Detection of parvovirus B19 infection in formalin-fixed and paraffin-embedded placenta and fetal tissues

Parvovirus B19 infection was first discovered in 1975 and it is implicated in fetal death from hydrops fetalis the world over. Diagnosis is usually made through histological identification of the intranuclear inclusion in placenta and fetal organs. However, these cells may be scarce or uncharacteristic, making definitive diagnosis difficult. We analyzed histologically placentas and fetal organs from 34 cases of non-immune hydrops fetalis, stained with Hematoxylin and Eosin (HE) and submitted to immunohistochemistry and polymerase chain reaction (PCR). Of 34 tissue samples, two (5.9%) presented typical intranuclear inclusion in circulating normoblasts seen in Hematoxylin and Eosin stained sections, confirmed by immunohistochemistry and PCR. However, PCR of fetal organs was negative in one case in which the placenta PCR was positive. We concluded that parvovirus B19 infection frequency is similar to the literature and that immunohistochemistry was the best detection method. It is highly specific and sensitive, preserves the morphology and reveals a larger number of positive cells than does HE with the advantage of showing cytoplasmic and nuclear positivity, making it more reliable. Although PCR is more specific and sensitive in fresh or ideally fixed material it is not so in formalin-fixed paraffin-embedded tissues, frequently the only one available in such cases.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

BIOFILM FORMATION OF Vibrio cholerae ON STAINLESS STEEL USED IN FOOD PROCESSING

Vibrio cholerae represents a significant threat to human health in developing countries. This pathogen forms biofilms which favors its attachment to surfaces and its survival and transmission by water or food. This work evaluated the in vitro biofilm formation of V. cholerae isolated from clinical and environmental sources on stainless steel of the type used in food processing by using the environmental scanning electron microscopy (ESEM). Results showed no cell adhesion at 4 h and scarce surface colonization at 24 h. Biofilms from the environmental strain were observed at 48 h with high cellular aggregations embedded in Vibrio exopolysaccharide (VPS), while less confluence and VPS production with microcolonies of elongated cells were observed in biofilms produced by the clinical strain. At 96 h the biofilms of the environmental strain were released from the surface leaving coccoid cells and residual structures, whereas biofilms of the clinical strain formed highly organized structures such as channels, mushroom-like and pillars. This is the first study that has shown the in vitro ability of V. cholerae to colonize and form biofilms on stainless steel used in food processing.

Intestinal intussusception and occlusion caused by small bowel polyps in the Peutz-Jeghers syndrome. Management by combined intraoperative enteroscopy and resection through minimal enterostomy: case report

The Peutz-Jeghers syndrome is a hereditary disease that requires frequent endoscopic and surgical intervention, leading to secondary complications such as short bowel syndrome. CASE REPORT: This paper reports on a 15-year-old male patient with a family history of the disease, who underwent surgery for treatment of an intestinal occlusion due to a small intestine intussusception. DISCUSSION: An intra-operative fiberscopic procedure was included for the detection and treatment of numerous polyps distributed along the small intestine. Enterotomy was performed to treat only the larger polyps, therefore limiting the intestinal resection to smaller segments. The postoperative follow-up was uneventful. CONCLUSION: We point out the importance of conservative treatment for patients with this syndrome, especially those who will undergo repeated surgical interventions because of clinical manifestation while they are still young.

A simple method for assessing the binding of concanavalin A to mononuclear cell surfaces: no interference of visceral leishmaniasis serum on this binding

We report a simple method for evaluating the binding of concanavalin A (ConA) to human peripheral blood mononuclear cells (PBMC). The binding is evidenced by an immunoenzymic assay using peroxidase-conjugated immunoglobulins of a rabbit anti-ConA serum. Using the method we show that sera from patients with American leishmaniasis do not interfere with binding of ConA to PBMC.

Detection of Hepatitis B Virus Antigens in Paraffin-embedded Liver Specimens from the Amazon Region, Brazil

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in different other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4%) histological samples were HBsAg reactive and 5 (6.3%) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

Disinfection with sodium hypochlorite in hospital environmental surfaces in the reduction of contamination and infection prevention: a systematic review

OBJECTIVETo search for evidence of the efficiency of sodium hypochlorite on environmental surfaces in reducing contamination and prevention of healthcare-associated infection HAIs.METHODSystematic review in accordance with the Cochrane Collaboration.RESULTSWe analyzed 14 studies, all controlled trials, published between 1989-2013. Most studies resulted in inhibition of microorganism growth. Some decreased infection, microorganism resistance and colonization, loss of efficiency in the presence of dirty and surface-dried viruses.CONCLUSIONThe hypochlorite is an effective disinfectant, however, the issue of the direct relation with the reduction of HAIs remains. The absence of control for confounding variables in the analyzed studies made the meta-analysis performance inadequate. The evaluation of internal validity using CONSORT and TREND was not possible because its contents were not appropriate to laboratory and microbiological studies. As a result, there is an urgent need for developing specific protocol for evaluating such studies.

Estimating soybean crop areas using spectral-temporal surfaces derived from MODIS images in Mato Grosso, Brazil

The objective of this work was to evaluate the application of the spectral-temporal response surface (STRS) classification method on Moderate Resolution Imaging Spectroradiometer (MODIS, 250 m) sensor images in order to estimate soybean areas in Mato Grosso state, Brazil. The classification was carried out using the maximum likelihood algorithm (MLA) adapted to the STRS method. Thirty segments of 30x30 km were chosen along the main agricultural regions of Mato Grosso state, using data from the summer season of 2005/2006 (from October to March), and were mapped based on fieldwork data, TM/Landsat-5 and CCD/CBERS-2 images. Five thematic classes were considered: Soybean, Forest, Cerrado, Pasture and Bare Soil. The classification by the STRS method was done over an area intersected with a subset of 30x30-km segments. In regions with soybean predominance, STRS classification overestimated in 21.31% of the reference values. In regions where soybean fields were less prevalent, the classifier overestimated 132.37% in the acreage of the reference. The overall classification accuracy was 80%. MODIS sensor images and the STRS algorithm showed to be promising for the classification of soybean areas in regions with the predominance of large farms. However, the results for fragmented areas and smaller farms were less efficient, overestimating soybean areas.

Probing the (empirical) quantum structure embedded in the periodic table with an effective Bohr model

The atomic shell structure can be observed by inspecting the experimental periodic properties of the Periodic Table. The (quantum) shell structure emerges from these properties and in this way quantum mechanics can be explicitly shown considering the (semi-)quantitative periodic properties. These periodic properties can be obtained with a simple effective Bohr model. An effective Bohr model with an effective quantum defect (u) was considered as a probe in order to show the quantum structure embedded in the Periodic Table. u(Z) shows a quasi-smoothed dependence of Z, i.e., u(Z) ≈ Z2/5 - 1.

Annual evolution of global, direct and diffuse radiation and fractions in tilted surfaces

It was evaluated the annual evolution of global, direct and diffuse components of incident solar radiation on tilted surfaces to 12.85, 22.85 and 32.85º, facing north, in Botucatu, state of São Paulo, Brazil. The radiometric fractions were obtained for each component of the radiation in the aforementioned surfaces, through the ratio with the global and top of the atmosphere radiations. Seasonality was evaluated based on monthly averages of daily values. The measures occurred between 04/1998 and 07/2001 at 22.85º; 08/2001 and 02/2003 at 12.85º; and from 03/2003 to 12/2007 at 32.85º, with concomitant measures in the horizontal surface (reference). The levels of global and direct radiation on tilted surfaces were lower in summer and higher in the equinoxes when compared with the horizontal. The diffuse radiation on tilted surfaces was lower in most months, with losses of up to 65%. A trend of increasing differences occurred between horizontal and tilted surfaces with the increase of the angle in all the components and fractions of incident radiation. The annual evolution of rainfall and cloud cover ratio directly affected the atmospheric transmissivity of direct and diffuse components in the region.