Coffea arabica and C. canephora discrimination in roasted and ground coffee from reference material candidates by real-time PCR.

To produce specific desirable coffee blends, Coffea arabica and C. canephora are mixed each other, in some cases to suit consumer preference, but in others to reduce production costs. In this scenario, the aim of this work was to evaluate standard candidate reference materials (RMc) for analysis of different blends of roasted and ground coffee. For this purpose, we analyzed different percentages of C. arabica and C. canephora (100:0; 50:50; 25:75; and 0:100, respectively). These RMc samples were developed in a previous study with green coffee beans submitted to medium roasting. In this work, coffee species differentiation (C. arabica and C. canephora) was analyzed by real-time PCR, using specific primers previously developed, called ARA primers. The RMc material with 100% C. canephora did not present amplification, in contrast with the samples containing C. arabica, which all presented amplification. These results indicate the specificity of ARA primers for C. arabica and that the detection system assay can be used as a promising molecular tool to identify and quantify percentages of C. arabica in different coffee blends.

A snapshot on prokaryotic diversity of the Solimões River basin (Amazon, Brazil).

The Amazon region has the largest hydrographic basin on the planet and is well known for its huge biodiversity of plants and animals. However, there is a lack of studies on aquatic microbial biodiversity in the Solimões River, one of its main water courses. To investigate the microbial biodiversity of this region, we performed 16S rRNA gene clone libraries from Solimões River and adjacent rivers and lakes. Our question was which microorganisms inhabit the different types of aquatic environments in this part of the basin, and how diversity varies among these environments (rivers and lakes). The microbial diversity generating 13 clone libraries of the bacterial 16S rRNA gene and 5 libraries of the archaeal 16S rRNA gene was assessed. Diversity measured by several alpha diversity indices (ACE, Chao, Shannon and Simpson) revealed significant differences in diversity indices between lake and river samples. The site with higher microbial diversity was in the Solimões River (4S), downstream the confluence with Purus River. The most common bacterial taxon was the cosmopolitan Polynucleobacter genus, widely observed in all samples. The phylum Thaumarchaeota was the prevailing archaeal taxon. Our results provide the first insight into the microbial diversity of the world's largest river basin.

Validation of a reaction volume reduction protocol for analysis of Y chromosome haplotypes targeting DNA databases.

The use of Y chromosome haplotypes, important for the detection of sexual crimes in forensics, has gained prominence with the use of databases that incorporate these genetic profiles in their system. Here, we optimized and validated an amplification protocol for Y chromosome profile retrieval in reference samples using lesser materials than those in commercial kits. FTA® cards (Flinders Technology Associates) were used to support the oral cells of male individuals, which were amplified directly using the SwabSolution reagent (Promega). First, we optimized and validated the process to define the volume and cycling conditions. Three reference samples and nineteen 1.2 mm-diameter perforated discs were used per sample. Amplification of one or two discs (samples) with the PowerPlex® Y23 kit (Promega) was performed using 25, 26, and 27 thermal cycles. Twenty percent, 32%, and 100% reagent volumes, one disc, and 26 cycles were used for the control per sample. Thereafter, all samples (N = 270) were amplified using 27 cycles, one disc, and 32% reagents (optimized conditions). Data was analyzed using a study of equilibrium values between fluorophore colors. In the samples analyzed with 20% volume, an imbalance was observed in peak heights, both inside and in-between each dye. In samples amplified with 32% reagents, the values obtained for the intra-color and inter-color standard balance calculations for verification of the quality of the analyzed peaks were similar to those of samples amplified with 100% of the recommended volume. The quality of the profiles obtained with 32% reagents was suitable for insertion into databases.

Human autosomal DNA and X chromosome STR profiles obtained from Chrysomya albiceps (Diptera: Calliphoridae) larvae used as a biological trace.

The use of insects to answer questions in criminal investigations, as well as a combination of forensic genetic techniques to obtain human DNA from the organisms, especially necrophagous dipterians, have gained ground in recent decades among researchers and professionals in this area. The objective of our study was to evaluate and compare two methods of human DNA extraction, commonly used for forensic samples, to obtain human autosomal DNA and X chromosome short tandem repeat profiles from the digestive tract of Chrysomya albiceps (Diptera: Calliphoridae) larvae. Immature specimens were collected from corpses at the Institute of Forensic Medicine of Pernambuco and raised in bovine ground meat to allow stabilization of the colony. Groups of larvae in the third instar were provided with bovine ground meat plus human blood for 48 h, dissected, and then subjected to DNA extraction. DNA was extracted using two methods: a DNA IQ™ kit and a phenol-chloroform method. Genomic DNA was amplified using AmpFℓSTR® Identifiler® Plus PCR and Argus-X-12® kits, and samples were sequenced to determine if the two extraction techniques generated reliable profiles that were compatible with a reference sample. The existence of comparable profiles from both techniques demonstrates the usefulness of dipteran larvae for obtaining human DNA from corpses, which can be further used to correlate genetic profiles in a crime scene when other traces are not available. However, several variables still require revision; thus, the technique should be further investigated for its validity, security, and, in particular, its reproducibility.

Detecting multiple DNA human profile from a mosquito blood meal.

Criminal traces commonly found at crime scenes may present mixtures from two or more individuals. The scene of the crime is important for the collection of various types of traces in order to find the perpetrator of the crime. Thus, we propose that hematophagous mosquitoes found at crime scenes can be used to perform genetic testing of human blood and aid in suspect investigation. The aim of the study was to obtain a single Aedes aegypti mosquito profile from a human DNA mixture containing genetic materials of four individuals. We also determined the effect of blood acquisition time by setting time intervals of 24, 48, and 72 h after the blood meal. STR loci and amelogenin were analyzed, and the results showed that human DNA profiles could be obtained from hematophagous mosquitos at 24 h following the blood meal. It is possible that hematophagous mosquitoes can be used as biological remains at the scene of the crime, and can be used to detect human DNA profiles of up to four individuals.

Prevalence and factors associated with oral potentially malignant disorders in Brazil's rural workers.

OBJECTIVES: To determine the prevalence of oral potentially malignant disorders (OPMDs) in a population of rural workers in the northeast of Brazil and to investigate the association with sociodemographic, occupational, and health factors. METHODS: A total of 1385 workers answered a validated questionnaire and were examined by calibrated dentists. A descriptive analysis, chi-square homogeneity test, and binary logistic regression were performed. RESULTS: The prevalence of OPMDs was 29.6%. Actinic cheilitis was the most predominant (28.4%), followed by leukoplakia (2.3%) and erythroplakia (0.3%). Gender, type of skin, and time exposed to the sun explained the presence of OPMD (P < 0.0001). The study found increased prevalence, especially for males over the age of 60 years and being exposed to the sun for more than 45 years. CONCLUSION: Rural workers showed high vulnerability to the presence of OPMDs, as estimated prevalence exhibited was high.

Quality of the Solimões River water for domestic use by the riverine community situated in Manacapuru-Amazonas-Brazil.

The Amazon has the largest watershed in the world, with abundant fresh water reserves. Such abundance contrasts with the quality of the water consumed in the riverine communities. This work highlights the importance of assessing the quality of water of the Solimões River in the stretch situated opposite the Community Nossa Senhora das Graças-Costa do Pesqueiro, in front of the town of Manacapuru-Amazonas-Brazil. The research aimed to evaluate environmental indicators for the quality of the river water as well as in households in this community, according to the regional seasonality. The monitored parameters such as conductivity, dissolved oxygen, turbidity, color, pH, nitrate, nitrite, and sulfate were compared with the water quality standards in force in Brazil. Values well above the maximum level allowed (MLA) for color and turbidity parameters were found in some households, indicating that the water collected from the river is not getting appropriate treatment. The analysis of the correlation matrix of the parameters in the flood period of the river evidenced high correlation among dissolved oxygen (D.O), NO3 (-), Cl(-), SO4 (2-), and color. In this study, by principal component analysis (PCA), it was observed that the characteristics of the water, obtained from the river to be consumed in the households, in the flood period showed similarities with the river water samples, indicating absence of efficient treatment for human consumption.

Trace samples of human blood in mosquitoes as a forensic investigation tool.

Investigations of any type of crime invariably starts at the crime scene by collecting evidence. Thus, the purpose of this research was to collect and analyze an entomological trace from an environment that is similar to those of indoor crime scenes. Hematophagous mosquitoes were collected from two residential units; saliva of volunteers that were residents in the units was also collected for genetic analysis as reference samples. We examined the allele frequencies of 15 short tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and amelogenin. A total of 26 female hematophagous mosquitoes were identified as Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus; we were able to obtain 11 forensically valid genetic profiles, with a minimum of 0.028203 ng/µL of human DNA. Thus, the results of this study showed that it was possible to correlate human genetic information from mosquitoes with the volunteer reference samples, which validates the use of this information as forensic evidence. Furthermore, we observed mixed genetic profiles from one mosquito. Therefore, it is clearly important to collect these insects indoors where crimes were committed, because it may be possible to find intact genetic profiles of suspects in the blood found in the digestive tract of hematophagous mosquitoes for later comparison to identify an offender and/or exclude suspects.

Distribution of forensic marker allelic frequencies in Pernambuco, Northestern Brazil.

Pernambuco is one of the 27 federal units of Brazil, ranking seventh in the number of inhabitants. We examined the allele frequencies of 13 short tandem repeat loci (CFS1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, vWA, and TPOX), the minimum recommended by the Federal Bureau of Investigation and commonly used in forensic genetics laboratories in Brazil, in a sample of 609 unrelated individuals from all geographic regions of Pernambuco. The allele frequencies ranged from 5 to 47.2%. No significant differences for any loci analyzed were observed compared with other publications in other various regions of Brazil. Most of the markers observed were in Hardy-Weinberg equilibrium. The occurrence of the allele 47.2 (locus FGA) and alleles 35.1 and 39 (locus D21S11), also described in a single study of the Brazilian population, was observed. The other forensic parameters analyzed (matching probability, power of discrimination, polymorphic information content, paternity exclusion, complement factor I, observed heterozygosity, expected heterozygosity) indicated that the studied markers are very informative for human forensic identification purposes in the Pernambuco population.

Evaluation of antimicrobial resistance of Campylobacter spp. isolated from broiler carcasses.

1. The aim of the present study was to evaluate the antimicrobial resistance of Campylobacter strains (C. jejuni, C. coli and C. lari) isolated from broiler carcasses processed in the State of Paraná, Brazil. 2. Rates of microbial resistance and susceptibility were assessed by both Disk Diffusion (DD) and Etest (Minimum Inhibitory Concentration) techniques. Antibiotics were tested using DD (12 antibiotics) and/or MIC (7 antibiotics) methods. 3. A total of 95.8% of the strains were resistant to at least two agents. In terms of multidrug resistance, 75% of strains were resistant to three or more groups of antibiotics. The highest rates of resistance were detected for cefalotin, ciprofloxacin, tetracycline and nalidixic acid. A high rate of susceptibility of the strains to erythromycin (95.8%) was found confirming that this is considered the agent of choice for treating campylobacteriosis. Comparison of the microbial resistance and susceptibility, as determined simultaneously by the two methods, found the techniques to be statistically equivalent for 5 out of the 6 antibiotics tested. 4. The results of this study suggest the need for adopting measures to control the use of antibiotics in broiler production to prevent multidrug resistance of Campylobacter strains and reduce the risk of serious human diseases caused by the consumption of contaminated chicken meat.

Giardia duodenalis: kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected.

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG(1), IgG(2a), IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10(1), 10(2), 10(3), 10(4), 10(5)) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1 response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.

Contaminação por Salmonella spp. em uma cadeia de produção de ovos de uma integração de postura comercial / Salmonella contamination in an egg production chain of a laying hens integration

Contamination of ISA BROWN layer hens by Salmonella spp. and serotyping of the isolated strains were performed in 30 farms. Salmonella detection was done from feces and cloaca swabs. Eggs produced in the farms contaminated by Salmonella spp. were analyzed by culturing the shell rinsing and egg yolk. Salmonella spp. was isolated in eight (23.0 percent) farms, but was not detected from eggs. S. Enteritidis, which is the most prevalent sorotype associated to human salmonelosis in Paraná State, was not isolated either from birds or eggs analyzed. The sorotypes isolated were Salmonella enterica subsp. enterica (0:3.10) (67.0 percent), Mbandaka (11.0 percent), Infantis (11.0 percent), and Newport (11.0 percent).

Evaluation of survival of Staphylococcus aureus and Clostridium botulinum in charqui meats.

Charqui meats were prepared in laboratory conditions in order to carry out experiments to observe the possibility of development of enterotoxigenic Staphylococcus aureus and Clostridium botulinum proteolytic type B spores and their toxins. Results demonstrated that the harsh processing conditions, high salt concentration, relative high temperature, a(w) values, inhibited the growth of both bacteria. Under our experimental conditions, S. aureus would survive throughout the sequence of salting steps i.e. brine followed by rock salting and the sunshine drying step. However, at final a(w) value of 0.70-0.75 would create conditions to inhibit its development. The other experiment revealed that C. botulinum spores germination also was impaired because of these low a(w) values. Under these conditions, charqui meats revealed to be safe products in relation to toxins from both enterotoxigenic S. aureus and C. botulinum.

Evaluation of fumonisin-aflatoxin co-occurrence in Brazilian corn hybrids by ELISA.

The natural co-occurrence of fumonisins and aflatoxins was investigated in freshly harvested corn kernels (150 samples, 62 hybrids), acquired from the Central-Southern (27 samples, 21 hybrids), Central-Western (86 samples, 51 hybrids) and Northern (37 samples, 18 hybrids) regions of the State of Paraná, Brazil using enzyme-linked immunosorbent assay (ELISA). Fumonisins were detected in 147 (98%) samples at a concentration range of 0.096 to 22.6 microg/g, while aflatoxins were detected in 17 (11.3%). All the aflatoxin-positive samples (range 38.0-460.0 ng/g) came from the Central-Western region and were co-contaminated with fumonisins. Fumonisin contamination was higher in corn from the Northern (9.85 microg/g) and Central-Western regions (5.08 microg/g), when compared with the Central-Southern region (1.14 microg/g). The overall evaluation detected 62% samples with fumonisin levels < or = 5.0 microg/g. Regional differences affected fumonisin levels in the same hybrid, regardless of Fusarium count and moisture content, suggesting interference from climatic conditions, in addition to the local predominance of toxigenic strains of the Fusarium biotype.

Infection control in a Brazilian regional multihospital system.

OBJECTIVE: This study describes the organization of infection control committees in a Brazilian multihospital system, identifying their major problems. Our goal is to discuss the main deficiencies in infection control and to target some interventions that can improve the efficiency of these actions in Brazilian hospitals. DESIGN AND SETTING: We used a descriptive epidemiologic design. We interviewed the chairs and the nurses of the infection control committees and visited the main areas to observe infection control in a multihospital system with 3146 beds. For analysis of the results, we performed a standardization process, establishing a score for each hospital by using infection control organization as a surrogate marker for quality outcome. The mean hospital scores for infection control, existence of policies, and infrastructure at each hospital were compared by using different stratification and multivariate analysis. RESULTS: Statistically significant differences were found among surveyed hospitals by using stratification by size, funding status, and presence of teaching activities. CONCLUSIONS: Diverse patterns of infection control organization were found among surveyed hospitals. Small hospitals represented the major problem in providing effective infection control. Chiefly for these hospitals, the epidemiologic indicators and the surveillance and control system proposed by the Brazilian Ministry of Health and based on the Centers for Disease Control and Prevention model showed poor suitability.

[The process if teaching-learning in blood pressure monitoring]. / O processo ensino-aprendizagem na verificação da pressão arterial.

The nurse is considered the professional that obtains more trustworthy data in the measures insinuations of arterial pressure for inducing less reactions of alert in the customer, generating of values falsely high. Even so, a lot of times, she presents spaces in the knowledge of the subject, so much in the technical aspects, as in the anátomo-physiologic ones. That prioritizes the development of teaching strategies, seeking to minimize flaws and to turn the effective learning process. Objectifying to promote the knowledge of the technique of measurement of the arterial pressure close to a research group, it took place a reciclation and avaliation questionnaire of the knowledge of the group was applied before and after the same. The effectiveness of that method was evidenced, because 100% of the sample (nine) they referred contribution for better of the fundamentation theoretical-practice, decreasing difficulties originating from of the verification of the arterial pressure. It is ended that, being the education actions in extremely valid and necessary health, the male nurse, as educator that is, needs to recycle its knowledge and its better techniques continually.

Reflectance in situ hybridization (RISH): detection, by confocal reflectance laser microscopy, of gold-labelled riboprobes in breast cancer cell lines and histological specimens.

A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1-nm colloidal-gold particles after RNA/RNA in situ hybridization, using digoxigenin-labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras-related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three-dimensional imaging were used for visualization. A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.

[Serologic study of experimental infections by Trypanosoma evansi, in guinea pigs]. / Estudo sorológico de infecções experimentais por Trypanosoma evansi, em cobaias.

Sera of 20 guinea-pigs experimentally infected with Trypanosoma evansi were obtained in order to compare the efficacy of gel diffusion, indirect immunofluorescence and agglutination tests, to detect antibodies to T. evansi. The fluorescent antibody test was positive in six (6) animals and the antibody titres were very low (1:4 to 1:16). The agglutination test detected trypanosomal antibodies in sera one (1) week after infection. After two (2) weeks all animals were positive with high titres (1:8.000 to 1:250.000). Agglutination was inhibited when sera were treated with 2-Mercapto-ethanol. This fact suggests that IgM is the principal class of antibodies in sera of infected guinea-pigs. Precipitating antibodies were not detected during the course of infection.

[Implementation of epidemiological surveillance actions at regional level]. / Implementação de ações de vigilância epidemiológica em nível regional.

An attempt at a reorganization of the Epidemiological Surveillance Service at regional level in the Central Health Region of Governador Valadares, MG, Brasil, undertaken during 1985 is reported on. This study discusses simple measures that may be taken with a view to increasing interest in Epidemiological Surveillance actions at the local level and emphasizes the fact that the of data to the local level is essential to this interest.

[Verrucous hemangioma (neviform verrucous acrohemangioma)]. / Hemangioma verrucoso (acrohemangioma verrucoso neviforme).

A case of verrucous haemangioma in a eighteen-years old patient, during her second pregnancy is reported. The lesions were localized on the dorsal aspect of the right foot and leg, simulating clinically the angiokeratoma circunscriptum naeviforme. The diagnosis was first firmed by the histologic features of the malformation on superficial, medial and deep dermis and a rough epidermic reactivity. It has been discussed the mean differential diagnosis and commented that those type of hemangiomatous lesions (macular and elevated lesions) plus varicosities, without hypertrophy of the limb, may represent a discrete form of Klippel-Trenaunay syndrome. The authors propose the name "Verrucous neviform acrohaemangioma" for those cases similar to this one and to make a difference from other type of haemangioma that became verrucous.