Diagnostic Performance of Tuberculosis-Specific IgG Antibody Profiles in Patients with Presumptive Tuberculosis from Two Continents.

BACKGROUND: Development of rapid diagnostic tests for tuberculosis is a global priority. A whole proteome screen identified Mycobacterium tuberculosis antigens associated with serological responses in tuberculosis patients. We used World Health Organization (WHO) target product profile (TPP) criteria for a detection test and triage test to evaluate these antigens. METHODS: Consecutive patients presenting to microscopy centers and district hospitals in Peru and to outpatient clinics at a tuberculosis reference center in Vietnam were recruited. We tested blood samples from 755 HIV-uninfected adults with presumptive pulmonary tuberculosis to measure IgG antibody responses to 57 M. tuberculosis antigens using a field-based multiplexed serological assay and a 132-antigen bead-based reference assay. We evaluated single antigen performance and models of all possible 3-antigen combinations and multiantigen combinations. RESULTS: Three-antigen and multiantigen models performed similarly and were superior to single antigens. With specificity set at 90% for a detection test, the best sensitivity of a 3-antigen model was 35% (95% confidence interval [CI], 31-40). With sensitivity set at 85% for a triage test, the specificity of the best 3-antigen model was 34% (95% CI, 29-40). The reference assay also did not meet study targets. Antigen performance differed significantly between the study sites for 7/22 of the best-performing antigens. CONCLUSIONS: Although M. tuberculosis antigens were recognized by the IgG response during tuberculosis, no single antigen or multiantigen set performance approached WHO TPP criteria for clinical utility among HIV-uninfected adults with presumed tuberculosis in high-volume, urban settings in tuberculosis-endemic countries.

Medication adherence in patients with cluster headache and migraine: an online survey.

To examine factors for adherent and non-adherent behavior in patients with cluster headache and migraine. Adults with cluster headache or migraine were included in this anonymous online survey using a questionnaire accessed via homepages of headache support groups. Medication adherence in preventive treatment was measured with the Medication Adherence Report Scale (MARS-D). Factors for non-adherent behavior were examined (subjective socioeconomic status, psychological comorbidities, self-efficacy, coping, side effects, expectations of treatment, information on medical treatment, and trust in the physician/treatment concept). 200 participants (n = 58 with cluster headache, n = 142 with migraine) were included. The rate of medication adherence in preventive treatment was 32.8% for participants with cluster headache and 20.4% for migraine. The most common reasons for low adherence in participants with cluster headache were altering the prescribed medication dose (34%) or taking less than instructed (14%), which was mostly due to insufficient benefit from the medication or side effects. Positive expectations of medical treatment (p ≤ 0.05) correlated significantly with adherent behavior in cluster headache. Furthermore, the adherence-promoting factors coping and self-efficacy were more pronounced in patients with cluster headache than in those with migraine (p < 0.05). This study is the first to comprehensively investigate medication adherence and factors influencing adherent/non-adherent behavior in patients with cluster headache. Patients with cluster headache had similar adherence levels to patients with migraine, but had higher resources of adherence-promoting factors.

Protein-protein-interactions in a multiplexed, miniaturized format a functional analysis of Rho GTPase activation and inhibition.

A miniaturized, bead-based protein-protein-interaction assay was developed to study the interaction of Rho GTPases with regulatory proteins. The setup, which uses only minute amounts of sample, was used to analyze small molecules that inhibit the interaction between Rho GTPases and RhoGDI alpha. Prenylcysteine analogues and the replacement of GDP by non-hydrolysable GTP analogues prevented the formation of Rho GTPase-RhoGDI alpha complexes in a concentration-dependent manner.

Bead-based protein-protein interaction assays for the analysis of Rho GTPase signaling.

Bead-based interaction assays are excellently suited to study protein-protein interactions, as they require only minimal amounts of sample material. Miniaturized protein-protein interaction assays were designed to analyze Rho GTPase activation based on its interaction with Rho GDI or p21-activated kinase (PAK).Rho GDI plays a key role in the regulation of a variety of cellular functions through its interaction with Rho GTPases. Rho GDI is frequently overexpressed in many human cancers. Therefore, there is a growing and as yet unfulfilled demand for screening assays to identify biologically active compounds that may inhibit the Rho GTPase-Rho GDI interaction. Bead-based interaction assays provide an interesting alternative that facilitate such assays to be performed faster with only small amounts of material compared to routinely used co-immunoprecipitation followed by Western Blot analysis.Bead-based protein interaction assays for overexpressed HA-tagged Rho GTPases were established to study the GTPγS-dependent interaction of five different Rho GTPases with the regulatory protein Rho GDIα and the downstream effector PAK1. In addition, it was demonstrated that the ability of Rho GTPases to interact with Rho GDI in this experimental system was markedly, but differentially sensitive to post-translational modification of their carboxyl terminus. Importantly, this modification also notably affected the ability of Rac1 and Rac2, but not of Cdc42, to interact with PAK1.

Serologic Responses in Childhood Pulmonary Tuberculosis.

BACKGROUND: Identification of the Mycobacterium tuberculosis immunoproteome and antigens associated with serologic responses in adults has renewed interest in developing a serologic test for childhood tuberculosis (TB). We investigated IgG antibody responses against M. tuberculosis antigens in children with well-characterized TB. METHODS: We studied archived sera obtained from hospitalized children with suspected pulmonary TB, and classified as having confirmed TB (culture-confirmed), unlikely TB (clinical improvement without TB treatment), or unconfirmed TB (all others). A multiplexed bead-based assay for IgG antibodies against 119 M. tuberculosis antigens was developed, validated and used to test sera. The area under the curves (AUCs) of the empiric receiver-operator characteristic curves were generated as measures of predictive ability. A cross-validated generalized linear model was used to select the most predictive combinations of antigens. RESULTS: For the confirmed TB versus unlikely TB comparison, the maximal single antigen AUC was 0.63, corresponding to sensitivity 0.60 and specificity 0.60. Older (age: 60+ months old) children's responses were better predictive of TB status than younger (age: 12-59 months old) children's, with a maximal single antigen AUC of -0.76. For the confirmed TB versus unlikely TB groups, the most predictive combinations of antigens assigned TB risk probabilities of 0.33 and 0.33, respectively, when all ages were considered, and 0.57 (interquartile range: 0.48-0.64) and 0.35 (interquartile range: 0.32-0.40) when only older children were considered. CONCLUSION: An antigen-based IgG test is unlikely to meet the performance characteristics required of a TB detection test applicable to all age groups.

Systematic reference sample generation for multiplexed serological assays.

Quality controls of serological assays have to contain defined amounts of human antibodies specific for the targeted antigen. A prevailing issue for array-based antigen assays is that dozens of antigens are targeted within the same assay. Commonly different patient sera are combined and optimal pools are empirically identified. Here, we report a mathematical approach how an optimal sample pool composition can be systematically calculated and accurately compiled. The approach was used to compose suitable quality controls for a 71 plex Tuberculosis antigen bead array using a limited number of positive human sera.

Functional analysis of Rho GTPase activation and inhibition in a bead-based miniaturized format.

Extensive knowledge about protein-protein interactions is fundamental to fully understand signaling pathways and for the development of new drugs. Rho GTPases are key molecules in cellular signaling processes and their deregulation is implicated in the development of a variety of diseases such as neurofibromatosis type 2 and cancer. Here, we describe a bead-based protein-protein interaction assay for overexpressed HA-tagged Rho GTPases to study the GTPγS-dependent interaction with the regulatory protein RhoGDIα. This assay provides a useful tool for the analysis of both macromolecular and small molecule activators and inhibitors of the protein-protein interactions of Rho GTPases with their regulatory proteins in a multiplexed miniaturized format.