Molecular analysis of adenoviruses from clinical samples

At present, 56 types of human adenovirus (HAdVs) have been identified and found to be associated with a variety of clinical features in the respiratory tract, eye, gastrointestinal tract, and other organs. In additions, HAdVs are able to establish persistent and latent infections in humans. Most of the work which has been carried out recently is related to adenovirus vectors and little has been done in other areas such as the nature and mechanisms of adenovirus persistence and latency in human tissues. Another area needing more investigation is the stability of the adenovirus genome which is useful for the development of adenovirus vectors and vaccines and for better understanding of adenovirus evolution especially with conflicting views about this issue.Recombination between two types of adenovirus can happen when the hexon epitope from one type and the fibre epitope from another type are found (intermediate strains). These recombinants can be detected by the conflicting results for serum neutralization (SN) and haemagglutination inhibition (HI) tests or by sequencing and phylogenetic analysis of the hexon and fibre regions of the adenovirus genome. The first part of this study is related to the stability and evolution of different adenovirus species. A total of 31 clinical isolates from AIDS patients previously typed in the hexon L2 region and the fibre knob region were analysed. These isolates were found to be from species D adenovirus (HAdV-D) and 28 of them had contradictory typing results in these two regions so they are clearly intermediate strains. Two isolates appear to be completely new and one isolate (Aids32) was typed as HAdV-D23 variant in both hexon L2 and fibre knob regions. Sequencing and phylogenetic analysis of the hexon L1, fibre shaft and penton regions of these adenoviruses revealed that no intragene recombination events occurred between the hexon L1 and L2 regions or between the fibre knob and fibre shaft regions. Sequencing and phylogenetic analysis of the penton showed that some of the intermediate strains had sequences from a third type of adenovirus in these regions. The penton analysis showed also that intragene recombination between penton HVR and RGD loop regions was common. New types of adenovirus were detected and sequential infection with different adenovirus variants was observed in some patients which indicates that the genome of HAdV-D from AIDS patients are not stable. Full genome sequencing and analysis was carried out for three isolates, two of them appeared to be new types of HAdV-D and the result of multi-recombination events and the third isolate appeared to be a variant of HAdV-D23.The stability of species B adenovirus (HAdV-B) was also analysed. A total of 96 isolates collected from the Manchester area typed previously by serum neutralization (SN) were analysed in five genome regions. Most of these isolates were HAdV-B3 and HAdV-B7 collected during a 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 collected in different years following the outbreak in addition to other adenovirus types isolated from different years. The phylogenetic analysis results of all the isolates in the structural regions: hexon L2, penton and fibre knob were found to be consistent and no mismatches (hexon from one type and fiber from another type) were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable in general. The last part of this study is investigating adenovirus persistence and latency in human tissues. Tonsils and adenoids (106 right and left tonsils and 10 adenoids) were obtained from 57 patients who underwent routine tonsillectomies and/or adenoidectomies. Eighty four (72.41%) tonsils and adenoids samples were positive for HAdV by real-time PCR. The viral load was not the same in the right and left tonsils in most of the cases and ranged from 280 to more than 2.6 x 106 copies/107 cells. Seventy eight of 84 positive samples could be typed by sequencing of the hexon L1 region. Species C types were detected in 82% of the samples followed by species B (7.7%), HAdV-E4 (7.7%) and HAdV-F41 (2.56%). No DNA methylation was detected in the major late promoter (MLP) and E1A promoter regions of six tonsils and adenoids samples and two clinical isolates.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Characterisation of acute respiratory infections at a United Kingdom paediatric teaching hospital: observational study assessing the impact of influenza A (2009 pdmH1N1) on predominant viral pathogens

Background According to the World Health Organisation, influenza A (2009 pdmH1N1) has moved into the post-pandemic phase, but there were still high numbers of infections occurring in the United Kingdom in 2010-11. It is therefore important to examine the burden of acute respiratory infections at a large children’s hospital to determine pathogen prevalence, occurrence of co-infection, prevalence of co-morbidities and diagnostic yield of sampling methods. Methods This was a retrospective study of respiratory virus aetiology in acute admissions to a paediatric teaching hospital in the North West of England between 1st April 2010 and 31st March 2011. Respiratory samples were analysed either with a rapid RSV test if the patient had symptoms suggestive of bronchiolitis, followed by multiplex PCR testing for ten respiratory viruses, or with multiplex PCR testing alone if the patient had suspected other ARI. Patient demographics and data regarding severity of illness, presence of co-morbidities and respiratory virus sampling method were retrieved from case notes. Results 645 patients were admitted during the study period. 82/645 (12.7%) patients were positive for 2009 pdmH1N1, of whom 24 (29.2%) required PICU admission, with 7.3% mortality rate. Viral co-infection occurred in 48/645 (7.4%) patients and was not associated with more severe disease. Co-morbidities were present more frequently in older children, but there was no significant difference in prevalence of co-morbidity between 2009 pdmH1N1 patients and those with other ARI. NPA samples had the highest diagnostic yield with 192/210 (91.4%) samples yielding an organism. Conclusions Influenza A (2009 pdmH1N1) is an ongoing cause of occasionally severe disease affecting both healthy children and those with co-morbidities. Surveillance of viral pathogens provides valuable information on patterns of disease

The Diagnostic and Prognostic Accuracy of Five Markers of Serious Bacterial Infection in Malawian Children with Signs of Severe Infection

Early recognition and prompt and appropriate antibiotic treatment can significantly reduce mortality from serious bacterial infections (SBI). The aim of this study was to evaluate the utility of five markers of infection: C-reactive protein (CRP), procalcitonin (PCT), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), CD163 and high mobility group box-1 (HMGB1), as markers of SBI in severely ill Malawian children.Children presenting with a signs of meningitis (n = 282) or pneumonia (n = 95), were prospectively recruited. Plasma samples were taken on admission for CRP, PCT, sTREM-1 CD163 and HMGB1 and the performance characteristics of each test to diagnose SBI and to predict mortality were determined. Of 377 children, 279 (74%) had SBI and 83 (22%) died. Plasma CRP, PCT, CD163 and HMGB1 and were higher in HIV-infected children than in HIV-uninfected children (p<0.01). In HIV-infected children, CRP and PCT were higher in children with SBI compared to those with no detectable bacterial infection (p<0.0005), and PCT and CD163 were higher in non-survivors (p = 0.001, p = 0.05 respectively). In HIV-uninfected children, CRP and PCT were also higher in children with SBI compared to those with no detectable bacterial infection (p<0.0005), and CD163 was higher in non-survivors (p = 0.05). The best predictors of SBI were CRP and PCT, and areas under the curve (AUCs) were 0.81 (95% CI 0.73–0.89) and 0.86 (95% CI 0.79–0.92) respectively. The best marker for predicting death was PCT, AUC 0.61 (95% CI 0.50–0.71).Admission PCT and CRP are useful markers of invasive bacterial infection in severely ill African children. The study of these markers using rapid tests in a less selected cohort would be important in this setting

Ultrasound-enhanced latex immunoagglutination and PCR as complementary methods for non-culture-based confirmation of meningococcal disease

Preadmission administration of antibiotics to patients with suspected meningococcal infection has decreased the likelihood of obtaining an isolate and has stimulated development of rapid and reliable non-culture-based diagnostic methods. The sensitivity of the conventional test card latex agglutination test (TCLAT) for detection of capsular polysaccharide has been reported to be suboptimal. In the United Kingdom meningococcal DNA detection by PCR has become readily available and is now used as a first-line investigation. Recently, the performance of latex antigen detection has been markedly improved by ultrasound enhancement. Three tests for laboratory confirmation of meningococcal infection, (i) PCR assays, (ii) TCLAT, and (iii) ultrasound-enhanced latex agglutination test (USELAT), were compared in a retrospective study of 125 specimens (serum, plasma, and cerebrospinal fluid specimens) from 90 patients in whom meningococcal disease was suspected on clinical grounds. Samples were from patients with (i) culture-confirmed meningococcal disease, (ii) culture-negative but PCR-confirmed meningococcal disease, and (iii) clinically suspected but non-laboratory-confirmed meningococcal disease. USELAT was found to be nearly five times more sensitive than TCLAT. Serogroup characterization was obtained by both PCR and USELAT for 44 samples; all results were concordant and agreed with the serogroups determined for the isolates when the serogroups were available. For 12 samples negative by USELAT, the serogroup was determined by PCR; however, for 12 other specimens for which PCR had failed to indicate the serogroup, USELAT gave a result. USELAT is a rapid, low-cost method which can confirm a diagnosis, identify serogroups, and guide appropriate management of meningococcal disease contacts. A complementary non-culture-based confirmation strategy of USELAT for local use supported by a centralized PCR assay service for detection of meningococci would give the benefits of timely information and improved epidemiological data

Epstein-Barr Virus Coinfection in Cerebrospinal Fluid Is Associated With Increased Mortality in Malawian Adults With Bacterial Meningitis

Mortality from adult bacterial meningitis exceeds 50% in sub-Saharan Africa. We postulated that—particularly in individuals infected with human immunodeficiency virus (HIV)—herpes simplex virus, varicella zoster virus, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) in the cerebrospinal fluid (CSF) contribute to poor outcome. CSF from 149 Malawian adults with bacterial meningitis and 39 controls were analyzed using polymerase chain reaction. EBV was detected in 79 of 149 bacterial meningitis patients. Mortality (54%) was associated with higher CSF EBV load when adjusted for HIV (P = .01). CMV was detected in 11 of 115 HIV-infected patients, 8 of whom died. The mechanisms by which EBV and CMV contribute to poor outcome require further investigation

PCR Improves Diagnostic Yield from Lung Aspiration in Malawian Children with Radiologically Confirmed Pneumonia

Accurate data on childhood pneumonia aetiology are essential especially from regions where mortality is high, in order to inform case-management guidelines and the potential of prevention strategies such as bacterial conjugate vaccines. Yield from blood culture is low, but lung aspirate culture provides a higher diagnostic yield. We aimed to determine if diagnostic yield could be increased further by polymerase chain reaction (PCR) detection of bacteria (Streptococcus pneumoniae and Haemophilus influenzae b) and viruses in lung aspirate fluid.A total of 95 children with radiological focal, lobar or segmental consolidation had lung aspirate performed and sent for bacterial culture and for PCR for detection of bacteria, viruses and Pneumocystis jirovecii. In children with a pneumococcal aetiology, pneumococcal bacterial loads were calculated in blood and lung aspirate fluid.Blood culture identified a bacterial pathogen in only 8 patients (8%). With the addition of PCR on lung aspirate samples, causative pathogens (bacterial, viral, pneumocystis) were identified singly or as co-infections in 59 children (62%). The commonest bacterial organism was S.pneumoniae (41%), followed by H. influenzae b (6%), and the commonest virus identified was adenovirus (16%), followed by human bocavirus (HBoV) (4%), either as single or co-infection.In a select group of African children, lung aspirate PCR significantly improves diagnostic yield. Our study confirms a major role of S.pneumoniae and viruses in the aetiology of childhood pneumonia in Africa

Genomic epidemiology of syphilis in England: a population-based study.

BACKGROUND: Syphilis is a sexually transmitted bacterial infection caused by Treponema pallidum subspecies pallidum. Since 2012, syphilis rates have risen dramatically in many high-income countries, including England. Although this increase in syphilis prevalence is known to be associated with high-risk sexual activity in gay, bisexual, and other men who have sex with men (GBMSM), cases are rising in heterosexual men and women. The transmission dynamics within and between sexual networks of GBMSM and heterosexual people are not well understood. We aimed to investigate if whole genome sequencing could be used to supplement or enhance epidemiological insights around syphilis transmission. METHODS: We linked national patient demographic, geospatial, and behavioural metadata to whole T pallidum genome sequences previously generated from patient samples collected from across England between Jan 1, 2012, and Oct 31, 2018, and performed detailed phylogenomic analyses. FINDINGS: Of 497 English samples submitted for sequencing, we recovered 240 genomes (198 from the UK Health Security Agency reference laboratory and 42 from other laboratories). Three duplicate samples (same patient and collection date) were included in the main phylogenies, but removed from further analyses of English populations, leaving 237 genomes. 220 (92·8%) of 237 samples were from men, nine (3·8%) were from women, and eight (3·4%) were of unknown gender. Samples were mostly from London (n=118 [49·8%]), followed by southeast England (n=29 [12·2%]), northeast England (n=24 [10·1%]), and southwest England (n=15 [6·3%]). 180 (76·0%) of 237 genomes came from GBMSM, compared with 25 (10·5%) from those identifying as men who have sex with women, 15 (6·3%) from men with unrecorded sexual orientation, nine (3·8%) from those identifying as women who have sex with men, and eight (3·4%) from people of unknown gender and sexual orientation. Phylogenomic analysis and clustering revealed two dominant T pallidum sublineages in England. Sublineage 1 was found throughout England and across all patient groups, whereas sublineage 14 occurred predominantly in GBMSM older than 34 years and was absent from samples sequenced from the north of England. These different spatiotemporal trends, linked to demography or behaviour in the dominant sublineages, suggest they represent different sexual networks. By focusing on different regions of England we were able to distinguish a local heterosexual transmission cluster from a background of transmission in GBMSM. INTERPRETATION: These findings show that, despite extremely close genetic relationships between T pallidum genomes globally, genomics can still be used to identify putative transmission clusters for epidemiological follow-up. This could be of value for deconvoluting putative outbreaks and for informing public health interventions. FUNDING: Wellcome funding to the Sanger Institute, UK Research and Innovation, National Institute for Health and Care Research, European and Developing Countries Clinical Trials Partnership, and UK Health Security Agency

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Funder: Queensland GovernmentSyphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides

Traffic reduction at visitor attractions: the case of Hadrian's Wall.

The paper addresses the issue of sustainable travel at a linear tourist destination, Hadrian’s Wall, in the north of England. It reports the findings of depth interviews with managers of visitor attractions on the wall and their lack of influence over who visits their sites and how they arrive. They also strongly believe that mode is chosen before destination. This paper reports the findings of research into the opinions and beliefs of the managers and discusses the consequences for planning more sustainable tourist travel. It suggests that methods adopted for making utility travel more environmentally sustainable would have to be modified to be effective for leisure travel