Plasma levels of NT-pro-BNP, a natriuretic peptide precursor, are raised in the presence of fluid retention of cardiac origin and can be used as markers of cardiac dysfunction. Recent studies showed high levels of NT pro BNP in patients with cirrhosis. We assessed NT pro-BNP and other parameters of cardiac dysfunction in patients with cirrhosis, with or without ascites, in order to determine whether the behaviour of NT pro BNP is linked to the stage of liver disease or to secondary cardiac dysfunction.Fifty eight consecutive hospitalized patients mostly with viral or NAFLD-related cirrhosis were studied. All underwent abdominal ultrasound and upper GI endoscopy. Cardiac morpho-functional changes were evaluated by echocardiography and NT-pro-BNP plasma levels determined upon admission. Twenty-eight hypertensive patients, without evidence of liver disease served as controls.Fifty eight cirrhotic patients (72% men) with a median age of 62 years  had a normal renal function . As compared to controls, cirrhotic patients had higher NT pro-BNP plasma levels . Left atrial volume (LAV) , and left ventricular ejection fraction  were also altered in cirrhotic patients that in controls. Patients with F2-F3 oesophageal varices as compared to F0/F1, showed higher e' velocity , and accordingly a higher E/A ratio .early decompensation of cirrhosis.NT-pro-BNP plasma levels are increased proportionally to the stage of chronic liver disease. Advanced cirrhosis and high NT-pro-BNP levels are significantly associated to increased LAV and to signs of cardiac diastolic dysfunction. NT pro-BNP levels could hence be an useful prognostic indicators of  The foremost hemodynamic feature of cirrhosis is the hyperdynamic syndrome, a circulatory state characterized by low arterial pressure, high cardiac output and decreased peripheral vascular resistance During the past few years, considerable attention has been paid to the role of markers of cardiac dysfunction, BNP and its pro-hormone, NT pro BNP, which both are secreted by heart ventricles in response to massive stretching of muscle cells or to mild cardiac damage and show capability to reduce blood pressure and cardiac hypertrophy In this study we aimed to assess in a well-characterized cohort of patients with cirrhosis of non-alcoholic aetiology, before or after the development of ascites, the expression of NT pro-BNP and of other parameters of cardiac dysfunction in order to determine whether the behaviour of NT pro BNP is linked to the stage of liver disease or to a cardiac dysfunction secondary to cirrhosis.The study was performed in accordance with the principles of the Declaration of Helsinki and its appendices, and with local and national laws. Approval was obtained from the AOUP Policlinico “ Paolo Giaccone” of Palermo, Institutional Review Board and Ethics Committee, and written informed consent was obtained from all patients and controls.From April 2010 to October 2011, we consecutively enrolled fifty eight patients with a clinical or histological diagnosis of cirrhosis, admitted to our liver Unit, which is a tertiary referral centre for chronic liver disease. All of them underwent, abdominal ultrasound and upper GI endoscopy in order to determine the stage of liver disease and the presence of ascites and indirect signs of portal hypertension.All patients with known cardiac or renal diseases  were excluded. The diagnosis of arterial hypertension was based on the following criteria: systolic blood pressure ≥135 mm Hg and/or diastolic blood pressure ≥85 mm Hg , or use of blood-pressure-lowering agents. The diagnosis of type 2 diabetes was based on the revised criteria of the American Diabetes Association, using a value of fasting blood glucose ≥126 mg/dL on at least two occasions We also evaluated a control group of 28 newly diagnosed hypertensive patients matched for age and sex and who were referred to Internal Medicine Unit for clinical investigation. All had a normal renal function without any signs of major organic disease, including liver disease. In particular, all were negative for HBsAg, anti-HCV, anti-HIV, and all of them had normal liver function tests (LFTs).All cirrhotic and control patients underwent echocardiographic examination to study diastolic and systolic cardiac function.One experienced observer performed all echocardiograms according to the recommendations of the American Society of Echocardiography (a) using a GE VIVID 7 Dimension Cardiac – Vascular Ultrasound, interfaced with a 2.5 MHz phased-array probe. All recordings were digitally stored for off-line analyses by dedicated software.http://www.ncbi.nlm.nih.gov/pubmed/2698218) and averaged over three cardiac cycles. The ejection fraction (EF) was calculated by using the area-length formula as previously described M-mode echocardiograms of the left ventricle were recorded from the parasternal long-axis view guided by two-dimensional image. We measured left ventricular internal diameter (LVID), interventricular septum (IVS), and posterior wall thickness (PWT) on this M-mode tracings, as described in the American Society of Echocardiography guidelines Assessment of diastolic dysfunction, according to Recommendations for the Evaluation of Left Ventricular Diastolic Function by ASE Plasma NT-pro-BNP levels were determined by a commercial assay . Blood samples from cirrhotic and controls have been drained and then centrifuged at 2000 rpm for 15 minutes at room temperature. The supernatant plasma was recovered and stored at −80°C until analysis.Continuous variables were summarized as mean ± SD; categorical variables as frequency and percentage. The ANOVA test, student's t-test, Kruskal-Wallis test and chi-square test were used when appropriate.Univariate linear regression analysis was used to study relationships between NT-pro-BNP and clinical, biochemical and echocardiographic variables of cirrhotic patients.Logistic regression model was used to identify the factors associated with the presence of ascites, by assessing the same independent variables included in the linear model. A two-tailed p value of p<0.05 was considered significant. STATA/SE, version 12.1 for Windows , was used to analyze the data.The baseline features of the 58 cirrhotic and 28 control patients are shown in Cirrhotic patients (72% men) had a median age of 62 years, a prevalence of arterial hypertension and diabetes of 11% and 31% respectively, and showed a renal function within the normal range (range 0.7–1.06) with a mean creatinine levels of 0.9 mg/dl. The liver disease was due to HCV infection (58.6%), HBV infection (13.7%), alcohol abuse (3.4%), NASH (13.7%), and cryptogenic liver damage 6 (10.4%). According to Child-Pugh classification, patients were divided as follows: 24 (41.4%) in A class; 23 (39.7%) and 11 (18.9%) in B and C classes, respectively. Further, all 58 patients were subgrouped according to the presence of ascites; 32 out of 58 (55.2%) were cirrhotic decompensated patients being on therapy with diuretics or paracentesis (refractory ascites); the remaining 44.8% were compensated (without ascites). For patients with refractory ascites, echocardiographic assessment was done after paracentesis. Upper GI endoscopy showed that 10 (17.3%) patients did not have signs of portal hypertension, while 24 (41.4%) had small (F1) oesophageal varices and 22 (37.9%) had medium-large (F2/F3) oesophageal varices. Only for two patients type and dimension of esophageal varices were not assessed. Twenty five patients (43.1%) were on beta blockers as prophylaxis of first variceal bleeding or rebleeding. Finally, in 25 (43.1%) cases a diagnosis of HCC was made during hospitalization, but none of these patients was on therapy with anti-angiogenic drugs.The control subjects  were comparable for diabetes prevalence to the cirrhotic population, and had similar creatinine serum levels.NT pro-BNP plasma levels and echocardiographic parameters of both cirrhotic and control patients are showed in Cirrhotic patients had significantly higher NT pro-BNP plasma levels compared to controls  .2; p = 0.08) and Systolic Pulmonary Arterial Pressure (PAPs) .Similarly, left atrial volume (LAV) , and left ventricular ejection fraction  were significantly altered in cirrhotic patients as compared to controls, and a trend was also observed for left ventricular mass , and to renal impairment (higher serum creatinine levels), and to larger atrial volumes .When comparing patients with F2-F3 oesophageal varices to those with F0/F1 varices , regardlIn cirrhotic patients, NT-pro-BNP serum levels as well as echocardiographic parameters such as left atrial volumes and ejection fraction are significantly altered. There is a significant association between NT-pro-BNP serum levels, parameter of kidney and liver function, and atrial volumes. We also observed that cirrhotic patients with ascites, compared to their counterpart without, had higher NT-pro-BNP serum levels and accordingly larger atrial volumes, and a paradoxical significant increase in E/A ratio also observed in patients with large esophageal varices.In an attempt to investigate the spectrum of cardiac abnormalities involving cirrhotic patients, we found that atrial volumes, ejection fraction, and partially left ventricular mass and PAPs are significantly altered, as compared to the hypertensive controls. These findings could be expression of the physio-pathological events that lead to the hyperdynamic syndrome, typical of cirrhosis, and characterized by increased heart rate and cardiac output and decreased systemic vascular resistance with normal or low blood arterial pressure Another relevant issue of our study, is that high levels of NT pro-BNP are increased in the presence of ascites as compared to cirrhotic patients without, probably as a result of increased fluid retention. We also observed a trend for larger atrial volumes in ascitic versus no ascitic patients. Overall these data suggest that NT pro-BNP is a marker of cardiac subclinical dysfunction participating to liver decompensation. Further studies are needed to assess the role of NT pro-BNP as a predictor of decompensation and prognosis in cirrhotic patients.In this study we also observed that patients with more advanced liver disease, i.e. those with large varices or with ascites, had a higher E/A ratio, and a relatively “better” diastolic cardiac function. This finding is explained by the fact that these patients usually need of periodic paracentesis and albumin infusion, determining an improvement of E/A ratio. In fact, it has been already described that after paracentesis and albumin infusion cirrhotic patients show an upgrading of diastolic dysfunction as demonstrated by an increase of E/A ratio The main limitation of this study, besides the unavoidable clinical heterogeneity and the relatively small number of patients, lies in its cross-sectional nature, unable to identify pathogenic mechanisms(s) linking cardiac dysfunction, NT pro BNP serum levels, and severity of liver disease. In addition the use of diuretics and beta-blockers in cirrhotics and of hypertensive patients as controls could also bias our results, underestimating the cardiac alterations of cirrhotic patients.early decompensation of cirrhosis.In conclusion, our study showed that cirrhotic patients have echocardiographic  and biochemical (higher NT pro BNP serum levels) alterations expression of cardiac dysfunction, leading as spectator or actor to liver decompensation and ascites development. While further physio-pathological studies should be performed in order to determine whether the high levels of NT-pro BNP are a consequence of the hyperdynamic syndrome and volume overload on heart chambers. It is clinically relevant that NT-pro-BNP plasma levels are increased proportionally to the severity of cirrhosis. In fact, advanced cirrhosis and high NT-pro-BNP levels are significantly associated to increased LAV, and to signs of cardiac diastolic dysfunction, which characterize morpho-functional changes usually found in end stage liver disease. The correlation between NT pro-BNP levels and cirrhosis stage could eventually, if confirmed by longitudinal studies, prove useful as a prognostic indicators of Table S1Demographic, clinical and laboratory features of 58 cirrhotic patients and 28 sex and age matched controls.(DOC)Click here for additional data file.


Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is the most common presentation of a disorder of sex development (DSD) in genetic females. A report of prenatal growth retardation in cases of 46,XY DSD, coupled with observations of below-optimal final height in both males and females with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, prompted us to investigate prenatal growth in the latter group. Additionally, because girls with congenital adrenal hyperplasia are exposed to increased levels of androgens in the absence of a male sex-chromosome complement, the presence or absence of typical sex differences in growth of newborns would support or refute a hormonal explanation for these differences.In total, 105 newborns with congenital adrenal hyperplasia were identified in our database. Gestational age (weeks), birth weight (kg), birth length (cm) and parental heights (cm) were obtained. Mid-parental height was considered in the analyses.P > 0.05). The expected sex difference in favor of heavier males was not seen (P > 0.05). Of the 105 subjects, 44  had birth length and gestational age recorded in their medical chart. Mean birth length for this subgroup was 50.90 cm (63rd percentile), which differed from the expected standard population median of 50th percentile (P = 0.0082). The expected sex difference in favor of longer males was also not seen (P > 0.05).Mean birth weight percentile for congenital adrenal hyperplasia was 49.26%, indicating no evidence of a difference in birth weight from the expected standard population median of 50th percentile (The prenatal growth retardation patterns reported in cases of 46,XY disorders of sex development do not generalize to people with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Sex differences in body weight and length typically seen in young infants were not seen in the subjects who participated in this study. We speculate that these differences were ameliorated in this study because of increased levels of prenatal androgens experienced by the females infants. For example, low birth weight is more common in newborns with isolated hypospadias -9 and innewborns .Low birth weight is of concern, independently of its relationship with atypical reproductive development, because of the association with development of metabolic, endocrine and cardiovascular diseases later in life . FurtherCongenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OH) is the commonest cause of DSD in 46,XX individuals. Females with this condition are exposed during early development to levels of androgens that can be similar to the atypically low levels of androgens experienced by some genetic males with DSD. Furthermore, both under- and over-treatment of CAH with glucocorticoids results in a final adult height that is shorter than that predicted from mean parental height (MPH) . A betteTo our knowledge, only three published studies have analyzed prenatal growth in newborns with CAH due to 21-hydroxylase (21-OH) deficiency. The first study reported normal birth weight , and theThe goal of the present study was to retrospectively evaluate birth weight and length in newborns with CAH due to 21-OH deficiency whereas controlling for the influence of parental height. Furthermore, because female newborns with CAH due to 21-OH deficiency are exposed to increased levels of androgens before treatment in the absence of a male sex-chromosome complement, their growth patterns allow us to discriminate between hormonal versus genetic influences in the context of sexually dimorphic anthropometric measures.The study was approved by the institutional review boards of the University of Oklahoma Health Sciences Center and the Johns Hopkins School of Medicine, informed consent was not required.A retrospective chart review was performed on all pediatric endocrine charts from both clinics collected over the past 50 years. Subjects with CAH due to 21-hydroxylase deficiency were identified from each institution and reviewed . Gestational age (weeks), birth weight (kg), birth length (cm) and parental heights (cm) were obtained when documented in the chart. Subjects of gestational age < 37 weeks or > 44 weeks were omitted from the analyses. A single instance of twin gestation was identified in our data, but was excluded from the current analyses based on delivery at 34 weeks gestational age.http://www.cdc.gov/epiinfo), yielding birth weight, length, and weight, for, height percentiles and z-scores. MPH was calculated based on the formula:  ± paternal height (cm) ± 13 cm  [Birth weight and length information were entered into a software program  .U-test and Student t-test were used to examine the difference in study variables between male and female subjects with CAH. Spearman correlation coefficients were also calculated to determine the strength of the relationship between MPH and both birth weight (kg) and birth length (cm).Wilcoxon signed-rank tests were used to test whether birth weight and length percentiles differed from the standard median (50th percentile), and the Mann-Whitney t (103) = -1.18, P > 0.05). Mean birth weight percentile for CAH was 49.26%, indicating no difference in birth weight from the expected standard population median of 50th percentile . There was no sex difference in birth weight percentile between male and females subjects .Of the 105 subjects, 91% were born between 37 and 40 weeks. Gestational age ranged from 37 to 43 weeks (mean ± SD 39.91 ± 1.04). Mean birth weight for CAH subjects was 3.42 kg, with an average female and male weight of 3.39 and 3.50 kg, respectively . The mean birth length percentile for all subjects was 63.11%, which differed from the expected standard population median of the 50th percentile . In total, 68 subjects had their MPH calculated, but only 30 subjects had four data points available  to analyze. There was no significant correlation between birth weight  or length  percentiles and MPH.Of the 105 subjects with CAH, 44  had birth length and gestational age recorded in their chart  system, adequate nutrient and oxygen supply to the fetus, and normal imprinting of genes involved in early growth. For example, children with IGF-1 deficiency have depressed patterns of prenatal growth despite their exposure to typical levels of androgens during gestation . Additioin utero, including 17-hydroxyprogesterone. Studies of non-human animal models indicate that maternal activation of the hypothalamic-pituitary-adrenal axis during pregnancy results in alterations in offspring body mass, but not skeletal growth [in utero have reduced body mass [For newborns with CAH due to 21-OH deficiency, increased birth length could be due to a relative deficiency of cortisol, which has well-known growth-suppressing effects, or to excess levels of adrenal hormone precursors l growth -39. Convody mass , limb leody mass ,42 and cIt seems that newborns with CAH have already experienced some alterations in linear growth compared with their unaffected counterparts. Possibly, some of the difficulty seen in effecting normal linear growth throughout childhood of individuals with this disorder may be independent of post-natal cortisol-replacement regimens.The prenatal growth retardation patterns reported in cases of 46,XY DSD do not generalize to individuals with CAH due to 21-OH deficiency. Sex differences in body weight and length typically seen in young infants were not observed in the subjects who participated in this study. We speculate that these differences were ameliorated in this study due to increased levels of prenatal androgens experienced by the female infants.The authors declare that they have no competing interests.The work presented here was carried out in collaboration between all the authors. All authors read and approved the final manuscript.


In recent years several new fastidious bacteria have been identified that display a high specificity for BV; however no previous studies have comprehensively assessed the behavioural risk associations of these bacterial vaginosis-candidate organisms (BV-COs).Atopobium vaginae, Gardnerella vaginalis, Leptotrichia spp., Megasphaera type I, Sneathia spp., and the Clostridia-like bacteria BVAB1, BVAB2 and BVAB3. Associations between BV-COs and BV and behaviours were examined by univariate and multivariable analyses.We examined the associations between 8 key previously described BV-COs and BV status established by Nugent's score (NS). We also examined the sexual practices associated with each BV-CO. We incorporated 2 study populations: 193 from a sexually-inexperienced university population and 146 from a highly sexually-active clinic population. Detailed behavioural data was collected by questionnaire and vaginal smears were scored by the Nugent method. Stored samples were tested by quantitative PCR assays for the 8 BV-COs: Megasphaera type I, BVAB2, A. vaginae and G. vaginalis were significantly independently associated with BV by multivariable analysis. Six of the eight BV-COs  were rare or absent in sexually-unexposed women, and demonstrated increasing odds of detection with increasing levels of sexual activity and/or numbers of lifetime sexual partners. Only G. vaginalis and A. vaginae were commonly detected in sexually-unexposed women. Megasphaera type I was independently associated with women-who-have-sex-with women (WSW) and lifetime sexual partner numbers, while unprotected penile-vaginal-sex was associated with BVAB2 detection by multivariate analysis.On univariate analysis, all BV-COs were more common in BV compared to normal flora. However, only Four of eight key BV-COs were significantly associated with BV after adjusting for the presence of other BV-COs. The majority of BV-COs were absent or rare in sexually-unexposed women, and associated with increasing sexual exposure, suggesting potential sexual transmission of BV-COs. Gardnerella vaginalis has been the bacterium most intensively studied in relation to BV; however it is not specific for BV and is commonly present in women with normal vaginal flora although usually in lower numbers than in those with BV G. vaginalis and Atopobium vaginae is associated with BV Leptotrichia and Sneathia genera, and the uncultivated Megasphaera-like phylotype (termed type I) Megasphaera type I had a sensitivity of 100% and specificity of 91.3% in predicting BV. The causal role of these BV-COs remains to be established and there are no published reports on their behavioural associations and whether they are sexually transmitted.Bacterial vaginosis (BV) is an enigmatic condition and despite over 50 years of medical research, its aetiology and pathogenesis remains unknown. It is the commonest cause of vaginal discharge globally Observational evidence shows a strong association between sexual activity and BV Using samples from two published studies that collected detailed behavioural data and included women with a broad range of sexual experience including sexually-naive women, we sought to examine the relative strength of associations between the aforementioned key BV-COs and BV status established by Nugent's score (NS), and to establish the behavioural characteristics associated with each BV-CO.The Human Research and Ethics Committee of the University of Melbourne approved both studies incorporated into this research. Informed written consent was obtained from all participants involved in the study.www.mshc.org.au/fuss/), which described the study in detail, and provided investigators' contact details. Research investigators provided a detailed explanation of the study upon contact, and a record was kept of all individuals who contacted investigators.In order to obtain a study population with a broad range of sexual exposure from no history of sexual activity to high levels of sexual activity, we combined populations from two published studies: the FUSS  FUSS contained 3 groups of women: sexually-inactive women with no history of any sexual-contact with another person, women with a history of non-coital contact but no penile-vaginal sex, and women with a history of penile-vaginal sex. Overall the FUSS population had limited sexual exposure. The clinic population was recruited at MSHC by clinicians and the participants were sexually-experienced women with higher numbers of lifetime partners. This group was included to act as a comparison group to the low risk FUSS population, to extend the breadth of sexual exposure in the overall study population and to provide generalisable findings to past studies. Most studies of the association of BV-COs with BV have been in women recruited from higher prevalence populations Participants in both studies completed questionnaires recording demographic, medical, sexual partnership and behavioural data. In FUSS, these data were collected via self-collected paper-based questionnaires with the option of online questionnaires, whereas in the clinic study women self-completed hard copy questionnaires. Questions were asked in a standardized way across both studies to allow these data to be aggregated where necessary. Both studies used the Nugent method to diagnose BV on Gram-stained vaginal smears. In FUSS they were self-collected, while they were clinician-collected in the clinic study. FUSS participants received clear diagrammatic instructions regarding self-collecting vaginal swabs and smears. Self-collected smears have been shown to be equivalent to practitioner-collected samples in published studies All vaginal smears were scored using the Nugent method by an experienced microscopist with no access to the behavioural data; a Nugent score (NS) of 0–3 was graded as normal flora (NF), 4–6 as intermediate flora, and 7–10 as BV. Ten percent of slides were re-read by a second microscopist, who was blind to the original results. If consensus was not reached, a third microscopist assessed the slide and a discussion followed until agreement was reached.Overall, 193 samples from FUSS were analysed including all samples with a NS of 7–10 (n = 24), all samples from women who reported no history of penile-vaginal sex ever (n = 82), and an equivalent random sample of women who reported penile-vaginal sex ever (n = 87). From the clinic study, 146 random samples were selected from participants reporting >10 lifetime partners . The two study populations were combined for this analysis in order to provide a uniquely diverse population of women that ranged from sexually-inexperienced (FUSS) to highly sexual experienced (clinic study). Having behavioural data and clinical samples from women with a broad range of sexual exposure was considered integral to investigating the associations between BV-COs and sexual activity. Only women with normal flora and BV were included in this analysis and women with intermediate flora were excluded. Women who were pregnant, HIV infected, postmenopausal, or non- fluent in English were excluded from participating in either study. All swabs samples used in this study were stored at −80°C until processing. The Human Research and Ethics Committee of the University of Melbourne approved both studies.Atopobium vaginae, Gardnerella vaginalis, Leptotrichia spp., Megasphaera type I, Sneathia spp. and the Clostridia-like bacteria BVAB1, BVAB2, and BVAB3; cost limited the inclusion of other BV-COs. Lactobacillus crispatus was also included as a key indicator of normal vaginal flora status. Total DNA was extracted from 200 µl volumes of the resuspended vaginal swab samples using the MagNA Pure LC system (Roche Diagnostics) with the DNA Isolation Kit I protocol, eluting in a final volume of 100 µl with DNA stored at −20°C until testing. Included in each DNA extraction run were controls that included human A549 cells and DNA-free phosphate buffered saline. To assess inhibition, sample adequacy and integrity, real-time PCR amplification and detection of the human β-globin gene was carried out as previously described A. vaginae, L. crispatus, Leptotrichia spp., Megasphaera type I, and the Clostridia-like bacteria BVAB1, BVAB2, and BVAB3 with the detection for Leptotrichia spp. and Sneathia spp. carried out as separate reactions. G. vaginalis detection was determined by an assay by Zariffard et al. Eight BV-COs were selected including The associations between BV-COs and BV were assessed by univariate and multivariate analyses using logistic regression. The presence of BV-COs according to sexual exposure variables were examined using Chi square tests or Fisher's exact where appropriate. To investigate the association between behavioural risk factors and BV-COs, a multivariate logistic regression model was constructed, including variables with a significance of p<0.05 from the univariate analysis and key risk factors for BV from published literature. These included: number of lifetime vaginal sexual partners (LSP), smoking, age, frequency of vaginal sex and frequency of receptive oral sex and unprotected vaginal sex (UPVSI) in the last 12 months. Samples with intermediate flora (Nugent's score 4–6) were not included in this analysis and all analysis was undertaken using PASW software (version 18).Overall, 339 specimens were analysed; 193 from FUSS and 146 from the clinic cohort. Among the study population, 233 women had NF and 106 women had BV. L. crispatus was significantly more common in NF than BV. In a logistic regression model, Megasphaera type I demonstrated the strongest independent association with BV of the 8 BV-COs [Adjusted Odds ratio (AOR) = 15.4; 95% CI 5.9–40.2, p<0.001, A. vaginae and G. vaginalis were also significantly independently associated with BV , nd/or BV . A. vagiTo further investigate the epidemiology of BV-COs, the association of BV sexual risk factors with each BV-CO was examined. BV risk factors included in the analysis included LSP number, UPVSI in the last 12 months, reporting a female partner (WSW) in the last year, age, smoking and frequent penile-vaginal sex and frequent receptive oral sex (>once weekly). The associations between each of these risk variables and each BV-CO was examined separately and adjusted for BV status .Sneathia, Leptotrichia, Megasphaera type I, G. vaginalis, BVAB2 and BVAB3 were significantly associated with having >10 LSP or UPVSI or both factors after adjustment for BV status. Frequent sexual activity  was related to increased detection of Sneathia, Leptotrichia, G. vaginalis, and BVAB3, while Megasphaera type I and Leptotrichia were significantly associated with WSW status. BVAB1 was uncommon and no associations could be examined with meaningful statistical power  in the last year, age, smoking, frequent penile-vaginal sex and frequent receptive oral sex (>once weekly) and BV status, Megasphaera type I was independently associated with WSW  and having more than 10 LSP , and BVAB2 showed a strong independent association with unprotected penile-vaginal sex in the last 12 months ; while no specific behaviours remained significantly associated with G. vaginalis or A. vaginae by multivariate analysis.As Megasphaera type I, BVAB2, A. vaginae and G. vaginalis) were significantly associated with BV after logistic regression analysis, while BVAB3, Sneathia and Leptotrichia spp. were not, and BVAB1 was too uncommon to meaningfully comment on. Six of the eight BV-COs demonstrated significant associations with increasing sexual exposure , and only two BV-COs, A. vaginae and G. vaginalis, were commonly detected in women with no sexual experience. Our findings suggest that there may be a gradient of importance among the eight BV-COs based on their strength of association with BV and their epidemiological association with increasing sexual exposure. The finding that the majority of BV-COs were absent or rare in sexually-unexposed women and associated with increasing sexual exposure suggest sexual transmission of BV-COs is occurring.Our paper explores the association between eight key BV-COs with BV and examines their sexual risk factors; Megasphaera type I and BVAB2 were both strongly associated with BV and were also rare or absent in sexually-naive women. The suggestion that Megasphaera type I and BVAB2 are key BV-COs is supported by Fredricks et al. who showed that the combination of BVAB2 and/or Megasphaera type I conferred the best BV diagnostic predictability for BV  For agents to be causative of BV one would anticipate that they would be absent in NF and universally present in BV; no single BV-CO in our study satisfied both these criteria. We have shown, however, that G. vaginalis and A. vaginae, bacteria known to be integrally related to BV disease; however these BV-COs were also detected at lower loads in women with NF (data not shown). G. vaginalis has long been implicated in the development of BV and recent published findings have suggested that G. vaginalis biofilms may be critical in BV pathogenesis and symptomatology G. vaginalis was ubiquitous in women with BV, and further analysis indicating a 6–7 log10 greater bacterial load in BV than NF (data not shown), are consistent with historical findings G. vaginalis was relatively common in truly virginal women ; however the prevalence of G. vaginalis increased dramatically with numbers of sexual partners, indicating that sexual activity or transmission contributes to increasing detection of G. vaginalis. Studies using enzyme assays to identify G. vaginalis biotypes and genomic comparisons of G. vaginalis isolates suggest particular strains of G. vaginalis may have differing pathogenic properties, such as biofilm formation G. vaginalis are present in sexually-inexperienced compared to experienced women is not known and would be of considerable interest.The other BV-COs which were significantly associated with BV were A. vaginae was independently associated with BV and whilst also commonly seen in those with NF (62%), it was generally at low loads . The relatively high prevalence of A. vaginae in NF samples was similar to that of Menard et al A. vaginae in 12% of NF A. vaginae, like G. vaginalis, was detected in sexually-inactive women, but unlike G. vaginalis, A. vaginae showed no significant association with increasing sexual exposure.Leptotrichia and Sneathia spp. are not associated with BV once adjusted for the presence of other BV-COs, and their strong relationship with increasing sexual risk was seen similarly in women with NF and BV. This could be important, because if a BV-CO is sexually transmitted but not associated with disease causation, then one would expect to see increasing prevalence with increasing sexual risk equally in both BV and NF. This was the case for Sneathia and Leptotrichia spp. but not for any of the other BV-COs. Collectively these findings suggest that Sneathia and Leptotrichia spp. could just be epidemiologically associated with BV, or sexually transmitted “passengers” rather than being involved in the development of BV; a similar scenario to that of some Ureaplasma and Mycoplasma spp. in NGU Sneathia and Leptotrichia spp. were detected in 74 and 73 percent of BV cases in our study respectively, their role in symptom/disease pathogenesis, especially in a polymicrobial disease context, cannot be excluded. Future work involving the exploration of metabolic interactions between BV-COs will assist in further understanding the contribution of these organisms to the development of BV.Importantly, our data seem to indicate that Megasphaera type I may have distinctive behavioural risk factors. Although we only had 12% of participants with a female partner in the last 12 months, these women demonstrated a strong association with Megasphaera type I detection. It is important to note that WSW have greater BV population prevalence than other women Megasphaera type I detection was highly sensitive for BV  in a population of women with a history of sexual contact with another woman. In our study Megasphaera type I was 91.3% (95%CI 70.4–98.4) sensitive for BV in WSW, but only 72.2% (95%CI 61.2–81.2) sensitive for BV in non-WSW. It is possible that Megasphaera type I may be an important BV-CO in the development of BV in WSW, and that WSW with BV differ in their microbiological BV profile to other women. Clearly larger prospective studies are needed to better understand this relationship, but an association between Megasphaera type I and WSW to our knowledge has not been previously described.Our data show that This study has several limitations. We used a study population that combined samples from two previous studies collected at different time points. One group was a symptomatic clinic-based population; the other a lower risk university population. By combining these populations investigators were able to obtain a uniquely diverse population of women who ranged from sexually-inexperienced to highly sexually-experienced, and we considered this necessary to be able to comprehensively address the contribution of BV-COs to sexual activity. Prior published studies investigating the association of BV-COs with BV have not contained women with limited or no sexual exposure. The fact that these populations substantially differ in their risk profile, however, is also limitation of the study. We have adjusted for lifetime sexual partner number in the multivariable analyses to control for the key differences in sexual exposure in the study groups, and although we describe known differences in the two study populations not all differences between the groups may not be accounted for. Questions were asked in a standardized way in both studies to allow these data to be aggregated where necessary, and self-collected paper-based questionnaires were used for both studies with the option of an identical self-completed online questionnaire for FUSS (40% of FUSS participants used online surveys). Self-sampling was employed in FUSS and clinician-collected samples were used in the clinic study; published studies confirm equivalency of self-collected to clinician-collected samples for the diagnosis of BV, and excellent agreement has been reported between self-collected versus clinician-collected swabs for molecular quantification (qPCR) of BV-associated bacteria Megasphaera spp. including the type II phylotype. Our data was cross-sectional, therefore we have no capacity to comment on temporal relationships between the detection of BV-COs and sexual behaviour, and we can only make inferences regarding BV causality and pathogenesis. Due to the complexity of the current analyses in this manuscript, we also did not include possible metabolic interactions between species in this study, and have limited ourselves to exploring the associations between individual BV-COs and behaviour and BV disease status. Given the polymicrobial nature of BV, assessing for metabolic interactions is the subject of further research by investigators. Another limitation of cross-sectional data is that fluctuations in vaginal flora are known to occur rapidly and to be influenced by various factors including hormonal variation and day-to-day behaviours Due to funding limitations, our use of molecular methods to characterise the vaginal bacterial communities was limited to a representative panel of eight of the most promising and specific BV-COs, acknowledging there is an increasing number of BV-COs being cited in the literature, particular from next-generation sequencing projects The strengths of our study design include the systematic objective assessment using Nugent's method of all vaginal smears by an experienced microbiologist and 10% being re-scored by an alternative microscopist. Not all studies examining the role of BV-COs in BV have applied methods such as the Nugent method to classify flora objectively and some have used more subjective clinical descriptions and/or patient history Megasphaera type I, BVAB2, A. vaginae and G. vaginalis were significantly associated with BV after adjusting for detection of other BV-COs; however, BVAB3, Sneathia and Leptotrichia spp. showed no independent association with BV, and BVAB1 was absent in NF and uncommon in BV. Our data show that six of the eight BV-CO were rare or absent in sexually-inactive women: Sneathia spp., Leptotrichia spp., Megasphaera type I, BVAB1, BVAB2 and BVAB3. G. vaginalis and A. vaginae were the only BV-COs detected commonly in sexually-inexperienced women. After multivariate-analysis Megasphaera type I was independently associated with WSW and lifetime sexual partner numbers, while unprotected vaginal sex was strongly related to detection of BVAB2. The finding that the majority of BV-COs were absent or rare in sexually-unexposed women, and associated with increasing sexual activity with other individuals, suggests sexual transmission of BV-COs is occurring. Longitudinal studies of BV and BV-COs, and consideration of metabolic interactions between BV-COs, will further elucidate the complex microbiology involved in the development of BV in different populations.These study data contribute to the developing collective understanding of BV-related bacteria and their role in BV pathogenesis. Table S1Demographic and behavioural characteristics of study population.(DOC)Click here for additional data file.Table S2Bacterial vaginosis candidate organisms in women with normal flora and BV.(DOC)Click here for additional data file.Table S3Associations between bacterial vaginosis candidate organisms and level of sexual exposure in women with normal flora and BV.(DOC)Click here for additional data file.Table S4Odds ratios for BV-COs behavioural risks factors adjusted for BV status (n = 339).(DOC)Click here for additional data file.Table S5Logistic regression analysis for BV and BV risk factors and Megasphaera type I, G. vaginalis, BVAB2 and A. vaginae (n = 339).(DOC)Click here for additional data file.Table S6Summary of major findings relating BV candidate organisms to BV status and sexual risk behaviours.(DOC)Click here for additional data file.


Several biomarkers and prognostic scores have been evaluated to predict prognosis in community-acquired pneumonia (CAP). Optimal risk stratification remains to be evaluated. The aim of this study was to evaluate serum cortisol as biomarker for the prediction of adverse outcomes independently of the CRB-65 score und inflammatory biomarkers in a large cohort of hospitalised patients with CAP.984 hospitalised CAP-patients were included. Serum cortisol was measured and its prognostic accuracy compared to the CRB-65 score, leucocyte count and C-reactive protein. Predefined endpoints were 30-day mortality and the combined endpoint critical pneumonia, defined as presence of death occurring during antibiotic treatment, mechanical ventilation, catecholamine treatment or ICU admission.64 patients died (6.5%) and 85 developed critical pneumonia (8.6%). Cortisol levels were significantly elevated in both adverse outcomes (p < 0.001) and predicted mortality  and critical pneumonia (AUC 0.71) independently of all other measured variables after logistic regression analysis . Prognostic accuracy of CRB-65 was significantly improved by adding cortisol levels (combined AUC 0.81 for both endpoints). In Kaplan-Meier analysis, cortisol predicted survival within different CRB-65 strata (p = 0.003). In subgroup analyses, cortisol independently predicted critical pneumonia when compared to procalcitonin, the CURB65 score and minor criteria for severe pneumonia according to the 2007 ATS/IDSA-guideline.Cortisol predicts mortality and critical disease in hospitalised CAP-patients independently of clinical scores and inflammatory biomarkers. It should be incorporated into trials assessing optimal combinations of clinical criteria and biomarkers to improve initial high risk prediction in CAP. A disease severity based approach with initial risk stratification is required to guide management and treatment decisions in CAP. International guidelines recommend prognostic scores to support clinical decision on management as outpatient, inpatient or admission to ICU. The most established score systems are the PSI-score and the CURB or CRB-65 scores, which perform comparably for mortality prediction and identification of low risk patients suitable for an outpatient management strategy -3. AmongAdditionally, biomarkers have been found to improve risk stratification and management decisions in CAP. Procalcitonin (PCT) has been demonstrated to provide additional information by identifying low risk patients , as paraHowever, accurate mortality prediction does not automatically lead to accurate identification of patients developing critical disease in need for intensive care treatment . Both thRecently, serum cortisol concentration was shown to be associated with severity and mortality in CAP in three small trials -23. AddiThe aim of this trial was, to evaluate serum cortisol as biomarker for the development of severe disease requiring more intensive monitoring and management strategies as well as 30-day mortality in a large cohort of hospitalised CAP-patients. Additionally, we studied the independent contribution of serum cortisol to the prognostic properties of the CRB-65 score and inflammatory biomarkers.Hospitalised CAP patients were recruited from a multicentre national CAP-study in Germany, the German Competence network for the study of CAP (CAPNETZ). Detailed description of the CAPNETZ methodology is given elsewhere . InclusiComorbidities in the present study were defined as presence of one or more of the following: congestive heart failure, COPD, chronic renal disease, chronic liver disease, cerebrovascular disease, malignancy, or diabetes mellitus.The CRB-65 score was determined in all patients as described previously .All patients were followed up according to a standardized data sheet for at least 180 days.Venous blood samples were collected within 24 h after first presentation and inclusion in the CAPNETZ study and stored centrally at -70°C. Leucocyte (WBC) count and urea were determined by the local hospital laboratories. Serum C-reactive protein (CRP) was centrally measured by nephelometry with a commercially available assay . Serum PCT was centrally determined by an immunofluorescent assay . Serum cortisol was centrally analysed by a commercially available chemiluminescence immunoassay . This assay has a measurement range of 0.5-1750 nmol/L, higher concentrations were analysed by dilution of the serum sample according to the manufacturers protocol.Predefined outcome variables were survival within 30 days after first contact and the combined endpoint critical pneumonia, defined as the presence of one of the following: death occurring during antibiotic treatment, mechanical ventilation, catecholamine treatment or ICU admission.U-test. Univariate and multivariate analyses were performed to predict the binary endpoints by including the CRB-65-score, the measured laboratory parameters WBC, CRP, cortisol, and all documented comorbid conditions and demographic factors given in Table Two group comparisons were calculated by the Mann-Whitney Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic properties of predictive parameters, optimal cut-off values were determined by Youden's index. To compare the predictive value of cortisol and the CRB-65 score, a binary logistic regression model of both parameters was used and the predicted probability derived from that model was included into the ROC-curve analysis. Odds ratios were calculated by using the Mantel-Haenszel estimate.To assess the influence of cortisol concentrations on mortality, we produced Kaplan-Meier survival curves according to cortisol quartiles or cut-off value and stratified by CRB-65 score. Comparison between the groups was performed by log-rank test. A p-value of ≤ 0.05 (two-sided) was considered statistically significant. Statistical analyses were performed with SPSS for Windows, version 17.0 .984 patients were included in the study, their baseline characteristics are summarised in Table Streptococcus pneumoniae was the most frequently isolated pathogen A causative microbiological pathogen was detected in 195 patients (20%). Of these, typical bacteria were found in 84 patients (43%), atypical bacteria in 64 (33%), viruses in 29 (15%) and mixed infection in 18 (9%). There were 64 (6.5%) non-survivors at day 30 after the first contact; their characteristics are shown in Table Serum cortisol levels on admission were significantly higher in non-survivors when compared to survivors  and in patients with critical pneumonia versus noncritical pneumonia . The optimal cut-off value was calculated with 795 nmol/l with a sensitivity of 66% and specificity of 71%  for 30-day mortality. Sensitivity and specificity for critical pneumonia were 64% and 72% .As shown in binary logistic regression analysis, the combined use of CRB-65 and cortisol significantly improved the prediction of 30-day mortality  and critical pneumonia  when compared to the CRB-65 score alone , and in this subgroup a post-hoc analysis of the CURB- and CURB65-scores was performed. The CURB-score predicted 30-day mortality and critical pneumonia equally to the CRB-65 score with an AUC of 0.77 and 0.78, respectively. The corresponding values for the CURB-65 score were 0.81 and 0.79, which was a superior to the CRB65 score (p = 0.001). If cortisol was added, further significant improvement of prediction resulted for both scores and endpoints .PCT was not routinely measured in this trial. However, in 643 patients (65.3%) initial PCT levels were available and post-hoc analysis of these patients was performed. Median PCT-concentration was 0.11 ng/ml (IQR 0.06-0.33), with significantly higher values in non-survivors (0.40 ng/ml [0.15-2.06]) vs. survivors . PCT predicted 30-day mortality (AUC = 0.73) and critical pneumonia (AUC = 0.74) by ROC-curve-analysis. In that subgroup, both PCT and cortisol tended to improve the predictive potential of CRB-65 score for 30-day-mortality, however in binary logistic regression analysis including all three variables, the added predictive value was not significant . If critical pneumonia was used as dependent variable, only cortisol added significantly and independently of PCT to the prognostic accuracy of CRB-65 . The combined addition of both biomarkers to CRB-65 showed no further improvement.2 < 52 mmHg instead of PaO2/FiO2 < 250 and without multilobar infiltrate), the resulting AUC after ROC curve analysis for predicting critical pneumonia was 0.71 with an optimal cut-off of ≥2 criteria . Cortisol added significantly to the predictive accuracy of this model in binary regression analysis .To assess the prognostic relevance of cortisol in comparison to the suggested minor criteria to identify patients in need of ICU-admission by the ATS/IDSA 2007 guideline , we addiThe main study result is that increased serum cortisol level is associated with the development of critical disease and increased 30-day-mortality in hospitalised CAP-patients and adds prognostic information independently of the CRB-65 score and the inflammatory biomarkers WBC and CRP. Serum cortisol level improves the predictive power of the CRB-65 score and shows independent prognostic significance within different CRB-65 strata. Its additional predictive value persisted, when cortisol was analysed in combination with urea within the CURB-65 score, PCT or minor criteria suggested by the recently revised ATS/IDSA-guideline  to prediOur study confirms and expands previous data from three smaller studies, including a total of 401 patients, suggesting cortisol as biomarker for predicting mortality in CAP patients -23. The In univariate analysis, we found the known risk factors age, male sex, nursing home residence, chronic comorbidities, CRB-65 score and the measured inflammatory biomarkers to be associated with both endpoints ,27. In cThe CAPNETZ study also records the cause of death as judged by the treating physician. This subjective statement certainly has a lower value than measured parameters. But, of the 22 patients who died within 30 days despite having an initial cortisol level below the cut-off of 795 nmol/l, only three died from pneumonia as judged by the treating physicians, and two of these three suffered from concomitant bronchial carcinoma.Mortality prediction might not be optimal to identify patients at risk for critical disease manifestations such as respiratory insufficiency or septic shock. These carry a high demand on and benefit of intensified management strategies, which makes their early detection crucial. On the other hand, CAP frequently occurs in elderly and multimorbid patients. Here mortality does not always reflect "dying from pneumonia" but rather "dying with pneumonia", and treatment restrictions might bias this endpoint further. By the inclusion of the predefined composite endpoint of critical pneumonia, our study provides even more accurate pneumonia-specific risk prediction than by mortality prediction alone. This dichotomy of endpoints is reflected by our data: Whereas mortality was predicted by concomitant malignancy, chronic renal failure and high age, critical pneumonia was predicted by higher CRP levels indicating systemic inflammation. CRB-65-score, congestive heart failure, male sex and serum cortisol as only biomarker independently predicted both endpoints, confirming the prognostic relevance of cortisol for CAP-related outcomes.Rapid and accurate identification of hospitalised CAP patients at risk of a critical disease course is a yet unmet need in CAP management. Several scores have been evaluated for predicting ICU-admission. The "classical" risk prediction tools, the CRB-65- and PSI-scores perform well in identifying patients with a low mortality risk, but are poor in predicting ICU-admission ,20. RecoTo account for the heterogeneous causes for deterioration in CAP, optimal risk prediction probably should incorporate clinical criteria plus biomarkers reflecting inflammatory, cardiovascular and other mechanisms of disease progression. Accordingly, although inflammatory markers like CRP  and PCT Cortisol is a biomarker reflecting additional mechanisms involved in critical disease development like stress response and immunomodulatory regulation of inflammatory processes . PhysiolRecently there have been conflicting reports on benefits and risks of steroid treatment in CAP ,38. GiveSeveral limitations of our study have to be mentioned:First, we were not able to correct for concomitant steroid medication, as this data were not documented. Systemic steroid medication of ≥ 20 mg prednisolone equivalent per day for more than 14 days excludes participation in CAPNETZ. In order to control for potential effects of steroid co-medication, which probably affect patients with chronic respiratory disease in particular, analysis after exclusion of all patients with chronic respiratory disease was performed and no relevant change of the diagnostic properties of cortisol could be detected.Second, as blood samples were taken at time of first contact, controlling for the time point of blood sampling could not be done. Cortisol exhibits diurnal concentration changes; however, during infectious diseases the circadian pattern is often lost .Third, we did not test for adrenal insufficiency based on the response to injection of synthetic adrenocorticotropin. However previous data show a very low rate of adrenal insufficiency in patients with CAP in the absence of septic shock , and theThus, deviation of our study results by concomitant steroid medication, adrenal insufficiency or different blood sampling time points cannot be ruled out. These limitations also might account for the slightly lower predictive performance of cortisol shown in our study when compared to some of the previous data ; acquisition of data ; analysis and interpretation ; substantial involvement in the writing and/or revision of the article . All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/90/prepub


Assumptions about survival of HIV-infected children in Africa without antiretroviral therapy need to be updated to inform ongoing UNAIDS modelling of paediatric HIV epidemics among children. Improved estimates of infant survival by timing of HIV-infection  are thus needed.A pooled analysis was conducted of individual data of all available intervention cohorts and randomized trials on prevention of HIV mother-to-child transmission in Africa. Studies were right-censored at the time of infant antiretroviral initiation. Overall mortality rate per 1000 child-years of follow-up was calculated by selected maternal and infant characteristics. The Kaplan-Meier method was used to estimate survival curves by child's HIV infection status and timing of HIV infection. Individual data from 12 studies were pooled, with 12,112 children of HIV-infected women. Mortality rates per 1,000 child-years follow-up were 39.3 and 381.6 for HIV-uninfected and infected children respectively. One year after acquisition of HIV infection, an estimated 26% postnatally and 52% perinatally infected children would have died; and 4% uninfected children by age 1 year. Mortality was independently associated with maternal death , maternal CD4<350 cells/ml , postnatal  or peri-partum HIV-infection .These results update previous work and inform future UNAIDS modelling by providing survival estimates for HIV-infected untreated African children by timing of infection. We highlight the urgent need for the prevention of peri-partum and postnatal transmission and timely assessment of HIV infection in infants to initiate antiretroviral care and support for HIV-infected children. Sub-Saharan Africa remains the region most heavily affected by HIV. In 2008, an estimated 1,800,000 HIV-infected children under 15 years were living in sub-Saharan Africa, and this continent accounted for 91% of new HIV infections among children More specifically, precise estimates of infant survival by timing of HIV-infection  are urgently needed. Such estimates will provide benchmarks for assessing the impacts of paediatric HIV treatment whilst allowing for the effect of background mortality on survival post-infection of children. We here present the results based on data with consistent information on timing of acquisition of infection, both before or during delivery, and postnatally to reliably estimate infant survival by timing of HIV-infection. We update the previously conducted individual patient meta-analysis on survival in HIV-exposed children Studies eligible for this pooled analysis were clinical trials or cohort studies in sub-Saharan African countries that investigated the effect of antiretroviral-based and/or infant feeding interventions in risk reduction of HIV vertical transmission with paediatric follow-up for 18–24 months and information about infant feeding. Overall, 18 trials and cohort studies aimed at the prevention of mother-to-child transmission of HIV in African settings were eligible for this pooled analysis and contacted . Of thesPaediatric HIV infection was defined as a positive plasma HIV-1 DNA/RNA PCR at any age. Children with a positive HIV-1 diagnostic test result before 4–6 weeks of age (depending on the timing of collection of blood samples in each study) were considered to be HIV-infected in peri-partum Antiretroviral treatment became available to children in the Mashi trial on the first of October 2002, so follow-up was rightly censored at this point. Infant antiretroviral treatment was not available during the time of the other trials. Maternal antiretroviral treatment was available in the Mitra Plus study only.Baseline characteristics were described for each study included in the pooled analysis. Overall mortality as a rate per 1000 child-years of follow-up was calculated by selected maternal and infant characteristics. The Kaplan-Meier method was used to estimate survival curves among HIV-infected children: children infected in peri-partum, infected in postnatal, and infected with unknown timing of infection. Estimated time of diagnosis of HIV-infection for those infected postnatally was taken as mid-way between the last HIV-negative test and the first HIV-positive test. The survival of children infected peri-partum or with unknown timing was estimated from birth. Studies were right-censored at the time of infant antiretroviral initiation. We used random-effects Weibull regression models to estimate mortality hazard ratios accounting for heterogeneity between trials and cohorts. Adjusted hazard ratios were obtained, both overall and for HIV-uninfected and HIV-infected children separately. The multivariable models included the following variables: region of origin , sex , maternal vital status , antenatal maternal CD4 count in cells/ml , breastfeeding practice in the first year of life , and child HIV-infection status . Age at time of infection was allowed for in the model.The characteristics of the datasets included in the first step analysis are detailed in Overall, 1,363 children acquired infection perinatally with median time of first positive DNA/RNA test at 19 days (interquartile range: 1–42 days), 581 children were negative at or after 4 weeks of age and acquired infection through breastfeeding at a median 24 weeks of age (interquartile range: 7–39 weeks), and 565 children were infected with unknown timing of infection and had a first positive DNA/RNA test at 80 days in median (interquartile range: 51–168 days).Crude mortality rates per 1,000 child-years of follow-up were 39.3 and 381.6 for HIV-uninfected and HIV-infected children, respectively. Mortality rate per 1000 child-years of follow-up by selected maternal and infant characteristics are detailed in Mortality was estimated for HIV-infected children by timing of transmission; initial time was the estimated date of acquisition of HIV-infection . OverallAdjusted mortality hazard ratios (aHR) with 95% CI by selected characteristics were estimated in regression analyses among HIV-infected and uninfected children . With South Africa as reference, mortality was significantly higher in East Africa, aHR 1.41 (1.14–1.74), but not West Africa, aHR 1.20 (0.88–1.63). Mortality did not differ significantly by sex: with boys as reference, aHR in girls was 1.02 (0.76–1.36). When the mother died during follow-up, children were more than twice likely to die (aHR 2.21 (1.63–3.00)) than when mothers survived. With mothers with CD4≥350 cells/ml as reference, children born to women with CD4<350 (aHR 1.36 (1.08–1.71)) or to women with unknown CD4 (aHR 1.30 (0.72–2.37)) were at significantly increased risk of dying. Children who had never been breastfed were at more than double the risk of dying than ever breastfed children (aHR 2.21 (1.62–3.01)). Mortality was substantially and significantly associated with child HIV infection status: compared to uninfected children, the aHRs were 12.45 (10.15–15.27) for perinatally infected children, 3.08 (2.29–4.14) for children infected through breastfeeding and 7.21 (5.53–9.39) for children with unknown timing of infection.Mortality of children born to HIV-infected mothers was assessed on a large dataset from three different regions in sub-Saharan Africa. Overall mortality of HIV-infected children was dramatically elevated, irrespective of timing of acquisition of infection. However, among HIV-infected children, survival from acquisition of infection in postnatally infected children was higher than in those with infection acquired around delivery: the 18-month post-infection mortality risks were 36% (30%–42%) and 60% (57%–63%), respectively. Most these children had been included in research studies offering relatively high standard of care compared to less supported field areas; a closed clinical follow-up adapted to the child's age with free provision of care was provided to these children. Hence, we assume the survival estimates presented here were higher than what would be expected in more operational field settings. Our results are in accordance with previous findings of high mortality in vertically-infected children Our analysis strategy implied that the survival was assessed from the estimated timing of infection in order to assess survival after infection for perinatally and postnatally infected children. This estimate would thus need to be interpreted in the light of the selection that occurs with children at risk of postnatal infection by definition being those who survived without infection the neonatal period which is one of high mortality risk. On the other hand, perinatally infected children will have to survive this risky period with the infection as an added exposure. However, there is no evidence of an increased neonatal mortality risk associated with perinatal infection We further also confirm the impact of both maternal health, as measured by her HIV immunological progression, and vital status on infant survival. Two-year infant mortality adjusted risk was one third higher among women with ante-partum CD4 count <350 cells/ml than among those with mothers a CD4 count above this threshold; and it was two times higher when the mother had died during the two years post-partum than when the mother was alive two years after birth. Lifelong antiretroviral treatment was not available to women followed-up in all studies selected for this pooled analysis but one. By lack of statistical power, it was therefore not possible to assess the impact of maternal antiretroviral treatment on the survival of children. Bearing in mind that maternal health is an important confounder of child survival, it would be crucial to investigate whether there is a positive impact of such a maternal treatment on infant survival. As maternal antiretroviral treatment keeps an infected mother alive, there would be an indirect effect of reducing child mortality in both her HIV-infected and uninfected children.We present estimates of risk of mortality in children born to HIV-infected mothers in Africa, on the basis of a large dataset with wide variations in the characteristics of the patients included in the meta-analysis. The individual meta-analysis and adjustments in the model account for some of these effects. We acknowledge that there may have been confounding factors that had not been taken into account by our design, but we consider that the most important ones were controlled for. Additionally, the science in the prevention of mother-to-child transmission of HIV has changed since the time of the studies included in this meta-analysis. Expanding the indication of use of potent antiretroviral drug combinations to all pregnant, delivering, and breast-feeding HIV-infected women is now seen as an unprecedented opportunity to radically reduce the burden of paediatric AIDS Our findings draw attention to the importance of comprehensive care for HIV-infected women. More specifically, our results stress the urgency of providing optimal antiretroviral treatment to women who need it for their own health (e.g. with ante-partum CD4 count <350 cell/ml according to the newly revised WHO guidelines Checklist S1PRISMA checklist.(DOC)Click here for additional data file.Table S1Probability with 95%CI of 18-month unadjusted mortality for HIV-infected children since acquisition of HIV infection .(DOCX)Click here for additional data file.


Haemophilus influenzae type b (Hib) is mediated by natural anti-capsular polysaccharide antibody. We hypothesized that non-vaccinated adults with CRF lack protective antibody against Hib, but may respond to the pediatric Hib vaccine.Patients with chronic renal failure (CRF) develop secondary immunodeficiency as a result of the uremic state and its metabolic consequences, along with a negative impact of the dialysis procedure on the immune system. Such patients are therefore at high risk for septicemia and other severe infections. In non-vaccinated adults, protection against Serum anti-Hib IgG and IgM were studied in 60 patients with CRF and 40 healthy controls. Thirty-two patients and 19 controls were immunized with one dose of pediatric Hib vaccine; serum antibody levels were assessed pre- and 1, 6, 9 months post-vaccine. Functional antibody activity was studied using a serum bactericidal assay.Almost 90% of controls, but only 43% of non-vaccinated CRF patients had protective anti-Hib antibody. Four week post-vaccination, all but one patient (97%) have developed protective antibody with a 14-fold increase (P<0.05); in 29 out of 32 (91%) the antibody exhibited bactericidal activity. In the majority of patients, protective antibody persisted 9 months post-vaccine. The vaccine response did not depend on the age, but was lower in CRF patients who had type 2 diabetes, COPD, or heart disease, compared to the rest of the group.Most adult patients with CRF are at an increased risk of acquiring invasive Hib disease as they lack protective antibody. The pediatric Hib vaccine is highly immunogenic in this group, with higher response compared to other vaccines administered to such patients . This study provides rationale for the immunization of individuals with secondary immnunodeficiency against Hib to achieve protective immunity.


The recently updated Durie/Salmon PLUS staging system published in 2006 highlights the many advances that have been made in the imaging of multiple myeloma, a common malignancy of plasma cells. In this article, we shall focus primarily on the more sensitive and specific whole-body imaging techniques, including whole-body computed tomography, whole-body magnetic resonance imaging, and positron emission computed tomography. We shall also discuss new and emerging imaging techniques and future developments in the radiological assessment of multiple myeloma. Multiple myeloma is a neoplastic disorder of plasma B cells characterised by bone marrow infiltration and overproduction of monoclonal immunoglobulins. It accounts for approximately 10% of all haematological malignancies and 1% of all cancers with an increasing incidence, affecting four in every 100,000 per year , 3.Standard investigations for multiple myeloma includes a complete blood count, serum biochemistry, serum and urine electrophoresis, and the gold standard for diagnosis: bone marrow aspirate and biopsy. The Durie/Salmon staging system introduced in 1975 used skeletal survey as its only radiological criterion . In an eThe role of radiological imaging in multiple myeloma is essentially three fold: in the initial staging of the disease, in the detection and characterisation of complications, and in the evaluation of patient's response to treatment. In the past decade, the role of the bone marrow microenvironment has been at the forefront of multiple myeloma research. The “seed and soil” hypothesis was first introduced in the late 1800s by an English surgeon, Dr. Stephen Paget, who proposed a neoplastic growth  will proliferate in an environment  that supports its' replication . Since tThe bony destructive lesions demonstrated by myeloma imaging techniques are caused by myeloma cell-mediated promotion of osteoclast-mediated bony destruction and inhibition of osteoblast-mediated bone anabolism. Myeloma cells attach to osteoclasts directly by numerous adhesion molecules, one example being vascular cell adhesion molecule-1 (VCAM-1), with resultant stimulation of osteoclastogenesis .α), soluble frizzled-related protein-2 (SFRP-2), and Activin A. DKK-1 and SFRP-2 act by inhibiting the Wnt pathway, a pathway that plays a significant role in osteoblastic maturation . Du. Du18]. Whole-body MR (WBMR) has emerged as the most sensitive imaging modality to date at detecting diffuse and focal multiple myeloma in the spine, as well as the extra-axial skeleton –21. Due The type of MR sequence applied greatly affects the MR images. Multiple sequences have been proposed for use in identifying focal or diffuse disease of the bone marrow. These include spin-echo (T1-weighted and T2-weighted), gradient-echo (T2*-weighted), STIR (short time inversion recovery), and contrast-enhanced spin-echo (with and without fat suppression) sequences . The terTypical myeloma lesions are marrow based and have low signal intensity on T1-weighted images , and a hWhole-body STIR imaging has gained wider acceptance in detecting occult malignant disease in the skeleton , 25. WitDedicated focal MR imaging of the skull, spine, pelvis, or of an extremity is also still a widely utilized technique. MR imaging is routinely required to evaluate the extent and characteristics of a solitary lytic lesion identified at plain radiography . There i99mTc) labelled diphosphonates relies on the osteoblastic response and activity of the skeletal system for uptake. Multiple myeloma, however, is primarily an osteolytic neoplasm. Lesions that are well defined on isotope bone scans are the result of complications of multiple myeloma, namely, osteoblastic response to a compression fracture of a vertebral body or pelvic insufficiency fracture. Bone scintigraphy may be helpful in evaluating areas not well demonstrated in plain radiography, such as ribs and sternum [Bone scintigraphy is of limited use in multiple myeloma. Detection of bone involvement using technetium 99-m ( sternum . In a re sternum .18flouro-deoxy-glucose (FDG) injected into the patient, followed by tomographic scanning approximately 10–40 minutes later. Tumour cells can be imaged with this technique due to their high metabolic rate and the resulting high glucose demand, allowing tumour cells to be distinguished from normal cells. Currently, PET/CT is being evaluated in patients with multiple myeloma and may detect early bone marrow involvement in patients with apparently solitary plasmacytoma [Positron emission computed, tomography (PET/CT) is a tomographic nuclear imaging technique that uses a labelled radiopharmaceutical such as macytoma , to assemacytoma .The main limitation of PET imaging (without the CT component) is limited spatial resolution, which may result in limitations in detecting subcentimetre lytic lesions seen on plain radiography. The advent of fusion scanning combining both the PET component and CT component addresses this issue. In PET/CT fusion scanning, the patient receives an injection of FDG about 1 hour before image acquisition. An initial topogram is acquired to define the range of image acquisition. A spiral CT is then performed. The actual scanning time is shorter for PET/CT (approximately 30 minutes) than for a PET scan alone (approximately 1 hour) as CT data is used to perform attenuation correction .One of the most significant advantages of PET/CT imaging is its ability to distinguish between active myeloma (FDG positive) and monoclonal gammopathy of undetermined significance (MGUS) or smouldering disease. MGUS is usually negative on PET/CT with neither diffuse marrow uptake nor focal disease in marrow sites . Active How does PET/CT compare with other imaging modalities? In a series of 43 patients with multiple myeloma and solitary plasmacytoma, Shirrmeister et al.  reportedFalse-positive PET/CT scans may arise from inflammatory changes due to active infection, chemotherapy within the preceding 4 weeks, or radiation therapy within the preceding 2-3 months .99Technitium sestamibi (MIBI) imaging is an alternative nuclear imaging modality that uses Tc-99m-2-methoxy-isobutyl-isonitrile as a tumour-seeking tracer to identify areas of active disease in a variety of tumours including plasma cell dyscrasias. Using MIBI imaging, it is possible to scan both skeletal lesions and soft tissue lesions and the overall sensitivity is approximately 92% and specificity is 96% [99Tc-MIBI is superior when compared with PET/CT for the visualisation of diffuse disease and despite its limited capacity in detecting focal lesions, this technique may be an alternative option when PET/CT is not available. In comparison to MRI, it has been shown that 99Tc-MIBI underestimates the extent of bone marrow infiltration in the spine, especially in patients with low disease stage [99Tc-MIBI may be useful in the detection of indolent disease, as very low-level myeloma is not detectable on PET/CT [99Tc-MIBI scanning is the enhanced uptake of 99Tc-MIBI by drug-resistant myeloma cells versus enhanced uptake of FDG by metabolically active myeloma cells [y is 96% . 99Tc-MIse stage . 99Tc-MIn PET/CT , 34. An Extramedullary multiple myeloma is uncommon. In one review of 432 patients with multiple myeloma, only 19 (4.4%) were identified as having extramedullary multiple myeloma . MultiplThe choice of imaging modality for assessing response to treatment or disease progression generally depends on the findings from the initial workup and the patients' specific treatment regime. Although new or enlarging lesions generally signify disease progression, lytic bone lesions rarely show evidence of healing on plain radiographs, and routine follow-up skeletal survey is of questionable benefit and not routinely indicated in monitoring disease progression or response to treatment. New vertebral compression fractures on plain radiography do not always signify disease progression and may occur even after effective treatment, due to the resolution of the tumour mass that was supporting the bony cortex . CT in the followup of treated disease may demonstrate the resolution of extramedullary disease, and the reappearance of a continuous cortical outline. Fatty marrow content may be seen in treated lytic disease in treated cases . As mentAlthough very accurate, MRI is not ideal for serial monitoring, as it takes 9–12 months for lesions evident on MRI to resolve and be clearly indicative of response . There aDiffuse or focal marrow changes may occur following treatment with synthetic growth factors including GCSF and erythropoietin that may simulate active disease on MR or PET/CT imaging . ArtificPET/CT imaging has been shown to be useful in evaluating response to therapy, particularly when other imaging techniques, such as MRI or CT have remained abnormal or inconclusive. PET/CT may identify new sites of disease as well as unsuspected extramedullary spread. Patients appear to have a particularly poor prognosis if abnormal FDG uptake is present following high-dose therapy or stem cell transplantation . In a reDespite the plethora of reports on the use of MIBI in the assessment of myeloma patients, the main limiting factor remains its limited sensitivity and specificity, the relatively low numbers of patients included in the reports, and the heterogeneity of clinical situations described .In the late 1990s in the USA, triggered by faster image acquisition, several groups of researchers revisited the earlier work of Dermadian's and Lauturbur's described techniques allowing rapid whole-body imaging using MR imaging. A relatively new whole body coil system is now available with 76 coil elements and 32 high-frequency channels on a 1.5-tesla system. This new system allows for coronal STIR imaging with a field-of-view of 205 cm within 14 minutes with high spatial resolution. The first whole-body MR study was performed by Eustace et al. in 1997 . Whole-b18F-FLT) which is taken up into cells in relation to the rate of DNA synthesis are being studied [New PET radiopharmaceutical tracers such as 3-flouro-3-deoxy-L-thymidine ( studied . They ma99Technitium-2-methoxy-isobutyl-isonitrile open up new avenues in assessing not only morphological disease activity, but also functional disease activity which may be of use in assessing response to treatment as well as tailoring treatment modalities to individual patients.It is clear that no one radiological technique in isolation is perfect in accurately staging and monitoring patients with multiple myeloma, and that there are nuances and pitfalls associated with even the most advanced techniques. Whole-body MR, CT, and PET/CT can provide valuable complimentary information when used in the correct setting. As the availability of these techniques increase, so too will their use. This is becoming increasingly important as clinicians strive to best assess the appropriateness and effectiveness of new and changing treatment regimes. Newer and more sensitive imaging techniques, including CT and MR whole-body imaging and functional imaging modalities including PET/CT and


A review of planning studies was undertaken to evaluate estimated risks of radiation induced second primary cancers (RISPC) associated with different prostate radiotherapy techniques for localised prostate cancer. A total of 83 publications were identified which employed a variety of methods to estimate RISPC risk. Of these, the 16 planning studies which specifically addressed absolute or relative second cancer risk using dose–response models were selected for inclusion within this review. There are uncertainties and limitations related to all the different methods for estimating RISPC risk. Whether or not dose models include the effects of the primary radiation beam, as well as out-of-field regions, influences estimated risks. Regarding the impact of IMRT compared to 3D-CRT, at equivalent energies, several studies suggest an increase in risk related to increased leakage contributing to out-of-field RISPC risk, although in absolute terms this increase in risk may be very small. IMRT also results in increased low dose normal tissue irradiation, but the extent to which this has been estimated to contribute to RISPC risk is variable, and may also be very small. IMRT is often delivered using 6MV photons while conventional radiotherapy often requires higher energies to achieve adequate tissue penetration, and so comparisons between IMRT and older techniques should not be restricted to equivalent energies. Proton and brachytherapy planning studies suggest very low RISPC risks associated with these techniques. Until there is sufficient clinical evidence regarding RISPC risks associated with modern irradiation techniques, the data produced from planning studies is relevant when considering which patients to irradiate, and which technique to employ. Prostate cancer (PCa) is the most common cancer in men in Europe and accounts for over one fifth of male cancer diagnoses . RadiothSome clinical data suggests that irradiated PCa patients may be at increased risk of RISPC, although the majority of clinical evidence concerns older EBRT techniques -12. In tRadiation to normal tissues consists of primary radiation, the direct result of the treatment beams, as well as secondary radiation, which largely affects out-of-field tissues.In photon treatments, secondary radiation results from scatter from within the patient and from the collimator, as well as leakage from the treatment machine -25. ClosFor proton treatments, secondary radiation consists of secondary photons and neutrons produced in the patient and treatment head, and which indirectly contribute to out-of-field dose . The rel-1) may also be applied when estimating out-of-field RISPC risk in these settings [In low dose out-of-field regions, radiation protection models are appropriate for estimating RISPC risk. A risk co-efficient, which reflects the likelihood of developing a second cancer in a specific organ, is applied to the equivalent dose received by that organ. The risk co-efficient is stated in per cent per Sievert and therefore, as dose increases, the risk of second malignancy increases in a linear fashion. The linear relationship is based on atomic bomb survivors and on the understanding that cells exposed to lower radiation doses are damaged, but not killed (or sterilised), by radiation, and so maintain the potential for malignant transformation . Risk cosettings ,30-35.In higher dose regions the relationship between dose and risk of second cancer is less certain and a number of dose–response models are proposed. Models consider the balance between radiation induced cell damage, which leaves cells with the potential for malignant transformation, and cell sterilisation which renders cells incapable of transformation. The most commonly adopted models include the linear-no-threshold model (LNT), the linear-plateau (LP) model and the linear-exponential (LE) model. All three display a linear dose–response relationship for about the initial 4Gy of fractionated radiotherapy ,36. At hA further model is the competitive risk model which also encompasses the effects of mutation induction at lower doses, and cell killing at higher doses. The relationship is approximately linear at lower doses, but then begins to fall at higher doses, according to a linear-quadratic function . This moThe concept of organ equivalent dose (OED) states that any dose distribution in an organ that results in the same RISPC incidence as an alternative dose distribution, has the same OED. This concept has employed LE, LP and LNT models. Estimations of RISPC incorporate age at irradiation, attained age, gender, and fractionation. The model considers radiation from the primary beam and out-of field components, which is considered a positive and more realistic move away from models which only consider out-of-field doses ,40. OED Planning studies were identified from literature review of Medline from 1946), EMBASE (from 1947) and CENTRAL (from 1974) databases. Search terms related to RISPC, radiotherapy and PCa. The precise search terms from the Medline search are provided as Additional file 946, EMBAOf the 16 studies, five evaluated out-of-field risk alone, without consideration of the impact of the primary beam. This approach has been criticised ,41 as byFactors of importance when considering the impact of IMRT on RISPC risk compared to 3D-CRT or conventional radiotherapy include the consequence of a change in dose distribution and the increase in monitor units (MU) required to deliver treatment Figure . The difWith two exceptions ,42, the Studies comparing RISPC risk at equivalent energies have consistently shown an increase in risk with IMRT. This has largely been attributed to the increase in leakage as a result of increased MU requirements ,45. In aRuben et al, as above, estimated that risks were similar between 18MV 3D-CRT and 6MV IMRT based on LP and LE models . The groStudies estimating RISPC risk following proton treatments have consistently shown a reduction in risk compared with 3D-CRT and IMRT, regardless of whether spot or passive scanning techniques are used Table . The redIt should be noted, however, that although RISPC risk from protons was lower compared to IMRT using both spot and passive scanning techniques, passive scanning techniques result in much greater neutron production and so any reduction in RISPC risk might be less with spot than passive scanning techniques. Of the studies reviewed here, none have directly compared RISPC risk from spot and passive scanning techniques. In addition, spot scanning has only been compared to 3D-CRT, while passive scanning has only been compared to IMRT. Accepting the limitations in the comparison, however, the reductions in RISPC risk using spot scanning compared to 3D-CRT are in the region of 40 to 50%, while when comparing passive scanning to IMRT , smaller RISPC risk reductions, in the region of 25 to 40%, are observed ,36,48,49Followill et al estimated out-of-field RISPC risk from tomotherapy. This study examined pelvic radiotherapy for rectal and gynaecological primary tumours, but remains relevant for PCa patients receiving pelvic treatments . Risk ap-4% ± 3x10-4 and 1.3x10-8% ± 7x10-8respectively, and for HDR monotherapy were 1.0x10-4% ±1x10-4 and 2.3x10-8% ±7x10-8. For EBRT-BT, rectal cancer risk was estimated at 0.06%. Overall, the lowest RISPC risks were associated with HDR or LDR BT monotherapy, and were attributed to the high (cell sterilising) equivalent doses received by small regions of neighbouring organs [RISPC risk following BT was estimated by Takam et al . The grog organs . Unfortug organs . In addiAlvarez Moret et al examined RISPC risk from quasiIMAT (intensity modulated arc therapy), a pseudo-rotational techniques employing 36 equally spaced step and shoot beams to simulate an arc . EstimatMore recently, albeit out with the time-frame of our search, but included as the only study to examine second cancer risks associated with actual arc treatements, is the work by Rechner et al . This grAs above, studies considering out-of-field doses alone, and thus neglecting the impact of the primary beam, have been criticised ,41. OmitOther factors which have been examined with regard to RISPC risk include dose escalation, hypofractionation, CTV-PTV margin width, collimator angles and photon energies -51,58,61All studies acknowledge that there are uncertainties and limitations in estimating RISPC risk. As such, absolute values for risk are perhaps less useful than comparisons between values obtained using the same method. Dose–response models which encompass the effects of the primary dose as well as out-of-field doses are considered more realistic than models only dealing with out-of-field risks which may over-estimate or under-estimate RISPC risks depending on the dose–response model employed and the actual doses received by tissues ,59. For Despite uncertainties, in general, the absolute risk of RISPC from IMRT appears small, particularly when estimated with dose models encompassing primary and out-of-field doses ,48,62. AStudies involving proton treatments have consistently shown reduced RISPC risks compared to 3D-CRT and IMRT, largely because a reduction in exit doses results in a reduction in the volume of normal tissues irradiated, thus resulting in improved conformity ,36,48,49The ALLEGRO project  is a collaborative project involving 13 European organisations which aims to clarify some of the uncertainties surrounding modelling and measurement of radiation doses . The proIn summary, multiple factors are involved when estimating RISPC risk, and there are uncertainties in all estimations. At present follow-up from clinical studies is too short, or patient numbers too small, to determine if the estimated changes in risk from more modern irradiation techniques translate into clinically significant changes in second primary cancer incidence in practice. Until then, the warnings produced from planning studies must be borne in mind when considering which patients to irradiate, and which technique to employ.3D-CRT: Three Dimensional Conformal Radiotherapy; BEIR: Biologic Effects of Ionizing Radiations; CI: Confidence interval; DDREF: Dose And Dose Rate Effectiveness Factor; IMAT: Intensity Modulated Arc Therapy; IMRT: Intensity Modulated Radiotherapy; LE: Linear Exponential; LNT: Linear No Threshold; LP: Linear Plateau; MU: Monitor Units; MV: Megavolts; NRCP: National Council on Radiation Protection and Measurements; OED: Organ Equivalent Dose; PCa: Prostate Cancer; PA: Passive Scattering; RISPC: Radiation Induced Second Primary Cancer; RR: Risk ratio; SS: Spot Scanning; TPS: Treatment Planning System; VMAT: Volumetric Modulated Arc Therapy.The authors have no competing interests to declare.AH, PH, FAS and JV conceived and designed the study. LM undertook the review. All authors participated in the drafting and revising of the manuscript.Search Strategy.Click here for file