Oral Fluid Specimens Research Articles

Polysubstance use patterns among individuals applying for opioid-use disorder treatment in the U.S.

ABSTRACT Objective The prevalence of polysubstance use is known to be high among individuals who use/misuse drugs. This study aims to extend existing research by (a) measuring polysubstance use through objective drug testing rather than fallible self-reports, (b) determining the most frequent three- and four-substance combinations instead of being limited to typical two-substance combinations, and (c) examining a comprehensive panel of substances beyond those commonly studied. Method Participants were a convenience sample of individuals applying for methadone maintenance treatment in 11 clinics across 7 states (n = 1098, 43.2% female). Participants voluntarily provided oral fluid and urine specimens for the study during clinic intake that were tested for 22 types of substances using liquid chromatography/tandem mass spectrometry (LC-MS-MS). Results Polysubstance use was high (89.6%), with the mean number of substances used by participants = 3.3, up to a maximum of 11. There were 10 three-substance combinations with prevalences of 5% or greater, the most frequent being opiates, fentanyl, and cocaine (10.5%). There were nine four-substance combinations with prevalences of 2% or greater, the most frequent being opiates, fentanyl, cocaine, and cannabis (4.5%). Many of these combinations can produce serious and even life-threatening interactions and side effects. The combination with perhaps the most severe potential consequence of concurrent use is the combination of opiates, fentanyl, and tramadol (5.1%), which all have sedative or central nervous system (CNS) depressive effects. Conclusion Clinicians should consider that symptoms of substance use are likely due to diverse combinations of substances, not only problematic use of a single presenting “substance of choice.”

Assessment of pheniramine in alternative biological matrices by liquid chromatography tandem mass spectrometry.

Pheniramine is an over-the-counter antihistamine drug. Its accessibility and low cost made it more popular among drug abusers in Pakistan. In this study, pheniramine was quantified in both conventional and alternative specimens of twenty chronic drug abusers, aged 16-50years, who were positive for pheniramine in comprehensive toxicological screening for drugs by gas chromatography with mass spectral detection in positive electron impact mode. Pheniramine was extracted from biological specimens using solid phase extraction and liquid chromatography tandem mass spectrometry was employed for quantification. Chromatographic separation was carried out on a Poroshell120EC-18 (2.1mm × 50mm × 2.7µm) column using water-acetonitrile in formic acid (0.1%) mobile phase in gradient elution mode with 500μL/min flow rate. Positive electrospray ionization mode and multi-reaction monitoring with ion transitionsm/z241.3 → 195.8 and 167.1 for pheniramine andm/zm/z247.6 → 173.1 for pheniramine-d6 were employed. The quantification method showed good linear ranges of 2-1000ng/mL in blood, urine, and oral fluid; 2-1000ng/mg in hair and 5-1000ng/mg in nail with ≥ 0.985% coefficient of linearity. The retention time of pheniramine was 3.0 ± 0.1min. The detection and lower quantification limits were 1ng/mL and 2ng/mL for blood, urine, oral fluid and hair whereas 2.5ng/mg and 5ng/mg for nail, respectively. Mean extraction recovery and ionization suppression ranged 86.3-95.1% and -4.6 to -14.4% in the studied matrices. Intra-day and inter-day precision were 4.1-9.3% and 2.8-11.2%, respectively. Pheniramine levels in specimens of drug abusers were 23-480ng/mL in blood, 72-735ng/mL in urine, 25-379ng/mL in oral fluid, 10-170ng/mg in hair and 8-86ng/mg in nail specimens. Alternative specimens are of utmost significance in clinical and medico-legal cases. In this study, authors compared matrix-matched calibration curves to blood calibration curve and obtained results within ± 10%; thereby justifying the use of blood calibration curve for urine, oral fluid, hair, and nail specimens.

A measles IgM rapid diagnostic test to address challenges with national measles surveillance and response in Malaysia.

A lateral flow rapid diagnostic test (RDT) enables detection of measles specific immunoglobulin M (IgM) antibody in serum, capillary blood, and oral fluid with accuracy consistent with enzyme immunoassay (EIA). The objectives of the study were: 1) to assess measles RDT inter-reader agreement between two clinic staff; 2) to assess the sensitivity and specificity of the measles RDT relative to standard surveillance testing in a low transmission setting; 3) to evaluate the knowledge, attitudes, and practices of staff in clinics using the RDT; and 4) to assess the impact of RDT testing on the measles public health response in Malaysia. The clinic-based prospective evaluation included all suspected measles cases captured by routine measles surveillance at 34 purposely selected clinics in 15 health districts in Malaysia between September 2019 and June 2020, following day-long regional trainings on RDT use. Following informed consent, four specimens were collected from each suspected case, including those routinely collected for standard surveillance [serum for EIA and throat swabs for quantitative reverse transcriptase polymerase chain reaction (RT-qPCR)] together with capillary blood and oral fluid tested with RDTs during the study. RDT impact was evaluated by comparing the rapidity of measles public health response between the pre-RDT implementation (December 2018 to August 2019) and RDT implementation periods (September 2019 to June 2020). To assess knowledge, attitudes, and practices of RDT use, staff involved in the public health management of measles at the selected sites were surveyed. Among the 436 suspect cases, agreement of direct visual readings of measles RDT devices between two health clinic staff was 99% for capillary blood (k = 0.94) and 97% for oral fluid (k = 0.90) specimens. Of the total, 45 (10%) were positive by measles IgM EIA (n = 44, including five also positive by RT-qPCR) or RT-qPCR only (n = 1), and 38 were positive by RDT (using either capillary blood or oral fluid). Using measles IgM EIA or RT-qPCR as reference, RDT sensitivity using capillary blood was 43% (95% CI: 30%-58%) and specificity was 98% (95% CI: 96%-99%); using oral fluid, sensitivity (26%, 95% CI: 15%-40%) and specificity (97%, 95% CI: 94%-98%) were lower. Nine months after training, RDT knowledge was high among staff involved with the public health management of measles (average quiz score of 80%) and was highest among those who received formal training (88%), followed by those trained during supervisory visits (83%). During the RDT implementation period, the number of days from case confirmation until initiation of public response decreased by about 5 days. The measles IgM RDT shows >95% inter-reader agreement, high retention of RDT knowledge, and a more rapid public health response. However, despite ≥95% RDT specificity using capillary blood or oral fluid, RDT sensitivity was <45%. Higher-powered studies using highly specific IgM assays and systematic RT-qPCR for case confirmation are needed to establish the role of RDT in measles elimination settings.

NPS–EQA PART I: Four years’ experience in external quality assessment program in Italy for classical and new psychoactive substances analysis in oral fluid

In 2019, Italian National Institute of Health established an external quality assessment program (EQA) to evaluate the performance of oral fluid testing for classical and new psychoactive substances by laboratories participating in the National Early Warning System collaborative centres. This report presents the results of four rounds between 2019 and 2023. Eleven oral fluid specimens, including 3 blank samples, were prepared by adding different classes of and new psychoactive drugs at known concentrations to pre-screened drug-free oral fluid. False-negative and false-positive results were calculated for the qualitative data evaluation. The quantitative evaluation measured the imprecision and accuracy of the results, in terms of coefficient of variation (CV%) and percent error (ERR%), respectively, with respect to a mean value obtained by reference laboratories. Z-score values were then calculated. Over the years, there has been a significant improvement in false-negative results (from 42.7% in the first year to 19.4% in the last year), but not in false-positive results (from 33.3% in the first year to 22.2% in the last one). In addition to the classic drugs of abuse (e.g. cocaine, amphetamine, methadone), the substances found in false positive samples belonged to the class of synthetic cannabinoids (e.g 5-fluoro CUMYL-PINACA and 5-fluoro-EDMB-PICA), synthetic opioids (e.g butyrylfentanyl) and tryptamines (e.g. 5-methoxy-N-methyl-N-isopropyltryptamine). The four rounds yielded a mean ERR% of approximately 22.1% and a mean CV% of around 41.5%. The participating laboratories demonstrated variable performances in relation to the class of analysed psychoactive substances, as evidenced by the calculated Z-scores. Between 25% and 60% of the reported results in all rounds should be considered satisfactory. EQA is a crucial element of laboratory quality management systems. It promotes continuous improvement and maintains high standards in the field of forensic and clinical drug testing.

Detection of Cannabinoids in Oral Fluid Specimens as the Preferred Biological Matrix for a Point-of-Care Biosensor Diagnostic Device.

An increasing number of countries have started to decriminalize or legalize the consumption of cannabis for recreational and medical purposes. The active ingredients in cannabis, termed cannabinoids, affect multiple functions in the human body, including coordination, motor skills, memory, response time to external stimuli, and even judgment. Cannabinoids are a unique class of terpeno-phenolic compounds, with 120 molecules discovered so far. There are certain situations when people under the influence of cannabis may be a risk to themselves or the public safety. Over the past two decades, there has been a growing research interest in detecting cannabinoids from various biological matrices. There is a need to develop a rapid, accurate, and reliable method of detecting cannabinoids in oral fluid as it can reveal the recent intake in comparison with urine specimens, which only show a history of consumption. Significant improvements are continuously made in the analytical formats of various technologies, mainly concerning improving their sensitivity, miniaturization, and making them more user-friendly. Additionally, sample collection and pretreatment have been extensively studied, and specific devices for collecting oral fluid specimens have been perfected to allow rapid and effective sample collection. This review presents the recent findings regarding the use of oral fluid specimens as the preferred biological matrix for cannabinoid detection in a point-of-care biosensor diagnostic device. A critical review is presented, discussing the findings from a collection of review and research articles, as well as publicly available data from companies that manufacture oral fluid screening devices. Firstly, the various conventional methods used to detect cannabinoids in biological matrices are presented. Secondly, the detection of cannabinoids using point-of-care biosensors is discussed, emphasizing oral fluid specimens. This review presents the current pressing technological challenges and highlights the gaps where new technological solutions can be implemented.

Pharmacokinetics and pharmacodynamics of five distinct commercially available hemp-derived topical cannabidiol products.

Products containing cannabidiol (CBD) have proliferated after the 2018 Farm Bill legalized hemp (cannabis with ≤0.3% delta-9-tetrahydrocannabinol (Δ9-THC)). CBD-containing topical products have surged in popularity, but controlled clinical studies on them are limited. This study characterized the effects of five commercially available hemp-derived high CBD/low Δ9-THC topical products. Healthy adults (N = 46) received one of six study drugs: a CBD-containing cream (N = 8), lotion (N = 8), patch (N = 7), balm (N = 8), gel (N = 6) or placebo (N = 9; matched to an active formulation). The protocol included three phases conducted over 17 days: (i) an acute drug application laboratory session, (ii) a 9-day outpatient phase with twice daily product application (visits occurred on Days 2, 3, 7 and 10) (iii) a 1-week washout phase. In each phase, whole blood, oral fluid and urine specimens were collected and analyzed via liquid chromatography with tandem mass spectrometry (LC-MS-MS) for CBD, Δ9-THC and primary metabolites of each and pharmacodynamic outcomes (subjective, cognitive/psychomotor and physiological effects) were assessed. Transdermal absorption of CBD was observed for three active products. On average, CBD/metabolite concentrations peaked after 7-10 days of product use and were highest for the lotion, which contained the most CBD and a permeation enhancer (vitamin E). Δ9-THC/metabolites were below the limit of detection in blood for all products, and no urine samples tested "positive" for cannabis using current US federal workplace drug testing criteria (immunoassay cut-off of 50 ng/mL and confirmatory LC-MS-MS cut-off of 15 ng/mL). Unexpectedly, nine participants (seven lotions, one patch and one gel) exhibited Δ9-THC oral fluid concentrations ≥2 ng/mL (current US federal workplace threshold for a "positive" test). Products did not produce discernable pharmacodynamic effects and were well-tolerated. This study provides important initial data on the acute/chronic effects of hemp-derived topical CBD products, but more research is needed given the diversity of products in this market.

Advances in HIV self-testing: Systematic review of current developments and the road ahead in high-burden countries of Africa.

Although HIV self-testing technologies have created new opportunities for achieving national and global HIV testing goals, current developments have not been compiled to inform policy and practice, especially in high HIV burden countries of Africa. We aimed to compile and synthesize the evidence about HIV self-testing technologies, strategies, and uptake in the top-10 high HIV burden countries of Africa. We searched CINAHL, PubMed, Web of Science, PsycINFO, Social Science Citation Index, and EMBASE to include eligible articles published in English between January 2012 and November 2022. In total, 865 articles were retrieved and only 16 studies conducted in five African countries were eligible and included in this review. The two types of HIV self-testing modalities presently being used in Africa are: The first is Home Self-Test which is done entirely at home or in another private location by using oral fluid or blood specimen. The second modality is Mail-In Self-Test (self-sampling), where the user collects their own sample and sends this to a laboratory for testing. Perceived opportunities for the uptake of HIV self-testing were autonomy and self-empowerment, privacy, suitability, creating a chance to test, and simplicity of use. The potential barriers to HIV self-testing included fear and worry of a positive test result, concern of the test results is not reliable, low literacy, and potential psychological and social harms. The oral-fluid self-testing is preferred by most users because it is easy to use, less invasive, and painless. The difficulty of instructions on how to use self-test kits, and the presence of different products of HIV self-testing kits, increase rates of user errors. Adopting HIV self-testing by overcoming the challenging potential barriers could enable early detection, care, treatment, and prevention of the disease to achieve the 95-95-95 goal by 2030. Further study is necessary to explore the actual practices related to HIV self-testing among different populations in Africa.

Prevalence and drug use correlates of inadvertent fentanyl exposure among individuals misusing drugs in seven U.S. states

Background: Fentanyl has emerged as the leading cause of fatal drug overdoses in the U.S. Individuals misusing drugs may not always be aware of exposure to fentanyl. Objectives: To determine the prevalence of fentanyl use and extent of awareness of fentanyl exposure among a national sample of treatment-seeking individuals with opioid use disorder (n = 1098). Methods: Participants provided oral fluid and urine specimens, which were tested for drugs by liquid chromatography/tandem mass spectrometry. Participants also provided self-reports of fentanyl use. Results: 49.5% tested positive for fentanyl in oral fluid, urine, or both. Of those testing positive for fentanyl, 29.8% were unaware that they had been exposed to fentanyl. Participants testing positive for opioids methadone, and specifically 6-monoacetylmorphine (6-MAM), a unique metabolite of heroin, were significantly more likely to be unaware of fentanyl exposure than participants testing negative for these substances, with a similar trend for oxycodone and tramadol. Conclusions: These findings may be due to fentanyl’s effect being difficult to distinguish from that of other opioids, whereas when other types of drugs are adulterated with fentanyl, the differences in effects are likely to be readily discernable. These results support the importance of expanded drug-checking services.

Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay.

Classical swine fever virus (CSFV) is an OIE-listed disease that requires effective surveillance tools for its detection and control. The aim of this study was to develop and evaluate the diagnostic performance of a novel CSFV Erns IgG AlphaLISA for both serum and oral fluid specimens that would likewise be compatible with the use of CSFV E2 DIVA vaccines. Test performance was evaluated using a panel of well-characterized serum (n = 760) and individual (n = 528) or pen-based (n = 30) oral fluid samples from four groups of animals: (1) negative controls (n = 60 pigs); (2) inoculated with ALD strain wild-type CSFV (n = 30 pigs); (3) vaccinated with LOM strain live CSFV vaccine (n = 30 pigs); and (4) vaccinated with live CSFV marker vaccine on commercial farms (n = 120 pigs). At a cutoff of S/P ≥ 0.7, the aggregate estimated diagnostic sensitivities and specificities of the assay were, respectively, 97.4% (95% CI 95.9%, 98.3%) and 100% for serum and 95.4% (95% CI 92.9%, 97.0%) and 100% for oral fluid. The Erns IgG antibody AlphaLISA combined DIVA capability with solid diagnostic performance, rapid turnaround, ease of use, and compatibility with both serum and oral fluid specimens.

B-327 Evidence of Missed Novel Psychoactive Substances (NPS) in Unexpected Fentanyl Positives

Abstract Background Recent data published by the CDC indicated that overdose deaths in the United States began to increase significantly in 2020 and continued throughout 2021. Synthetic opioids, such as illicitly-manufactured fentanyl (IMF), have been a primary driver in overdose deaths. There has been a concomitant increase in both the prevalence and diversification of novel psychoactive substances (NPS) and IMF. We performed a blinded secondary analysis of remnant samples to further characterize the incidence in which individuals are encountering NPS when using non-prescribed fentanyl. Various classes of NPS compounds (e.g., designer opioids, designer benzodiazepines, xylazine) were evaluated in samples that were originally positive for fentanyl but did not include a request from the provider for NPS testing to be performed. Methods This study was IRB approved. A sampling of 200 urine or oral fluid specimens collected from individuals as part of their routine medical treatment, wherein definitive testing for fentanyl and norfentanyl was performed and the drug and metabolite were both identified above the upper limit of quantitation of the assay, underwent additional analysis for NPS. Samples were excluded from blinded secondary analysis if any NPS testing, regardless of drug class, was originally ordered and performed. Additional testing that was performed included the following classes of drugs: designer opioids, designer benzodiazepines, synthetic cannabinoids, synthetic stimulants, hallucinogens/dissociatives, xylazine, tianeptine, and phenibut. Results Specimens within the sample set (N: 200) that were originally positive for fentanyl without a previous request for NPS testing originated from 30 different states within the US. These samples were originally collected between 9/19/2022–1/12/2023. Of those that underwent additional analysis after initial reporting of positive fentanyl results, 117 (58.5%) had at least one analyte within an NPS class identified. Designer opioids (N: 93, 47%), and xylazine (N: 76, 38%), followed by designer benzodiazepines (N: 16, 8%), synthetic stimulants (N: 4, 2%), and hallucinogens/dissociatives (N:3, 2%) were the most frequent compounds detected on a per sample basis. No synthetic cannabinoids were detected within this sample set. Conclusion Although much of the national focus around fatal and non-fatal overdoses centers on intentional and unintentional exposures to IMF, healthcare providers and the general public must be made aware of the prevalence of NPS. These compounds, which are often intentionally added to drugs sold as fentanyl to enhance the drug potency, can increase risk for an adverse event. Of paramount importance is that the effects of substances such as xylazine and designer benzodiazepines, which contribute to central nervous system depression, are not effectively reversed by administration of naloxone, a mainstay of harm reduction protocols. Knowledge of the prevalence of these compounds should impact clinical decision-making and individual risk mitigation plans. From a broader perspective, improving understanding of the incidence in which these drugs are found within communities could improve overall surveillance efforts on current drug use trends.

B-326 Home Collected Oral Fluid Specimens Are a Viable Option for Telemedicine Based Toxicology Testing

Abstract Background A standard treatment for Substance Use Disorder (SUD) is Medication Assisted Therapy (MAT) consisting of a buprenorphine-naloxone combination medication. Typically, a urine specimen is collected during the in-office visit for the patient to receive MAT. Urine drug testing (UDT) ensures compliance and adherence with the treatment protocol and is often mandated for MAT. However, access to in-office visits and the ability to provide a specimen for UDT can be problematic for individuals undergoing MAT for SUD due to factors such as limited access to reliable transportation or distance to facilities. The COVID-19 pandemic has eased restrictions around telemedicine, including for the administration of MAT. Telemedicine can be a valuable tool to provide greater access to MAT for individuals by removing transportation barriers to treatment. Gravity Diagnostics partnered with various facilities to provide a telemedicine observed oral fluid toxicology (OFT) collection process. To understand if telemedicine OFT could provide the same information as in-office collected specimens, we compared telemedicine OFT to in-office OFT specimens and for one facility telemedicine-OFT to in-office UDT. Methods Gravity Diagnostics provides OFT through LC-MS/MS quantitative testing for over 72 analytes. Using a deidentified data set from our LIS, we identified OFT specimens that were collected either in-office or through telemedicine during a 6 month period. The ability to determine the effectiveness of telemedicine OFT was measured by examining the unexpected positivity rate of 72 analytes when compared to in-office OFT. We used 2-way ANOVA to determine if the detection rate of unexpected analytes between two collection methods were significantly different. Additionally, we identified a facility that conducted in-office UDT and telemedicine OFT on the same patient population. These data were compared in a similar manner with the unexpected positivity rate for 47 analytes subjected to 2-way ANOVA. Results When the unexpected detection rates between the in-office and telemedicine OFT were compared with a 2-way ANOVA, a P-value of 0.554 was found between the collection methods. This result indicated that the detection rates of unexpected analytes between the in-office and telemedicine collections were not significantly different. Similarly, when the unexpected detection rates between the UDT and the telemedicine-OFT were compared, a P-value of 0.391 was found between the collection methods. This result indicated that the detection rates between in office UDT and telemedicine OFT were not significantly different when compared within the same patient population. Conclusion Our data showed that telemedicine-OFT are comparable to in-office OFT for the detection of unexpected positive analytes. In addition, when the same patient population was tested by either telemedicine-OFT or in-office UDT, no significant difference in detection rates of unexpected positives is observed. Therefore, we are confident that we can utilize telemedicine-OFT to provide greater access to health care for patients undergoing MAT for SUD.

Evaluating the impact of point-of-care HIV viral load assessment on linkage to care in Baltimore, MD: a randomized controlled trial

BackgroundIntegration of a sensitive point-of-care (POC) HIV viral load (VL) test into screening algorithms may help detect acute HIV infection earlier, identify people with HIV (PWH) who are not virally suppressed, and facilitate earlier referral to antiretroviral therapy (ART), or evaluation for pre-exposure prophylaxis (PrEP). This report describes a randomized clinical trial sponsored by the Centers for Disease Control and Prevention (CDC): “Ending the HIV Epidemic Through Point-of-Care Technologies” (EHPOC). The study’s primary aim is to evaluate the use of a POC HIV VL test as part of a testing approach and assess the impact on time to linkage to ART or PrEP. The study will recruit people in Baltimore, Maryland, including patients attending a hospital emergency department, patients attending an infectious disease clinic, and people recruited via community outreach. The secondary aim is to evaluate the performance characteristics of two rapid HIV antibody tests approved by the United States Food and Drug Administration (FDA).MethodsThe study will recruit people 18 years or older who have risk factors for HIV acquisition and are not on PrEP, or PWH who are not taking ART. Participants will be randomly assigned to either the control arm or the intervention arm. Participants randomized to the control arm will only receive the standard-of-care (SOC) HIV screening tests. Intervention arm participants will receive a POC HIV VL test in addition to the SOC HIV diagnostic screening tests. Follow up will consist of an interim phone survey conducted at week-4 and an in-person week-12 visit. Demographic and behavioral information, and oral fluid and blood specimens will be collected at enrollment and at week-12. Survey data will be captured in a Research Electronic Data Capture (REDCap) database. Participants in both arms will be referred for either ART or PrEP based on their HIV test results.DiscussionThe EHPOC trial will explore a novel HIV diagnostic technology that can be performed at the POC and provide viral assessment. The study may help inform HIV testing algorithms and contribute to the evidence to support same day ART and PrEP recommendations.Trial registrationNIH ClinicalTrials.gov NCT04793750. Date: 11 March 2021.

Comparing presumptive with direct-to-definitive drug testing in oral fluid vs. urine for a U.S. national sample of individuals misusing drugs

BackgroundThe aims are to compare the results of presumptive drug testing with confirmation of positives vs. direct-to-definitive drug testing, combined with investigation of urine vs. oral fluid as test matrices. Methods: Paired oral fluid and urine specimens were collected voluntarily and anonymously from 1098 individuals applying for methadone treatment in 11 clinics across 7 U.S. states. All specimens were analyzed by immunoassay (IA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). Results: Confirmed IA prevalences for urine were significantly higher than for oral fluid for 7 out of 10 drug classes - benzodiazepines, cannabis, cocaine, methadone, opiates, oxycodone and tramadol. Drug prevalences by direct-to-definitive LC-MS-MS were either the same or higher than prevalences by confirmed IA. Drug prevalences by LC-MS-MS were higher in urine for two drug classes (cocaine, methadone) and higher in oral fluid for two drug classes (buprenorphine, tramadol), but were equivalent in urine and oral fluid when averaged over all 10 drug classes. Certain drugs of special concern such as heroin and buprenorphine were more frequently detected in oral fluid than urine. Conclusions: Urine analysis showed some technical advantage over oral fluid in sensitivity to several drug classes within a confirmed IA testing protocol, but this may be outweighed if there is reason to believe that tampering with urine specimens is a significant problem. Overall drug detection by direct-to-definitive testing was similar for oral fluid and urine, but one matrix may be preferable if there is a particular drug of clinical or epidemiological interest.

Effect of extrinsic factors on the detection of PRRSV and a porcine-specific internal sample control in serum, oral fluid, and fecal specimens tested by RT-rtPCR.

We characterized the effect of 1) temperature × time, 2) freeze-thaw cycles, and 3) high porcine reproductive and respiratory syndrome virus (PRRSV) RNA concentrations on the detection of PRRSV and a porcine-specific internal sample control (ISC) in serum, oral fluid, and fecal samples using a commercial PRRSV RT-rtPCR assay (Idexx). In study 1, the effect of temperature × time on PRRSV and ISC detection was shown to be specimen dependent. In serum stored at 4, 10, or 20°C, PRRSV detection was consistent for up to 168 h, but storage at 30°C reduced detectable PRRSV RNA. ISC RNA was stable in serum held at 4 and 10°C, but not at 20 and 30°C. In contrast, PRRSV and ISC RNAs in oral fluid and fecal samples continuously decreased at all temperature × time treatments. Based on these data, serum samples should be stored at ≤ 20°C to optimize PRRSV RNA detection. Oral fluid and fecal samples should be frozen in a non-self-defrosting freezer until tested. In study 2, freeze-thaw cycles had little impact on PRRSV and ISC detection, but more so in oral fluids than serum or fecal samples. Thus, freeze-thaw cycles in oral fluids should be minimized before RT-rtPCR testing. In study 3, the ISC was not affected by high concentrations of PRRSV RNA in serum, oral fluid, or fecal samples. It should not be assumed that data from our PRRSV study are applicable to other pathogens; additional pathogen-specific studies are required.

Internal reference genes with the potential for normalizing quantitative PCR results for oral fluid specimens.

In basic research, testing of oral fluid specimens by real-time quantitative polymerase chain reaction (qPCR) has been used to evaluate changes in gene expression levels following experimental treatments. In diagnostic medicine, qPCR has been used to detect DNA/RNA transcripts indicative of bacterial or viral infections. Normalization of qPCR using endogenous and exogenous reference genes is a well-established strategy for ensuring result comparability by controlling sample-to-sample variation introduced during sampling, storage, and qPCR testing. In this review, the majority of recent publications in human (n = 136) and veterinary (n = 179) medicine did not describe the use of internal reference genes in qPCRs for oral fluid specimens (52.9% animal studies; 57.0% human studies). However, the use of endogenous reference genes has not been fully explored or validated for oral fluid specimens. The lack of valid internal reference genes inherent to the oral fluid matrix will continue to hamper the reliability, reproducibility, and generalizability of oral fluid qPCR assays until this issue is addressed.

Local monitoring of SARS-CoV-2 variants in two large California counties in 2021

Coronavirus Disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to persist due to mutations resulting in newer, more infectious variants of concern. We aimed to leverage an ongoing private SARS-CoV-2 testing laboratory’s infrastructure to monitor SARS-CoV-2 variants in two large California counties. Study enrollment was offered to adults aged 18 years or older in Los Angeles County and Riverside County who recently tested positive for SARS-CoV-2 with a polymerase chain reaction (PCR) assay. A cycle threshold value less than or equal to 30 cycles was considered a positive test for sequencing purposes. Within 5 days of study enrollment, clinician-monitored, self-collected oral fluid and anterior nares swab specimens were obtained from participants. Specimens were transported and stored at 8 °C or cooler. Samples underwent ribonucleic acid extraction, library preparation, and sequencing. SARS-CoV-2 lineages were identified using sequencing data. Participant and genomic data were analyzed using statistical tools and visualized with toolkits. The study was approved by Advarra Institutional Review Board (Pro00053729). From May 27, 2021 to September 9, 2021, 503 individuals were enrolled and underwent specimen collection. Of the 503 participants, 238 (47.3%) participants were women, 329 (63.6%) participants were vaccinated, and 221 (43.9%) participants were of Hispanic or Spanish origin. Of the cohort, 496 (98.6%) participants had symptoms at the time of collection. Among the 503 samples, 443 (88.1%) nasal specimens and 353 (70.2%) oral specimens yielded positive sequencing results. Over our study period, the prevalence of the Alpha variant of SARS-CoV-2 decreased (initially 23.1% [95% confidence interval (95% CI): 0–0.49%] to 0% [95% CI 0.0–0.0%]) as the prevalence of the Delta variant of SARS-CoV-2 increased (initially 33.3% [95% CI 0.0–100.0%] to 100.0% [95% CI 100.0–100.0%]). A strain that carried mutations of both Delta and Mu was identified. We found that outpatient SARS-CoV-2 variant surveillance could be conducted in a timely and accurate manner. The prevalence of different variants changed over time. A higher proportion of nasal specimens yielded results versus oral specimens. Timely and regional outpatient SARS-CoV-2 variant surveillance could be used for public health efforts to identify changes in SARS-CoV-2 strain epidemiology.

The analysis of drugs in neonates using an ad hoc oral fluid collection device to develop a prediction model for neonatal abstinence syndrome and provide maternal support

To use oral fluid specimens collected in an ad hoc device to test for opioid exposure and to help assess the risk of withdrawal in potential neonatal abstinence syndrome (NAS) cases. Testing for Neonatal drug exposure typical uses meconium, urine, blood, hair, or umbilical cord tissue/blood specimens. Due to the invasiveness, challenges, and limitations of collection, and/or analytical difficulties of these matrices, oral fluid may be a more desirable specimen when testing for opioid exposure and assessing the risk of neonatal abstinence syndrome (NAS). Traditional oral fluid collection devices are not viable options for neonatal specimen collection due to their large size and potential chemicals additives that induce salivation. Unstimulated and stimulated infant oral fluid samples have been used for therapeutic drug monitoring as an alternative matrix to blood. Oral fluid samples were collected under Virginia Commonwealth University's Institutional Review Board from ten neonates born to mothers in methadone or buprenorphine treatment programs. Oral fluid samples were collected by rolling three foam-tipped swabs along the mouth, inner cheeks, and tongue until saturated. The three swabs containing approximately 300 μL oral fluid were placed in 2.0 mL of phosphate-buffered saline, centrifuged, and analyzed by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for 26 drugs and/or metabolites after solid phase extraction (SPE) with OFXQ columns. Results were compared to maternal and the neonatal medical history. The following analytes were detected in the neonatal oral fluid: 2-Ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 6-acetylmorphine, amphetamine, benzoylecgonine, cocaine, cotinine, ethyl sulfate, methadone, methamphetamine, morphine, nicotine, and norfentanyl. Maternal history and infant medications were available for 9 of 10 neonates. The detection of norfentanyl was consistent with epidurals given during birth. The following analytes detected were inconsistent with patient histories; 6-acetylmorphine, amphetamines, benzoylecgonine, ethyl sulfate, and nicotine. Three of the ten neonates were treated for withdrawal symptoms by an oral suspension of methadone given twice daily. These neonates were released from the hospital on day of life 18, 20, or 24. Prior to treatment, four to eleven drugs and/or metabolites were identified in the three neonates, including 6-acetlymorphine, amphetamine, benzoylecgonine, cocaine, cotinine, EDDP, EMDP, ethyl sulfate, methadone, morphine, nicotine, and/or norfentanyl. In two neonates, methadone oral fluid concentrations increased in the first few days of life then significantly decreased in concentration prior to treatment. No methadone was detected in the third neonate, but a variety of other drugs were identified. None of the other neonates presented symptoms of withdrawal and were released from the hospital on day 3 or 4 of life, except for one neonate born pre-term (35 weeks gestation) was not released until day of life 9. An easy-to-use collection device for neonatal oral fluid must be developed. Identifying trends between neonates who demonstrate withdrawal symptoms and those who do not could be vital to developing a predictive model for the determination of NAS. When analyzing neonatal samples, complete clinical histories of mother and neonate are necessary to prevent the misinterpretation of data, which could lead to unwarranted criminal or legal complications.

Evaluation of Alcohol Markers in Urine and Oral Fluid after Regular and Hard Kombucha Consumption.

Although kombucha is a popular fermented beverage, the presence of alcohol markers has not been well studied despite being potential indicators of unintentional impairment. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) were measured in oral fluid and urine collected after consumption of regular or hard kombucha. Participants drank within 20 min and provided all urine voids for 12 h, the first urine voids on days 2 and 3 and oral fluid specimens at fixed time points for 48 h. Screening employed liquid chromatography-tandem mass spectrometry (LC-MS-MS; EtS, 25 ng/mL cutoff [oral]; 100 ng/mL cutoff [urine]; EtG, 500 ng/mL cutoff [urine] and immunoassay (IA; EtG, 500 ng/mL cutoff [urine]). After consuming regular kombucha (n = 12 participants), EtS was not detected in oral fluid but both markers were detected by LC-MS-MS in urine specimens within the first five voids from 83% of participants with median (range) concentrations of 240 (100-3,700) ng/mL for EtS and 830 (530-2,200) ng/mL for EtG. Neither marker was positive by IA nor LC-MS-MS after day 1. After consuming hard kombucha (n = 7 participants), 2 (2.8%) of the 70 collected oral fluid specimens tested positive for EtS 3 h after consumption; however, 21 (30%) had EtS levels above the limit of detection (LOD, 10 ng/mL) after 0.5-8 h. Both markers were detected in urine specimens from all participants with median (range) concentrations of 3,381 (559-70,250) ng/mL for EtS and 763 (104-12,864) ng/mL For EtG. Urine specimens were negative for EtG and EtS by the end of the 48-hour study.

SARS-CoV-2 RNA and antibody dynamics in a Dutch household study with dense sampling frame

This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in 242 household members of different ages and with different symptom severity after SARS-CoV-2 exposure early in the pandemic (March–April 2020). Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable.

Validation study of an automated chemiluminiscence assay to detect HIV antibodies in oral fluid specimens.

Oral fluid specimens (OF) have been widely used to know the HIV prevalence in several key populations. Here, we aim to validate in OF specimens an existing HIV chemiluminiscence assay for serum specimens. Paired OF and serum specimens were collected from 83 known HIV-positives and 83 known HIV-negatives in order to validate the performance characteristics of the automated chemiluminiscence Liaison XL Murex HIV Ag/Ab assay (Diasorin Inc, Iberia) for HIV antibody detection in OF specimens. Among the previously known HIV-seropositive group, HIV antibodies were detected in 69 out of 83 OF specimens. All serum and OF specimens collected from 83 HIV seronegative individuals were negative. The sensitivity and specificity of this assay were 83.13% and 100% respectively in OF. The PPV and NPV values were 100% and 85.57% respectively. The correlation obtained between both specimens was (K: 0.83, [95% CI: 0.748–0.915]) according to the kappa index. The ROC curve analysing the optimal cut-off of the Liaison XL Murex HIV Ag/Ab to detect positive OF specimens revealed that a cut-off of 0.497 showed sensitivity and specificity values of 98.8% and 97.59% respectively. Taking into account this cut-off, the overall sensitivity and NPV of the Liaison XL Murex HIV Ag/Ab assay could rise from 83.1 to 98.8% and from 85.5 to 97.7%, respectively. Our results suggest that the Liaison XL HIV Ag/Ab assay is suitable for the detection of HIV antibodies in OF specimens.