Prospective analysis between the therapy of immunosuppressive medication and allogeneic microchimerism after liver transplantation

After liver transplantation, migration of donor-derived hematopoietic cells to recipient can be detected in pheripheral blood. This state is termed microchimerism. The aim of this study was to investigate prospectively the presence of allogeneic microchimerism, the occurrence of acute cellular rejection and the level of immunosuppression in transplanted patients. Microchimerism occurrence between 10 days and 12 months after liver transplantation was analyzed in 47 patients aged between 15 and 65 by a two-stage nested PCR/SSP technique to detect donor MHC HLA-DR gene specifically. A pre-transplant blood sample was colleted from each patient to serve as individual negative control. Microchimerism was demonstrated in 32 (68%) of the 47 patients; of these, only 10 patients (31.2%) presented rejection. Early microchimerism was observed in 25 patients (78.12%) and late microchimerism in 7 patients (21.8%). Among the patients with microchimerism, 14 were given CyA and 18 were given FK506. In the group without microchimerism, 12 patients were given CyA and 03 were given FK506. There was a significant association between the presence of microchimerism and the absence of rejection (p=0.02) and also between microchimerism and the type of immunosuppression used. Our data indicate that microchimerism and probably differentiation of donor-derived leukocytes can have relevant immunologic effects both in terms of sensitization of recipient and in terms of immunomodulation toward tolerance induction. (C) 2008 Elsevier B.V. All rights reserved.

Occurrence of TRGV-BJ hybrid gene in SV40-transformed fibroblast cell lines

Illegitimate V(D)J-recombination in lymphoid malignancies involves rearrangements in immunoglobulin or T-cell receptor genes, and these rearrangements may play a role in oncogenic events. High frequencies of TRGV-BJ hybrid gene (rearrangement between the TRB and TRG loci at 7q35 and 7p14-15, respectively) have been detected in lymphocytes from patients with ataxia telangiectasia (AT), and also in patients with lymphoid malignancies. Although the TRGV-BJ gene has been described only in T-lymphocytes, we previously detected the presence of TRGV-BJ hybrid gene in the genomic DNA extracted from SV40-transformed AT5BIVA fibroblasts from an AT patient. Aiming to determine whether the AT phenotype or the SV40 transformation could be responsible for the production of the hybrid gene by illegitimate V(D)J-recombination, DNA samples were extracted from primary and SV40-transformed (normal and AT) cell lines, following Nested-PCR with TRGV- and TRBJ-specific primers. The hybrid gene was only detected in SV40-transformed fibroblasts (AT-5BIVA and MRC-5). Sequence alignment of the cloned PCR products using the BLAST program confirmed that the fragments corresponded to the TRGV-BJ hybrid gene. The present results indicate that the rearrangement can be produced in nonlymphoid cells, probably as a consequence of the genomic instability caused by the SV40-transformation, and independently of ATM gene mutation.

First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America

The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica. (C) 2010 Elsevier Ltd. All rights reserved.

Analysis of marine bivalve shellfish from the fish market in Santos city, Sao Paulo state, Brazil, for Toxoplasma gondii

The aim of this study was to determine if Toxoplasma gondii are present in oysters (Crassostrea rhizophorae) and mussels (Mytella guyanensis) under natural conditions using a bioassay in mice and molecular detection methods. We first compared two standard protocols for DNA extraction, phenol-chloroform (PC) and guanidine-thiocyanate (GT), for both molluscs. A total of 300 oysters and 300 mussels were then acquired from the fish market in Santos city, Sao Paulo state, Brazil, between March and August of 2008 and divided into 60 groups of 5 oysters and 20 groups of 15 mussels. To isolate the parasite, five mice were orally inoculated with sieved tissue homogenates from each group of oysters or mussels. For molecular detection of T. gondii, DNA from mussels was extracted using the PC method and DNA from oysters was extracted using the GT method. A nested-PCR (Polymerase Chain Reaction) based on the amplification of a 155 bp fragment from the B1 gene of T. gondii was then performed. Eleven PCR-RFLP (Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 and Apico, were used to genotype positive samples. There was no isolation of the parasite by bioassay in mice. T. gondii was not detected in any of the groups of mussels by nested-PCR. DNA of T. gondii was apparently detected by nested-PCR in 2 groups of oysters (3.3%). Genotyping of these two positive samples was not successful. The results suggest that oysters of the species C. rhizophorae, the most common species from the coast of Sao Paulo, can filter and retain T. gondii oocysts from the marine environment. Ingestion of raw oysters as a potential transmission source of T. gondii to humans and marine mammals should be further investigated. (C) 2010 Elsevier B.V. All rights reserved.

Crab-eating fox (Cerdocyon thous), a South American canid, as a definitive host for Hammondia heydorni

Hammondia heydorni is a cyst forming coccidia closely related to other apicomplexans, such as Toxoplasma gondii, Neospora caninum and Hammondia hammondi with a two-host life cycle. Dogs and other canids as red foxes (Vulpes vulpes) and coyotes (Canis latrans) may serve as definitive hosts for H. heydorni. Sporulated oocysts are infective for cattle, sheep and goats, which may serve as intermediate hosts. Herein, we describe the ability of crab-eating fox (Cerdocyon thous), a wild carnivore that is commonly found from northern Argentina to northern South America, to serve as definitive host of H. heydorni. The whole masseter muscle and brain from two 2-year-old bovines were collected, minced and pooled together for the fox infection. The bovine pooled tissues were equally administered to four foxes, in two consecutive days. Two foxes shed subspherical unsporulated oocysts measuring 10-15 mu m, after 8 and 9 days post-infection, respectively. One of the foxes eliminated oocysts for 5 days, while the other fox shed oocysts for 9 days. A DNA sample of oocysts detected at each day of oocyst elimination was tested by two PCRs, one of them carried out employing primers directed to the common toxoplasmatiid 18S and 5.8S ribosomal RNA coding genes (PCR-ITS1) and the other based on heat-shock protein 70 kDa coding gene (PCR-HSP70). These samples were also submitted to a N. caninum specific nested-PCR protocol based on a N. caninum specific gene (Nc5-nPCR). All of them were positive by PCR-ITS1 and PCR-HSP70 but negative by Nc5-nPCR. The PCR-ITS1 and PCR-HSP70 nucleotide sequences amplified from the oocysts shed by the foxes revealed 100% identity with homologous sequences of H. heydorni. In conclusion, it is clear that H. heydorni also uses the crab-eating fox as a definitive host. The crab-eating fox is usually reported to live in close contact with livestock in several regions of Brazil. Therefore, it is reasonable to infer that such carnivores may play an important role in the sylvatic and domestic cycles of H. heydorni infection. (C) 2009 Elsevier B.V. All rights reserved.

Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

The aim of this study was to determine the prevalence of Cryptosporidium species and genotypes in birds kept in captivity in Brazil. A total of 966 samples from 18 families of birds was collected and stored in 5% potassium dichromate solution at 4 degrees C until processing. Oocysts were purified in Sheather sugar solution following extraction of genomic DNA. Molecular analyses were performed using nested-PCR for amplification of fragments of the 18S subunit of rRNA gene and of the actin gene. Amplification of Cryptosporidium DNA fragments was obtained in 47 (4.86%) samples. Sequencing of amplified fragments and phylogenetic analyses allowed the identification of Cryptosporidium baileyi in a black vulture (Coragyps atratus), a domestic chicken (Gallus gallus domesticus) and a saffron finch (Sicalis flaveola); Cryptosporidium galli in canaries (Serinus canaria), a cockatiel (Nymphicus hollandicus) and lesser seed-finches (Oryzoborus angolensis); Cryptosporidium meleagridis in a domestic chicken (G. g. domesticus); Cryptosporidium parvum in a cockatiel (N. hollandicus); Cryptosporidium avian genotype I in a canary (S. canaria) and an Indian peafowl (Pavo cristatus); Cryptosporidium avian genotype II in ostriches (Struthio camelus) and Cryptosporidium avian genotype III in a cockatiel (N. hollandicurs) and a peach-faced lovebird (Agapornis roseicolis). (C) 2009 Elsevier B.V. All rights reserved.

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 mu l of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Detection and stability of Japanese encephalitis virus RNA and virus viability in dead infected mosquitoes under different storage conditions

A semi-nested polymerase chain reaction (PCR) was evaluated for detection of Japanese encephalitis (JE) virus in infected mosquitoes stored under simulated northern Australian summer conditions. The effect of silica gel, thymol, and a combination of the two on RNA stability and virus viability in dead mosquitoes were also examined. While JE virus RNA was relatively stable in mosquitoes held for up to 14 days after death, viable virus was not detected after day 1. Thymol vapor inhibited fungal contamination. Detection of single mosquitoes infected with JE virus in large pools of mosquitoes was also investigated. Single laboratory-infected mosquitoes were detected in pools of less than or equal to200 mosquitoes and in pools diluted to 0.2/100 and 0.1/100 mosquitoes, using the semi-nested PCR. However, the ability to detect live virus decreased as pool size increased. The semi-nested PCR proved more expensive than virus isolation for pools of 100 mosquitoes. However, the semi-nested PCR was faster and more economical using larger pools. Results indicate that surveillance of JE virus in mosquitoes using the semi-nested PCR is an alternative to monitoring seroconversions in sentinel pigs.

Distribution of Chlamydia pneumoniae DNA in atherosclerotic carotid arteries: significance for sampling procedures

Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates. In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C. pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium. A sensitive nested PCR assay detected C. pneumoniae DNA in all patients at one or more locations within the plaque. On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C. pneumoniae DNA present. A patchy distribution of C. pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C. pneumoniae DNA present. If a single random 30-mum-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C. pneumoniae DNA in a patient with carotid artery disease. A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives. The low concentration and patchy distribution of C. pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.

V��rus dengue em larvas de Aedes aegypti e sua din��mica de infesta����o, Roraima, Brasil

OBJETIVO: Identificar a presen��a do v��rus dengue em formas larvais de Aedes aegypti e relacionar a presen��a do vetor com ��ndice pluviom��trico e n��mero de casos de dengue. M��TODOS: Dezoito domic��lios foram selecionados aleatoriamente para coleta de ovos em um bairro da cidade de Boa Vista (RR). Foram instaladas duas ovitrampas por domic��lio e removidas ap��s uma semana, mensalmente, de novembro de 2006 a maio de 2007. Foram calculados o ��ndice de positividade de ovitrampa e o ��ndice de densidade dos ovos. Ap��s eclos��o de 1.422 ovos coletados, foram formados 44 pools de no m��ximo 30 larvas para teste de presen��a do v��rus dengue por meio de RT-PCR e hemi-nested PCR. O ��ndice de incid��ncia de dengue no per��odo foi correlacionado com a precipita����o pluvial. A associa����o entre essas vari��veis e n��mero de ovos coletados foi analisada pelo coeficiente de Pearson. RESULTADOS: Nenhum dos pools apresentou positividade para o v��rus dengue, apesar do bairro ter apresentado elevados ��ndices de incid��ncia de dengue no per��odo estudado. A densidade da popula����o de Ae. aegypti aumentou conforme a pluviosidade, mas n��o apresentou correla����o com ��ndices de incid��ncia de casos de dengue. CONCLUS��ES: Os resultados sugerem que a transmiss��o transovariana do v��rus em mosquitos ocorre a uma freq����ncia muito baixa e por isso sua persist��ncia em meio urbano pode n��o depender desse fen��meno. A popula����o do mosquito aumentou no per��odo de chuvas devido �� forma����o de criadouros; a n��o-correla����o com o ��ndice de incid��ncia de dengue deve-se �� possibilidade desse dado ser subestimado em per��odos de epidemia.

Implementa����o de m��todos moleculares para o diagn��stico precoce de leptospirose humana

Disserta����o apresentada na Faculdade de Ci��ncias e Tecnologia da Universidade Nova de Lisboa para a obten����o do grau de Mestre em Biotecnologia

Contributo para o estudo da infec����o cong��nita pelo v��rus citomeg��lico em Portugal

Resumo O v��rus citomeg��lico humano (CMV) �� o principal agente de infec����o cong��nita, atingindo cerca de 0.2 a 2.2% de todos os rec��m-nascidos. As crian��as que nascem infectadas por este v��rus t��m cerca de 11% a 12.7% de probabilidades de apresentarem sintomas e sinais de doen��a citomeg��lica ao nascimento, podendo cerca de 40 a 58% destas virem a apresentar sequelas neurol��gicas permanentes. Das crian��as infectadas que ter��o infec����o assintom��tica no per��odo neo-natal, 5 a 15% poder��o vir igualmente a sofrer de sequelas tardias, sobretudo a surdez ou o atraso mental. Em Portugal, desconhece-se a dimens��o deste problema. O primeiro objectivo desta disserta����o foi, desta forma, a determina����o da preval��ncia atrav��s do recurso aos cart��es do diagn��stico precoce (���Guthrie cards���), utilizando uma t��cnica de nested-PCR dirigida para o v��rus. Foram estudados 3600 cart��es, seleccionados de todo o territ��rio nacional (continente e ilhas), de uma forma proporcional ao n��mero de nascimentos em cada distrito, dos quais 38 foram positivos, o que d�� uma preval��ncia de 1.05% (intervalo de confian��a para 95%: 0.748-1.446). A revis��o sobre a experi��ncia acumulada nos ��ltimos 15 anos, na ��rea do diagn��stico pr��-natal, juntamente com um estudo adicional sobre a t��cnica da avidez, permitiu retirar algumas ila����es, nomeadamente que este diagn��stico constitui uma arma diagnostica fi��vel para a avalia����o pr��-natal desta infec����o cong��nita e que a selec����o dos casos para amniocentese dever�� obedecer a indica����es serol��gicas precisas, como a ���seroconvers��o para IgG��� ou a ���IgM confirmada��� (devendo o m��todo de confirma����o ser a avidez das IgG com um ��ndice <0,6) e as altera����es ecogr��ficas de etiologia n��o esclarecida. A possibilidade de utilizar pools de urinas para detectar a infec����o cong��nita por CMV foi abordada na terceira parte do trabalho experimental. A metodologia a�� descrita teve correla����o total com o m��todo de refer��ncia, permitindo uma redu����o bastante significativa nos tempos de execu����o e nos custos em consum��veis, pelo que abre a possibilidade da sua utiliza����o para o rastreio da infec����o cong��nita por CMV nos rec��m-nascidos.

Chlamydia trachomatis Infec����o na gr��vida e no rec��m-nascido

A infec����o urogenital causada por Chlamydia trachomatis �� a doen��a bacteriana sexualmente transmiss��vel mais comum na Europa, tanto em homens como em mulheres. Constitui um grave problema de sa��de p��blica devido �� elevada percentagem de portadores assintom��ticos, ��s complica����es cl��nicas que da�� podem resultar e �� possibilidade de transmiss��o vertical. O presente trabalho teve como principais objectivos: i) avaliar a preval��ncia da infec����o por C. trachomatis e por Neisseria gonorrhoeae num grupo de gr��vidas de 36 semanas atendidas na consulta externa de Obstetr��cia do Hospital Amadora Sintra e nos rec��m-nascidos de m��es infectadas, ii) identificar os serovares respons��veis pelas infec����es por C. trachomatis, iii) verificar a distribui����o da preval��ncia da infec����o por C. trachomatis em fun����o da idade e iv) avaliar a utilidade de uma t��cnica de PCR multiplex e de PCR multiplex em tempo real no diagn��stico desta infec����o. Foram testadas 1201 amostras de urina do primeiro jacto de gr��vidas e 18 exsudados oculares provenientes de rec��m-nascidos cujas m��es estavam infectadas com C. trachomatis. Cada amostra foi testada pelas t��cnicas de PCR multiplex e de PCR multiplex em tempo real, tendo como alvos de amplifica����o um fragmento do plasm��dio cr��ptico e outro do gene omp1. Todos os resultados positivos foram confirmados com uma t��cnica de nested PCR e posteriormente enviados para sequencia����o para identifica����o dos serovares envolvidos. Em todas as amostras foi ainda pesquisada a presen��a de ADN de N. gonorrhoeae atrav��s de t��cnicas de PCR e de PCR em tempo real sendo que, na primeira, o alvo a amplificar foi um fragmento do gene ccpB do plasm��dio pJDI e na segunda o pseudogene porA. Os resultados positivos foram confirmados por RFLP. A preval��ncia da infec����o por C. trachomatis e por N. gonorrhoeae foi de 3,7% (45/1201) e de 0,08% (1/1201), respectivamente. Nos rec��m-nascidos, a preval��ncia foi de 0% para ambas as infec����es, embora o n��mero de rec��m-nascidos estudados (18/45) dificilmente seja representativo. O serovar mais prevalente foi o E (31,1%), seguido do G (15,6%), do D/Da (13,3%), do F, I/Ia e do J (11,1%). O serovar K foi identificado em 4,4% das amostras infectadas e o H em apenas 2,2%. A t��cnica de PCR multiplex em tempo real parece ser mais adequada para o diagn��stico da infec����o por C. trachomatis do que a t��cnica de PCR multiplex, tendo a primeira detectado 100% dos casos de infec����o por este microrganismo (45/45), enquanto que a segunda detectou apenas 71% (32/45) dos mesmos.

Cytomegalovirus infection in children with Down syndrome in a day-care center in Brazil

This study evaluates the transmission of CMV infection in 120 children aged 1 to 15 years with Down syndrome who attended a day-care center for handicapped children in S��o Paulo, Brazil. A blood sample was obtained from each children at the beginning of the study for detection of IgG and IgM cytomegalovirus (CMV) antibodies by an immunofluorescence assay. Samples of saliva and urine were obtained every 3 months from the children with CMV antibodies to detect shedding of the virus by culture in human foreskin fibroblasts, by detection of pp65 CMV-antigen and by a nested PCR assay. The prevalence of anti CMV-IgG antibodies was 76.6% (92/120), and IgM anti-CMV antibodies were detected in 13% (12/92) of the seropositive children. During the first viral evaluation, CMV was detected in the urine and/or saliva in 39/90 (43.3%) of the seropositive children. In the second and third evaluations, CMV was detected in 41/89 (46%) and in 35/89 (39.3%) children, respectively. Detection of CMV was shown both in urine and saliva in 28/39 (71.8%), 19/41(46.3%) and 20/35 (57.1%) of the children excreting the virus, respectively. Additionally, in 33/49 (67.4%) of the excreters CMV could be demonstrated in urine or saliva in at least two out of the three virological evaluations carried out sequentially in a six month period. Of the 28 initially seronegative children, 26 were re-examined for anti-CMV IgG antibodies about 18 months after the negative sample; seroconversion was found in 10/26 (38.5%). Taking all 536 samples of urine or saliva examined by virus culture and pp65 antigen detection during the study into account, 159 (29.6%) were positive by virus culture and 59 (11%) gave a positive result with the pp65 assay. These data demonstrate the high prevalence of CMV shedding and the high risk of CMV infection in children with Down syndrome attending a day-care center for mentally handicapped patients. The virus culture was more sensitive than the pp65 CMV antigen assay for CMV detection in both urine and saliva samples.

Outbreaks of human-herpes virus 6 (HHV-6) infection in day-care centers in Bel��m, Par��, Brazil

A total of 730 children aged less than 7 years, attending 8 day-care centers (DCCs) in Bel��m, Brazil were followed-up from January to December 1997 to investigate the occurrence of human-herpes virus 6 (HHV-6) infection in these institutional settings. Between October and December 1997 there have been outbreaks of a febrile- and -exanthematous disease, affecting at least 15-20% of children in each of the DCCs. Both serum- and- plasma samples were obtained from 401 (55%) of the 730 participating children for the detection of HHV-6 antibodies by enzyme-linked immunosorbent assay (ELISA), and viral DNA amplification through the nested-PCR. Recent HHV-6 infection was diagnosed in 63.8% (256/401) of them, as defined by the presence of both IgM and IgG-specific antibodies (IgM+/IgG+); of these, 114 (44.5%) were symptomatic and 142 (55.5%) had no symptoms (p = 0.03). A subgroup of 123 (30.7%) children were found to be IgM-/IgG+, whereas the remaining 22 (5.5%) children had neither IgM nor IgG HHV-6- antibodies (IgM-/IgG-). Of the 118 children reacting strongly IgM-positive ( > or = 30 PANBIO units), 26 (22.0%) were found to harbour the HHV-6 DNA, as demonstrated by nested-PCR. Taken the ELISA-IgM- and- nested PCR-positive results together, HHV-6 infection was shown to have occurred in 5 of the 8 DCCs under follow-up. Serological evidence of recent infections by Epstein-Barr virus (EBV) and parvovirus B19 were identified in 2.0% (8/401) and 1.5% (6/401) of the children, respectively. Our data provide strong evidence that HHV-6 is a common cause of outbreaks of febrile/exanthematous diseases among children attending DCCs in the Bel��m area.