Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1beta and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1beta and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal-Wallis analysis of variance with Dunn's multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences ( P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1beta determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.

HIV testing in patients presenting with indicator conditions in outpatient settings: offer and uptake rates, and educational and active interventions.

Approximately 13% of people living with HIV in the UK are undiagnosed which has significant implications in terms of onward transmission and late diagnosis. HIV testing guidelines recommend routine screening in anyone presenting to healthcare with an HIV indicator condition (IC); however, this does not occur routinely. This study aimed to assess the feasibility and effectiveness of using case note prompts highlighting the presence of an IC to increase HIV testing. Clinicians in three outpatient departments received case note prompts either before or after a period of clinician-led identification. Test offer and uptake rates were assessed. A parallel anonymous seroprevalence study estimated the prevalence of undiagnosed HIV. A total of 4191 patients had an appointment during the study period; 608 (14.5%) had an IC. HIV test offer was significantly higher when a prompt was inserted into notes (34.3% versus 3.2%, p < 0.001). The prevalence of diagnosed HIV in the cohort was 4.1%. No cases of undiagnosed HIV infection were identified. Despite guidelines, offer of HIV testing is low. Strategies to increase routine screening of patients presenting with an IC are needed. Individual case note prompts significantly increase HIV test offer; however, the effect is lost if the strategy is withdrawn.

Fungal burden, early fungicidal activity, and outcome in cryptococcal meningitis in antiretroviral-naive or antiretroviral-experienced patients treated with amphotericin B or fluconazole.

In a prospective observational study of 54 patients with human immunodeficiency virus-associated cryptococcal meningitis, the early fungicidal activity of amphotericin B (1 mg/kg/day) was significantly greater than that of fluconazole (400 mg/day). Compared with antiretroviral therapy-naive patients, patients developing cryptococcal meningitis while already receiving antiretroviral therapy had lower baseline fungal burdens and a longer median duration of survival, but there were no differences observed in fungal clearance, cerebrospinal fluid proinflammatory cytokines, or 10-week mortality.

Immune reconstitution inflammatory syndrome in HIV-associated cryptococcal meningitis: a prospective study.

BACKGROUND: Prospective data on incidence, characteristics, and risk factors for cryptococcal meningitis immune reconstitution inflammatory syndrome (CM-IRIS) are lacking. METHODS: Prospective study of 65 antiretroviral therapy (ART)-naive HIV-infected cryptococcal meningitis (CM) patients, who started ART after initiation of antifungal treatment. CM-IRIS definition: (1) cerebrospinal fluid (CSF) culture-confirmed CM, (2) symptom resolution before starting ART, (3) adherence to fluconazole and ART, (4) recurrence of CM symptoms after starting ART, (5) immunologic and/or virologic response to ART, (6) no alternative diagnosis. RESULTS: ART was started at a median of 47 days from CM diagnosis. CM-IRIS developed in 11 of 65 (17%), at a median 29 days from starting ART. No factors at first CM episode (fungal burden, rate of clearance, CSF, or HIV parameters) predicted those at risk of CM-IRIS. At 6 months on ART, IRIS patients had greater CD4 rise from baseline (220 vs. 124 x 10 cells /L in non-IRIS, P = 0.01), and 4 of 11 CM-IRIS patients died compared with 14 of 54 non-IRIS patients (P = 0.5). For those developing CM-IRIS, CSF proinflammatory cytokines interferon gamma, tumour necrosis factor alpha, and interleukin 6, did not differ between first CM and CM-IRIS episode. CONCLUSIONS: Patients with CM-IRIS had greater immune restoration in response to ART. Although common and potentially fatal, larger prospective studies are needed to determine whether CM-IRIS, in patients treated initially with amphotericin B, is associated with any increase in overall mortality.