Development of a generic microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids.

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive "sample-to-result" diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

Development of an ultrasensitive immunochromatography test to detect nicotine metabolites in oral fluids.

Passive exposure to tobacco smoke causes a variety of illnesses ranging from allergic responses to cancer. Assessment of exposure to second-hand tobacco smoke (SHS), particularly among vulnerable populations enables intervention and prevention of future disease. A minimally invasive oral fluids-based onsite test to detect such exposure would create a valuable tool for researchers and clinicians. Here we describe the development of a test that uses an inexpensive reader that utilizes a CMOS image sensor to reliably quantify a reporter signal and determine nicotine exposure. The rapid lateral flow test consists of a nitrocellulose strip with a control line containing goat anti-rabbit IgG, used as an internal standard, and a test line containing BSA-cotinine conjugate. To run the test, diluted sample containing antibodies against cotinine, the major metabolite of nicotine, is mixed with protein A-gold nanoparticles and placed on the sample pad. As the sample runs up to the nitrocellulose pad, antibodies in the running buffer bind to available cotinine. If cotinine is absent, the antibodies will bind to the BSA-cotinine derivative immobilized on the test line, resulting in an intense purple-red band. The concentration of cotinine equivalents in the sample can be estimated from interpretation of the test line. In this article we describe the effect of different cotinine derivatives, oral fluid pretreatment, and application and running buffers on assay sensitivity. The test can reliably detect as little as 2 ng mL(-1) cotinine equivalents. The assay is sensitive, simple, rapid, inexpensive, and easily implementable in point-of-care facilities to detect second-hand smoke exposure.

Biomarker discovery: validation and decision-making in product development.

The conference on which this supplement reports was organized to bring together microbicide researchers with researchers in the areas of drug prophylaxis and therapeutics development. The goal of session 1 was to share methods used to validate markers of disease, elucidate the logic used to substantiate the performance of those markers, and identify ways to translate this experience into practical steps for developing microbicides. The experiences discussed ranged from de novo discovery of new therapeutics to qualification of biomarkers across all stages of development, and covered the complexity of biomarker identification, use, and assessment in the clinical areas of cancer and infectious disease. This review is based on the presentations of Drs. Harsukh Parmar (AstraZeneca), Sudhir Srivastava (National Cancer Institute [NCI]), and Juan Leal (Exelixis Inc.), and the subsequent discussion led by Drs. Sam Niedbala (Lehigh University) and Thomas Moench (ReProtect) during a background session held at the conference entitled "Biomarkers for evaluation of vaginal microbicides and contraceptives: Discovery and early validation," organized by CONRAD and the Alliance for Microbicide Development in November 2006.

Rapid prototyping of lateral flow assays.

Principles and characteristics of lateral flow strip assays are reviewed. Recent technology developments permit the use of inexpensive electronic readers for interrogating lateral flow strip test results, thus avoiding the inevitable variation and subjectivity of visual inspection to assess the capture of reporter-labeled analyte on test lines of the strip. Protocols for developing lateral flow assays are described, including two specific case studies for assaying cotinine (a small-molecule metabolite of nicotine) in a competitive format, and assaying HIV antibodies in a sandwich-type assay format.

A microfluidic system for saliva-based detection of infectious diseases.

A "lab-on-a-chip" system for detecting bacterial pathogens in oral fluid samples is described. The system comprises: (1) an oral fluid sample collector; (2) a disposable, plastic microfluidic cassette ("chip") for sample processing including immunochromatographic assay with a nitrocellulose lateral flow strip; (3) a platform that controls the cassette operation by providing metered quantities of reagents, temperature regulation, valve actuation; and (4) a laser scanner to interrogate the lateral flow strip. The microfluidic chip hosts a fluidic network for cell lysis, nucleic acid extraction and isolation, PCR, and labeling of the PCR product with bioconjugated, upconverting phosphor particles for detection on the lateral flow strip.

Passive cannabis smoke exposure and oral fluid testing. II. Two studies of extreme cannabis smoke exposure in a motor vehicle.

Two studies were conducted to determine if extreme passive exposure to cannabis smoke in a motor vehicle would produce positive results for delta-tetrahydrocannabinol (THC) in oral fluid. Passive exposure to cannabis smoke in an unventilated room has been shown to produce a transient appearance of THC in oral fluid for up to 30 min. However, it is well known that such factors as room size and extent of smoke exposure can affect results. Questions have also been raised concerning the effects of tobacco when mixed with marijuana and THC content. We conducted two passive cannabis studies under severe passive smoke exposure conditions in an unventilated eight-passenger van. Four passive subjects sat alongside four active cannabis smokers who each smoked a single cannabis cigarette containing either 5.4%, 39.5 mg THC (Study 1) or 10.4%, 83.2 mg THC (Study 2). The cigarettes in Study 1 contained tobacco mixed with cannabis; cigarettes in Study 2 contained only cannabis. Oral fluid specimens were collected from passive and active subjects with the Intercept Oral Specimen Collection Device for 1 h after smoking cessation while inside the van (Study 1) and up to 72 h (passive) or 8 h (active) outside the van. Additionally in Study 1, Intercept collectors were exposed to smoke in the van to assess environmental contamination during collection procedures. For Study 2, all oral fluid collections were outside the van following smoking cessation to minimize environmental contamination. Oral samples were analyzed with the Cannabinoids Intercept MICRO-PLATE EIA and quantitatively by gas chromatography-tandem mass spectrometry (GC-MS-MS). THC concentrations were adjusted for dilution (x 3). The screening and confirmation cutoff concentrations for THC in neat oral fluid were 3 ng/mL and 1.5 ng/mL, respectively. The limits of detection (LOD) and quantitation (LOQ) for THC in the GC-MS-MS assay were 0.3 and 0.75 ng/mL, respectively. Urine specimens were collected, screened (EMIT, 50 ng/mL cutoff), and analyzed by GC-MS-MS for THCCOOH (LOD/LOQ = 1.0 ng/mL). Peak oral fluid THC concentrations in passive subjects recorded at the end of cannabis smoke exposure were up to 7.5 ng/mL (Study 1) and 1.2 ng/mL (Study 2). Thereafter, THC concentrations quickly declined to negative levels within 30-45 min in Study 1. It was found that environmentally exposed Collectors contained 3-14 ng/mL in Study 1. When potential contamination during collection was eliminated in Study 2, all passive subjects were negative at screening/confirmation cutoff concentrations throughout the study. Oral fluid specimens from active smokers had peak concentrations of THC approximately 100-fold greater than passive subjects in both studies. Positive oral fluid results were observed for active smokers 0-8 h. Urine analysis confirmed oral fluid results. These studies clarify earlier findings on the effects of passive cannabis smoke on oral fluid results. Oral fluid specimens collected in the presence of cannabis smoke appear to have been contaminated, thereby falsely elevating THC concentrations in oral fluid. The risk of a positive test for THC was virtually eliminated when specimens were collected in the absence of THC smoke.

High prevalence of 6-acetylmorphine in morphine-positive oral fluid specimens.

Identification of 6-acetylmorphine, a specific metabolite of heroin, is considered to be definitive evidence of heroin use. Although 6-acetylmorphine has been identified in oral fluid following controlled heroin administration, no prevalence data is available for oral fluid specimens collected in the workplace. We evaluated the prevalence of positive test results for 6-acetylmorphine in 77,218 oral fluid specimens collected over a 10-month period (January-October 2001) from private workplace testing programs. Specimens were analyzed by Intercept immunoassay (cutoff concentration=30 ng/ml) and confirmed by GC-MS-MS (cutoff concentrations=30 ng/ml for morphine and codeine, and 3 ng/ml for 6-acetylmorphine). Only morphine-positive oral fluid specimens were tested by GC-MS-MS for 6-acetylmorphine. A total of 48 confirmed positive morphine results were identified. An additional 107 specimens were confirmed for codeine only. Of the 48 morphine-positive specimens, 32 (66.7%) specimens were positive for 6-acetylmorphine. Mean concentrations (+/-S.E.M.) of morphine, 6-acetylmorphine and codeine in the 32 specimens were 755+/-201, 416+/-168 and 196+/-36 ng/ml, respectively. Concentrations of 6-acetylmorphine in oral fluid ranged from 3 to 4095 ng/ml. The mean ratio (+/-S.E.M.) of 6-acetylmorphine/morphine was 0.33+/-0.06. It is suggested that, based on controlled dose studies of heroin administration, ratios >1 of 6-acetylmorphine/morphine in oral fluid are consistent with heroin use within the last hour before specimen collection. The confirmation of 6-acetylmorphine in 66.7% of morphine-positive oral fluid specimens indicates that oral fluid testing for opioids may offer advantages over urine in workplace drug testing programs and in testing drugged drivers for recent heroin use.

Oral fluid testing for drugs of abuse: positive prevalence rates by Intercept immunoassay screening and GC-MS-MS confirmation and suggested cutoff concentrations.

Draft guidelines for the use of oral fluid for workplace drug testing are under development by the Substance Abuse and Mental Health Services Administration (SAMHSA) in cooperation with industry and researchers. Comparison studies of the effectiveness of oral fluid testing versus urine testing are needed to establish scientifically reliable cutoff concentrations for oral fluid testing. We present the results of the first large scale database on oral fluid testing in private industry. A total of 77,218 oral fluid specimens were tested over the period of January through October 2001 at LabOne. Specimens were screened by Intercept immunoassay at manufacturer's recommended cutoff concentrations for the five SAMHSA drug categories (marijuana, cocaine, opiates, phencyclidine, and amphetamines). Presumptive positive specimens were confirmed by gas chromatography-tandem mass spectrometry. A total of 3908 positive tests were reported over the 10-month period, representing a positive rate of 5.06%. Of the five drug categories, marijuana and cocaine accounted for 85.75% of the positives. The pattern and frequency of drug positives showed remarkable similarity to urine drug prevalence rates reported for the general workforce according to the Quest Diagnostics' Drug Testing Index over the same general period, suggesting that oral fluid testing produces equivalent results to urine testing. The data on oral fluid testing also revealed a surprisingly high 66.7% prevalence of 6-acetylmorphine confirmations for morphine positives suggesting that oral fluid testing may be superior in some cases to urine testing. Comparison of oral fluid drug concentrations to SAMHSA-recommended cutoff concentrations in Draft Guidelines indicated that adoption of the screening and confirmation cutoff concentrations of Draft Guidelines #3 would produce the most consistent reporting results for all drug classes except amphetamines. Consequently, it is suggested that the final Guidelines adopt the screening and cutoff concentrations listed in Draft Guidelines #3 with the exception of lowering the amphetamines cutoff concentrations (screening/confirmation) to 50/50 ng/mL for amphetamine and methamphetamine.

Varying results for immunoassay screening kits for cotinine level.

Our earlier study found that although enzyme-linked immunosorbent analysis (ELISA) screening assays for urine cotinine indicated use in former smoking treatment patients who reported abstinence, this finding was sometimes incorrect when validated against gas chromatography/mass spectrometry (GC/ MS; P. Gariti, A. I. Alterman, R. Ehrmann, F. D. Mulvaney, & C. P. O'Brien, 2002). In the current validation study, separate urine samples of 71 of these same patients were reanalyzed by an independent laboratory in blinded fashion using a screening enzyme immunoassay (EIA) analysis and GC/MS confirmation. EIA results showed almost total agreement with confirmatory testing. The findings indicate that use of screening ELISA/EIA for urine cotinine can detect unreported cases of smoking in former patients, but that care is needed in selecting a laboratory for conducting these tests.

Automated homogeneous immunoassay analysis of cotinine in urine.

A study was conducted to evaluate the performance comparison of a homogeneous enzyme immunoassay (EIA) designed to detect cotinine in urine and carbon monoxide (CO) breath measurements to determine smoking status. The clinical comparison was done using urine and breath specimens from 218 volunteers. Urine samples were analyzed by immunoassay and confirmed by gas chromatography-mass spectrometry (GC-MS). Breath carbon monoxide was determined by a commercial analyzer. Using cutoffs of 10 ppm for CO and 500 ng/mL for urinary cotinine, the relative sensitivity/specificity was 93.6%/74.0%. The positive predictive value was 86.8%, and the negative predictive value was 86.5%. However, comparison of the EIA to GC-MS showed a sensitivity/specificity of 96.2%/98.4% and a positive predictive value of 99.3%. The EIA was also evaluated non-clinically for precision, stability, recovery, and interferences. In addition, the non-clinical evaluation demonstrated coefficients of variation from 0.37 to 1.09% across cotinine concentrations ranging from 0 to 5000 ng/mL. The assay was found to be highly specific for cotinine and cross-reacted to a limited degree with 3-hydroxycotinine. Finally, multiple freeze-thaw cycles of urines containing cotinine showed no degradation of the drug in the specimen when tested in the EIA. Thus, the EIA tested is a rapid, lab-based test that can reliably determine cotinine levels and their relation to smoking status.