Diagnosis of Helicobacter pylori: comparison of an urease test, histological visualization of curved bacteria and culture

Helicobacter pylori was investigated in 189 patients for culture, microscopic visualization of campylobacter-like organisms (CLO) and a ten minute urease test. In 136 (72%) the bacteria was isolated, and in 98 of them CLO were histologically detected. Specificity, sensitivity, positive and negative predictive values of microscopic visualization of CLO were: 0.77, 0.73, 0.97 and 0.51, respectively; 98 culture-positive patients were urease test positive. Specificity, sensitivity, positive and negative predictive values of the urease test were: 0.83, 0.72, 0.92 and 0.54, respectively. Comparing the urease test with culture of H. pylori combined with microscopic visualization of CLO, its specificity, sensitivity, positive and negative predictive values were: 0.95, 0.71, 0.98 and 0.48, respectively. Probably, these values are not real, since bacteria different from H. pylori could be misclassified as CLO.

Adenovirus respiratory infection: significant increase in diagnosis using PCR comparing with antigen detection and culture methods

Adenovirus (AdV) respiratory infections are usually described as being associated with high mortality rates. Laboratory diagnosis is essential for the establishment of the appropriate therapy, and for guiding the implementation of preventive measures in order to prevent the spread of the infection. Aiming to analyze the sensitivity and specificity of the laboratorial diagnosis methods available, we compared antigen detection by indirect immunofluorescence assay (IF), and a specific nested polymerase chain reaction (PCR), to detect AdV in respiratory samples collected from patients admitted to hospital with acute respiratory disease. Positive samples were inoculated into a cell culture to confirm the results. We analyzed 381 samples from the nasopharyngeal aspirates collected during the year 2008; of these, 2.6% tested were positive for adenovirus through IF and 10% through PCR; positive isolation was obtained in 40% and 26% of these cases, respectively. Most infected patients were children under six months of age, and despite of the fact that a significant number of patients required intensive care, the mortality rate was low (5%). In conclusion, molecular methods were found to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection; their introduction permitted a significant increase in diagnoses of adenovirus infections.

Standardization of bovine macrophage monolayers and isolation and culture of trypanosomes

We describe a method for culturing over 90% pure bovine macrophages from peripheral blood mononuclear cells separated with Nycoprep. The cells were cultured for 12 days and then stained with esterase and with anti CD14 to test for purity. The method is reproducible and ensures an adequate number of cells for immunological research. Additionally, we report the unexpected finding of Trypanosoma trypomastigotes in our macrophage cultures from bovines belonging to a geographic area from which no bovine trypanosomes had been reported before.

Isolation and culture of umbilical vein mesenchymal stem cells

Bone marrow contains a population of stem cells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymal stem cells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentiation characteristics similar to those of mesenchymal stem cells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipocytes and osteocytes in culture. Immunophenotypically, this cell population was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The characteristics described are the same as those presented by bone marrow mesenchymal stem cells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source of mesenchymal stem cells that could be used in cell therapy protocols.

Linking customer satisfaction, quality, and strategic planning

By acknowledging and dissecting the interconnected roles of customer satisfaction, quality, and strategic planning, this paper provides an analytical framework for creating a customer-driven organization and culture. It shows how quality starts and ends with the customer. Companies that are achieving long-term continuous improvement in quality tailored to customer satisfaction possess lasting characteristics such as customer orientation, customer consciousness, and customer responsiveness. In doing so, they liberate the quality concept from the narrow product or service focus to encompass total conformance to customer requirements in spite of the existing functionalization and departmentalization of modern complex structures. In addition to these key components, a customer-driven organization demands building and nurturing a customer satisfaction culture and value system that makes quality improvement and heightened concern for customer satisfaction a permanent aspect of organizational life.

Aspergillosis in immunocompromised children acute myeloid leukemia and bone marrow aplasia.: Report of two cases

Two cases of Aspergillosis in immunocompromised children are reported. Both were caused by Aspergillns flavus. Early diagnosis and treatment led to the remission of the process. One patient had acute myeloid leukemia; the fungus was isolated from the blood. The other patient with bone marrow aplasia, presented an invasive aspergillosis of the paranasal sinuses with dissemination of fungal infection; the diagnosis was obtained by histology and culture of biopsied tissue from a palatal ulceration.

Prevalence of putative periodontopathogens from periodontal patients and healthy subjects in São Paulo, SP, Brazil

A. actinomycetemcomitans, B. forsythus, P. gingivalis, C. rectus, E. corrodens, P. intermedia, F. nucleatum, and T. denticola were identified from subgingival plaque from 50 periodontal patients and 50 healthy subjects. PCR products from each species showed a specific band and could be used to identify periodontal organisms from clinical specimens. Identical negative or positive results between PCR and culture occurred in 66% (A. actinomycetemcomitans) to 93% (F. nucleatum) of the samples. PCR detection odds ratio values for A. actinomycetemcomitans, B. forsythus, C. rectus, E. corrodens, P. intermedia, and T. denticola were significantly associated with disease having a higher OR values for B. forsythus (2.97, 95% CI 1.88 - 4.70). Cultures showed that A. actinomycetemcomitans, B. forsythus and P. intermedia were associated with periodontitis, however, P. gingivalis, C. rectus, E. corrodens and F. nucleatum were not significantly associated with the disease.

Fonsecaea pedrosoi cerebral phaeohyphomycosis ("chromoblastomycosis"): first human culture-proven case reported in Brazil

Cerebral phaeohyphomycosis ("chromoblastomycosis") is a rare intracranial lesion. We report the first human culture-proven case of brain abscesses due to Fonsecaea pedrosoi in Brazil. The patient, a 28 year-old immunocompetent white male, had ocular manifestations and a hypertensive intracranial syndrome. Magnetic resonance imaging (MRI) of the brain revealed a main tumoral mass involving the right temporo-occipital area and another smaller apparently healed lesion at the left occipital lobe. A cerebral biopsy was performed and the pathological report was cerebral chromoblastomycosis. The main lesion was enucleated surgically and culture of the necrotic and suppurative mass grew a fungus identified as Fonsecaea pedrosoi. The patient had received a knife wound sixteen years prior to his hospitalization and, more recently, manifested a pulmonary granulomatous lesion in the right lung with a single non-pigmented form of a fungus present. It was speculated that the fungus might have gained entrance to the host through the skin lesion, although a primary respiratory lesion was not excluded. The patient was discharged from the hospital still with ocular manifestations and on antimycotic therapy and was followed for eight months without disease recurrence. Few months after he had complications of the previous neuro-surgery and died. A complete autopsy was performed and no residual fungal disease was found.

Immunoprecipitation techniques and Elisa in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), counterimmunoelectrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

Methylene blue vital staining for Trypanosoma cruzi trypomastigotes and epimastigotes

The morphological identification of Trypanosoma cruzi is currently considered to have a high specificity, but its sensitivity, which depends on the volume of the sample examined, is rather low. Trypanosome developmental stages suspended in blood, reduviid feces, and culture media are routinely searched for by means of fresh film examination (about 2 µL). High speed centrifugation of blood samples separates the buffy coat, where most trypomastigotes concentrate. As the parasites are transparent and colorless, their detection is mostly dependent on their motility. The fluorescent vital stain acridine orange has been used to enhance image contrast, as exemplified by the QBC (Quantitative Buffy Coat) technique. Staining blood, buffy coat, reduviid feces, and culture media samples with methylene blue (also a vital dye) is a means of producing sharp, well contrasted images of motile or non-motile T. cruzi developmental stages, only standard laboratory microscopes being required. Slides previously coated with a thin layer of methylene blue are used to stain fresh blood films. Photomicrographs exemplify the results of methylene blue staining applied to living and fixed parasites.

Improving tuberculosis control through the partnership between university and the health system

INTRODUCTION: Tuberculosis (TB) control is linked to the availability of qualified methods for microbiological diagnostics; however, microscopy with limited sensitivity is the only method available in many locations. The objective of this study was to evaluate the introduction of culture, drug susceptibility testing (DST), and genotyping in the routine of a Municipal Program of Tuberculosis Control. METHODS: Direct microscopy of sputum and culture in Ogawa-Kudoh were performed on 1,636 samples from 787 patients. DST of positive cultures was performed by resazurin microtiter assay and genotyping by mycobacterial interspersed repetitive units-variable number tandem repeat. RESULTS: A total 91 patients with TB were identified. The culture increased case detection by 32% compared with the microscopy; acquired resistance was 3.3% and the genotyping showed high genetic diversity. CONCLUSIONS: Ogawa-Kudoh contributed significantly to the increase in case detection and is suitable for implementation in poor-resource locations. The acquired resistance rate was lower than that reported in a recent Brazilian survey. The high genetic diversity is possibly related to the high TB prevalence in the population, as well as to early detection and suitable treatment of patients. The interaction between research and health care is important for reorienting the practice, transferring technology, and improving TB control.

Comparative study of lymphocytes from individuals that were vaccinated and unvaccinated against the pandemic 2009-2011 H1N1 influenza virus in Southern Brazil

ABSTRACTINTRODUCTION:While no single factor is sufficient to guarantee the success of influenza vaccine programs, knowledge of the levels of immunity in local populations is critical. Here, we analyzed influenza immunity in a population from Southern Brazil, a region with weather conditions that are distinct from those in the rest of country, where influenza infections are endemic, and where greater than 50% of the population is vaccinated annually.METHODS:Peripheral blood mononuclear cells were isolated from 40 individuals. Of these, 20 had received the H1N1 vaccine, while the remaining 20 were unvaccinated against the disease. Cells were stimulated in vitro with the trivalent post-pandemic influenza vaccine or with conserved major histocompatibility complex I (MHC I) peptides derived from hemagglutinin and neuraminidase. Cell viability was then analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)]-based colorimetric assay (MTT), and culture supernatants were assayed for helper T type 1 (Th1) and Th2-specific cytokine levels.RESULTS:Peripheral blood lymphocytes from vaccinated, but not unvaccinated, individuals exhibited significant proliferation in vitro in the presence of a cognate influenza antigen. After culturing with vaccine antigens, cells from vaccinated individuals produced similar levels of interleukin (IL)-10 and interferon (IFN)-γ, while those from unvaccinated individuals produced higher levels of IFN-γ than of IL-10.CONCLUSIONS:Our data indicate that peripheral blood lymphocytes from vaccinated individuals are stimulated upon encountering a cognate antigen, but did not support the hypothesis that cross-reactive responses related to previous infections can ameliorate the immune response. Moreover, monitoring IL-10 production in vaccinated individuals could comprise a valuable tool for predicting disease evolution.

Helicobacter pylori in dyspeptic children and adults: endoscopic, bacteriologic and histologic correlations

Using different bacteriological (urease test, Gram staining and culture) and histological (Steiner staining and modified Giemsa staining) techniques, we searched for the presence of Helicobacter pylori in the gastric antrum of 200 dyspeptic Brazilian patients (106 females and 94 males aged 19 days to 81 years). The presence of bacteria was then correlated with the endoscopic and histological findings. H. pylori was present in 59.5 of the population studied. In Brazil, colonization occurs early, involving 37 of the dyspeptic population by 20 years of age. The presence of H. pylori in the gastric antrum was strongly associated with duodenal ulcer (P < 0.001) and a normal endoscopic examination did not exclude the possibility of colonization of the gastric antrum by H. pylori. The most sensitive test was the preformed urease test (89). We conclude that more than one diagnostic method should preferably be used for the detection of H. pylori and that the presence of H. pylori is closely correlated with active chronic gastritis (P < 0.001).

Distribution and pathogenicity of Trypanosoma cruzi isolated from peridomestic populations of Triatoma infestans and Triatoma guasayana from rural Western Argentina

We assessed the distribution of Trypanosoma cruzi infection in peridomestic triatomines collected manually at a district-wide scale in rural villages around Olta, Western Argentina, and typed the isolated strains according to their pathogenicity to laboratory mice. Of 1623 triatomines examined, only 14 (0.9%) were infected with T. cruzi based on microscopical examination of feces. The prevalence of T. cruzi infection was 0.8% in Triatoma infestans, 2.3% in T. guasayana, and nil in T. garciabesi, T. platensis, and T. eratyrusiformis. Local transmission occurred in kitchens, store-rooms and goat corrals or nearby, though at very low levels. T. cruzi was detected by at least one parasitological method in 11 (79%) of 14 microscope-positive bugs. Hemoculture was the most sensitive method (67%) followed by culture of organ homogenates, histopathology or xenodiagnosis of inoculated suckling mice (55-58%), and culture of microscope-positive bug feces (46%). The evidence suggests that most of the isolated T. cruzi strains would be myotropic type III. Our study establishes for the first time that peridomestic, microscope-positive T. guasayana nymphs were actually infected with T. cruzi, and may be implicated as a putative secondary vector of T. cruzi in domestic or peridomestic sites.

Onychomycosis in Ceará (Northeast Brazil): epidemiological and laboratory aspects

Knowledge of epidemiological and mycological characteristics of onychomycosis has been noted by many authors as being an important tool for control of these fungal infections. This study seeks to improve knowledge of onychomycosis epidemiology and mycological features. Samples were taken from infected fingernails and toenails of 976 patients undergoing treatment at a respected Dermatology Center in Ceará, Fortaleza, CE, Brazil. Specimens from 512 patients (52%) were positive for onychomycosis. From the culture-positive samples, yeasts of the genus Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis) were dominant. The dermatophytes isolated (Trichophyton rubrum, T. tonsurans, T. mentagrophytes var. mentagrophytes) were dominant in 46 patients (12.99%). The mould Fusarium spp. was isolated from 29 patients (8.19%). Yeast of the genus Candida is the main causal factor in onychomycosis in our region. Also, the study showed the importance of performing direct examination and culture in diagnosis of onychomycosis.