Differentiation of banding patterns between Streptococcus mutans and Streptococcus sobrinus isolates in rep-PCR using ERIC primer

Streptococcus mutans and Streptococcus sobrinus are considered to be important bacterial species in the initiation of human dental caries. Therefore, the establishment of a reliable genotyping method to distinguish S. mutans from S. sobrinus is of central importance.We assessed the usefulness of repetitive extragenic palindromic polymerase chain reaction (rep-PCR) using ERIC primer banding patterns in differentiating S. mutans and S. sobrinus.Five S. mutans and two S. sobrinus prototype strains and 50 clinical isolates (38 S. mutans serotype c, 4 S. sobrinus serotype d, and 8 S. sobrinus serotype g) were examined. The banding patterns of amplicons generated were compared among the prototype strains and clinical isolates, to find common bands that distinguish S. mutans and S. sobrinus.Multiple banding patterns were seen with all strains tested. The representative strains of S. mutans tested revealed six unique, strong bands at 2,000 bp, 1,700 bp, 1,400 bp, 1,100 bp, 850 bp, and 250 bp, whereas S. sobrinus had seven strong bands at 2,000 bp, 1,800 bp, 1,100 bp, 900 bp, 800 bp, 600 bp, and 550 bp. The band at 1,100 bp was the only band that was observed in both S. mutans and S. sobrinus. Furthermore, most clinical S. mutans isolates revealed identical banding patterns. All S. mutans had amplicons at 1,700 bp, 850 bp, and 250 bp, whereas those of S. sobrinus were at 1,100 bp, 900 bp, and 800 bp.These results indicate that using rep-PCR with the ERIC primers can distinguish between S. mutans and S. sobrinus

Response and resilience of Spartina alterniflora to sudden dieback

We measured an array of biophysical and spectral variables to evaluate the response and recovery of Spartina alterniflora to a sudden dieback event in spring and summer 2004 within a low marsh in coastal Virginia, USA. S. alterniflora is a foundation species, whose loss decreases ecosystem services and potentiates ecosystem state change. Long-term records of the potential environmental drivers of dieback such as precipitation and tidal inundation did not evidence any particular anomalies, although Hurricane Isabel in fall 2003 may have been related to dieback. Transects were established across the interface between the dieback area and apparently healthy areas of marsh. Plant condition was classified based on ground cover within transects as dieback, intermediate and healthy. Numerous characteristics of S. alterniflora culms within each condition class were assessed including biomass, morphology and spectral attributes associated with photosynthetic pigments. Plants demonstrated evidence of stress in 2004 and 2005 beyond areas of obvious dieback and resilience at a multi-year scale. Resilience of the plants was evident in recovery of ground cover and biomass largely within 3 y, although a small remnant of dieback persisted for 8 y. Culms surviving within the dieback and areas of intermediate impact had modified morphological traits and spectral response that reflected stress. These morphometric and spectral differences among plant cover condition classes serve as guidelines for monitoring of dieback initiation, effects and subsequent recovery. Although a number of environmental and biotic parameters were assessed relative to causation, the reason for this particular dieback remains largely unknown, however

Skeleton of an unusual cat-sized marsupial relative (Metatheria: Marsupialiformes) from the middle Eocene (Lutetian: 44-43 million years ago) of Turkey

We describe a near-complete, three-dimensionally preserved skeleton of a metatherian (relative of modern marsupials) from the middle Eocene (Lutetian: 44–43 million years ago) Lülük member of the Uzunçarşıdere Formation, central Turkey. With an estimated body mass of 3–4 kg, about the size of a domestic cat (Felis catus) or spotted quoll (Dasyurus maculatus), it is an order of magnitude larger than the largest fossil metatherians previously known from the Cenozoic of the northern hemisphere. This new taxon is characterised by large, broad third premolars that probably represent adaptations for hard object feeding (durophagy), and its craniodental morphology suggests the capacity to generate high bite forces. Qualitative and quantitative functional analyses of its postcranial skeleton indicate that it was probably scansorial and relatively agile, perhaps broadly similar in locomotor mode to the spotted quoll, but with a greater capacity for climbing and grasping. Bayesian phylogenetic analysis of a total evidence dataset comprising 259 morphological characters and 9kb of DNA sequence data from five nuclear protein-coding genes, using both undated and “tip-and-node dating” approaches, place the new taxon outside the marsupial crown-clade, but within the clade Marsupialiformes. It demonstrates that at least one metatherian lineage evolved to occupy the small-medium, meso- or hypo-carnivore niche in the northern hemisphere during the early Cenozoic, at a time when there were numerous eutherians (placentals and their fossil relatives) filling similar niches. However, the known mammal fauna from Uzunçarşıdere Formation appears highly endemic, and geological evidence suggests that this region of Turkey was an island for at least part of the early Cenozoic, and so the new taxon may have evolved in isolation from potential eutherian competitors. Nevertheless, the new taxon reveals previously unsuspected ecomorphological disparity among northern hemisphere metatherians during the first half of the Cenozoic

Mapping diphtheria-pertussis-tetanus vaccine coverage in Africa, 2000-2016: a spatial and temporal modelling study.

BACKGROUND: Routine childhood vaccination is among the most cost-effective, successful public health interventions available. Amid substantial investments to expand vaccine delivery throughout Africa and strengthen administrative reporting systems, most countries still require robust measures of local routine vaccine coverage and changes in geographical inequalities over time. METHODS: This analysis drew from 183 surveys done between 2000 and 2016, including data from 881 268 children in 49 African countries. We used a Bayesian geostatistical model calibrated to results from the Global Burden of Diseases, Injuries, and Risk Factors Study 2017, to produce annual estimates with high-spatial resolution (5 ×    5 km) of diphtheria-pertussis-tetanus (DPT) vaccine coverage and dropout for children aged 12-23 months in 52 African countries from 2000 to 2016. FINDINGS: Estimated third-dose (DPT3) coverage increased in 72·3% (95% uncertainty interval [UI] 64·6-80·3) of second-level administrative units in Africa from 2000 to 2016, but substantial geographical inequalities in DPT coverage remained across and within African countries. In 2016, DPT3 coverage at the second administrative (ie, district) level varied by more than 25% in 29 of 52 countries, with only two (Morocco and Rwanda) of 52 countries meeting the Global Vaccine Action Plan target of 80% DPT3 coverage or higher in all second-level administrative units with high confidence (posterior probability ≥95%). Large areas of low DPT3 coverage (≤50%) were identified in the Sahel, Somalia, eastern Ethiopia, and in Angola. Low first-dose (DPT1) coverage (≤50%) and high relative dropout (≥30%) together drove low DPT3 coverage across the Sahel, Somalia, eastern Ethiopia, Guinea, and Angola. INTERPRETATION: Despite substantial progress in Africa, marked national and subnational inequalities in DPT coverage persist throughout the continent. These results can help identify areas of low coverage and vaccine delivery system vulnerabilities and can ultimately support more precise targeting of resources to improve vaccine coverage and health outcomes for African children. FUNDING: Bill & Melinda Gates Foundation

The association between Zika virus infection and microcephaly in Brazil 2015-2017: An observational analysis of over 4 million births.

BACKGROUND: In 2015, high rates of microcephaly were reported in Northeast Brazil following the first South American Zika virus (ZIKV) outbreak. Reported microcephaly rates in other Zika-affected areas were significantly lower, suggesting alternate causes or the involvement of arboviral cofactors in exacerbating microcephaly rates. METHODS AND FINDINGS: We merged data from multiple national reporting databases in Brazil to estimate exposure to 9 known or hypothesized causes of microcephaly for every pregnancy nationwide since the beginning of the ZIKV outbreak; this generated between 3.6 and 5.4 million cases (depending on analysis) over the time period 1 January 2015-23 May 2017. The association between ZIKV and microcephaly was statistically tested against models with alternative causes or with effect modifiers. We found no evidence for alternative non-ZIKV causes of the 2015-2017 microcephaly outbreak, nor that concurrent exposure to arbovirus infection or vaccination modified risk. We estimate an absolute risk of microcephaly of 40.8 (95% CI 34.2-49.3) per 10,000 births and a relative risk of 16.8 (95% CI 3.2-369.1) given ZIKV infection in the first or second trimester of pregnancy; however, because ZIKV infection rates were highly variable, most pregnant women in Brazil during the ZIKV outbreak will have been subject to lower risk levels. Statistically significant associations of ZIKV with other birth defects were also detected, but at lower relative risks than that of microcephaly (relative risk < 1.5). Our analysis was limited by missing data prior to the establishment of nationwide ZIKV surveillance, and its findings may be affected by unmeasured confounding causes of microcephaly not available in routinely collected surveillance data. CONCLUSIONS: This study strengthens the evidence that congenital ZIKV infection, particularly in the first 2 trimesters of pregnancy, is associated with microcephaly and less frequently with other birth defects. The finding of no alternative causes for geographic differences in microcephaly rate leads us to hypothesize that the Northeast region was disproportionately affected by this Zika outbreak, with 94% of an estimated 8.5 million total cases occurring in this region, suggesting a need for seroprevalence surveys to determine the underlying reason

Variation in Childhood Diarrheal Morbidity and Mortality in Africa, 2000-2015.

BACKGROUND: Diarrheal diseases are the third leading cause of disease and death in children younger than 5 years of age in Africa and were responsible for an estimated 30 million cases of severe diarrhea (95% credible interval, 27 million to 33 million) and 330,000 deaths (95% credible interval, 270,000 to 380,000) in 2015. The development of targeted approaches to address this burden has been hampered by a paucity of comprehensive, fine-scale estimates of diarrhea-related disease and death among and within countries. METHODS: We produced annual estimates of the prevalence and incidence of diarrhea and diarrhea-related mortality with high geographic detail (5 km2) across Africa from 2000 through 2015. Estimates were created with the use of Bayesian geostatistical techniques and were calibrated to the results from the Global Burden of Diseases, Injuries, and Risk Factors Study 2016. RESULTS: The results revealed geographic inequality with regard to diarrhea risk in Africa. Of the estimated 330,000 childhood deaths that were attributable to diarrhea in 2015, more than 50% occurred in 55 of the 782 first-level administrative subdivisions (e.g., states). In 2015, mortality rates among first-level administrative subdivisions in Nigeria differed by up to a factor of 6. The case fatality rates were highly varied at the national level across Africa, with the highest values observed in Benin, Lesotho, Mali, Nigeria, and Sierra Leone. CONCLUSIONS: Our findings showed concentrated areas of diarrheal disease and diarrhea-related death in countries that had a consistently high burden as well as in countries that had considerable national-level reductions in diarrhea burden. (Funded by the Bill and Melinda Gates Foundation.)

Global, regional, and national burden of tuberculosis, 1990–2016: results from the Global Burden of Diseases, Injuries, and Risk Factors 2016 Study

Background Although a preventable and treatable disease, tuberculosis causes more than a million deaths each year. As countries work towards achieving the Sustainable Development Goal (SDG) target to end the tuberculosis epidemic by 2030, robust assessments of the levels and trends of the burden of tuberculosis are crucial to inform policy and programme decision making. We assessed the levels and trends in the fatal and non-fatal burden of tuberculosis by drug resistance and HIV status for 195 countries and territories from 1990 to 2016. Methods We analysed 15 943 site-years of vital registration data, 1710 site-years of verbal autopsy data, 764 site-years of sample-based vital registration data, and 361 site-years of mortality surveillance data to estimate mortality due to tuberculosis using the Cause of Death Ensemble model. We analysed all available data sources, including annual case notifications, prevalence surveys, population-based tuberculin surveys, and estimated tuberculosis cause-specific mortality to generate internally consistent estimates of incidence, prevalence, and mortality using DisMod-MR 2.1, a Bayesian meta-regression tool. We assessed how the burden of tuberculosis differed from the burden predicted by the Socio-demographic Index (SDI), a composite indicator of income per capita, average years of schooling, and total fertility rate. Findings Globally in 2016, among HIV-negative individuals, the number of incident cases of tuberculosis was 9·02 million (95% uncertainty interval [UI] 8·05–10·16) and the number of tuberculosis deaths was 1·21 million (1·16–1·27). Among HIV-positive individuals, the number of incident cases was 1·40 million (1·01–1·89) and the number of tuberculosis deaths was 0·24 million (0·16–0·31). Globally, among HIV-negative individuals the age-standardised incidence of tuberculosis decreased annually at a slower rate (–1·3% [–1·5 to −1·2]) than mortality did (–4·5% [–5·0 to −4·1]) from 2006 to 2016. Among HIV-positive individuals during the same period, the rate of change in annualised age-standardised incidence was −4·0% (–4·5 to −3·7) and mortality was −8·9% (–9·5 to −8·4). Several regions had higher rates of age-standardised incidence and mortality than expected on the basis of their SDI levels in 2016. For drug-susceptible tuberculosis, the highest observed-to-expected ratios were in southern sub-Saharan Africa (13·7 for incidence and 14·9 for mortality), and the lowest ratios were in high-income North America (0·4 for incidence) and Oceania (0·3 for mortality). For multidrug-resistant tuberculosis, eastern Europe had the highest observed-to-expected ratios (67·3 for incidence and 73·0 for mortality), and high-income North America had the lowest ratios (0·4 for incidence and 0·5 for mortality). Interpretation If current trends in tuberculosis incidence continue, few countries are likely to meet the SDG target to end the tuberculosis epidemic by 2030. Progress needs to be accelerated by improving the quality of and access to tuberculosis diagnosis and care, by developing new tools, scaling up interventions to prevent risk factors for tuberculosis, and integrating control programmes for tuberculosis and HIV

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes