Reduced Cardiac Index Reserve and Hypovolemia in Severe Falciparum Malaria.

BACKGROUND: Impaired microvascular perfusion is central to the development of coma and lactic acidosis in severe falciparum malaria. Refractory hypotension is rare on admission but develops frequently in fatal cases. We assessed cardiac function and volume status in severe falciparum malaria and its prognostic significance. METHODS: Patients with severe (N = 101) or acute uncomplicated falciparum malaria (N = 83) were recruited from 2 hospitals in India and Bangladesh, and healthy participants (N = 44) underwent echocardiography. RESULTS: Patients with severe malaria had 38% shorter left ventricular (LV) filling times and 25% shorter LV ejection times than healthy participants because of tachycardia; however, stroke volume, LV internal diameter in diastole (LVIDd), and LV internal diameter in systole (LVIDs) indices were similar. A low endocardial fraction shortening (eFS) was present in 17% (9 of 52) of severe malaria patients. Adjusting for preload and afterload, eFS was similar in health and severe malaria. Fatal cases had smaller baseline LVIDd and LVIDs indices, more collapsible inferior vena cavae (IVC), and higher heart rates than survivors. The LVIDs and IVC collapsibility were independent predictors for mortality, together with base excess and Glasgow Coma Scale. CONCLUSIONS: Patients with severe malaria have rapid ejection of a normal stroke volume. Fatal cases had features of relative hypovolemia and reduced cardiac index reserve.

Associations Between Restrictive Fluid Management and Renal Function and Tissue Perfusion in Adults With Severe Falciparum Malaria: A Prospective Observational Study.

BACKGROUND: Liberal fluid resuscitation has proved harmful in adults with severe malaria, but the level of restriction has not been defined. METHODS: In a prospective observational study in adults with severe falciparum malaria, restrictive fluid management was provided at the discretion of the treating physician. The relationships between the volume of fluid and changes in renal function or tissue perfusion were evaluated. RESULTS: A total of 154 patients were studied, 41 (26.6%) of whom died. Median total fluid intake during the first 6 and 24 hours from enrollment was 3.3 (interquartile range [IQR], 1.8-5.1) mL/kg per hour and 2.2 (IQR, 1.6-3.2) mL/kg per hour, respectively. Total fluid intake at 6 hours was not correlated with changes in plasma creatinine at 24 hours (n = 116; rs = 0.16; P = .089) or lactate at 6 hours (n = 94; rs = -0.05; P = .660). Development of hypotensive shock or pulmonary edema within 24 hours after enrollment were not related to the volume of fluid administration. CONCLUSIONS: Restrictive fluid management did not worsen kidney function and tissue perfusion in adult patients with severe falciparum malaria. We suggest crystalloid administration of 2-3 mL/kg per hour during the first 24 hours without bolus therapy, unless the patient is hypotensive.

Cell-Free Hemoglobin Is Associated With Increased Vascular Resistance and Reduced Peripheral Perfusion in Severe Malaria.

BACKGROUND: In severe falciparum malaria, unlike sepsis, hypotension on admission is uncommon. We hypothesized that low nitric oxide bioavailability due to the presence of cell-free hemoglobin (CFH) increases vascular tone in severe malaria. METHODS: Patients with severe malaria (n = 119), uncomplicated malaria (n = 91), or suspected bacterial sepsis (n = 56), as well as healthy participants (n = 50), were recruited. The systemic vascular resistance index (SVRI) was estimated from the echocardiographic cardiac index and the mean arterial pressure. RESULTS: SVRI and hematocrit levels were lower and plasma CFH and asymmetric dimethylarginine levels were higher in patients with malaria, compared with healthy participants. In multivariate linear regression models for mean arterial pressure or SVRI in patients with severe malaria, hematocrit and CFH but not asymmetric dimethylarginine were significant predictors. The SVRI was lower in patients with suspected bacterial sepsis than in those with severe malaria, after adjustment for hematocrit and age. Plasma CFH levels correlated positively with the core-peripheral temperature gradient and plasma lactate levels and inversely with the perfusion index. Impaired peripheral perfusion, as reflected by a low perfusion index or a high core-peripheral temperature gradient, predicted mortality in patients with severe malaria. CONCLUSIONS: CFH is associated with mean arterial pressure, SVRI, and peripheral perfusion in patients with severe malaria. This may be mediated through the nitric oxide scavenging potency of CFH, increasing basal vascular tone and impairing tissue perfusion.

Rhombencephalitis and Myeloradiculitis Caused by a European Subtype of Tick-Borne Encephalitis Virus.

We report a case of a previously healthy man returning to the United Kingdom from Lithuania who developed rhombencephalitis and myeloradiculitis due to tick-borne encephalitis. These findings add to sparse data on tick-borne encephalitis virus phylogeny and associated neurologic syndromes and underscore the importance of vaccinating people traveling to endemic regions.

Does reduced oxygen delivery cause lactic acidosis in falciparum malaria? An observational study.

BACKGROUND: Lactic acidosis with an elevated lactate-pyruvate ratio suggesting anoxia is a common feature of severe falciparum malaria. High lactate levels are associated with parasitized erythrocyte sequestration in the microcirculation. To assess if there is an additional contribution to hyperlactataemia from relatively inadequate total oxygen delivery, oxygen consumption and delivery were investigated in patients with malaria. METHODS: Adult Bangladeshi and Indian patients with uncomplicated (N = 50) or severe (N = 46) falciparum malaria or suspected bacterial sepsis (N = 27) and healthy participants as controls (N = 26) were recruited at Chittagong Medical College Hospital, Chittagong, Bangladesh and Ispat General Hospital, Rourkela, India. Oxygen delivery (DO2I) was estimated from pulse oximetry, echocardiographic estimates of cardiac index and haematocrit. Oxygen consumption (VO2I) was estimated by expired gas collection. RESULTS: VO2I was elevated in uncomplicated median (IQR) 185.1 ml/min/m2 (135-215.9) and severe malaria 192 ml/min/m2 (140.7-227.9) relative to healthy persons 107.9 ml/min/m2 (69.9-138.1) (both p < 0.001). Median DO2I was similar in uncomplicated 515 ml/min/m2 (432-612) and severe 487 ml/min/m2 (382-601) malaria and healthy persons 503 ml/min/m2 (447-517) (p = 0.27 and 0.89, respectively). The VO2/DO2 ratio was, therefore, increased by similar amounts in both uncomplicated 0.35 (0.28-0.44) and severe malaria 0.38 (0.29-0.48) relative to healthy participants 0.23 (0.17-0.28) (both p < 0.001). VO2I, DO2I and VO2/DO2 did not correlate with plasma lactate concentrations in severe malaria. CONCLUSIONS: Reduced total oxygen delivery is not a major contributor to lactic acidosis in severe falciparum malaria.

Acetaminophen as a Renoprotective Adjunctive Treatment in Patients With Severe and Moderately Severe Falciparum Malaria: A Randomized, Controlled, Open-Label Trial.

Background: Acute kidney injury independently predicts mortality in falciparum malaria. It is unknown whether acetaminophen's capacity to inhibit plasma hemoglobin-mediated oxidation is renoprotective in severe malaria. Methods: This phase 2, open-label, randomized controlled trial conducted at two hospitals in Bangladesh assessed effects on renal function, safety, pharmacokinetic (PK) properties and pharmacodynamic (PD) effects of acetaminophen. Febrile patients (>12 years) with severe falciparum malaria were randomly assigned to receive acetaminophen (1 g 6-hourly for 72 hours) or no acetaminophen, in addition to intravenous artesunate. Primary outcome was the proportional change in creatinine after 72 hours stratified by median plasma hemoglobin. Results: Between 2012 and 2014, 62 patients were randomly assigned to receive acetaminophen (n = 31) or no acetaminophen (n = 31). Median (interquartile range) reduction in creatinine after 72 hours was 23% (37% to 18%) in patients assigned to acetaminophen, versus 14% (29% to 0%) in patients assigned to no acetaminophen (P = .043). This difference in reduction was 37% (48% to 22%) versus 14% (30% to -71%) in patients with hemoglobin ≥45000 ng/mL (P = .010). The proportion with progressing kidney injury was higher among controls (subdistribution hazard ratio, 3.0; 95% confidence interval, 1.1 to 8.5; P = .034). PK-PD analyses showed that higher exposure to acetaminophen increased the probability of creatinine improvement. No patient fulfilled Hy's law for hepatotoxicity. Conclusions: In this proof-of-principle study, acetaminophen showed renoprotection without evidence of safety concerns in patients with severe falciparum malaria, particularly in those with prominent intravascular hemolysis. Clinical Trials Registration: NCT01641289.

Genotypic and phenotypic characterization of G6PD deficiency in Bengali adults with severe and uncomplicated malaria.

BACKGROUND: Control of malaria increasingly involves administration of 8-aminoquinolines, with accompanying risk of haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Few data on the prevalence and genotypic basis of G6PD deficiency are available from Bangladesh, where malaria remains a major problem in the South (Chittagong Division). The aim of this study was to determine the prevalence of G6PD deficiency, and associated G6PD genotypes, in adults with falciparum malaria in southern Bangladesh. METHODS: G6PD status was assessed via a combination of fluorescent spot testing (FST) and genotyping in 141 Bengali patients admitted with falciparum malaria to two centres in Chittagong Division from 2012 to 2014. In addition, an analysis of genomic data from 1000 Genomes Project was carried out among five healthy Indian subcontinent populations. RESULTS: One male patient with uncomplicated malaria was found to have G6PD deficiency on FST and a genotype associated with deficiency (hemizygous Orissa variant). In addition, there were two female patients heterozygous for deficiency variants (Orissa and Kerala-Kalyan). These three patients had a relatively long duration of symptoms prior to admission compared to G6PD normal cases, possibly suggesting an interaction with parasite multiplication rate. In addition, one of 27 healthy local controls was deficient on FST and hemizygous for the Mahidol variant of G6PD deficiency. Examination of 1000 Genomes Project sequencing data across the Indian subcontinent showed that 19/723 chromosomes (2.63%) carried a variant associated with deficiency. In the Bengali from Bangladesh 1000 Genomes population, three of 130 chromosomes (2.31%) carried deficient alleles; this included single chromosomes carrying the Kerala-Kalyan and Orissa variants. CONCLUSIONS: In line with other recent work, G6PD deficiency is uncommon in Bengalis in Bangladesh. Further studies of particular ethnic groups are needed to evaluate the potential risk of wide deployment of primaquine in malaria control efforts in Bangladesh.

Cell-free hemoglobin mediated oxidative stress is associated with acute kidney injury and renal replacement therapy in severe falciparum malaria: an observational study.

BACKGROUND: Intravascular hemolysis is an intrinsic feature of severe malaria pathophysiology but the pathogenic role of cell-free hemoglobin-mediated oxidative stress in severe malaria associated acute kidney injury (AKI) is unknown. METHODS: As part of a prospective observational study, enrolment plasma cell-free hemoglobin (CFH), lipid peroxidation markers (F2-isoprostanes (F2-IsoPs) and isofurans (IsoFs)), red cell deformability, and serum creatinine were quantified in Bangladeshi patients with severe falciparum malaria (n = 107), uncomplicated malaria (n = 80) and sepsis (n = 28). The relationships between these indices and kidney function and clinical outcomes were examined. RESULTS: AKI was diagnosed at enrolment in 58% (62/107) of consecutive patients with severe malaria, defined by an increase in creatinine ≥1.5 times expected baseline. Severe malaria patients with AKI had significantly higher plasma cell-free hemoglobin (geometric mean CFH: 8.8 µM; 95% CI, 6.2-12.3 µM), F2-isoprostane (56.7 pg/ml; 95% CI, 45.3-71.0 pg/ml) and isofuran (109.2 pg/ml; 95% CI, 85.1-140.1 pg/ml) concentrations on enrolment compared to those without AKI (CFH: 5.1 µM; 95% CI, 4.0-6.6 µM; P = 0.018; F2-IsoPs: 27.8 pg/ml; 95% CI, 23.7-32.7 pg/ml; P < 0.001; IsoFs: 41.7 pg/ml; 95% CI, 30.2-57.6 pg/ml; P < 0.001). Cell-free hemoglobin correlated with markers of hemolysis, parasite burden (P. falciparum histidine rich protein 2 (PfHRP2)), and F2-IsoPs. Plasma F2-IsoPs and IsoFs inversely correlated with pH, positively correlated with creatinine, PfHRP2 and fractional excretion of sodium, and were higher in patients later requiring hemodialysis. Plasma F2-IsoP concentrations also inversely correlated with red cell deformability and were higher in fatal cases. Mixed effects modeling including an interaction term for CFH and time showed that F2-IsoPs, IsoFs, PfHRP2, CFH, and red cell rigidity were independently associated with increasing creatinine over 72 h. Multivariable logistic regression showed that admission F2-IsoPs, IsoFs and red cell deformability were associated with the need for subsequent hemodialysis. CONCLUSIONS: Cell-free hemoglobin and lipid peroxidation are associated with acute kidney injury and disease severity in falciparum malaria, suggesting a pathophysiological role in renal tubular injury. Evaluation of adjunctive therapies targeting cell-free hemoglobin-mediated oxidative stress is warranted.

The role of previously unmeasured organic acids in the pathogenesis of severe malaria.

INTRODUCTION: Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria. METHODS: In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death. RESULTS: Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean ± SD) was elevated in severe malaria (8.2 mEq/L ± 4.5) and severe sepsis (8.6 mEq/L ± 7.7) compared with uncomplicated malaria (6.0 mEq/L ± 5.1) and encephalopathy (6.6 mEq/L ± 4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and ß-hydroxybutyric acid (all P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death (odds ratio [OR] 3.5, 95 % confidence interval [CI] 1.6-7.5, P=0.001), comparable to LA (OR 3.5, 95 % CI 1.5-7.8, P=0.003) (combined area under the receiver operating characteristic curve 0.81). CONCLUSIONS: Newly identified acids, in addition to LA, are elevated in patients with severe malaria and are highly predictive of fatal outcome. Further characterisation of their sources and metabolic pathways is now needed.

Field-based assessments of the seasonality of Culex pipiens sensu lato in England: an important enzootic vector of Usutu and West Nile viruses.

BACKGROUND: Usutu virus (USUV), which is closely related to West Nile virus (WNV), sharing a similar ecology and transmission cycle, was first reported in the UK in the southeast of England in 2020. Both USUV and WNV are emerging zoonotic viruses hosted by wild birds. The 2020 finding of USUV in England raised awareness of this virus and highlighted the importance of understanding the seasonality of Culex pipiens sensu lato (Cx. pipiens s.l.), the main enzootic vector of these viruses. Zoos are prime locations for trapping mosquitoes because of their infrastructure, security, and range of vertebrate hosts and aquatic habitats. METHODS: Three independent zoo-based case studies at four locations that cover the seasonality of Cx. pipiens s.l. in England were undertaken: (i) London Zoo (Zoological Society London [ZSL]) and surrounding areas, London; (ii) Chester Zoo (Cheshire); (ii) Twycross Zoo (Leicestershire); and (iv) Flamingo Land (zoo; North Yorkshire). Various adult mosquito traps were used to catch adult Cx. pipiens s.l. across seasons. RESULTS: High yields of Cx. pipiens s.l./Culex torrentium were observed in Biogents-Mosquitaire and Center for Disease Control and Prevention Gravid traps in all studies where these traps were used. Mosquito counts varied between sites and between years. Observations of adult Cx. pipiens s.l./Cx. torrentium abundance and modelling studies demonstrated peak adult abundance between late July and early August, with active adult female Cx. pipiens s.l./Cx. torrentium populations between May and September. CONCLUSIONS: The information collated in this study illustrates the value of multiple mosquito monitoring approaches in zoos to describe the seasonality of this UK vector across multiple sites in England and provides a framework that can be used for ongoing and future surveillance programmes and disease risk management strategies.

The effectiveness of primary series CoronaVac vaccine in preventing COVID-19 illness: A prospective cohort study among healthcare workers in Azerbaijan, May-November 2021.

Background: Healthcare workers (HCWs) have suffered considerable morbidity and mortality during the COVID-19 pandemic. Few studies have evaluated the CoronaVac vaccine effectiveness (VE), particularly in Eastern Europe, where the vaccine has been widely used. Methods: We conducted a prospective cohort study among HCWs in seven hospitals in Baku, Azerbaijan between May 17 and November 30, 2021, to evaluate primary series (two-dose) CoronaVac VE against symptomatic SARS-CoV-2 infection. Participants completed weekly symptom questionnaires, provided nasopharyngeal swabs for SARS-CoV-2 RT-PCR testing when symptomatic, and provided serology samples at enrollment that were tested for anti-spike and anti-nucleocapsid antibodies. We estimated VE as (1 - hazard ratio)*100 using a Cox proportional hazards model with vaccination status as a time-varying exposure, adjusting for hospital and previous SARS-CoV-2 infection status. Results: We enrolled 1582 HCWs. At enrollment, 1040 (66%) had received two doses of CoronaVac; 421 (27%) were unvaccinated. During the study period, 72 PCR-positive SARS-CoV-2 infections occurred; 36/39 (92%) sequenced samples were classified as Delta variants. Primary series VE against COVID-19 illness was 29% (95% CI: -51%; 67%) for the entire analysis period. For the Delta-only period (July 1-November 30, 2021), primary series VE was 19% (95% CI: -81%; 64%). For the entire analysis period, primary series VE was 39% (95% CI: -40%; 73%) for HCWs vaccinated within 14-149 days and 19% (95% CI: -81%; 63%) for those vaccinated ≥150 days. Conclusions: During a period in Azerbaijan characterized by mostly Delta circulation, VE point estimates suggested that primary series CoronaVac protected nearly 1 in 3 HCWs against COVID-19, but 95% confidence intervals were wide, with lower bounds that crossed zero, reflecting the limited precision of our VE estimates. Our findings underscore the need to consider booster doses for individuals who have received the primary series of CoronaVac.

Experience of using a regional network of hospitals in the care of older inpatients with COVID-19 in spring 2020.

Introduction: Frail, older patients with COVID-19 have an increased risk of hospital admission and death. Methods: We studied a regional model of care used for older patients with COVID-19 in spring 2020 across three settings: an acute teaching hospital, a district general hospital and a temporary emergency hospital. We also studied demographic and outcome data for these patients. Results: Increasing bed capacity in non-acute sites freed up beds in acute hospitals. Strict admission criteria and multidisciplinary team involvement allowed for the safe delivery of care in step-down sites. Conclusion: This model of care allowed for patient flow out of acute sites following the acute stage of their illness allowing for an increase in bed capacity while providing a safe setting for ongoing management.

Levels and genotypes of Salmonella and levels of Escherichia coli in frozen ready-to-cook chicken and turkey products in England tested in 2020 in relation to an outbreak of S. Enteritidis.

Frozen reformulated (FR) breaded chicken products have previously been implicated in causing human salmonellosis. A multi-country Salmonella enterica serovar Enteritidis outbreak involving several strains with >400 reported human cases in the UK occurred in 2020. Initially S. Infantis was detected in one sample from a case home but S. Enteritidis was then also isolated using a S. Enteritidis specific PCR in combination with isolation via a Craigie-tube. This prompted a survey to examine the presence and levels of Salmonella and E. coli in ready-to-cook FR poultry products in England in 2020. From a total of 483 samples, including two from cases' homes, Salmonella was detected in 42 chicken samples, these originated from six out of 53 production plants recorded. Salmonella detection was associated with elevated levels of generic E. coli (OR = 6.63). S. Enteritidis was detected in 17 samples, S. Infantis in 25, S. Newport in four and S. Java, S. Livingstone and S. Senftenberg in one each. The highest levels of Salmonella were 54 MPN/g for S. Infantis and 28 MPN/g for S. Enteritidis; 60% of the Salmonella-positive samples had <1.0 MPN/g. S. Enteritidis was detected together with S. Infantis in five samples and with S. Livingstone in one. Where S. Enteritidis was detected with other Salmonella, the former was present at between 2 and 100-fold lower concentrations. The Salmonella contamination was homogeneously distributed amongst chicken pieces from a single pack and present in both the outer coating and inner content. The S. Enteritidis were all outbreak strains and detected in six products that were linked to four production plants which implicated a Polish origin of contamination. Despite S. Infantis being most prevalent in these products, S. Infantis from only two contemporaneous human cases in the UK fell into the same cluster as isolates detected in one product. Except for one human case falling into the same cluster as one of the S. Newport strains from the chicken, no further isolates from human cases fell into clusters with any of the other serovars detected in the chicken samples. This study found that higher E. coli levels indicated a higher probability of Salmonella contamination in FR chicken products. The results also highlight the importance of recognising co-contamination of foods with multiple Salmonella types and has provided essential information for detecting and understanding outbreaks where multiple strains are involved.

Increasingly limited options for the treatment of enteric fever in travellers returning to England, 2014-2019: a cross-sectional analytical study.

Introduction. Enteric fever (caused by Salmonella enterica serovars Typhi and Paratyphi) frequently presents as an acute, undifferentiated febrile illness in returning travellers, requiring timely empirical antibiotics.Gap Statement. Determining which empirical antibiotics to prescribe for enteric fever requires up-to-date knowledge of susceptibility patterns.Aim. By characterising factors associated with antimicrobial resistance in cases of S. Typhi and S. Paratyphi imported to England, we aim to guide effective empirical treatment.Methodology. All English isolates of S. Typhi and S. Paratyphi 2014-2019 underwent antimicrobial susceptibility testing; results were compared to a previous survey in London 2005-2012. Risk factors for antimicrobial resistance were analysed with logistic regression models to predict adjusted odds ratios (aOR) for resistance to individual antibiotics and multi-drug resistance.Results. We identified 1088 cases of S. Typhi, 729 S. Paratyphi A, 93 S. Paratyphi B, and one S. Paratyphi C. In total, 93 % were imported. Overall, 90 % of S. Typhi and 97 % of S. Paratyphi A isolates were resistant to ciprofloxacin; 26 % of S. Typhi were multidrug resistant to ciprofloxacin, amoxicillin, co-trimoxazole, and chloramphenicol (MDR+FQ). Of the isolates, 4 % of S. Typhi showed an extended drug resistance (XDR) phenotype of MDR+FQ plus resistance to third-generation cephalosporins, with cases of XDR rising sharply in recent years (none before 2017, one in 2017, six in 2018, 32 in 2019). For S. Typhi isolates, resistance to ciprofloxacin was associated with travel to Pakistan (aOR=32.0, 95 % CI: 15.4-66.4), India (aOR=21.8, 95 % CI: 11.6-41.2), and Bangladesh (aOR=6.2, 95 % CI: 2.8-13.6) compared to travel elsewhere, after adjusting for rising prevalence of resistance over time. MDR+FQ resistance in S. Typhi isolates was associated with travel to Pakistan (aOR=3.5, 95 % CI: 2.4-5.2) and less likely with travel to India (aOR=0.07, 95 % CI 0.04-0.15) compared to travel elsewhere. All XDR cases were imported from Pakistan. No isolate was resistant to azithromycin. Comparison with the 2005-2012 London survey indicates substantial increases in the prevalence of resistance of S. Typhi isolates to ciprofloxacin associated with travel to Pakistan (from 79-98 %) and Africa (from 12-60 %).Conclusion. Third-generation cephalosporins and azithromycin remain appropriate choices for empirical treatment of enteric fever in most returning travellers to the UK from endemic countries, except from Pakistan, where XDR represents a significant risk.

Clinical application of real-time PCR to screening critically ill and emergency-care surgical patients for methicillin-resistant Staphylococcus aureus: a quantitative analytical study.

The clinical utility of real-time PCR screening assays for methicillin (methicillin)-resistant Staphylococcus aureus (MRSA) colonization is constrained by the predictive values of their results: as MRSA prevalence falls, the assay's positive predictive value (PPV) drops, and a rising proportion of positive PCR assays will not be confirmed by culture. We provide a quantitative analysis of universal PCR screening of critical care and emergency surgical patients using the BD GeneOhm MRSA PCR system, involving 3,294 assays over six months. A total of 248 PCR assays (7.7%) were positive; however, 88 failed to be confirmed by culture, giving a PPV of 65%. Multivariate analysis was performed to compare PCR-positive culture-positive (P+C+) and PCR-positive culture-negative (P+C-) assays. P+C- results were positively associated with a history of methicillin-sensitive Staphylococcus aureus infection or colonization (odds ratio [OR], 3.15; 95% confidence interval [CI], 1.32 to 7.54) and high PCR thresholds of signal intensity, indicative of a low concentration of target DNA (OR, 1.19 per cycle; 95% CI, 1.11 to 1.26). P+C- results were negatively associated with a history of MRSA infection or colonization (OR, 0.19; 95% CI, 0.09 to 0.42) and male sex (OR, 0.40; 95% CI, 0.20 to 0.81). P+C+ patients were significantly more likely to have subsequent positive MRSA culture assays and microbiological evidence of clinical MRSA infection. The risk of subsequent MRSA infection in P+C- patients was not significantly different from that in case-matched PCR-negative controls. We conclude that, given the low PPV and poor correlation between a PCR-positive assay and the clinical outcome, it would be prudent to await culture confirmation before altering infection control measures on the basis of a positive PCR result.

Defining Surrogate Endpoints for Clinical Trials in Severe Falciparum Malaria.

BACKGROUND: Clinical trials in severe falciparum malaria require a large sample size to detect clinically meaningful differences in mortality. This means few interventions can be evaluated at any time. Using a validated surrogate endpoint for mortality would provide a useful alternative allowing a smaller sample size. Here we evaluate changes in coma score and plasma lactate as surrogate endpoints for mortality in severe falciparum malaria. METHODS: Three datasets of clinical studies in severe malaria were re-evaluated: studies from Chittagong, Bangladesh (adults), the African 'AQUAMAT' trial comparing artesunate and quinine (children), and the Vietnamese 'AQ' study (adults) comparing artemether with quinine. The absolute change, relative change, slope of the normalization over time, and time to normalization were derived from sequential measurements of plasma lactate and coma score, and validated for their use as surrogate endpoint, including the proportion of treatment effect on mortality explained (PTE) by these surrogate measures. RESULTS: Improvements in lactate concentration or coma scores over the first 24 hours of admission, were strongly prognostic for survival in all datasets. In hyperlactataemic patients in the AQ study (n = 173), lower mortality with artemether compared to quinine closely correlated with faster reduction in plasma lactate concentration, with a high PTE of the relative change in plasma lactate at 8 and 12 hours of 0.81 and 0.75, respectively. In paediatric patients enrolled in the 'AQUAMAT' study with cerebral malaria (n = 785), mortality was lower with artesunate compared to quinine, but this was not associated with faster coma recovery. CONCLUSIONS: The relative changes in plasma lactate concentration assessed at 8 or 12 hours after admission are valid surrogate endpoints for severe malaria studies on antimalarial drugs or adjuvant treatments aiming at improving the microcirculation. Measures of coma recovery are not valid surrogate endpoints for mortality.

The Relationship between Poverty and Healthcare Seeking among Patients Hospitalized with Acute Febrile Illnesses in Chittagong, Bangladesh.

Delays in seeking appropriate healthcare can increase the case fatality of acute febrile illnesses, and circuitous routes of care-seeking can have a catastrophic financial impact upon patients in low-income settings. To investigate the relationship between poverty and pre-hospital delays for patients with acute febrile illnesses, we recruited a cross-sectional, convenience sample of 527 acutely ill adults and children aged over 6 months, with a documented fever ≥38.0 °C and symptoms of up to 14 days' duration, presenting to a tertiary referral hospital in Chittagong, Bangladesh, over the course of one year from September 2011 to September 2012. Participants were classified according to the socioeconomic status of their households, defined by the Oxford Poverty and Human Development Initiative's multidimensional poverty index (MPI). 51% of participants were classified as multidimensionally poor (MPI>0.33). Median time from onset of any symptoms to arrival at hospital was 22 hours longer for MPI poor adults compared to non-poor adults (123 vs. 101 hours) rising to a difference of 26 hours with adjustment in a multivariate regression model (95% confidence interval 7 to 46 hours; P = 0.009). There was no difference in delays for children from poor and non-poor households (97 vs. 119 hours; P = 0.394). Case fatality was 5.9% vs. 0.8% in poor and non-poor individuals respectively (P = 0.001)-5.1% vs. 0.0% for poor and non-poor adults (P = 0.010) and 6.4% vs. 1.8% for poor and non-poor children (P = 0.083). Deaths were attributed to central nervous system infection (11), malaria (3), urinary tract infection (2), gastrointestinal infection (1) and undifferentiated sepsis (1). Both poor and non-poor households relied predominantly upon the (often informal) private sector for medical advice before reaching the referral hospital, but MPI poor participants were less likely to have consulted a qualified doctor. Poor participants were more likely to attribute delays in decision-making and travel to a lack of money (P<0.001), and more likely to face catastrophic expenditure of more than 25% of monthly household income (P<0.001). We conclude that multidimensional poverty is associated with greater pre-hospital delays and expenditure in this setting. Closer links between health and development agendas could address these consequences of poverty and streamline access to adequate healthcare.

Relative sensitivities of repair-deficient mammalian cells for clonogenic survival after alpha-particle irradiation.

The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after alpha-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single alpha-particle traversal, the average number of lethal lesions per track and per unit dose, along with the "intrinsic radiosensitivity" of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, alpha particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to alpha particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for alpha-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of alpha-particle-induced damage, although a large proportion of damage is not repaired by either pathway.

Characterization of naturally occurring HPV16 integration sites isolated from cervical keratinocytes under noncompetitive conditions.

As the high-risk human papillomavirus (HPV) integrants seen in anogenital carcinomas represent the end-point of a clonal selection process, we used the W12 model to study the naturally occurring integration events that exist in HPV16-infected cervical keratinocytes before integrant selection. We performed limiting dilution cloning to identify integrants present in cells that also maintain episomes. Such integrants arise in a natural context and exist in a noncompetitive environment, as they are transcriptionally repressed by episome-derived E2. We found that integration can occur at any time during episome maintenance, providing biological support for epidemiologic observations that persistent HPV infection is a major risk factor in cervical carcinogenesis. Of 24 different integration sites isolated from a single nonclonal population of W12, 12 (50%) occurred within chromosome bands containing a common fragile site (CFS), similar to observations for selected integrants in vivo. This suggests that such regions represent relatively accessible sites for insertion of foreign DNA, rather than conferring a selective advantage when disrupted. Interestingly, however, integrants and CFSs did not accurately colocalize. We further observed that local DNA rearrangements occur frequently and rapidly after the integration event. The majority of integrants were in chromosome bands containing a cancer-associated coding gene or microRNA, indicating that integration occurs commonly in these regions, regardless of selective pressure. The cancer-associated genes were generally a considerable distance from the integration site, and there was no evidence for altered expression of nine strong candidate genes. These latter observations do not support an important role for HPV16 integration in causing insertional mutagenesis.