PCR analysis of egyptian respiratory adenovirus isolates, including identification of species, serotypes, and coinfections

Eighty-eight adenovirus (Ad) isolates and associated clinical data were collected from walk-in patients with influenza-like illness in Egypt during routine influenza surveillance from 1999 through 2002. Respiratory Ad distributions are geographically variable, and serotype prevalence has not been previously characterized in this region. Serotype identity is clinically relevant because it predicts vaccine efficacy and correlates strongly with both clinical presentation and epidemiological pattern. Species and serotype identities were determined using several well-validated multiplex PCR protocols culled from the literature and supplemented with a few novel primer sets designed to identify rare types. The isolates included common species B1 serotypes (Ad3 and Ad7), common species C serotypes (Ad1, Ad2, and Ad5), the less common species B2 serotype Ad11, and three isolates of the rare species B1 serotype Ad16. Two isolates that appear to be variant Ad16 were also identified. Fifteen coinfections of multiple adenoviral types, primarily AdB/AdC and Ad3/Ad7 dual infections, were detected. The majority of these were verified using redundant PCR tests targeted at multiple genes. PCR is able to resolve coinfections, in contrast to traditional serum neutralization tests. PCR is also comparatively rapid and requires very little equipment. Application of the method allowed an inclusive determination of the serotypes found in the Egyptian respiratory sample set and demonstrated that coinfections are common and may play a previously unrecognized role in adenovirus pathogenesis, evolution, and epidemiology. In particular, coinfections may influence adenoviral evolution, as interserotypic recombination has been identified as a source of emerging strains.     

Epidemiology of Cyclospora cayetanensis and other intestinal parasites in a community in Haiti

We conducted an exploratory investigation in a community in Haiti to determine the prevalence of Cyclospora cayetanensis infection and to identify potential risk factors for C. cayetanensis infection. In 2001, two cross-sectional stool surveys and a nested case-control study were conducted. In 2002, a follow-up cross-sectional stool survey was conducted among children < or =10 years of age. Stool specimens from study participants and water samples from their wells were examined for Cyclospora and other intestinal parasites. In stools, the prevalence of infection with Cyclospora in persons of all ages decreased from 12% (20 of 167 persons) in February 2001 to 1.1% (4 of 352 persons) in April 2001, a 90.8% decrease. For children < or =10 years of age, the prevalence rates were 22.5% (16 of 71 children) in February 2001, 3.0% (4 of 135 children) in April 2001, and 2.5% (2 of 81 children) in January 2002. Use of the water from the artesian well in the northern region of the community versus the one in the south was the only risk factor associated with Cyclospora infection in multivariate analyses (odds ratio, 18.5; 95% confidence interval, 2.4 to 143.1). The water sample from one of the nine wells or water sources tested (one sample per source) in January 2001, shortly before the investigation began, was positive for Cyclospora by UV fluorescence microscopy and PCR. None of the water samples from the 46 wells or water sources tested during the investigation (one sample per source per testing period, including the artesian wells) were positive for Cyclospora. Further studies are needed to assess the role of water as a possible risk factor for Cyclospora infection in Haiti and other developing countries.     

Pemphigus Vulgaris Autoantibody Response is Linked to HLA-DQB10503 in Pakistani Patients

ABSTRACT: Pemphigus vulgaris (PV) is an autoimmune disease of the skin and mucous membranes characterized by an autoantibody response against an epidermal cadherin. We performed high resolution HLA class II typing in 19 patients with PV from Rawalpindi, Pakistan and 19 non-Jewish European PV patients from Boston by sequence-specific oligonucleotide probe hybridization. The results were compared with two separate ethnically matched control populations. We found that PV patients from Pakistan had significantly increased frequencies of DRB1*1404 ( p = 0.01), DQA1*0101 ( p = 0.02), and DQB1*0503 ( p = 0.01). Among the patients of non-Jewish European ancestry, DRB1*1401 ( p < 10⁻⁶), DQA1*0101 ( p < 10⁻⁵) and DQB1*0503 ( p < 10⁻⁶), were increased in PV patients. Formal linkage analysis between the major histocompatibility complex and the PV antibody was performed in 67 relatives of the 19 Pakistani patients. The results showed strong evidence for linkage of HLA-DRB1*1404, DQA1*0101, DQB1*0503, with the presence of PV antibody in relatives’ families with a significant logarithm of the odds score of 6.06. Based on the three dimensional structure of class II molecules, we propose that HLA-DQA1*0101 and DQB1*0503, encode a negatively charged P9 peptide binding pocket of the DQ molecule and are significantly associated with susceptibility to PV in non-Jewish populations.     

Oral hairy leukoplakia. Histopathologic and cytopathologic features of a subclinical phase

Accurate diagnosis of oral hairy leukoplakia (OHL) is important because it may be an early indicator of undiagnosed HIV infection; moreover, it may be a prognostic indicator. Our purpose was to investigate the histopathologic features of subclinical OHL and to evaluate and support the rationale of detecting subclinical OHL with cytopathology. The Epstein-Barr virus (EBV) was detected by immunohistochemistry and in situ hybridization in 4 cases of macroscopically normal lateral borders of tongue mucosa from 8 AIDS necropsies and in none of 8 controls. The histopathologic features were specific when based on nuclear changes: Cowdry type A inclusion, ground glass, and nuclear beading. Smears were obtained from 50 patients with AIDS, without OHL, from the scraping of lateral borders of the tongue. Numerous clusters of the cells were associated with Candida organisms (30% of cases). Nuclear changes were observed in 12 patients (24%) on both sides of the tongue. We describe the histopathologic features of subclinical OHL, and our observations suggest that cytopathology can detect OHL in the subclinical phase.     

The telescoping suture--Part II: A novel method to improve the mechanical behavior of a new biomaterial: ostrich pericardium

Ostrich pericardium, sutured using a telescoping or overlapping technique, was studied to determine its mechanical behavior. From each of 12 pericardial sacs, four contiguous strips were cut longitudinally, from root to apex, and another four contiguous strips were cut in transverse direction. One of the strips in each set of four was used as an unsutured control and the remaining three were sutured by overlapping 0.5 cm of the tissue and sewing with Gore-tex, Prolene or Pronova. These 96 samples were then subjected to tensile testing along their major axes until rupture. The tensile stresses recorded in the suture materials at the moment tears appeared in the pericardium ranged between 55.99 MPa and 70.23 MPa for Gore-tex in samples cut in the two directions. Shear stress became ostensible at 56 MPa, with clearly evident tears. However, microfracture of the collagen fibers must be produced at much lower stress levels. The comparison of the resistance in kilograms (machine-imposed), without taking into account the sections in which the load was applied, demonstrated only a slight loss of load when the telescoping suture was employed in ostrich pericardium samples. Ostrich pericardium may continue to be an alternative biological material for the construction of heart valve leaflets.     

Lethal encephalitis in myeloid differentiation factor 88-deficient mice infected with herpes simplex virus 1

Herpes simplex virus 1 (HSV-1), a large DNA virus from the Herpesviridae family, is the major cause of sporadic lethal encephalitis and blindness in humans. Recent studies have shown the importance of Toll-like receptors (TLRs) in the immune response to HSV-1 infection. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein that is downstream to mediated TLR activation and is essential for the production of inflammatory cytokines. Here, we studied the relationship between MyD88 and HSV-1 using a purified HSV-1 isolated from a natural oral recurrent human infection. We observed the activation of TLR-2 by HSV-1 in vitro using Chinese hamster ovary cells stably transfected with a reporter gene. Interestingly, we found that only peritoneal macrophages from MyD88-/- mice, but not macrophages from TRL2-/- or from wild-type mice, were unable to produce tumor necrosis factor-alpha in response to HSV-1 exposure. Additionally, although TLR2-/- mice showed no enhanced susceptibility to intranasal infection with HSV-1, MyD88-/- mice were highly susceptible to infection and displayed viral migration to the brain, severe neuropathological signs of encephalitis, and 100% mortality by day 10 after infection. Together, our results suggest that innate resistance to HSV-1 is mediated by MyD88 and may rely on activation of multiple TLRs.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Microsatellite instability, mitochondrial DNA large deletions, and mitochondrial DNA mutations in gastric carcinoma

Mitochondrial DNA (mtDNA) large deletions and mtDNA mutations have been demonstrated in various types of human cancer. The relationship between the occurrence of such alterations and the nuclear microsatellite instability (MSI) status of the neoplastic cells remains controversial. In an attempt to clarify the situation in gastric carcinoma, we studied, by PCR/SSCP and sequencing, five mitochondrial genes and two D-loop regions in 32 gastric carcinomas that had been previously screened for MSI and mitochondrial common deletion. MtDNA alterations were detected in 26 carcinomas (81%). All the mtDNA mutations, which occurred mainly in the D-loop and ND1 and ND5 genes, were transitions. D-loop alterations (insertions and/or deletions) were not significantly associated with mutations in the coding regions. There was a trend towards an inverse relationship between the occurrence of mitochondrial common deletion and mtDNA mutations. No significant relationship was observed between MSI status and mtDNA mutations, whereas the mitochondrial common deletion appeared to be almost exclusively restricted to MSI-negative tumors. The latter finding--almost no gastric carcinoma with MSI-positive phenotype has large deletions of mtDNA--needs to be confirmed in a larger series and in tumors from other organs.     

Inhibin alpha-subunit (INHA) gene and locus changes in paediatric adrenocortical tumours from TP53 R337H mutation heterozygote carriers

The R337H TP53 mutation is a low-penetrance molecular defect that predisposes to adrenocortical tumour (ACT) formation in Brazilian and possibly other populations. Additional genetic defects may be responsible for the variable expression of ACTs in these cases. The inhibin α-subunit gene (INHA) on 2q33-qter has been implicated in mouse adrenocortical tumourigenesis. We studied 46 pediatric patients with ACTs from Brazil for INHA genetic alterations; 39 of these patients were heterozygous carriers of the R337H TP53 mutation. We first mapped the INHA gene by radiation hybrid analysis and determined 10 linked microsatellite markers in an area flanked by D2S1371 and D2S206 on 2q33-qter. These markers were then used for loss of heterozygozity (LOH) studies in nine paired germline and tumour DNA samples. Mapping placed the INHA gene in close proximity to D2S2848 (SHGC11864) with a log of odds (LOD) score of 5.84. LOH for at least one marker in the region was identified in 8/9 tumours (89%). Six patients were heterozygous for three INHA mutations: one in exon 1, 127C>G, and two in exon 2, 3998G>A and 4088G>A, all leading to amino acid substitutions (P43A, G227R, and A257T, respectively). A257T is located in a conserved INHA region, highly homologous to transforming growth factor-ß; both G227R and A257T change polarity, and, in addition, G227R changes the pH. We conclude that these sequence alterations and the detected 2q allelic changes suggest that INHA may be one of the contributing factors needed for ACT formation in pediatric patient carriers of the R337H TP53 mutation.     

Xylitol formation by Candida guilliermondii in media containing different nitrogen sources

The xylose conversion into xylitol by Candida guilliermondii was evaluated in semi-synthetic media supplemented with different nitrogen sources in a ratio C/N equal 25.6. It was noticed that the xylitol yield was around 80% and also that the type of nitrogen source did not influence this bioconversion.