Irma Rodriguez

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)-1β 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-1β 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-1β 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.

Detection of Hepatitis C Virus (HCV) in Serum and Peripheral-Blood Mononuclear Cells from HCV-Monoinfected and HIV/HCV–Coinfected Persons

It has been speculated that hepatitis C virus (HCV) replicates in peripheral-blood mononuclear cells (PBMCs), which, therefore, may be a site for interaction with human immunodeficiency virus (HIV). We used strand-specific real-time polymerase chain reaction to detect HCV RNA in 28 HCV-monoinfected and 20 HIV/HCV-coinfected women. At the first visit, positive-strand HCV RNA was detected in serum samples from 89% of the women, whereas positive-strand HCV RNA was detected in PBMC samples from 32% and 55% of the HCV-monoinfected and HIV/HCV-coinfected women, respectively. After initiation of antiretroviral therapy, the HIV/HCV-coinfected women were significantly more likely to have detectable positive- and negative-strand HCV RNA in the PBMC compartment than were the HCV-monoinfected women. HIV and HCV RNA levels were not correlated. Serum HCV RNA levels were correlated over time; HCV RNA levels in the serum and PBMC compartments were not. These data suggest differential regulation of HCV RNA in the serum and PBMC compartments and may partially explain the limited HCV antiviral response rates observed in coinfected persons.

Compartmentalization of Hepatitis C Virus (HCV) during HCV/HIV Coinfection

Extrahepatic replication has important implications for the transmission and treatment of hepatitis C virus (HCV). We analyzed longitudinal HCV diversity in peripheral-blood mononuclear cells (PBMCs) and serum during HCV monoinfection and HCV/HIV coinfection to determine whether distinct amino acid signatures characterized HCV replicating within PBMCs. Analysis of E1-HVR1 sequences demonstrated higher serum genetic distances among HCV/human immunodeficiency virus (HIV)-coinfected persons. Moreover, consensus PBMC sequences were rarely identical to those in the corresponding serum, suggesting divergence in these 2 compartments. Three of 5 HCV/HIV-coinfected participants showed evidence of HCV compartmentalization in PBMCs. Additionally, signature sequence analysis identified PBMC-specific amino acids in all HCV/HIV-coinfected persons. To our knowledge, this is the first study to identify specific amino acids that may distinguish HCV variants replicating in PBMCs. It is provocative to speculate that extrahepatic HCV diversity may be an important determinant of treatment response and thus warrants additional study, particularly during HCV/HIV coinfection.

Dried blood spots (DBS): a valuable tool for HIV surveillance in developing/tropical countries

Dried blood spots (DBS) on filter paper have been used as a practical method of sample collection in sero-surveillance studies of numerous diseases. DBS may be particularly useful for HIV screening in remote areas, in which unrefrigerated transfer time to a laboratory may take a number of days. In this study, we evaluate the ability to detect human immunodeficiency virus (HIV) type-1 antibodies from DBS that have been subjected to a tropical climate in southern India for 6 days. DBS were prepared from blood samples of 59 known HIV-positive individuals and 30 known HIV-negative individuals. ELISA and Western blot results from DBS that were subjected to a mean temperature of 35.8°C and humidity of 73% for 6 days had a sensitivity of 100% and 92%, respectively, and a specificity of 100% and 100%, respectively. Based on these findings, we conclude that DBS sampling could serve as a cost-effective and convenient tool for widespread HIV sero-surveillance in remote areas within tropical countries.

UNPROTECTED SEX, UNDERESTIMATED RISK, UNDIAGNOSED HIV AND SEXUALLY TRANSMITTED DISEASES AMONG MEN WHO HAVE SEX WITH MEN ACCESSING TESTING SERVICES IN A NEW ENGLAND BATHHOUSE

Abstract:American men who have sex with men (MSM) continue to have increased rates of HIV and sexually transmitted diseases (STD). Between 2004 and 2010, 1155 MSM were tested for HIV and/or STDs at Providence, RI bathhouse. The prevalence of HIV was 2.3%; syphilis, 2.0%; urethral gonorrhea, 0.1%; urethral chlamydia, 1.3%; 2.2% of the men had hepatitis C antibodies. Although 43.2% of the men engaged in unprotected anal intercourse in the prior 2 months, the majority of the men thought that their behaviors did not put them at increased risk for HIV or STDs. Multivariate analyses found that men who engaged in unprotected anal intercourse were more likely to have had sex with unknown status or HIV-infected partners; have sex although under the influence of drugs; tended to find partners on the internet; and were more likely to have a primary male partner. Men who were newly diagnosed with HIV or syphilis tended to be older than 30 years; had sex with an HIV-infected partner; had a prior STD diagnosis; and met partners on the internet. For 10.5% of the men, bathhouse testing was the first time that they had ever been screened for HIV. Of 24 men who were newly diagnosed with HIV infection, only 1 was not successfully linked to care. These data suggest that offering HIV and STD testing in a bathhouse setting is effective in attracting MSM who are at increased risk for HIV and/or STD acquisition or transmission.

Dried blood spots are an acceptable and useful HIV surveillance tool in a remote developing world setting.

Enzyme-linked immunosorbent assay and Western blot analysis of dried blood spots (DBS) on filter paper have been shown to be as sensitive and specific as analysis of serum, and therefore may be a cost-effective and culturally appropriate HIV seroprevalence tool in remote areas. This study examines the acceptability of DBS in a tropical, rural population from an outpatient clinic in Andhra Pradesh, India, where participation was offered to every fifth patient seeking general medical care between March and April 2001. All 1413 patients approached for the study agreed to participate and provide a DBS for examination. The overall HIV seroprevalence in this sample was 2.8%. Of the participants, 51.7% were male, 93.2% were between the ages of 18 and 40, 85.3% were married, 29.7% were employed, 47.6% had no education and 73.1% resided in a rural setting. In the univariate analysis, history of genital warts (P=0.01), sexually transmitted disease (P=0.001), premarital sexual intercourse (P=0.002), sexual contact with a commercial sex worker (P=0.003), being employed (P=0.011) and having more than 10 injections for medical purposes (P=0.006) all correlated with being HIV-infected. Given the uniform willingness of these clinic attendees to be tested, we conclude that DBS is a useful, cost-effective tool in HIV serosurveillance in a rural, tropical setting.

Genital Tract Interleukin-8 but not Interleukin-1 or Interleukin-6 Concentration is Associated with Bacterial Vaginosis and Its Clearance in HIV-Infected and HIV-Uninfected Women

Genital tract infections and cytokine perturbations are associated with increased HIV acquisition and transmission. We measured the relationship between bacterial vaginosis (BV) and concentrations of Interleukin-8 (IL-8), Interleukin-1β (IL-1β), and Interleukin-6 (IL-6) in cervicovaginal lavage (CVL) specimens collected longitudinally from 16 HIV-infected and 8 HIV-uninfected high-risk women. CVL samples were analyzed when women presented with BV, and at their next visit, after successful treatment, when BV was cleared. A subset of participants had cytokine levels evaluated at three consecutive clinic visits: before developing BV, at the time of BV diagnosis, and after clearing BV. Significantly higher IL-8, but not IL-1β or IL-6 levels were present when women had active BV compared to when BV was absent. Trends in cytokine levels were similar for HIV-infected and HIV-uninfected women. BV in these women was associated with significantly higher concentrations of genital tract IL-8 which decreased 2.4 fold when BV was cleared.

A Pilot Study of Treatment of Bacterial Vaginosis with a Buffering Vaginal Microbicide

BACKGROUND Bacterial vaginosis (BV) is an extremely common problem for women and is associated with adverse pregnancy outcomes and HIV infection. Currently available antibiotic treatments are moderately effective but may need to be repeated frequently because of the recurrent nature of the disease. We undertook a pilot study of a buffering vaginal microbicide in the treatment of BV. METHODS Women with clinically diagnosed BV were recruited to receive seven applications (5 g per application) of BufferGel trade mark (ReProtect, LLC, Baltimore, MD), a topical vaginal microbicide, and had clinical and gram stain evaluation of response. Subjects were evaluated at 2-3 days after the last application of BufferGel as a test of cure and again at 1 month to assess for relapse. Subjects with BV at test of cure were offered oral metronidazole. RESULTS Thirty-one women were screened, 16 were offered enrollment, and 10 completed the study. Treatment with BufferGel was clinically effective in 70% of women at 2-3 days after treatment and in 40% of women by 1-month follow-up. CONCLUSIONS These results suggest that 5 g of BufferGel vaginally once a day appears to be a moderately effective treatment for BV.