Molecular identification of adenoviruses associated with respiratory infection in Egypt from 2003 to 2010.

BACKGROUND: Human adenoviruses of species B, C, and E (HAdV-B, -C, -E) are frequent causative agents of acute respiratory infections worldwide. As part of a surveillance program aimed at identifying the etiology of influenza-like illness (ILI) in Egypt, we characterized 105 adenovirus isolates from clinical samples collected between 2003 and 2010. METHODS: Identification of the isolates as HAdV was accomplished by an immunofluorescence assay (IFA) and confirmed by a set of species and type specific polymerase chain reactions (PCR). RESULTS: Of the 105 isolates, 42% were identified as belonging to HAdV-B, 60% as HAdV-C, and 1% as HAdV-E. We identified a total of six co-infections by PCR, of which five were HAdV-B/HAdV-C co-infections, and one was a co-infection of two HAdV-C types: HAdV-5/HAdV-6. Molecular typing by PCR enabled the identification of eight genotypes of human adenoviruses; HAdV-3 (n = 22), HAdV-7 (n = 14), HAdV-11 (n = 8), HAdV-1 (n = 22), HAdV-2 (20), HAdV-5 (n = 15), HAdV-6 (n = 3) and HAdV-4 (n = 1). The most abundant species in the characterized collection of isolates was HAdV-C, which is concordant with existing data for worldwide epidemiology of HAdV respiratory infections. CONCLUSIONS: We identified three species, HAdV-B, -C and -E, among patients with ILI over the course of 7 years in Egypt, with at least eight diverse types circulating

Dipstick for Rapid Diagnosis of Shigella flexneri 2a in Stool

BACKGROUND: Shigellosis or bacillary dysentery, an acute bloody diarrhoea, is a major public health burden in developing countries. In the absence of prompt and appropriate treatment, the infection is often fatal, particularly in young malnourished children. Here, we describe a new diagnostic test for rapid detection, in stool, at the bedside of patients, of Shigella flexneri 2a, the most predominant agent of the endemic form of the disease. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S.flexneri 2a lipopolysaccharide (LPS) using serotype 2a-specific monoclonal antibodies coupled to gold particles and displayed on one-step immunochromatographic dipstick. A concentration as low as 20 ng/ml of LPS is detected in distilled water and in reconstituted stools in under 15 minutes. The threshold of detection corresponds to a concentration of 5×10(7) CFU/ml of S. flexneri 2a, which provides an unequivocal positive reaction in three minutes in distilled water and reconstituted stools. The specificity is 100% when tested with a battery of Shigella and unrelated strains, in culture. When tested in Vietnam, on clinical samples, the specificity and sensitivity were 99.2 and 91.5%, respectively. A decrease of the sensitivity during the evaluation on stool samples was observed after five weeks at room temperature and was due to moistening of the dipsticks caused by the humidity of the air during the fifth week of the evaluation. This drawback is now overcome by improving the packaging and providing dipsticks individually wrapped in waterproof bags. CONCLUSION: This simple dipstick-bases test represents a powerful tool for case management and epidemiological surveys

Use of Saliva for Early Dengue Diagnosis

The importance of laboratory diagnosis of dengue cannot be undermined. In recent years, many dengue diagnostic tools have become available for various stages of the disease, but the one limitation is that they require blood as a specimen for testing. In many incidences, phlebotomy in needle-phobic febrile individuals, especially children, can be challenging, and the tendency to forgo a dengue blood test is high. To circumvent this, we decided to work toward a saliva-based assay (antigen-capture anti-DENV IgA ELISA, ACA-ELISA) that has the necessary sensitivity and specificity to detect dengue early. Overall sensitivity of the ACA-ELISA, when tested on saliva collected from dengue-confirmed patients (EDEN study) at three time points, was 70% in the first 3 days after fever onset and 93% between 4 to 8 days after fever onset. In patients with secondary dengue infections, salivary IgA was detected on the first day of fever onset in all the dengue confirmed patients. This demonstrates the utility of saliva in the ACA-ELISA for early dengue diagnostics. This technique is easy to perform, cost effective, and is especially useful in dengue endemic countries

A New Approach to Dengue Fatal Cases Diagnosis: NS1 Antigen Capture in Tissues

Dengue manifestations may vary from asymptomatic to potentially fatal complications. With an increasing number of Dengue Hemorrhagic fever (DHF) and fatal cases, the availability of new approaches useful for cases confirmation plays an important role for the disease surveillance. The diagnosis of fatal cases in frozen and fixed tissues from autopsies can be determined by techniques such as viral RT-PCR, in situ hybridization, viral proteins detection by immunohistochemistry and NS3 specific immunostaining. We aimed to assess for the first time the usefulness of NS1 capture tests as a diagnostic technique to demonstrate DENV antigens in human tissue specimens. The highest sensitivity was obtained by a rapid ICT which was also the most sensitive in liver, lung, kidney, brain, spleen and thymus. Despite a number of studies demonstrating the usefulness of DENV NS1 antigen detection by different ELISAs in plasma and/or sera of dengue patients, no research has been done previously to demonstrate NS1 presence in tissues of fatal dengue cases. Moreover, the application of NS1 kits to demonstrate the presence of DENV may provide a better understanding of viral tropism in fatal cases and may be useful for studies of pathogenesis in vivo and in experimental animals

Structure-Based High-Throughput Epitope Analysis of Hexon Proteins in B and C Species Human Adenoviruses (HAdVs)

Human adenoviruses (HAdVs) are the etiologic agent of many human infectious diseases. The existence of at least 54 different serotypes of HAdVs has resulted in difficulties in clinical diagnosis. Acute respiratory tract disease (ARD) caused by some serotypes from B and C species is particularly serious. Hexon, the main coat protein of HAdV, contains the major serotype-specific B cell epitopes; however, few studies have addressed epitope mapping in most HAdV serotypes. In this study, we utilized a novel and rapid method for the modeling of homologous proteins based on the phylogenetic tree of protein families and built three-dimensional (3D) models of hexon proteins in B and C species HAdVs. Based on refined hexon structures, we used reverse evolutionary trace (RET) bioinformatics analysis combined with a specially designed hexon epitope screening algorithm to achieve high-throughput epitope mapping of all 13 hexon proteins in B and C species HAdVs. This study has demonstrated that all of the epitopes from the 13 hexon proteins are located in the proteins' tower regions; however, the exact number, location, and size of the epitopes differ among the HAdV serotypes