Identification of Streptococcus suis Meningitis through Population-Based Surveillance, Togo, 2010-2014.

During 2010-2014, we enrolled 511 patients with suspected bacterial meningitis into surveillance in 2 districts of northern Togo. We identified 15 persons with Streptococcus suis infection; 10 had occupational contact with pigs, and 12 suffered neurologic sequelae. S. suis testing should be considered in rural areas of the African meningitis belt.

Effectiveness of rapid SARS-CoV-2 genome sequencing in supporting infection control for hospital-onset COVID-19 infection: Multicentre, prospective study.

Background: Viral sequencing of SARS-CoV-2 has been used for outbreak investigation, but there is limited evidence supporting routine use for infection prevention and control (IPC) within hospital settings. Methods: We conducted a prospective non-randomised trial of sequencing at 14 acute UK hospital trusts. Sites each had a 4-week baseline data collection period, followed by intervention periods comprising 8 weeks of 'rapid' (<48 hr) and 4 weeks of 'longer-turnaround' (5-10 days) sequencing using a sequence reporting tool (SRT). Data were collected on all hospital-onset COVID-19 infections (HOCIs; detected ≥48 hr from admission). The impact of the sequencing intervention on IPC knowledge and actions, and on the incidence of probable/definite hospital-acquired infections (HAIs), was evaluated. Results: A total of 2170 HOCI cases were recorded from October 2020 to April 2021, corresponding to a period of extreme strain on the health service, with sequence reports returned for 650/1320 (49.2%) during intervention phases. We did not detect a statistically significant change in weekly incidence of HAIs in longer-turnaround (incidence rate ratio 1.60, 95% CI 0.85-3.01; p=0.14) or rapid (0.85, 0.48-1.50; p=0.54) intervention phases compared to baseline phase. However, IPC practice was changed in 7.8 and 7.4% of all HOCI cases in rapid and longer-turnaround phases, respectively, and 17.2 and 11.6% of cases where the report was returned. In a 'per-protocol' sensitivity analysis, there was an impact on IPC actions in 20.7% of HOCI cases when the SRT report was returned within 5 days. Capacity to respond effectively to insights from sequencing was breached in most sites by the volume of cases and limited resources. Conclusions: While we did not demonstrate a direct impact of sequencing on the incidence of nosocomial transmission, our results suggest that sequencing can inform IPC response to HOCIs, particularly when returned within 5 days. Funding: COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) (grant code: MC_PC_19027), and Genome Research Limited, operating as the Wellcome Sanger Institute. Clinical trial number: NCT04405934.

Antibiotic Susceptibility, Virulome, and Clinical Outcomes in European Infants with Bloodstream Infections Caused by Enterobacterales.

Mortality in neonates with Gram-negative bloodstream infections has remained unacceptably high. Very few data are available on the impact of resistance profiles, virulence factors, appropriateness of empirical treatment and clinical characteristics on patients' mortality. A survival analysis to investigate 28-day mortality probability and predictors was performed including (I) infants <90 days (II) with an available Enterobacterales blood isolate with (III) clinical, treatment and 28-day outcome data. Eighty-seven patients were included. Overall, 299 virulence genes were identified among all the pathogens. Escherichia coli had significantly more virulence genes identified compared with other species. A strong positive correlation between the number of resistance and virulence genes carried by each isolate was found. The cumulative probability of death obtained by the Kaplan-Meier survival analysis was 19.5%. In the descriptive analysis, early age at onset, gestational age at onset, culture positive for E. coli and number of classes of virulence genes carried by each isolate were significantly associated with mortality. By Cox multivariate regression, none of the investigated variables was significant. This pilot study has demonstrated the feasibility of investigating the association between neonatal sepsis mortality and the causative Enterobacterales isolates virulome. This relationship needs further exploration in larger studies, ideally including host immunopathological response, in order to develop a tailor-made therapeutic strategy.

Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay.

BACKGROUND: Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. METHODOLOGY AND PRINCIPAL FINDINGS: A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95). CONCLUSION: The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.

Gene expression profile of abdominal aortic aneurysm rupture.

To search for novel transcriptional pathways that are activated in abdominal aortic aneurysm rupture, cDNA microarrays were used to compare global mRNA expression at the aneurysm rupture edge to anterior sac, and selected results were confirmed using quantitative real-time-polymerase chain reaction (QRT-PCR). This study identified apoptosis, angiogenesis, and inflammation as potentially important participants during the process of aneurysm rupture.

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

BACKGROUND: The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. METHODS: The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. FINDINGS: Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. INTERPRETATION: Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. RELEVANCE TO PRACTICE: Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

Ribosomal protein L22 inhibits regulation of cellular activities by the Epstein-Barr virus small RNA EBER-1.

Epstein-Barr virus (EBV) is a potent mitogenic and antiapoptotic agent for B lymphocytes and is associated with several different types of human tumour. The abundantly expressed small viral RNA, EBER-1, binds to the growth inhibitory and pro-apoptotic protein kinase R (PKR) and blocks activation of the latter by double-stranded RNA. Recent evidence has suggested that expression of EBER-1 alone in EBV-negative B cells promotes a tumorigenic phenotype and that this may be related to inhibition of the pro-apoptotic effects of PKR. The ribosomal protein L22 binds to EBER-1 in virus-infected cells, but the significance of this has not previously been established. We report here that L22 and PKR compete for a common binding site on EBER-1. As a result of this competition, L22 interferes with the ability of the small RNA to inhibit the activation of PKR by dsRNA. Transient expression of EBER-1 in murine embryonic fibroblasts stimulates reporter gene expression and partially reverses the inhibitory effect of PKR. However, EBER-1 is also stimulatory when transfected into PKR knockout cells, suggesting an additional, PKR-independent, mode of action of the small RNA. Expression of L22 prevents both the PKR-dependent and -independent effects of EBER-1 in vivo. These results suggest that the association of L22 with EBER-1 in EBV-infected cells can attenuate the biological effects of the viral RNA. Such effects include both the inhibition of PKR and additional mechanism(s) by which EBER-1 stimulates gene expression.

Fidelity and reproducibility of antisense RNA amplification for the study of gene expression in human CD34+ haemopoietic stem and progenitor cells.

Microarrays provide a powerful tool for the study of haemopoietic stem and progenitor cells (HSC). Because of the low frequency of HSC, it is rarely feasible to obtain enough mRNA for microarray hybridizations, and amplification will be necessary. Antisense RNA (aRNA) amplification is reported to give high-fidelity amplification, but most studies have used only qualitative validation. Before applying aRNA amplification to the study of HSC, we wished to determine its fidelity and reproducibility, and whether statistically significant results can be obtained. We found that aRNA amplification introduced biases into relative RNA abundance. However, these biases were extremely consistent, and valid comparisons could be made, if amplified RNA was compared with amplified RNA. By applying this method to the effect of interferon-gamma and tumour necrosis factor-alpha on normal primary CD34+ HSC, biologically significant differences could be detected, including potential mechanisms for resistance of CD34+ cells to CD95-mediated apoptosis and evidence of the differentiating effects of the cytokines. Differences of twofold or less were detected, and most of these differences attained statistical significance after triplicate experiments. These data demonstrate that aRNA amplification can be used with microarray hybridization to study the transcriptional profiles of small numbers of primary CD34+ HSC.

In vivo effects of the Epstein-Barr virus small RNA EBER-1 on protein synthesis and cell growth regulation.

Recent studies have suggested a role for the Epstein-Barr virus-encoded RNA EBER-1 in malignant transformation. EBER-1 inhibits the activity of the protein kinase PKR, an inhibitor of protein synthesis with tumour suppressor properties. In human 293 cells and murine embryonic fibroblasts, transient expression of EBER-1 promoted total protein synthesis and enhanced the expression of cotransfected reporter genes. However reporter gene expression was stimulated equally well in cells from control and PKR knockout mice. NIH 3T3 cells stably expressing EBER-1 exhibited a greatly increased frequency of colony formation in soft agar, and protein synthesis in these cells was relatively resistant to inhibition by the calcium ionophore A23187. Nevertheless clones containing a high concentration of EBER-1 were not invariably tumourigenic. We conclude that EBER-1 can enhance protein synthesis by a PKR-independent mechanism and that, although this RNA may contribute to the oncogenic potential of Epstein-Barr virus, its expression is not always sufficient for malignant transformation.

The mRNA of the translationally controlled tumor protein P23/TCTP is a highly structured RNA, which activates the dsRNA-dependent protein kinase PKR.

The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible elF2alpha kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a beta-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase.