuropathology features unspecific syndromes with disputed relation to HIV infection: myelin pallor, vacuolar myelopathy, vacuolar leukoencephalopathy, lymphocytic meningitis, and diffuse poliodystrophy. All types of neuropathology may contribute to clinical manifestation according to severity, extent, and distribution of lesions, but clinico-pathologic correlation may be poor in the individual case. Neuropathologic and other data suggest two major pathogenetic pathways of HIV-associated CNS damage: First, systemic and local increase of the virus load leads to HIV encephalitis or HIV leukoencephalopathy; this is corroborated by prominent HIV production within such lesions. Second, neuronotoxicity by HIV proteins or factors secreted from infected cells is supported by histological changes of diffuse poliodystrophy and by morphometric loss of frontocortical neurons.