Carol T. Schnizlein-Bick

Evaluation of TruCount absolute-count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus-positive adults.

ABSTRACT A single-platform technology that uses an internal bead standard and three-color flow cytometry to determine CD4 and CD8 absolute counts was evaluated for reproducibility and agreement. Values obtained using TruCount absolute-count tubes were compared to those obtained using a two-color predicate methodology. Sixty specimens from human immunodeficiency virus type 1-infected donors were shipped to five laboratories. Each site also analyzed replicates of 14 human immunodeficiency virus type 1-infected local specimens at 6 h and again at 24 h. The interlaboratory variability was significantly less with TruCount (median difference in percent coefficient of variation [%CV] between the two methods was −8% and −3% for CD4 and CD8, respectively) than with the predicate method. Intralaboratory variability was smaller, with a median difference in %CV of −1% for both CD4 and CD8 with 6-h samples and −2% and −3% for CD4 and CD8, respectively, with 24-h samples. Use of TruCount for shipped samples resulted in a median CD4 count change of 7 cells (50th estimated percentile) when all laboratories and CD4 strata were combined. For on-site samples, the median CD4 count change was 10 CD4 cells for 6-h samples and 2 CD4 cells for 24-h samples. Individual site biases occurred in both directions and cancelled each other when the data were combined for all laboratories. Thus, the combined data showed a smaller change in median CD4 count than what may have occurred at an individual site. In summary, the use of TruCount decreased both the inter- and intralaboratory variability in determining absolute CD4 and CD8 counts.

Safety of Discontinuation of Maintenance Therapy for Disseminated Histoplasmosis after Immunologic Response to Antiretroviral Therapy

We performed a prospective observational study to assess the safety of stopping maintenance therapy for disseminated histoplasmosis among human immunodeficiency virus infected patients after response to antiretroviral therapy. All subjects received at least 12 months of antifungal therapy and 6 months of antiretroviral therapy before entry. Negative results of fungal blood cultures, urine and serum Histoplasma antigen level of <4.1 units, and CD4+ T cell count of >150 cells/mm3 were required for eligibility. Thirty-two subjects were enrolled; the median CD4+ T cell count at study entry was 289 cells/mm3. No relapses of histoplasmosis occurred after a median duration of follow-up of 24 months. This corresponded to an observed relapse rate of 0 cases per 65 person-years. The median CD4+ T cell count at final study visit was 338 cells/mm3. Discontinuation of antifungal maintenance therapy appears to be safe for patients with acquired immunodeficiency syndrome with previously treated disseminated histoplasmosis and sustained immunologic improvement in response to antiretroviral therapy.

Comparison of the echinocandin caspofungin with amphotericin B for treatment of histoplasmosis following pulmonary challenge in a murine model.

ABSTRACT Twenty clinical isolates of Histoplasma capsulatum were tested for their in vitro susceptibilities to caspofungin in comparison to those to amphotericin B by following National Committee for Clinical Laboratory Standards guidelines for yeasts. The mean MICs were 16.6 μg/ml (range, 8 to 32 μg/ml) for caspofungin and 0.56 μg/ml (range, 0.5 to 1.0 μg/ml) for amphotericin B. Survival experiments used a 105 dose in a pulmonary challenge model with B6C3F1 mice. All mice that received amphotericin B at 2 mg/kg of body weight every other day (q.o.d.), 30% of mice that received caspofungin at 8 mg/kg/day, and 20% of mice that received caspofungin at 4 mg/kg/day survived to day 15, while mice that received caspofungin at 2 mg/kg/day and all control mice that received the vehicle died by day 14. Amphotericin B at 2 mg/kg q.o.d. markedly reduced the fungal burden in the lungs and spleens, as measured byHistoplasma antigen detection techniques and quantitative cultures, for each comparison. Caspofungin at 10 mg/kg twice a day (b.i.d.) did not reduce the fungal burden, as measured by antigen detection techniques, but slightly reduced the levels of fungi in both the lungs and spleens, as determined by quantitative cultures. Caspofungin at 5 mg/kg b.i.d. did not affect fungal burden. Overall, caspofungin had only a slight effect on survival or fungal burden.

Comparison of a new triazole, posaconazole, with itraconazole and amphotericin B for treatment of histoplasmosis following pulmonary challenge in immunocompromised mice

ABSTRACT A murine model of intratracheally induced histoplasmosis in immunocompromised B6C3F1 mice was used to evaluate a new triazole antifungal agent, posaconazole. This compound was previously shown to be comparable to amphotericin B and superior to itraconazole for the treatment of histoplasmosis in immunocompetent mice. The current study used mice that were depleted of T lymphocytes by intraperitoneal injection of anti-CD4 and anti-CD8 monoclonal antibodies beginning 2 days before infection and continuing at 5-day intervals until completion of the study. Groups of B6C3F1mice that were depleted of CD4 and CD8 T cells were infected with an inoculum of 104Histoplasma capsulatum yeasts. All mice receiving posaconazole at 1 or 0.1 mg/kg of body weight/day, amphotericin B at 2 mg/kg every other day (qod), or itraconazole at 75 mg/kg/day survived to day 29. Only 60% of mice receiving itraconazole at 10 mg/kg/day and none receiving amphotericin B at 0.2 mg/kg qod survived to that date. Fungal burdens were determined at day 14 of infection, 1 day after discontinuation of therapy. Quantitative colony counts and Histoplasma antigen levels in lung and spleen tissues declined following treatment with amphotericin B at 2 mg/kg qod, posaconazole at 5 and 1 mg/kg/day, and itraconazole at 75 mg/kg/day but not in mice treated with amphotericin B at 0.2 mg/kg qod or itraconazole at 10 mg/kg/day. Posaconazole at 0.1 mg/kg/day reduced fungal colony counts and antigen levels in spleens but not in lungs. This study shows posaconazole activity for the treatment of histoplasmosis in immunosuppressed animals.

The Immune Response to Haemophilus ducreyi Resembles a Delayed-Type Hypersensitivity Reaction throughout Experimental Infection of Human Subjects

Previous work in 3 subjects infected for 2 weeks indicated that experimental infection with Haemophilus ducreyi recruits CD4 cells to the skin at the pustular stage of disease. In order to describe the kinetics of the host response, 23 subjects were infected at 2 sites with a standardized dose of H. ducreyi. Subjects were biopsied 1 or 4 days after inoculation or when they developed a painful pustular lesion (days 7-14). Papules and pustules contained a predominant T cell infiltrate that consisted of CD45RO and CD4 cells of the alpha beta lineage. Both papules and pustules contained mixed or T helper 1 type cytokine mRNA and interleukin-8 and tumor necrosis factor-alpha mRNA. Although the subjects had no history of chancroid, their immune responses resembled delayed-type hypersensitivity reactions that occurred within 24 h of inoculation and persisted throughout the course of experimental infection.

Lower CD4+ T Lymphocyte Nadirs May Indicate Limited Immune Reconstitution in HIV-1 Infected Individuals on Potent Antiretroviral Therapy: Analysis of Immunophenotypic Marker Results of AACTG 5067

Background: Although initiation of potent antiretroviral therapy (ART) has significantly improved immune perturbations in individuals with AIDS, it is unclear which factors are most important in determining the degree of immune reconstitution.Methods: Whole blood was analyzed at baseline and week 12 in six groups of subjects (n = 81): those with acute or following immune reconstitution after Pneumocystis jirovecii (previously known as Pneumocystis carinii) pneumonia (PcP) (two groups) and cytomegalovirus (CMV) retinitis (two groups), HIV-infection without AIDS (one group), and healthy volunteers (one group). Absolute CD4+ and CD8+ T lymphocytes, naíve (CD45RA+) and memory (CD45RO+) CD4+ T lymphocytes and percentages of activated CD8+ T lymphocytes (CD8+/CD38+/HLA-DR), and CD28 expression on CD4+ and CD8+ T lymphocytes were enumerated.Results: The reconstituted CMV group, which had a history of a lower CD4+ T lymphocyte nadir compared to the reconstituted PcP group (15 cells/mm3 versus 48 cells/mm3; p = .013), had significantly lower absolute CD4+, CD8+ and naíve CD4+ T lymphocytes and a trend toward lower memory CD4+ T lymphocytes compared to the reconstituted PcP group. Moreover, no difference was noted between the reconstituted groups in the proportion of subjects with undetected HIV-1 RNA. The reconstituted subjects had significantly lower absolute CD4+, memory CD4+ and naíve CD4+ T lymphocytes than the HIV-positive controls and a significantly higher percentage of activated CD8+ T lymphocytes with a lower percentage of CD8+CD28 expression than the HIV-negative controls.Conclusion: The association of CD4+ T lymphocyte nadir with the extent of immune reconstitution in HIV-infected individuals suggests that HIV-1 may cause irreparable immune system damage despite potent ART.

Evidence of immune reconstitution in antiretroviral drug-experienced patients with advanced HIV disease

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.

Amphotericin B combined with itraconazole or fluconazole for treatment of histoplasmosis.

To investigate the efficacy of combined treatment with fluconazole (Flu) and amphotericin B (AmB) for Histoplasma capsulatum meningitis, MICs were determined for 10 clinical isolates, following National Committee for Clinical Laboratory Standards guidelines. Weak synergy was observed for 6 of the 10 isolates. For the in vivo models, mice either were sham treated or were given Flu (75 mg/kg/day), AmB (2 mg/kg every other day), itraconazole (Itra; 75 mg/kg/day), AmB+Flu, or AmB+Itra. Following infection with 5x105 yeasts, Flu antagonized AmBs reduction of fungal burden without reducing its effect on survival. When in vivo antagonism was reproduced following infection with 1x104 yeasts, a higher fungal burden was observed in the lungs. Itra had no effect on AmBs activity and was more effective than Flu for clearance of fungal burden. These findings caution against use of AmB+Flu for treatment of histoplasmosis, but studies of the effect of treatment on the fungal burden in the brain are needed to assess combination therapy for meningitis.

Evolution of the Cutaneous Immune Response to Experimental Haemophilus ducreyi Infection and Its Relevance to HIV-1 Acquisition

Haemophilus ducreyi causes the sexually transmitted disease chancroid, which facilitates HIV-1 transmission. Skin biopsies were obtained from subjects experimentally infected with H. ducreyi to study the evolution of the immune response and immunophenotypes relevant to transmission of HIV-1. Compared with peripheral blood, there was an enrichment of T cells and macrophages after 48 h of infection in the skin. Neutrophils became the predominant cell type by 7–9 days. By immunohistochemistry, macrophage-inflammatory protein-1α was not present early in infection, but was abundant at later stages. RANTES was present throughout the papular and pustular stages of experimental infection, but not present in uninfected control skin. Stromal cell-derived factor-1 was present at low levels in all samples examined. Macrophages in lesions had significantly increased expression of CCR5 and CXCR4 compared with peripheral blood cells, and CD4 T cells had significant up-regulation of CCR5. The magnitude of increased expression of these receptors was not replicated when PBMCs were incubated with H. ducreyi or H. ducreyi lipooligosaccharide in vitro. Together with the disruption of mucosal and skin barriers, the presence of cells with up-regulated HIV-1 coreceptors in H. ducreyi-infected lesions may provide an environment that facilitates the acquisition of R5 (CCR5), X4 (CXCR4), and dual-tropic HIV-1 strains.

Production of interferon-γ by lung lymphocytes in HIV-infected individuals

A CD8+ lymphocytic alveolitis occurs in up to 60% of asymptomatic human immunodeficiency virus (HIV)-infected individuals. Early in HIV infection, lymphocytes consist predominantly of cytotoxic T lymphocytes directed against HIV-infected targets. As HIV disease progresses, they are replaced by CD8+CD57+suppressor cells. Virus-specific cytotoxic T lymphocytes secrete interferon-γ (IFN-γ), an important cytokine in upregulating immune responses, primarily through macrophage activation. We examined the ability of lung and blood lymphocytes from HIV-positive patients at various stages of HIV infection to secrete IFN-γ spontaneously and in response to phytohemagglutinin A. IFN-γ production and secretion were determined with ELISA, Western blot, immunoprecipitation, and Northern blot techniques. Lung lymphocytes from HIV-infected individuals secreted large amounts of IFN-γ. However, this ability was lost in patients with late-stage disease. Correlation between blood and lung lymphocyte IFN-γ secretion was poor, suggesting regional differences in lymphocyte function. These data suggest that lung levels of IFN-γ are high until late in HIV disease. These findings support the concept of administering exogenous IFN-γ to patients with late-stage HIV disease and opportunistic infections.