Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Performance of direct immunofluorescence assay for the detection of human metapneumovirus under clinical laboratory settings

ABSTRACT INTRODUCTION: Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofluorescence (DIF) to detect hMPV in a clinical laboratory setting. METHODS: Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. RESULTS: In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofluorescence specificity was 99% and sensitivity was 38%. CONCLUSIONS: DIF is not very sensitive under clinical laboratory settings.

Analysis of classification algorithms for crop detection using LANDSAT 8 images

Remote sensing - the acquisition of information about an object or phenomenon without making physical contact with the object - is applied in a multitude of different areas, ranging from agriculture, forestry, cartography, hydrology, geology, meteorology, aerial traffic control, among many others. Regarding agriculture, an example of application of this information is regarding crop detection, to monitor existing crops easily and help in the region’s strategic planning. In any of these areas, there is always an ongoing search for better methods that allow us to obtain better results. For over forty years, the Landsat program has utilized satellites to collect spectral information from Earth’s surface, creating a historical archive unmatched in quality, detail, coverage, and length. The most recent one was launched on February 11, 2013, having a number of improvements regarding its predecessors. This project aims to compare classification methods in Portugal’s Ribatejo region, specifically regarding crop detection. The state of the art algorithms will be used in this region and their performance will be analyzed.

Molecular detection of Trypanosoma sp. and Blastocrithidia sp. (Trypanosomatidae) in phlebotomine sand flies (Psychodidae) in the Federal District of Brazil

Abstract: INTRODUCTION : This study describes the occurrence of trypanosomatids in phlebotomines in Brasília, Brazil. METHODS : Two hundred and ten females of 13 sand fly species were analyzed by polymerase chain reaction (PCR) using different molecular markers (D7 24Sα rRNA, kDNA, and ITS1) and sequencing. RESULTS : PCR revealed trypanosomatid-positive samples from Nyssomyia whitmani and Evandromyia evandroi, which were negative by kDNA and ITS1 Leishmania-specific PCRs. DNA sequence analysis of D7 24Sα rRNA amplicons indicated the occurrence of Blastocrithidia sp. and Trypanosoma sp. in Nyssomyia whitmani and Evandromyia evandroi, respectively. CONCLUSIONS : Two trypanosomatid species other than Leishmania sp. were found to circulate in sand flies in Central Brazil.

Detection of canine visceral leishmaniasis by conjunctival swab PCR

Abstract: INTRODUCTION: Conjunctival swab PCR was evaluated as a tool to diagnose visceral leishmaniasis in dogs. METHODS: Conjunctival swab PCR was compared to indirect immunofluorescence antibody test and blood PCR. RESULTS: Indirect immunofluorescence was significantly correlated with conjunctival swab PCR (p < 0.05), but not with blood PCR (p > 0.05). In addition, conjunctival swab PCR was significantly associated with presence of clinical symptoms (p < 0.05), whereas blood PCR was associated with absence of clinical symptoms (p < 0.05). CONCLUSIONS: Results indicate that conjunctival swab PCR is useful in epidemiological surveys of canine visceral leishmaniasis.

Detection of codon 12 mutation in the k-ras oncogene in pancreatic tumors

Mutations at codons 12, 13, or 61 of the H-ras, K-ras, and N-ras have been detected in human neoplasias by a variety of techniques. Some of these techniques are very sensitive and can detect K-ras mutation in 90% of the cases of pancreatic adenocarcinomas. We analyzed 11 samples of pancreatic adenocarcinoma, three samples of pancreatic mucinous cystadenoma, and two samples without tumors in formalin-fixed paraffin embedded tissue sections. K-ras mutations at codon 12 were detected by a two-step PCR-enriched technique in all the samples of pancreatic adenocarcinoma, but not in cystadenoma or control samples. This technique may be useful for early detection of pancreatic cancer.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

Implementation of membrane processes for improvement of the detection of food contaminants

The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities (http://www.em3e.eu)

Detection of testicular cancer in men presenting with infertility

PURPOSE: Infertility is one of the less common presenting features associated with testicular tumors. We evaluated the histologic and biochemical findings, and pregnancy outcome in patients presenting with infertility who were found to have testicular tumors. METHODS: Seven patients with infertility were found to have testicular cancer over a 15-year period. All patients had a testicular ultrasound evaluation. The indications for the ultrasound were testicular pain in 2 patients, suspicious palpable mass in 4, and to rule out the presence of germ cell neoplasia in a patient with carcinoma in situ detected on a previous biopsy. Physical exam, histological findings, hormonal levels, tumor markers, and pregnancy outcome results were recorded from the patients medical charts. RESULTS: Two men had elevated serum follicle stimulant hormone and luteinizing hormone levels, 1 of them had an abnormally low serum testosterone level. Tumor markers were normal in all patients. In 4 patients the tumor was on the right side and in 3 on the left. The histological diagnoses were seminoma (n = 5), Leydig cell tumor (n = 1), and carcinoma in situ (n = 1). Of the 7 patients, 5 underwent adjuvant radiation therapy. Two patients had sperm cryopreserved. Follow up on fertility status was available in 6 cases. One patient has established a pregnancy and 5 did not achieve a pregnancy after treatment for their cancer. CONCLUSIONS: Most of the men who have testicular cancer and male infertility have a seminona. Therefore, men who present with infertility should be thoroughly investigated to rule out such serious, concomitant diseases along with their infertility.

Development and validation of gold nanoprobes for human SNP detection towards commercial application

Conventional molecular techniques for detection and characterization of relevant nucleic acid (i.e. DNA) sequences are, nowadays, cumbersome, expensive and with reduced portability. The main objective of this dissertation consisted in the optimization and validation of a fast and low-cost colorimetric nanodiagnostic methodology for the detection of single nucleotide polymorphisms (SNPs). This was done considering SNPs associated to obesity of commercial interest for STAB VIDA, and subsequent evaluation of other clinically relevant targets. Also, integration of this methodology into a microfluidic platform envisaging portability and application on points-of-care (POC) was achieved. To warrant success in pursuing these objectives, the experimental work was divided in four sections: i) genetic association of SNPs to obesity in the Portuguese population; ii) optimization and validation of the non-cross-linking approach for complete genotype characterization of these SNPs; iii) incorporation into a microfluidic platform; and iv) translation to other relevant commercial targets. FTO dbSNP rs#:9939609 carriers had higher body mass index (BMI), total body fat mass, waist perimeter and 2.5 times higher risk to obesity. AuNPs functionalized with thiolated oligonucleotides (Au-nanoprobes) were used via the non-cross-linking to validate a diagnostics approach against the gold standard technique - Sanger Sequencing - with high levels of sensitivity (87.50%) and specificity (91.67%). A proof-of-concept POC microfluidic device was assembled towards incorporation of the molecular detection strategy. In conclusion a successful framework was developed and validated for the detection of SNPs with commercial interest for STAB VIDA, towards future translation into a POC device.

Prevalência da C.trachomatis entre homens que fazem sexo com homens (HSH) que frequentam um centro clínico comunitário alternativo em Barcelona

RESUMO - Enquadramento: A infeção por Chlamydia trachomatis (CT) é considerada um grave problema de Saúde Pública. É causa de infeções sintomáticas tanto no homem como na mulher, bem como, de infeções assintomáticas que podem ter consequências muito graves a longo prazo. A presença de CT sem tratamento aumenta o risco de transmissão do vírus da imunodeficiência humana. Existem vários estudos publicados de prevalência da CT por todo o mundo, no entanto, estudos de prevalência da CT em populações assintomáticas, envolvendo homens que fazem sexo com homens (HSH) são raros na Europa. Assim, o presente estudo, ao estimar a prevalência e ao descrever as caraterísticas epidemiológicas e de conduta dos HSH, pretende contribuir para o plano de ação contra as IST´s e VIH na Catalunha, através de estratégias concretas para deteção e prevenção da CT. Métodos: Trata-se de um estudo observacional, transversal de prevalência da CT em HSH utentes de um serviço comunitário em Barcelona, com recurso a técnicas de diagnóstico de biologia molecular e a um questionário. Durante Março e Junho de 2015, foram recrutados 200 voluntários de um serviço comunitário em Barcelona que foram testados para CT e NG em três locais anatómicos através de uma PCR em tempo real utilizando o ensaio Anyplex™ CT/NG Real-time Detection. Resultados: O presente estudo permitiu uma caraterização da situação atual quanto á prevalência e aos fatores de risco associados á infeção por CT em HSH. A prevalência nesta população específica foi de 12,6% e os fatores risco associados foram o facto de serem VIH e praticarem sexo anal insertivo/recetivo com parceiro estável. Conclusões: Os resultados obtidos reforçam a necessidade do desenvolvimento de estratégias adequadas de controlo e prevenção da CT nesta população de risco, tais como: rastreios frequentes e tratamento dos casos positivos para quebrar a cadeia de transmissão, promoção da saúde, educação e notificação dos parceiros sexuais. Também demonstram a importância destes rastreios nos três locais anatómicos: uretra, reto e faringe.

Lightweight concrete masonry units based on processed granulate of corn cob as aggregate

A research work was performed in order to assess the potential application of processed granulate of corn cob (PCC) as an alternative lightweight aggregate for the manufacturing process of lightweight concrete masonry units (CMU). Therefore, CMU-PCC were prepared in a factory using a typical lightweight concrete mixture for non-structural purposes. Additionally, lightweight concrete masonry units based on a currently applied lightweight aggregate such as expanded clay (CMU-EC) were also manufactured. An experimental work allowed achieving a set of results that suggest that the proposed building product presents interesting material properties within the masonry wall context. Therefore, this unit is promising for both interior and exterior applications. This conclusion is even more relevant considering that corn cob is an agricultural waste product.

The influence of fibre orientation on the post-cracking tensile behaviour of steel fibre reinforced self-compacting concrete

Adding fibres to concrete provides several advantages, especially in terms of controlling the crack opening width and propagation after the cracking onset. However, distribution and orientation of the fibres toward the active crack plane are significantly important in order to maximize its benefits. Therefore, in this study, the effect of the fibre distribution and orientation on the post-cracking tensile behaviour of the steel fibre reinforced self-compacting concrete (SFRSCC) specimens is investigated. For this purpose, several cores were extracted from distinct locations of a panel and were subjected to indirect (splitting) and direct tensile tests. The local stress-crack opening relationship (σ-w) was obtained by modelling the splitting tensile test under the finite element framework and by performing an Inverse Analysis (IA) procedure. Afterwards the σ-w law obtained from IA is then compared with the one ascertained directly from the uniaxial tensile tests. Finally, the fibre distribution/orientation parameters were determined adopting an image analysis technique.

Bone turnover markers for early detection of fracture healing disturbances: A review of the scientific literature

Imaging techniques are the standard method for assessment of fracture healing processes. However, these methods are perhaps not entirely reliable for early detection of complications, the most frequent of these being delayed union and non-union. A prompt diagnosis of such disorders could prevent prolonged patient distress and disability. Efforts should be directed towards the development of new technologies for improving accuracy in diagnosing complications following bone fractures. The variation in the levels of bone turnover markers (BTMs) have been assessed with regard to there ability to predict impaired fracture healing at an early stage, nevertheless the conclusions of some studies are not consensual. In this article the authors have revised the potential of BTMs as early predictors of prognosis in adult patients presenting traumatic bone fractures but who did not suffer from osteopenia or postmenopausal osteoporosis. The available information from the different studies performed in this field was systematized in order to highlight the most promising BTMs for the assessment of fracture healing outcome.

Self-monitoring of freeze–thaw damage using triphasic electric conductive concrete

The effect of freeze–thaw cycles on concrete is of great importance for durability evaluation of concrete structures in cold regions. In this paper, damage accumulation was studied by following the fractional change of impedance (FCI) with number of freeze–thaw cycles (N). The nano-carbon black (NCB), carbon fiber (CF) and steel fiber (SF) were added to plain concrete to produce the triphasic electrical conductive (TEC) and ductile concrete. The effects of NCB, CF and SF on the compressive strength, flexural properties, electrical impedance were investigated. The concrete beams with different dosages of conductive materials were studied for FCI, N and mass loss (ML), the relationship between FCI and N of conductive concrete can be well defined by a first order exponential decay curve. It is noted that this nondestructive and sensitive real-time testing method is meaningful for evaluating of freeze–thaw damage in concrete.