Serological survey for IgG antibodies against SARS-CoV-2 from newborns and their mothers / Inquérito sorológico para anticorpos IgG contra SARS-CoV-2 de recém-nascidos e suas mães / Encuesta serológica de anticuerpos IgG contra el SARS-CoV-2 de recién nacidos y sus madres

This serological survey, conducted in five Brazilian municipalities, evaluated the use of dried blood spots (DBS), obtained from newborns and their mothers, to detect SARS-CoV-2 IgG antibodies. DBS were obtained from 4,803 neonates aged up to seven days and their mothers, both asymptomatic, at public health care clinics during newborn screening. DBS were processed by ELISA to detect IgG antibodies against SARS-CoV-2 nucleocapsid antigen. Mothers of seropositive neonates were interviewed about sociodemographic characteristics and clinical and laboratory antecedents. Non-satisfactory samples, dyads with incomplete data, and vaccinated mothers were excluded. Of the 1,917 DBS dyads samples analyzed, 14.7% of neonates showed IgG antibodies against SARS-CoV-2. Among seropositive neonates, 73.2% of their mothers were also seropositive. More than half of the mothers with seropositive neonates denied clinical or laboratory suspicion of COVID-19 during pregnancy. Suspicion occurred in the third trimester for 24.6% of the mothers. This study tested an innovative strategy to improve the understanding of COVID-19 antibody dynamics during pregnancy and suggests the feasibility of a universal serological survey in puerperal women and neonates.

Este inquérito sorológico, realizado em cinco municípios brasileiros, avaliou o uso de sangue seco em papel filtro (DBS), obtidas de recém-nascidos e suas mães, para detectar anticorpos IgG SARS-CoV-2. DBS foram obtidas de 4.803 neonatos com até sete dias de vida e suas mães, ambos assintomáticos, em unidades de saúde pública durante a triagem neonatal. DBS foram processadas ​​por ELISA para detectar anticorpos IgG contra o antígeno do nucleocapsídeo SARS-CoV-2. As mães de neonatos soropositivos foram entrevistadas quanto às características sociodemográficas e antecedentes clínicos e laboratoriais. Foram excluídas amostras insatisfatórias, díades com dados incompletos e mães vacinadas. Das 1.917 amostras analisadas, 14,7% dos neonatos apresentaram anticorpos IgG contra SARS-CoV-2. Entre os recém-nascidos soropositivos, 73,2% era filho de mulheres também soropositivas. Mais da metade das mães com recém-nascidos soropositivos negaram suspeita clínica ou laboratorial de COVID-19 durante a gravidez. A suspeita de COVID-19 ocorreu no terceiro trimestre para 24,6% das mães. Este estudo testou uma estratégia inovadora para melhorar a compreensão da dinâmica de anticorpos contra SARS-CoV-2 durante a gravidez e sugere a viabilidade de realização de um inquérito sorológico universal em puérperas e neonatos.

Esta encuesta serológica, realizada en cinco municipios brasileños, evaluó el uso de manchas de sangre seca (DBS), obtenidas de recién nacidos y sus madres, para detectar anticuerpos IgG contra el SARS-CoV-2. Se obtuvieron DBS de 4.803 recién nacidos de hasta siete días de edad y sus madres, ambos asintomáticos, en clínicas de salud pública durante el cribado neonatal. Las DBS se procesaron mediante ELISA para detectar anticuerpos IgG contra el antígeno de la nucleocápside del SARS-CoV-2. Se entrevistó a madres de recién nacidos seropositivos sobre características sociodemográficas y antecedentes clínicos y de laboratorio. Se excluyeron muestras no satisfactorias, díadas con datos incompletos y madres vacunadas. De las 1.917 muestras de díadas DBS analizadas, el 14,7 % de los recién nacidos mostró anticuerpos IgG contra el SARS-CoV-2. Entre los recién nacidos seropositivos, el 73,2% de sus madres también eran seropositivas. Más de la mitad de las madres con recién nacidos seropositivos negaron sospecha clínica o de laboratorio de COVID-19 durante el embarazo. La sospecha ocurrió en el tercer trimestre para el 24,6% de las madres. Este estudio probó una estrategia innovadora para mejorar la comprensión de la dinámica de anticuerpos de COVID-19 durante el embarazo y sugiere la viabilidad de una encuesta serológica universal en mujeres puérperas y recién nacidos.

Current Scenario and Future Direction of Newborn Screening and Management Program for Phenylketonuria in Bangladesh

Abstract Phenylketonuria (PKU) is a correctable inborn error of metabolism which causes lethal intellectual delay and neurobehavioral anomalies. A screening package, especially for early recognition can support to regulate the PKU process of most patients. New-born screening program in any country focuses at the earliest detection of inheritance deficiency disorders in order to avoid the most severe repercussion by appropriate medication. This screening program needs a concomitant diagnosis and involves additional clinical research. Strategies from developed countries recommend that new-born screening should be done as soon as possible after birth before hospital/clinic discharge because if detected later, it conveys to significantly increase in disability as well as morbidity. Although exact protocol differs among different countries, testing procedures for PKU should be followed universally recognized in the developed world. Unfortunately, new-born screening program in Bangladesh is in lying-in room or possibly in pilot study in particular hospital, because the health-care system is classically targeted mortality (like childbirth complications) and transmittable morbidities (such as COVID-19) but not inborn frailties. Although policies and management of childbirth complications have been successfully lowered infant and mother mortality rates, the number of disabled babies increased tremendously. The study aims to investigate the current status of new-born screening (NBS) program of PKU in the Rajshahi Division Bangladesh, and focus on future plans to manage with life-long treatment. The primary challenges such as financial support for newborn screening, publicity, should be identified and implemented for national PKU-NBS policy as a role model of Bangladesh for developing countries.

Current Status and Prospects of Screening for Newborn Hereditary Metaboolic Disease / 罕见病研究

Newborn screening is an effective measure for early detection and early treatment of rare genetic diseases. Among the three-level preventive measures to reduce birth defects, newborn screening has a significant preventive effect, and continues to develop with the advancement of new therapies and new technologies. Newborn screening is also relatively more reliable to obtain data on the prevalence of rare diseases. This article introduces the history and current status of neonatal screening for newborn hereditary metabolic disease in China, presents the disease spectrum and prevalence of 7 819 662 cases of neonatal screening by tandem mass spectrometry, and proposes 12 rare diseases as the primary targeting diseases for newborn screening by tandem mass spectrometry in China. At last, the article raises and discusses the issues of requirement for technology development and ethics of newborn screening.

Comparison between Enzyme Immunoassays Performed on Samples of Dried Blood and Serum for Toxoplasmosis Prenatal Screening: Population-based Study / Comparação entre ensaios imunoenzimáticos realizados em amostras de sangue seco e soro para triagem prénatal da toxoplasmose: Estudo populacional

Abstract Objective Most prenatal screening programs for toxoplasmosis use immunoassays in serum samples of pregnant women. Few studies assess the accuracy of screening tests in dried blood spots, which are of easy collection, storage, and transportation. The goals of the present study are to determine the performance and evaluate the agreement between an immunoassay of dried blood spots and a reference test in the serum of pregnant women from a population-based prenatal screening program for toxoplasmosis in Brazil. Methods A cross-sectional study was performed to compare the immunoassays Imunoscreen Toxoplasmose IgM and Imunoscreen Toxoplasmose IgG (Mbiolog Diagnósticos, Ltda., Contagem, Minas Gerais, Brazil)in dried blood spots with the enzymelinked fluorescent assay (ELFA, BioMérieux S.A., Lyon, France) reference standard in the serum of pregnant women from Minas Gerais Congenital Toxoplasmosis Control Program. Results The dried blood spot test was able to discriminate positive and negative results of pregnant women when comparedwith the reference test, with an accuracy of 98.2% for immunoglobulin G (IgG), and of 95.8% for immunoglobulin M (IgM). Conclusion Dried blood samples are easy to collect, store, and transport, and they have a good performance,making this a promisingmethod for prenatal toxoplasmosis screening programs in countries with continental dimensions, limited resources, and a high prevalence of toxoplasmosis, as is the case of Brazil.

Resumo Objetivo A maioria dos programas de triagem pré-natal para toxoplasmose utiliza imunoensaios em amostras de soro de gestantes. Poucos estudos avaliam a acurácia dos testes de triagem em amostras de sangue seco, que são de fácil coleta, armazenamento e transporte. Este estudo teve como objetivo determinar o desempenho e avaliar a concordância entre um imunoensaio em sangue seco e um teste de referência em soro de gestantes de um programa de rastreamento pré-natal de base populacional para toxoplasmose no Brasil. Métodos Realizou-se um estudo transversal para comparar os imunoensaios Imunoscreen Toxoplasmose IgM e Imunoscreen Toxoplasmose IgG (Mbiolog Diagnósticos, Ltda., Contagem, Minas Gerais, Brazil) em sangue seco com o padrão de referência ensaio fluorescente ligado a enzimas (enzyme-linked fluorescent assay, ELFA, BioMérieux S.A., Lion, França) no soro de gestantes do Programa de Controle de Toxoplasmose Congênita de Minas Gerais. Resultados O exame em sangue seco foi capaz de discriminar os resultados positivos e negativos das gestantes quando comparado ao teste de referência, com acurácia de 98,2% para imunoglobulina G (IgG), e de 95,8% para imunoglobulina M (IgM). Conclusão O sangue seco apresenta bom desempenho e é uma amostra de fácil coleta, armazenamento e transporte, o que o torna um método promissor para programas de triagem pré-natal de toxoplasmose em países com dimensões continentais, recursos limitados, e alta prevalência de toxoplasmose, como é o caso do Brasil.

Exploration of an Efficient Simultaneous Molecular Detection Method of HIV, HCV, and Syphilis from a Single Dried Blood Spot / 生物医学与环境科学（英文）

Objective@#The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma.@*Method@#A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma.@*Results@#Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log @*Conclusion@#The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.

Diagnóstico temprano de enfermedad de gaucher mediante detección de manifestaciones óseas / Early diagnosis of Gaucher disease based on bone symptoms

Resumen La enfermedad de Gaucher (EG) es causada por una deficiencia genética de la glucocerebrosidasa (GCasa) que provoca acumulación de glucocerebrósido en hígado, bazo y médula ósea. La terapia temprana de reemplazo enzimático revierte citopenias, visceromegalias y previene lesiones óseas irreversibles, por lo cual el diagnóstico precoz es fundamental. Los algoritmos diagnósticos en uso apuntan a manifestaciones hematológicas clásicas. Los síntomas óseos están presentes en 25-32% de los pacientes pero no suelen despertar sospecha de EG. Diseñamos un programa educativo sobre la afectación ósea de la EG y un algoritmo focalizado en la presentación con manifestaciones óseas para facilitar su diagnóstico precoz (proyecto BIG: Bone Involvement in Gaucher Disease). El objetivo del trabajo es describir el proyecto BIG y los resultados de su aplicación en nuestra consulta. Entre marzo de 2017 y diciembre de 2018 se recibieron 38 muestras de sangre seca de pacientes con alguna manifestación ósea sospechosa de EG para cuantificar la actividad de GCasa. Una muestra no cumplía los criterios de inclusión y en 3 de las 37 restantes se observó actividad deficiente de GCasa. El diagnóstico de EG se confirmó por medición de GCasa en leucocitos en dos niñas con manifestaciones óseas de 4 y 2 años de evolución, respectivamente, sin citopenia ni visceromegalia clínicamente evidentes. En el otro paciente con baja actividad la medición en leucocitos fue normal. Los casos detectados muestran la efectividad de un programa educacional de difusión y la utilidad de un algoritmo de detección precoz basado en síntomas óseos que facilitaría el diagnóstico de EG.

Abstract Gaucher disease (GD) is caused by a genetic deficiency of the lysosomal enzyme glucocerebrosidase (GCase) leading to the accumulation of glucocerebroside in the liver, spleen, and bone marrow. The early diagnosis allows a prompt enzyme replacement therapy reversing cytopenias and visceromegaly and preventing irreversible bone lesions. Current diagnostic algorithms are based on well-recognized hematological manifestations. Although bone symptoms are present in 25-32% of the patients, they are not usually suspected as associated with Gaucher disease at clinical presentation. We designed an educational program aimed to give advice on the skeletal involvement in GD and a new diagnostic algorithm that considers bone symptoms to facilitate its early diagnosis (BIG project: Bone Involvement in Gaucher Disease). The study aims at describing the BIG project and the results of its application in our clinic in various cities in Argentina. Within the frame of this project, between March 2017 and December 2018, 38 dry blood spot samples from patients with bone manifestations suspected of having GD were submitted to quantification of GCase activity. One sample did not meet the inclusion criteria. Deficient GCase activity was detected in three of the remaining 37 samples. The diagnosis of GD was confirmed in two girls who presented bone manifestations of 4 and 2 years of evolution, respectively, without hematological alterations. The third patient with low enzyme activity had normal leukocyte GCase. The two newly diagnosed cases of GD show the efficacy of our dual strategy aimed to facilitate the early diagnosis of this rare disease.

Molecular analysis of the CYP21A2 gene in dried blood spot samples

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.

La hiperplasia suprarrenal congénita (HSC) es un desorden autosómico recesivo producido por la deficiencia de alguna de las enzimas involucradas en la biosíntesis de cortisol. Más del 90% se debe a mutaciones en el gen CYP21A2 que genera deficiencia de 21 hidroxilasa (21OHD). Este gen se encuentra en el brazo corto del cromosoma 6 (6p21·3) y codifica para la enzima citocromo P450C21. Los programas de pesquisa neonatal detectan la forma clásica de la HSC-21OHD cuantificando 17OH-progesterona en gota de sangre en papel de filtro (GSPF). Este test es muy sensible, pero tiene baja especificidad , por lo que se utiliza una segunda muestra para confirmar el resultado. En estos casos, una segunda determinación en la misma muestra podría ser de utilidad. Nuestro objetivo fue evaluar el método de extracción de ADN y posterior análisis molecular del gen CYP21A2 en muestras de GSPF. Analizamos doce individuos presumiblemente afectados por HSC en la pesquisa neonatal usando ADN extraído de sangre fresca recolectada sobre EDTA y de GSPF. Realizamos el análisis del gen CYP21A2 mediante secuenciación automática de todos los exones y regiones intrónicas flanqueantes y MLPA en GSPF, y comparamos los resultados con ambos métodos de extracción. En este estudio demostramos que el ADN extraído de GSPF es una herramienta muy útil para analizar las mutaciones del gen CYP21A2 en la confirmación diagnóstica de 21-OHD para los programas de pesquisa neonatal y que los resultados son comparables con la genotipificación tradicional.

Analysis Of Doxorubicin And Doxorubicinol In Dried Blood Spot Of Breast Cancer Patients For Monitoring The Cardiotoxicity Of Doxorubicin

Objective: This study aimed to analyze doxorubicin and doxorubicinol levels in Dried Blood Spot (DBS) from 25 breast cancer patients who received doxorubicin in their therapeutic regiment. Methods: DBS samples were extracted by protein precipitation method and analyzed using Ultra Performance Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS), with the Acquity UPLC BEH C18 Waters chromatography column (2.1 x 100 mm x 1.7 μm). The mobile phase consisted of 0.1% acetic acid (eluent A) and acetonitrile (eluent B) with gradient elution; the flow rate was 0.15 ml/min and runtime, 7 min. This method was linear within the concentration range of 10–200 ng/ml for doxorubicin and 4–100 ng/ml for doxorubicinol. Result: The analysis results showed that doxorubicin levels were in the range of 11.01 ng/ml to 93.75 ng/ml and doxorubicinol was 5.80 ng/ml to 58.57 ng/ml. Conclusion: Cumulative doses of all patients were in the range of 49.11 mg/m2to 303.70 mg/m2, which have cardiomyopathy incidence rates<4%.

Effects of delivery and storage conditions on concentrations of amino acids and carnitines in neonatal dried blood spots / 浙江大学学报·医学版

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, @*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 @*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.

Hormona de crecimiento en sangre de papel filtro para el diagnóstico de deficiencia de hormona de crecimiento / Growth hormone of dried blood spot for the diagnosis of growth hormone deficiency

INTRODUCCIÓN: El diagnóstico de deficiencia de hormona de crecimiento (DHC) es difícil de establecer, y se puede asociar a serias complicaciones, especialmente en el período neonatal. La prueba de estímulo de secreción de hormona de crecimiento (HC) se considera de elección para el diagnóstico, pero presenta complicaciones metodológicas y se asocia a efectos adversos. Los neonatos presentan aumento de la secreción de HC de forma fisiológica, siendo una ventana diagnóstica. OBJETIVO: Evaluar si la muestra de sangre en papel filtro tomada en el período neonatal, en contexto del tamizaje neonatal de hipotiroidismo congénito y fenilcetonuria, permite diferenciar pacientes con DHC, de los que no la presentan. PACIENTES Y MÉTODO: Estudio de casos y controles mediante determinación de concentración de HC en sangre de papel filtro extraída en período neonatal, comparando controles con DHC con casos con deficiencia descartada. Se realizó extracción de la muestra del papel filtro, obteniendo dos discos de 0,125 pulgada por cada uno de los pacientes desde el centro de la mancha de sangre del papel, para un ELISA de HC humana altamente sensible basado en el uso de anticuerpos policlonales dirigidos contra la HC humana recombinante de 22kDa de peso molecular. RESULTADOS: Se obtuvo un total de 7 casos de DHC y 10 controles. La mediana de concentración de HC de papel filtro en los casos es 2,0 ng/ml (Rango intercuartil 3,6 ng/ml) y controles 2,05 ng/mL (RIC 2,0 ng/ml), U de Mann-Withney 30,5 (p = 0,68). Los dos casos con deficiencia de hormonas hipofisarias múltiples (DHHM) presentan concentraciones menores a 1 ng/ml. CONCLUSIÓN: La muestra de papel filtro no permitió diferenciar a los pacientes con DHC de los casos controles, aunque los casos con DHHM presentaron concentraciones mucho menores, en comparación a la deficiencia de hormona de crecimiento aislada (DHCA).

INTRODUCTION: The diagnosis of growth hormone deficiency (GHD) is difficult to determine, and could be associated with severe complications, especially in the neonatal period. The stimulation test of growth hormone (GH) secretion is considered the gold standard for diagnosis, but it has methodological complications and is associated with adverse effects. Neonates present physiological increased secretion of GH, representing a diagnostic window. OBJECTIVE: To evaluate if the dried blood spot on filter paper obtained in the neonatal period, as part of a neonatal screening for con genital hypothyroidism and phenylketonuria, allows differentiating patients with GHD from those who do not have it. PATIENTS AND METHOD: Study of cases and controls by measuring the GH concen tration in dried blood spot on filter paper obtained in the neonatal period, comparing controls with GHD with cases with discarded deficiency. The sample was extracted from the filter paper, obtaining two 0.125 inch discs per each patient from the center of the blood spot on the paper, for a highly sen sitive ELISA assay for human GH based on the use of polyclonal antibodies against 22 kDa recom binant human GH. RESULTS: Seven cases of GHD and ten controls were obtained. The median GH concentration of the dried blood spot in the cases is 2.0 ng/ml (Interquartile range 3.6 ng/ml) and 2.05 ng/ml (Interquartile range 2.0 ng/ml) in the controls, Mann-Whitney U test 30.5 (p = 0.68). The two cases with multiple pituitary-hormone deficiency (MPHD) present concentrations lower than 1 ng/ml. CONCLUSION: The dried blood spot sample did not differentiate GHD patients from control cases, although MPHD cases present much lower concentrations compared to isolated growth hor mone deficiency (IGHD).

Pseudodeficiency alleles affect the newborn screening of glycogen storage disease typeⅡ / 中华检验医学杂志

Objective To investigate the effect of pseudodeficiency alleles on the newborn screening of glycogen storage disease typeⅡ(GSDⅡ) by using afluorometric enzymatic assay to determine acidα-glucosidase (GAA) activity in dried blood spot (DBS). Methods A total of 30507 newborns' DBSs, obtained from Newborn Screening Center of Xinhua Hospital Shanghai Jiao Tong University School of Medicine from May to December 2017, were screened for GSDⅡby fluorometric enzymatic assay of GAA activity. The suspected positive DBSs after the first and second screening were directly analyzed by Sanger sequencing of GAA to confirm the diagnosis. Retrospective analysis of 3172 controls without GSDⅡand 36 GSDⅡpatients were conducted to investigate the carrier status of pseudodeficiency alleles. Statistical analysis of frequency of pseudodeficiency alleles were carried out by Chi-square test or Fisher exact probability test. Results GAA activity of 30507 newborns showed a positively skewed distribution. Twenty-nine cases of newborns, suspected to be GSDⅡwere confirmed to be normal with genetic analysis of the original DBSs. Among the 29 suspected positive cases, 24 cases were homozygous for pseudodeficiency alleles c. [1726A/A; 2065A/A], and the other 5 cases were c. [1726G/A; 2065G/A] heterozygote. The frequency of c. 1726G>Ahomozygote in 3172 non-GSDⅡcontrols was 2.08％(66/3172), and c. 1726G>A homozygote occurred in allelic conjunction with c. 2065G>Ahomozygote. Frequency of c. [1726A; 2065A] haplotype in 3172 controls was 3.2％(206/6344). Frequency of c. [1726A/A;2065A/A] homozygote in 36 GSDⅡpatients (16.67％, 6/36) was significantly higher than that in non-GSDⅡcontrols(2.08％, 66/3172) (χ2=34.517, P<0.001). Conclusions Pseudodeficiency alleles show a high frequency in Chinese, which leads to a high false positive rate in the newborns screening of GSDⅡ.The afterword genetic analysis of the original DBS after the GAA activity screening could reduce the effect of pseudodeficiency alleles on the newborns screening of GSDⅡ.

Pseudodeficiency alleles affect the newborn screening of glycogen storage disease typeⅡ / 中华检验医学杂志

Objective@#To investigate the effect of pseudodeficiency alleles on the newborn screening of glycogen storage disease type Ⅱ(GSDⅡ) by using afluorometric enzymatic assay to determine acid α-glucosidase (GAA) activity in dried blood spot (DBS).@*Methods@#A total of 30 507 newborns′ DBSs, obtained from Newborn Screening Center of Xinhua Hospital Shanghai Jiao Tong University School of Medicine from May to December 2017, were screened for GSD Ⅱ by fluorometric enzymatic assay of GAA activity. The suspected positive DBSs after the first and second screening were directly analyzed by Sanger sequencing of GAA to confirm the diagnosis. Retrospective analysis of 3 172 controls without GSDⅡand 36 GSD Ⅱ patients were conducted to investigate the carrier status of pseudodeficiency alleles. Statistical analysis of frequency of pseudodeficiency alleles were carried out by Chi-square test or Fisher exact probability test.@*Results@#GAA activity of 30 507 newborns showed a positively skewed distribution.Twenty-nine cases of newborns, suspected to be GSDⅡwere confirmed to be normal with genetic analysis of the original DBSs. Among the 29 suspected positive cases, 24 cases were homozygous for pseudodeficiency alleles c.[1726A/A; 2065A/A], and the other 5 cases were c.[1726G/A; 2065G/A] heterozygote. The frequency of c.1726G>Ahomozygote in 3 172 non-GSD Ⅱcontrols was 2.08% (66/3 172), and c.1726G>A homozygote occurred in allelic conjunction with c.2065G>Ahomozygote. Frequency of c.[1726A; 2065A] haplotype in 3 172 controls was 3.2%(206/6 344). Frequency of c.[1726A/A; 2065A/A] homozygote in 36 GSDⅡpatients (16.67%, 6/36) was significantly higher than that in non-GSD Ⅱcontrols(2.08%, 66/3 172) (χ2=34.517, P<0.001).@*Conclusions@#Pseudodeficiency alleles show a high frequency in Chinese, which leads to a high false positive rate in the newborns screening of GSDⅡ.The afterword genetic analysis of the original DBS after the GAA activity screening could reduce the effect of pseudodeficiency alleles on the newborns screening of GSDⅡ.

Initial evaluation of universal immunization with a single dose against hepatitis A virus in Central Brazil

ABSTRACT Vaccination against the hepatitis A virus (HAV) administered in two doses has been used effectively in universal child immunization programs in several countries. A single-dose vaccination was adopted in some low-income countries in an attempt to reduce costs without losing effectiveness. In 2014, single-dose universal vaccination was introduced in Brazil for children aged two years. Since such strategy is still not universally accepted, its efficacy should be compared to the two-dose strategy. To assess the humoral response after the single-dose HAV vaccination schedule, a cross-sectional study was conducted in Primavera do Leste, in Mato Grosso state, Central Brazil, including 265 children vaccinated through the National Immunization Program. Blood was collected by using a digital puncture and further applied to filter paper cards. Anti-HAV was detected in 218 out of 265 dried blood spots (DBS). Blood venous samples were collected from 34 out of 47 children who were not anti-HAV positive in DBS samples. Eighteen of them tested positive for anti-HAV, giving a final score of 93.6% (236/252) of seropositivity. In conclusion, this study demonstrated a high rate of anti-HAV positivity in the short term after single-dose hepatitis A vaccination in the population investigated. Moreover, the DBS was shown to be a reliable tool for detecting anti-HAV antibodies.

Detection of anti-hepatitis C virus and hepatitis C virus RNA in dried blood spot specimens using Whatman No. 1 filter paper

Purpose: Dried blood spot (DBS) specimen simplifies blood collection, processing, storage and shipment and may reduce the cost of testing for hepatitis C virus (HCV) infection. We wanted to see if DBS using a cheap filter paper is reliable alternative to serum for detection of anti-HCV and HCV RNA. Materials and Methods: At a tertiary care hospital in Northeast India, we collected 91 paired DBS and serum specimens from patients at risk of HCV infection from July 2014 to June 2015. DBS was collected on Whatman No. 1 filter paper. After processing, the specimens were subjected to anti-HCV detection by a third-generation Enzyme-Linked Immunosorbent Assay (ELISA). The reactive DBS and serum specimens were further subjected to HCV RNA detection by polymerase chain reaction. The results were analysed in paired screen-positive study design. Results: Anti-HCV was detected in 9 (9.9%) DBS specimens and 10 (10.9%) serum specimens. There was statistically significant (P < 0.0001) correlation between the optical density values of DBS and serum specimens (Pearson r = 0.9181, 95% confidence interval: 0.8781–0.9453). HCV RNA was detected in 5/9 (55.6%) reactive DBS and 9/10 (90.0%) reactive serum specimens. There was no correlation between HCV RNA levels in the DBS and the serum specimens. The relative sensitivity rate and the relative false-positive rate of DBS anti-HCV ELISA were 0.89 and 1.00, respectively. Conclusions: DBS using Whatman No. 1 filter paper is quite reliable as serum for detection of anti-HCV. It can be useful in effective surveillance. However, it is not suitable for confirmation of chronic HCV infection.

A New Integrated Newborn Screening Workflow Can Provide a Shortcut to Differential Diagnosis and Confirmation of Inherited Metabolic Diseases

PURPOSE: We developed a new workflow design which included results from both biochemical and targeted gene sequencing analysis interpreted comprehensively. We then conducted a pilot study to evaluate the benefit of this new approach in newborn screening (NBS) and demonstrated the efficiency of this workflow in detecting causative genetic variants. MATERIALS AND METHODS: Ten patients in Group 1 were diagnosed clinically using biochemical assays only, and 10 newborns in Group 2 were diagnosed with suspected inherited metabolic disease (IMD) in NBS. We applied NewbornDiscovery (SD Genomics), an integrated workflow design that encompasses analyte-phenotype-gene, single nucleotide variant/small insertion and deletion/copy number variation analyses along with clinical interpretation of genetic variants related to each participant's condition. RESULTS: A molecular genetic diagnosis was established in 95% (19/20) of individuals. In Group 1, 13 and 7 of 20 alleles were classified as pathogenic and likely pathogenic, respectively. In Group 2, 11 and 6 of 17 alleles with identified causative variants were pathogenic and likely pathogenic, respectively. There were no variants of uncertain significance. For each individual, the NewbornDiscovery and biochemical analysis results reached 100% concordance, since the single newborn testing negative for causative genetic variant in Group 2 showed a benign clinical course. CONCLUSION: This integrated diagnostic workflow resulted in a high yield. This approach not only enabled early confirmation of specific IMD, but also detected conditions not included in the current NBS.

High resolution melting real-time PCR detect and identify filarial parasites in domestic cats

Objective:To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR).Methods:A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected.Results:Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences.Conclusions:This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis.

Comparison of different methods for measuring the phenylalanine concentration of phenylketonuria patients in dried blood spots / 中华检验医学杂志

Objective To compare the phenylalanine(Phe)concentration in the sample of dried blood spot,measured by four different methods:fluorescence assay,tandem mass spectrometry(MS/MS), including MS/MS Derivatized,MS/MS Non-Derivatized and MS/MS-Standard Curve.Methods A total of 204 dried blood spot(DBS)samples of phenylketonuria(PKU)patients from Shanghai Xinhua Hospital were collected in this study.Phe concentration in DBS was measured by fluorescence assay and MS /MS assay,including MS/MS Derivatized, MS/MS Non-Derivatized and MS/MS-Standard Curve.The samples were divided into low, middle and high concentration groups according to Phe concentration, which were under the 360 μmol/L,between 360 and 600 μmol/L,and over 600 μmol/L respectively.The differences among the groups were analyzed by consistency check and non-parametric test.Results The within-day and between-day precisions of MS/MS-Standard Curve assay were 4.0%-7.2%,the recoveries were 93.2%-97.3%.Consistency check showed that less than 8.1%of Phe value were out of the 95%limit of agreement among these four methods.In the low and middle concentration groups,the quartile of Phe value measured by fluorescence assay,MS/MS Standard Curve and MS/MS Derivatized were 176(118,251)μmol/L,174 (94,273)μmol/L,153(94,242)μmol/L; and 540(478,578)μmol/L,485(414,529)μmol/L,466(402,513)μmol/L,respectively.Phe value measured by fluorescence assay was significantly higher than that by MS/MS Standard Curve,the difference was 4.8%-9.0%,(Z=-3.787 to -2.674,P<0.01). Phe value obtained from MS/MS Non-Derivatized was less than that by MS/MS-Standard Curve, the difference was 3.9%-5.2%,(Z=-7.474 to -5.747,P<0.01).In the high concentration group,the quartile of Phe value measured by MS/MS-Standard Curve, MS/MS Derivatized, MS/MS Non-Derivatized and fluorescence assay were 807(695,924)μmol/L,700(575,785)μmol/L,680(623,771)μmol/L and 711(674, 794)μmol/L, respectively.Phe value obtained from MS/MS Standard Curve was significantly higher than those from the other methods and the difference was 10.9% -16.2%,(Z=-4.458 to-4.356,P<0.01).Conclusions The MS/MS Standard Curve assay showed good specificity and accuracy.These four assays displayed good agreement.Although there was difference in measuring the Phe concentration among these methods, they could be used in blood Phe concentration monitoring for PKU patients.

Population screening of thalassemia using neonatal heel dried blood spot specimens on filter paper / 中华检验医学杂志

Objective To investigate the neonatal genetic carrier rate and genotype composition of thalassemia in Haikou city,and to evaluate the application of using dried blood spot specimens of neonates for population screening of thalassemia.Methods From January 2016 to December 2016,25% to 50% of all dried blood spot specimens of registered neonates were collected randomly by mechanical samping method in 30 hospitals of Haikou city everyday.Total of 6 864 samples were primarily analyzed by hemoglobin electrophoresis,and those with positive results were secondarily recalled for venous blood collection and subsequent genotyping tests.Feasibility of using neonatal dried blood spot specimens for population screening of thalassemia was retrospectively analyzed.Results A total of 6 864 neonatal dried blood spot specimens were analyzed.Out of 604 positive samples primarily screened by Hb electrophoresis, the positive rate was 8.80%,343 samples carried thalassemia gene mutations secondarily confirmed by genotyping test, the carrying rate was 5%(343/6 864),among which 81.92 %(281/343)were α-thalassemia,16.62%(57/343)were β-thalassemia and 1.46%(5/343)were both α-and β-thalassemia.In those α-thalassemia,the deleted α-gene types occupied 89.68%(252/281),the major genotype was --SEA/αα; the mutated α-gene types occupied 4.98%(14/281),the major genotype was αQS α/αα.In those β-thalassemia,there were 9 genotypes,the major genotype was CD41-42/N, occupying 61.40%(35/57).Conclusions The neonates have a relatively high carrying rate of thalassemia gene mutations in Haikou city, the genotype composition has geographical characteristics,and the major type is α-thalassemia.The method of using heel dried blood spot specimens on filter paper to screen thalassemia among neonates is advantageous and worthy of advocation.

High resolution melting real-time PCR detect and identify filarial parasites in domestic cats

Objective: To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR). Methods: A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected. Results: Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences. Conclusions: This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis.

Performance validation of western blot for anti-HIV antibody detection in blood samples collected on filter paper (DBS) / Validação de desempenho do western blot para detecção de anticorpos anti-HIV em amostras de sangue coletadas em papel-filtro (DBS)

ABSTRACT Introduction: For conducting field studies on human immunodeficiency virus (HIV) serodiagnosis, the use of samples collected on filter paper [dried blood spot (DBS)] is profitable owing to the simplicity in handling and delivering them. Because of these characteristics, the use of DBS is recommended for studies in which the goal is to increase the population access to diagnostic testing, including HIV infection screening. For HIV diagnosis, the conventional strategy firstly uses an enzyme immunoassay as screening test, and the positive sample is confirmed on a complementary assay, as western blotting (WB). Objective: This study aimed at evaluating the analytical performance of WB assay for analyzing DSB samples. Method: One hundred eighteen blood samples collected in filter paper were analyzed by a modified WB. These samples derived from the SampaCentro Study, and they were HIV-antibody-positive in a screening test. In order to assess the reliability of these results, the assay performance was previously certified by employing the sample panel (reference panel) of Instituto Adolfo Lutz (IAL), which consisted of whole blood and plasma paired samples. Results: All of the DBS samples [100% (118/118)] showed the band pattern compatible with the criterion for HIV positivity established by the Ministry of Health. To validate the WB testing, the reciprocal relationship between antibody reactivity of DBS samples and of the respective plasma samples was investigated. Conclusion: WB optimization allowed the achievement of accurate results, and the easy DBS sample collection, transportation and storage will support the use of this immunodiagnostic technique for conducting HIV/acquired immunodeficiency syndrome (Aids) epidemiological surveys.

RESUMO Introdução: Em estudos de campo é vantajoso o emprego de amostras coletadas em papel de filtro (DBS) pela simplicidade em manuseá-las e transportá-las. Essas características colocam a tecnologia de DBS em posição de destaque nas pesquisas que buscam aumentar o acesso da população aos testes diagnósticos, incluindo os testes sorológicos para vírus da imunodeficiência humana (HIV). A estratégia convencional para o diagnóstico da infecção pelo HIV é utilizar um teste imunoenzimático como primeiro teste e, quando este for reagente, um teste complementar, como o western blot (WB), para confirmar a reatividade da amostra. Objetivo: Neste estudo foi avaliado o desempenho analítico do ensaio WB em amostras DBS. Método: As 118 amostras de sangue coletadas em papel-filtro, provenientes do estudo SampaCentro e reagentes para anticorpos anti-HIV no ensaio de triagem, foram analisadas no WB modificado. Para conferir a confiabilidade a esses resultados, o desempenho do ensaio foi previamente validado por meio de painel de amostras do Instituto Adolfo Lutz (IAL) (painel de referência), composto por amostras pareadas de sangue total e plasma. Resultados: Todas as amostras DBS [100% (118/118)] apresentaram o padrão de bandas compatível com o critério de positividade para HIV preconizado pelo Ministério da Saúde. Na validação do WB, foi averiguada a reciprocidade da reatividade dos anticorpos das amostras DBS e de suas respectivas amostras de plasma. Conclusão: A otimização do WB possibilitou a obtenção de resultados confiáveis, e a facilidade de execução, coleta, transporte e armazenamento de amostra DBS poderá viabilizar a utilização dessa técnica imunodiagnóstica em inquéritos epidemiológicos de HIV/síndrome da imunodeficiência adquirida (Aids).