Detection of Toxoplasma gondii DNA in the milk of naturally infected ewes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of Total Sulfur in Agricultural Samples by High-Resolution Continuum Source Flame Molecular Absorption Spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against babesia boris in cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65 (PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.

Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens.

Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.

Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples

Background. The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology. Findings. The aim of this work was to evaluate the RT-PCR and hnRT-PCR for rabies virus detection in original tissues stored at -20°C for different periods considering their use for rabies virus detection in stored and decomposed samples. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition. The RT-PCR and hnRT-PCR results were compared with previous results from Direct Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples evaluated to the molecular techniques. Conclusion. These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR demonstrated efficacy in rabies virus detection in stored and decomposed materials suggesting it's application for rabies virus retrospective epidemiological studies. © 2008 Arajo et al; licensee BioMed Central Ltd.

Development and application of a highly selective biomimetic sensor for detection of captopril, an important ally in hypertension control

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Application of molecular markers on genetic improvement and reproduction in the post-genomic Era: Emerging biotechnologies and perspectives in livestock

Background: New challenges are rising in the animal protein market, and one of the main world challenges is to produce more in shorter time, with better quality and in a sustainable way. Brazil is the largest beef exporter in volume hence the factors affecting the beef meat chain are of major concern in countrýs economy. An emerging class of biotechnological approaches, the molecular markers, is bringing new perspectives to face these challenges, particularly after the publication of the first complete livestock genome (bovine), which has triggered a massive initiative to put in practice the benefits of the so called the Post-Genomic Era. Review: This article aimed at showing the directions and insights in the application of molecular markers on livestock genetic improvement and reproduction as well at organizing the progress so far, pointing some perspectives of these emerging technologies in Brazilian ruminant production context. An overview on the nature of the main molecular markers explored in ruminant production is provided, which describes the molecular bases and detection approaches available for microsatellites (STR) and single nucleotide polymorphisms (SNP). A topic is dedicated to review the history of association studies between markers and important trait variation in livestock, showing the timeline starting on quantitative trait loci (QTL) identification using STR markers and ending in high resolution SNP panels to proceed whole genome scans for phenotype/genotype association. Also the article organizes this information to reveal how QTL prospection using STR could open ground to the feasibility of marker-assisted selection and why this approach is quickly being replaced by studies involving the application of genome-wide association using SNP research in a new concept called genomic selection. Conclusion: The world's scientific community is dedicating effort and resources to apply SNP information in livestock selection through the development of high density panels for genomic association studies, connecting molecular genetic data with phenotypes of economic interest. Once generated, this information can be used to take decisions in genetic improvement programs by selecting animals with the assistance of molecular markers.

Detection of classical 17p11.2 deletions, an atypical deletion and RAI1 alterations in patients with features suggestive of Smith-Magenis syndrome

Smith-Magenis syndrome (SMS) is a complex disorder whose clinical features include mild to severe intellectual disability with speech delay, growth failure, brachycephaly, flat midface, short broad hands, and behavioral problems. SMS is typically caused by a large deletion on 17p11.2 that encompasses multiple genes including the retinoic acid induced 1, RAI1, gene or a mutation in the RAI1 gene. Here we have evaluated 30 patients with suspected SMS and identified SMS-associated classical 17p11.2 deletions in six patients, an atypical deletion of ∼139 kb that partially deletes the RAI1 gene in one patient, and RAI1 gene nonsynonymous alterations of unknown significance in two unrelated patients. The RAI1 mutant proteins showed no significant alterations in molecular weight, subcellular localization and transcriptional activity. Clinical features of patients with or without 17p11.2 deletions and mutations involving the RAI1 gene were compared to identify phenotypes that may be useful in diagnosing patients with SMS. © 2012 Macmillan Publishers Limited All rights reserved.

Circulation of Different Lineages of Dengue Virus 2, Genotype American/Asian in Brazil: Dynamics and Molecular and Phylogenetic Characterization

The American/Asian genotype of Dengue virus type 2 (DENV-2) was introduced into the Americas in the 80′s. Although there is no data showing when this genotype was first introduced into Brazil, it was first detected in Brazil in 1990. After which the virus spread throughout the country and major epidemics occurred in 1998, 2007/08 and 2010. In this study we sequenced 12 DENV-2 genomes obtained from serum samples of patients with dengue fever residing in São José do Rio Preto, São Paulo (SJRP/SP), Brazil, in 2008. The whole open reading frame or envelope sequences were used to perform phylogenetic, phylogeographic and evolutionary analyses. Isolates from SJRP/SP were grouped within one lineage (BR3) close to isolates from Rio de Janeiro, Brazil. Isolates from SJRP were probably introduced there at least in 2007, prior to its detection in the 2008 outbreak. DENV-2 circulation in Brazil is characterized by the introduction, displacement and circulation of three well-defined lineages in different times, most probably from the Caribbean. Thirty-seven unique amino acid substitutions were observed among the lineages, including seven amino acid differences in domains I to III of the envelope protein. Moreover, we dated here, for the first time, the introduction of American/Asian genotype into Brazil (lineage BR1) to 1988/89, followed by the introduction of lineages BR2 (1998-2000) and BR3 (2003-05). Our results show a delay between the introduction and detection of DENV-2 lineages in Brazil, reinforcing the importance and need for surveillance programs to detect and trace the evolution of these viruses. Additionally, Brazilian DENV-2 differed in genetic diversity, date of introduction and geographic origin and distribution in Brazil, and these are important factors for the evolution, dynamics and control of dengue. © 2013 Drumond et al.

Outbreak of fungemia caused by Candida parapsilosis in a neonatal intensive care unit: Molecular investigation through microsatellite analysis

Background: Opportunistic infections are an increasingly common problem in hospitals, and the yeast Candida parapsilosis has emerged as an important nosocomial pathogen, especially in neonatal intensive care units (NICUs) where it has been responsible for outbreak cases. Risk factors for C. parapsilosis infection in neonates include prematurity, very low birth weight, prolonged hospitalization, indwelling central venous catheters, hyperalimentation, intravenous fatty emulsions and broad spectrum antibiotic therapy. Molecular methods are widely used to elucidate these hospital outbreaks, establishing genetic variations among strains of yeast. Aims: The aim of this study was to detect an outbreak of C. parapsilosis in an NICU at the Hospital das Clinicas , Faculty of Medicine of Botucatu, a tertiary hospital located in São Paulo, Brazil, using the molecular genotyping by the microsatellite markers analysis. Methods: A total of 11 cases of fungemia caused by C. parapsilosis were identified during a period of 43 days in the NICU. To confirm the outbreak all strains were molecularly typed using the technique of microsatellites. Results: Out of the 11 yeast samples studied, nine showed the same genotypic profile using the technique of microsatellites. Conclusions: Our study shows that the technique of microsatellites can be useful for these purposes. In conclusion, we detected the presence of an outbreak of C. parapsilosis in the NICU of the hospital analyzed, emphasizing the importance of using molecular tools, for the early detection of hospital outbreaks, and for the introduction of effective preventive measures, especially in NICUs. © 2012 Revista Iberoamericana de Micología.

SERS mapping in langmuir-blodgett films and single-molecule detection

Plasmon-enhanced spectroscopic techniques have expanded single-molecule detection (SMD) and are revolutionizing areas such as bio-imaging and single-cell manipulation. Surface-enhanced (resonance) Raman scattering (SERS or SERRS) combines high sensitivity with molecularfingerprint information at the single-molecule level. Spectra originating from single-molecule SERS experiments are rare events, which occur only if a single molecule is located in a hot-spot zone. In this spot, the molecule is selectively exposed to a significant enhancement associated with a high, local electromagnetic field in the plasmonic substrate. Here, we report an SMD study with an electrostatic approach in which a Langmuir film of a phospholipid with anionic polar head groups (PO 4 -) was doped with cationic methylene blue (MB), creating a homogeneous, two-dimensional distribution of dyes in the monolayer. The number of dyes in the probed area of the Langmuir-Blodgett (LB) film coating the Ag nanostructures established a regime in which single-molecule events were observed, with the identification based on direct matching of the observed spectrum at each point of the mapping with a reference spectrum for the MB molecule. In addition, advanced fitting techniques were tested with the data obtained from micro-Raman mapping, thus achieving real-time processing to extract the MB single-molecule spectra. © 2013 Society for Applied Spectroscopy.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in a burn unit from Brazil

Methicillin-resistant Staphylococcus aureus (MRSA) poses a threat for patients in burn units. Studies that mix epidemiological designs with molecular typing may contribute to the development of strategies for MRSA control. We conducted a study including: molecular characterization of Staphylococcal Chromosome Cassette mecA (SCCmec), strain typing with pulsed field gel electrophoresis (PFGE) and detection of virulence genes, altogether with a case-case-control study that assessed risk factors for MRSA and for methicillin-susceptible S. aureus (MSSA), using S. aureus negative patients as controls. Strains were collected from clinical and surveillance cultures from October 2006 through March 2009. MRSA was isolated from 96 patients. Most isolates (94.8%) harbored SCCmec type III. SCCmec type IV was identified in isolates from four patients. In only one case it could be epidemiologically characterized as community-associated. PFGE typing identified 36 coexisting MRSA clones. When compared to MSSA (38 isolates), MRSA isolates were more likely to harbor two virulence genes: tst and lukPV. Previous stay in other hospital and admission to Intensive Care Unit were independent risk factors for both MRSA and MSSA, while the number of burn wound excisions was significantly related with the former (OR = 6.80, 95%CI = 3.54-13.07). In conclusion, our study found polyclonal endemicity of MRSA in a burn unit, possibly related to importing of strains from other hospitals. Also, it pointed out to a role of surgical procedures in the dissemination of MRSA strains. © 2013 Elsevier Ltd and ISBI. All rights reserved.

Molecular epidemiology and extended-spectrum β-lactamases production of klebsiella pneumoniae isolated from three dairy herds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)