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PURPOSE: Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology. METHODS: An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation. RESULTS: The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified. CONCLUSION: The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.
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Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Triagem Neonatal/métodos , Análise de Sequência de DNA/métodos , Fibrose Cística/genética , Teste em Amostras de Sangue Seco , Estudos de Viabilidade , Predisposição Genética para Doença , Humanos , Recém-Nascido , Mutação , Estudos RetrospectivosRESUMO
Although inherited blood disorders are common among children in many parts of Africa, limited data are available about their prevalence or contribution to childhood anaemia. We conducted a cross-sectional survey of 858 children aged 6-35 months who were randomly selected from 60 villages in western Kenya. Haemoglobin (Hb), ferritin, malaria, C-reactive protein (CRP) and retinol binding protein (RBP) were measured from capillary blood. Using polymerase chain reaction (PCR), Hb type, -3.7 kb alpha-globin chain deletion, glucose-6-phosphate dehydrogenase (G6PD) genotype and haptoglobin (Hp) genotype were determined. More than 2 out of 3 children had at least one measured blood disorder. Sickle cell trait (HbAS) and disease (HbSS) were found in 17.1% and 1.6% of children, respectively; 38.5% were heterozygotes and 9.6% were homozygotes for α(+) -thalassaemia. The Hp 2-2 genotype was found in 20.4% of children, whereas 8.2% of males and 6.8% of children overall had G6PD deficiency. There were no significant differences in the distribution of malaria by the measured blood disorders, except among males with G6PD deficiency who had a lower prevalence of clinical malaria than males of normal G6PD genotype (P = 0.005). After excluding children with malaria parasitaemia, inflammation (CRP > 5 mg L(-1) ), iron deficiency (ferritin < 12 µg L(-1) ) or vitamin A deficiency (RBP < 0.7 µg L(-1) ), the prevalence of anaemia among those without α(+) -thalassaemia (43.0%) remained significantly lower than that among children who were either heterozygotes (53.5%) or homozygotes (67.7%, P = 0.03). Inherited blood disorders are common among pre-school children in western Kenya and are important contributors to anaemia.
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Anemia Falciforme/epidemiologia , Efeitos Psicossociais da Doença , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Malária/epidemiologia , Talassemia/epidemiologia , Anemia Falciforme/genética , Proteína C-Reativa/metabolismo , Pré-Escolar , Estudos Transversais , Feminino , Ferritinas/sangue , Deleção de Genes , Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/genética , Haptoglobinas , Hemoglobinas/metabolismo , Heterozigoto , Homozigoto , Humanos , Lactente , Quênia/epidemiologia , Malária/genética , Masculino , Prevalência , Proteínas de Ligação ao Retinol/metabolismo , Traço Falciforme/epidemiologia , Traço Falciforme/genética , Talassemia/genéticaRESUMO
BACKGROUND: Proficiency testing (PT) can have regulatory and nonregulatory uses, providing an effective tool for quality improvement. Clinical laboratories were surveyed to determine how they perceive PT and how they use PT results and materials to improve laboratory testing quality. METHODS: All laboratories certified to perform nonwaived testing under the CLIA regulations expected to perform required PT were invited to participate in the survey. We examined respondents' use of PT from 5 laboratory types: hospital, independent, public health, physician office, and "all other." Respondents' awareness of resources about PT was also examined. Several questions allowed responses on a categorical scale. RESULTS: Varying proportions of the respondents (n = 769) used PT to identify problems in the preanalytic (48%), analytic (86%), and postanalytic (76%) phases of testing. Responses also showed that PT was important for demonstrating personnel competency (93%), inappropriate specimen handling (80%), incorrect result interpretation (84%), and other uses. Respodents purchased PT even when not required to do so (77%). Based on all responses, most considered PT worth the cost (65%). CONCLUSIONS: Laboratories, regardless of type, have found ways of using leftover PT samples and the information from PT event summaries to help improve laboratory quality. Our findings suggest many laboratories are not taking full advantage of PT to improve testing quality. Additionally, the study suggests a need to improve awareness of resources about PT.
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BACKGROUND: Certain alleles among the genes that code for the human leukocyte antigens (HLA) confer susceptibility or resistance to the development of autoimmunity that causes type 1 diabetes (T1D). A number of ongoing diabetes research studies analyze dried blood spots (DBS) from newborn infants for HLA-D alleles to identify higher-risk children as early as possible. A commercially available assay to detect such alleles has recently become available using a dissociation- enhanced lanthanide fluorescence system found in many newborn screening laboratories. METHODS: We adapted the system for use with DBS and improved the sample set-up for greater efficiency. We also developed an independent system for data analysis based on a spreadsheet program. These modifications were applied to HLA-DQB1 gene locus (DQB) analysis of 117 newborn DBS, and the results we obtained were compared with independent reference values. RESULTS: Our assay modifications and independent data analysis improved sample throughput and result tabulation. DQB results from the modified assay were consistent with the reference values in all but one sample, which showed a partial match. CONCLUSIONS: The modifications described here make this commercially available assay more suitable for high-throughput applications such as newborn screening. Our results show that this system allows highly accurate detection of DQB alleles that influence T1D risk.
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Diabetes Mellitus Tipo 1/genética , Antígenos HLA-DQ/genética , Recém-Nascido/sangue , Triagem Neonatal/métodos , Análise Química do Sangue , Fluorometria/métodos , Marcadores Genéticos , Cadeias beta de HLA-DQ , Humanos , Triagem Neonatal/instrumentação , Sondas de Oligonucleotídeos , Fatores de RiscoRESUMO
OBJECTIVES: In response to increasing numbers of states in the US that test newborn babies for cystic fibrosis (CF), the Newborn Screening Quality Assurance Programme initiated a pilot proficiency testing programme for immunoreactive trypsinogen (IRT), the biomarker for CF. Dried blood spot specimens (DBS) were used to evaluate the performance of laboratories that screen babies for CF. METHODS: DBS were prepared from human whole blood enriched with physiologically relevant levels of IRT. Various methods of making IRT-enriched DBS were used to optimize the recovery and stability of the biomarker, including preparation of DBS from either intact or lysed red blood cells, varying the timing of IRT addition to blood before dispensing onto filter paper, adding a protease inhibitor cocktail, and treating serum with charcoal before IRT enrichment. The recovery and stability of IRT in DBS were assessed. Newborn screening laboratories were offered the opportunity to test blind-coded DBS in the pilot PT programme. RESULTS: IRT was stable in the filter paper matrix when stored for one year at either -20 degrees C or 4 degrees C. Fifty percent more IRT was recovered from DBS prepared with lysed red blood cells where the IRT was added to blood just before dispensing; however, protease inhibitors did not improve IRT recovery. CONCLUSIONS: IRT in the DBS matrix is stable and can be shipped worldwide under ambient conditions. Optimal IRT recovery was achieved by adjustment of DBS production practices. Laboratories receiving specimens accurately measured IRT by a variety of commercial and in-house methods.
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Fibrose Cística/diagnóstico , Triagem Neonatal/instrumentação , Triagem Neonatal/métodos , Tripsinogênio/química , Carvão Vegetal/farmacologia , Fibrose Cística/sangue , Humanos , Recém-Nascido , Programas de Rastreamento/métodos , Mutação , Projetos Piloto , Inibidores de Proteases/farmacologia , Controle de Qualidade , Manejo de Espécimes , Temperatura , Tripsinogênio/imunologia , Estados UnidosRESUMO
BACKGROUND: During an October 2005 algal bloom (i.e., a rapid increase or accumulation in the population of algae) off the coast of Nicaragua, 45 people developed symptoms of paralytic shellfish poisoning (PSP) and one person died. PSP in humans is caused by ingestion of saxitoxin, which is a neurotoxin often associated with shellfish contaminated by algal blooms. To explore the relationship between the algal bloom and human illnesses, we performed a case-control study of residents living in a coastal island. We administered a standardized clinical questionnaire, sampled locally harvested seafood and algae, and obtained urine samples for saxitoxin testing from symptomatic and asymptomatic persons. PSP case-patients were defined as island residents who developed at least one neurological symptom during the November 4-16 intoxication period. Seafood and algal samples were analyzed for saxitoxins using the receptor-binding assay and high-performance liquid chromatography. Two urine samples were analyzed for saxitoxins using a newly developed immunoassay. FINDINGS: Three shellfish and two algal samples tested positive for saxitoxins. Ten (9%) of 107 participants developed neurological symptoms during the specified time period and five required hospitalization. While 6 (67%) of 9 possible case-patients and 21 (21%) of 98 controls had eaten fish (p=0.008), all case-patients and 17 (17%) of controls had eaten clams (P<0.0001). The saxitoxin concentration in the urine of a hospitalized case-patient was 21 ng saxitoxin/g creatinine compared to 0.16 ng saxitoxin/g creatinine in the single control patient's urine. CONCLUSIONS: These findings suggest that a bloom of saxitoxin-producing algae resulted in saxitoxin accumulation in local clams and was responsible for the PSP intoxication.
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Proliferação Nociva de Algas , Intoxicação por Frutos do Mar/epidemiologia , Adolescente , Adulto , Criança , Estudos Transversais , Surtos de Doenças , Feminino , Humanos , Masculino , Nicarágua/epidemiologia , Alimentos Marinhos , Intoxicação por Frutos do Mar/etiologia , Adulto JovemRESUMO
BACKGROUND: CDC's Newborn Screening Quality Assurance Program collaborated with several U.S. Cystic Fibrosis Care Centers to collect specimens for development of a molecular CFTR proficiency testing program using dried-blood spots for newborn screening laboratories. METHODS: Adult and adolescent patients or carriers donated whole blood that was aliquoted onto filter paper cards. Five blind-coded specimens were sent to participating newborn screening laboratories quarterly. Proficiency testing results were evaluated based on presumptive clinical assessment. Individual evaluations and summary reports were sent to each participating laboratory and technical consultations were offered if incorrect assessments were reported. RESULTS: The current CDC repository contains specimens with 39 different CFTR mutations. Up to 45 laboratories have participated in the program. Three years of data showed that correct assessments were reported 97.7% of the time overall when both mutations could be determined. Incorrect assessments that could have lead to a missed case occurred 0.9% of the time, and no information was reported 1.1% of the time due to sample failure. CONCLUSIONS: Results show that laboratories using molecular assays to detect CFTR mutations are performing satisfactorily. The programmatic results presented demonstrate the importance and complexity of providing proficiency testing for DNA-based assays.
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Técnicas de Laboratório Clínico , Fibrose Cística/genética , Garantia da Qualidade dos Cuidados de Saúde , Adolescente , Adulto , Fibrose Cística/sangue , Genótipo , Humanos , Mutação , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: Residual dried blood spots (DBS) from newborn screening programs are often stored for years and are sometimes used for epidemiological studies. Because there is potential for DNA cross-contamination from specimen-to-specimen contact, we determined contamination levels following intentional contact and assessed archival DBS DNA degradation after storage in an uncontrolled environment. METHODS: DBS from healthy adult females were rubbed with DBS from healthy or cystic fibrosis (CF)-affected adult males. Total human and male DNA was measured from the female DBS. Contamination levels were assessed using short tandem repeats (STRs). Female DBS contaminated with CF male DNA containing the F508del were analyzed for presence of this mutation. Archival DBS DNA amplification efficiency was determined using STR analysis. RESULTS: Most female DBS were contaminated, however only one specimen showed an incomplete STR profile consistent with contaminating CF-affected male DNA. Further testing by CF mutation screening was negative. DNA extracted from archival DBS showed robust amplification (range 100 bp-320 bp). CONCLUSIONS: Lightly abrasive contact between DBS resulted in DNA cross-contamination. The contaminating DNA did not interfere in CF-mutation tests; however this should be determined for individual assays. Since DNA from archival DBS robustly amplifies, newborn DBS could provide an invaluable resource for public health studies.
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Fibrose Cística/sangue , DNA/sangue , Manejo de Espécimes , Adulto , Sequência de Bases , Bancos de Sangue , Fibrose Cística/genética , DNA/isolamento & purificação , Dano ao DNA , Feminino , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
To study genetic risk factors for common diseases, researchers have begun collecting DNA specimens in large epidemiologic studies and surveys. However, little information is available to guide researchers in selecting the most appropriate specimens. In an effort to gather the best information for the selection of specimens for these studies, we convened a meeting of scientists engaged in DNA banking for large epidemiologic studies. In this discussion, we review the information presented at that meeting in the context of recent published information. Factors to be considered in choosing the appropriate specimens for epidemiologic studies include quality and quantity of DNA, convenience of collection and storage, cost, and ability to accommodate future needs for genotyping. We focus on four types of specimens that are stored in these banks: (1) whole blood preserved as dried blood spots; (2) whole blood from which genomic DNA is isolated, (3) immortalized lymphocytes from whole blood or separated lymphocytes, prepared immediately or subsequent to cryopreservation; and (4) buccal epithelial cells. Each of the specimens discussed is useful for epidemiologic studies according to specific needs, which we enumerate in our conclusions.
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Bancos de Espécimes Biológicos/normas , DNA/sangue , DNA/isolamento & purificação , Linfócitos/sangue , Mucosa Bucal/citologia , Manejo de Espécimes/métodos , Bochecha , Criopreservação , Estudos Epidemiológicos , Genótipo , Humanos , Controle de QualidadeRESUMO
Multiplexed assays using fluorescent microspheres is an exciting technique that has been gaining popularity among researchers, particularly those in the public health field. Part of its popularity is due to its flexibility, as both immunoassays and oligonucleotide hybridization assays can be developed on this platform. This report summarizes a workshop held by the Centers for Disease Control and Prevention that discussed issues surrounding these assays and the Luminex 100 xMAP instrument. Topics included instrumentation, assay design, sample matrix and volume, quality control, and development of commercial applications.