Detecção de Histophilus somni (Haemophilus somnus) no sêmen bovino mediante reação em cadeia pela polimerase (PCR)

The present study evaluated the use of PCR for Histophilus somni detection in bovine semen. Semen samples were experimentally infected with H. somni at dilutions ranging from 107 to 101 bacteria/mL and subjected to DNA extraction by the phenol/chloroform method, followed by PCR amplification. The amplification products were analyzed by electrophoresis in 8% acrylamide gel. The oligonucleotide primers used yielded an amplification fragment of 400 base pairs from the bacterial DNA. Positive amplification was obtained even for the 101 bacteria/mL dilution. PCR proved to be an efficient method for the detection of H. somni. The results obtained in this study have brought relevant information for the diagnosis of H. somni, justifying the need for the diagnosis of this bacterium in bulls, especially in semen samples that should be free of contamination. The PCR method has shown to be a useful tool for the quality control of semen produced in artificial insemination centers.

Phylogeographic analysis in populations of Protonectarina sylveirae (Saussure, 1854) (Hymenoptera, Vespidae, Polistinae)

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

Investigação de genes de resistência a carbapenêmicos, cefalosporinas e quinolonas e tipagem molecular de Enterobacter spp isolados de pacientes internados em um hospital terciário do Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Phylogeographic analysis in populations of Protonectarina sylveirae (Saussure, 1854) (Hymenoptera, Vespidae, Polistinae)

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

Investigação de genes de resistência a carbapenêmicos, cefalosporinas e quinolonas e tipagem molecular de Enterobacter spp isolados de pacientes internados em um hospital terciário do Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Epidemiological study of HPV in oral mucosa through PCR

The Human Papillomavirus (HPV) belongs to the Papillomaviridae family and has a capsid and a single DNA strand. Its infection occurs mainly through sexual intercourse, having an important tropism for skin and mucosal cells. Aim: To evaluate the HPV presence in normal oral mucosa of asymptomatic subjects and; in parallel, to correlate social behavioral habits with the virus. Materials and Methods: Contemporary cohort cross-sectional study. The HPV was found by PCR, using general primers MY09/11 in 125 oral mucosa samples submitted to DNA extraction and PCR to search for the beta-globin gene in order to assess the quality of the extracted DNA. In parallel, we carried out a study of behavioral issues associated with the patients. Results: All the samples had a positive diagnosis of the beta-hemoglobin gene. HPV was diagnosed in 23.2% of the samples analyzed. Conclusion: The virus was present in 29 of the 125 patients, without them having any clinical-pathological manifestation associated with the HPV. As to the social behavior of the patients, we concluded that oral sex is statistically correlated to the virus, and besides the HPV has been statistically more present in female patients.

Detection of Hepatitis B virus subgenotype A1 in a Quilombo community from Maranhão, Brazil

Abstract Background The Brazilian population is mainly descendant from European colonizers, Africans and Native Americans. Some Afro-descendants lived in small isolated communities since the slavery period. The epidemiological status of HBV infection in Quilombos communities from northeast of Brazil remains unknown. The aim of this study was to characterize the HBV genotypes circulating inside a Quilombo isolated community from Maranhão State, Brazil. Methods Seventy-two samples from Frechal Quilombo community at Maranhão were collected. All serum samples were screened by enzyme-linked immunosorbent assays for the presence of hepatitis B surface antigen (HBsAg). HBsAg positive samples were submitted to DNA extraction and a fragment of 1306 bp partially comprising HBsAg and polymerase coding regions (S/POL) was amplified by nested PCR and its nucleotide sequence was determined. Viral isolates were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 320). Sequences were aligned using Muscle software and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted using Markov Chain Monte Carlo (MCMC) method to obtain the MCC tree using BEAST v.1.5.3. Results Of the 72 individuals, 9 (12.5%) were HBsAg-positive and 4 of them were successfully sequenced for the 1306 bp fragment. All these samples were genotype A1 and grouped together with other sequences reported from Brazil. Conclusions The present study represents the first report on the HBV genotypes characterization of this community in the Maranhão state in Brazil where a high HBsAg frequency was found. In this study, we reported a high frequency of HBV infection and the exclusive presence of subgenotype A1 in an Afro-descendent community in the Maranhão State, Brazil.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Ornithodoros peropteryx (Acari: Argasidae) in Bolivia: an argasid tick with a single nymphal stage

By the end of the 1960s, the argasid tick Ornithodoros peropteryx was described from larval specimens collected from the bat Peropteryx macrotis in Colombia. Since its original description, no additional record of O. peropteryx has been reported, and its post-larval stages have remained unknown. During July 2010, 18 larvae were collected from 9 bats (Centronycteris maximiliani), resulting in a mean infestation of 2.0 ± 2.2 ticks per bat (range 1–8). These bats were captured in a farm in northeastern Bolivia close to Guaporé River in the border with Brazil. Morphological examinations of the larvae revealed them to represent the species O. peropteryx. One engorged larva that was kept alive in the laboratory moulted to a nymph after 9 days. Fourteen days after the larval moulting, the nymph moulted to an adult female without taking any blood meal during the nymphal period. This adult female was used for a morphological description of the female stage of O. peropteryx. In addition, the larvae were used for a morphological redescription of this stage. One larva and two legs extirpated from the adult female were submitted to DNA extraction and PCR targeting a fragment of the mitochondrial 16S rDNA gene, which yielded DNA sequences at least 11 % divergent from any available argasid sequence in Genbank. We show that O. peropteryx ontogeny is characterized by a single, non-feeding, nymphal stage. This condition has never been reported for ticks.

Ornithodoros peropteryx (Acari Argasidae) in Bolivia an argasid tick with a single nymphal stage

By the end of the 1960s, the argasid tick Ornithodoros peropteryx was described from larval specimens collected from the bat Peropteryx macrotis in Colombia. Since its original description, no additional record of O. peropteryx has been reported, and its post-larval stages have remained unknown. During July 2010, 18 larvae were collected from 9 bats (Centronycteris maximiliani), resulting in a mean infestation of 2.0 ± 2.2 ticks per bat (range 1–8). These bats were captured in a farm in northeastern Bolivia close to Guapore´ River in the border with Brazil. Morphological examinations of the larvae revealed them to represent the species O. peropteryx. One engorged larva that was kept alive in the laboratory moulted to a nymph after 9 days. Fourteen days after the larval moulting, the nymph moulted to an adult female without taking any blood meal during the nymphal period. This adult female was used for a morphological description of the female stage of O. peropteryx. In addition, the larvae were used for a morphological redescription of this stage. One larva and two legs extirpated from the adult female were submitted to DNA extraction and PCR targeting a fragment of the mitochondrial 16S rDNA gene, which yielded DNA sequences at least 11 % divergent from any available argasid sequence in Genbank. We show that O. peropteryx ontogeny is characterized by a single, non-feeding, nymphal stage. This condition has never been reported for ticks.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (â^ž), NLR (0.017), and Ef (99%).

Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT

1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA. Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G). A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed. Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values. Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR. Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous. 2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy. Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy. Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV. Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.

Identification of stock units of Mullus barbatus (Linnaeus, 1758) in the Italian seas: development of genomic 2b-RAD markers and biological variation analysis.

This thesis is settled within the STOCKMAPPING project, which represents one of the studies that were developed in the framework of RITMARE Flagship project. The main goals of STOCKMAPPING were the creation of a genomic mapping for stocks of demersal target species and the assembling of a database of population genomic, in order to identify stocks and stocks boundaries. The thesis focuses on three main objectives representing the core for the initial assessment of the methodologies and structure that would be applied to the entire STOCKMAPPING project: individuation of an analytical design to identify and locate stocks and stocks boundaries of Mullus barbatus, application of a multidisciplinary approach to validate biological methods and an initial assessment and improvement for the genotyping by sequencing technique utilized (2b-RAD). The first step is the individuation of an analytical design that has to take in to account the biological characteristics of red mullet and being representative for STOCKMAPPING commitments. In this framework a reduction and selection steps was needed due to budget reduction. Sampling areas were ranked according the individuation of four priorities. To guarantee a multidisciplinary approach the biological data associated to the collected samples were used to investigate differences between sampling areas and GSAs. Genomic techniques were applied to red mullet for the first time so an initial assessment of molecular protocols for DNA extraction and 2b-RAD processing were needed. At the end 192 good quality DNAs have been extracted and eight samples have been processed with 2b-RAD. Utilizing the software Stacks for sequences analyses a great number of SNPs markers among the eight samples have been identified. Several tests have been performed changing the main parameter of the Stacks pipeline in order to identify the most explicative and functional sets of parameters.

Stock boundaries of Parapenaeus longirostris (Lucas, 1846) populations in the Italian seas: Development of genomic 2b-RAD markers and biological variation analysis.

This thesis is developed in the contest of Ritmare project WP1, which main objective is the development of a sustainable fishery through the identification of populations boundaries in commercially important species in Italian Seas. Three main objectives are discussed in order to help reach the main purpose of identification of stock boundaries in Parapenaeus longirostris: 1 -Development of a representative sampling design for Italian seas; 2 -Evaluation of 2b-RAD protocol; 3 -Investigation of populations through biological data analysis. First of all we defined and accomplished a sampling design which properly represents all Italian seas. Then we used information and data about nursery areas distribution, abundance of populations and importance of P. longirostris in local fishery, to develop an experimental design that prioritize the most important areas to maximize the results with actual project funds. We introduced for the first time the use of 2b-RAD on this species, a genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Thanks to this method we were able to move from genetics to the more complex genomics. In order to proceed with 2b-RAD we performed several tests to identify the best DNA extraction kit and protocol and finally we were able to extract 192 high quality DNA extracts ready to be processed. We tested 2b-RAD with five samples and after high-throughput sequencing of libraries we used the software “Stacks” to analyze the sequences. We obtained positive results identifying a great number of SNP markers among the five samples. To guarantee a multidisciplinary approach we used the biological data associated to the collected samples to investigate differences between geographical samples. Such approach assures continuity with other project, for instance STOCKMED, which utilize a combination of molecular and biological analysis as well.

Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.