Hantaviruses in Central South America: phylogenetic analysis of the S segment from HPS cases in Paraná, Brazil

We sequenced the complete S segments of hantaviruses detected from 12 HPS patients living in southern of Brazil. Samples were obtained from patients diagnosed in different years, in distinct areas, and with a broad spectrum of clinical signs. Despite these differences, all the S proteins of hantavirus from Paraná were identical, except for one amino acid substitution. Phylogenetic analyses of the complete S segment nucleotide and amino acid sequences indicated that hantaviruses from Paraná form a distinct clade from those circulating in South and North America. Other hantaviruses from Brazil were not placed in the same clade. The Oligoryzomys nigripes-associated strains ITA37 and ITA38 from Paraguay were found to belong to the same clade as the hantaviruses from Paraná. Paraguay and Paraná state are located at the same latitude and some ecosystems are similar in both places. The geographic position and common rodent hosts could explain this phylogenetic relationship.     

Comparison of latex agglutination with enzyme immunoassay for detection of rotavirus in fecal specimens

Human rotaviruses are the most important etiologic agents of acquired diarrhea in infants and young children worldwide. Early diagnosis is essentialfor effective patient treatment. The latex agglutination (LA) assays for rotavirus diagnosis are rapid, inexpensive, and the most widely used to screen specimens. The performance of the LA Rotagen (Biokit S.A., Barcelona, Spain) was evaluated for rotavirus detection infecal samples of outpatients with acute gastroenteritis. This assay was compared with the enzyme immunoassay (EIA) EIARA (Bio-Manguinhos, Rio de Janeiro, Brazil). From January to October 2000, 285 fecal specimens were analyzed. Forty-four samples (15.4%) were reactive, 214 (75.4%) were nonreactive, and 27 (9.5%) were indeterminate by LA. All LA-positive samples were positive by EIA, and 2 LA-negative samples were positive by EIA. Of specimens indeterminate by LA, 67% were positive by EIA. The sensitivity, specificity, and accuracy of LA were 69%, 100%, and 93%, respectively. These results indicate that assay is as sensitive and specific as the EIA, and it could be applied on a large scale for screening stool specimens in suspected rotavirus diarrhea. However, the indeterminate results must be confirmed by other methods, such as EIA.     

RT-PCR-RFLP for genetic diversity analysis of Tunisian Grapevine fanleaf virus isolates in their natural host plants

Genetic diversity was characterized in 20 isolates of Grapevine fanleaf virus (GFLV) recovered from naturally infected grapevine plants (Vitis vinifera) in the North of Tunisia. Viral RNAs were isolated by oligoprobe capture, and a 605 bp fragment containing a part of the viral coat protein gene was amplified by RT-PCR. Sequence variation among isolates was characterized by restriction fragment length polymorphism (RFLP) analysis and confirmed by sequencing. The GFLV infections are found as a complex mixture of closely related genomes. In further studies, RFLP analyses of virus isolates using AluI showed that GFLV populations in Tunisian vineyards consist of two restrictotypes corresponding to distinct sub-populations Sp1 and Sp2. The relative field distribution of these sub-populations showed that Sp2 was more abundant. Individual genomes were recovered by cloning the RT-PCR products. The sequences were found to vary from each other by as much as 11%. Cloning from mixed infections showed that Sp2 are also predominant.     

Notch/Delta4 interaction in human embryonic liver CD34+ CD38- cells: positive influence on BFU-E production and LTC-IC potential maintenance

We investigated whether Notch signaling pathways have a role in human developmental hematopoiesis. In situ histochemistry analysis revealed that Notch1, 2, and 4 and Notch ligand (Delta1-4, and Jagged1) proteins were not expressed in the yolk sac blood islands, the para-aortic splanchnopleure, the hematopoietic aortic clusters, and at the early stages of embryonic liver hematopoiesis. Notch1-2, and Delta4 were eventually detected in the embryonic liver, from 34 until 38 days postconception. Fluorescence-activated cell sorter analysis showed that first-trimester embryonic liver CD34(+)CD38(low) cells expressed both Notch1 and Notch2. When these cells were cultured on S17 stroma stably expressing Delta4, a 2.6-fold increase in BFU-E number was observed at day 7, as compared with cultures with control stroma, and this effect was maintained for 2 weeks. Importantly, exposure of these cells to Delta4 under these conditions maintained the original frequency and quality of long-term culture-initiating cells (LTC-ICs), while control cultures quickly resulted in the extinction of this LTC-IC potential. Furthermore, short-term exposure of embryonic liver adherent cells to erythropoietin resulted in a dose-dependent increase in Delta4 expression, almost doubling the expression observed with untreated stroma. This suggests that Delta4 has a role in the regulation of hematopoiesis after a hypoxic stress in the fetus.     

Bilateral abscessed orchiepididymitis associated with sepsis caused by Veillonella parvula and Clostridium perfringens: case report and review of the literature.

Veillonella species is a gram-negative coccus which is part of the anaerobic normal flora in the oral cavity, small intestine, upper respiratory tract, vagina, and urinary tract. The role that this organism plays in infection is not well known, and it is generally associated with other bacteria. We present a case of bilateral abscessed orchiepididymitis associated with septicemia due to Veillonella parvula and, later, to Clostridium perfringens, with the development of severe renal insufficiency and septic shock, which resolved favorably with antibiotic therapy, treatment of shock, and hyperbaric oxygen therapy. In reviewing the literature, we have not found any other case of sepsis due to Veillonella sp. associated with urological disorders.     

Coxiella burnetii Induces Reorganization of the Actin Cytoskeleton in Human Monocytes

Coxiella burnetii, an obligate intracellular bacterium which survives in myeloid cells, causes Q fever in humans. We previously demonstrated that virulent C. burnetii organisms are poorly internalized by monocytes compared to avirulent variants. We hypothesized that a differential mobilization of the actin cytoskeleton may account for this distinct phagocytic behavior. Scanning electron microscopy demonstrated that virulent C. burnetii stimulated profound and polymorphic changes in the morphology of THP-1 monocytes, consisting of membrane protrusions and polarized projections. These changes were transient, requiring 5 min to reach their maximum extent and vanishing after 60 min of incubation. In contrast, avirulent variants of C. burnetii did not induce any significant changes in cell morphology. The distribution of filamentous actin (F-actin) was then studied with a specific probe, bodipy phallacidin. Virulent C. burnetii induced a profound and transient reorganization of F-actin, accompanied by an increase in the F-actin content of THP-1 cells. F-actin was colocalized with myosin in cell protrusions, suggesting that actin polymerization and the tension of actin-myosin filaments play a role in C. burnetii-induced morphological changes. In addition, contact between the cell and the bacterium seems to be necessary to induce cytoskeleton reorganization. Bacterial supernatants did not stimulate actin remodeling, and virulent C. burnetii organisms were found in close apposition with F-actin protrusions. The manipulation of the actin cytoskeleton by C. burnetii may therefore play a critical role in the internalization strategy of this bacterium.     

Development and comparison of enzyme immunoassays for diagnosis of Chagas' disease using fixed forms of Trypanosoma cruzi (Epimastigotes, Amastigotes, and Trypomastigotes) and assessment of antigen stability for the three assays

Three enzyme immunoassays (EIAs) for diagnosis of Chagas' disease were developed with fixed forms of Trypanosoma cruzi using a panel of 435 sera from the following groups: Venezuelan subjects positive by immunofluorescence (n = 70), Venezuelan healthy controls (n = 85), healthy Canadians (n = 166), and subjects with other parasitic diseases (n = 114). All assays achieved 100% sensitivity and reasonable specificity for amastigotes (97.6%), epimastigotes (98.3%), and trypomastigotes (99.3%). The fixed-trypomastigote assay was stable over 4 months at 4 degrees C and room temperature. These data suggest that a fixed-trypomastigote EIA may be a suitable candidate for blood bank screening.     

Study of CYP2D6 gene in children with autoimmune hepatitis and P450 IID6 autoantibodies.

Cytochrome P450 IID6 is an autoantigen recognized by the sera of children affected with a subtype of autoimmune hepatitis. It was hypothesized that a mutation in the CYP2D6 gene could explain the autoimmune response in these patients. To examine this question, genomic DNA from peripheral lymphocytes (n = 9) and liver (n = 1) of 10 patients with anti-LKM-1 antibody was analysed by Southern blot for genetic association studies between a particular CYP2D6 haplotype and autoimmune hepatitis. In addition, a region of CYP2D6, from the same genomic DNA, was amplified by polymerase chain reaction (PCR) and digested by BstNI, in a search for the most prevalent 29B mutation, described in subjects who do not express the P450 IID6. Total RNA and proteins, prepared from the liver of an anti-LKM-1+ patient, were analysed by Northern and Western (immunoblot) blots respectively. Our results do not reveal any major structural change in the DNA of this patient at the CYP2D6 locus that could explain their autoimmune response. Corroborating this observation, no changes were noted either in P450 IID6 mRNA size or in the corresponding protein. However, these data do not exclude the possibility of subtle changes in the protein due to point mutations in critical regions that might trigger an autoimmune response.     

Threshold effect of urinary glycosaminoglycans and the walk test as indicators of disease progression in a survey of subjects with Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome)

A cross-sectional survey in individuals affected with the lysosomal storage disease Mucopolysaccharidosis VI (MPS VI) was conducted to establish demographics, urinary glycosaminoglycan (GAG) levels, and clinical progression of the disease. The survey evaluated 121 bona fide MPS VI-affected individuals over the age of 4 years from 15 countries across the Americas, Europe, and Australasia representing greater than 10% of the estimated world prevalence of the disease. A medical history, complete physical exam, urinary GAG determination, and assessment of several clinical measures related to physical endurance, pulmonary function, joint range of motion, strength, and quality of life were completed for each participant. Although a wide variation in clinical presentation was observed, several general findings were obtained reflecting progression of the disease. Impaired physical endurance, as measured by the distance achieved in a 6-min walk, could be demonstrated across all age groups of MPS VI-affected individuals. High urinary GAG values (>200 mug/mg creatinine) were associated with an accelerated clinical course comprised of age-adjusted short stature and low body weight, impaired endurance, compromised pulmonary function, and reduced joint range of motion. An unexpected result was the predominance of urinary GAG values <100 mug/mg creatinine for those participants over the age of 20 years. Pending the collection of longitudinal data, these results suggest that urinary GAG levels predict clinical morbidity, and longer-term survival is associated with urinary GAG levels below a threshold of 100 mug/mg creatinine.