Sífilis congénita : caracterização da infecção e avaliação de técnicas laboratoriais para o seu diagnóstico

RESUMO: Um dos maiores problemas da sífilis é a infecção intra-uterina do feto, que pode resultar em morte fetal com aborto espontâneo. Os objectivos desta tese foram comparar os cinco testes serológicos para o diagnóstico de sífilis congénita e optimizar e aplicar a várias amostras clínicas, colhidas de indivíduos com suspeita de infecção por Treponema pallidum, uma técnica de PCR-Multiplex e uma técnica de PCR em tempo real. Na globalidade dos soros estudados obtiveram-se os seguintes resultados: o RPR reactivo em 87/517; TPHA reactivo em 135/517; e EIA reactivo em 127/517. A pesquisa de anticorpos específicos de tipo IgM foi efectuada em 33 soros sendo estudada pelas técnicas de imunofluorescência indirecta e de westernblot. Quanto aos resultados obtidos pelas duas técnicas, o teste FTA-Abs-IgM demonstrou reactividade em 3/33, enquanto que a técnica de westernblot-M, apresentou reactividade em 18/33. Para a avaliação de uma técnica de PCR-TR, foram estudadas 318 amostras de 236 indivíduos classificados, com base em critérios clínicos e laboratoriais, em diferentes estádios de infecção por Treponema pallidum e em indivíduos sem infecção por aquele microrganismo. Relativamente a estas técnicas foi possível observar amplificação de ADN de Treponema pallidum em 133/318 pela técnica de PCR-TR e 90/318 pela técnica de PCR-Multiplex. Mediante os resultados obtidos e de outros estudos efectuados parece poder concluir-se que o teste EIA é o mais indicado para o rastreio da infecção por Treponema pallidum, devendo um resultado reactivo por esta técnica, ser confirmado com a realização de um teste não treponémico (RPR). Relativamente ao diagnóstico de infecção congénita, o teste westernblot para pesquisa de anticorpos específicos de tipo IgM, parece ser o mais apropriado. A técnica PCR-TR, parece ser a mais indicada, pois apresenta uma maior sensibilidade e especificidade que a PCR-Multiplex.----------------- ABSTRACT:The syphilis major problem is the intrauterine infection of the fetus, which may result in fetal death with spontaneous abortion. The objectives of this thesis were to compare the five serological tests for the diagnosis of congenital syphilis and optimize and apply several clinical samples taken from individuals suspected of infection with Treponema pallidum, a PCR-Multiplex and real-time PCR. In all sera studied were obtained the following results: the RPR reactive in 87/517; reactive TPHA in 135/517, and 127/517 in reactive EIA. The search for specific IgM antibodies was performed on 33 sera that were studied by indirect immunofluorescence and westernblot. The results obtained by both techniques, the test FTA-Abs-IgM showed reactivity in 3/33, while the technique westernblot-M, showed reactivity in 18/33. For an RT-PCR evaluation, we studied 318 samples from 236 individuals classified based on clinical and laboratory criteria at different stages of infection by Treponema pallidum and in individuals without infection by that organism. For these techniques it was possible to observe ADN amplification of Treponema pallidum in 133/318 by RT-PCR and 90/318 by PCR-Multiplex. Based on obtained results and in other studies seems that we can conclude that the EIA test is the most suitable for Treponema pallidum screening infection, and a reactive result by this technique, should be confirmed with the realization of a non-treponemal test (RPR). For the congenital infection diagnosis, testing for antibodies westernblot specific IgM appears to be the most appropriate. The RT-PCR seems to be the most suitable, since it has a higher sensitivity and specificity than the PCR-Multiplex.

Serological, clinical and epidemiological evaluation of toxocariasis in urban areas of south Brazil

Toxocariasis is a worldwide public-health problem that poses major risks to children who may accidentally ingest embryonated eggs of Toxocara. The objectives of this study were to investigate the occurrence of anti-Toxocara spp. antibodies in children and adolescents and the variables that may be involved, as well as environmental contamination by Toxocara spp. eggs, in urban recreation areas of north central mesoregion, Paraná State, Brazil. From June 2005 to March 2007. a total of 376 blood samples were collected by the Public Health Service from children and adolescents one to 12 years old, of both genders. Samples were analyzed by the indirect ELISA method for detection of anti-Toxocara antibodies. Serum samples were previously absorbed with Ascaris suum antigens, and considered positive with a reagent reactivity index >1. Soil samples from all of the public squares and schools located in the four evaluated municipalities that had sand surfaces (n = 19) or lawns (n = 15) were analyzed. Of the 376 serum samples, 194 (51.6%) were positive. The seroprevalence rate was substantially higher among children aging one to five years (p = 0.001) and six to eight years (p = 0.022). The clinical signs and symptoms investigated did not show a statistical difference between seropositive and seronegative individuals (p > 0.05). In 76.5% of the investigated recreation places, eggs of Toxocara were detected in at least one of the five collected samples. Recreation areas from public schools were 2.8 times more contaminated than from public squares. It is important to institute educational programs to inform families and educators, as well as to improve sanitary control of animals and cleaning of the areas intended for recreation in order to prevent toxocariasis.

Peritubular Capillaries C4d Deposits in Renal Allograft Biopsies and Anti HLA I/II Alloantibodies Screening. Incidence and Clinical Importance

Aim: To characterise clinically the patients with C4d in peritubular capillaries deposits (C4dPTCD) and/or circulating anti-HLA class I/II alloantibodies. To determine the correlation between positive C4dPTCD and circulating anti-HLA class I/II alloantibodies during episodes of graft dysfunction. Subjects and Methods: C4d staining was performed in biopsies with available frozen tissue obtained between January 2004 and December 2006. The study was prospective from March 2005, when a serum sample was obtained at the time of biopsy to detect circulating anti-HLA class I/II alloantibodies. Results: We studied 109 biopsies in 86 cadaver renal transplant patients. Sixteen of these (14.7%) presented diffuse positive C4dPTCD. There was a 13.5% rate of +C4dPTCD incidence within the first six months of transplantation and 16% after six months (p>0.05). Half of the +C4dPTCD in the first six months was associated with acute humoral rejection. After six months, the majority of +C4dPTCD (n=7/8) was present in biopsies with evidence of interstitial fibrosis/tubular atrophy and/or transplant glomerulopathy. The C4dPTCD was more frequent in patients with positive anti-HCV antibodies(p<0.0001), a previous renal transplant (p=0.007), and with a panel reactivity antibody (PRA) ≥ 50%(p=0.0098). The anti-HCV+ patients had longer time on dialysis (p=0.0019) and higher PRA(p=0.005). Circulating anti-HLA I/II alloantibodies were screened in 46 serum samples. They were positive in 10.9% of samples, all obtained after six months post transplant. Circulating alloantibodies were absent in 92.5% of the C4d negative biopsies. Conclusion: We found an association between the presence of C4dPTCD and 2nd transplant recipients,higher PRA and the presence of anti-HCV antibodies. The presence of HCV antibodies is not a risk factor for C4dPTCD per se, but appears to reflect longer time on dialysis and presensitisation. In renal dysfunction a negative alloantibody screening is associated with a reduced risk of C4dPTCD (<10%).

Anemia de Células Falciformes e Acidente Vascular Cerebral: Uma História Evitável

Introdução: Na criança, a etiologia do acidente vascular cerebral (AVC) é conhecida em 75% dos casos, sendo a anemia de células falciformes (ACF) a mais frequente na criança de raça negra. O interesse deste caso clínico reside na forma de apresentação pouco habitual e curso evitável. Caso clínico: Criança de raça negra com 27 meses de idade, sem antecedentes relevantes, admitida por sinais neurológicos focais de instalação súbita. A tomografia computorizada cranio-encefálica e ressonância magnética evidenciaram lesão isquémica aguda extensa e alterações compatíveis com AVC silencioso prévio. Analiticamente apresentava anemia normocítica, muitos drepanocitos de formação espontânea e 87% de hemoglobina S. Neste contexto, foi submetida a transfusão-permuta. Conclusão: O AVC como complicação da ACF pode acontecer em idades precoces e surgir como quadro inaugural. Pensamos que se justifica divulgar o rastreio pré-natal e realizar um estudo da relação custo-benefício para a implementação de um rastreio neonatal desta patologia em Portugal.

Potential immunological markers for diagnosis and therapeutic assessment of toxocariasis

In human toxocariasis, there are few approaches using immunological markers for diagnosis and therapeutic assessment. An immunoblot (IB) assay using excretory-secretory Toxocara canis antigen was standardized for monitoring IgG, IgE and IgA antibodies in 27 children with toxocariasis (23 visceral, three mixed visceral and ocular, and one ocular form) for 22-116 months after chemotherapy. IB sensitivity was 100% for IgG antibodies to bands of molecular weight 29-38, 48-54, 95-116, 121-162, >205 kDa, 80.8% for IgE to 29-38, 48-54, 95-121, > 205 kDa, and 65.4% for IgA to 29-38, 48-54, 81-93 kDa. Candidates for diagnostic markers should be IgG antibodies to bands of low molecular weight (29-38 and 48-54 kDa). One group of patients presented the same antibody reactivity to all bands throughout the follow-up study; in the other group, antibodies decayed partially or completely to some or all bands, but these changes were not correlated with time after chemotherapy. Candidates for monitoring patients after chemotherapy may be IgG antibodies to > 205 kDa fractions, IgA to 29-38, 48-54, 81-93 kDa and IgE to 95-121 kDa. Further identification of antigen epitopes related to these markers will allow the development of sensitive and specific immunoassays for the diagnosis and therapeutic assessment of toxocariasis.

Necrólise Epidérmica Tóxica – Valproato de Sódio e Vancomicina?

A prevalência de reacções adversas a medicamentos (RAM) em doentes hospitalizados é estimada em 10 -20% e estas podem ser potencialmente fatais. A necrólise epidérmica tóxica (NET) é uma das apresentações de RAM mais severa, com baixa incidência mas mortalidade elevada. Os autores apresentam o caso de uma mulher de 79 anos, com doença cerebrovascular hemorrágica grave, pós -traumática, com necessidade de internamento em cuidados intensivos que, sob terapêutica com meropenem, vancomicina e valproato de sódio, desenvolveu um quadro de NET. Para identificação do fármaco responsável realizou -se teste de transformação linfoblástica (TTL). Os índices de estimulação obtidos foram < 2,0 para o meropenem, 7,4 para vancomicina e 6,4 para o valproato de sódio; a sua valorização foi efectuada com cut -off >3. Apesar de ser ainda um instrumento de investigação, o TTL foi decisivo na confirmação da base imunológica da reacção. Vancomicina e valproato de sódio estão totalmente contraindicados nesta doente.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

IgG Antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.

Variability in Galactomannan detection by platelia Aspergillus EIA™ according to the Aspergillus species

Here we investigate the extent to which different Aspergillus species release galactomannan (GM) in vitro. Marked variability was observed in GM reactivity between and within Aspergillus species, with A. terreus strains showing the highest GM indexes. The in vivo significance of these findings remains to be determined.

Evaluation of the ELISA-F29 test as an early marker of therapeutic efficacy in adults with chronic Chagas disease

This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.

Reactividade Cutânea Inespecífica em Estudos Populacionais

Os testes cutâneos por prick constituem o método diagnóstico de eleição no estudo da sensibilização alergénica, sendo no entanto influenciados por diversos factores. Não se encontram disponíveis dados sobre a quantificação da reacti-vidade cutânea inespecífica em estudos populacionais pediátricos, incluindo amostras de raças diversas. Objectivo: Avaliar e correlacionar a reactividade cutânea à histamina em crianças de diferentes raças,bem como a possível influência de factores como o sexo e a atopia. Material e Métodos: Englobado no Estudo Português de Doenças Alérgicas na Criança (PAC Study), foram estudadas 1710 crianças em idade escolar (6 a 12 anos), 244 de raça Negra na República de Cabo Verde, 756 Caucasianas na Ilha da Madeira e 710 de raça Chinesa em Macau. A todas as crianças foram efectuados testes cutâneos por prick com aeroalergenos, utilizando como referência positiva um extracto de histamina a 10mg/ml (Merck Allergopharma). Foi efectuada análise comparativa do diâmetro médio da pápula induzida pela histamina.

MONTENEGRO SKIN TEST AND AGE OF SKIN LESION AS PREDICTORS OF TREATMENT FAILURE IN CUTANEOUS LEISHMANIASIS

A case-control study was conducted to examine the association among the Montenegro skin test (MST), age of skin lesion and therapeutic response in patients with cutaneous leishmaniasis (CL) treated at Evandro Chagas National Institute of Infectious Diseases (INI), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil. For each treatment failure (case), two controls showing skin lesion healing following treatment, paired by sex and age, were randomly selected. All patients were treated with 5 mg Sb5+/kg/day of intramuscular meglumine antimoniate (Sb5+) for 30 successive days. Patients with CL were approximately five times more likely to fail when lesions were less than two months old at the first appointment. Patients with treatment failure showed less intense MST reactions than patients progressing to clinical cure. For each 10 mm of increase in MST response, there was a 26% reduction in the chance of treatment failure. An early treatment - defined as a treatment applied for skin lesions, which starts when they are less than two months old at the first appointment -, as well as a poor cellular immune response, reflected by lower reactivity in MST, were associated with treatment failure in cutaneous leishmaniasis.

The Role of the Humoral Immune Response in the Molecular Evolution of the Envelope C2, V3 and C3 Regions in Chronically HIV-2 Infected Patients

BACKGROUND: This study was designed to investigate, for the first time, the short-term molecular evolution of the HIV-2 C2, V3 and C3 envelope regions and its association with the immune response. Clonal sequences of the env C2V3C3 region were obtained from a cohort of eighteen HIV-2 chronically infected patients followed prospectively during 2-4 years. Genetic diversity, divergence, positive selection and glycosylation in the C2V3C3 region were analysed as a function of the number of CD4+ T cells and the anti-C2V3C3 IgG and IgA antibody reactivity RESULTS: The mean intra-host nucleotide diversity was 2.1% (SD, 1.1%), increasing along the course of infection in most patients. Diversity at the amino acid level was significantly lower for the V3 region and higher for the C2 region. The average divergence rate was 0.014 substitutions/site/year, which is similar to that reported in chronic HIV-1 infection. The number and position of positively selected sites was highly variable, except for codons 267 and 270 in C2 that were under strong and persistent positive selection in most patients. N-glycosylation sites located in C2 and V3 were conserved in all patients along the course of infection. Intra-host variation of C2V3C3-specific IgG response over time was inversely associated with the variation in nucleotide and amino acid diversity of the C2V3C3 region. Variation of the C2V3C3-specific IgA response was inversely associated with variation in the number of N-glycosylation sites. CONCLUSION: The evolutionary dynamics of HIV-2 envelope during chronic aviremic infection is similar to HIV-1 implying that the virus should be actively replicating in cellular compartments. Convergent evolution of N-glycosylation in C2 and V3, and the limited diversification of V3, indicates that there are important functional constraints to the potential diversity of the HIV-2 envelope. C2V3C3-specific IgG antibodies are effective at reducing viral population size limiting the number of virus escape mutants. The C3 region seems to be a target for IgA antibodies and increasing N-linked glycosylation may prevent HIV-2 envelope recognition by these antibodies. Our results provide new insights into the biology of HIV-2 and its relation with the human host and may have important implications for vaccine design.

Acidente Vascular Cerebral no Jovem e Déficit de Proteína S

Os autores descrevem o caso clínico de uma mulher de 33 anos, internada por hemiparésia direita e afasia global de instalação súbita, havendo referência a um episódio prévio de afasia global 7 anos antes, do qual recuperou sem sequelas. É referida a investigação complementar a que foi sujeita, especialmente vocacionada para o despiste das diversas causas de AVC no jovem. Discute-se a relação entre déficit de proteínas inibidoras da coagulação e patologia arterial, fazendo-se ainda uma breve referência à bibliografia existente sobre a matéria.

Biological and Physiological Markers of Tactile Sensorial Processing in Healthy Newborns

The main objective of this review is to provide a descriptive analysis of the biological and physiological markers of tactile sensorial processing in healthy, full-term newborns. Research articles were selected according to the following study design criteria: (a) tactile stimulation for touch sense as an independent variable; (b) having at least one biological or physiological variable as a dependent variable; and (c) the group of participants were characterized as full-term and healthy newborns; a mixed group of full-term newborns and preterm newborns; or premature newborns with appropriate-weight-for-gestational age and without clinical differences or considered to have a normal, healthy somatosensory system. Studies were then grouped according to the dependent variable type, and only those that met the aforementioned three major criteria were described. Cortisol level, growth measures, and urinary catecholamine, serotonin, and melatonin levels were reported as biological-marker candidates for tactile sensorial processing. Heart rate, body temperature, skin-conductance activity, and vagal reactivity were described as neurovegetative-marker candidates. Somatosensory evoked potentials, somatosensory evoked magnetic fields, and functional neuroimaging data also were included.