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Thalidomide is used to treat human immunodeficiency virus (HIV)-associated conditions, including aphthous ulcers and wasting syndrome. The safety, tolerability, and pharmacokinetics of a formulation of thalidomide with improved bioavailability in HIV-infected persons was examined in a placebo-controlled, dose-escalating phase 1 study. Subjects with CD4 cell counts of 200-500 cells/mm(3) were enrolled and randomized 3:1 in groups of 12 to receive 50, 100, or 150 mg of thalidomide or matching placebo. Two subjects who received 150 mg of drug and 2 subjects assigned placebo experienced dose-limiting toxicity. Concentrations of thalidomide in the blood increased with escalating dose, but the time to maximum concentration and clearance did not differ across dose cohorts. Previous suggestions of autoinduction of drug metabolism were not confirmed by this study. At the doses studied, thalidomide was tolerated well and had linear pharmacokinetics.

Highly active antiretroviral therapy (HAART) of HIV disease is associated with effective virologic control, immune reconstitution, and clinical improvements. This study addresses the potential for improvements in lymphocyte phenotype and virologic responses of HIV-infected persons with extensive experience with dual nucleoside reverse transcriptase (NRTI) treatment and advanced HIV disease after a change to a potent antiretroviral therapy (NRTI + protease inhibitor). The majority of participants achieved virologic success. There was a median rise in CD4+ lymphocytes of 99 cells/mm(3) by 48 weeks, because of an increase in memory CD4+ cells at 4 and 16 weeks, followed by a later increase in naive CD4+ cells between weeks 16 and 48. The proportion of activated, DR+ CD38+ CD8+ lymphocytes decreased during the 48 weeks of follow-up. The immunologic findings (increased memory and naive T cells and reduced activation levels) were significantly improved in participants with persistent suppression of viral replication over the 48 weeks of the study. Baseline HIV RNA copy number was lower (median, 14,784 copies/ml) in persons who responded virologically than in those not suppressing viral replication (median, 49,454 copies/ml). CD4+ cell counts above the median (125/mm(3)) at time 0 for the participants, was the only baseline immunologic marker significantly associated with viral suppression at week 48. Participants older than 40 years of age demonstrated less immunologic recovery. The results of the study show that patients with extensive experience with NRTIs respond both virologically and immunologically during the first 48 weeks of therapy with a potent antiretroviral regimen.

Evaluation of fetomaternal hemorrhage (FMH) in the immediate postpartum period is critical for the timely administration of Rh immunoglobulin (RhIG) prophylaxis to minimize the risk of alloimmunization in D-negative mothers of D-positive newborns. We report a series of two clinically-unsuspected cases of massive FMHs identified at our university medical center. Retrospective records of two cases of massive FMH were investigated using the electronic medical record. After positive fetal bleed screens, flow cytometric analysis for hemoglobin F was performed to quantify the volume of the hemorrhages in both cases. Flow cytometric enumeration with anti-D was also performed in one case. The two patients had 209.5 and 75 mL of fetal blood in circulation, resulting in 8 and 4 doses of RhIG administered, respectively. For the former patient, flow cytometric analysis with anti-D ruled out hereditary persistence of fetal hemoglobin and supported the fetal origin of the red cells. Due to the clinically-silent nature of both hemorrhages, further evaluation of the newborns' blood was not performed. These cases highlight the importance of rapidly obtaining accurate measurements of fetal blood loss via flow cytometric analysis in cases of FMH, particularly in clinically-unsuspected cases, to ensure timely administration of adequate immunoprophylaxis to D-negative mothers.

B cell depletion is an effective remission induction and maintenance therapy in patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV).1–6 Rituximab targets both pathogenic effector B cells and protective regulatory B cells. To avoid infections and adverse events from therapy, clinicians require improved markers of disease activity and impending relapse to guide immunosuppression strategies following B cell depletion. We reported that CD5+ B cells, as a surrogate marker of B regulatory cells, are decreased in patients with active AAV and normalise during disease remission.7 After B cell depletion, patients who repopulated with a low or decreasing percentage of CD5+ B cells and were on low maintenance immunosuppression had a shorter time to relapse than patients on similar levels of immunosuppression with normalised CD5+ B cells or patients with similarly low CD5+ B cells but higher immunosuppression. The CD5+CD24hiCD38hi B cell subpopulation correlates inversely with active disease but parallels both interleukin (IL)-10 production and suppression of ANCA.8 CD5 may identify B cells enriched in IL-10 production, the defining cytokine of B regulatory cells.8 ,9 Whether CD5+ B cells can serve as an indicator of time to relapse without considering remission maintenance immunosuppression dose is not known. We sought to address this question and confirm our previous findings in a larger cohort by separating patients solely based on their CD5+ B cells at repopulation. We examined B cell phenotype in 50 patients with AAV following rituximab therapy by flow cytometry (table …

Clinical laboratory immunology affects practically every aspect of medicine. Accordingly, appropriately trained, board-certified clinical laboratory immunologists are key contributors to the diagnosis and management of patients with various immune-mediated conditions. This review highlights the availability of postdoctoral level training programs for clinical laboratory immunology and identifies possible career tracks.Fundamental elements for doctoral level clinical laboratory immunologists are identified and the critical components of diagnostic immunology training as well as career opportunities in and out of academia are described.Relative to other disciplines in laboratory medicine, little emphasis has been given to clinical laboratory immunology in medical, graduate, and postgraduate training. Formal postgraduate fellowship programs and board certification examinations are available, yet there remains a significant lack of awareness in the medical education community about the value and necessity of training in this field.It is anticipated that sharing this knowledge will increase awareness of the discipline of clinical laboratory immunology at the postdoctoral level with implications for the practice of laboratory medicine.

We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174–179, 2000. © 2000 Wiley-Liss, Inc.

Immunology has a crucial role in microbiology. This is evident in that antibody is a key component of the test methods used to diagnose infectious disease. Antibody is used to detect pathogens with techniques such as latex agglutination, enzyme-linked immunosorbent assay, and immunofluorescent antibody test. In addition, antibody may be quantitated and identified to determine approximate time of exposure to a particular pathogen. An understanding of the basic assays used in microbiology, as well as their strengths and weaknesses, is important for the microbiologist or immunologist.

Accurate enumeration of CD34+ stem cells is important in assessing the need for continued mobilization and subsequent apheresis collections. We compared two new analysis systems, ProCOUNT (Becton Dickinson Immunocytometry Systems) and IMAGN 2000 STELLer (Biometric Imaging, Inc.) with our current (3-Color) flow cytometry-based method. The ProCOUNT system uses an absolute counting tube, which contains reference beads and a specific (multiple) gating strategy to determine an absolute count. The STELLer assay combines microvolume fluorimetry and automated analysis software to determine an absolute count. To evaluate linearity and reproducibility, peripheral blood was spiked with CD34+ cells (KG1a cell line). Three dilution series (measured at approximately equal to 0, 5, 10, 25, 50, and 100 CD34+ cells/microliter) were analyzed by each method. Analysis of predicted versus actual CD34+ concentration showed excellent correlation with all methods (r2or = 0.97, slope 0.98-1.04). To further assess precision, two PBSC samples, at approximately 200 and 800 CD34+ cells/microliter, respectively, were analyzed 10 times by each method. Coefficients of variation for the precision analysis of these samples were 5.1%-6.4% and 5.4%-12.3%, respectively. To assess overall performance, 75 patient specimens were analyzed. Excellent correlation (r2 values of 0.89-0.98) was observed among all three methods. We conclude that the three methods provide comparable linearity and reproducibility.

Summary Is a molecular diagnostic method needed for the laboratory diagnosis of syphilis? The answer is that in several instances a sensitive and specific method for the direct demonstration of T. pallidum would be beneficial. To date, the clearest progress has been made in the area of GUD and congenital diagnosis. The multiplex PCR method promises to be useful in GUD. It may be particularly useful in large scale epidemiological studies of GUD. For diagnostic purposes, its use will likely be limited to larger centers seeing sufficient numbers of patients. Congenital syphilis diagnostics could certainly use a tool that would help distinguish truly infected from noninfected asymptomatic infants born to seropositive mothers. IgM serologies are useful tools in symptomatic infants but their role, and that of PCR, in asymptomatics is not clear at this time. Certainly, prospective studies with adequate follow-up of infants to clearly document infection are needed to resolve this. Finally, neurosyphilis patients would also benefit from a sensitive and specific test to identify the presence of T. pallidum in the CSF and monitor effective therapy. To date, the experience with PCR is not consistent and additional investigations are needed. The use of molecular diagnostics in syphilis is not as well established as it is for other STDs. However, there is a need and it is hoped that work in this area will continue.

Background In HIV-1-infected patients receiving anti-retroviral therapy (ART), the relationship between residual viraemia and ex vivo recovery of infectious virus from latently infected CD4 cells is uncertain. Methods We measured residual viraemia (HIV-1 RNA copies/ml) by single-copy assay (SCA) and the latent reservoir by infectious virus recovery from resting memory CD4 cells (infectious units per million cells [IUPM]) in patients who initiated ART. We assessed immune activation by measuring CD38 expression on T-cells. Results Ten patients who initiated ART and maintained a plasma HIV-1 RNA level 10 HIV-1 RNA copies/ml per 1 log10 higher IUPM; P=0.005). Residual viraemia level was positively associated with CD38 density and percentage on CD8+ T-cells in concurrent samples and with pre-ART HIV-1 RNA levels. Conclusions In patients with HIV-1 RNA levels

Transplant centers often rely on CD34+ cell quantitation by flow cytometry to ensure adequacy of hematopoietic progenitor cell collection. Because of variation in interpretation, a lack of interlaboratory proficiency studies, and no generally accepted methodology, comparison of CD34 data from site to site is difficult. Twenty-one samples from marrow and peripheral blood stem cell collections were shipped to 10 participating North American laboratories for analysis. Duplicate samples were included to assess reproducibility. Participants were surveyed for methodology. Three centers had previously attempted to standardize their methodology among themselves. The variability observed in the CD34 values ranged from a max/min. reported value per sample of 2.9 to 749 (median 76). Exclusion of two outlying sites reduced the variability of results to 1.2 to 27 (median 3.1). Variation among the three standardized sites ranged from 1.2 to 4.4 (median 1.6). Overall reproducibility (excluding the outlying sites B and G) ranged from a minimum of 0-16.5 (percent mean difference) for site C to a maximum of 4.1-133 for site H. Strategies for gating were found to largely influence results. We observed an alarming variation among the CD34 cell counts reported from different laboratories. Standardization substantially reduced observed variation. The need for standardized methodology, reporting, quality control, and proficiency testing is underscored by these findings.

Preview What are the main causes of genital ulcer disease in the United States? Why is clinical diagnosis so difficult? What role will molecular diagnostics have in the future? The authors answer these questions and provide a functional approach to successful management of three common types of sexually transmitted genital ulcers.

A 42 year old African American female with a history of hypertensive nephrosclerosis secondary to diabetes mullitus type I and previous kidney/pancreas transplant was seen at our institution for relisting due to graft failure. Initial HLA typing (One Lambda LabType SSO) demonstrated a Class II typing of HLA-DRB1 ∗ 07, 11; DRB4 ∗ 01; DQB1 ∗ 02, 03. Of note was the lack of the typical associated DRB3 ∗ gene. The patient’s HLA typing was concordant with the previous HLA typing obtained from the original transplant (including the apparent lack of the DRB3 gene). Next Generation Sequencing was performed (Illumina TruSight HLA) with concordant results at low resolution. The high resolution class II result (HLA-DRB1 ∗ 07:01, 11:xx; DRB4 ∗ 01:01; DQB1 ∗ 02:02, 03:01) confirmed the absence of the DRB3 gene and additionally documented a novel DRB1 ∗ 11:04 variant. A single nucleotide polymorphism (SNP) was detected in exon 4 at base pair 12226 resulting in a change of amino acid from Glutamic acid to Valine. Since this SNP is in exon 4 which is not part of the antigen binding domain, it is considered a variant of the DRB1 ∗ 11:04 allele. This is, to our knowledge, the first description of an HLA-DRB3 negative HLA-DR ∗ 11 haplotype with a new DRB1 ∗ 11 allele.

We compared the ability of direct immunofluorescent staining (DFA) and the PCR to detect Treponema pallidum in specimens from patients with genital ulcer disease. Touch preparations from 156 patients with genital lesions were fixed in acetone and stained with a fluorescein-labeled monoclonal antibody specific for the 37-kDa antigen of T. pallidum. After microscopic examination, the smear was removed from the slide with a swab. DNA was extracted with phenol-chloroform and precipitated with isopropanol. Ten microliters of the extracted DNA was amplified by PCR using primers for the gene encoding the 47-kDa protein of T. pallidum and hybridized to an internal probe. Twenty-two of 156 specimens were positive for T. pallidum by DFA and PCR, while 127 were negative by both methods, yielding a concordance of 95.5% (kappa = 0.84). Four specimens were positive by PCR and negative by DFA, while three specimens were negative by PCR and positive by DFA. The DFA-negative, PCR-positive specimens may have resulted from the presence of large numbers of leukocytes on the slides, obscuring visualization of treponemes. The DFA-positive, PCR-negative results were not due to inhibition of the PCR since purified T. pallidum DNA was amplified when added to aliquots of these specimens. Negative results in these specimens were most likely due to inefficient recovery of their DNA. These data suggest that DFA and PCR are equivalent methods for detection of T. pallidum on touch preparations of genital lesions. Further refinements of the PCR assay are necessary for it to significantly improve the detection of T. pallidum in genital lesions.

Background Lung transplantation is limited by chronic lung allograft dysfunction. Acute cellular rejection (ACR) is a risk factor for allograft dysfunction; however, the role of antibody-mediated rejection (AMR) is not well characterized. Methods This was a retrospective review from 2007 to 2011 of lung transplant recipients with human leukocyte antigen (HLA) antibody testing using Luminex (Luminex Corp, Austin, TX) single-antigen beads. Statistics included Fisher's exact test for significance. Results Donor-specific antibodies (DSA) developed in 13 of 44 patients. Of the 13 with DSA, 12 had cystic fibrosis compared with 18 of 31 in the non-DSA group ( p = 0.035). Of those with DSAs, 23.1% occurred within the first year, and 69.2% occurred between 1 and 3 years. Twelve of 13 DSA patients had anti-HLA DQ specificity compared with 2 of 31 non-DSA patients ( p = 0.0007). AMR developed in 10 of the 13 DSA patients compared with 1 of 31 non-DSA patients ( p = 0.0001). The DSA group experienced 2.6 episodes/patient of cellular rejection vs 1.7 episodes/patient in the non-DSA group ( p = 0.059). Bronchiolitis obliterans syndrome developed in 11 of 13 in the DSA group vs 10 of 31 in the non-DSA group ( p = 0.0024). In the DSA group, 11.5% HLAs matched compared with 20.4% in the non-DSA group ( p = 0.093). AMR developed in 11 of 22 patients in the non-DSA HLA group compared with 0 of 22 in the group without non-DSA HLA antibodies ( p = 0.002). Survival at 1 and 3 years was 92% and 36% in the DSA group, respectively, and 97% and 65% in the non-DSA group. Conclusions DSAs and non-DSAs occur frequently after lung transplantation. DSAs are prevalent in the cystic fibrosis population and are associated with AMR, bronchiolitis obliterans syndrome, and possibly, ACR.

We screened cord blood or serum samples from 101 infants at risk for congenital syphilis and serum samples from their mothers for immunoglobulin G (IgG), IgM, and IgA antibodies to Treponema pallidum by western blotting (immunoblotting). Clinical evaluation showed that six infants had signs and/or symptoms consistent with congenital syphilis. The sera from five of these infants were IgM blot positive, and four were IgA blot positive. Four asymptomatic infants had serologic evidence of congenital syphilis. The sera from three of these infants were IgM blot positive, and two were IgA blot positive. However, the IgM reactivity of the serum from one asymptomatic infant, which was also IgA positive, was abolished by protein G treatment. An IgM capture enzyme-linked immunosorbent assay corroborated the presence of IgM antibodies in six of seven IgM blot-reactive sera. Overall, for detection of symptomatic congenital syphilis, a sensitivity of 83% for IgM blotting and 67% for IgA blotting was obtained. The significance of positive IgM or IgA Western blots for asymptomatic infants requires further study to confirm infection in these infants.

This prospective, cross-sectional study of HIV testing at two sexually transmitted infection clinics compares testing results from the ARCHITECT HIV Ag/Ab Combo fourth generation assay against the current standard of care in North Carolina (third generation enzyme immunoassay testing with western blot confirmation and reflex nucleic acid amplification testing of pooled seronegative samples). In this setting, the assay reported a sensitivity of 100%, a specificity of 99.9%, and a median turn-around time of 26.1 h.

Anti-neutrophil cytoplasmic autoantibody (ANCA) disease rarely occurs in African Americans and risk factors for the disease in this population are unknown. Here, we genotyped MHC class II alleles and found that, among African Americans, those with proteinase 3–ANCA (PR3-ANCA) had 73.3-fold higher odds of having HLA-DRB1 * 15 alleles than community-based controls (OR 73.3; 95% CI 9.1 to 591). In addition, a disproportionate number of African American patients carried the DRB1 * 1501 allelic variant of Caucasian descent rather than the DRB1 * 1503 allelic variant of African descent. Among Caucasians, those with PR3-ANCA had 2.2-fold higher odds of carrying DRB1 * 1501 than controls (OR 2.2; 95% CI 1.2 to 4.0). A validation study supported by the Vasculitis Clinical Research Consortium confirmed the strong association between the DRB1 * 15 allele and PR3-ANCA disease, among African Americans. Furthermore, we found that DRB1*1501 protein binds with high affinity to amino acid sequences of sense-PR3, purportedly an antigenic epitope, and to the amino acid sequence complementary to this epitope in vitro . Peptides of sense-PR3 and complementary-PR3 also bound to TNF-α–induced surface expression of DRB1 * 1501 on peripheral neutrophils. Taken together, these data suggest HLA-DRB1 * 15 alleles contribute to the pathogenesis of PR3-ANCA disease.

Some patients with proteinase 3 specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA) also have antibodies that react to complementary-PR3 (cPR3), a protein encoded by the antisense RNA of the PR3 gene. To study whether patients with anti-cPR3 antibodies have cPR3-responsive memory T cells we selected conditions that allowed cultivation of memory cells but not naïve cells. About half of the patients were found to have CD4+TH1 memory cells responsive to the cPR3(138-169)-peptide; while only a third of the patients had HI-PR3 protein responsive T cells. A significant number of T cells from patients responded to cPR3(138-169) peptide and to HI-PR3 protein by proliferation and/or secretion of IFN-gamma, compared to healthy controls while there was no response to scrambled peptide. Cells responsive to cPR3(138-169)-peptide were not detected in MPO-ANCA patients suggesting that this response is specific. The HLADRB1(*) 15 allele was significantly overrepresented in our patient group and is predicted to bind cPR3(138-169) peptide with high affinity. Regression analysis showed a significant likelihood that anti-cPR3 antibodies and cPR3-specific T cells coexist in individuals, consistent with an immunological history of encounter with a PR3-complementary protein. We suggest that the presence of cells reacting to potential complementary protein pairs might provide an alternative mechanism for auto-immune diseases.

Valproic acid and intensified antiretroviral therapy may deplete resting CD4+ T-cell HIV infection. We tested the ability of valproic acid to deplete resting CD4+ T-cell infection in patients receiving standard antiretroviral therapy.Resting CD4+ T-cell infection was measured in 11 stably aviremic volunteers twice prior to, and twice after Depakote ER 1000 mg was added to standard antiretroviral therapy. Resting CD4+ T-cell infection frequency was measured by outgrowth assay. Low-level viremia was quantitated by single copy plasma HIV RNA assay.A decrease in resting CD4+ T-cell infection was observed in only four of the 11 patients. Levels of immune activation and HIV-specific T-cell response were low and stable. Valproic acid levels ranged from 26 to 96 microg/ml when measured near trough. Single copy assay was performed in nine patients. In three patients with depletion of resting CD4+ T-cell infection following valproic acid, single copy assay ranged from less than 1-5 copies/ml. Continuous low-level viremia was observed in three patients with stable resting CD4+ T-cell infection (24-87, 8-87, and 1-7 copies/ml respectively) in whom multiple samples were analyzed.The prospective addition of valproic acid to stable antiretroviral therapy reduced the frequency of resting CD4+ T-cell infection in a minority of volunteers. In patients in whom resting CD4+ T-cell infection depletion was observed, viremia was rarely detectable by single copy assay.

Background Concern about costs and antiretroviral therapy (ART)-associated toxicities led to the consideration of CD4 driven strategies for the management of HIV. That approach was evaluated in the SMART trial that reported an unexpected increase of cardiovascular events after treatment interruption (TI). Our goal was to evaluate fasting metabolic changes associated with interruption of antiretroviral therapy and relate them to changes of immune activation markers and cardiovascular risk. Methodology ACTG 5102 enrolled 47 HIV-1-infected subjects on stable ART, with

Aim The presence of the genotype HLA-B∗57:01 is highly predictive of Abacavir hypersensitivity. The need to identify the presence of this antigen in the HIV population is pertinent to treatment. Traditional molecular methods of HLA-B∗57:01 with high specificity come often with long turn-around times and high costs. Our laboratory developed a flow cytometric screening method to identify the HLA-B∗57 antigen for an expedited negative result. Methods 59 random patients were tested for the HLA-B∗57 antigen utilizing a two-step flow cytometry staining protocol. Whole blood was incubated with CD3-APC and a biotinylated B17 monoclonal antibody. The monoclonal HLA-B∗17 antibody detects the presence of the HLA- B∗57 or B∗58 antigens on the cell surface. To enable detection of the B57 antibody, streptavidin-PE was added. An internal positive/negative control was developed to confirm assay performance. 10 μL of Flow PRA 1 screening beads (One Lambda) were added to the patient sample. Based on SSC and FSC, beads are easily distinguishable from patient cells. All samples were confirmed by molecular HLA typing using real time PCR or SSOP. Results Of the 59 patient samples, 12 were considered positive with a Mean MFI of 8622 ± 3962. 2 of the 12 positive screens were positive for HLA-B∗57:01 by molecular typing. Based on the data, a cutoff was set with a mean MFI of 500. None of the negative screens had either HLA-B∗57 or B∗58 present by molecular typing. No cross-reactivity was observed from the 5 CREG group. Thus there is a lower likelihood of detecting antigens outside of HLA-B∗57 and HLA-B∗58. Conclusions The screening of patients for the presence of HLA-B∗57:01 is important prior to initiation of Abacavir therapy. An estimated 0.1–2.5% of the US population is positive for HLA-B∗57:01, so the majority of patients will be negative. This rapid flow cytometry assay allows for faster turn-around time for B∗57:01 negative patients. The introduction of an internal positive control into the assay provides quality control that previous HLA-B∗57 screening assays failed to provide.

Although an intermittent antiviral treatment (ART) strategy may limit long-term toxicity and cost, there is concern about the risk for virologic failure, selection of drug resistance mutations, and disease progression. By boosting CD4 T-cell counts, interleukin 2 (IL-2) could safely prolong the duration of treatment interruption (TI) in a CD4-driven strategy.The AIDS Clinical Trials Group (ACTG) study A5102 evaluated 3 cycles of IL-2 before TI, on clinical and immunologic outcomes, using a CD4 T-cell count of350 cells/mm as the threshold for restarting ART. Forty-seven HIV-infected subjects on potent ART with CD4 T-cell counts ofor =500 cells/mm or more and HIV RNA levels of less than 200 copies/mL were randomized to arm A (ART + three 5-day cycles of IL-2 at 4.5 million U, Sc, BID every 8 weeks, n = 23) or arm B (ART alone, n = 24) for 18 weeks (step 1). At the end of step 1, subjects with a CD4 T-cell count ofor =500 cells/mm or more stopped ART until a CD4 count of350 cells/mm (step 2). CD4 T-cell count, time to return of viremia, and the emergence of drug resistance mutations after TI were compared between study arms.IL-2 recipients maintained higher CD4 counts during TI for 48 weeks with a waning of the CD4 effect by 72 weeks. A sustained CD4 T-cell count of more than 350 cells/mm and more durable TI were associated with a higher nadir CD4 T-cell count before ART and higher naive CD4 T-cell count at entry. After TI, a higher viral set point and drug resistance mutations at virologic rebound were associated with a shorter time to CD4 T-cell count of less than 350 cell/mm. There were no differences in the magnitude of virologic rebound (at week 8 of step 2, median log10 HIV RNA level was 4.23 for arm A and 4.21 for arm B) or the steady-state HIV-1 RNA level after week 8.IL-2 before TI did not prolong time to CD4 of less than 350 cells/mm. A TI strategy utilizing a CD4 T-cell threshold of less than 350 cells/mm for restarting ART appears generally safe with most subjects in both arms remaining off ART for more than 1 year. Implications of our results for TI strategies include the potential advantage of starting ART at higher CD4 T-cell levels while avoiding any drug resistance and evaluating immunomodulators or drugs to reduce T-cell activation and HIV-1 RNA rebound during the TI.

Background and Objectives: The prevalence of trichomoniasis in the general population of the United States is unknown. This study provides the first population-based prevalence estimates of trichomoniasis among young adults in the United States. Methods: The National Longitudinal Study of Adolescent Health (Add Health) is an ongoing prospective cohort study. In a cross-sectional analysis of Wave III of Add Health (N = 12,449), we determined the prevalence of trichomoniasis using a polymerase chain reaction assay. Results: The estimated overall prevalence of trichomoniasis in U.S. young adults was 2.3% (95% confidence interval [CI], 1.8–2.7%). The prevalence was slightly higher among women (2.8%; 95% CI, 2.2–3.6%) than men (1.7%; 95% CI, 1.3–2.2%). The prevalence increased with age and varied by region, with the south having the highest prevalence (2.8%; 95% CI, 2.2–3.5%). The prevalence was highest among black women (10.5%; 95% CI, 8.3–13.3%) and lowest among white women (1.1%; 95% CI, 0.8–1.6%). Among men, the prevalence was highest among Native Americans (4.1%; 95% CI, 0.4–29.3%) and blacks (3.3%; 95% CI, 2.2–4.9%), and lowest among white men (1.3%; 95% CI, 0.9–1.8%). Conclusions: Trichomoniasis is moderately prevalent among the general U.S. population of young adults and disturbingly high among certain racial/ethnic groups.

The cost for the Roche Monitor assay kit can be reduced 50% by using only the 1:1, 1:25, and 1:625 human immunodeficiency virus (HIV) and the 1:1 quantitation standard dilutions. This abbreviated test applied to 1,774 mostly African samples had results nearly identical to those obtained following the package insert instructions. To make this approach feasible, Roche would have to provide additional lysis buffer and master mix.

Context: Chlamydial and gonococcal infections are important causes of pelvic inflammatory disease, ectopic pregnancy, and infertility. Although screening for Chlamydia trachomatis is widely recommended among young adult women, little information is available regarding the prevalence of chlamydial and gonococcal infections in the general young adult population. Objective: To determine the prevalence of chlamydial and gonoccoccal infections in a nationally representative sample of young adults living in the United States. Design, Setting, and Participants: Cross-sectional analyses of a prospective cohort study of a nationally representative sample of 14322 young adults aged 18 to 26 years. In-home interviews were conducted across the United States for Wave III of The National Longitudinal Study of Adolescent Health (Add Health) from April 2, 2001, to May 9, 2002. This study sample represented 66.3% of the original 18924 participants in Wave I of Add Health. First-void urine specimens using ligase chain reaction assay were available for 12548 (87.6%) of the Wave III participants. Main Outcome Measures: Prevalences of chlamydial and gonococcal infections in the general young adult population, and by age, self-reported race/ethnicity, and geographic region of current residence. Results: Overall prevalence of chlamydial infection was 4.19% (95% confidence interval [CI], 3.48%-4.90%). Women (4.74%; 95% CI, 3.93%-5.71%) were more likely to be infected than men (3.67%; 95% CI, 2.93%-4.58%; prevalence ratio, 1.29; 95% CI, 1.03-1.63). The prevalence of chlamydial infection was highest among black women (13.95%; 95% CI, 11.25%-17.18%) and black men (11.12%; 95% CI, 8.51%- 14.42%); lowest prevalences were among Asian men (1.14%; 95% CI, 0.40%-3.21%), white men (1.38%; 95% CI, 0.93%-2.03%), and white women (2.52%; 95% CI, 1.90%-3.34%). Prevalence of chlamydial infection was highest in the south (5.39%; 95% CI, 4.24%-6.83%) and lowest in the northeast (2.39%; 95% CI, 1.56%-3.65%). Overall prevalence of gonorrhea was 0.43% (95% CI, 0.29%-0.63%). Among black men and women, the prevalence was 2.13% (95% CI, 1.46%-3.10%) and among white young adults, 0.10% (95% CI, 0.03%-0.27%). Prevalence of coinfection with both chlamydial and gonococcal infections was 0.030% (95% CI, 0.18%-0.49%). Conclusions: The prevalence of chlamydial infection is high among young adults in the United States. Substantial racial/ethnic disparities are present in the prevalence of both chlamydial and gonococcal infections.

Detection and specificity of autoantibodies against extractable nuclear antigens (ENA) play a critical role in the diagnosis and management of autoimmune disease. Historically, the detection of these antibodies has employed double immunodiffusion (DID). Autoantibody specificity was correlated with diagnoses by this technique. Enzyme immunoassays have been developed by multiple manufacturers to detect and identify the specificity ENA autoantibodies. To address the relationship of ENA detection by DID and enzyme immunoassay, the performances of five immunoassays were compared. These included two DID and three enzyme-linked immunoassays (ELISA) (both screening and individual antigen profile kits). The sample set included 83 ENA-positive, antinuclear-antibody (ANA)-positive specimens, 77 ENA-negative, ANA-positive specimens, and 20 ENA- and ANA-negative specimens. Sensitivity and specificity were calculated by two methods: first, by using the in-house DID result as the reference standard, and second, by using latent class analysis, which evaluates each kit result independently. Overall, the results showed that the ELISA methods were more sensitive for detection of ENA autoantibodies than DID techniques, but presence and/or specific type of ENA autoantibody did not always correlate with the patient's clinical presentation. Regardless of the testing strategy an individual laboratory uses, clear communication with the clinical staff regarding the significance of a positive result is imperative. The laboratory and the clinician must both be aware of the sensitivity and specificity of each testing method in use in the clinical laboratory.

CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 ( gag ) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads 350 CD4 T lymphocytes/mm 3 , and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.

Background White blood cells on endocervical Gram stain and vaginal wet mount are frequently used to predict chlamydial and gonococcal infections. Previous studies provide conflicting evidence for the clinical utility of these tests. Goal To evaluate the clinical utility of measuring white blood cells on vaginal wet mount and endocervical Gram stain for the prediction of chlamydial infection and gonorrhea. Study design Women undergoing pelvic examinations at 10 county health department family planning and sexually transmitted disease clinics were tested for chlamydial infection by ligase chain reaction assay (n = 4550) and for gonorrhea by culture (n = 4402). Vaginal wet mount and endocervical Gram stains were performed in county laboratories at the time of examination. Results The prevalences of chlamydial infection and gonorrhea were 8.8% and 3.2%, respectively. For detection of chlamydial or gonococcal infection, the likelihood ratio was 2.85 (95% CI, 2.10-3.87) for > 30 white blood cells on vaginal wet mount and 2.91 (95% CI, 2.07-4.09) for > 30 white blood cells on endocervical Gram stain. Similar results were seen for individual diagnoses either of chlamydial infection or of gonorrhea. Conclusion Vaginal wet mount and endocervical Gram stain white blood cells are useful for the presumptive diagnosis of chlamydial infection or gonorrhea only in settings with a relatively high prevalence of infection or when other predictors can increase the likelihood of infection.

Lymphocyte proliferation assays (LPAs) are widely used to assess T-lymphocyte function of patients with human immunodeficiency virus infection and other primary and secondary immunodeficiency disorders. Since these assays require expertise not readily available at all clinical sites, specimens may be shipped to central labs for testing. We conducted a large multicenter study to evaluate the effects of shipping on assay performance and found significant loss of LPA activity. This may lead to erroneous results for individual subjects and introduce bias into multicenter trials.

Background: Screening sexually active women for Chlamydia trachomatis is necessary to detect asymptomatic infections. Selective screening is a common strategy because universal screening is too costly in many settings. In order to guide local programs in the choice of selective screening criteria, we examined the performance of previously proposed screening criteria for C trachomatis . Methods: A clinic-based, cross-sectional study was conducted in public family planning and sexually transmitted disease (STD) clinics in ten counties in North Carolina. Women ( n = 4471 in family planning and n = 2201 in STD clinics) undergoing pelvic examination were enrolled consecutively. Nine sets of screening criteria, including age alone, were compared using sensitivity, specificity, number of tests required and receiver-operator characteristic (ROC) analysis. All women underwent testing with ligase chain reaction assay of cervical specimens to identify C trachomatis infection. Results: The prevalence of C trachomatis was 7.8% and 11.0% in family planning and STD clinics, respectively. The sensitivities of published criteria ranged from 0.50 to 0.97. Specificities ranged from 0.05 to 0.66. In family planning clinics, the best performing criteria would detect 84% of infections while screening 51% of women. In STD clinics, the same criteria would detect 83% of infections but require testing 67% of women. Testing women aged ≤22 would detect 77% of infections in family planning and 74% of infections in STD clinics, while testing 51% and 48% of the women, respectively. Conclusions: When site-specific criteria cannot be developed, age alone is an acceptable strategy for selective screening for chlamydial infection.

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Iβ, soluble TNF receptor II, soluble IL-2 receptor alpha, β 2 -microglobulin, and neopterin. Twelve laboratories associated with the Adult and Pediatric AIDS Clinical Trial Groups participated in the study. The performance features that were evaluated included intralaboratory variability, interlaboratory variability, comparison of reagent sources, and ability to detect CSM in the plasma of normal subjects as well as the changes occurring in disease. The principal findings were as follows: (i) on initial testing, i.e., before participating in the program, laboratories frequently differed markedly in their analytic results; (ii) the quality of testing of a CSM in individual participating laboratories could be assessed; (iii) most commercial kits allowed distinction between normal and abnormal plasma CSM levels and between supernatants of stimulated and unstimulated cells; (iv) different sources of reagents and reference standards frequently provided different absolute values; (v) inexperienced laboratories can benefit from participating in the program; (vi) laboratory performance improved during active participation in the program; and (vii) comparability between analyses conducted at different sites can be ensured by an external proficiency testing program.

Of 400 cases of urethritis in male soldiers enrolled in a behavioral intervention project, the etiology of 69% was defined at study enrollment, as well as the etiology of 72% of 25 repeated episodes involving 21 men during the first 78 days of active follow-up (5% of the cohort). Chlamydia trachomatis (36%), Neisseria gonorrhoeae (34%), and Ureaplasma urealyticum (19%) were the most common causes of infection identified at enrollment and during subsequent visits (44%, 28%, and 12%, respectively). By univariate analysis, patients with repeated infection ("repeaters") were significantly more likely to report a history of sexually transmitted disease (STD; relative risk [RR], 3) and sex with sex workers (RR, 4) than were nonrepeaters. By multivariate analysis, only STD history was significant (RR, 2.8). Characteristics of repeaters in this cohort suggest that specific patterns may be used to establish screening "profiles" of potential repeaters, by which such individuals might be targeted for aggressive intervention at the time of the initial diagnosis.

Antineutrophil cytoplasmic autoantibodies (ANCAs) are increasingly used as serologic markers for pauci-immune crescentic glomerulonephritis and small vessel vasculitis. Many hospital laboratories and referral laboratories use commercial assay kits to detect ANCAs, despite inadequate documentation in the medical literature of kit performance. We evaluated the diagnostic sensitivity, specificity, and predictive value of 3 commercial indirect immunofluorescence assay (IFA) kits and 7 commercial enzyme immunoassay (EIA) kits for several ANCA subtypes. Serum samples from 396 patients with a variety of renal diseases were analyzed, including 146 patients with pauci-immune crescentic glomerulo-nephritis with or without systemic vasculitis. With 1 exception, the kits had more than 90% agreement with the reference standard and gave results similar to those of research laboratories. IFA diagnostic sensitivity ranged from 81% to 91% and EIA sensitivity from 75% to 84%. Maximum specificity was obtained with combined IFA and EIA. Diagnostic specificity was more than 70% for 2 of 3 IFA kits and at least 90% for 5 of 7 EIA kits. Predictive values varied with clinical manifestations. Most commercial IFA and EIA kits that were evaluated provide acceptably accurate analytic results.

A whole-blood flow cytometry-based assay was utilized to assess CD4 and CD8 T-lymphocyte activation in response to phytohemagglutinin (PHA) stimulation. T-lymphocyte activation was assessed by qualitative (percent CD69) and semiquantitative (anti-CD69 antibody binding capacity) measurements of CD69 surface expression. Whole-blood samples from 21 healthy and 21 human immunodeficiency virus (HIV)-infected (3 ) individuals were stimulated with 20 μg of PHA per ml for 18 to 24 h. The proportions of activated CD4 and CD8 T lymphocytes expressing CD69 (percent CD69) and the levels of CD69 expression on each T-lymphocyte subset (anti-CD69 antibody binding capacity) were measured. By using this assay system, T-lymphocyte activation was impaired in both CD4 and CD8 T-lymphocyte subsets of HIV-infected individuals. The proportions of CD69-positive CD4 and CD8 T lymphocytes were 43 and 27% lower, respectively, in samples from HIV-infected individuals compared to samples from healthy individuals. Similarly, the levels of CD69 expression on each activated CD4 and CD8 T-lymphocyte subset were 48 and 51% lower, respectively. These results suggest that both qualitative and semiquantitative measurements of CD69 surface expression by flow cytometry can be used to assess T-lymphocyte activation.

Aim Recently our renal transplant (Tx) program has implemented routine HLA antibody monitoring of allograft recipients. We determined the incidence of donor specific antibody (DSA) in the 6 months – 1 year post-Tx period. Methods 155 renal allograft recipients (Tx in 2011-2012) were tested for HLA antibody prior to Tx and 6 – 12 months post-Tx. Pre and post-Tx monitoring of antibodies was determined using flow cytometry screening beads and a Luminex-based solid-phase class I and II single antigen bead assay with a positive cutoff of 1000 MFI. Crossmatches (XM) were performed using pronase treated cells and flow cytometry. Correlations between DSA development and serum creatinine were analyzed. Results Overall, 67 of 155 renal Tx recipients were sensitized at time of Tx of which 41 were tested for DSA post-transplant. Of 88 non-sensitized patients, 50 were tested post-Tx. 5 of 41 sensitized patients developed de novo DSA compared to 4 of 50 patients in the non-sensitized group. Of the 9 patients that developed DSA, 7 had DSA to HLA-II antigens while 2 had antibodies against HLA-I antigens. In patients with DSA, serum creatinine level ranged from 1.39 – 5.87. However, no correlation was observed between DSA MFI and serum creatinine level. Conclusions Institution of routine testing for DSA following renal Tx has allowed identification of patients with HLA DSA that may not have been tested in an indication based scheme. A higher percentage of sensitized patients received routine monitoring than non-sensitized patients. More monitoring in sensitized patients is consistent with sensitized patients increased susceptibility to develop DSA. Development of DSA does not correlate with a positive XM but does correlate with sensitization. Consistent with previous reports, the majority of patients developed DSA to HLA-II antigens. Routine testing for DSA will enable a better understanding of possible connections between development of DSA and clinical outcomes in our cohort.

Natural killer cells (NK cells) are a subset of peripheral blood lymphocytes that mediate non-major histocompatibility complex-restricted cytotoxicity of foreign target cells. The "gold standard" assay for NK cell activity has been the chromium release assay. This method is not easily performed in the clinical laboratory because of difficulties with disposal of radioactive and hazardous materials, short reagent half-lives, expense, and difficulties with assay standardization. We describe a flow cytometric assay for the clinical measurement of NK cell activity. This study compared the chromium release assay and the flow cytometric assay by using clinically relevant specimens. There were no significant differences between the two assays in the measurement of lytic activity for 17 peripheral blood specimens or in reproducibility in repeated samplings of healthy individuals. We also established a normal range of values for NK activity in healthy adults and identified a small cluster of individuals who have exceptionally high or low levels of NK activity. The flow cytometric assay was validated by testing specimens from subjects expected to have abnormally low levels of NK activity (pregnant women) and specimens from healthy individuals in whom the activity of NK cells was enhanced by exposure to interleukin-2 or alpha interferon. Treatment with these agents was associated with a significant increase in NK activity. These results confirm and extend those of others, showing that the flow cytometric assay is a viable alternative to the chromium release assay for measuring NK cell activity.

We compared flow cytometric immunophenotyping results obtained by using the lysed whole blood method of sample preparation with those obtained by using Ficoll-Hypaque-separated cells on 44 consecutive specimens from patients with various hematologic malignancies. When the samples were analyzed as a group, seven antigens (CD2, CD3, CD5, CD11c, CD20, CD22, and CD34) demonstrated significantly different percentages of positively staining cells. When the samples were grouped by disease, results for patients with acute lymphocytic leukemia were discordant for CD22 and HLA-DR and results for patients with hairy cell leukemia were discordant for CD34. Most of the differences, however, were not with antigens critical to the evaluation of the malignancy. Additionally, the most frequent reason for differences in the percentage of positive cells was due to isotype control-based placement of the quadrant markers and not an actual discrepancy in staining. However, analysis of the CD34 antigen yielded eight instances in which staining of Ficoll-Hypaque-separated cells was essentially negative, but a clearly positive population was evident with the lysed preparation. This finding has important implications because of the prognostic significance of this antigen. Further studies are needed to determine the cause of this phenomenon.