Liquid-Liquid Extraction Solvent Selection for Comparing Illegal Drugs in Whole Blood and Dried Blood Spot with LC-MS/MS.

AIM: This study focused on the simultaneous detection of amphetamine, 3,4-methyl enedioxy methamphetamine, morphine, benzoylecgonine, and 11-nor-9-carboxy- tetrahydrocannabinol (Δ9-THC-COOH) in whole blood and DBS. It is aimed to select a solvent mixture for liquid-liquid extraction (LLE) technique employing LC-MS/MS. The obtained DBS results were compared with the whole blood samples results. METHODS: A simple, rapid, and reliable LC-MS/MS method was developed and validated for all analytes in whole blood and DBS. LC was performed on a Hypersil Gold C18 column an initial step with a gradient of 0.01 % formic acid, 5 mM ammonium format buffer in water, and acetonitrile at 0.3 ml/min with 7.5-min runtime. RESULTS: A methanol:acetonitrile (40:60 v/v) mixture was selected for both matrices. LOQ values were 10-25 ng/mL; linear ranges were LOQ-500 ng/ml for all analytes; correlation coefficients were greater than 0.99, and all calibrator concentrations were within 20%. Analytical recovery in blood and DBS ranged from 84.9-113.2% of the expected concentration for both intra and-inter day. Analytes were stable for 1, 10, and 30 days after three freeze/thaw cycles. It was determined that the variances of the results obtained with the two matrices in the comparison study were equal for each analyte, and the results were highly correlated (r=0.9625). CONCLUSION: A sensitive, accurate, and reliable chromatographic method was developed to determine amphetamine, MDMA, morphine, benzoylecgonine, and cannabis, by performing the same preliminary steps with whole blood and dried blood spots. It was observed that the results obtained in these two matrices were compatible and interchangeable when statistically compared.

Acceptability of dried blood spot collection by primary caregivers of Filipino patients with maple syrup urine disease (MSUD) and phenylketonuria (PKU).

MSUD and PKU require lifetime management hence, regular monitoring of amino acid levels is needed to achieve good metabolic control. Ideally, plasma amino acid analysis (PLAA) is used to monitor concentrations but is expensive and not widely available in local laboratories. The newborn screening program in the Philippines uses dried blood spot (DBS) analysis as an alternative where only trained healthcare providers are allowed to perform the collection at selected facilities. With the increasing number of patients, DBS monitoring has been noted to be delayed due to multiple factors. This issue became even more evident during the COVID-19 pandemic where high-risk patients need to travel outside for blood collection. The study used a cross-sectional study design to determine the primary caregivers' perspective on DBS self-sampling for patients with MSUD and PKU and the acceptability of the samples collected. This was done through a series of collection training, pre-/post- surveys, and 10-item questionnaire, and an in-depth 1-on-1 interview for thematic analysis. The acceptability of samples was processed and evaluated by the newborn screening laboratory. At-home DBS collection by primary caregivers was found to be acceptable. The provision of knowledge and routine collection training by the medical team aids in the increase of sample acceptability as well as a source of empowerment in being equipped to take care of their child. It is highly recommended that DBS samples collected by caregivers be considered acceptable for more time and cost-saving monitoring of the patients' metabolites. This practice also promotes timely and appropriate management which can lead to better patient health outcomes.

Evaluating vitamin D status in Chinese pre-school children using dried blood spots coupled with liquid chromatograph coupled with mass spectrometer.

AIM: Vitamin D is an essential micronutrient for multiple physiological processes, and its deficiency remains a world-wide public health problem that cannot be ignored. Dried blood spot (DBS) is a convenient tool in large-scale epidemiological studies, but its application in evaluating vitamin D status in Chinese population is still scarce. Herein, we aimed to determine the vitamin D status in Chinese pre-school children using DBS coupled with LC-MS/MS method. METHODS: We first developed a sensitive and reliable method for the determination of 25-hydroxyvitamin D (25(OH)D) in DBS samples using an ultra-high-performance liquid chromatograph coupled with triple quadrupole mass spectrometer (UHPLC-QQQ-MS/MS). Next, we conducted a pilot study to compare the 25(OH)D concentration in DBS and serum samples. Finally, the assay method was used to evaluate vitamin D status in Chinese pre-school children. RESULTS: The present method was validated to be reliable and robust for the determination of 25(OH)D in DBS samples. Comparable consistency was observed between the 25(OH)D concentration in DBS and serum samples. A total of 3826 DBS samples collected from children aged 1-7 years were determined. The median concentration of 25(OH)D was 19.57 ng/mL (interquartile range 14.73-24.36 ng/mL), and decreased from 1 to 7 years of age. In addition, 13.51% of male children and 15.12% female children are found to be deficient in 25(OH)D. CONCLUSIONS: DBS coupled with LC-MS/MS is a feasible strategy to evaluate vitamin D status in epidemiological studies. And vitamin D deficiency remains a common health problem in Chinese pre-school children.

Quantification of Letrozole, Palbociclib, Ribociclib, Abemaciclib, and Metabolites in Volumetric Dried Blood Spots: Development and Validation of an LC-MS/MS Method for Therapeutic Drug Monitoring.

Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure-toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95-106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22-55%),with high recovery rates (81-93%) and minimal matrix effects (ME 0.9-1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented.

Infants with biliary atresia exhibit an altered amino acid profile in their newborn screening.

INTRODUCTION: Biliary atresia (BA) is a rare progressive neonatal cholangiopathy with unknown pathophysiology and time of onset. Newborn Screening (NBS) in Germany is routinely performed in the first days of life to identify rare congenital diseases utilizing dried blood spot (DBS) card analyses. Infants with biliary atresia (BA) are known to have altered amino acid profiles (AAP) at the time point of diagnosis, but it is unclear whether these alterations are present at the time point of NBS. OBJECTIVES: We aimed to analyze amino acid profiles in NBS-DBS of infants with Biliary Atresia. METHODS: Original NBS-DBS cards of 41 infants who were later on diagnosed with BA were retrospectively obtained. NBS-DBS cards from healthy newborns (n = 40) served as controls. In some BA infants (n = 14) a second DBS card was obtained at time of Kasai surgery. AAP in DBS cards were analyzed by targeted metabolomics. RESULTS: DBS metabolomics in the NBS of at that time point seemingly healthy infants later diagnosed with BA revealed significantly higher levels of Methionine (14.6 ± 8.6 µmol/l), Histidine (23.5 ± 50.3 µmol/l), Threonine (123.9 ± 72.8 µmol/l) and Arginine (14.1 ± 11.8 µmol/l) compared to healthy controls (Met: 8.1 ± 2.6 µmol/l, His: 18.6 ± 10.1 µmol/l, Thr: 98.1 ± 34.3 µmol/l, Arg: 9.3 ± 6.6 µmol/l). Methionine, Arginine and Histidine showed a further increase at time point of Kasai procedure. No correlation between amino acid levels and clinical course was observed. CONCLUSION: Our data demonstrate that BA patients exhibit an altered AAP within 72 h after birth, long before the infants become symptomatic. This supports the theory of a prenatal onset of the disease and, thus, the possibility of developing a sensitive and specific NBS. Methionine might be particularly relevant due to its involvement in glutathione metabolism. Further investigation of AAP in BA may help in understanding the underlying pathophysiology.

A Risk Correlative Model for Sleep Disorders in Chinese Older Adults Based on Blood Micronutrient Levels: A Matched Case-Control Study.

BACKGROUND: The physical abilities of older adults decline with age, making them more susceptible to micronutrient deficiency, which may affect their sleep quality. OBJECTIVES: This study aimed to construct a risk correlative model for sleep disorders in Chinese older adults based on blood micronutrient levels. METHODS: In this matched case-control study, we recruited 124 participants with sleep disorders and 124 matched controls from the Tianjin Elderly Nutrition and Cognition cohort in China. Micronutrient levels in whole blood were measured using the dried blood spot technique. We compared the differences in micronutrient levels between the two groups and also constructed a receiver operating characteristic (ROC) model and nomogram for sleep disorders. RESULTS: In comparison to the control group, the sleep disorders group showed lower levels of blood vitamin A, vitamin E (VE), folate, magnesium, copper, iron, and selenium (Se) in the univariate analysis (p < 0.05). The ROC curve analysis indicated that the combination of VE + folate + Se may have an excellent diagnostic effect on sleep disorders, with an area under the curve of 0.964. This VE + folate + Se was integrated into a nomogram model to demonstrate their relationship with sleep disorders. The consistency index of the model was 0.88, suggesting that the model assessed sleep disorders well. CONCLUSIONS: The sleep disorders risk correlative model constructed by the levels of VE, folate, and Se in whole blood might show good performance in assessing the risk of sleep disorders in older adults.

Utility of an Archival Dried Blood Spot (DBS) Collection from HIV-Infected Individuals with and without Cancer in a Resource-Limited Setting.

The frequency of virus-associated cancers is growing worldwide, especially in resource-limited settings. One of the biggest challenges in cancer research among people living with HIV (PLWH) has been understanding how infection with both HIV and Kaposi sarcoma-associated herpesvirus (KSHV) promotes the pathogenesis of Kaposi sarcoma (KS), the most common cancer among PLWH worldwide and a significant public health problem in regions with high prevalence of HIV such as Sub-Saharan Africa (SSA). The AIDS and Cancer Specimen Resource (ACSR) provides samples for research, including dried blood spots (DBS) that were collected from large clinical epidemiology studies of KSHV and KS in PLWH conducted more than a decade ago in SSA. Here, we validated the quality of DNA derived from DBS samples from SSA studies and provided evidence of quantitative recovery of inflammatory cytokines using these DBS samples through comparison with paired frozen plasma. Significant differences in DNA, protein yields, and inflammatory biomarker levels were also observed between PLWH with/without KS. Establishing the fitness of DBS samples for studies of KS pathogenesis extends the number of projects that can be supported by these ACSR special collections and provides evidence that DBS collection for future KS research is a practical option in resource-limited settings.

Methods established for the detection of mineral levels in whole blood by the dried blood spot technique.

Mineral are intimately related to human health and disease, and detection of mineral content in the body is of great significance for the diagnosis and prevention of diseases. In this study, we validated the method developed to detect magnesium (Mg), copper (Cu), iron (Fe), zinc (Zn), and selenium (Se) levels in dried blood spots (DBS). In accordance with the requirements of the guidelines for the Bioanalytical Method Validation Guidance for Industry, we evaluate the linearity, sensitivity, precision, accuracy and selectivity of the developed methods. In addition, Mg, Cu, Fe, Zn and Se were quantified in 195 older adults using DBS technique, and its accuracy was assessed by comparing the results to those detected by inductively coupled plasma-mass spectrometry (ICP-MS). The method has excellent sensitivity and linear range to cover the concentration range of mineral elements in the general population with the required precision, accuracy and selectivity. The correlation coefficients of Mg, Cu, Fe, Zn and Se levels in blood detected by the DBS technique and ICP-MS were 0.638, 0.823, 0.463, 0.728 and 0.751, respectively (all P < 0.05), which indicated that there was a strong correlation between the detection results of the two methods. More than 95 % of the sample results in the Bland-Altman consistency test were within the acceptable limits of agreement (LOA) range, indicating that they had good consistency. DBS technique has good accuracy and reliability in detecting blood mineral levels in the elderly, suggesting potential in the quantification of mineral level in blood.

Screening for Fabry disease in patients with left ventricular hypertrophy in China: A multicentre and prospective study.

AIMS: Left ventricular hypertrophy (LVH) is frequently detected via echocardiography in individuals with Fabry disease (FD), sometimes leading to confusion with hypertrophic cardiomyopathy (HCM) of other aetiologies. Considering this diagnosis challenge, FD should be included in the list of differential diagnosis for patients presenting with LVH. To address this concern, we conducted a prospective screening study in China, using dried blood spot (DBS) testing, to evaluate patients with unexplained LVH. METHODS: Our study was designed as a nationwide, multicentre prospective investigation. A total of 1015 patients from 55 different centres who were diagnosed with LVH by echocardiography were screened in the study from September 2022 to December 2023. Demographic information, biochemistry data, echocardiography parameters and clinical observations were meticulously collected from all participants. The DBS method was used to assess α-galactosidase A (α-Gal A) activity in males and both α-Gal A and globotriaosylsphingosine (lyso-Gb3) levels in females. RESULTS: The final screening population included 906 patients (589 males, 65%) with LVH, characterized by a mean maximal myocardial thickness of 14.8 ± 4.6 mm and an average age of 56.9 ± 17.2 years. In total, 43 patients (38 males, 5 females) exhibited low α-Gal A activity measurement (<2.2 µmol/L), while 21 patients (10 males, 11 females) presented low α-Gal A activity or elevated lyso-Gb3 levels (>1.1 ng/mL). Among these patients, eight individuals (7 males and 1 female) were genetically confirmed to harbour pathogenic GLA mutations, resulting in a total prevalence of 0.88%. Compared with patients without FD, patients with FD tended to have proteinuria (75% vs. 21.2%, P = 0.001), family history of HCM (37.5% vs. 2.3%, P < 0.01) and neuropathic pain (37.5% vs. 4.4%, P < 0.01) but lower systolic blood pressure (118.5 ± 12.5 vs. 143.3 ± 29.3 mmHg, P = 0.017). Five mutations were previously recognized as associated with FD while the remaining two, p.Asp313Val (c.938A>T) and c.547+3A>G, were deemed potentially pathogenic. Subsequent familial validation post-diagnosis identified an additional 14 confirmed cases. CONCLUSIONS: This pioneering screening study for FD among Chinese patients with unexplained LVH using DBS measurement, revealed an FD detection rate of 0.88%. Our findings confirmed that the combined measurement of lyso-Gb3 and α-Gal A activity is beneficial for primary screening of FD in patients with LVH. Given the availability of efficacious therapies and the value of cascade screening in extended families, early detection of FD in LVH patients is clinically important.

Monitoring SARS-CoV-2 IgA, IgM and IgG antibodies in dried blood and saliva samples using antibody proximity extension assays (AbPEA).

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Diagnostic Value of Menstrual Blood Lipidomics in Endometriosis: A Pilot Study.

Endometriosis is a prevalent chronic inflammatory disease characterized by a considerable delay between initial symptoms and diagnosis through surgery. The pressing need for a timely, non-invasive diagnostic solution underscores the focus of current research efforts. This study examines the diagnostic potential of the menstrual blood lipidome. The lipid profile of 39 samples (23 women with endometriosis and 16 patients in a control group) was acquired using reverse-phase high-performance liquid chromatography-mass spectrometry with LipidMatch processing and identification. Profiles were normalized based on total ion counts. Significant differences in lipids were determined using the Mann-Whitney test. Lipids for the diagnostic model, based on logistic regression, were selected using a combination of variance importance projection filters and Akaike information criteria. Levels of ceramides, sphingomyelins, cardiolipins, triacylglycerols, acyl- and alkenyl-phosphatidylethanolamines, and alkenyl-phosphatidylcholines increased, while acyl- and alkyl-phosphatidylcholines decreased in cases of endometriosis. Plasmenylphosphatidylethanolamine PE P-16:0/18:1 and cardiolipin CL 16:0_18:0_22:5_22:6 serve as marker lipids in the diagnostic model, exhibiting a sensitivity of 81% and specificity of 85%. The diagnostic approach based on dried spots of menstrual blood holds promise as an alternative to traditional non-invasive methods for endometriosis screening.

Challenges and Opportunities with at-Home Blood Collection for HIV-1 Viral Load Monitoring among Sexual Minoritized Men who use Stimulants.

Sexually minoritized men (SMM) with HIV who use stimulants experience difficulties achieving and maintaining an undetectable viral load (VL). Home-based VL monitoring could augment HIV care by supporting interim, early identification of detectable VL. We describe implementation challenges associated with a home-collection device for laboratory-based VL testing among SMM with HIV who use stimulants. From March-May 2022, cisgender SMM with HIV reporting moderate-to-severe stimulant use disorder and suboptimal (< 90%) past-month antiretroviral therapy (ART) adherence were recruited via a consent-to-contact participant registry. Eligible men completed teleconference-based informed consent and were mailed a HemaSpot-HD blood collection device (volume capacity 160 µL; lower limit of detection 839 copies/mL) with detailed instructions for home blood self-collection and return shipment. Implementation process measures included estimated blood volume and VL quantification. Among 24 participants, 21 (88%) returned specimens with a median duration of 23 days (range: 10-71 days) between sending devices to participants and receiving specimens. Of these, 13/21 (62%) included enough blood (≥ 40 µL) for confidence in detectable/undetectable results; 10/13 (77%) had detectable VL, with 4/10 (40%) were quantifiable at ≥ 839 copies/mL. The remaining 8/21 had low blood volume (< 40 µL), but 3/8 (38%) still had detectable VL, with 1/3 (33%) quantifiable at ≥ 839 copies/mL. Home blood collection of ≥ 40 µL using HemaSpot-HD was feasible among this high-priority population, with > 50% having a VL detected. However, interim VL monitoring using HemaSpot-HD among those experiencing difficulties with ART adherence may be strengthened by building rapport via teleconferencing and providing detailed instructions to achieve adequate sample volume.

Determination of Normal Range of Acylcarnitine in Neonatal Dried Blood Spots using LC-MS/MS.

Background: Acylcarnitine is one of the crucial markers of fatty acid metabolism, and examination of their level in infants can reveal several Inherited Metabolic Disorders (IDM) or Inborn errors of Metabolism (IEM). Because of the great importance of hereditary, metabolic, and other inherited disorders early diagnosis before the appearance of clinical symptoms, this study was carried out to establish a reference range for carnitine analytes and to identify acylcarnitine profiles in normal weight neonatal dried blood spots (DBS) specimens. Methods: By using liquid chromatography tandem mass spectrometry (LC-MS/MS) for neonatal screening and eventually the examination and analysis of LC-MS/MS results, 34 acylcarnitine derivatives were identified. Results: The normal range for acylcarnitine analytes with carbon numbers ranging from zero to 18, both main and the branched ones, were ultimately measured. Afterward, they were compared with the results of some other diagnostic laboratories to be verified. Conclusions: This study differed from the other findings, which could be due to diversity in population and work methods. However, the reference range of most acylcarnitine derivatives in Tehran closely aligned with this study's findings.

Quantitative analysis of nonsteroidal anti-inflammatory drugs in dried blood spot from mountain ultra-trail runners. Contribution of pharmacokinetic models for the interpretation of the results.

Monitoring of drug use in athletes is of interest both for health and competition-related issues. Considering the advantages of Dried Blood Sampling (low invasiveness, easy sampling, long term storage), we have validated a quantitative LC-MS/HRMS method for the screening of 16 nonsteroidal anti-inflammatory drugs. For all drugs, accuracy and imprecision were within 15% for the 3 levels of quality control and lower than 20% for the lower limit of quantification. Application was performed from samples obtained for Ultra-Trail du Mont-Blanc® 2021 and 2022. A focus on ibuprofen and its metabolites (hydroxyibuprofen, carboxyibuprofen, ibuprofen glucuronide and hydroxyibuprofen glucuronide) was made because the results showed that it was the most detected nonsteroidal anti-inflammatory drug. Further, an interpretation of the ibuprofen concentrations was proposed either from experimental data obtained after an intake of ibuprofen by 10 control subjects, or from a pharmacokinetic modelling and simulations. Depending on the analytical performances of the method, we proposed possible detection windows for ibuprofen in runners. The pharmacokinetic model made it possible to consider two scenarios with and without modification of the total clearance of ibuprofen linked to a modification of the pharmacokinetics of the drugs due to the practice of a long and intense physical activity.

Clozapine levels and outcomes in Serbian patients with treatment-resistant psychotic disorders previously treated without measuring clozapine levels (CLOSER).

Clozapine remains the only pharmacological treatment option for treatment-resistant schizophrenia. Therapeutic drug monitoring (TDM) of clozapine is recommended, although evidence for the therapeutic range of 350-600 ng/ml is limited. In various countries including Serbia, TDM of clozapine is not routinely performed. This study evaluated the distribution of clozapine levels and their relationship with clinical outcomes in Serbian patients who had not undergone prior TDM. 140 Patients with treatment-resistant schizophrenia and schizo-affective disorder were enrolled. Clozapine levels were measured by dried blood spot (DBS) analysis. Side effects were evaluated by GASS-c, severity of symptoms and functional impairment with WHODAS, CGI-S and GAF. Of the patients, 51.2% had subtherapeutic levels, 24.8% were in the therapeutic window, and 24% had supratherapeutic levels. Clozapine levels showed no association with side effects and a weak positive association with symptom severity and functional impairment. No serious side effects were observed in patients with clozapine levels surpassing 1000 ng/ml (n = 8). Based on these findings, we propose that the upper limit of the therapeutic range should not be regarded as an absolute barrier, and guidelines should allow for a personalized approach when prescribing clozapine.

HCV prevalence and phylogenetic characteristics in a cross-sectional, community study of young people who inject drugs in New York City: Opportunity for and threats to HCV elimination.

Background and Aims: In the United States, the opioid epidemic has led many young people who use opioids to initiate injection drug use, putting them at risk for hepatitis C virus (HCV) infection. However, community surveys to monitor HCV prevalence among young people who inject drugs (YPWID) are rare. Methods: As part of Staying Safe (Ssafe), a trial to evaluate an HCV-prevention intervention, a community-recruited sample of 439 young people who use opioids (ages 18-30) in New York City (NYC) were screened from 2018 to 2021. Screening procedures included a brief verbal questionnaire, a visual check for injection marks, onsite urine drug testing, rapid HCV antibody (Ab) testing, and dried blood spot (DBS) collection. DBS specimens were sent to a laboratory for HCV RNA testing and phylogenetic analysis to identify genetic linkages among HCV RNA-positive specimens. Multivariable logistic regression was used to assess associations between HCV status (Ab and RNA) and demographics and drug use patterns. Results: Among the 330 participants who reported injecting drugs (past 6 months), 33% (n = 110) tested HCV Ab-positive, 58% of whom (n = 64) had HCV RNA-positive DBS specimens, indicating active infection. In multivariable analysis, visible injection marks (AOR = 3.02; p < 0.001), older age (AOR = 1.38; p < 0.05), and female gender (AOR = 1.69; p = 0.052) were associated with HCV Ab-positive status. Visible injection marks were also associated with HCV RNA-positive status (AOR = 5.24; p < 0.01). Twenty-five percent of RNA-positive specimens (14/57) were genetically linked. Conclusion: The relatively low prevalence of active infection suggests the potential impact of treatment-as-prevention in reducing HCV prevalence among YPWID. Targeted community serosurveys could help identify actively infected YPWID for treatment, thereby reducing HCV incidence and future transmissions.

Pharmacokinetic profile of phenytoin in dried blood spot with high-performance liquid chromatography-photodiode array.

Phenytoin is a first-line antiepileptic drug with narrow therapeutic range and follows non-linear pharmacokinetics. Pharmacokinetics of phenytoin have been studied in plasma matrix before, however, there were several disadvantages. This study aimed to obtain partial validation data of the analytical method and the pharmacokinetic profile of phenytoin in Dried Blood Spot (DBS) of six healthy subjects. DBS has the advantage of only requiring small sample volumes and could be transported more efficiently. Phenytoin along with carbamazepine as the chosen internal standard was analyzed with a reversed-phase high performance-liquid chromatography system and a photodiode array detector at 205 nm. The results of partial validation, which evaluated the linearity, within-run accuracy, and precision, were within the criteria acceptance range. The pharmacokinetic profile showed that average AUC0-t was 83.81 ± 37.32 µg.h/mL and AUC0-∞ was 83.65 ± 38.89 µg.h/mL with an average ratio of 93%. Previous study quantifying phenytoin in the plasma matrix found the average AUC0-t was 39.41 ± 8.57 µg.h/mL and AUC0-∞ was 42.94 ± 9.55 µg.h/mL. Despite the difference between parameters of phenytoin analyzed in DBS and plasma matrices, the pharmacokinetic profiles obtained from both matrices were similar indicated by comparable concentration-time curves, thus, proving that DBS matrix can be used interchangeably with the plasma matrix as a more comfortable and effective alternative to phenytoin quantification in blood.

Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control.

Due to the ease of collection, transport and storage, the use of dried blood spots (DBS) offers an attractive alternative matrix for detection of the abuse of gene therapy, otherwise known as gene doping. This study evaluated the recovery, extraction efficiency and resulting detection capability of DNA from DBS by evaluating different target types, DNA extraction kits, the number of punches and blood tube preservatives. The long-term storage stability of low-copy-number transgene targets in DBS was not assessed in this study but would be noteworthy to investigate further. DNA was quantified using two detection methods: qPCR and digital PCR (dPCR). Using six punches with the Qiagen Investigator kit gave the best overall DNA yield compared with other extraction methods. Including three punches, however, gave better DNA extraction efficiency. Reference material could be detected using qPCR and dPCR in DBS spiked with 5000 copies/mL of blood (approximately 15 copies per 3 mm of punch). The optimal DNA extraction protocol was used on DBS samples from a custom recombinant adeno-associated virus administration study and showed successful detection of vector targets in DBS samples.

Patterns of daily oral HIV PrEP adherence among people who inject drugs in Ukraine: an analysis of biomarkers.

INTRODUCTION: Daily oral HIV pre-exposure prophylaxis (PrEP) with tenofovir/emtricitabine (TDF/FTC) is recommended for people who inject drugs (PWID) but coverage is low. The real-life effectiveness of PrEP among PWID is unknown as previous studies were conducted in controlled settings and mainly relied on self-report. Analysis of PrEP metabolites-tenofovir diphosphate (TFVdp) and emtricitabine triphosphate (FTCtp)-offers an objective measure of adherence. METHODS: To analyse longitudinal patterns of PrEP adherence among PWID in Ukraine, we used data from a community-based implementation trial conducted in Kyiv between July 2020 and March 2021 to test the efficacy of SMS reminders to improve adherence. Among 199 enrolled participants, 156 (78.4%) were retained through 6 months. Based on TFVdp/FTCtp levels assessed at 3 and 6 months, we identified groups with various adherence patterns (adherent at ≥2 doses/week, improved, worsened, non-adherent). Correlates of adherence were analysed using multinomial logistic regression. RESULTS: Most participants (53.8%, n = 84/156) had no detectable metabolites at both assessments; 7.1% (n = 11/156) were consistently taking ≥2 doses/week; 1.3% (n = 2/156) were consistently taking ≥4 doses/week; 13.5% (n = 21/156) exhibited improved and 21.8% (n = 34/156) had worsened adherence at 6 compared to 3 months. "White coat compliance" (increased dosing prior to assessment) was common. Consistent adherence was associated with SMS reminders, younger age, employment, lower income, longer injection drug use duration, recent high-risk injecting (receptive syringe sharing, using pre-filled syringe, back- or front-loading, container sharing), absence of overdose in the past 6 months, perceived HIV risk through sexual intercourse and higher PrEP self-efficacy. Alcohol consumption was associated with inconsistent PrEP use. Groups with improved and worsened adherence did not differ. CONCLUSIONS: Daily oral PrEP may not achieve the desired effectiveness among PWID as a standalone intervention, calling for testing of alternative PrEP formulations and innovative integrated risk reduction strategies, especially in the context of HIV epidemics associated with injection drug use in eastern Europe and central Asia and the public health crisis in Ukraine caused by the war with Russia. SMS reminders may be effective among PWID who prioritize PrEP. Our findings offer practical guidance in identifying PWID who are likely to benefit from PrEP and those who need additional support.

Unraveling the metabolomic architecture of autism in a large Danish population-based cohort.

BACKGROUND: The prevalence of autism in Denmark has been increasing, reaching 1.65% among 10-year-old children, and similar trends are seen elsewhere. Although there are several factors associated with autism, including genetic, environmental, and prenatal factors, the molecular etiology of autism is largely unknown. Here, we use untargeted metabolomics to characterize the neonatal metabolome from dried blood spots collected shortly after birth. METHODS: We analyze the metabolomic profiles of a subset of a large Danish population-based cohort (iPSYCH2015) consisting of over 1400 newborns, who later are diagnosed with autism and matching controls and in two Swedish population-based cohorts comprising over 7000 adult participants. Mass spectrometry analysis was performed by a timsTOF Pro operated in QTOF mode, using data-dependent acquisition. By applying an untargeted metabolomics approach, we could reproducibly measure over 800 metabolite features. RESULTS: We detected underlying molecular perturbations across several metabolite classes that precede autism. In particular, the cyclic dipeptide cyclo-leucine-proline (FDR-adjusted p = 0.003) and the carnitine-related 5-aminovaleric acid betaine (5-AVAB) (FDR-adjusted p = 0.03), were associated with an increased probability for autism, independently of known prenatal and genetic risk factors. Analysis of genetic and dietary data in adults revealed that 5-AVAB was associated with increased habitual dietary intake of dairy (FDR-adjusted p < 0.05) and with variants near SLC22A4 and SLC22A5 (p < 5.0e - 8), coding for a transmembrane carnitine transporter protein involved in controlling intracellular carnitine levels. CONCLUSIONS: Cyclo-leucine-proline and 5-AVAB are associated with future diagnosis of autism in Danish neonates, both representing novel early biomarkers for autism. 5-AVAB is potentially modifiable and may influence carnitine homeostasis.