Immunodeficiency and the risk of cervical intraepithelial neoplasia 2/3 and cervical cancer: A nested case-control study in the Swiss HIV cohort study.

HIV-infected women are at increased risk of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC), but it has been difficult to disentangle the influences of heavy exposure to HPV infection, inadequate screening and immunodeficiency. A case-control study including 364 CIN2/3 and 20 ICC cases matched to 1,147 controls was nested in the Swiss HIV Cohort Study (1985-2013). CIN2/3 risk was significantly associated with low CD4+ cell counts, whether measured as nadir [odds ratio (OR) per 100-cell/μL decrease = 1.15, 95% CI: 1.08, 1.22], or at CIN2/3 diagnosis (1.10, 95% CI: 1.04, 1.16). An association was evident even for nadir CD4+ 200-349 versus ≥350 cells/μL (OR = 1.57, 95% CI: 1.09, 2.25). After adjustment for nadir CD4+, a protective effect of >2-year cART use was seen against CIN2/3 (OR versus never cART use = 0.64, 95% CI: 0.42, 0.98). Despite low study power, similar associations were seen for ICC, notably with nadir CD4+ (OR for 50 vs. >350 cells/μL= 11.10, 95% CI: 1.24, 100). HPV16-L1 antibodies were significantly associated with CIN2/3, but HPV16-E6 antibodies were nearly exclusively detected in ICC. In conclusion, worsening immunodeficiency, even at only moderately decreased CD4+ cell counts, is a significant risk factor for CIN2/3 and cervical cancer.

A rapid and sensitive diagnosis of bovine leukaemia virus infection using the nested shuttle polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). In Argentina, where a program to eradicate EBL has been introduced, sensitive and reliable diagnosis has attained high priority. Although the importance of the agar gel immunodiffusion test remains unchanged for routine work, an additional diagnostic technique is necessary to confirm cases of sera with equivocal results or of calves carrying maternal antibodies.Utilizing a nested shuttle polymerase chain reaction, the proviral DNA was detected from cows experimentally infected with as little as 5 ml of whole blood from BLV seropositive cows that were nonetheless normal in haematological terms. It proved to be a very sensitive technique, since it rapidly revealed the presence of the provirus, frequently at 2 weeks postinoculation and using a two-round procedure of nested PCR taking only 3 hours. Additionally, the primers used flanked a portion of the viral genome often employed to differentiate BLV type applying BamHI digestion. It is concluded that this method might offer a highly promising diagnostic tool for BLV infection.

Um protocolo de "nested-PCR" para detecção do vírus da anemia das galinhas

Este trabalho descreve o estabelecimento de um pro!tocolo de "nested-PCR" para a detecção do vírus da anemia das galinhas (CAV, chicken anemia virus), agente causador da anemia infecciosa das galinhas. Para a extração de DNA a partir de amostras clínicas um método baseado no uso de tiocianato de guanidina mostrou-se mais sensível e prático, do que os demais avaliados. Para a PCR inicial foi selecionado um par de primers que amplifica uma região de 664 pares de bases (pb) do gene VP1. Para a "nested-PCR" propriamente dita, foi selecionado um segundo par que amplifica uma região interna de 520 pb. A especificidade dos primers foi avaliada utilizando amostras de lotes controlados para CAV. Outras trinta amostras vírus e bactérias, causadoras de doenças em aves, não geraram produto de amplificação. A sensibilidade do teste foi determinada a partir de diluições seriadas de uma amostra vacinal de CAV. A "nested-PCR" mostrou ser mais sensível do que a PCR e foi capaz de detectar pelo menos 0,16 TCID50% da cepa vacinal. Além disso, detectou DNA viral em tecidos, soro e cama aviária de lotes com e sem sinais clínicos. Conclui-se que, como técnica para a detecção do CAV, o protocolo de "nested-PCR" aqui descrito, é mais sensível, rápido e menos trabalhoso do que o isolamento viral em cultivo celular.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

Conventional PCR (PCRTeq) for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc) for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq) for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128), but did not differ significantly from the M/PCRTeq-Bc (1:64). In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780) and moderate agreement with N/PCR-Teq (k = 0.562) and M/PCRTeq-Bc (k = 0.488). PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05), and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05). PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeirão Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2%) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2% of the patients with CMV infection were symptomatic.

Typing class I HLA-A gene using a nested PCR-RFLP procedure

In order to detect several new HLA-A class I alleles that have been described since 1998, the original PCR-RFLP method developed to identify the 78 alleles recognized at that time at high resolution level was adapted by us for low and medium resolution levels using a nested PCR-RFLP approach. The results obtained from blood samples of 23 subjects using both the PCR-RFLP method and a commercial kit (MicroSSP1A®, One Lambda Inc.) showed an agreement higher than 95%. The PCR-RFLP adapted method was effective in low and medium resolution histocompatibility evaluations.

Message Integrity in the World Wide Web: Use of Nested Hash Function and a Fast Stream Cipher

The focus of this work is to provide authentication and confidentiality of messages in a swift and cost effective manner to suit the fast growing Internet applications. A nested hash function with lower computational and storage demands is designed with a view to providing authentication as also to encrypt the message as well as the hash code using a fast stream cipher MAJE4 with a variable key size of 128-bit or 256-bit for achieving confidentiality. Both nested Hash function and MAJE4 stream cipher algorithm use primitive computational operators commonly found in microprocessors; this makes the method simple and fast to implement both in hardware and software. Since the memory requirement is less, it can be used for handheld devices for security purposes.

Virtualization of seed research: perhaps a continuity model.

The research nurseries serve as a initiative designed for the purpose of contribute to the breakdown of seizure paradigms of knowledge, supported by a theoretical model of discernment that exceed the traditional method of learning in the classroom, to transcend critical insights into transforming the environment under an autonomous space to provide tools essentially scientific, but with ethical criterion and social commitment. Such a conception, however, could be perceived as talkative, and even metaphorical, at the contrast of reality purely versatile in which the neophyte not only tries to get enough of the fruit of knowledge, but also requires that appropriate strategies are put forward to achieve seizure. Then arises, from the formative entelechy the use of technological tools based on virtual learning environments, in order to enhance motivation as an alternative against the blurring of perspectives and the emerging business in some types of nurseries, stranded attitude, that sometimes not far from the master model.

Estimating the spatial scales of regionalized variables by nested sampling, hierarchical analysis of variance and residual maximum likelihood

The variogram is essential for local estimation and mapping of any variable by kriging. The variogram itself must usually be estimated from sample data. The sampling density is a compromise between precision and cost, but it must be sufficiently dense to encompass the principal spatial sources of variance. A nested, multi-stage, sampling with separating distances increasing in geometric progression from stage to stage will do that. The data may then be analyzed by a hierarchical analysis of variance to estimate the components of variance for every stage, and hence lag. By accumulating the components starting from the shortest lag one obtains a rough variogram for modest effort. For balanced designs the analysis of variance is optimal; for unbalanced ones, however, these estimators are not necessarily the best, and the analysis by residual maximum likelihood (REML) will usually be preferable. The paper summarizes the underlying theory and illustrates its application with data from three surveys, one in which the design had four stages and was balanced and two implemented with unbalanced designs to economize when there were more stages. A Fortran program is available for the analysis of variance, and code for the REML analysis is listed in the paper. (c) 2005 Elsevier Ltd. All rights reserved.

Estimating the spatial scale of herbicide and soil interactions by nested sampling, hierarchical analysis of variance and residual maximum likelihood

An unbalanced nested sampling design was used to investigate the spatial scale of soil and herbicide interactions at the field scale. A hierarchical analysis of variance based on residual maximum likelihood (REML) was used to analyse the data and provide a first estimate of the variogram. Soil samples were taken at 108 locations at a range of separating distances in a 9 ha field to explore small and medium scale spatial variation. Soil organic matter content, pH, particle size distribution, microbial biomass and the degradation and sorption of the herbicide, isoproturon, were determined for each soil sample. A large proportion of the spatial variation in isoproturon degradation and sorption occurred at sampling intervals less than 60 m, however, the sampling design did not resolve the variation present at scales greater than this. A sampling interval of 20-25 m should ensure that the main spatial structures are identified for isoproturon degradation rate and sorption without too great a loss of information in this field.