Dried Blood Samples Research Articles

A comprehensive HADHA c.1528G>C frequency study reveals high prevalence of long‐chain 3‐hydroxyacyl‐CoA dehydrogenase deficiency in Poland

Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is associated with c.1528G>C substitution in the HADHA gene, since most patients have the prevalent mutation on at least one allele. As it is known that the disease is relatively frequent in Europe, especially around the Baltic Sea, and that the majority of Polish LCHADD patients originate from the coastal Pomeranian province, partly inhabited by an ancient ethnic group, the Kashubians, we aimed to determine the carrier frequency of the prevalent HADHA mutation in various districts of Poland with special focus on the Kashubian district. A total of 6,854 neonatal dried blood samples from the entire country, including 2,976 Pomeranian neonates of Kashubian origin, were c.1528G>C genotyped. Fifty-nine heterozygous carriers for the prevalent c.1528G>C substitution (41 Pomeranian children) were detected in the studied group. Our data reveal a geographically skewed distribution of the c.1528C allele in the Polish population; in the northern Pomeranian province the carrier frequency is 1:73, which is the highest frequency ever reported, whereas in the remaining regions it is 1:217. Hence, the incidence of LCHADD in Poland is predicted to be 1:118,336 versus 1:16,900 in the Pomeranian district. Despite the relative rarity of the disease, screening for LCHADD in neonates born in the northern part of Poland, especially those of Kashubian origin, is justified. Our data allow us to suggest a probable Kashubian origin of the prevalent c.1528G>C mutation.

Neonatal Vitamin D Status and Risk of Schizophrenia

Clues from the epidemiology of schizophrenia suggest that low levels of developmental vitamin D may be associated with increased risk of schizophrenia. To directly examine the association between neonatal vitamin D status and risk of schizophrenia. Individually matched case-control study drawn from a population-based cohort. Danish national health registers and neonatal biobank. A total of 424 individuals with schizophrenia and 424 controls matched for sex and date of birth. The concentration of 25 hydroxyvitamin D(3) (25[OH]D3) was assessed from neonatal dried blood samples using a highly sensitive liquid chromatography tandem mass spectroscopy method. Relative risks were calculated for the matched pairs when examined for quintiles of 25(OH)D3. Compared with neonates in the fourth quintile (with 25[OH]D3 concentrations between 40.5 and 50.9 nmol/L), those in each of the lower 3 quintiles had a significantly increased risk of schizophrenia (2-fold elevated risk). Unexpectedly, those in the highest quintile also had a significantly increased risk of schizophrenia. Based on this analysis, the population-attributable fraction associated with neonatal vitamin D status was 44%. The relationship was not explained by a wide range of potential confounding or interacting variables. Both low and high concentrations of neonatal vitamin D are associated with increased risk of schizophrenia, and it is feasible that this exposure could contribute to a sizeable proportion of cases in Denmark. In light of the substantial public health implications of this finding, there is an urgent need to further explore the effect of vitamin D status on brain development and later mental health.

Detection of HBV-DNA in Dried Bloodstains on Filter Paper by Nested Polymerase Chain Reaction

To describe the technical performance of nested PCR for identifying hepatitis B viral (HBV) DNA. Hepatitis B viral DNA was extracted from a dried bloodstain on filter paper by a Chelex-100 method. Then the DNA fragment was amplified by nested polymerase chain reaction (PCR). The sensitivity and specificity of this method were also analyzed. The positive rate of the nested PCR-based method was compared with that of the enzyme-linked immunosorbent assays (ELISA) method. The lowest detection limit of the test was 5 copies of HBV DNA per μL by this method. McNemar’s test showed that the difference between the positive rates of these 2 methods was not statistically significant (P=0.289, P>0.05). Hepatitis B viral test results showed a good concordance between these 2 methods (kappa=0.727). A very small amount of the dried blood sample is required for the detection, which could overcome the shortage of the normal ELISA method that requires a relatively large amount of fresh blood samples.

Postprandial changes of amino acid and acylcarnitine concentrations in dried blood samples

Blood sampling for newborn screening cannot be standardized as for example blood collection in adults after an overnight fast. Therefore the influence of postprandial changes and individual variation is valuable information for the assessment of sensitivity and specificity of newborn screening for certain disorders. We have analyzed 92 pairs of dried blood samples taken pre- and one hour postprandially, respectively. We have determined the mean increase in metabolite concentration and calculated its significance. Individual variation after an overnight fast in healthy adults (n = 3) was between 12 and 32% (SD). Postprandial increases of acylcarnitines were mostly not significant and not exceeding 10%. Postprandial increase of amino acids was highly significant for most proteinogenic amino acids, but not for all. With the collected data we were able to estimate that mainly decreased levels of methionine and, to a lesser extent, of free carnitine could be "masked" by postprandial increases of the respective metabolites, and could therefore lead to false negative results for the detection of disorders of cobalamin metabolism and carnitine transporter deficiency.

Newborn screening for hemoglobinopathies using capillary electrophoresis technology: Testing the Capillarys® Neonat Fast Hb device

Objectives To diagnose hemoglobinopathies in newborns by separating and measuring the Hb fractions on high throughput capillary electrophoresis. To test and validate the Capillarys Neonat Fast Hb device (Sebia) on fresh and dry blood samples. Design and methods The Hb fractions in 1.600 cord blood samples from the multi ethnic Dutch population were separated and measured. Further, the sensitivity, specificity and reproducibility of the device in detecting abnormalities and measuring the Hb fractions were estimated. Results The apparatus separated all significant Hb fractions that should be detected during newborn screening (NBS) with 100% sensitivity. The reproducibility of the migrations guaranteed putative specificity for the few relevant frequent variants observed (HbS, C, and E). The estimation of the HbA and F fractions proved reliable using a well-designed integration mode. Discussion Due to the limited number of samples no cases with sickle cell disease or β-thalassemia major were found in this cohort. However, the heterozygous state for the common variants associated with these diseases was clearly recognizable. The measurements were sufficiently precise to recognize sickle cell disease, β-thalassemia major and intermedia and to identify carriers including possible β-thalassemia. Therefore, Capillarys Neonat Fast Hb (Sebia) can be considered as a valid instrument for NBS of the Hemoglobinopathies on fresh and dry blood samples.

Rapid quantification of conjugated and unconjugated bile acids and C27 precursors in dried blood spots and small volumes of serum

The aim of the study was to develop a method for fast and reliable diagnosis of peroxisomal diseases and to facilitate differential diagnosis of cholestatic hepatopathy. For the quantification of bile acids and their conjugates as well as C(27) precursors di- and trihydroxycholestanoic acid (DHCA, THCA), in small pediatric blood samples we combined HPLC separation on a reverse-phase C18 column with ESI-MS/MS analysis in the negative ion mode. Analysis was done with good precision (CV 3,7%-11.1%) and sufficient sensitivity (LOQ: 11-91 nmol/L) without derivatization. Complete analysis of 17 free and conjugated bile acids from dried blood spots and 10 microL serum samples, respectively, was performed within 12 min. Measurement of conjugated primary bile acids plus DHCA and THCA as well as ursodeoxycholic acid was done in 4.5 min. In blood spots of healthy newborns, conjugated primary bile acids were found in the range of 0.01 to 2.01 micromol/L. Concentrations of C(27) precursors were below the detection limit in normal controls. DHCA and THCA were specifically elevated in cases of peroxysomal defects and one Zellweger patient.

Genotypic testing for HIV-1 drug resistance using dried blood samples

In third-world countries, dried blood samples (DBS) are a convenient alternative to plasma for monitoring viral load during HIV-1 therapy. In this study, we evaluated the feasibility of using DBS to perform HIV-1 drug resistance genotyping in a ViroSeq assay in which the protease and reverse transcriptase regions of the pol gene are analyzed. Fifty-seven antiretroviral genotypes from plasma samples were tested, and drug resistance genotypes were determined. Only 38.6% paired DBS samples were sequenced. Failure to amplify DNA from DBS samples generally correlated with plasma viral loads below log(10) 5.1. The majority of the mutations identified in plasma pol sequences were also found in their DBS counterpart, with a concordance in genotype interpretation of 96.4%. Several factors were identified that could potentially improve both the sensitivity and the quality of genotype data, such as sample storage conditions and sequence analysis. Therefore, DBS sampling is useful to determine viral load and drug resistance genotypes in HIV patients.

Lessons from 30 years of selective screening for tetrahydrobiopterin deficiency

In addition to being hyperphenylalaninemic, patients lacking tetrahydrobiopterin (BH4) are deficient in the neurotransmitters whose synthesis depends on the normal activity of tetrahydrobiopterin-dependent tyrosine and tryptophan hydroxylases. Consequently, these patients have to be rapidly recognized among hyperphenylalaninemic babies, since they need specific and early substitutive therapy. Since 1980, BH4 metabolism has been investigated in 2,186 hyperphenylalaninemic babies, using HPLC measurement of pteridines in urine to recognize tetrahydrobiopterin synthesis deficiency (GTP cyclohydrolase and PTPS deficiency) and direct DHPR assay in dried blood samples to recognize DHPR deficiency. A total of 73 tetrahydrobiopterin deficient patients have been detected. Considering the group of neonates born in France (1,342), out of the 32 BH4 deficient patients which have been detected, only 8 were from caucasian families. The lessons from that experience are: (1) tests on blood and urine collected on filter paper cards commend itself by their convenience and simplicity, and samples can be collected on the first visit of the screened infants to the out-patient clinic; and (2) the preconceaved idea that newborns with moderate elevation of blood phenylalanine are false positives of the screening or mild forms of hyperphenylalaninemia explains that a significant number of cases were investigated after 1month of age; however, in half of BH4-deficient babies, blood phenylalanine was below 10mg/dl (0.6mmol/l).

Geographic distribution of human T-lymphotropic virus types 1 and 2 among mothers of newborns tested during neonatal screening, Minas Gerais, Brazil

To evaluate the geographic distribution of human T-lymphotropic virus types 1 and 2 (HTLV-1/2) in the State of Minas Gerais, Brazil, in puerperal women whose newborns were tested for HTLV-1/2 during neonatal screening, and to overlap seropositivity with social and economic status determinants. During September-November 2007, the dry-blood samples taken from newborns on filter paper for routine screening were also tested for maternal IgG anti-HTLV-1/2 antibodies. For reactive samples, the mothers of the newborns had blood drawn to test for these viruses. The study analyzed 55,293 specimens taken from newborns. Of these, 52 (9.4 per 10,000) were reactive and 42 mothers (7.6 per 10,000) were confirmed with HTLV-1/2 infection. HTLV-1/2 geographic distribution was heterogeneous, with a tendency to be higher in the North and North-East parts of Minas Gerais. The highest rates of seropositivity were observed in Vale do Mucuri (55.9 per 10,000) and in Jequitinhonha (16.0 per 10,000), overlapping with the State's worst social and economic indicators. To our knowledge this was the first time that neonatal screening for HTLV-1/2 was performed in Brazil. This model could be used in other areas with high HTLV-1/2 prevalence rates. The detection of carrier mothers can enable intervention measures, such as providing infant formula to newborns, to be implemented expeditiously to reduce vertical transmission.

Laboratory testing of individuals with severe α1-antitrypsin deficiency in three European centres

alpha(1)-Antitrypsin (AT) deficiency is a hereditary disorder that may lead to early-onset emphysema, and chronic liver disease later in life. Although there are validated methods for testing, the vast majority of alpha(1)-AT-deficient individuals remain undiagnosed. Recommendations have been published for the testing and diagnosis of alpha( 1)-AT deficiency; however, guidelines on best practice are not well established. In our article, we review the developments in diagnostic techniques that have taken place in recent years, and describe the practices used in our three European centres. The determination of the level of alpha(1)-AT and genotyping are reported as the main diagnostic steps, whereas isoelectric focusing (also referred to as phenotyping) is reserved for confirmatory analysis. The following recommendations for best practice are put forward: detection of all PiZZ and other severe deficiency individuals; automated genotyping; preparation of reference standards; quality control programmes; development of standard operating procedure documents; and standardised methods for the collection of dried blood samples. Closer cooperation between laboratories and the sharing of knowledge are recommended, with the objectives of improving the efficiency of the diagnosis of severe alpha(1)-AT deficiency, increasing the numbers of individuals who are detected with the disorder, and assisting the establishment of new patient identification programmes.

The first case of mitochondrial acetoacetyl‐CoA thiolase deficiency identified by expanded newborn metabolic screening in Italy: the importance of an integrated diagnostic approach

A pilot expanded newborn screening programme to detect inherited metabolic disorders by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) began in the Campania region, southern Italy, in 2007. By October 2009, >8,800 dried blood samples on filter paper from 11 hospitals had been screened. Within this screening programme, we identified a case of mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency [β-ketothiolase (β-KT) deficiency] by analysing the acylcarnitine profile from a dried blood spot with LC-MS/MS. Gas chromatography coupled with mass spectrometry analysis of urinary organic acids and LC-MS/MS analysis of urinary acylcarnitines were in line with this disorder. In fact, concentrations were well beyond the cut-off values of tiglyl carnitine, 3-hydroxybutyrylcarnitine and 2-methyl-3-hydroxybutyrylcarnitine, 2-methyl-3-hydroxybutyric acid and tiglyl glycine. The absence of 2-methylacetoacetic acid in urine may be attributed to: (i) the instability of this β-ketoacid because it undergoes spontaneous decarboxylation to 2-butanone, which is highly volatile and thus difficult to detect, and (ii) the good health of the patient in the first days of life. β-KT deficiency was subsequently diagnosed in the patient's older sister, who showed increased levels of the same metabolites but also small amounts of 2-methylacetoacetic acid, which is considered a key marker for β-KT diagnosis. Genomic analysis revealed mutation c.1189C >G in exon 12 of the ACAT1 gene, which results in a severe defect because of the p.H397D amino acid change in both alleles of both patients.

Pp65 antigenemia, plasma real-time PCR and DBS test in symptomatic and asymptomatic cytomegalovirus congenitally infected newborns

BackgroundMany congenitally cytomegalovirus-infected (cCMV) neonates are at risk for severe consequences, even if they are asymptomatic at birth. The assessment of the viral load in neonatal blood could help in identifying the babies at risk of sequelae.MethodsIn the present study, we elaborated the results obtained on blood samples collected in the first two weeks of life from 22 symptomatic and 48 asymptomatic newborns with cCMV diagnosed through urine testing. We evaluated the performances of two quantitative methods (pp65 antigenemia test and plasma Real-time PCR) and the semi-quantitative results of dried blood sample (DBS) test in the aim of identifying a valid method for measuring viral load.ResultsPlasma qPCR and DBS tests were positive in 100% of cases, antigenemia in 81%. Only the latter test gave quantitatively different results in symptomatic versus asymptomatic children. qPCR values of 103 copies/ml were found in 52% of newborn. "Strong" DBS test positivity cases had higher median values of both pp65 positive PBL and DNA copies/ml than cases with a "weak" positivity.ConclusionsAs expected antigenemia test was less sensitive than molecular tests and DBS test performed better on samples with higher rates of pp65 positive PBL and higher numbers of DNA copies/ml. The prognostic significance of the results of these tests will be evaluated on completion of the ongoing collection of follow-up data of these children.

Frequency of Fabry disease in male and female haemodialysis patients in Spain

BackgroundFabry disease (FD), an X-linked lysosomal storage disorder, is caused by a reduced activity of the lysosomal enzyme α-galactosidase A. The disorder ultimately leads to organ damage (including renal failure) in males and females. However, heterozygous females usually present a milder phenotype with a later onset and a slower progression.MethodsA combined enzymatic and genetic strategy was used, measuring the activity of α-galactosidase A and genotyping the α-galactosidase A gene (GLA) in dried blood samples (DBS) of 911 patients undergoing haemodialysis in centers across Spain.ResultsGLA alterations were found in seven unrelated patients (4 males and 3 females). Two novel mutations (p.Gly346AlafsX347 and p.Val199GlyfsX203) were identified as well as a previously described mutation, R118C. The R118C mutation was present in 60% of unrelated patients with GLA causal mutations. The D313Y alteration, considered by some authors as a pseudo-deficiency allele, was also found in two out of seven patients.ConclusionsExcluding the controversial D313Y alteration, FD presents a frequency of one in 182 individuals (0.55%) within this population of males and females undergoing haemodialysis. Moreover, our findings suggest that a number of patients with unexplained and atypical symptoms of renal disease may have FD. Screening programmes for FD in populations of individuals presenting severe kidney dysfunction, cardiac alterations or cerebrovascular disease may lead to the diagnosis of FD in those patients, the study of their families and eventually the implementation of a specific therapy.

Policy Issues and Stakeholder Concerns Regarding the Storage and Use of Residual Newborn Dried Blood Samples for Research

Newborn screening is an important public health programs in the United States. Over 4 million infants are screened each year for a number of conditions. There is a growing need for more explicit state policies governing the storage and research use of residual newborn samples. This paper provides an overview of newborn screening and issues related to policies of residual newborn samples as well as attitudes and opinions from stakeholders. Three groups (n = 21) were conducted with stakeholders: an African American group, a Pediatrician group and a Mothers of young children group. Despite the differences between these groups, consistent themes emerged from all groups that may be relevant for policy development governing the storage and use of residual newborn samples. The data from this exploratory study suggest that future policy developments with the newborn screening program warrant further public input on these topics.

Potential of Dried Blood Self-Sampling for CyclosporineC2Monitoring in Transplant Outpatients

Background. Close therapeutic drug monitoring of Cyclosporine (CsA) in transplant outpatients is a favourable procedure to maintain the long-term blood drug levels within their respective narrow therapeutic ranges. Compared to basal levels (C0), CsA peak levels (C2) are more predictive for transplant rejection. However, the application of C2 levels is hampered by the precise time of blood sampling and the need of qualified personnel. Therefore, we evaluated a new C2 self-obtained blood sampling in transplant outpatients using dried capillary and venous blood samples and compared the CsA levels, stability, and clinical practicability of the different procedures. Methods. 55 solid organ transplant recipients were instructed to use single-handed sampling of each 50 μL capillary blood and dried blood spots by finger prick using standard finger prick devices. We used standardized EDTA-coated capillary blood collection systems and standardized filter paper WS 903. CsA was determined by LC-MS/MS. The patients and technicians also answered a questionnaire on the procedure and sample quality. Results. The C0 and C2 levels from capillary blood collection systems (C0 [ng/mL]: 114.5 ± 44.5; C2: 578.2 ± 222.2) and capillary dried blood (C0 [ng/mL]: 175.4 ± 137.7; C2: 743.1 ± 368.1) significantly (P < .01) correlated with the drug levels of the venous blood samples (C0 [ng/mL]: 97.8 ± 37.4; C2: 511.2 ± 201.5). The correlation at C0 was ρ cap.-ven. = 0.749, and ρ dried blood-ven = 0.432; at C2: ρ cap.-ven. = 0.861 and ρ dried blood-ven = 0.711. The patients preferred the dried blood sampling because of the more simple and less painful procedure. Additionally, the sample quality of self-obtained dried blood spots for LC-MS/MS analytics was superior to the respective capillary blood samples. Conclusions. C2 self-obtained dried blood sampling can easily be performed by transplant outpatients and is therefore suitable and cost-effective for close therapeutic drug monitoring.

Measles in Papua New Guinea: An age-specific serological survey

We aimed to determine the proportion of the population in Madang (Papua New Guinea) immune to measles infection by age groups, with respect to immunization status and study location, using dried blood sampling technology. We performed a prospective cross-sectional sero-survey. Population immunity against measles was sub-optimal (77%) and reported measles vaccine coverage in children <10 years of age was low (41%). The urban population was more susceptible to measles infection, compared with the rural population (66% vs 79% immune, aOR = 0.6, p = 0.05). Sero-conversion and long term protection rates appeared to be higher when at least one dose of vaccine was provided at or after 12 months of age (84% vs 59%, aOR = 4.3, p = 0.004). Such a dose is, however, not currently prescribed by the national immunization schedule.

Determination of phenylalanine and tyrosine in plasma and dried blood samples using HPLC with fluorescence detection

The determination of phenylalanine and tyrosine is presently the most reliable direct approach to the diagnosis of phenylketonuria. An HPLC method for the simultaneous measurement of phenylalanine and tyrosine in samples of dried blood spots and plasma has been developed and evaluated. We have used an inherent fluorescence of both phenylalanine and tyrosine. For the separation, a reverse-phase column LiChroCart 125-4, Purospher RP-18e, 5 μm, was used. The mixture of ethanol and deionized water (5:95, v/v) was used as a mobile phase. Analytical performance of this method is satisfactory for both phenylalanine and tyrosine: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma and blood samples were between 92.0 and 102.9%. The limit of detection was 10.0 and 5.0 μmol/L, respectively. The preliminary reference ranges of phenylalanine and tyrosine in a group of newborns are 69.3 ± 13.1 and 42.7 ± 12.9 μmol/L, in a group of blood donors are 68.4 ± 9.9 and 52.1 ± 10.9 μmol/L. The presented method is inexpensive and suitable for diagnosis of phenylketonuria.

Cytomegalovirus DNA Detection in Guthrie Cards

Cytomegalovirus (CMV) infection is the leading cause of congenital nongenetic sensorineural hearing loss (SNHL) and a major cause of prelingual SNHL that is not present at birth. Polymerase chain reaction (PCR) analysis of dried blood samples on the Guthrie card has been proposed as a sensitive and specific method to screen for congenital CMV infection. Prospectively, consecutive infants who failed universal neonatal hearing screening and children referred for a noncongenital SNHL (NCHL) were included and underwent a standard audiometric and etiologic work-up. DNA was extracted from dried blood spots on neonatal Guthrie cards and amplified by real-time PCR. Data were available for 96 cases. Mean age of the universal neonatal hearing screening group was 3.8 +/- 2.4 months (n = 41). Auditory brain stem response thresholds were 72.9 +/- 20.2 dB nHL. A CMV-positive PCR was obtained in 4 babies. One test was considered false-positive. This resulted in a 7.3% prevalence of congenital CMV infections.Mean age of the NCHL group was 4.9 +/- 3.2 years (n = 55). Hearing loss was moderate in 37, severe in 5, and profound in 13 children. A CMV-positive PCR was obtained in 4 children (7.3%). Other causes of SNHL were excluded in the PCR positive cases of both study groups. We advocate PCR for CMV DNA detection on Guthrie cards in the etiologic work-up of childhood SNHL and recommend serologic confirmation to exclude false-positive PCR results. 7.3% of SNHL in babies with congenital hearing loss and children with NCHL could be attributed with this technique to congenital CMV infection.

Use of dried blood samples for monitoring hepatitis B virus infection

BackgroundHepatitis B virus (HBV) infection is a problem in several regions of the world with limited resources. Blood samples dried on filter paper (DBS) have been successfully used to diagnose and monitor several infectious diseases. In Mexico there is an urgent need for an affordable and easy sampling method for viral load (VL) testing and monitoring of chronic HBV infection. The purpose of this work was to validate the utility of DBS samples for monitoring HBV infection in patients from Mexico City.MethodsMatched samples of plasma and DBS on filter paper from 47 HBV infected patients from the Instituto Mexicano del Seguro Social (IMSS), were included. To evaluate the DNA stability and purity from DBS stored at different temperature conditions, samples from ten patients were stored at 4 degree, 25 degree, and 37 degree C for 7 days. After DBS elution and DNA extraction, the purity of these samples was determined measuring the O.D. rate 260/280. The DBS utility for molecular studies was assessed with PCR assays to amplify a 322 bp fragment from the "a" determinant region of the HBV "S" gene. The VL from all samples was determined to evaluate the correlation between plasma and DBS matched samples.ResultsThe quality of the DNA from DBS specimen is not adversely affected by storage at 4 degree, 25 degree and 37 degree C for up 7 days. Statistical ANOVA analyses did not show any significant difference. The same amplification efficiency was observed between DNA templates from samples stored at different temperatures. The Pearson correlation between the VL from DBS and plasma matched samples was 0.93 (p = 0.01). The SD was 1.48 for DBS vs.1.32 for Plasma, and an average of log10 copies/mL of 5.32 vs. 5.53. ANOVA analysis did not show any statistically significant difference between the analyzed groups (p = 0.92).ConclusionThe results provide strong evidence that the isolation and quantification of DNA-HBV from DBS is a viable alternative for patient monitoring, and molecular characterization of the virus variants circulating in Mexico.

Congenital Toxoplasmosis in Southeastern Brazil: Results of Early Ophthalmologic Examination of a Large Cohort of Neonates

To report results of early ophthalmologic examinations in a large cohort of newborns with congenital toxoplasmosis (CT) after neonatal screening. Cross-sectional analysis of a cohort. A total of 178 newborns with confirmed CT from 146,307 screened babies (95% of live births) from Minas Gerais state, southeastern Brazil. From November 2006 to May 2007, newborns underwent neonatal screening by immunoglobulin (Ig)M capture of dried blood samples. On all positive or suspected cases, confirmative serology was performed on babies and their mothers. Congenital toxoplasmosis was confirmed in newborns who had IgM and/or IgA and IgG, or IgG associated with suggestive ocular lesions (with IgM and IgG in the mother). Ophthalmologic evaluation consisted of indirect ophthalmoscopy with a lid speculum. Pediatric examination and radiologic studies of the central nervous system were also performed. In selected cases, biomicroscopy of the anterior segment, fundus photographs, or ultrasonography (B-scan) was performed. Prevalence of retinochoroidal lesions, either cicatricial or active, and their location and associated findings, such as vascular sheathing, hemorrhage, vitreous opacities, and retinal detachment, were evaluated. The occurrence of cataract, microphthalmia, microcephaly, intracranial calcification, and hydrocephalus was also recorded. Of 146,307 neonates screened, 190 had CT, yielding a prevalence of 1 in 770 live births, of whom 178 (93.7%) underwent standardized ophthalmologic examination at an average age of 55.6+/-16.6 days. Of these 178 infants, 142 (79.8%) had retinochoroidal lesions consistent with CT in at least 1 eye. Bilateral involvement was noted in 113 patients (63.5%). Macular involvement was seen in 165 eyes (46.3%) of 111 patients (62.4%). Active lesions were observed in 142 eyes (39.9%) of 85 patients (47.8%). These lesions were located in the macula of 75 eyes (21.1%) and were associated with retinal vascular sheathing in 44 eyes (12.4%). A high prevalence of CT was encountered (1/770) with high rates of early retinochoroidal involvement ( approximately 80%) and many active lesions (in approximately 50%), indicating a possibly more severe ocular involvement by CT in Brazil than in other parts of the world. The hypotheses of higher parasite virulence and increased individual susceptibility are being currently investigated.