José Nélio Januário

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

OBJECTIVE To report results of early ophthalmologic examinations in a large cohort of newborns with congenital toxoplasmosis (CT) after neonatal screening. DESIGN Cross-sectional analysis of a cohort. PARTICIPANTS A total of 178 newborns with confirmed CT from 146,307 screened babies (95% of live births) from Minas Gerais state, southeastern Brazil. METHODS 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. MAIN OUTCOME MEASURES 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. RESULTS 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%). CONCLUSIONS 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.

Genetic Characterization of Toxoplasma gondii Revealed Highly Diverse Genotypes for Isolates from Newborns with Congenital Toxoplasmosis in Southeastern Brazil

ABSTRACT Recent studies of Toxoplasma gondii isolates from animals in Brazil have revealed high genetic diversity. Many of these isolates are virulent to mice. It is speculated that these isolates may also be virulent to humans. However, there is very limited data regarding T. gondii strains from human infection. Therefore, it is not clear whether there is any association between parasite genotypes and disease phenotypes. In this study, a total of 27 T. gondii strains were isolated from humans with congenital toxoplasmosis in Minas Gerais state, Brazil. The genetic variability was assessed by restricted fragment length polymorphism in 11 loci (SAG1, 5′ plus 3′ SAG2, alternative [alt.] SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Genetic analysis of 24 strains revealed 14 different genotypes, including 7 previously identified from animals and 7 new types. The widespread genotype BrII accounted for 29% (7/24) of the isolates and was the dominant genotype involved in this study. This is the first report of genotyping of T. gondii isolates obtained from blood samples from newborns with congenital toxoplasmosis. Genotypic characterization of these isolates suggests high genetic diversity of T. gondii in this human population in Brazil. Future studies are needed to determine the source of contamination of this human population.

Mortalidade de crianças com doença falciforme: um estudo de base populacional

OBJETIVO: Caracterizar os obitos das criancas com doenca falciforme (DF) triadas no estado de Minas Gerais e acompanhadas na Fundacao Hemominas. METODOS: Coorte de criancas diagnosticadas pelo Programa de Triagem Neonatal de Minas Gerais (marco/1998 - fevereiro/2005). Os obitos foram identificados pela busca ativa das criancas ausentes nas consultas agendadas nos hemocentros. Dados clinicos e epidemiologicos foram coletados dos documentos de obito, banco de dados da triagem neonatal, prontuarios medicos e em entrevistas com as familias. RESULTADOS: Foram triadas 1.833.030 criancas no periodo, sendo 1.396 com DF (1:1.300). Ocorreram 78 obitos: 63 em criancas com genotipo SS, 12 em criancas com genotipo SC e tres em criancas com genotipo S/β+ talassemia. Cinquenta e seis criancas (71,8%) morreram antes dos 2 anos de idade; 59 morreram em hospitais e 18 no domicilio ou trânsito. Causas de obito pelo atestado (n = 78): 38,5% infeccao; 16,6% sequestro esplenico agudo; 9% outras causas; 15,4% sem assistencia medica; e 20,5% indeterminada. Segundo as entrevistas (n = 52), o sequestro esplenico foi responsavel por quase 1/3 dos obitos, contrastando com a porcentagem de apenas 14% registrada nos atestados de obito. As probabilidades de sobrevida aos 5 anos (erro padrao da media) para criancas SS, SC e Sβ+ talassemia foram: 89,4 (1,4), 97,7 (0,7) e 94,7% (3,0), respectivamente (SS versus SC, p < 0,0001). CONCLUSOES: Mesmo em um programa de triagem neonatal com rigoroso controle do tratamento, a probabilidade de obito em criancas com genotipo SS ainda e elevada. Os obitos com causa indeterminada indicam dificuldades no reconhecimento da DF e das suas complicacoes. Esforcos educativos dirigidos a profissionais da saude e familiares devem ser incrementados para diminuir a mortalidade pela DF.

Congenital toxoplasmosis from a chronically infected woman with reactivation of retinochoroiditis during pregnancy.

OBJECTIVE To report a rare case of congenital toxoplasmosis from an immunocompetent mother with chronic infection who had reactivation of ocular disease during pregnancy. DESCRIPTION The newborn was asymptomatic at birth and identified by neonatal screening (IgM anti-Toxoplasma gondii in dried blood) among other 190 infants with congenital toxoplasmosis during a 7-month period. His mother had had a non-treated episode of reactivation of toxoplasmic retinochoroiditis during pregnancy, with stable IgG titers and negative IgM results. Results of IgM and IgG in the newborns serum, as well as IgG immunoblotting were positive and active retinochoroidal lesions were detected in his peripheral retina. The neonate was treated with sulfadiazine, pyrimethamine and folinic acid. At 14 months of life, the child remained asymptomatic, with regression of retinochoroidal lesions and persistence of IgG. COMMENTS It is possible that systematic neonatal screening in areas with high prevalence of infection may identify these cases.

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

OBJECTIVE 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. METHODS 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. RESULTS 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 States worst social and economic indicators. CONCLUSIONS 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.

Comparison of HPV genotyping by type-specific PCR and sequencing

Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide and there is a strong link between certain high-risk viral types and cervical carcinogenesis. Although there are several typing methods, it is still unclear which test is the best. This study compared the effectiveness of type-specific PCR (TS-PCR) and sequencing, with a focus on their clinical application. A total of 260 cervical samples from HPV-positive patients were tested for types 6, 11, 16, 18, 31, 33 and 35 using TS-PCR and sequencing. The genotype was identified in 36% of cases by TS-PCR and in 75% by sequencing. Sequencing was four times more likely to identify the viral type in positive samples than TS-PCR (p = 0.00). Despite being more effective for virus genotyping, sequencing was unable to identify viral types in multiple infections. Combining both techniques resulted in highly sensitive detection (87% of cases), showing that they are complementary methods. HPV genotyping is an important step in HPV management, helping to identify patients with a higher risk of developing cervical cancer and contributing to the development of type-specific vaccines.

Variations in genotype-phenotype correlations in phenylketonuria patients.

Phenylalanine hydroxylase deficiency is a trait inherited in an autosomal recessive pattern; the associated phenotype varies considerably. This variation is mainly due to the considerable allelic heterogeneity in the phenylalanine hydroxylase enzyme locus. We examined the genotype-phenotype correlation in 54 phenylketonuria (PKU) patients from Minas Gerais, Brazil. Two systems were used. The first was a phenotype prediction system based on arbitrary values (AV) attributed to each mutation and the second was a correlation analysis. An AV was assigned to each mutation: AV = 1 for classical PKU mutation; AV = 2 for moderate PKU mutation; AV = 4 for mild PKU mutation, and AV = 8 for non-PKU hyperphenylalaninemia mutation. The observed phenotype for AV analysis was the clinical diagnosis established by the overloading phenylalanine test. Among the 51 PKU patients that we analyzed based on this trait, in 51% the predicted phenotype did not match the observed phenotype; the highest degree of concordance was found in patients with null/null genotypes. The genotype was observed to be a good predictor of the clinical course of the patients and significant correlations were found between phenylalanine values at first interview and predicted residual activity, genotype and arbitrary value sum.

Biomarker analysis revealed distinct profiles of innate and adaptive immunity in infants with ocular lesions of congenital toxoplasmosis.

Toxoplasma gondii is the main infectious cause of human posterior retinochoroiditis, the most frequent clinical manifestation of congenital toxoplasmosis. This investigation was performed after neonatal screening to identify biomarkers of immunity associated with immunopathological features of the disease by flow cytometry. The study included infected infants without NRL and with retinochoroidal lesions (ARL, ACRL, and CRL) as well as noninfected individuals (NI). Our data demonstrated that leukocytosis, with increased monocytes and lymphocytes, was a relevant hematological biomarker of ARL. Immunophenotypic analysis also revealed expansion of CD14+CD16+HLA-DRhigh monocytes and CD56dim cytotoxic NK-cells in ARL. Moreover, augmented TCRγ δ + and CD8+ T-cell counts were apparently good biomarkers of morbidity. Biomarker network analysis revealed that complex and intricated networks underscored the negative correlation of monocytes with NK- and B-cells in NRL. The remarkable lack of connections involving B-cells and a relevant shift of NK-cell connections from B-cells toward T-cells observed in ARL were outstanding. A tightly connected biomarker network was observed in CRL, with relevant connections of NK- and CD8+ T-cells with a broad range of cell subsets. Our findings add novel elements to the current knowledge on the innate and adaptive immune responses in congenital toxoplasmosis.

Hearing loss in congenital toxoplasmosis detected by newborn screening

UNLABELLED Congenital toxoplasmosis may cause sensorineural deficit in up to 20% of the patients and proper treatment in the first year improves prognosis. In Brazil, this infections impact on hearing impairment is unknown. AIM To evaluate hearing of newborns with congenital toxoplasmosis identified by the newborn screening service. METHOD This prospective study analyzed children with congenital toxoplasmosis identified by newborn screening (IgM anti-T.gondii) in Belo Horizonte during 2003/2004. The presence of IgM and/or IgA in the first 6 months or IgG at 12 months-of age in serology was used as case definition. Hearing tests were carried out at the time of diagnosis and 12 months later, including behavioral audiometry, evoked otoacoustic emission and brainstem evoked responses audiometry. RESULTS Among 30.808 screened children (97% of live births), 20 had congenital toxoplasmosis, 15 (75%) were asymptomatic at birth. Nineteen children were evaluated by hearing tests. Four had sensorineural impairment (21.1%). One child had other risk factors for hearing impairment; the other three had no other risk factors but toxoplasmosis. Two properly children treated still had hearing loss, in disagreement with current literature. CONCLUSION Results suggest that congenital toxoplasmosis, common in Brazil, is a risk factor for hearing impairment and its impact on hearing loss deserves further studies.

Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening

The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.