Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

hsp65 PCR-restriction enzyme analysis (PRA) for identification of mycobacteria in the clinical laboratory

More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to four weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.

PRETERM BIRTH AND FETAL GROWTH RESTRICTION IN HIV-INFECTED BRAZILIAN PREGNANT WOMEN

Introduction: Maternal HIV infection and related co-morbidities may have two outstanding consequences to fetal health: mother-to-child transmission (MTCT) and adverse perinatal outcomes. After Brazilian success in reducing MTCT, the attention must now be diverted to the potentially increased risk for preterm birth (PTB) and intrauterine fetal growth restriction (IUGR). Objective: To determine the prevalence of PTB and IUGR in low income, antiretroviral users, publicly assisted, HIV-infected women and to verify its relation to the HIV infection stage. Patients and Methods: Out of 250 deliveries from HIV-infected mothers that delivered at a tertiary public university hospital in the city of Vitória, state of Espírito Santo, Southeastern Brazil, from November 2001 to May 2012, 74 single pregnancies were selected for study, with ultrasound validated gestational age (GA) and data on birth dimensions: fetal weight (FW), birth length (BL), head and abdominal circumferences (HC, AC). The data were extracted from clinical and pathological records, and the outcomes summarized as proportions of preterm birth (PTB, < 37 weeks), low birth weight (LBW, < 2500g) and small (SGA), adequate (AGA) and large (LGA) for GA, defined as having a value below, between or beyond the ±1.28 z/GA score, the usual clinical cut-off to demarcate the 10th and 90th percentiles. Results: PTB was observed in 17.5%, LBW in 20.2% and SGA FW, BL, HC and AC in 16.2%, 19.1%, 13.8%, and 17.4% respectively. The proportions in HIV-only and AIDS cases were: PTB: 5.9 versus 27.5%, LBW: 14.7% versus 25.0%, SGA BW: 17.6% versus 15.0%, BL: 6.0% versus 30.0%, HC: 9.0% versus 17.9%, and AC: 13.3% versus 21.2%; only SGA BL attained a significant difference. Out of 15 cases of LBW, eight (53.3%) were preterm only, four (26.7%) were SGA only, and three (20.0%) were both PTB and SGA cases. A concomitant presence of, at least, two SGA dimensions in the same fetus was frequent. Conclusions: The proportions of preterm birth and low birth weight were higher than the local and Brazilian prevalence and a trend was observed for higher proportions of SGA fetal dimensions than the expected population distribution in this small casuistry of newborn from the HIV-infected, low income, antiretroviral users, and publicly assisted pregnant women. A trend for higher prevalence of PTB, LBW and SGA fetal dimensions was also observed in infants born to mothers with AIDS compared to HIV-infected mothers without AIDS.

Effects of severe protein restriction in levels of parasitemia and in mortality of mice accutely infected with Trypanosoma cruzi

Adult mice were submitted to different degrees of protein restriction for five weeks (4.75, 9.5,14.25 and 19% of protein in isocaloric diets with normal content of mineral and vitamins), being subsequently infected with two strains of Trypanosoma cruzi: 10(5) trypomastigotes of Y strain or 14(5) trypomastigotes of CL strain. The same diet was maintained for all animals and the infection wasfollowed up by evaluation of blood parasites, mortality and intensity of lesions in the heart and skeleton muscle. Only severe protein restriction (4.75%) induced decrease in resistance to the infection with both the Y and CL strains of T. cruzi, which resulted in higher parasitemia and mortality. The inflammatory lesions in heart and skeleton muscle were less extensive in groups with severe protein restriction despite the increased number of parasite in muscle cells. Depression of immune mechanisms could be responsiblefor the reduced resistance and reduced inflammatory reaction after T. cruzi infection in severely protein restricted animals.

Polymerase chain reaction and restriction fragment length polymorphism analysis of the ITS2 region for differatiation of Brazilian Biomphalaria intermediate hosts of the Schistosoma mansoni

We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the three Schistosoma mansoni intermediate hosts in Brazil: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. Analysis of a restriction map from those sequences allowed us to select putative restriction enzymes able to identify the snail species under study. Four restriction enzymes were used and HpaII provided simple species-specific profiles easily visualized in polyacrylamide gels. The use of ITS2 is advantageous as it provides a small fragment of 460 bp which may be easily amplified by PCR. In the current work, we showed that the amplification of ITS2-DNAr together with HpaII enzyme restriction is an auxiliary molecular tool for the morphological identification of such snails as well as for taxonomic and phylogenetic studies of neotropical planorbids.

Restriction enzyme analysis of the human cytomegalovirus genome in specimens collected from immunodeficient patients in Belém, State of Pará, Brazil

INTRODUCTION: Human cytomegalovirus is an opportunistic betaherpesvirus that causes persistent and serious infections in immunodeficient patients. Recurrent infections occur due to the presence of the virus in a latent state in some cell types. It is possible to examine the virus using molecular methods to aid in the immunological diagnosis and to generate a molecular viral profile in immunodeficient patients. The objective of this study was to characterize cytomegalovirus genotypes and to generate the epidemiological and molecular viral profile in immunodeficient patients. METHODS: A total of 105 samples were collected from immunodeficient patients from the City of Belém, including newborns, hemodialysis patients, transplant recipients and HIV+ patients. An IgG and IgM antibody study was completed using ELISA, and enzymatic analysis by restriction fragment length polymorphism (RFLP) was performed to characterize viral genotypes. RESULTS: It was observed that 100% of the patients had IgG antibodies, 87% of which were IgG+/IgM-, consistent with a prior infection profile, 13% were IgG+/IgM+, suggestive of recent infection. The newborn group had the highest frequency (27%) of the IgG+/IgM+ profile. By RFLP analysis, only one genotype was observed, gB2, which corresponded to the standard AD169 strain. CONCLUSIONS: The presence of IgM antibodies in new borns indicates that HCMV continues to be an important cause of congenital infection. The low observed genotypic diversity could be attributed to the small sample size because newborns were excluded from the RFLP analysis. This study will be continued including samples from newborns to extend the knowledge of the general and molecular epidemiology of HCMV in immunodeficient patients.

Molecular characterization of Mycobacterium tuberculosis isolates from Tehran, Iran by restriction fragment length polymorphism analysis and spoligotyping

Abstract: INTRODUCTION Characterization of Mycobacterium tuberculosis (MTB) isolates by DNA fingerprinting has contributed to tuberculosis (TB) control. The aim of this study was to determine the genetic diversity of MTB isolates from Tehran province in Iran. METHODS MTB isolates from 60 Iranian and 10 Afghan TB patients were fingerprinted by standard IS6110-restriction fragment length polymorphism (RFLP) analysis and spoligotyping. RESULTS The copy number of IS6110 ranged from 10-24 per isolate. The isolates were classified into 22 clusters showing ≥ 80% similarity by RFLP analysis. Fourteen multidrug-resistant (MDR) isolates were grouped into 4 IS6110-RFLP clusters, with 10 isolates [71% (95% CI: 45-89%)] in 1 cluster, suggesting a possible epidemiological linkage. Eighteen Iranian isolates showed ≥ 80% similarity with Afghan isolates. There were no strains with identical fingerprints. Spoligotyping of 70 isolates produced 23 distinct patterns. Sixty (85.7%) isolates were grouped into 13 clusters, while the remaining 10 isolates (14.2%) were not clustered. Ural (formerly Haarlem4) (n = 22, 31.4%) was the most common family followed by Central Asian strain (CAS) (n = 18, 25.7%) and T (n = 9, 12.8%) families. Only 1strain was characterized as having the Beijing genotype. Among 60 Iranian and 10 Afghan MTB isolates, 25% (95% CI: 16-37) and 70% (95% CI: 39-89) were categorized as Ural lineage, respectively. CONCLUSIONS A higher prevalence of Ural family MTB isolates among Afghan patients than among Iranian patients suggests the possible transmission of this lineage following the immigration of Afghans to Iran.

Effects of protein-calorie restriction on mechanical function of hypertrophied cardiac muscle

OBJECTIVE: To assess the effect of food restriction (FR) on hypertrophied cardiac muscle in spontaneously hypertensive rats (SHR). METHODS: Isolated papillary muscle preparations of the left ventricle (LV) of 60-day-old SHR and of normotensive Wistar-Kyoto (WKY) rats were studied. The rats were fed either an unrestricted diet or FR diet (50% of the intake of the control diet) for 30 days. The mechanical function of the muscles was evaluated through monitoring isometric and isotonic contractions. RESULTS: FR caused: 1) reduction in the body weight and LV weight of SHR and WKY rats; 2) increase in the time to peak shortening and the time to peak developed tension (DT) in the hypertrophied myocardium of the SHR; 3) diverging changes in the mechanical function of the normal cardiac muscles of WKY rats with reduction in maximum velocity of isotonic shortening and of the time for DT to decrease 50% of its maximum value, and increase of the resting tension and of the rate of tension decline. CONCLUSION: Short-term FR causes prolongation of the contraction time of hypertrophied muscles and paradoxal changes in mechanical performance of normal cardiac fibers, with worsening of the shortening indices and of the resting tension, and improvement of the isometric relaxation.

Adaptação transcultural para o Brasil do Dietary Sodium Restriction Questionnaire (Questionário Restritivo da Dieta de Sódio) (DSRQ)

FUNDAMENTO: A restrição de sódio é uma medida não farmacológica frequentemente orientada aos pacientes com Insuficiência Cardíaca (IC). No entanto, a adesão é de baixa prevalência, ficando entre as causas mais frequentes de descompensação da IC. O Dietary Sodium Restriction Questionnaire (DSRQ) tem como objetivo identificar fatores que afetam a adesão à restrição dietética de sódio para pacientes com IC. No Brasil, não existem instrumentos que avaliem tais fatores. OBJETIVO: Realizar a adaptação transcultural do DSRQ. MÉTODOS: Estudo metodológico que envolveu as seguintes etapas: tradução, síntese, retrotradução, revisão por um comitê de especialistas, pré-teste da versão final e análise de concordância interobservador. No pré-teste foram avaliados os itens e sua compreensão, além da consistência interna pelo coeficiente alfa de Cronbach. O instrumento foi aplicado por dois pesquisadores simultânea e independentemente, sendo utilizado o teste Kappa para análise da concordância. RESULTADOS: Apenas uma questão sofreu alterações semânticas e/ou culturais maiores. No pré-teste, o alfa de Cronbach obtido para o total foi de 0,77, e para as escalas de Atitude, Norma subjetiva e Controle Comportamental obtiveram-se, respectivamente, 0,66, 0,50 e 0,85. Na etapa de concordância, o Kappa foi calculado para 12 das 16 questões, com valores que variaram de 0,62 a 1,00. Nos itens em que o cálculo não foi possível, a incidência de respostas iguais variou de 95% a 97,5%. CONCLUSÃO: A partir da adaptação transcultural do DSRQ foi possível propor uma versão do questionário para posterior avaliação das propriedades psicométricas.

Restriction in the repertoire of detectable autoantibodies in polyclonal B cell activations and the mimicry of autoantigens by idiotopes

Despite the existence of erythrocyte-autoreactive B cells in normal animals, erythrocyte-autoantibodies could not be detected during polyclonal B-cell activation (PBA) both in patients with visceral leishmaniasis and in bacterial lipopolysacharide (LPS) - injected mice. The failure to detect these autoantibodies in mice with PBA di not seem to be due to suppressor-cell activity, since (1) transfer of spleen cells from LPS-treated mice to naive recipients did not affect the erythrocyte-autoantibody response elicited by subsequent injections of rat erythrocytes and (2) low doses of X-radiation did no lead to erythrocyte-autoantibody detection in LPS-treated mice. The possibility that the detection of erytrocyte-autoantibodies could be affected by autoantibodies with idiotopes mimicring erythrocyte epitopes, the synthesis of which would also be triggerred in PBA, is discussed. Indirect evidence for the existence in normal animal of an expanded lymphocyte population with DNP-binding. Ia-mimicring antigen receptors is presented.

Human antibody responses to Schistosoma mansoni: does antigen directed, isotype restriction result in the production of blocking antibodies?

After treatment young Kenyan schoolchildren are highly susceptible to reinfection with Schistosoma mansoni. Older children and adults are resistant to reinfection. There is no evidence that this age related resistance is due to a slow development of protective immunological mechanisms, rather, it appears that young children are susceptible because of the presence of blocking antibodies which decline with age, thus allowing the expression of protective responses. Correlations between antibody responses to different stages of the parasite life-cycle suggest that, in young children, antigen directed, isotype restriction of the response against cross-reactive polysaccharide egg antigens results in an ineffectual, or even blocking antibody response to the schistosomulum.

Hemagglutinating and fusogenic activities of Newcastle disease virus: studies on receptor binding specificity and pH-induced conformational changes

Vaccinal and wild strains of Newcastle Disease virus (NDV) were analyzed for cell receptor binding and fusogenic biological properties associated with their HN (hemagglutinin-neuraminidase) and F (fusion protein) surface structures respectively. The evaluation of the biological activities of HN and F was carried out respectively by determination of hemagglutinating titers and hemolysis percentages, using erythrocytes from various animal origins at different pH values. Significant differences in hemagglutination titers for some strains of NDV were detected, when interacting with goose, sheep, guinea-pip and human "O" group erythrocytes at neutral pH. Diversity of hemolysis percentagens was observed between different NDV strains at acid pH. These analysis were developed to evaluate particular aspects of the actual influence of the receptor specifity and pH on the receptor binding and fusogenic processes of Newcastle Disease viruses.