Spot Samples Research Articles

Nutritional status of khat (Catha-edulis) chewing adult males in Nakawa division, Kampala city, Uganda

Khat (Catha edulis) also known as “Mairungi” in Uganda, is a green shrub whose leaves are chewed in social gatherings and designated hotspots mainly for its euphoric effect. The socio-economic and health impacts of khat have been previously reported. However, the nutritional implications of khat chewing need to be investigated. A cross-sectional study with a descriptive approach was done to assess the nutritional status of adult male users of khat in the Nakawa division, Kampala city. Using chain sampling, khat users and hot spots were mapped, identified, and listed. Using simple random sampling, 100 khat users were targeted to consent for structured interviews and an assessment of nutritional status over a 3-month persuasion period. Anthropometric data were used to compute the Body Mass Index (BMI) and dietary information was assessed based on food groups consumed and thresholds of Recommended Dietary Allowances (RDA). The degree of relationship among variables was statistically evaluated using, chi-square analyses and the ANOVA test. Statistical significance was considered at P < 0.05. Confidentiality was observed by the use of anonymous questionnaires and coded datasheets. The majority of khat users chewed khat daily 56.0% and mixed khat chewing with other intoxicants such as alcohol 35.0%, cigarettes 15.0%, and cannabis 12.0%. The main driver towards khat chewing in Nakawa was peer pressure 60.0%. The Dietary Diversity Score (DDS) was low, medium and high among 20.0%, 57.0% and 23.0% of khat users, respectively. The DDS was significantly associated with the hours spent on khat chewing X2(4) 10.156, P = 0.025 with most users spending more than four hours, and the frequency of khat chewing X2(4)14.423, P = 0.038. Of the 100 respondents, 26.0% were underweight and 3.0% were overweight. The BMI was significantly associated with age P = 0.029, marital status P = 0.023, frequency of khat chewing P = 0.038, the number of meals P = 0.013 and the dietary diversity scores P < 0.001 in khat users. Khat chewing and its associated habits predispose khat users to a double jeopardy of malnutrition and non-communicable diseases (NCDs). There should be tighter control of khat use through policy and legislation with community-based measures to regulate its demand and supply. Key words: Khat Chewing, Mairungi, Nutritional Status, Body Mass Index, Nakawa, Uganda

Clinical, Biochemical, and Molecular Characteristics of Filipino Patients with Tyrosinemia Type 1.

Hereditary tyrosinemia type I (HT1), or hepatorenal tyrosinemia, is an amino acid disorder which may cause hepatic failure as well as renal and neurologic comorbidities. Early detection of this disorder is possible with newborn screening (NBS). The objective of this study is to describe the clinical, biochemical, and molecular characteristics of Filipino patients diagnosed with HT1 through the expansion of the Philippine NBS program in 2014. There were a total of 16 patients with confirmed HT1 from then until September 2022. Clinical and biochemical data during confirmation and initial evaluation, as well as molecular data, were obtained from the patients' medical records. The cohort included children between the ages of 18 and 54 months at the time of data collection. The mean age at treatment initiation was 26.8 days. The mean succinylacetone level from dried blood spot sampling using tandem mass spectrometry (MS) was 11.1 µmol/L. Biochemical confirmatory tests via plasma amino acid analysis showed mean levels of tyrosine, phenylalanine, and methionine of 506.1 µmol/L, 111.5 µmol/L, and 125.4 µmol/L, respectively. Upon urine organic acid (UOA) analysis, succinylacetone was detected in all except for one patient, who was managed prior to UOA analysis. The most common clinical characteristics were abnormal clotting times (62.5%), elevated alpha fetoprotein (37.5%), anemia (31.3%), and metabolic acidosis (31.3%). The most common genotype was homozygous c.122T>C p.Leu41Pro in 64.3% of patients. The allelic frequency of this pathogenic variant is 71.4%. The inclusion of HT1 in the Philippine NBS program allowed early diagnosis and management of HT1 patients.

Pharmacokinetics of extended-release clarithromycin in patients with Mycobacterium ulcerans infection

Clarithromycin extended-release (CLA-ER) was used as companion drug to rifampicin (RIF) for Mycobacterium ulcerans infection in the intervention arm of a WHO drug trial. RIF enhances CYP3A4 metabolism, thereby reducing CLA serum concentrations, and RIF concentrations might be increased by CLA co-administration. We studied the pharmacokinetics of CLA-ER at a daily dose of 15 mg/kg combined with RIF at a dose of 10 mg/kg in a subset of trial participants, and compared these to previously obtained pharmacokinetic data. Serial dried blood spot samples were obtained over a period of ten hours, and analyzed by LC–MS/MS in 30 study participants—20 in the RIF-CLA study arm, and 10 in the RIF-streptomycin study arm. Median CLA Cmax was 0.4 mg/L—and median AUC 3.9 mg*h/L, following 15 mg/kg CLA-ER. Compared to standard CLA dosed at 7.5 mg/kg previously, CLA-ER resulted in a non-significant 58% decrease in Cmax and a non-significant 30% increase in AUC. CLA co-administration did not alter RIF Cmax or AUC. Treatment was successful in all study participants. No effect of CLA co-administration on RIF pharmacokinetics was observed. Based on our serum concentration studies, the benefits CLA-ER over CLA immediate release are unclear.

PCR Detection of Bartonella spp. and Borreliella spp. DNA in Dry Blood Spot Samples from Human Patients.

Lyme disease is the most commonly reported vector-borne disease in the United States. Bartonella constitute an additional zoonotic pathogen whose public health impact and diversity continue to emerge. Rapid, sensitive, and specific detection of these and other vector-borne pathogens remains challenging, especially for patients with persistent infections. This report describes an approach for DNA extraction and PCR testing for the detection of Bartonella spp. and Borreliella spp. from dry blood spot (DBS) specimens from human patients. The present study included extraction of DNA and PCR testing of DBS samples from 105 patients with poorly defined, chronic symptoms labeled as Lyme-Like Syndromic Illness (LLSI). Bartonella spp. DNA was detected in 20/105 (19%) and Borreliella spp. DNA was detected in 41/105 (39%) patients with LLSI. Neither group of organisms was detected in DBS samples from 42 healthy control subjects. Bartonella spp. 16S-23S rRNA internal transcribed spacer sequences were highly similar to ones previously identified in yellow flies, lone star ticks, a human patient from Florida, mosquitoes in Europe, or B. apihabitans and choladocola strains from honeybees. These human strains may represent new genetic strains or groups of human pathogenic species of Bartonella. The 41 Borreliella spp. flaB gene sequences obtained from human patients suggested the presence of four different species, including B. burgdorferi, B. americana, B. andersonii, and B. bissettiae/carolinensis-like strains. These results suggest that specific aspects of the DBS DNA extraction and PCR approach enabled the detection of Bartonella spp. and Borreliella spp. DNA from very small amounts of human whole blood from some patients, including specimens stored on filter paper for 17 years.

Biological Reference Intervals for 17α-Hydroxyprogesterone Immunoreactive Trypsinogen, and Biotinidase in Indian Newborns

Neonatal deaths, which usually occur in the first week after delivery, account for nearly 75 percent of all deaths of children under 5 years of age. Prematurity, birth difficulties, infections, and birth defects are responsible for about 40 percent of these deaths. Although mortality rates have declined since 2000, access to quality healthcare remains a major problem for mothers and infants worldwide. In perspective, the present study aimed to establish clear biological reference intervals for 17α-hydroxyprogesterone (17-OHP), immunoreactive trypsinogen (IRT), and biotinidase in Indian neonates. The statistical analysis of data from up to 3200 dried blood spot (DBS) samples of Indian newborns provided valuable information for the new cut-off values in newborn screening (NBS) programs. We applied correlation analysis to fix the relationship for NBS parameters such as 17-OHP, IRT, and biotinidase. This study provided important information about the distribution and comparison of key cut-offs for biomarkers considering body weights and gestational age in the Indian newborn population for the first time, which can help healthcare experts make easier treatment decisions.

Efficient Green Solvent Extraction and Bioassay Studies of Favipiravir in Rat Dried Blood Spots Using Ionic Liquid-Based Dispersive Liquid-Liquid Microextraction (IL-DLLME) Coupled with HPLC-PDA: Application to Bioequivalence Studies

Background: Favipiravir is an antiviral drug having pyrazine group moiety. It is a reliable and an efficient green solvent, and a highly recoverable bio-sampling method is required for it. It is widely used in COVID-19 treatment for the prevention of the spread of viral infections in the body Objective: This proposed green solvent (ionic liquid)-based bioassay study aimed to quantify favipiravir in rat-dried blood spots using Dispersive Liquid-liquid Microextraction (IL-DLLME). Methods: The proposed bioassay separation was achieved through an isocratic elution mode using a hybrid silica-based ODS column (250 × 4.6 mm; 5 μm), a column temperature of 25°C, an injection volume of 10 μL, a flow rate of 1.0 mL/min, and a detector wavelength set at 310 nm. The run time was less than 10 minutes. Mobile phase was delivered with acetonitrile, methanol, and 10 mM Na2HPO4 at pH 4.0 ± 0.5, (15: 20: 65, v/v/v). In a microtube, 50 mL of Ionic Liquid (IL), 500 μL of disperser ACN, 50 μL of 10% NaCl, and IS (20 ng mL-1) were added to perform the Dried Blood Spot (DBS) sample extraction methodology. The advantages of the proposed methodology have been found to include minimum hematocrit effect and an adequate blood volume for testing, easy transportation, and significant extraction recovery. The sample analysis has been carried out using HPLC-PDA with oseltamivir used as an internal standard. Results: We have investigated the important variables, i.e., salt concentration (10% NaCl, analyte recovery being higher) and disperser solvent [50μL of BMIHP plus 500 μL of ACN (v/v) yielding the highest recovery], in the extraction process. Extraction Recovery (ER) and Enrichment Factor (EF) were also evaluated using the IL-DLLME method. The calibration curve examined a range of 0.5-150 μg/mL, with a lower Limit of Quantification (LOQ) being 0.5 μg/mL in QC as well as calibration samples, respectively. Conclusion: Significant bioanalytical validation has been performed and all the parameters have been evaluated systematically as per bioanalytical method validation protocols, i.e., US FDA- 2018 guidelines. The following analytical parameters have been covered: standard curve, limit of quantification, range, recovery, stability, accuracy, precision, sensitivity, ER, EF, and selectivity. The developed bioassay method has been successfully applied in the pharmacokinetic studies of rats and successfully applied in bulk drugs.

Analytical and clinical validation of an LC-MS/MS method for carbamazepine, lamotrigine and levetiracetam in dried blood spots

ObjectivesTherapeutic drug monitoring is performed routinely in patients on anti-epileptic drugs (AEDs) for optimisation and individualisation of therapy. The dried blood spot (DBS) sampling technique is a suitable, more patient-friendly...

Circulation of Non-falciparum Species in Niger: Implications for Malaria Diagnosis.

Niger's National Malaria Control Programme and its partners use histidine-rich protein 2-based RDTs, which are specific to Plasmodium falciparum diagnosis. This study aimed to screen for the circulation of non-falciparum species in Zinder, a region of Niger, West Africa. A cross-sectional study was carried out from July to December 2022 at the district hospital of the Zinder region of Niger. P falciparum histidine-rich protein 2-based rapid diagnostic tests were performed, and dried blood spot samples were collected for further laboratory multiplexed photo-induced electron transfer-polymerase chain reaction (PET-PCR) analysis on positive light microscopy from all patients with fever who attended the Zinder district hospital during the study period. In total, 340 dried blood spots were collected and analyzed by PET-PCR. Overall, 73.2% (95% CI, 68.2%-77.9%; 249/340) were positive for Plasmodium genus and species and represented the study population. Plasmodium species proportions were 89.5% (95% CI, 85.1%-93.1%; 223/249) for P falciparum, 38.5% (95% CI, 32.5%-44.9%; 96/249) for P malariae, 10.8% (95% CI, 7.3%-15.4%; 27/249) for P vivax, and 1.6% (95% CI, .4%-4.1%; 4/249) for P ovale. Single infection with Plasmodium species counted for 61.8% (95% CI, 55.5%-67.9%; 154/249), and the mixed infections rate, with at least 2 Plasmodium species, was 38.1% (95% CI, 32.1%-44.5%; 95/249). Single non-falciparum infections represented a rate of 10.0% (95% CI, 6.6%-14.5%; 25/249). This study confirms the first evidence of Plasmodium vivax by PET-PCR in Niger in addition to the other 3 Plasmodium species. These findings underline the need to adapt malaria diagnostic tools and therapeutic management, as well as the training of microscopists, for recognition of non-falciparum plasmodial species circulating in the country. This will better inform the strategies toward malaria control and elimination, as well as the decision making of the health authorities of Niger.

Publisher's Note

BackgroundPrevalence of hepatitis C virus (HCV) antibody (Ab) on dried blood spot (DBS) samples in the Australian Needle and Syringe Program Survey (ANSPS) decreased nationally from 57% in 2015 to 32% in 2022. We aimed to investigate potential explanations for this decline. MethodsChanges in DBS HCV Ab prevalence were investigated by redefining positive cases as those with those with either a positive HCV Ab test result or a self-reported history of ever having HCV treatment (modified prevalence), examining HCV Ab prevalence by birth and age cohorts, and assessing trends in key risk behaviours. ResultsOverall prevalence of DBS HCV Ab declined rapidly and significantly from 57% in 2015 to 32% in 2022 (p<0.001) however modified HCV Ab prevalence remained stable over time (85% and 88% in 2015 and 2022, respectively, p=0.357). The proportion of participants with negative HCV Ab and self-reported HCV infection increased from 20% in 1995 to 40% in 2022 (p<0.001) and the proportion with negative HCV Ab and lifetime HCV treatment increased from 3% in 1999 to 67% in 2022 (p<0.001). We also observed a decreasing trend in DBS HCV Ab prevalence in all birth and age cohorts with a noticeable acceleration in the decline commensurate with the advent of HCV DAA treatment. A long-term decreasing trend was also observed for key risk behaviours (p<0.001) however the short-term trend was not significant for recent receptive syringe sharing. ConclusionThe temporal decline in HCV Ab prevalence appears related to reduced sensitivity of DBS HCV Ab detection with viral clearance following treatment. Since 2016, HCV treatment uptake has increased markedly including among people who inject drugs. In this context, continuing to monitor HCV Ab prevalence by DBS testing is problematic, with a shift to surveillance of active infection the most relevant to guide policy and practice in this setting.

Prevalence of mutations associated with artemisinin partial resistance and sulfadoxine–pyrimethamine resistance in 13 regions in Tanzania in 2021: a cross-sectional survey

The emergence of the artemisinin partial resistance (ART-R) mutation in the Plasmodium falciparum kelch13 gene (k13), Arg561His, in Rwanda and the regional presence of polymorphisms affecting sulfadoxine-pyrimethamine have raised concern in neighbouring Tanzania. The goal of this study was to assess the status of antimalarial resistance in Tanzania, with a focus on the border with Rwanda, to understand the distribution of the Arg561His mutation, partner drug resistance, and resistance to chemoprevention drugs. In this cross-sectional survey, capillary dried blood spots were collected from malaria positive asymptomatic individuals in the community and symptomatic individuals in health facilities aged 6months and older, in 13regions of mainland Tanzania from Jan 31to June 26, 2021. Exclusion criteria included residence of the areas other than the target sites, presenting to the health facility for care and treatment of conditions other than malaria, and not providing informed consent. Samples were assessed for antimalarial resistance polymorphisms and genetic relatedness using molecular inversion probes targeting Pfalciparum and short-read whole-genome sequencing. The primary outcome was the prevalence of molecular markers of antimalarial resistance at the region level, as well as at the district level in Kagera, a region in the northwest of the country at the border with Rwanda. 6855 (88·1%) of 7782capillary dried blood spot samples collected were successfully genotyped. The overall prevalence of k13 Arg561His in Kagera was 7·7% (90% CI 6·0-9·4; 50of 649), with the highest prevalence in the districts near the Rwandan border (22·8% [31 of 136] in Karagwe, 14·4% [17 of 118]) in Kyerwa, and 1·4% [two of 144] in Ngara). k13 Arg561His was uncommon in the other regions. Haplotype analysis suggested that some of these parasites are related to isolates collected in Rwanda in 2015, supporting regional spread of Arg561His. However, a novel k13 Arg561His haplotype was observed, potentially indicating a second origin in the region. Other validated k13 resistance polymorphisms (one Arg622Ile and two Ala675Val isolates) were also identified. Aregion of prevalent dihydrofolate reductase Ile164Leu mutation, associated with sulfadoxine-pyrimethamine resistance, was also identified in Kagera (15·2% [12·6-17·8%]; 80of 526). The mutant crt Lys76Thr mutation, associated with chloroquine and amodiaquine resistance, was uncommon, occurring only in 75of 2861genotyped isolates, whereases the wild-type mdr1 Asn86Tyr allele, associated with reduced sensitivity to lumefantrine, was found in 99·7% (3819 of 3830) of samples countrywide. These findings show that the k13 Arg561His mutation is common in northwest Tanzania and that multiple emergences of ART-R, similar as to what was seen in southeast Asia, have occurred. Mutations associated with high levels of sulfadoxine-pyrimethamine resistance are common. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Understanding how multiple emergences interact with drivers of regional spread is essential for combating ART-R in Africa. This study was funded by the Bill & Melinda Gates Foundation and the National Institutes of Health.

Dried Blood Spot Metabolome Features of Ischemic-Hypoxic Encephalopathy: A Neonatal Rat Model.

Hypoxic-ischemic encephalopathy (HIE) is a severe neurological disorder caused by perinatal asphyxia with significant consequences. Early recognition and intervention are crucial, with therapeutic hypothermia (TH) being the primary treatment, but its efficacy depends on early initiation of treatment. Accurately assessing the HIE severity in neonatal care poses challenges, but omics approaches have made significant contribution to understanding its complex pathophysiology. Our study further explores the impact of HIE on the blood metabolome over time and investigated changes associated with hypothermia's therapeutic effects. Using a rat model of hypoxic-ischemic brain injury, we comprehensively analyzed dried blood spot samples for fat-soluble compounds using HPLC-MS. Our research shows significant changes in the blood metabolome after HIE, with a particularly rapid recovery of lipid metabolism observed. Significant changes in lipid metabolites were observed after 3 h of HIE, including increases in ceramides, carnitines, certain fatty acids, phosphocholines, and phosphoethanolamines, while sphingomyelins and N-acylethanolamines (NAEs) decreased (p < 0.05). Furthermore, NAEs were found to be significant features in the OPLS-DA model for HIE diagnosis, with an area under the curve of 0.812. TH showed a notable association with decreased concentrations of ceramides. Enrichment analysis further corroborated these observations, showing modulation in several key metabolic pathways, including arachidonic acid oxylipin metabolism, eicosanoid metabolism via lipooxygenases, and leukotriene C4 synthesis deficiency. Our study reveals dynamic changes in the blood metabolome after HIE and the therapeutic effects of hypothermia, which improves our understanding of the pathophysiology of HIE and could lead to the development of new rapid diagnostic approaches for neonatal HIE.

TEAM: Triangular-mEsh Adaptive and Multiscale proton spot generation method.

Proton therapy is preferred for its dose conformality to spare normal tissues and organs-at-risk (OAR) via Bragg peaks with negligible exit dose. However, proton dose conformality can be further optimized: (1) the spot placement is based on the structured (e.g., Cartesian) grid, which may not offer conformal shaping to complex tumor targets; (2) the spot sampling pattern is uniform, which may be insufficient at the tumor boundary to provide the sharp dose falloff, and at the same time may be redundant at the tumor interior to provide the uniform dose coverage, for example, due to multiple Coulomb scattering (MCS); and (3) the lateral spot penumbra increases with respect to the depth due to MCS, which blurs the lateral dose falloff. On the other hand, while (1) the deliverable spots are subject to the minimum-monitor-unit (MMU) constraint, and (2) the dose rate is proportional to the MMU threshold, the current spot sampling method is sensitive to the MMU threshold and can fail to provide satisfactory plan quality for a large MMU threshold (i.e., high-dose-rate delivery). This work will develop a novel Triangular-mEsh-based Adaptive and Multiscale (TEAM) proton spot generation method to address these issues for optimizing proton dose conformality and plan delivery efficiency. Compared to the standard clinically-used spot placement method, three key elements of TEAM are as follows: (1) a triangular mesh instead of a structured grid: the triangular mesh is geometrically more conformal to complex target shapes and therefore more efficient and accurate for dose shaping inside and around the target; (2) adaptive sampling instead of uniform sampling: the adaptive sampling consists of relatively dense sampling at the tumor boundary to create the sharp dose falloff, which is more accurate, and coarse sampling at the tumor interior to uniformly cover the target, which is more efficient; and (3) depth-dependent sampling instead of depth-independent sampling: the depth-dependent sampling is used to compensate for MCS, that is, with increasingly dense sampling at the tumor boundary to improve dose shaping accuracy, and increasingly coarse sampling at the tumor interior to improve dose shaping efficiency, as the depth increases. In the TEAM method the spot locations are generated for each energy layer and layer-by-layer in the multiscale fashion; and then the spot weights are derived by solving the IMPT problem of dose-volume planning objectives, MMU constraints, and robustness optimization with respect to range and setup uncertainties. Compared to the standard clinically-used spot placement method UNIFORM, TEAM achieved (1) better plan quality using <60% number of spots of UNIFORM; (2) better robustness to the number of spots; (3) better robustness to a large MMU threshold. Furthermore, TEAM provided better plan quality with fewer spots than other adaptive methods (Cartesian-grid or triangular-mesh). A novel triangular-mesh-based proton spot placement method called TEAM is proposed, and it is demonstrated to improve plan quality, robustness to the number of spots, and robustness to the MMU threshold, compared to the clinically-used spot placement method and other adaptive methods.

Post-Omicron SARS-CoV-2 serostatus in Sierra Leone: A cross-sectional study in a maternity hospital setting in Freetown, November/December 2022

BackgroundSpread of SARS-CoV-2 in Sub-Saharan African countries has been poorly investigated, especially in the later pandemic stages. We aimed to assess the post-Omicron situation in Sierra Leone in November/December 2022 considering SARS-CoV-2 serostatus, vaccinations, and behavioral factors. MethodsIn a cross-sectional study conducted in a maternity hospital in Freetown, Sierra Leone, both patients and staff provided dried blood spot samples for analysis of anti-S and anti-N IgG prevalence using Anti-SARS-CoV-2-ELISA. Additionally, we collected sociodemographic and infection-related information through questionnaires. Outcome parameters included seropositivity, infection-related seroprevalence, and self-reported vaccination status. We used logistic regression to identify associations with prior infection and with vaccination status. ResultsOut of 791 participants (389 patients, 402 staff), 670 (84.7 %) displayed a positive SARS-CoV-2 serostatus resulting from either infection or vaccination. Among a sub-sample of 514 participants within which determination of prior natural infection was possible, 441individuals (85.8 %) were affected. Prior infection was associated with female sex and tertiary education level. Overall, 60.3 % reported having been vaccinated. Staff as opposed to patients, and individuals with higher socioeconomic status were more likely to report vaccination. Individuals who assessed their risk of COVID-19 as either higher or lower compared to a medium-level risk were more likely to have contracted the virus and less likely to have received vaccination. ConclusionOur findings suggest that since the Omicron wave in 2022, the Sierra Leonean population has almost universally been exposed to SARS-CoV-2. While this is encouraging in the light of relatively low excess mortality in the country, future investigations on the long-term effect of high viral exposure on epidemic resilience and public health impact will be crucial.

Feeding or pen surface application of clinoptilolite with different particle sizes: impact on nitrogen utilization and manure ammonia emissions in feedlot cattle.

This study investigated the effects of feeding clinoptilolite (CLN; 2.5% of diet dry matter) with a particle size of either 30- or 400-µm on ruminal fermentation characteristics, measures of nitrogen (N) utilization, and manure ammonia-N (NH3) emissions in feedlot cattle. The impact of directly applying 30- or 400-µm CLN to the pen surface (2,250kg/ha) on manure NH3-N emissions was also evaluated. Six beef heifers were used in a replicated 3 × 3 Latin square design with 21-d periods. Dietary treatments were 1) finishing ration with no supplement (CON), 2) CON + 30-µm CLN (CLN-30), and 3) CON + 400-µm CL (CLN-400). Intake was measured daily. To evaluate fermentation characteristics, ruminal fluid was collected on day 19. Indwelling pH loggers were used to measure ruminal pH from days 15 to 21. Blood was collected 3-h post-feeding on day 21 for metabolite analysis. Fecal grab and urine spot samples were also collected from days 19 to 21 to measure nutrient digestibility, route of N excretion, and in vitro NH3 emissions. There was no diet effect (P ≥ 0.12) on nutrient intake and apparent total tract digestibility, and ruminal short-chain fatty acid profile and pH. Ruminal NH3 concentration, which was lower (P = 0.04) for CLN-30 than CON heifers, did not differ between CON and CLN-400 heifers. Although there was no diet effect (P = 0.50) on plasma urea-N (PUN) concentration, proportion of urea-N excreted in urine was lower (P = 0.01) for CLN-30 than CON and CLN-400 heifers. Urinary NH3-N excretion, which was greater (P ≤ 0.04) for CLN-400 than CON heifers, did not differ between CLN-30 and CLN-400 heifers. Feeding CLN also increased (P ≤ 0.02) fecal excretion of potassium (K) and iron (Fe) and reduced (P = 0.01) urinary excretion of calcium (Ca). There was a treatment × time interaction (P = 0.01) for NH3 emission rate, which was greatest within the first 36h of incubation and was lower for manure from CLN-400 compared to CON and CLN-30 heifers and pen surface application treatments. Cumulative NH3 emissions were lower (P < 0.01) for manure from CLN-400 compared to CON and CLN-30 heifers and the pen surface application treatments. Although surface application was ineffective, feeding 400-µm CLN to finishing cattle could result in a beneficial decrease in manure NH3 emissions. However, changes in fecal and urine excretion of minerals like K and Ca, which suggest a decrease in bioavailability, need to be considered when feeding CLN in finishing cattle diets.

Antiretroviral therapy adherence among peripartum women with HIV in Kenya: an explanatory mixed methods study using dry blood spot measures and narrative interviews

ABSTRACT Adherence to antiretroviral therapy (ART) remains sub-optimal among pregnant and postpartum women with HIV (PPWH) in high HIV prevalence low resource settings with few effective behavioral interventions. A large body of qualitative literature has established general barriers and facilitators to ART adherence in PPWH at various levels (individual, interpersonal, structural). However, research exploring the underlying behavioral mechanisms of ART adherence in PPWH with objectively verified adherence biomarkers is extremely limited. We conducted 24 in-depth interviews with postpartum women in western Kenya who had linked ART drug concentrations obtained from three dried blood spot samples across the peripartum period. Among PPWH with a low drug concentration (n = 13) compared to those with continuously high drug concentrations (n = 11), distinct themes emerged related to HIV status disclosure, social support, interactions with the health system, and health beliefs. By combining ART biomarkers with patient reported challenges, there is the potential for real-time interventions to support sustained ART adherence among PPWH and improve maternal and infant health outcomes.

Formula-led methods using first morning fasting spot urine to assess usual salt intake: a secondary analysis of PURE study data.

Observational studies that assess the relationship between salt intake and long-term outcomes require a valid estimate of usual salt intake. The gold-standard measure in individuals is sodium excretion in multiple nonconsecutive 24-h urines. Multiple studies have demonstrated that random spot urine samples are not valid for estimating usual salt intake; however, some researchers believe that fasting morning spot urine samples produce a better measure of usual salt intake than random spot samples. We have used publicly available data from a PURE China validation study to compare estimates of usual salt intake from morning spot urine samples and three published formulae with mean of two 24-h urine samples (reference). We estimated the means and 95% confidence intervals of absolute and relative errors for each formula-led method and the degree to which estimates were able to be classified into the correct quartile of intake. Bland-Altman plots were used to test the level of agreement. The results show that compared with the reference method, all formulae-led estimates from spot urine collections have high error rates: both random and systematic. This is demonstrated for individual estimates, as well as by quartiles of reference salt intake. This study conclusively demonstrates the unsuitability of morning spot urine formula-led estimates of usual salt intake. Our findings support international recommendations to not conduct, fund, or publish research studies that use spot urine samples with estimating equations to assess individuals' salt intake in association with health outcomes.

Prevalence of Alpha-1 Antitrypsin Deficiency Alleles in a Lithuanian Cohort of Wheezing Small Children.

Severe inherited alpha-1 antitrypsin deficiency (AATD) is an autosomal genetic condition linked to chronic obstructive pulmonary disease (COPD). The significance of heterozygous, milder deficiency variants (PiSZ, PiMZ, PiMS) is less clear. We studied AATD genotypes in 145 children (up to 72 months old) with assessed wheezing severity using the Pediatric Respiratory Assessment Measure (BCCH PRAM score). A control group of 74 children without airway obstruction was included. AAT concentration and Pi phenotype were determined from dry blood spot samples using nephelometry and real-time PCR; PiS and PiZ alleles were identified by isoelectrofocusing. Among the wheezers, the Pi*S allele incidence was 2.07% (3 cases) and the Pi*Z allele was 6.9% (10 cases). The Pi*Z allele frequency was higher in wheezers compared to controls (44.8% vs. 20.27%) and the general Lithuanian population (44.8% vs. 13.6%) and was similar to adult COPD patients in Lithuania: Pi*S 10.3% vs. 15.8% and Pi*Z 44.8% vs. 46.1%. No association was found between AAT genotypes and wheezing severity. Finding that wheezer children exhibit a frequency of Z* and S* alleles like that found in adults with COPD suggests a potential genetic predisposition that links early wheezing in children to the development of COPD in adulthood. Larger cohort studies are needed to confirm this finding.

Longevity of Schistosoma mansoni circulating cathodic antigens in filter paper dried urine spots

ObjectivesThis study aims to determine the temporal stability of Schistosoma mansoni circulating cathodic antigens (CCA) in filter paper-based dried urine spot (FP-DUS) samples under varying temperatures condition. MethodsUrine from 20 children confirmed to have S. mansoni infection using Kato-Katz (at least 1 egg per gram of stool) and Schisto POC-CCA (2+ and 3+) methods were stored in form of FP-DUS and urine at room temperature (RT), 4 °C and −20 °C. Standard urine and FP-DUS Schisto POC-CCA methods were employed to detect CCA in urine and FP-DUS samples respectively, at weeks 4, 8 and 12. The results were reported as negative or positive (trace, 1+. 2+, and 3+). ResultsIn FP-DUS samples, POC-CCA scores initially increase after 4–8 weeks, but then showed a decrease in intensity while still remaining positive, independent of temperature condition. From week 4 to week 12, at least 80 % of urine samples had POC-CCA score of 3+, independent of temperature condition. However, 2 urine samples at RT tested negative at weeks 8 and 12. ConclusionsDespite the decrease in the intensity of test line in many samples, S. mansoni CCA remains stable and detectable in urine samples stored in FP-DUS.

Dried Blood Spot Sampling for Monitoring Children With Immune-Mediated Glomerulopathies and After Kidney Transplantation

BackgroundMonitoring kidney function and immunosuppressant levels in children post-kidney transplantation or those with glomerulopathies is challenging due to frequent venipunctures and clinic visits. Capillary dried blood spot sampling (DBS) offers a potential alternative. MethodsIn this prospective single-center study, 89 children (38 % female, 62 % male) requiring therapeutic drug monitoring (TDM) and kidney function assessment were enrolled. 79 % of the patients were kidney transplant recipients, and 21 % suffered from immune-mediated glomerulopathies. The mean age was 13.4 years (range 5.7-18.0). DBS and standard venous serum samples were collected simultaneously for tacrolimus (TAC), cyclosporine A (CsA), everolimus (EVR), and creatinine levels. Further, patient feedback on pain perception and feasibility was collected via questionnaire. ResultsNo significant differences in parameter values between DBS and standard methods were found (creatinine -1.7±14.5 μmol/l; EVR 0.1±1.2 μg/L; TAC 0.3±1.1μg/L; CsA 2.8±9.8 μg/L). DBS demonstrated sufficient accuracy compared to standard methods. Patients favored DBS and telehealth consultations, especially due to less travel and school absences. Patients preferred finger pricking over ear pricking. ConclusionsCapillary DBS proves reliable for TDM and kidney function assessment in pediatric kidney disease. It reduces patient and family burden compared to venous blood collection, while also enabling telehealth consultations.

Metabolism study of 3-chloromethcathinone (3-CMC) by dried blood spot (DBS) sampling after controlled administration using a murine model.

The metabolism of 3-chloromethcathinone (3-CMC) was studied after controlled administration in a murine model using the dried blood spot (DBS) technique for the sampling, storage and purification of blood samples. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was used for the identification of metabolites and investigation of their fragmentation pattern. Subsequently, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for their identification and 3-CMC quantification in routine workload. The main metabolites identified were two stereoisomers of dihydro-CMC, N-demethyl-CMC, and dihydro-N-demethyl-CMC. The stability of 3-CMC and of its metabolites deposited on DBS was evaluated by replicate analyses after 30, 50, and 90 days, demonstrating a decrease in concentration. It was more pronounced for 3-CMC, with -67% and -82% percentage deviation from the initial concentrations, and for N-demethyl 3-CMC (decrease comprised between -48% and -88%) than for the di-hydro metabolites, ranging from -5% to -37%. Regardless, all of them were detectable till 90 days after deposition as DBS. The possibility of identifying 3-CMC and its metabolites with high sensitivity is an invaluable tool for the diagnosis of exposure to the substance, also in low doses or after some hours, and for various applications in clinical and forensic toxicology, such as driving under the influence, drug-facilitated crimes, and addiction to intoxications. DBS demonstrated to be a reliable technique for the sampling, storage, and purification of the blood specimen for 3-CMC and metabolite detection.