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Molecular Genetics and Metabolism... Jun 2024Each year thousands of babies are born with rare genetic disorders not identified by current NBS panels, due to programs which are not yet optimal. Next-generation...
Each year thousands of babies are born with rare genetic disorders not identified by current NBS panels, due to programs which are not yet optimal. Next-generation sequencing technologies have the potential to overcome many NBS drawbacks and provide large amounts of molecular data, broadening the number of diseases investigated. Here, we design and set up an NGS-based approach to evaluate the feasibility of NGS from dried blood spot starting from 34 DBSs. After assessing gDNA yield and integrity, libraries were performed using three target enrichment approaches, sequenced on NS500 platform, and analyzed on commercial platform. Specifically, we focus on virtual gene panels related to highly actionable neonatal/pediatric disorders. WES show that amount and quality of DBS-extracted gDNA are suitable for high-throughput sequencing. We obtain 500-1500 ng for each specimen, 1.7-1.8 260/280 wavelength, and DIN of 7 resulting DNA integrity, on par with traditional venous blood collection. A high read depth with 94.3% coverage uniformity is achieved for all samples. Data results on mean coverage are comparable among the different workflows tested and demonstrate that DBS from newborn collected at birth is a suitable material for the developing of gNBS programs.
PubMed: 38544910
DOI: 10.1016/j.ymgmr.2024.101074 -
Clinica Chimica Acta; International... Apr 2024Volumetric Absorptive Microsampling (VAMS) is emerging as a valuable technique in the collection of dried biological specimens, offering a potential alternative to...
BACKGROUND
Volumetric Absorptive Microsampling (VAMS) is emerging as a valuable technique in the collection of dried biological specimens, offering a potential alternative to traditional sampling methods. The objective of this study was to assess the suitability of 30 μL VAMS for the measurement of endogenous steroid hormones.
METHODS
A novel LC-MS/MS method was developed for the quantification of 18 analytes in VAMS samples, including main endogenous free steroids and phase II metabolites of androgens. The method underwent validation in accordance with ISO/IEC 17025:2017 and World Anti-Doping Agency (WADA) requirements. Subsequently, it was applied to authentic VAMS samples obtained from 20 healthy volunteers to assess the stability of target analytes under varying storage conditions.
RESULTS
The validation protocol assessed method's selectivity, matrix effect, extraction recovery, quantitative performance, carry-over and robustness. The analysis of authentic samples demonstrated the satisfactory stability of monitored steroids in VAMS stored at room temperature, 4 °C, -20 °C and -80 °C for up to 100 days and subjected to up to 3 freezing-thawing cycles.
CONCLUSIONS
The validated LC-MS/MS method demonstrated its suitability for the measurement of steroids in dried blood VAMS. The observed stability of steroidal compounds suggests promising prospects for future applications of VAMS, both in anti-doping contexts and clinical research.
Topics: Humans; Androgens; Blood Specimen Collection; Chromatography, Liquid; Doping in Sports; Dried Blood Spot Testing; Liquid Chromatography-Mass Spectrometry; Steroids; Tandem Mass Spectrometry
PubMed: 38537673
DOI: 10.1016/j.cca.2024.117890 -
Data in Brief Jun 2024This dataset presents a comprehensive collection of images representing both dried and live samples from eight distinct Thai cannabis classes. The dataset includes a...
This dataset presents a comprehensive collection of images representing both dried and live samples from eight distinct Thai cannabis classes. The dataset includes a total of 14,094 images, with images depicting dried and healthy specimens. These images serve as a valuable resource for researchers engaged in botanical exploration, machine learning, and computer vision studies. Additionally, the dataset facilitates investigations into the medicinal properties of Thai cannabis. Interdisciplinary collaboration is encouraged, providing opportunities for innovative insights spanning biology, horticulture, and data science. Beyond fundamental research, this dataset holds practical implications for agriculture, technology development, and disease prevention, offering insights into both dried and live states of Thai cannabis plants across various strains.
PubMed: 38516281
DOI: 10.1016/j.dib.2024.110292 -
Clinical and Experimental Dental... Apr 2024The use of silver fluoride followed by stannous fluoride was designed for the treatment of open carious lesions in primary molars in dental outreach programs. However,...
OBJECTIVES
The use of silver fluoride followed by stannous fluoride was designed for the treatment of open carious lesions in primary molars in dental outreach programs. However, during the COVID-19 pandemic when aerosol-producing procedures were inadvisable, one dental location started using it as the first stage in a two-visit restorative procedure for carious primary molars. If the gap between the fluoride application and the restoration placement stages was around 3-5 weeks it was noticed that a black friable crust appeared on the caries surface. To investigate further a normally discarded crust from one patient was retrieved and sent for analysis.
MATERIALS AND METHODS
Two techniques suitable for identification and preliminary analysis of material of unknown composition, scanning electron microscopy and energy dispersive spectroscopy (EDS) were used. The only preparation was that the specimen was dried and coated beforehand.
RESULTS AND CONCLUSIONS
This preliminary examination showed two unexpected findings. The first was that the crust surface indicated a possible dentine derivation as it was covered with reasonably evenly spaced holes. In addition, the EDS spectrum showed it to be, at least, partially mineralized. The second unexpected finding was that the surface was coated with electron-dense particles. The size of the particles and the EDS spectrum pointed to the likelihood of the majority of them being nanosilver. These unexpected findings suggest a possible new direction for research.
Topics: Humans; Tin Fluorides; Pandemics; Dental Caries; Molar; Fluorides; Silver Compounds
PubMed: 38506304
DOI: 10.1002/cre2.838 -
Plant Disease Mar 2024In February 2022, leaf zonate spot disease afflicted Aloe vera L. in Yunnan, China, endangering the $39 billion industry with 0.33ha under cultivation (Wan 2015). The...
In February 2022, leaf zonate spot disease afflicted Aloe vera L. in Yunnan, China, endangering the $39 billion industry with 0.33ha under cultivation (Wan 2015). The disease manifested with watery spots progressing into oval or circular necrosis lesions, characterized by a dark center surrounded by a gray-brown zone. In the late stage of the disease, lesions regress in size and several small dark picnidia dots appeared on the gray-brown zone. The disease incidence ranged from 10% to 15% in three commercial plantations. If left uncontrolled, the disease could diminish the commercial value of Aloe vera plants. Eighteen symptomatic leaf samples underwent morphological and genetic identification. The samples were carefully washed with distilled water and 1×1 cm2 sections of tissue were excised using a sterile scalpel. The sections underwent surface-disinfection with 3% NaOCl for 3 min and 75% ethanol for 30 s. After three sterile water rinses the sections were air-dried. Subsequently, they were transferred to potato dextrose agar (PDA) before being incubated at 25 ℃ in the dark. Of the 18 samples, eight produced the colonies with similar morphological characteristics, named LH7. Isolate LH7 had downy to woolly aerial mycelia, initially pinkish white on the surface, and gradually turned greenish-olivaceous from the middle, and eventually turned dark brown to black after seven days. The fungus formed arthric chains in the aerial mycelium on PDA but did not produce conidiomata. The conidia, which occurred in arthric chains were 5.50-9.9 × 4.08-7.51 μm (mean 7.09× 5.26 μm, n=50) in size, cylindrical, brown, and 0-1 septate. To ascertain LH7's pathogenicity, three healthy one-year old aloe plants were surface-sanitized with a 1% aqueous chlorine solution, rinsed with sterile water, and dried. Three leaves from each plant were punctuated and inoculated using conidial suspension (100 μl of 1x 106 conidial mL-1), while three control plants were inoculated with sterile distilled water. The pathogenicity tests were repeated twice. The inoculated plants were kept at 25 ℃ with a 12-hour light/12-hour dark cycle. After seven days, symptoms observed in the field appeared in the plants, while no disease occurred in the control plants. After 21 days, conidiomata formed on the inoculated leaves, averaging 116.92 μm (n=20) in diameter. These conidiomata were globose to subglobose, and brown to sub-brown. The fungus was successfully re-isolated from symptomatic tissue and the resulting colonies were morphologically consistent with isolate LH7. Based on the characteristics, the fungus was identified as Neoscytalidium dimidiatum (Philips et al. 2013). The specimen was deposited in China Center for Type Culture Collection ( CCTCC AF 2024001). This identification was confirmed through sequencing of ITS gene region of rDNA using ITS1/ITS4 (Imran et al. 2022). The sequence was submitted into GenBank database (ON878059). BLAST analysis of the LH7's ITS amplicon showed 100% similarity with that of JN093303.1. A phylogenetic tree constructed using the maximum likelihood method revealed that ON878059 was clustered with JN093303.1. Previous studies have documented that pathogens such as Colletotrichum gloeosporioides (Penz.), Fusarium spp. and Rhizopus oryzae can also cause diseases in A. vera in China (Zhou et al. 2008; Ding et al. 2015). Additinonally, Cladosporium sphaerospermum, Pseudopestalotiopsis theae, and Lasiodiplodia theobromae have been identified as causal agents of aloe leaf spot diseases in India, Bangladesh and Malaysia (Avasthi et al. 2016; Ahmmed et al. 2022; Khoo et al. 2022). To our knowledge, this is the first report of N. dimidiatum causing leaf necrosis of aloe in China. Vigilant surveillance and disease control measures are imperative to mitigate potential losses in this region.
PubMed: 38499972
DOI: 10.1094/PDIS-09-23-1911-PDN -
Journal of Clinical Microbiology Apr 2024Scaling up of newer innovations that address the limitations of the dried blood spot and the logistics of plasma monitoring is needed. We employed a multi-site,...
UNLABELLED
Scaling up of newer innovations that address the limitations of the dried blood spot and the logistics of plasma monitoring is needed. We employed a multi-site, cross-sectional assessment of the plasma separation card (PSC) on blood specimens collected from all consenting adults, assenting young and pediatric patients living with HIV from 10 primary healthcare clinics in South Africa. Venous blood for EDTA-plasma samples was collected and analyzed according to the standard of care assay, while collected capillary blood for the PSC samples was analyzed using the Roche COBAS AmpliPrep/Cobas TaqMan (CAP/CTM) HIV-1 Test at the National Reference laboratories. McNemar tests assessed the differences in concordance between the centrifuged plasma and dried plasma spots. The usability of PSC by blood spotting, PSC preparation, and pre-analytical work was assessed by collecting seven-point Likert-scale data from healthcare and laboratory workers. We enrolled 538 patients, mostly adults [ = 515, 95.7% (95% CI: 93.7%-97.1%)] and females [ = 322, 64.2% (95% CI: 60.0%-68.1%)]. Overall, 536 paired samples were collected using both PSC- and EDTA-plasma diagnostics, and 502 paired PSC- and EDTA-plasma samples assessed. Concordance between the paired samples was obtained for 446 samples. Analysis of these 446 paired samples at 1,000 copies per milliliter threshold yielded an overall sensitivity of 87.5% [95% CI: 73.2%-95.8%] and specificity of 99.3% [95% CI: 97.9%-99.8%]. Laboratory staff reported technical difficulties in most tasks. The usability of the PSC by healthcare workers was favorable. For policymakers to consider PSC scale-up for viral load monitoring, technical challenges around using PSC at the clinic and laboratory level need to be addressed.
IMPORTANCE
Findings from this manuscript emphasize the reliability of the plasma separation card (PSC), a novel diagnostic method that can be implemented in healthcare facilities in resource-constrained settings. The agreement of the PSC with the standard of care EDTA plasma for viral load monitoring is high. Since the findings showed that these tests were highly specific, we recommend a scale-up of PSC in South Africa for diagnosis of treatment failure.
Topics: Adult; Female; Humans; Child; Sensitivity and Specificity; HIV-1; Viral Load; South Africa; Cross-Sectional Studies; Edetic Acid; Reproducibility of Results; HIV Infections; RNA, Viral
PubMed: 38470024
DOI: 10.1128/jcm.01649-23 -
Journal of Pharmaceutical and... Jun 2024Drug testing commonly use urine as a specimen and immunoassays for screening. The need for supervised urine collection has led to an interest in alternative specimens...
BACKGROUND
Drug testing commonly use urine as a specimen and immunoassays for screening. The need for supervised urine collection has led to an interest in alternative specimens and a need for using mass spectrometry methods already for screening. In addition, mass spectrometry methods allow for broad multipanel screening which of great value because of the increased number of substances that needs to be covered has increased over time. One alternative specimen of interest for drugs of abuse testing is dried blood spots (DBS) and this work aimed at developing multipanel screening methods based on selected reaction monitoring liquid chromatography - mass spectrometry for both urine and dried finger blood as specimens.
MATERIALS AND METHODS
The urine method comprised 37 analytes and utilised salted out liquid/liquid extraction in 96-well format, respectively, and the blood method comprised 35 analytes, a 10 µL volumetric DBS device and a two-step solvent extraction procedure. In both cases stable isotope labelled internal standards were used for almost all analytes.
RESULTS
The methods were validated according to forensic standard. The lowest reporting limits were generally set at 100 ng/mL for urine and 1 ng/mL for blood and the accuracy and imprecision were within limits of 15 and 20%. The methods were applied in a clinical study on patients receiving methadone maintenance treatment for opioid dependence. Methadone was detected in all urine and DBS samples, for urine sometimes below the commonly applied screening cutoff limit of 300 ng/mL. In 20 out of 99 cases no other drug was detected in any specimen. The most commonly other detected substances were pregabalin, amphetamine, alprazolam, zopiclone and THCCOOH. Findings in urine and DBS generally agreed well but more positives were detected in DBS.
CONCLUSION
Multipanel methods using liquid chromatography - mass spectrometry suitable for clinical drug screening were successfully developed for urine and blood collected by finger-pricking and stored as DBS.
Topics: Humans; Chromatography, Liquid; Liquid Chromatography-Mass Spectrometry; Tandem Mass Spectrometry; Methadone; Dried Blood Spot Testing
PubMed: 38457867
DOI: 10.1016/j.jpba.2024.116075 -
Molecular Genetics and Metabolism May 2024Phenylketonuria (PKU) requires regular phenylalanine monitoring to ensure optimal outcome. However, home sampling methods used for monitoring suffer high pre-analytical...
INTRODUCTION
Phenylketonuria (PKU) requires regular phenylalanine monitoring to ensure optimal outcome. However, home sampling methods used for monitoring suffer high pre-analytical variability, inter-laboratory variability and turn-around-times, highlighting the need for alternative methods of home sampling or monitoring.
METHODS
A survey was distributed through email and social media to (parents of) PKU patients and professionals working in inherited metabolic diseases in Denmark, The Netherlands, and United Kingdom regarding satisfaction with current home sampling methods and expectations for future point-of-care testing (POCT).
RESULTS
210 parents, 156 patients and 95 professionals completed the survey. Countries, and parents and patients were analysed together, in absence of significant group differences for most questions. Important results are: 1) Many patients take less home samples than advised. 2) The majority of (parents of) PKU patients are (somewhat) dissatisfied with their home sampling method, especially with turn-around-times (3-5 days). 3) 37% of professionals are dissatisfied with their home sampling method and 45% with the turn-around-times. 4) All responders are positive towards developments for POCT: 97% (n = 332) of (parents of) patients is willing to use a POC-device and 76% (n = 61) of professionals would recommend their patients to use a POC-device. 5) Concerns from all participants for future POC-devices are costs/reimbursements and accuracy, and to professionals specifically, accessibility to results, over-testing, patient anxiety, and patients adjusting their diet without consultation.
CONCLUSION
The PKU community is (somewhat) dissatisfied with current home sampling methods, highlighting the need for alternatives of Phe monitoring. POCT might be such an alternative and the community is eager for its arrival.
Topics: Humans; Phenylketonurias; Point-of-Care Testing; Male; Female; Surveys and Questionnaires; Parents; Blood Specimen Collection; United Kingdom; Netherlands; Adult; Patient Satisfaction; Phenylalanine; Denmark; Child; Adolescent
PubMed: 38442492
DOI: 10.1016/j.ymgme.2024.108361 -
Royal Society Open Science Feb 2024The article's main aim is to assess the mechanical behaviour of linden under high-rate loadings (impact) and its change due to changes in moisture content (MC) over...
The article's main aim is to assess the mechanical behaviour of linden under high-rate loadings (impact) and its change due to changes in moisture content (MC) over fibre saturation point. For assessing the mechanical properties of green wood, mainly the data of the dried wood is not applicable since the moisture content can drastically affect the mechanical properties of the wood. By testing both dried and high-moisture-content wood, we can understand a general viewpoint toward the effect of the moisture content on the impact behaviour of the wood. Several test samples were made of linden wood with different moisture content levels of 11%, 60% and 160%. A drop-weight impact machine tested the specimens to measure the reaction force of the hammer during a very short impact period. The results of the tests were parameters such as force-time chart, the maximum force required for crack initiation, the impact bending strength (IBS) and the work needed for crack initiation. The results indicated an increase in MC decreases the maximum force, work required for crack initiation and IBS drastically. However, when MC exceeded the fibre saturation point (FSP), there was no further influence on the force pattern and maximum required force.
PubMed: 38420630
DOI: 10.1098/rsos.231685 -
BMC Infectious Diseases Feb 2024Sexually transmitted infections (STIs) such as syphilis and HIV remain to be a significant public health issue worldwide. Dual rapid point-of-care tests (POCTs) have...
BACKGROUND
Sexually transmitted infections (STIs) such as syphilis and HIV remain to be a significant public health issue worldwide. Dual rapid point-of-care tests (POCTs) have shown promise for detecting antibodies to HIV and syphilis but have not been fully evaluated in the field. Our study supported the WHO ProSPeRo study on Sexually Transmitted Infection Point-of-Care Testing (STI POCT) by providing external quality assessment (EQA) for HIV and syphilis testing in reference laboratories and their associated clinical sites in seven countries.
METHODS
HIV/syphilis serum liquid and dried tube specimen (DTS) panels were prepared by CDC. Liquid panels were distributed to the reference laboratories for three rounds of testing using commercially and locally available laboratory-based serological tests. DTS panels were sent to the clinical testing sites for 8 rounds of POC testing using the Abbott SD BIOLINE HIV/Syphilis Duo test (hereafter referred to as SD BIOLINE) and the Chembio Dual Path Platform (DPP) HIV-Syphilis assay. EQA panels were tested at CDC using the Rapid Plasma Reagin (RPR) test and the Treponema pallidum Particle Agglutination assay (TP-PA) for syphilis antibodies. Genetic Systems HIV-1/HIV-2 Plus O EIA, Geenius HIV Supplemental Assay and the Oraquick Advance HIV test were used to detect HIV antibodies in the EQA panels. Results from the reference laboratories and POCT sites were compared to those obtained at the CDC and a percentage agreement was calculated.
RESULTS
Qualitative RPR and TP-PA performed at the reference laboratories demonstrated 95.4-100% agreement with CDC results while quantitative RPR and TP-PA tests demonstrated 87.7% and 89.2% agreement, respectively. A 93.8% concordance rate was observed for qualitative HIV testing in laboratories. EQA testing at clinical sites using dual tests showed 98.7% and 99.1% agreement for detection of HIV antibodies and eight out of 10 sites had > 95.8% agreement for syphilis testing. However, two clinical sites showed only 65.0-66.7% agreement for SD BIOLINE and 84.0-86.7% for DPP, respectively, for syphilis testing.
CONCLUSIONS
Overall, laboratories demonstrated high EQA performance in this study. Both HIV/syphilis POCTs gave expected results in the clinic-based evaluations using DTS. However, testing errors were identified in a few testing sites suggesting the necessity for continuous training and monitoring the quality of POC testing.
Topics: Humans; Syphilis; Treponema pallidum; HIV Antibodies; HIV Infections; Sensitivity and Specificity; Antibodies, Bacterial; Point-of-Care Testing; Syphilis Serodiagnosis; HIV-2; HIV-1; World Health Organization; Point-of-Care Systems
PubMed: 38418989
DOI: 10.1186/s12879-024-09027-3