RESUMO
The need for an accurate, rapid, simple and affordable point-of-care (POC) test for Tuberculosis (TB) that can be implemented in microscopy centers and other peripheral health-care settings in the TB-endemic countries remains unmet. This manuscript describes preliminary results of a new prototype rapid lateral flow TB test based on detection of antibodies to immunodominant epitopes (peptides) derived from carefully selected, highly immunogenic M. tuberculosis cell-wall proteins. Peptide selection was initially based on recognition by antibodies in sera from TB patients but not in PPD-/PPD+/BCG-vaccinated individuals from TB-endemic settings. The peptides were conjugated to BSA; the purified peptide-BSA conjugates striped onto nitrocellulose membrane and adsorbed onto colloidal gold particles to devise the prototype test, and evaluated for reactivity with sera from 3 PPD-, 29 PPD+, 15 PPD-unknown healthy subjects, 10 patients with non-TB lung disease and 124 smear-positive TB patients. The assay parameters were adjusted to determine positive/negative status within 15 minutes via visual or instrumented assessment. There was minimal or no reactivity of sera from non-TB subjects with the striped BSA-peptides demonstrating the lack of anti-peptide antibodies in subjects with latent TB and/or BCG vaccination. Sera from most TB patients demonstrated reactivity with one or more peptides. The sensitivity of antibody detection ranged from 28-85% with the 9 BSA-peptides. Three peptides were further evaluated with sera from 400 subjects, including additional PPD-/PPD+/PPD-unknown healthy contacts, close hospital contacts and household contacts of untreated TB patients, patients with non-TB lung disease, and HIV+TB- patients. Combination of the 3 peptides provided sensitivity and specificity>90%. While the final fully optimized lateral flow POC test for TB is under development, these preliminary results demonstrate that an antibody-detection based rapid POC lateral flow test based on select combinations of immunodominant M. tb-specific epitopes may potentially replace microscopy for TB diagnosis in TB-endemic settings.
Assuntos
Proteínas de Bactérias/imunologia , Epitopos Imunodominantes/isolamento & purificação , Mycobacterium tuberculosis/metabolismo , Teste Tuberculínico/métodos , Tuberculose Pulmonar/diagnóstico , Parede Celular/imunologia , Doenças Endêmicas/prevenção & controle , Humanos , Epitopos Imunodominantes/química , Mycobacterium tuberculosis/imunologia , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Soroalbumina Bovina/química , Teste Tuberculínico/economia , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologiaRESUMO
Saliva and other types of oral samples can readily be used for noninvasive diagnosis of diseases of the oral cavity and systemic diseases. Following an introduction outlining the types of oral samples and the analytes that can be measured in these samples, a detailed description of a novel oral-based diagnostic system to detect multiple bacterial and/or viral pathogens is presented. A reasonably priced, portable, point-of-care diagnostic system should be available within five years.
Assuntos
Doenças Transmissíveis/diagnóstico , Diagnóstico Bucal/métodos , Saliva/química , DNA/análise , Diagnóstico Bucal/instrumentação , Técnicas e Procedimentos Diagnósticos/instrumentação , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , RNA/análiseRESUMO
Oral fluid testing for Delta(9)-tetrahydrocannabinol (THC) provides a convenient means of detection of recent cannabis usage. In this study, the risk of positive oral fluid tests from passive cannabis smoke exposure was investigated by housing four cannabis-free volunteers in a small, unventilated, and sealed room with an approximate volume of 36 m(3). Five active cannabis smokers were also present in the room, and each smoked a single cannabis cigarette (1.75% THC). Cannabis smoking occurred over the first 20 min of the study session. All subjects remained in the room for approximately 4 h. Oral fluid specimens were collected with the Intercept DOA Oral Specimen Collection Device. Three urine specimens were collected (0, 20, and 245 min). In addition, three air samples were collected for measurement of THC content. All oral fluid specimens were screened by enzyme immunoassay (EIA) for cannabinoids (cutoff concentration = 3 ng/mL) and tested by gas chromatography-tandem mass spectrometry (GC-MS-MS) for THC (LOQ/LOD = 0.75 ng/mL). All urine specimens were screened by EIA for cannabinoids (cutoff concentration = 50 ng/mL) and tested by GC-MS-MS for THCCOOH (LOQ/LOD = 1 ng/mL). Air samples were measured for THC by GC-MS (LOD = 1 ng/L). A total of eight oral fluid specimens (collected 20 to 50 min following initiation of smoking) from the four passive subjects screened and confirmed positive for THC at concentrations ranging from 3.6 to 26.4 ng/mL. Two additional specimens from one passive subject, collected at 50 and 65 min, screened negative but contained THC in concentrations of 4.2 and 1.1 ng/mL, respectively. All subsequent specimens for passive participants tested negative by EIA and GC-MS-MS for the remainder of the 4-h session. In contrast, oral fluid specimens collected from the five cannabis smokers generally screened and confirmed positive for THC throughout the session at concentrations substantially higher than observed for passive subjects. Urine specimens from active cannabis smokers also screened and confirmed positive at conventional cutoff concentrations. A biphasic pattern of decline for THC was observed in oral fluid specimens collected from cannabis smokers, whereas a linear decline was seen for passive subjects suggesting that initial oral fluid contamination is cleared rapidly and is followed by THC sequestration in the oral mucosa. It is concluded that the risk of positive oral fluid tests from passive cannabis smoke inhalation is limited to a period of approximately 30 min following exposure.