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1.
J Immunol Methods ; 492: 112996, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33582147

RESUMO

Dried blood spots (DBS) are routinely used in screening newborns for treatable disorders. Immunoglobulin extraction from DBS, serum or other biological fluids loaded on filter paper cards could represent a valuable method of specimen preservation in monitoring immune response against pathogens as well as vaccination efficiency. In this study using different sources including serum, and monoclonal antibodies we established parameters for antibody extraction from the filter cards to assess antibody reactivity against Helicobacter pylori, measles virus (MV) and the novel coronavirus SARS-CoV-2 antigens. We demonstrated that DBS and dried undiluted serum result in completely preserved antibody activity for immunoassays, including in virus neutralization assays against MV. Extraction efficiency was determined by IgG concentration measurements. The plaque-reduction neutralization titer 50% of dried human serum spots remained stable after more than 10-day storage - 1:359 vs. 1:345 for the corresponding frozen sample. DBSs could be used to monitor immune response to bacterial and viral antigens following natural exposure or immunization. Mice immunized with recombinant spike protein receptor-binding domain of SARS-CoV-2 developed a strong antibody response by day 14 and reached titers above 1:64,000 on day 21 following the secondary boost immunization as measured on DBS samples in antigen-mediated ELISA. Variability in IgG concentration of eluted DBS could be influenced by factors involved in sample application, extraction process and sample characteristics. Adjustment of antibody specific activity to the eluted IgG concentration can increase accuracy of the result interpretation, including in SARS-CoV-2 serological diagnostics.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Teste em Amostras de Sangue Seco , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/fisiologia , Imunoensaio/métodos , Vírus do Sarampo/fisiologia , Sarampo/diagnóstico , Monitorização Imunológica/métodos , /fisiologia , Animais , Anticorpos Monoclonais , Formação de Anticorpos , Teste em Amostras de Sangue Seco/métodos , Feminino , Humanos , Imunidade Humoral , Camundongos , Camundongos Endogâmicos BALB C , Testes Sorológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação
2.
Diabetes Metab Syndr ; 15(1): 433-437, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33588200

RESUMO

BACKGROUND AND AIMS: The COVID-19 pandemic has led to a rapid growth in the use of telemedicine for delivery of ambulatory diabetes care. This study evaluated the feasibility of remote HbA1c monitoring via dried blood spot (DBS) testing to support assessment of glycemic control for telemedicine visits and examined clinical and demographic characteristics associated with patient completion of DBS testing. METHODS: Providers could place orders for DBS HbA1c 3 weeks prior to telemedicine visits. Feasibility was assessed by examining DBS completion rates, time to completion, and availability of DBS results prior to telemedicine visits. Chi-square tests and Mann Whitney tests were used to assess whether completion rates were associated with participant characteristics. RESULTS: Of 303 DBS orders placed for telemedicine visits in June 2020, 162 patients completed the DBS test for a completion rate of (53.4%). Average time from collection at home to result being reported was 6.9 (3.8) days. The DBS result was available in 67.6% of patients who completed successful DBS, before the telemedicine clinic visit. HbA1c was lower in the DBS completion group as compared to the non-completion group (8.2% vs. 8.9%, p = 0.01). No other clinical or demographic characteristics were significantly different between the two groups. CONCLUSION: Remote HbA1c monitoring via DBS is feasible and offers an avenue to support assessment of glycemic control for patients seen via telemedicine. Future work should focus on improving clinic and laboratory processes to support remote DBS collection.


Assuntos
/epidemiologia , Diabetes Mellitus/sangue , Diabetes Mellitus/epidemiologia , Teste em Amostras de Sangue Seco/métodos , Hemoglobina A Glicada/metabolismo , Telemedicina/métodos , Adaptação Psicológica , Adolescente , Criança , Diabetes Mellitus/diagnóstico , Teste em Amostras de Sangue Seco/tendências , Estudos de Viabilidade , Feminino , Humanos , Masculino , Telemedicina/tendências
3.
J Clin Virol ; 136: 104739, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33588354

RESUMO

Dried blood spot samples (DBS) provide an alternative sample type to venous blood samples for antibody testing. DBS are used by NHS for diagnosing Hepatitis C and by Public Health England for large scale HIV and Hepatitis C serosurveillance; the applicability of DBS based approaches for SARS-CoV-2 antibody detection is uncertain. The study aimed to compare antibody detection in DBS eluates using the Roche Elecsys ® immunoassay with antibody detection in paired plasma samples, using the same assay. The study was in one Police and one Fire & Rescue facility in England; it comprised of 195 participants within a larger sample COVID-19 serodiagnostics study of keyworkers, EDSAB-HOME. Outcome measures were sensitivity and specificity of DBS (the index test) relative to plasma (the reference test), at an experimental cut-off; quality of DBS sample collected; estimates of relative sensitivity of DBS vs. plasma immunoassay in a larger population. 18/195 (9.2%) participants tested positive using plasma samples. DBS sample quality varied markedly by phlebotomist, and low sample volume significantly reduced immunoassay signals. Using an experimental cut-off, sensitivity and specificity of DBS were 89.0% (95% CI 67.2, 96.9%) and 100.0% (95% CI 97.9, 100%) respectively compared with using plasma. The limit of detection for DBS is about 30 times higher than for plasma. DBS use for SARS-CoV-2 serology, though feasible, is insensitive relative to immunoassays on plasma. Sample quality impacts on assay performance. Alternatives, including the collection of capillary blood samples, should be considered for screening programs.


Assuntos
Anticorpos Antivirais/sangue , /diagnóstico , Teste em Amostras de Sangue Seco/métodos , /imunologia , Adolescente , Adulto , Inglaterra , Monitoramento Epidemiológico , Feminino , Humanos , Imunoensaio , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Adulto Jovem
4.
Methods Mol Biol ; 2237: 199-206, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237419

RESUMO

Dried blood samples have been increasingly considered for clinical applications in recent years. The main disadvantages that limit DBS utility in clinical applications are the small sample volume collected, area bias and homogeneity issues, and sample preparation requirements for the necessary sensitivity and reproducibility required for clinical assessment. The recent advances in antibody array technology overcome the common disadvantages of immunoassay approaches by increasing the multiplex capabilities and decreasing the sample volume requirements as well as minimizing the expense and technical expertise required with many alternative high-density approaches like mass spectrometry.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Análise Serial de Proteínas/métodos , Proteômica/métodos , Animais , Humanos , Imunoensaio/métodos
5.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 49(5): 565-573, 2020 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-33210481

RESUMO

OBJECTIVE: To explore effects of different delivery and storage conditions on concentrations of amino acids and carnitines in neonatal dried blood spots (DBS), so as to provide evidence for improving accurate and reliable detection by tandem mass spectrometry. METHODS: A total of 1 254 616 newborn DBS samples in Newborn Screening Center of Zhejiang Province were delivered and stored at room temperature (group A, n=338 467), delivered by cold-chain logistics system and stored at low temperature (group B, n=480 021), or delivered by cold-chain logistics system and stored at low temperature and low humidity (group C, n= 436 128), respectively. The concentrations of amino acids and carnitines in DBS were detected by tandem mass spectrometry. Data analysis was performed by SPSS 24.0 to explore the influence of temperature and humidity on the concentrations of amino acids and carnitines. RESULTS: The concentrations of amino acids and carnitines in the three groups were skewed, and the differences in amino acid and carnitine concentrations among groups were statistically significant (all P<0.01). The median concentration of tyrosine was lower in group A than those in group B and group C by 18%and 16%respectively, while there was no significant difference between the last two groups. The median concentrations of methionine were lower in group A and group B than that in group C by 15%and 11%, respectively. The median concentrations of arginine were lower in group A and group B than that in group C by 12%and 25%, respectively. The median concentration of free carnitine (C0) was higher in group A than that in group C by 12%, while there was no significant difference between group A and group B. The median concentrations of acetylcarnitine (C2), propionyl carnitine (C3), C3DC+C4OH, C5DC+C6OH and hexadecanoyl carnitine (C16) were lower in group A than those in group B and group C by 21%-64%. The concentrations of other amino acids and acylcarnitines differed little among three groups. The monthly median coefficients of variation of other amino acids and carnitines in group A were higher than those in group B and group C except for citrulline, C4DC+C5OH and isovalerylcarnitine (C5). CONCLUSIONS: Cold-chain logistics system and storage in low temperature and low humidity can effectively reduce degradation of some amino acids and carnitines in DBS, improve the accuracy and reliability of detection, and thus ensures the quality of screening for neonatal metabolic diseases.


Assuntos
Aminoácidos , Teste em Amostras de Sangue Seco , Triagem Neonatal , Aminoácidos/análise , Carnitina/análise , Teste em Amostras de Sangue Seco/métodos , Teste em Amostras de Sangue Seco/normas , Humanos , Umidade , Recém-Nascido , Reprodutibilidade dos Testes , Manejo de Espécimes/normas , Espectrometria de Massas em Tandem , Temperatura , Fatores de Tempo
6.
PLoS One ; 15(8): e0236775, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756585

RESUMO

BACKGROUND: Options to increase the ease of testing for SARS-CoV-2 infection and immune response are needed. Self-collection of diagnostic specimens at home offers an avenue to allow people to test for SARS-CoV-2 infection or immune response without traveling to a clinic or laboratory. Before this study, survey respondents indicated willingness to self-collect specimens for COVID-related tests, but hypothetical willingness can differ from post-collection acceptability after participants collect specimens. METHODS: 153 US adults were enrolled in a study of the willingness and feasibility of patients to self-collect three diagnostic specimens (saliva, oropharyngeal swab (OPS) and dried blood spot (DBS) card) while observed by a clinician through a telehealth session. After the specimens were collected, 148 participants participated in a survey about the acceptability of the collection, packing and shipping process, and their confidence in the samples collected for COVID-related laboratory testing. RESULTS: A large majority of participants (>84%) reported that collecting, packing and shipping of saliva, OPS, and DBS specimens were acceptable. Nearly nine in 10 (87%) reported being confident or very confident that the specimens they collected were sufficient for laboratory analysis.There were no differences in acceptability for any specimen type, packing and shipping, or confidence in samples, by gender, age, race/ethnicity, or educational level. CONCLUSIONS: Self-collection of specimens for SARS-CoV-2 testing, and preparing and shipping specimens for analysis, were acceptable in a diverse group of US adults. Further refinement of materials and instructions to support self-collection of saliva, OPS and DBS specimens for COVID-related testing is needed.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Orofaringe/virologia , Cooperação do Paciente , Pneumonia Viral/diagnóstico , Saliva/virologia , Manejo de Espécimes/métodos , Adulto , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Teste em Amostras de Sangue Seco/métodos , Escolaridade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Cooperação do Paciente/etnologia , Pneumonia Viral/virologia , Autocuidado , Inquéritos e Questionários , Telemedicina
7.
J Chromatogr A ; 1628: 461445, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822984

RESUMO

Anabolic androgenic steroids (AAS) have been the most commonly abused substances taken by not only professional sportsmen but also recreational bodybuilders. The detection of micro-dose testosterone (T) misuse is particularly challenging as it possesses pseudo-endogenous origin and is sometimes impossible to be identified in urine samples. Dried blood (DB) obtained by finger pricking has been proven to be an alternative matrix for better correlating to physiological responses. Moreover, the introduction of the volumetric absorptive microsampling (VAMS) technology allows overcoming some major limitations of spotting blood onto a filter paper card. In this work, a fast and sensitive GC-MS/MS method was developed and validated for the quantification of AAS in DB collected by means of VAMS. T and the eight top abused synthetic AAS, namely nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and dehydrochloromethyl T were selected as the target analytes. The method based on VAMS exhibited good precision, accuracy as well as stability, and superior extraction recoveries over the punched DB spots reported in the literature. The chromatographic separation was achieved within 6.4 min and the detection limit is as little as 50 fg (i.e. able to detect 0.10 ng mL-1 in 20 µL of DB). Confirmed by forty real blood samples, the Deming regression and Bland-Altman analysis revealed that the VAMS DB could be employed for quantifying blood T level in agreement with using the serum specimen. The feasibility of the method was then successfully proven by the analysis of samples collected from a three-arm T administration trial. Our results highlighted that DB total T was a sensitive indicator for identifying transdermal micro-dosing of T. In the groups of receiving T gel administration, T concentrations could rise up to ten times higher than the baseline at 9 h after the application. As a future step, this approach is being expanded to a large cohort screening of bodybuilders at gym and ultimately may allow universal applications on monitoring sports drug misuse.


Assuntos
Androgênios/sangue , Teste em Amostras de Sangue Seco/métodos , Monitoramento de Medicamentos/métodos , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de Massas em Tandem , Congêneres da Testosterona/sangue , Testosterona/análise , Humanos
8.
Zhonghua Liu Xing Bing Xue Za Zhi ; 41(6): 913-918, 2020 Jun 10.
Artigo em Chinês | MEDLINE | ID: mdl-32564559

RESUMO

Objectives: To understand the prevalence of HIV nucleic acid using internet-based dry blood spots HIV testing strategy in men who had sex with men (MSM) and to probe the factors associated with HIV infection. Methods: Using convenient sampling method, 1 375 MSM were recruited and their dry blood spots samples were collected before being mailed to the laboratories for HIV nucleic acid testing. Results were showed to these MSM on a specific website by inputting their codes to it. Non-conditional binary logistic regression method was used to identify the associated factors on HIV infection. Results: The overall proportions of HIV nucleic acid positives appeared as 9.7% (131/1 349) and HIV antibody positives as 8.3% (112/1 349). Fresh infections accounted for 14.5% (19/131) among the newly-identified HIV nucleic acid positives, and the interval was ranging from 6 to 120 days, between the laboratory testings and the closest date that experiencing high risk behavior. Risk factors that related to HIV infection would include: 30 to 39 years of age (comparing to those under the age of 30, OR=1.88, 95%CI: 1.07-3.29), ≥8 000 Yuan of monthly income (comparing to those without income, OR=0.42, 95%CI: 0.19-0.96), inconsistent condom use during anal sexual contacts in the last six months (compared with those who had not anal sex or used condoms consistently in anal sex in the past six months, OR=2.22, 95%CI: 1.45-3.40), ever use of Rush Poppers (compared with those who never used Rush Poppers, OR=2.33, 95%CI: 1.49-3.64), addictive drug abuse (compared with those who never abused addictive drugs, OR=5.43, 95%CI: 2.32-12.69), and not having regular sexual partners (compared with having regular sexual partners, OR=1.74, 95%CI: 1.13-2.68) etc.. Conclusions: Dry blood spots HIV nucleic acid testing could help to identify the fresh HIV infections at an early stage, so as to prevent further transmission in the MSM population, among which fresh HIV infections accounted for a fairly large proportion. It is necessary to set up programs in reducing the abuse of drugs or Rush Poppers, and to promote condom use and advocate on stable sexual partnership etc., among the MSM population.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Infecções por HIV/epidemiologia , Homossexualidade Masculina/estatística & dados numéricos , Internet , Ácidos Nucleicos/sangue , Adulto , Pequim/epidemiologia , Estudos de Viabilidade , Humanos , Masculino , Fatores de Risco
9.
Medicina (B Aires) ; 80(3): 197-202, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32442933

RESUMO

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder due to a deficiency of enzymes involved in cortisol biosynthesis. In more than 90% of cases, CAH is secondary to deleterious mutations in the CYP21A2 gene leading to 21-hydroxilase deficiency (21OHD). The CYP21A2 gene is located on the short arm of chromosome 6 (6p21·3) and encodes the cytochrome P450C21 enzyme. Neonatal screening programs detect the classic forms of CAH-21OHD quantifying 17OH-progesterone in dried blood spots (DBS). This test is very sensitive, but it has a low specificity, requiring a second sample to confirm the result. In these cases, a second-tier test in the same sample may be useful. Our aim was to evaluate a DNA extraction method from DBS and assess the performance of such DNA in the molecular analysis of the CYP21A2 gene mutations. Twelve individuals, who presumably had CAH based on the initial neonatal screening results, were analyzed using DNA extracted from freshly collected blood on EDTA and DBS. The CYP21A2 gene was analyzed by automated sequencing of all exons and intron boundaries and MLPA analysis in DBS. Molecular analysis results from both extraction methods were compared. In this study, we show that DNA extracted from neonatal screening DBS is a useful tool to define CYP21A2 gene mutations in 21-OHD diagnostic confirmation for the newborn screening program and that its results are comparable to traditional genotyping.


Assuntos
Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Teste em Amostras de Sangue Seco/métodos , Mutação , Triagem Neonatal/métodos , Esteroide 21-Hidroxilase/genética , 17-alfa-Hidroxiprogesterona/análise , Alelos , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase , Valores de Referência , Reprodutibilidade dos Testes , Espectrofotometria
10.
PLoS One ; 15(5): e0232345, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469947

RESUMO

BACKGROUND: In remote settings, timely plasma separation and transportation to testing laboratories is an impediment to the access of HIV viral load (VL) testing. Potential solutions are whole blood testing through point of care (POC) assays or dried blood spots (DBS). METHODS: We evaluated the performance of a prototype Alere q whole blood VL protocol and compared it against plasma (Abbott RealTime HIV-1) and DBS VL (Abbott RealTime HIV-1 DBS revised prototype protocol and Roche CAP/CTM HIV-1 v2.0 DBS free virus elution protocol). Virological failure (VF) was defined at >1000 copies/ml. RESULTS: Of 299 samples, Alere q correctly classified VF in 61% versus 87% by Abbott DBS and 76% by Roche FVE. Performance varied across plasma VL categories. Alere q showed 100% sensitivity. Below 1000 copies/ml of plasma, Alere q demonstrated over-quantification, with 19% specificity. Abbott DBS had 91% sensitivity and the best overall correlation with plasma (r2 = 0.72). Roche FVE had the best specificity of 99% but reduced sensitivity of 52%, especially between 1000-10,000 copies/ml of plasma. Correlation was best for all assays at >10,000 copies/ml. CONCLUSION: Variability was prominent between the assays. Each method requires optimization to facilitate the implementation of a cut-off with optimal sensitivity and specificity for VF. Although Alere q whole blood assay exhibited excellent sensitivity, the poor specificity of only 19% would lead to unnecessary switching of regimens. Thus any VF detected would need to be confirmed by a more specific assay. Both the Abbott DBS and Roche FVE protocols showed good specificity, however sensitivity was reduced when the plasma VL was 1000-10,000 copies/ml. This could result in delays in detecting VF and accumulation of drug resistance. Field evaluation in settings that have adopted these DBS protocols are necessary.


Assuntos
Teste em Amostras de Sangue Seco/métodos , HIV-1/fisiologia , HIV-2/fisiologia , Carga Viral , Humanos , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao Leito
11.
BMC Infect Dis ; 20(1): 305, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-32334529

RESUMO

BACKGROUND: This study aimed to detect Hepatitis B virus (HBV) genome sequences and their variants as of nationwide scale using dried blood spot (DBS) samples and to provide up-to-date reference data for infection control and surveillance in Cambodia. METHOD: Among 2518 children age 5-7 years and their 2023 mothers participated in 2017 Cambodia nationwide sero-survey on hepatitis B surface antigen (HBsAg) prevalence using multistage random sampling strategy, 95 mothers and 13 children positive to HBsAg were included in this study. HBV DNA was extracted from DBS, then performed polymerase chain reaction. HBV genotypes and potential variants were examined by partial and full length genomic analysis. RESULTS: HBsAg positive rate was 4.7% (95/2023) in mothers and 0.52% (13/2518) in their children. Genotype C (80.49%) was abundantly found throughout the whole Cambodia whilst genotype B (19.51%) was exclusively found in regions bordering Vietnam. S gene mutants of HBV were found in 24.29% of mothers and 16.67% of children with HBV DNA positive sera. Full-length genome analysis revealed the homology of 99.62-100% in each mother-child pair. Genotype B was clarified to recombinant genotype B4/C2 and B2/C2. Double (48.39%) and combination mutation (32.26%) were observed in core promoter region of HBV C1 strains. CONCLUSIONS: This study showed the capable of DBS for large-scale molecular epidemiological study of HBV in resource limited countries. Full-genome sequences yield the better understanding of sub-genotypes, their variants and the degree of homology between strains isolated from mother-child pairs calls for effective strategies on prevention, control and surveillance of mother-to-child HBV transmission in Cambodia.


Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B/epidemiologia , Mutação , Adulto , Camboja/epidemiologia , Criança , Pré-Escolar , Teste em Amostras de Sangue Seco/métodos , Feminino , Genoma Viral , Genótipo , Hepatite B/transmissão , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/sangue , Humanos , Transmissão Vertical de Doença Infecciosa , Mães , Filogenia , Reação em Cadeia da Polimerase , Prevalência
12.
PLoS One ; 15(4): e0230968, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271796

RESUMO

BACKGROUND: Access to HIV viral load is crucial to efficiently monitor patients on antiretroviral treatment (ART) and prevent HIV drug resistance acquisition. However, in some remote settings, access to viral load monitoring is still complex due to logistical and financial constraints. Use of dried blood spots (DBS) for blood collection could overcome these difficulties. This study aims to describe feasibility and operability of DBS use for routine viral load monitoring. METHODS: From June 2017 to April 2018, HIV-infected adults who initiated ART were enrolled in a prospective cohort in 43 clinical sites across 6 provinces in North Vietnam. Following national guidelines, the first viral load monitoring was planned 6 months after ART initiation. DBS were collected at the clinical site and sent by post to a central laboratory in Hanoi for viral load measurement. RESULTS: Of the 578 patients enrolled, 537 were still followed 6 months after ART initiation, of which DBS was collected for 397 (73.9%). The median (inter quartile range) delay between DBS collection at site level and reception at the central laboratory was 8 (6-19) days and for 70.0% viral load was measured ≤30 days after blood collection. The proportion of patients with viral load ≥1000 copies/mL at the 6 month evaluation was 15.9% (n = 59). Of these, a DBS was collected again to confirm virological failure in 15 (24.4%) of which virological failure was confirmed in 11 (73.3%). CONCLUSION: Delay of DBS transfer to the central laboratory was acceptable and most viral loads were measured in ≤30 days, in-line with routine follow-up. However, the level of DBS coverage and the proportion of patients in failure for whom a confirmatory viral load was available were suboptimal, indicating that integration of viral load monitoring in the field requires, among other things, careful training and strong involvement of the local teams. The proportion of patients experiencing virological failure was in line with other reports; interestingly those who reported being non-adherent and those with a low BMI were more at risk of failure.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1 , Carga Viral , Adulto , Antirretrovirais/uso terapêutico , Estudos de Coortes , Farmacorresistência Viral , Estudos de Viabilidade , Feminino , Infecções por HIV/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Tempo , Falha de Tratamento , Vietnã/epidemiologia
13.
PLoS One ; 15(4): e0231385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287293

RESUMO

To facilitate HCV diagnosis, we developed an HCV-RNA testing service, which involved home-sampled dried blood spots (DBS). The main objective of this study was to evaluate the feasibility of self-sampling at home. Furthermore, to optimise the processing of DBS samples for RNA detection, we evaluated two elution buffers: phosphate-buffered saline (PBS) and L6-buffer. 27 HCV-RNA and 12 HIV-1 RNA positive patients were included. Laboratory spotted DBS (LabDBS) were made by a technician from blood samples drawn at inclusion. Patients received a DBS home-sampling kit and were requested to return their self-sampled DBS (ssDBS) by mail. We compared the RNA load of PBS and L6-eluted labDBS, and of L6-eluted ssDBS, L6-eluted labDBS and plasma. LabDBS load measurements were repeated after 7-13 and 14-21 days to evaluate RNA stability. All 39 plasma samples provided quantifiable RNA loads. In 1/39 labDBS sample, RNA could not be detected (plasma HCV load: 2.98 log10 IU/ml). L6-eluted samples gave a 0.7 log10 and 0.6 log10 higher viral load for HCV and HIV-1 respectively, compared to PBS-eluted samples. Strong correlations were found between labDBS and ssDBS HCV RNA (r = 0.833; mean difference 0.3 log10 IU/mL) and HIV-1 RNA results (r = 0.857; mean difference 0.1 log10 copies/mL). Correlations between labDBS and plasma values were high for HCV (r = 0.958) and HIV-1 (r = 0.844). RNA loads in DBS remained stable over 21 days. Our study demonstrates that self-sampling dried blood spots at home is a feasible strategy for the detection of HCV and HIV-1 RNA. This could facilitate one-step diagnostics and treatment monitoring in communities with high HCV prevalence.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Hepacivirus/genética , Hepatite C/diagnóstico , RNA Viral/sangue , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Estabilidade de RNA , RNA Viral/metabolismo , Kit de Reagentes para Diagnóstico , Manejo de Espécimes , Carga Viral
14.
BMC Infect Dis ; 20(1): 217, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164599

RESUMO

BACKGROUND: Congenital Cytomegalovirus (cCMV) is a serious global public health issue that can cause irreversible fetal and neonatal congenital defects in symptomatic or asymptomatic newborns at birth. In absence of universal cCMV screening, the retrospective diagnosis of cCMV infection in children is only possible by examining Dried Blood Spot (DBS) samples routinely collected at birth and stored for different time spans depending on the newborn screening regulations in force in different countries. In this article, we summarize the arguments in favor of long-term DBS sample storage for detecting cCMV infection. MAIN TEXT: CMV infection is the most common cause of congenital infection resulting in severe defects and anomalies that can be apparent at birth or develop in early childhood. Sensorineural hearing loss is the most frequent consequence of cCMV infection and may have a late onset and progress in the first years of life. The virological diagnosis of cCMV is essential for clinical research and public health practices. In fact, in order to assess the natural history of CMV infection and distinguish between congenital or acquired infection, children should be diagnosed early by analyzing biological samples collected in the first weeks of life (3 weeks by using viral culture and 2 weeks by molecular assays), which, unfortunately, are not always available for asymptomatic or mildly symptomatic children. It now seems possible to overcome this problem since the CMV-DNA present in the blood of congenitally infected newborns can be easily retrieved from the DBS samples on the Guthrie cards routinely collected and stored within 3 days from birth in the neonatal screening program for genetic and congenital diseases. Early collection and long-term storage are inexpensive methods for long-term bio-banking and are the key points of DBS testing for the detection of cCMV. CONCLUSION: DBS sampling is a reliable and inexpensive method for long-term bio-banking, which enables to diagnose known infectious diseases - including cCMV - as well as diseases not jet recognized, therefore their storage sites and long-term storage conditions and durations should be the subject of political decision-making.


Assuntos
Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/diagnóstico , Teste em Amostras de Sangue Seco/métodos , Perda Auditiva Neurossensorial/virologia , Humanos , Recém-Nascido , Triagem Neonatal/métodos , Estudos Retrospectivos
15.
Biochim Biophys Acta Mol Basis Dis ; 1866(6): 165725, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061778

RESUMO

PURPOSE: Newborns who test positive for very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) in newborn screening may have a severe phenotype with early onset of life-threatening symptoms but may also have an attenuated phenotype and never become symptomatic. The objective of this study is to investigate whether metabolomic profiles in dried bloodspots (DBS) of newborns allow early phenotypic prediction, permitting tailored treatment and follow-up. METHODS: A metabolic fingerprint was generated by direct infusion high resolution mass spectrometry in DBS of VLCADD patients (n = 15) and matched controls. Multivariate analysis of the metabolomic profiles was applied to differentiate subgroups. RESULTS: Concentration of six acylcarnitine species differed significantly between patients and controls. The concentration of C18:2- and C20:0-carnitine, 13,14-dihydroretinol and deoxycytidine monophosphate allowed separation between mild and severe patients. Two patients who could not be prognosticated on early clinical symptoms, were correctly fitted for severity in the score plot based on the untargeted metabolomics. CONCLUSION: Distinctive metabolomic profiles in DBS of newborns with VLCADD may allow phenotypic prognostication. The full potential of this approach as well as the underlying biochemical mechanisms need further investigation.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Carnitina/análogos & derivados , Síndrome Congênita de Insuficiência da Medula Óssea/sangue , Erros Inatos do Metabolismo Lipídico/sangue , Metabolômica , Doenças Mitocondriais/sangue , Doenças Musculares/sangue , Triagem Neonatal , Acil-CoA Desidrogenase de Cadeia Longa/sangue , Acil-CoA Desidrogenase de Cadeia Longa/genética , Carnitina/metabolismo , Criança , Pré-Escolar , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Teste em Amostras de Sangue Seco/métodos , Feminino , Humanos , Lactente , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/patologia , Masculino , Espectrometria de Massas , Doenças Mitocondriais/patologia , Doenças Musculares/patologia , Fenótipo
16.
Rapid Commun Mass Spectrom ; 34(7): e8601, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32043669

RESUMO

RATIONALE: Paper spray mass spectrometry (PS-MS) was used to analyze and quantify ampicillin, a hydrophilic compound and frequently utilized antibiotic. Hydrophilic molecules are difficult to analyze via PS-MS due to their strong binding affinity to paper substrates and low ionization efficiency, among other reasons. METHODS: Solvent and paper parameters were optimized to increase the extraction of ampicillin from the paper substrate. After optimizing these key parameters, a Resolution IV 1/16 fractional factorial design with two center points was employed to screen eight different design parameters simultaneously. RESULTS: Pore size, sample volume, and solvent volume were the most significant factors affecting average peak area under the curve (AUC) and the signal-to-blank (S/B) ratio for the 1 µg/mL ampicillin calibrant. After optimizing the key parameters, a linear calibration curve with a range of 0.2 µg/mL to 100 µg/mL was generated (R2  = 0.98) and the limit of detection (LOD) and lower limit of quantification (LLOQ) were calculated to be 0.07 µg/mL and 0.25 µg/mL, respectively. CONCLUSIONS: The statistical optimization procedure undertaken here increased the mass spectral signal intensity by more than a factor of 40. This statistical method of screening followed by optimization experiments proved faster and more efficient, and produced more drastic improvements than typical one-factor-at-a-time experiments.


Assuntos
Ampicilina/sangue , Antibacterianos/sangue , Ampicilina/análise , Antibacterianos/análise , Área Sob a Curva , Teste em Amostras de Sangue Seco/métodos , Humanos , Limite de Detecção , Espectrometria de Massas/métodos , Papel , Solventes/química
17.
Toxicol Lett ; 324: 65-74, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32035980

RESUMO

Paracetamol (acetaminophen, APAP) is the most frequently used analgesic drug worldwide. However, patients in several specific populations can have an increased exposure to toxic APAP metabolites. Therefore, APAP-protein adducts have been proposed as an alternative marker for the assessment of APAP intoxications and as an effective tool to study and steer APAP treatment in patients with an increased risk of APAP-induced liver damage. These adducts have been determined in plasma or serum as a matrix. Blood microsampling allows the determination of a variety of analytes, including protein adducts, in a drop of blood, facilitating convenient follow-up of patients in a home-sampling context, as well as repeated sampling of pediatric patients. We therefore evaluated the use of blood-based volumetric microsamples for the quantification of APAP-protein adducts. Quantitative methods for the determination of APAP-protein adducts in dried blood and dried plasma volumetric absorptive microsamples were developed and validated. Also a preliminary evaluation of pediatric patient dried blood microsamples was conducted. Method validation encompassed the evaluation of selectivity, carry over, calibration model, accuracy and precision, matrix effect, recovery and the effect of the hematocrit on the recovery, dilution integrity, and stability. All pre-set acceptance criteria were met, except for stability. Spiking of blank blood with APAP revealed a concentration-dependent ex vivo formation of APAP-protein adducts, resulting in a response for the measurand APAP-Cys, with an apparent role for the red blood cell fraction. Analysis of authentic samples, following intake of APAP at therapeutic dosing, revealed much higher APAP-Cys concentrations in dried blood vs. dried plasma samples, making interpretation of the results in the context of published intervals difficult. In addition, in contrast to what was observed during method validation, the data obtained for the patient samples showed a high and unacceptable variation. We conclude that, for a combination of reasons, dried blood is not a suitable matrix for the quantification of APAP-protein adducts via the measurement of the APAP-Cys digestion product. The collection of plasma or serum, either in the form of a liquid sample or a dried microsample for this purpose is advised.


Assuntos
Acetaminofen/análogos & derivados , Acetaminofen/sangue , Analgésicos não Entorpecentes/sangue , Teste em Amostras de Sangue Seco/métodos , Coleta de Amostras Sanguíneas , Cisteína/análogos & derivados , Cisteína/sangue , Hematócrito , Humanos , Ligação Proteica , Controle de Qualidade
18.
PLoS One ; 15(2): e0229352, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084225

RESUMO

INTRODUCTION: Heel pricks are performed on newborns for diagnostic screenings of various pre-symptomatic metabolic and genetic diseases. Excess blood is spotted on Guthrie cards and archived by many states in biobanks for follow-up diagnoses and public health research. However, storage environment may vary across biobanks and across time within biobanks. With increased applications of DNA extracted from spots for genetic studies, identifying factors associated with genotyping success is critical to maximize DNA quality for future studies. METHOD: We evaluated 399 blood spots, which were part of a genome-wide association study of childhood leukemia risk in children with Down syndrome, archived at the Michigan Neonatal Biobank between 1992 and 2008. High quality DNA was defined as having post-quality control call rate ≥ 99.0% based on the Illumina GenomeStudio 2.0 GenCall algorithm after processing the samples on the Illumina Infinium Global Screening Array. Bivariate analyses and multivariable logistic regression models were applied to evaluate effects of storage environment and storage duration on DNA genotyping quality. RESULTS: Both storage environment and duration were associated with sample genotyping call rates (p-values < 0.001). Sample call rates were associated with storage duration independent of storage environment (p-trend = 0.006 for DBS archived in an uncontrolled environment and p-trend = 0.002 in a controlled environment). However, 95% of the total sample had high genotyping quality with a call rate ≥ 95.0%, a standard threshold for acceptable sample quality in many genetic studies. CONCLUSION: Blood spot DNA quality was lower in samples archived in uncontrolled storage environments and for samples archived for longer durations. Still, regardless of storage environment or duration, neonatal biobanks including the Michigan Neonatal Biobanks can provide access to large collections of spots with DNA quality acceptable for most genotyping studies.


Assuntos
DNA/análise , Teste em Amostras de Sangue Seco/métodos , Genoma Humano , Estudo de Associação Genômica Ampla/métodos , Leucemia/diagnóstico , Triagem Neonatal/métodos , Feminino , Genótipo , Humanos , Recém-Nascido , Leucemia/genética , Masculino , Polimorfismo de Nucleotídeo Único , Manejo de Espécimes
19.
Curr Opin Pediatr ; 32(2): 300-307, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31913157

RESUMO

PURPOSE OF REVIEW: Exposomics studies can measure health-relevant chemical exposures during a lifetime and estimate the 'internal' environment. However, sampling limitations make these features difficult to capture directly during the critical neonatal time period. RECENT FINDINGS: We review the use of newborn dried bloodspots (DBS) archived from newborn screening programs for exposomic analysis in epidemiological children's health studies. Emerging 'omics technologies such as adductomics and metabolomics have been adapted for DBS analysis, and these technologies can now provide valuable etiological information on the complex interplay between exposures, biological response, and population phenotypes. SUMMARY: Adductomics and metabolomics of DBS can provide robust measurements for retrospective epidemiological investigations. With extensive bioarchiving programs in the United States and other countries, DBS are poised to substantially aid epidemiological studies, particularly for rare and low-frequency childhood diseases and disorders.


Assuntos
Adutos de DNA/análise , Teste em Amostras de Sangue Seco/métodos , Exposição Ambiental/efeitos adversos , Expossoma , Metabolômica , Criança , DNA/metabolismo , Predisposição Genética para Doença , Humanos , Recém-Nascido , Proteômica
20.
Artigo em Inglês | MEDLINE | ID: mdl-31891859

RESUMO

This study aims to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for vancomycin and creatinine measurement in dried blood spots (DBS) and to evaluate its clinical application. The analytes were extracted from DBS and analyzed by LC-MS/MS. Vancomycin and creatinine DBS and plasma concentrations were compared in 54 and 35 samples, respectively, from 29 patients. Accuracy was 94.4-102.6%, intra-assay precision was 2.1-5.6%, and inter-assay precision was 3.5-7.0%. Patients vancomycin plasma to DBS concentration ratios were highly variable (1.148-5.022), differently from creatinine (0.800-1.283). The assay has adequate analytical performance. Plasma concentrations can be satisfactorily predicted from DBS measurements for creatinine, but not for vancomycin, which limits its clinical application.


Assuntos
Creatinina/sangue , Teste em Amostras de Sangue Seco/métodos , Vancomicina/sangue , Adulto , Cromatografia Líquida/métodos , Humanos , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos
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