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-1 target cells in foreskins of African men with varying histories of sexually transmitted infections.

Numerous epidemiologic studies have found significant associations between lack of circumcision and HIV-1 acquisition in men. To our knowledge, this is the first study of human foreskin tissue that examines biologic mechanisms that increase susceptibility of uncircumcised African men to HIV-1. Foreskin specimens from 20 men with and 19 men with no history of sexually transmitted infections were examined for HIV-1 target cells. Most Langerhans cells were found in the epithelium; most CD4+ T cells and macrophages were in the submucosa. There were no differences in HIV-1 target cells between men with and those without history of sexually transmitted infections. However Langerhans cells and macrophages were more abundant in the group with a history of infection. The densities and positions of HIV-1 target cells in the foreskin tissue of these Kenyan men indicate that the inner mucosal surface of the human foreskin contains cells that make it highly susceptible to HIV infection.

Cervicovaginal levels of lactoferrin, secretory leukocyte protease inhibitor, and RANTES and the effects of coexisting vaginoses in human immunodeficiency virus (HIV)-seronegative women with a high risk of heterosexual acquisition of HIV infection.

Innate immune factors in mucosal secretions may influence human immunodeficiency virus type 1 (HIV-1) transmission. This study examined the levels of three such factors, genital tract lactoferrin [Lf], secretory leukocyte protease inhibitor [SLPI], and RANTES, in women at risk for acquiring HIV infection, as well as cofactors that may be associated with their presence. Women at high risk for HIV infection meeting established criteria (n = 62) and low-risk controls (n = 33) underwent cervicovaginal lavage (CVL), and the CVL fluid samples were assayed for Lf and SLPI. Subsets of 26 and 10 samples, respectively, were assayed for RANTES. Coexisting sexually transmitted infections and vaginoses were also assessed, and detailed behavioral information was collected. Lf levels were higher in high-risk (mean, 204 ng/ml) versus low-risk (mean, 160 ng/ml, P = 0.007) women, but SLPI levels did not differ, and RANTES levels were higher in only the highest-risk subset. Lf was positively associated only with the presence of leukocytes in the CVL fluid (P < 0.0001). SLPI levels were lower in women with bacterial vaginosis [BV] than in those without BV (P = 0.04). Treatment of BV reduced RANTES levels (P = 0.05). The influence, if any, of these three cofactors on HIV transmission in women cannot be determined from this study. The higher Lf concentrations observed in high-risk women were strongly associated with the presence of leukocytes, suggesting a leukocyte source and consistent with greater genital tract inflammation in the high-risk group. Reduced SLPI levels during BV infection are consistent with an increased risk of HIV infection, which has been associated with BV. However, the increased RANTES levels in a higher-risk subset of high-risk women were reduced after BV treatment.

The public health imperative for a neonatal herpes simplex virus infection surveillance system.

About 1 in 5 sexually active adults in the United States has serologic evidence of genital herpes caused by herpes simplex virus type-2. Neonatal herpes simplex virus infection is a serious consequence of genital herpes infection. Herpes infection in neonates causes significant morbidity and neurologic damage and generally has a case-fatality ratio untreated of 60%. It is estimated that 440 to 1,320 cases of neonatal herpes infections occur in the United States per year (11-33 cases occur per 100,000 live births). Given the challenges in surveillance for genital herpes due to the large number of asymptomatic infections and infrequent laboratory-based diagnosis, we recommend that to begin an effective national control program for herpes infections, a mandatory national surveillance system for neonatal herpes be implemented. Such a system would help assure appropriate therapy, help monitor trends and understand the burden of disease, identify risk determinants, and evaluate prevention efforts.

HIV-1-infected antiretroviral-treated patients with prolonged partial viral suppression: clinical, virologic, and immunologic course.

The long-term feasibility of a drug conservation strategy that allows low-level viral replication is unknown. We performed a retrospective study of treated HIV-infected patients with stable detectable viral replication (<10000 copies/mL [low-level viremia]) and compared their clinical, virologic and immunologic courses with those of treated patients with undetectable viremia and viremia (>or=10000 copies/mL [high-level viremia]). Viral reverse transcriptase and protease genotype and HIV-specific CD4 T-cell responses were determined using patient-derived samples. Clinical and immunologic benefits were maintained in patients with partial virologic suppression (or=2 classes of antiretroviral medications. HIV-specific CD4+ T-cell immunity was detected in most subjects with low-level and undetectable viremia and may have a role in controlling viremia in the setting of partial suppression.