Associations of the correlates of protection and implication on the statistical power for demonstrating non-inferiority: application of a re-sampling method on a large phase III influenza vaccine clinical trial.

BACKGROUND: In the later stage of the clinical development of new vaccines it is required to demonstrate their efficacy with the immunogenicity measures established as correlates for disease protection. Evaluation of interpandemic trivalent influenza vaccines is commonly assessed by three immunogenicity measures for each strain in different age groups: Seroprotection, Seroconversion and Geometric Mean Titers. US and European guidelines with respect to this topic have been issued for the licensure of new influenza vaccines. The statistical power of comparative trials, which consider these endpoint variables, could be affected to the extent that these measures are correlated. Results from a large non-inferiority trial in the clinical development of a novel cell-derived influenza vaccine have been analyzed with the aim of evaluating how statistical dependency between the above-mentioned three immunogenicity measures might affect the power to demonstrate non-inferiority. METHODS: The statistical non-inferiority criteria, which were met in the trial, were applied to different subsets (n=250, n=370 and n=500) using a re-sampling method from the original dataset (re-samples=10,000). RESULTS: The measures of immunogenicity were highly correlated, and the fulfillment or failure of any of the non-inferiority criteria for a specific measure partially predicted the same outcome for the other measures. Due to this dependency within each strain, the levels of power obtained by re-sampling methods were always higher than those obtained by theoretical calculations, which were based on the assumptions of independency between the three measures of immunogenicity. Seroconversion and Geometric Mean Ratio (GMR) showed a higher correlation. A failure in the fulfillment of the non-inferiority criteria for GMR predicted the failure for Seroconversion in >76% of cases. CONCLUSIONS: The correlation between different measures of immunogenicity should be taken into account when evaluating statistical power for non-inferiority in influenza vaccine trials and in establishing sample sizes. Statistical approaches that include either all three measures of immunogenicity or both Seroconversion and the ratio of GMTs as co-primary non-inferiority endpoints might create redundancy and could increase the probability of not meeting at least one non-inferiority criterion by chance, due to multiplicity.

Safety, tolerability and immunogenicity of a mammalian cell-culture-derived influenza vaccine: a sequential Phase I and Phase II clinical trial.

This sequential, observer-blind, randomised, single-centre, combined Phase I and Phase II clinical trial compared the tolerability and immunogenicity of a single intramuscular dose of a novel cell-culture-derived influenza vaccine (CCIV), produced in Madin-Darby canine kidney cells, with a conventional egg-based vaccine. The immunogenicity of both vaccines was assessed by SRH assay, a well-recognized test by EMEA, in compliance with the requirements of the EU Committee for Medicinal Products for Human Use (CHMP). The Phase I part of the trial comprised 40 healthy adults (18-40 years of age); the subsequent Phase II part involved 200 healthy adult (n=80, 18-60 years of age) and elderly (n=120, > or =61 years of age) subjects. Both vaccines showed similar reactogenicity and any solicited local or systemic reactions were mostly mild or moderate. Regarding immunogenicity, both the CCIV and the control vaccine met all of the EU Committee for Medicinal Products for Human Use criteria for influenza vaccines for each strain and in both age groups. In conclusion, the CCIV produced in mammalian cell-culture is as well tolerated and as immunogenic as the control egg-based vaccine in non-elderly and elderly adults.

Blood profiles, body fat and mood state in healthy subjects on different diets supplemented with Omega-3 polyunsaturated fatty acids.

BACKGROUND: Diets and Omega-3 polyunsaturated fatty acids have been considered as important factors to reduce the risk of cardiovascular and inflammatory diseases, but there are few details on the effects on healthy subjects. The aim of the present study was to examine the variation of several physiological parameters in healthy subjects on different diets supplemented with Omega-3 fatty acids. MATERIALS AND METHODS: The experiment was carried out on 33 subjects divided into four groups according to a double-blind cross-over design with a 1 : 1 ratio for Omega-3 (vs. placebo) and open-label parallel-groups with a 1 : 1 ratio for the Zone diet (vs. the diet suggested by the Italian National Research Institute for Nutrition and Foods). Blood samples were collected at the beginning of the experiment and after 35 (cross-over) and 70 days. The Profile of Mood States test (POMS) was also performed. RESULTS: The arachidonic acid/eicosapentaenoic acid ratio (AA/EPA) was strongly reduced by Omega-3 with a supplementary effect of the diet and in particular the Zone diet. The AA/EPA reduction was correlated with a concomitant decrease of insulin and homocysteine levels. The Zone diet reduced skinfold thickness and body fat percentage and also showed antioxidant effects. The mood state changed after Omega-3 supplementation, with an increased POMS index. This was related to a concomitant reduction of AA/EPA and was particularly evident in the Zone diet. CONCLUSION: AA/EPA and mood state are differently influenced by diet and Omega-3, body fat is particularly reduced by Zone diet, while blood parameters such as triglycerides/HDL ratio, insulin and homocysteine are related to AA/EPA variations. These findings are discussed in terms of differences in the composition of the diets and the influences of Omega-3 on physiological functions.

Prevalence and risk factors for herpes simplex virus type 2 infection among adolescents and adults in northern Cameroon.

BACKGROUND: A number of surveys have reported a high prevalence of herpes simplex virus type 2 (HSV-2) infection in south central and east central Africa. GOAL: To evaluate the distribution and modes of HSV-2 transmission in northern Cameroon. STUDY DESIGN: A serosurvey was conducted among 194 adolescents and adults in a Cameroon hospital. Antibodies against HSV-2 were detected by a gG2-specific enzyme-linked immunoabsorbent assay. RESULTS: The prevalence of HSV-2 antibodies was found to be 37.1%. In the multivariate analysis, female gender, no condom use, a positive syphilis serology, and HIV infection were significantly associated with HSV-2 infection. CONCLUSIONS: The current findings show that the HSV-2 seroprevalence in northern Cameroon is high and similar to that found in other African countries. HSV-2 infection represents a marker for at-risk sexual behavior and may be a common cofactor for HIV transmission in this geographic area.

Low frequency of severe hepatotoxicity and association with HCV coinfection in HIV-positive patients treated with HAART.

BACKGROUND: Highly active antiretroviral therapy (HAART) is strongly effective in reducing morbidity and mortality in HIV-1-positive individuals. Its main drawback is the potential toxicity. Data on the frequency and determinants of severe hepatotoxicity in a clinical setting are still sparse. METHODS: This is a prospective study of HIV-1-positive individuals with known HBsAg and HCV-Ab serology. The study end point was progression to alanine aminotransferase (ALT) levels > or =200 IU/L after HAART initiation. Cumulative probability of progression to this end point was estimated by the Kaplan-Meier method. Crude and adjusted hazard ratios (HR) were estimated by proportional hazards regression model. RESULTS: One thousand two hundred fifty-five patients were included. HBsAg was found in 91 (7.2%), HCV-Ab in 578 (46.5%) patients; almost all injection drug users (451 of 482; 93.6%) were HCV-Ab positive. Sixty-one individuals progressed to the end point with a probability of 7.9% (95% confidence interval [CI], 5.6-10.0) of progression at 24 months from starting. Independent factors predicting progression to the end point were baseline ALT levels (HR, 5.29; 95% CI, 3.24-8.65; every 10 IU/L higher), HCV-Ab positivity (HR, 4.01; 95% CI, 1.48-10.85) or both HBsAg and HCV Ab positivity (HR, 3.85, 95% CI, 1.01-14.61), and previous non-HAART therapy (HR, 1.84, 95% CI, 1.04-3.42). Patients receiving stavudine-containing regimens had a lower risk than those receiving zidovudine-containing regimens (HR, 0.30, 95% CI, 0.12-0.71). CONCLUSIONS: There was a low risk of ALT > or =200 IU/L in our cohort. Hepatitis C coinfection and elevated ALT levels at HAART initiation are important predictors of progression to ALT > or =200 IU/L; stavudine-containing regimens were associated with a lower risk compared with zidovudine-containing regimens.

Markers of cell death-activation in lymphocytes of vertically HIV-infected children naive to highly active antiretroviral therapy: the role of age.

BACKGROUND: Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear. OBJECTIVES: We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection. METHODS: Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential. RESULTS: Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage. CONCLUSION: Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.

Kaposi's sarcoma-associated herpesvirus serology in Europe and Uganda: multicentre study with multiple and novel assays.

A multicentre study was undertaken to define novel assays with increased inter-assay concordance, sensitivity, specificity and predictive value for serological diagnosis of human herpesvirus type 8 (HHV-8) infection. A total of 562 sera from European and Ugandan human immunodeficiency virus (HIV)-infected or uninfected individuals with or without Kaposi's sarcoma (KS) and blood donors were examined under code by 18 different assays in seven European laboratories. Sera from KS patients and all non-KS sera found positive by at least 70%, 80%, or 90% of the assays were considered "true positive." The validity of the assays was then evaluated by univariate logistic regression analysis. Two immunofluorescence assays (IFA) for detection of antibodies against HHV-8 lytic (Rlyt) or latent (LLANA) antigens and two enzyme-linked-immunosorbent assays (ELISA) (M2, EK8.1) for detection of antibodies against HHV-8 structural proteins were found to be highly concordant, specific, and sensitive, with odds ratios that indicated a high predictive value. When used together, the two IFA (Rlyt-LLANA) showed the best combination of sensitivity (89.1%) and specificity (94.9%). The performance of these assays indicate that they may be used for the clinical management of individuals at risk of developing HHV-8 associated tumours such as allograft recipients.

Serum concentrations of fibroblast growth factor 2 are increased in HIV type 1-infected patients and inversely related to survival probability.

HIV-1-infected patients develop a generalized vasculopathy that is clinically most evident as Kaposi's sarcoma (KS), a multifocally appearing endothelial cell-derived tumor. Fibroblast growth factor 2 (FGF-2) is a potent autocrine and paracrine mitogen of endothelial cells and has been implicated in the cell proliferation and angiogenesis observed in KS. Here we determined by ELISA the FGF-2 serum concentrations in different clinical groups of HIV-1-infected patients. AIDS-KS patients (n = 53) and HIV-1-infected patients without KS (n = 39) revealed significantly increased FGF-2 serum concentrations (median, 4.5 and 4.6 pg/ml, respectively), as compared with the healthy control group (n = 22; median, 2.2 pg/ml; p < 0.01). FGF-2 concentrations were highest in untreated HIV-1-infected patients (median, 8.6 pg/ml) and were significantly decreased in patients undergoing antiretroviral therapy (AZT-median, 4.5 pg/ml; HAART-median, 2.5 pg/ml; p < 0.01). In addition, FGF-2 serum concentrations above 5.2 pg/ml were associated with a statistically significant higher risk of death in HIV-1-infected patients. Multivariate analysis showed that this effect is independent of CD4 levels, localization of KS (cutaneous or visceral), AIDS-defining opportunistic diseases, and therapy. Circulating FGF-2 may contribute to AIDS-associated vasculopathy and may be a sensitive and easily accessible surrogate marker to determine the survival time of HIV-1-infected patients and the efficacy of antiretroviral therapy.

Prevalence and risk factors for human herpesvirus 8 infection in northern Cameroon.

BACKGROUND: The modes of transmission of HHV-8 are still unclear. GOAL: To evaluate the distribution and transmission of HHV-8 infection. DESIGN: Serosurvey conducted in a Cameroon hospital among 292 persons, including children (5-10 years), adolescents (15-20 years), and adults (30-40 years). Antibodies against lytic and latent antigens to HHV-8 were detected by immunofluorescence assay; antibodies against Epstein-Barr virus viral antigens were detected by enzyme-linked immunoabsorbent assay. RESULTS: The prevalence of HHV-8 antilytic antibodies remained stable and was 39.8% among children, 51.5% among adolescents, and 61.8% among adults. Epstein-Barr virus seroprevalence was high among children, and remained stable among adolescents and adults. A history of sexually transmitted diseases was an independent determinant of HHV-8 infection (adjusted odds ratio 2.47; 95% CI 1.09-4.91). CONCLUSION: The high prevalence of HHV-8 infection among children indicates nonsexual modes of transmission in Cameroon, with sexual transmission occurring among adolescents and adults.

PIP: The modes of transmission of HHV-8 are still unclear. To evaluate the distribution and transmission of HHV-8 infection serosurvey was conducted in a Cameroon hospital among 292 persons, including children (5-10 years), adolescents (15-20 years), and adults (30-40 years). Antibodies against lytic and latent antigens to HHV-8 were detected by immunofluorescence assay; antibodies against Epstein-Barr virus viral antigens were detected by enzyme-linked immunoabsorbent assay. The prevalence of HHV-8 antilytic antibodies remained stable and was 39.8% among children, 51.5% among adolescents, and 61.8% among adults. Epstein-Barr virus seroprevalence was high among children, and remained stable among adolescents and adults. A history of sexually transmitted diseases was an independent determinant of HHV-8 infection (adjusted odds ratio, 2.47; 95% CI, 1.09-4.91). The high prevalence of HHV-8 infection among children indicates nonsexual modes of transmission in Cameroon, with sexual transmission occurring among adolescents and adults.

Antiretroviral therapy in HIV-infected individuals in clinical practice: are the criteria for initiating and choosing the type of drug regimen based only on immunologic and virologic values?

OBJECTIVES: To determine factors associated with beginning antiretroviral therapy and with the number of drugs used. METHODS: Longitudinal study of 3169 HIV-infected individuals naïve from antiretroviral drugs at enrollment in 65 infectious disease clinics in Italy. Initiation of antiretroviral therapy and number of drugs used (i.e., < 3 vs. > or = 3 drugs) were the main outcome measures. Adjusted odds ratios were calculated by logistic models to establish cofactors of these two measures. RESULTS: From January 1997 to December 1998, 1288 (40.6%) individuals started therapy, 58.0% of whom were given a triple combination regimen. This regimen became more frequent over time. By multivariate analysis, high levels of HIV-RNA and low CD4 counts were the most important independent predictors of starting any type of therapy. A significant association was also found with HIV exposure category, reason for being antiretroviral-naïve, presence/absence of liver disease, presence/absence of a new AIDS-defining disease, and clinical centre. High levels of HIV-RNA and low CD4 counts were also the most important predictors of starting with > or = 3 drugs, compared to < 3 drugs, and men had an independent higher probability of starting with > or = 3 drugs, compared to women. The probability of starting with > or = 3 drugs significantly increased with calendar time. CONCLUSIONS: CD4 and HIV-RNA were the main cofactors of initiating both any type of therapy and therapy with > or = 3 drugs. The large variability among clinical centres suggests that clinicians are uncertain as to the exact timing of beginning therapy and the specific regimen, especially among women.

Predilection sites of Trichinella spiralis larvae in naturally infected horses.

A total of 120 muscle tissues from three horses naturally infected with Trichinella spiralis were examined. The head was the most infected site. In particular, the muscles harbouring the highest number of larvae were: musculus buccinator (12, 411 and 1183 larvae g-1), the tongue (11, 615 and 1749 larvae g-1), m. levator labii maxillaris (17,582 and 1676 larvae g-1), and the masseter (4.9, 289 and 821 larvae g-1). Compared with the diaphragm, the number of larvae per gram was from 3.5 to 6.8 times higher in the tongue, from 3.5 to 6.5 higher in m. levator labii maxillaris, and from 2.5 to 4.6 higher in m. buccinator. Of the examined muscles, the diaphragm had from the 6th to the 15th highest level of infection (3.1, 166 and 256 larvae g-1). Published data from experimentally infected horses confirm these results, suggesting that efforts to detect predilection sites should focus on the head muscles.