Avaliação do valor prognóstico da detecção do status quimérico de pacientes após o transplante alogênico de células progenitoras hematopoéticas

Este trabalho teve por objetivo correlacionar o status quimérico de pacientes pós -TCPH alogênico com parâmetros clínicos, para avaliar o valor preditivo dos achados laboratorias de quimerismo. Amostras de sangue de 98 pacientes (67 em seguimento e 31 novos casos) foram submetidas à análise do status quimérico pós-TCPH. Os locianalisados por biologia molecular foram CS1PO, TPOX, F13A1, FESFPS, HUMTH01, VWA, SE33, HUMARA, HUMD21S11 e Amelogenina. Precocidade da evidência laboratorial de quimerismo misto (QM), em relação ao aparecimento dos sintomas clínicos de recaída, foi observada em 9 dos 12 pacientes nas LA, ou seja, nesses casos, a primeira manifestação de QM foi detectada pelo exame laboratorial antes de qualquer evidência citológica ou clínica de recaída. em todos eles, houve uma mudança terapêutica relacionada com esse momento do aparecimento do QM. em 100% dos pacientes com QM na LMC, a detecção do quimerismo pelo exame laboratorial foi anterior a qualquer evidência citológica ou clínica de recaída. de uma maneira geral, o exame laboratorial da avaliação do status quimérico pós-TCPH alogênico pela análise dos locihipervariáveis do genoma, mostrou ser um exame sensível, com detecção de até 1% de QM e precoce, visto que, muitas vezes, foi a primeira manifestação de doença residual antes de qualquer evidência citológica ou clínica da mesma. A associação da existência de QM e a recaída clínica e/ou óbito fica mais evidente nos casos de LA do que nos casos de LMC e AAS.

A STR mutation in a heteropaternal twin case

A heteropaternal male twin case with two men being alleged fathers was investigated as requested by the Court. Up to 37 PCR-based polymorphic DNA systems were studied in this case which was complicated by a paternal ACTBP2 mutation detected in one twin. This is the first report on a STR mutation in a double paternity case where both biological fathers were indisputably identified. The STR systems enable the resolution of these complex genetic relationships even in a case where a mutation in one STR locus was encountered.

DIP-STR: A new marker for resolving unbalanced DNA mixtures

The genetic characterization of unbalanced mixed stains remains an important area where improvementis imperative. Most cases of aggression, homicide and sexual assault produce biological traces withrelatively large amount of the victim's DNA and small amount of the aggressor's DNA. If this ratio issmaller than 1:10 it is currently not possible to obtain a conventional autosomal DNA profile of the minorcontributor, with potential loss of crucial DNA evidence. Y-STR analysis represents a solution for somecases but has several limitations. We propose here a method based on a new compound genetic markerformed by a Deletion/Insertion Polymorphism (DIP) linked to a Short Tandem Repeat polymorphism(STR), that we name DIP-STR. By means of allele-specific amplifications of DIP-STR haplotypes, we canproduce a high resolution autosomal DNA profile of a donor that contributes less than 0.1% to a DNAmixture. Based on these features DIP-STR markers may outperform conventional Y-STR markers inmixed stain analysis.

DIP-STR: Highly Sensitive Markers for the Analysis of Unbalanced Genomic Mixtures.

Samples containing highly unbalanced DNA mixtures from two individuals commonly occur both in forensic mixed stains and in peripheral blood DNA microchimerism induced by pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred as short tandem repeats (STR), and single-nucleotide polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP-STR that relies on pairing deletion-insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP-STR genotypes of the minor were successfully procured at ratios up to 1:1,000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP-STR approach on an initial set of nine markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures.

DNA extraction using the QIAsymphony: Evaluation of PCR inhibitor removal

The goal of this study was to compare the quantity and purity of DNA extracted from biological tracesusing the QIAsymphony robot with that of the manual QIAamp DNA mini kit currently in use in ourlaboratory. We found that the DNA yield of robot was 1.6-3.5 times lower than that of the manualprotocol. This resulted in a loss of 8% and 29% of the alleles correctly scored when analyzing 1/400 and 1/800 diluted saliva samples, respectively. Specific tests showed that the QIAsymphony was at least 2-16times more efficient at removing PCR inhibitors. The higher purity of the DNA may therefore partlycompensate for the lower DNA yield obtained. No case of cross-contamination was observed amongsamples. After purification with the robot, DNA extracts can be automatically transferred in 96-wellsplates, which is an ideal format for subsequent RT-qPCR quantification and DNA amplification. Lesshands-on time and reduced risk of operational errors represent additional advantages of the robotic platform.

Aplicação de microssatélites (STR) de bovinos em animais de raças zebuínas

O gado zebu (Bos indicus) é predominante em todo o mundo. Atualmente, no Brasil, o rebanho de bovinos e zebuínos é composto por aproximadamente 170 milhões de indivíduos, no qual, 80% são zebuínos ou animais de raças cruzadas com zebu. O principal objetivo deste estudo foi determinar a possibilidade da utilização de marcadores moleculares de bovinos na identificação genética de zebuínos. A confirmação desta possibilidade seria de extrema valia, já que a quantidade de informações sobre o genoma zebuíno é limitada e metodologias específicas de identificação de marcadores genéticos para raças zebuínas são raras. Assim, o DNA de 65 zebus, de 4 raças diferentes (Gir, Tabapuã, Nelore e Brahman), foi extraído a partir de sangue aplicado em papel filtro, utilizando o “kit” DNA IQTM SYSTEM (Promega®). Após a extração de DNA, foi feito um PCR Multiplex utilizando o “kit” StockMarks® (Applied Biosystems®), o qual amplifica um total de 11 loci (BM1824, BM2113, ETH10, ETH225, ETH3, INRA023, SPS115, TGLA122, TGLA126, TGLA227 e TGLA53). Os fragmentos gerados pela PCR foram analisados por eletroforese capilar utilizando o analisador genético ABI Prism 3100 (Applied Biosystems®) e o programa GeneScan® v.3.7 (Applied Biosystems®). A freqüência dos marcadores e os índices de variabilidade genética foram calculados utilizando o programa Microsatellite Toolkit v.3.1 e o equilíbrio de Hardy-Weinberg foi determinado através do programa GENEPOP v.3.4. Através destas técnicas foi possível demonstrar que os marcadores moleculares utilizados para identificação genética de bovinos podem também ser utilizados para a realização da técnica de identificação genética de zebuínos Além disso, foi possível demonstrar a freqüência alélica na população de zebuínos analisada como um todo, assim como foi determinada a freqüência alélica para os diferentes marcadores moleculares dentro das 4 raças estudas. Considerando o total de 100 diferentes alelos identificados entre os 11 STR (Short Tandem Repeats) analisados, foi possível observar que a heterozigosidade esperada (He) foi relativamente alta na população analisada, assim como na análise por raça. Estes dados indicam alto grau de diversidade genética nas diferentes raças. Neste sentido, a raça Brahman apresentou a menor diversidade (0,60) e a raça Tabapuã, a maior (0,69). Contudo, estudos complementares são necessários, a fim de confirmar as freqüências alélicas estabelecidas, uma vez que os resultados encontrados no presente trabalho referem-se a um número relativamente pequeno de zebuínos, quando comparados com o total de zebus encontrados no rebanho brasileiro.

Análise da frequência alélica de 15 LOCI STR na população do Rio Grande do Norte

Human population have a significant number of polymorphic loci, whose use and applications range from construction of linkage maps, to study the evolution of populations, through the determination of paternity, forensic medicine and migration. Currently, STRs (Short Tanden Repeats) markers are considered the major markers for human identification, mainly due to its abundance and high variability because of the fact that they are easily amplifiable by PCR (Polymerase Chain Reaction), work with low amounts of DNA and be capable of automation processes involving fluorescence detection. The creation of regional databases containing allele frequencies of population provide subsidies to increase the reliability of the results of determining the genetic link. This paper aims to obtain a database of allele frequencies of 15 polymorphic molecular loci (D8S1179, D21S11, D7S820, CSF1PO, D19S433, vWA, TPOX, D18S51, D3S1358, TH01, D13S317, D16S539, D2S1338, D5S818 e FGA) in a population classifies as born in the State of Rio Grande do Norte, Brazil, totaling 1100 unrelated individuals. To evaluate the frequency, DNA samples were submitted to PCR amplification, followed by capilarry electrophoresis genetic sequencer. The frequencies identified in this study were compared with brazilian population in general and other states in Brazil. Except for the loci D21S11, D19S433 and D2D1338, the genotypes found were in Hardy-Weinberg equilibrium and no significant differences among the frequencies were found in the populations studied. The most informative loci was D2S1338 and D18S51, and the less informative is the locus TPOX

Results of the GEP-ISFG collaborative study on an X-STR Decaplex

A collaborative work was carried out by the Spanish and Portuguese ISFG Working Group with a PCR multiplex for X chromosome STRs. Markers were selected among those described as polymorphic in humans and that have been used by some laboratories in forensics. Primers and various technical methods were investigated with the aim of optimizing a multiplex for the 10 selected X-STRs. Primer mix stock solutions were sent to the laboratories that were asked to analyse two female bloodstains, taking as reference the genetic profiles from 9947A, 9948 and NA3657 samples. In this work, we report the results obtained by 30 GEP-ISFG laboratories, using this Decaplex, as well as alternative technical conditions that also produced good results. © 2008 Elsevier B.V. All rights reserved.

Assessment of STR Typing Success Rate in Soft Tissues from Putrefied Bodies Based on a Quantitative Grading System for Putrefaction.

To date, there is no systematic investigation of the association of short tandem repeat (STR) typing success rate in soft tissues with different signs of putrefaction. Herein, putrefaction was rated using a newly developed 19-parameter system in soft tissues from a collective of 68 decaying bodies, and DNA yield was determined in 408 samples. DNA integrity was rated using a self-devised pentaplex PCR generating an "integrity score" (Si ). STR typing success rate was then assessed for selected cases. DNA yield and Si differed significantly between tissues with kidney on average exhibiting the highest Si values. Statistical analysis revealed that nine parameters were significantly and positively correlated with Si . The observed values for each of these nine parameters were summed up to generate a putrefaction score (Sp ) for each sample. Our results show that STR typing success rate can be predicted based on Sp before expensive multiplex STR profiling is performed.

Phylogenetics, Ancestral State Reconstruction, And A New Infrafamilial Classification Of The Pantropical Ochnaceae (medusagynaceae, Ochnaceae S.str., Quiinaceae) Based On Five Dna Regions.

Ochnaceae s.str. (Malpighiales) are a pantropical family of about 500 species and 27 genera of almost exclusively woody plants. Infrafamilial classification and relationships have been controversial partially due to the lack of a robust phylogenetic framework. Including all genera except Indosinia and Perissocarpa and DNA sequence data for five DNA regions (ITS, matK, ndhF, rbcL, trnL-F), we provide for the first time a nearly complete molecular phylogenetic analysis of Ochnaceae s.l. resolving most of the phylogenetic backbone of the family. Based on this, we present a new classification of Ochnaceae s.l., with Medusagynoideae and Quiinoideae included as subfamilies and the former subfamilies Ochnoideae and Sauvagesioideae recognized at the rank of tribe. Our data support a monophyletic Ochneae, but Sauvagesieae in the traditional circumscription is paraphyletic because Testulea emerges as sister to the rest of Ochnoideae, and the next clade shows Luxemburgia+Philacra as sister group to the remaining Ochnoideae. To avoid paraphyly, we classify Luxemburgieae and Testuleeae as new tribes. The African genus Lophira, which has switched between subfamilies (here tribes) in past classifications, emerges as sister to all other Ochneae. Thus, endosperm-free seeds and ovules with partly to completely united integuments (resulting in an apparently single integument) are characters that unite all members of that tribe. The relationships within its largest clade, Ochnineae (former Ochneae), are poorly resolved, but former Ochninae (Brackenridgea, Ochna) are polyphyletic. Within Sauvagesieae, the genus Sauvagesia in its broad circumscription is polyphyletic as Sauvagesia serrata is sister to a clade of Adenarake, Sauvagesia spp., and three other genera. Within Quiinoideae, in contrast to former phylogenetic hypotheses, Lacunaria and Touroulia form a clade that is sister to Quiina. Bayesian ancestral state reconstructions showed that zygomorphic flowers with adaptations to buzz-pollination (poricidal anthers), a syncarpous gynoecium (a near-apocarpous gynoecium evolved independently in Quiinoideae and Ochninae), numerous ovules, septicidal capsules, and winged seeds with endosperm are the ancestral condition in Ochnoideae. Although in some lineages poricidal anthers were lost secondarily, the evolution of poricidal superstructures secured the maintenance of buzz-pollination in some of these genera, indicating a strong selective pressure on keeping that specialized pollination system.

Post Hoc Analysis Of The Patricia Randomized Trial Of The Efficacy Of Human Papillomavirus Type 16 (hpv-16)/hpv-18 As04-adjuvanted Vaccine Against Incident And Persistent Infection With Nonvaccine Oncogenic Hpv Types Using An Alternative Multiplex Type-specific Pcr Assay For Hpv Dna.

The efficacy of the human papillomavirus type 16 (HPV-16)/HPV-18 AS04-adjuvanted vaccine against cervical infections with HPV in the Papilloma Trial against Cancer in Young Adults (PATRICIA) was evaluated using a combination of the broad-spectrum L1-based SPF10 PCR-DNA enzyme immunoassay (DEIA)/line probe assay (LiPA25) system with type-specific PCRs for HPV-16 and -18. Broad-spectrum PCR assays may underestimate the presence of HPV genotypes present at relatively low concentrations in multiple infections, due to competition between genotypes. Therefore, samples were retrospectively reanalyzed using a testing algorithm incorporating the SPF10 PCR-DEIA/LiPA25 plus a novel E6-based multiplex type-specific PCR and reverse hybridization assay (MPTS12 RHA), which permits detection of a panel of nine oncogenic HPV genotypes (types 16, 18, 31, 33, 35, 45, 52, 58, and 59). For the vaccine against HPV types 16 and 18, there was no major impact on estimates of vaccine efficacy (VE) for incident or 6-month or 12-month persistent infections when the MPTS12 RHA was included in the testing algorithm versus estimates with the protocol-specified algorithm. However, the alternative testing algorithm showed greater sensitivity than the protocol-specified algorithm for detection of some nonvaccine oncogenic HPV types. More cases were gained in the control group than in the vaccine group, leading to higher point estimates of VE for 6-month and 12-month persistent infections for the nonvaccine oncogenic types included in the MPTS12 RHA assay (types 31, 33, 35, 45, 52, 58, and 59). This post hoc analysis indicates that the per-protocol testing algorithm used in PATRICIA underestimated the VE against some nonvaccine oncogenic HPV types and that the choice of the HPV DNA testing methodology is important for the evaluation of VE in clinical trials. (This study has been registered at ClinicalTrials.gov under registration no. NCT00122681.).

Human Herpesvirus Infections Of The Central Nervous System: Laboratory Diagnosis Based On Dna Detection By Nested Pcr In Plasma And Cerebrospinal Fluid Samples.

Infections of the central nervous systems (CNS) present a diagnostic problem for which an accurate laboratory diagnosis is essential. Invasive practices, such as cerebral biopsy, have been replaced by obtaining a polymerase chain reaction (PCR) diagnosis using cerebral spinal fluid (CSF) as a reference method. Tests on DNA extracted from plasma are noninvasive, thus avoiding all of the collateral effects and patient risks associated with CSF collection. This study aimed to determine whether plasma can replace CSF in nested PCR analysis for the detection of CNS human herpesvirus (HHV) diseases by analysing the proportion of patients whose CSF nested PCR results were positive for CNS HHV who also had the same organism identified by plasma nested PCR. In this study, CSF DNA was used as the gold standard, and nested PCR was performed on both types of samples. Fifty-two patients with symptoms of nervous system infection were submitted to CSF and blood collection. For the eight HHV, one positive DNA result-in plasma and/or CSF nested PCR-was considered an active HHV infection, whereas the occurrence of two or more HHVs in the same sample was considered a coinfection. HHV infections were positively detected in 27/52 (51.9%) of the CSF and in 32/52 (61.5%) of the plasma, difference not significant, thus nested PCR can be performed on plasma instead of CSF. In conclusion, this findings suggest that plasma as a useful material for the diagnosis of cases where there is any difficulty to perform a CSF puncture.

As Apocynaceae s. str. da região de Bauru, São Paulo, Brasil

The present study describes a survey of the Apocynaceae in different types of vegetation, in the region of Bauru, in the Center-West of São Paulo State. Of the 25 species encountered, distributed into 15 genera, representation ranges as follows: Forsteronia G. Mey, five species; Aspidosperma Mart., four species; Mandevilla Lindl., three species; Prestonia R. Br., two species; and Condylocarpon Desf., Hancornia Gomez, Himatanthus Willd. ex Roem. et Schult., Macrosiphonia Müll. Arg., Mesechites Müll. Arg., Odontadenia Benth, Peltastes Woodson, Rhodocalyx Müll Arg., Secondatia A. DC., Tabernaemontana L. and Temnadenia Miers, one species each. In addition to both distribution data, and flowering and fruiting times, identifications keys, descriptions and illustrations of the species are presented.

Revisão taxonômica de Lonchocarpus s. str. (Leguminosae, Papilionoideae) do Brasil

A revision of the Brazilian species of Lonchocarpus s. str. is presented. This study is based on field observation and an analysis of approximately 1,200 herbarium collections. Nine species are recognized, L. cultratus, L. hedyosmus, L. latifolius, L. macrocarpus, L. nitidus, L. pluvialis, L. sericeus, L. spiciflorus, and L. violaceus, which grow in forests and are usually associated with river banks. Lonchocarpus sericeus and L. cultratus have a wide distribution throughout Brazil, whereas L. hedyosmus, L. macrocarpus, L. spiciflorus, and L. latifolius are restricted to the Amazonian domain. Lonchocarpus pluvialis occurs in the Central-West (Mato Grosso do Sul and Goiás) and Southeast (São Paulo) regions. Lonchocarpus violaceus is found in the states of Bahia and Espírito Santo, and is reported for the first time for Brazil. Identification keys, descriptions, and illustrations, in addition to information about habitat, geographic distribution and taxonomic and nomenclatural comments, are provided for the species. Four new synonyms and five lectotypifications are proposed.

Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308

The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p<0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and viceversa. Therefore, the best strategy for B. abortus detection in the organs of aborted fetuses or calves born from infected cows is the use, in parallel, of isolation by classical bacteriology and the PCR, with the DNA extraction performed by the Boom protocol.