Screening and monitoring in pregnancy are strategies used by healthcare providers to identify high-risk pregnancies so that they can provide more targeted and appropriate treatment and follow-up care, and to monitor fetal well-being in both low- and high-risk pregnancies. The use of many of these techniques is controversial and their ability to detect fetal compromise often unknown. Theoretically, appropriate management of maternal and fetal risk factors and complications that are detected in pregnancy and labour could prevent a large proportion of the world's 3.2 million estimated annual stillbirths, as well as minimise maternal and neonatal morbidity and mortality.The fourth in a series of papers assessing the evidence base for prevention of stillbirths, this paper reviews available published evidence for the impact of 14 screening and monitoring interventions in pregnancy on stillbirth, including identification and management of high-risk pregnancies, advanced monitoring techniques, and monitoring of labour. Using broad and specific strategies to search PubMed and the Cochrane Library, we identified 221 relevant reviews and studies testing screening and monitoring interventions during the antenatal and intrapartum periods and reporting stillbirth or perinatal mortality as an outcome.We found a dearth of rigorous evidence of direct impact of any of these screening procedures and interventions on stillbirth incidence. Observational studies testing some interventions, including fetal movement monitoring and Doppler monitoring, showed some evidence of impact on stillbirths in selected high-risk populations, but require larger rigourous trials to confirm impact. Other interventions, such as amniotic fluid assessment for oligohydramnios, appear predictive of stillbirth risk, but studies are lacking which assess the impact on perinatal mortality of subsequent intervention based on test findings. Few rigorous studies of cardiotocography have reported stillbirth outcomes, but steep declines in stillbirth rates have been observed in high-income settings such as the U.S., where cardiotocography is used in conjunction with Caesarean section for fetal distress.There are numerous research gaps and large, adequately controlled trials are still needed for most of the interventions we considered. The impact of monitoring interventions on stillbirth relies on use of effective and timely intervention should problems be detected. Numerous studies indicated that positive tests were associated with increased perinatal mortality, but while some tests had good sensitivity in detecting distress, false-positive rates were high for most tests, and questions remain about optimal timing, frequency, and implications of testing. Few studies included assessments of impact of subsequent intervention needed before recommending particular monitoring strategies as a means to decrease stillbirth incidence. In high-income countries such as the US, observational evidence suggests that widespread use of cardiotocography with Caesarean section for fetal distress has led to significant declines in stillbirth rates. Efforts to increase availability of Caesarean section in low-/middle-income countries should be coupled with intrapartum monitoring technologies where resources and provider skills permit. Although most pregnancies progress normally, some are more complex because of antenatal or intrapartum conditions that place the mother, the developing fetus, or both at a higher risk for complications than pregnancies without these conditions. Pre-existing chronic conditions, as well as conditions that arise during pregnancy, can threaten the life and health of the fetus or the mother. Maternal hypertension, diabetes mellitus, renal disease, and autoimmune disorders, as well as placentation abnormalities and congenital anomalies, are examples of conditions that can place the pregnancy at high risk of fetal compromise. Fetal growth restriction arising from placental insufficiency is a significant cause of perinatal mortality  and morbidity (complications of prematurity) internationally . AdditioRelatively non-invasive techniques exist to screen for a number of these conditions during the antenatal and intrapartum periods. These screening tools can also be used to monitor fetal well-being via assessment of fetal movement, heart rate, and/or growth; and feto-placental and/or uteroplacental circulatory dynamics, whether routinely at antenatal care (ANC) visits or via more complex screening tests in high-risk and post-term pregnancies . DespiteMost studies of fetal screening and monitoring to date have been conducted in high-resource settings. Theoretically, evidence-based screening and monitoring techniques that are already in widespread use in high-income countries could be promoted to prevent stillbirth and other adverse pregnancy outcomes in low-/middle-income countries. We focus here on monitoring methods during pregnancy and the intrapartum periods, including identification and care of high-risk pregnancies and advanced monitoring techniques, with attention given where relevant to the feasibility and potential impact of implementing these techniques in low-resource settings where most stillbirths occur.This is the fourth in a series of papers on the evidence for interventions that impact stillbirths. Details of the search strategy and review procedures for this paper are described in detail in Paper 1 of this series . Each stWe reviewed 14 screening and monitoring interventions for evidence of impact which are included in this paper  by Lema  found th Muylder  evaluatek charts ,26.2 = 6.8; P < 0.01). A Cochrane review by Mangesi et al [[LOE: 1++].Other observational studies, RCTs, and reviews assessed impact on perinatal mortality of interventions using kick charts. A before-after study by Moore et al  introducsi et al  included[LOE: 1+]. However, of the 17 women in the study randomised to kick charts who alerted their provider about decreased fetal movement and subsequently delivered a stillborn baby, none had an emergency delivery, as follow-up testing using cardiotocography resulted in false negatives for all 17 womenAnother Cochrane review by Neilson  reviewedA second Cochrane review by Bricker et al.  assessed[LOE: 2+]. In another RCT by Proud et al [[LOE: 2+].An additional RCT (N = 1528 women) from New Zealand by Duff et al.  documentud et al  where plWe also conducted an independent, new meta-analysis for the purposes of this review, as we identified 9 RCTs  reporting an impact on perinatal mortality rate of ultrasound in early pregnancy versus no or selective use of ultrasound in early pregnancy (before 24 weeks) Figure . We founThere is no clear evidence that ultrasound examination during pregnancy is harmful , and itsBased on the results of 2 Cochrane reviews, our meta-analysis, and other RCTs , there is no evidence that routine ultrasonography has any impact on perinatal mortality compared to the selective use of ultrasonography based on clinician judgement. It may be that routine ultrasound cannot reliably detect complications, or that high rates of false positives expose higher numbers of babies to iatrogenic intervention . The routine use of early ultrasonography in pregnancy cannot be recommended to prevent stillbirth, as there is no evidence of its benefit in preventing stillbirth or perinatal mortality. There is a need for decision analysis studies subsequent to diagnostic ultrasound, like the study of indicated Caesarean section for fetal macrosomia diagnosed by ultrasound by Rouse et al , but whiIn many high-income countries, Doppler ultrasound studies are used as a non-invasive means to assess the sufficiency of uterine and umbilical cord blood flow. These velocimetry studies can improve management of pregnancies by aiding identification of fetuses at highest risk of adverse outcomes associated with pre-eclampsia, fetal growth restriction, and congenital malformations. Management of pre-term pregnancies with signs of fetal growth restriction and pre-eclampsia is complex, especially before 32 weeks gestation. The risk of prolonged hypoxia and acidaemia leading to stillbirth or neonatal death if the pregnancy is allowed to progress must be balanced against the risks of neonatal morbidity and mortality associated with prematurity if early delivery is chosen. Doppler ultrasonographic evaluation may aid determinations of the degree to which the fetus may be or become compromised.Early in normal pregnancy, trophoblasts invade the maternal uterine spiral arteries and reduce resistance to uterine blood flow. Impeded flow measured by uterine artery Doppler suggests a failure of this trophoblastic invasion, which is associated with subsequent pre-eclampsia, fetal growth restriction, and stillbirth . UterineObservational Doppler studies of the umbilical artery, first conducted in the 1970s, consistently showed a correlation between extremely abnormal waveforms and adverse outcomes, including fetal growth restriction and stillbirth -42. In gDespite these well-established markers of fetal compromise in Doppler testing, it is not clear whether abnormal results of different modalities of Doppler ultrasound lead to improved perinatal outcomes and prevention of stillbirths, nor are the most appropriate indications and timing of testing known. Additionally, if a fetus is not seriously compromised, Doppler ultrasound may potentially cause iatrogenic harm in suggesting the need for inappropriate early delivery.The literature search identified three systematic reviews, one Cochrane protocol, and 10 other intervention/observational studies Table .[LOE: 1+]  Collaborative Group  randomis2 = 0.49, P < 0.001) and stillbirth . In fetuses with absent or reversed umbilical end flow, prediction of asphyxia and stillbirth was significantly enhanced by venous Doppler. Umbilical artery waveform analysis offered the highest sensitivity and negative predictive value, whereas ductus venosus and umbilical vein flow studies had the best specificity and positive predictive values for perinatal death [LOE: 1+].Baschat et al  performe[LOE: 3].Using personal computer-based, continuous-wave Doppler machines to assess umbilical artery flow velocity waveforms in women referred for suspected fetal growth restriction, Hugo et al.  found a [LOE: 1+].Baschat et al.  conducte[LOE: 1+]. A Cochrane protocol by Alfirevic et al indicates that a review is in progress to assess whether the use of umbilical artery, middle cerebral artery, and ductus venosus Doppler velocimetry improves subsequent obstetric care and fetal outcome  , Langer et al  observedr et al.  randomly[LOE: 2-]. A somewhat less rigorous intervention study in India (N = 240 mothers) by Banerjee et al. [[LOE: 1-].In the US, Gonzalez-Quintero et al.  conductee et al.  sought t[LOE: 1-]. In Soweto, South Africa, Huddle et al [[LOE: 3].In the US, McElvy et al.  conductele et al  reported[LOE: 3].A descriptive study by Landon et al.  of non-s[LOE: 1++] , suggests that pregnant women who do not have vascular complications and with good glycaemic control do not as a group have an increased risk of stillbirth above the general population . In settAntenatal fetal surveillance studies are widely used to assess fetal well-being and to identify the compromised fetus. Fetal heart rate patterns are a principal component of the majority of tests of fetal well-being, and cardiotocography, where it is available, is the method frequently used to electronically record the fetal heart rate. In high-income countries, beginning in the third trimester, cardiotocographic monitoring  has largely replaced intermittent auscultation  for monitoring fetal heart rate. During the antenatal period, external cardiotocography is used, which employs a Doppler ultrasound transducer; the mother's uterine contractions are also monitored using a pressure transducer, with both transducers strapped to the mother's abdomen while recording is in process.Antenatal fetal heart monitoring includes both non-stress tests (NST) and contraction stress tests (CST). Fetal heart rate patterns are classified as either reassuring (reactive), non-reassuring (non-reactive) or abnormal, considering heart rate baseline, variability, and decelerations. In high-income countries, particularly the US, the NST is widely accepted as a primary fetal surveillance tool in high-risk pregnancies. It may be used in combination with ultrasound testing to observe fetal movement and amniotic fluid indices. When an NST is non-reactive, its poor predictive value (< 50%) warrants confirmatory testing with CST or a complete BPP . During Variation between practitioners in interpretation of cardiotocographic tracings can potentially lead to inappropriate intervention, or false reassurance without appropriate intervention. Tracings are notoriously difficult to interpret, as fetal sleep patterns, labour progress, external stimuli, and opiate administration to the mother can alter fetal heart rate changes in the absence of fetal hypoxia or distress . AdditioWe identified 1 Cochrane review and 4 observational studies testing the impact of antepartum cardiotocography (including both NST and CST) on perinatal outcomes Table .[LOE: 1+]. An observational study of high-risk pregnant women (N = 561) by Rayburn et al [[LOE: 2-]. Evertson et al [Several studies described how well NST and CST results correlated with subsequent perinatal outcome. An RCT by Flynn et al  identifirn et al  assessedon et al  assessed[LOE: 1++].A review by Pattison and McCowan  included[LOE: 2+].Freeman et al  comparedThere are relatively few RCTs of antepartum fetal heart rate surveillance (Grade C evidence), though observational studies clearly indicate a correlation between non-reassuring cardiotocographic traces and adverse perinatal outcomes, including stillbirth. Although there is a Cochrane review on antepartum cardiotocography  showing When used experimentally as a primary fetal surveillance tool, CST use was associated with lower rates of antepartum stillbirth , suggestThe BPP was conceptually derived from the Apgar score that is used to rate the condition of the newborn . Using NWe identified one Cochrane review and four other observational studies of BPP reporting perinatal mortality outcomes Table . A multi[LOE: 1+].Lalor et al.  undertooCompared with conventional fetal monitoring, which is based primarily on cardiotocography/NST, BPP appears to offer no improvement in pregnancy outcomes (Grade C evidence). Although rates of false negative test results of the BPP are low, rates of false positives are high. The total number of subjects included in the meta-analysis remains small (N = 2974), and some of the studies carry a high risk of allocation bias. Although there is no consensus on optimal protocol for fetal monitoring, studies demonstrate that it is reasonable to implement the modified BPP (which consists of an NST and amniotic fluid volume measurement only), with full BPP or other tests such as the CST reserved for abnormal test results . This paAdjunctive vibroacoustic stimulation with the NST is used less often than CST or BPP after non-reassuring NST results. Vibroacoustic stimulation provokes fetal response using a sound-emitting device placed on the maternal abdomen near the fetal head, along with simultaneous cardiotocography to document fetal movements and heart traces. Theoretically, the resultant startle reflex in the fetus and subsequent fetal heart rate acceleration or transient tachycardia following vibroacoustic stimulation provide reassurance of fetal well-being and minimise unnecessary intervention for nonreactive tests .[LOE: 1+]  of pregnancies monitored by vibroacoustic stimulation compared to mock or no stimulation  , the non-significant impact on perinatal mortality does not support including this intervention presently. Further RCTs are needed to determine optimal intensity, frequency, duration and position of vibroacoustic stimulation, as well as efficacy, predictive reliability, safety  and perinatal outcome.Amniotic fluid protects and supports the fetus during pregnancy. Both low amniotic fluid (oligohydramnios) and high amniotic fluid (polyhydramnios) are abnormal and potentially place the fetus at risk of adverse outcomes. Oligohydramnios can be associated with maternal conditions including pre-eclampsia, or with placental membrane rupture, fetal growth restriction, post-term pregnancy, fetal kidney problems, or fetal or placental abnormalities ,106. PolOur literature search identified two Cochrane reviews, one other review, and 15 other interventional or observational trials of amniotic fluid assessment to identify polyhydramnios or oligohydramnios that reported perinatal outcomes Table .Polyhydramnios[LOE: 2-]. A retrospective cohort study by Dashe et al. [[LOE: 2-]. Magann et al. conducted a review of idiopathic polyhydramnios [[LOE: 2+]  were no more likely to have Caesarean section for fetal distress, neonatal acidosis, or Apgar score < 7 at 5 minutes than untested controls or women with normal AFI results; no perinatal mortality statistics were reported [LOE: 1+].Only one intervention RCT compared AFI to no amniotic fluid assessment. Comparing AFI on admission (intervention) to no AFI (controls), Chauhan et al  found thOne intervention strategy to improve perinatal outcomes is to augment amniotic fluid volume in cases of oligohydroamnios. Hofmeyr and Gülmezoglu  undertooPolyhydramnios is a clear risk factor for perinatal mortality, whether associated with congenital malformations, placental insufficiency, or of idiopathic origin. However, no studies reported the impact of interventions subsequent to a diagnosis of polyhydramnios after amniotic fluid assessment, so the impact on stillbirth of amniotic fluid screening for polyhydramnios remains unclear. Although robust RCTs are limited, very low AFI values (oligohydramnios) are frequently associated with poor pregnancy outcomes, and in these cases a reactive NST loses its usual reassuring value (Grade C evidence). Where feasible, amniotic fluid volume estimation may be helpful for identifying severe oligohydramnios, but further research is needed to document subsequent intervention and perinatal mortality outcomes to determine the cost-benefit ratio of utilizing amniotic fluid assessment procedures. Amniotic fluid assessment is complicated by high variability of sequential measurements and use of different measurement methods, which can compromise the accuracy of the test. No particular method of amniotic fluid volume assessment appears superior to another ,124, thoWomen with high-risk pregnancies, especially multiple pregnancies and pregnancies complicated by hypertensive disorders, are frequently admitted to hospital for bed rest and monitoring. However, in some instances, more limited monitoring in facilities and home-based bed rest or reduced physical activity may be as effective as hospital-based monitoring. Home-based activity modification, accompanied by outpatient surveillance and hospital admissions only for complications, would offer cost savings over hospital admission, reduced burden on hospital resources and personnel, and reduced disruption to the life of the mother posed by lengthy hospital stays.[LOE: 1++].The literature search identified two Cochrane reviews and two other interventional studies comparing home-based versus hospital-based methods of care for high-risk pregnancies Table . The fir[LOE: 1++]Another Cochrane review  evaluate[LOE: 1-]. A large RCT from China . The Cochrane protocol, for a review currently in progress, proposes to assess the impact of different specified regimens of fetal surveillance for impaired fetal growth on maternal and perinatal outcomes and length of hospital admission [[LOE: 2+], suggesting that fetal surveillance on an outpatient basis was equally effective as hospital admission in managing high-risk pregnancy. Menzies et al. [[LOE: 2-].Our literature search identified 1 Cochrane review, 1 Cochrane protocol and 2 other intervention studies Table . A Cochrdmission . The 2 idmission  conductes et al.  performeIf patients could be consistently monitored on an outpatient basis, obstetricians would be more willing to admit patients only for delivery, and to perform more fetal assessments and maternal tests as outpatient services. Outpatient fetal surveillance offers benefits in terms of cost and convenience, and could improve health facility organisation, and improved record-keeping would streamline obstetric and perinatal audit, data quality for research, and teaching opportunities . HoweverA partograph, alternatively called a partogram, is a simple pre-printed paper form on which midwives and obstetricians record labour observations. The tool provides a continuous pictorial overview of the progress of labour, while monitoring maternal and fetal well-being. The partograph distinguishes between the latent and active phases of labour. The active labour section has 2 straight lines called the alert and action lines. The alert line reflects a modification of the mean rate of cervical dilatation of the slowest 10% of primigravid women in the active phase of labour (1 cm per hour). Slower progress than this crosses the alert line on the partograph, which may prompt initiation of the process of transfer to a facility with emergency obstetric capacity in preparation for intervention for prolonged labour. Depending on the partograph version, the 'action line' is 2 to 4 hours to the right of the alert line. Labour crossing this line suggests primary inefficient uterine activity and prompts immediate appropriate management of slow progress of labour, usually via amniotomy, oxytocin infusion, or both.Some evidence suggests that midwives and physicians find the partograph practical in terms of ease of use, time resourcefulness, continuity of care and educational assistance , which mWe identified 1 Cochrane review including 5 RCTs and quasi-RCTs (N = 6963 women), and 3 intervention/observational studies Table . The CocThe Cochrane review  also comThe review also pooled the results from 3 RCTs to compare shorter  versus longer time-to-intervention  and found no differences between the groups for Caesarean section rate, Apgar score or instrumental delivery, but early intervention in the low-resource setting reduced the Caesarean section rate.A large multicentre study  comparedIn Indonesia, Fahdhy et al  comparedOverall, there were no significant differences in maternal or perinatal outcomes with the use of partograph versus no partograph, and no evidence that any particular version of the partograph is better than another in preventing perinatal mortality. Partographs may be comparatively more effective in low-resource settings, as the studies from Africa and Mexico in the Lavender review , as wellMethods to assess fetal heart rate and levels of oxygenation of fetal blood are monitoring strategies intended to identify early signs of fetal compromise as a result of oxygen shortage, or fetal hypoxia . Severe In many hospitals in high-income countries including the US and Canada, cardiotocography is widely used to monitor fetal heart rate in labour, though intermittent auscultation  is occasionally performed. In addition to external cardiotocography, which can be employed continuously or intermittently during labour, internal cardiotocography can be performed by attaching a sensor to the fetal presenting part, usually its head. This can be done only if the membranes are ruptured, as electrode must be embedded in the baby's scalp. Intermittent auscultation via stethoscope or handheld Doppler is more common than electronic fetal monitoring methods in low-resource settings where fetal heart rate monitoring is available.Currently, cardiotocography is recommended in high-risk pregnancy and labours induced or augmented with oxytocin . BecauseThere is some danger that improper interpretation of cardiotocographic tracings can lead to inappropriate intervention, or false reassurance that delays necessary intervention. There is a need for sensitive and specific methods of using cardiotocography, potentially in conjunction with pulse oximetry, to improve detection of fetal compromise due to hypoxia. Improved detection of hypoxia, primarily by expediting delivery, could improve outcomes and prevent stillbirth in these instances.[LOE: 1+]. A new Cochrane review assessing cardiotocography versus intermittent auscultation of fetal heart for assessment of fetal well-being is in progress .Another Cochrane review  4 RCTs,. In comp[LOE: 1++].A third Cochrane review  assessedAlthough there are several studies of fetal pulse oximetry and intrapartum cardiotocography, few reported stillbirth or perinatal mortality as outcomes. The available results show no statistically significant impact on stillbirths or perinatal mortality, whether cardiotocography is used alone or in conjunction with fetal pulse oximetry in labour. Continuous cardiotocography appears associated with increased rates of operative delivery and lower rates of neonatal seizures compared to no or intermittent cardiotocography, but has no demonstrated impact on rates of perinatal mortality acidosis, dystocia, or long-term physical or developmental outcomes. There is currently no evidence of benefit from randomised or quasi-randomised studies for intrapartum cardiotocography alone or in conjunction with electrocardiogram or fetal pulse oximetry in preventing stillbirth.One drawback of intrapartum cardiotocography is that its poor predictive value of true fetal distress and hypoxia is not sufficiently enhanced by any adjunctive technologies at this time. Pulse oximetry appears safe, though long-term developmental studies have not been performed, but it does not have any clear impact on stillbirths or perinatal mortality . AdjunctThere are robust studies and Cochrane reviews on fetal pulse oximetry and intrapartum cardiotocography (Grade B evidence). The non-significant impact of cardiotocography with or without fetal pulse oximetry on stillbirth/perinatal mortality in these studies suggests no evidence of benefit. In high-income countries, documented stillbirth rates, particularly stillbirths associated with intrauterine asphyxia, in high-income countries have followed increasing prevalence of cardiotocographic fetal monitoring with Caesarean section for fetal distress -157, thoAlthough a reasonable number of Cochrane reviews and RCTs were available which assessed the impact of one or more implementation strategies for many screening and monitoring interventions, none of the interventions we reviewed demonstrated convincing evidence of impact on stillbirths or perinatal mortality. A number of studies that did report statistically significant impact on stillbirth or perinatal mortality, or which suggested large magnitude differences between intervention and control/comparison groups, lacked sufficient rigor to justify recommending the routine use of the interventions evaluated.The evidence for all interventions reviewed in this paper is summarised in Table Despite the existence of many tools, devices, and techniques for monitoring pregnancy for complications, there is a dearth of rigorous evidence that any screening and monitoring intervention has a direct impact on stillbirth rates in unselected and low-risk populations. This stems from several common shortcomings in the design and interpretation of studies investigating the use of screening and monitoring techniques. First, the small size and insufficient rigor of many screening and monitoring studies to date renders them underpowered to detect significant differences in perinatal mortality subsequent to their implementation. Many studies assessing the impact of the interventions covered in this review did not report stillbirths or perinatal mortality at all. For virtually all the interventions included in this review (with the exception of x-ray pelvimetry), there is a need for RCTs sufficiently powered to detect statistically significant impact on stillbirth or perinatal mortality, if such differences exist. Second, for screening and monitoring interventions to reduce stillbirth incidence, they must effectively identify women at higher risk of stillbirth in time for an appropriate and effective intervention to be provided. Most studies we reviewed limited their analysis to the first step of successful screening or monitoring: detection of women at increased risk of stillbirth or perinatal death. A number of studies showed that positive test results corresponded fairly well with increased risk of adverse pregnancy outcome. However, a trial of an otherwise effective screening method will show no impact on stillbirths if the subsequent intervention fails to prevent stillbirth, either because intervention is ineffective or provided too late. In most of the studies we reviewed, few women who screened positive were treated according to an established protocol. This flaw precludes assessment of whether the lack of impact of any given screening technique is attributable to a failed screening method or a failed intervention. This fact highlights the need for time-to-decision and time-to-intervention studies, as well as analyses that consider which interventions are employed, and protocols for employing them, subsequent to adverse findings of screening and monitoring studies. The quality, appropriateness, and timeliness of the intervention care provided must also be considered along with the effectiveness of screening or monitoring in detecting true complications.In addition to these tests of diagnostic accuracy and intervention effectiveness, other types of studies are also needed, including innovative pilot studies, safety studies, and effectiveness trials, to bolster the weak evidence base for screening and monitoring interventions during labour Table . In highAdditional questions surround which screening and monitoring interventions could be most effective in low-/middle-income country settings where 98% or more of the global burden of stillbirths occurs . ScreeniPracticality, logistical feasibility, and cost-effectiveness are chief concerns in many low-resource areas. Where ANC services are available and widely utilised, the prospects for identifying and monitoring high-risk pregnancy improve. Along with interventions that improve detection of risk factors and complications, improved management and referral of women with complications are critical to manage obstetric risk in low-/middle-income countries. Unfortunately, in areas where ANC attendance is poor, few of these screening and monitoring techniques are likely to be implementable.A few monitoring approaches showed promise for use in high-risk pregnancies, including fetal movement monitoring and umbilical Doppler velocimetry. Before recommending these interventions for widespread use, further large RCTs of sufficient rigour to detect differences in stillbirth and perinatal outcomes are needed to determine the utility and effectiveness of these monitoring approaches and interventions used after positive tests.This review identified several interventions – for example, amniotic fluid measurement for signs of oligohydramnios, especially as pregnancy approaches or exceeds term; routine ultrasound scanning, and intrapartum cardiotocography – that are in widespread use in high-resource settings, but for which rigorous evidence is lacking. For these interventions, their continued use is reasonable even though the evidence base for impact on stillbirths is lacking. Intrapartum cardiotocography in particular has been shown in RCTs to elevate the risk of operative delivery. Prevailing wisdom credits cardiotocography and available Caesarean section with the diminution of stillbirth rates in high-income countries in recent decades; however, the role of cardiotocography in reducing stillbirth should be confirmed with more rigorous evidence. Efforts to achieve glycaemic control in cases of maternal diabetes mellitus are also encouraged, despite the lack of strong evidence showing an impact of such interventions on stillbirth, on the grounds that women with good glycaemic control experience fewer complications and negative outcomes of pregnancy.Particularly in pregnancies deemed high-risk based on screening test results, clinical progression of pregnancy, or reproductive history, multiple antepartum testing modalities can minimise deficiencies in sensitivity and predictive value when used in combination to assess fetal well-being and abnormalities. The BPP reflects one standardised strategy by which tests of fetal well-being have been packaged to permit a more comprehensive assessment of fetal well-being. In cases where one test raises the possibility that a pregnancy is high-risk, multiple tests of fetal well-being should be considered if time allows, along with the patient's overall clinical history, to determine the appropriateness of intervention. Multiple tests are recommended in many protocols for identifying and making clinical decisions about management of pregnancies with suspected fetal growth restriction . In thesRecommendations for clinical decision-making protocols and introduction of interventions in low-/middle-income countries are more difficult. Resource constraints preclude the implementation of many of the interventions reviewed in this paper. The interventions requiring the lowest investment in resources and training – fetal movement monitoring and the partograph – show some evidence of benefit in high-risk pregnancies, but more studies are needed. Where safe Caesarean section is available, ensuring that intermittent or continuous intrapartum fetal heart rate monitoring is performed may improve targeting of Caesarean section while improving birth outcomes.Screening and monitoring interventions cannot be effective without responsive, quality care and careful clinical management, particularly for high-risk pregnancies. Health systems quality improvement activities, including boosting provider skill in interpreting test results and determining appropriate intervention, timely response including swift referral, and evaluative strategies to document outcomes after intervention are essential complementary activities to ensure that an effective screening or monitoring technique translates to measurable improvements in perinatal mortality outcomes.Screening pregnancies to identify risk factors, complications, or indications of fetal distress and providing appropriate surveillance for identified risk factors theoretically encourages appropriate use of interventions, including induction of labour, Caesarean section, and pharmacological treatment, to prevent fetal loss and adverse maternal or neonatal outcomes. Screening and monitoring are futile, however, in the absence of effective interventions to act promptly and appropriately to manage identified risk factors and complications. The weak evidence in this review for impact of screening and monitoring interventions on stillbirth incidence is due at least in part to limitations in study designs that result in uninterpretable data. Screening tests may be ineffective, inappropriate, or occur too late to prevent adverse outcomes, but more problematically, most studies did not report the impact of intervention (particularly standardised intervention) following positive screening test results. Effective interventions exist to prevent stillbirth associated with many maternal infections or conditions  and placental dysfunction . Because screening and monitoring techniques during pregnancy also pose the risk of inappropriate or unnecessary use of drugs, induction of labour, iatrogenic preterm birth, or Caesarean section, it is practically, ethically, and economically important to validate the sensitivity, specificity, and positive predictive value of available screening and monitoring techniques to protect women and their babies from iatrogenic harm; and to ensure the wise use of scarce medical resources in low- and middle-income settings.AFI: amniotic fluid index; ANC: antenatal care; BPP: biophysical profile; CI: confidence interval; CST: contraction stress test; CTG: cardiotocography; ECG: electrocardiogram; FHR: fetal heart rate; HR: hazard ratio; IU: international units; LOE: level of evidence; NS: non-significant; NST: non-stress test; OR: odds ratio; PMR: perinatal mortality rate; PPV: positive predictive value; PROM: premature rupture of membranes; PPROM: pre-term premature rupture of membranes; RCT: randomised clinical/controlled trial; RD: risk difference; RI: resistance index; RR: relative risk/risk ratio; SB: stillbirth; SBR: stillbirth rate; SD: standard deviation; SGA: small for gestational age; TBA: traditional birth attendant; UK: United Kingdom; USA: United States of America; WHO: World Health Organisation; WMD: weighted mean difference.The authors declare that they have no competing interests.The paper was written and reviewed by all the authors.Web Table 1. Component studies in Mangesi et al. 2007 meta-analysis: Impact of fetal movement monitoring. Component studies in Mangesi et al. 2007 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 2. Component studies in Neilson 1998 meta-analysis: Impact of routine ultrasound in pregnancy. Component studies in Neilson 1998 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 3. Component studies in Bricker et al. 2008 meta-analysis: Impact of ultrasound in late pregnancy. Component studies in Bricker et al. 2008 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 4. Component studies in Papageorghiou et al. 2002: Impact of second trimester uterine artery Doppler on perinatal mortality. Component studies in Papageorghiou et al. 2002 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 5. Component studies in Baschat 2004: impact of Doppler velocimetry on stillbirth and perinatal mortality in pre-term growth-restricted fetuses. Component studies in Baschat 2004 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 6. Component studies in Neilson et al. 2000 meta-analysis: Impact of Doppler screening during pregnancy. Component studies in Neilson et al. 2000 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 7. Component studies in Pattinson et al. 1997 meta-analysis: Impact of pelvimetry during pregnancy. Component studies in Pattinson et al. 1997 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 8. Component studies in Russell et al. 2007 meta-analysis: Impact of management of gestational diabetes on stillbirth and perinatal outcomes. Component studies in Russell et al. 2007 showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 9. Component studies in Mukhopadhyay et al. 2007: Impact of continuous subcutaneous insulin infusion on intrauterine fetal death. Component studies in Mukhopadhyay et al. 2007 showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 10. Component studies in Boulvain et al. 2001 meta-analysis: Impact of elective delivery on perinatal mortality. Component studies in Boulvain et al. 2001 showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 11. Component studies in Pattison and McCowan 1999 meta-analysis: Impact of cardiotocography for antepartum fetal assessment on perinatal mortality. Component studies in Pattison and McCowan 1999 showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 12. Component studies in Lalor et al. 2008 meta-analysis: Impact of fetal biophysical profile. Component studies in Lalor et al. 2008 showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 13. Component studies in Tan et al. 2001 meta-analysis: Impact of vibroacoustic stimulation. Component studies in Tan et al. 2001 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 14. Component studies in Magann et al 2007 review: impact of idiopathic polyhydramnios on stillbirth and perinatal mortality. Component studies in Magann et al. 2007 review showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 15. Component studies in Nabhan and Abdelmoula 2008 [meta-analysis: comparison of single deepest vertical pocket vs. AFI in predicting perinatal outcome. Component studies in Nabhan and Abdelmoula 2008 review showing impact on stillbirths/perinatal mortalityula 2008 meta-analClick here for fileWeb Table 16. Component studies in Hofmeyr and Gulmezoglu 2002 [meta-analysis: impact of maternal hydration on amniotic fluid volume and perinatal outcomes. Component studies in Hofmeyr and Gulmezoglu 2002 review showing impact on stillbirths/perinatal mortalityglu 2002 meta-analClick here for fileWeb Table 17. Component studies in Crowther 2001 meta-analysis: Impact of hospitalisation for bed rest for women with a multiple pregnancy on stillbirth and perinatal mortality. Component studies in Crowther 2001 review showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 18. Component studies in Meher et al. 2005: Impact of some rest in hospital vs. routine activity at home on stillbirth and perinatal mortality. Component studies in Meher et al. 2005 review showing impact on stillbirths/perinatal mortalityClick here for fileComponent studies in Kroner et al. 2001: Impact of antenatal day care units on perinatal mortality. Component studies in Kroner et al. 2001 review showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 20. Component studies in Lavender et al. 2008 meta-analysis: Impact of different designs of partogram on neonatal morbidity or perinatal mortality. Component studies in Lavender et al. 2008 review showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 21. Component studies in Alfirevic and Devane 2006 meta-analysis: Impact of continuous cardiotocography on stillbirth and perinatal mortality. Component studies in Lavender et al. 2008 review showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 22. Component studies in Neilson 2006 meta-analysis: Impact of fetal electrocardiogram (ECG) on perinatal mortality. Component studies in Neilson meta-analysis showing impact on stillbirths/perinatal mortalityClick here for fileWeb Table 23. Component studies in East et al. 2007 meta-analysis: Impact of fetal pulse oximetry on fetal/neonatal death. Component studies in East et al. 2007 meta-analysis showing impact on stillbirths/perinatal mortalityClick here for file


We suggest that sustained arousal can originate from different precipitating factors  interacting with predisposing factors  and learned expectancies . Furthermore, sustained arousal may explain documented alterations by establishing vicious circles within immunology  vs Th1 (cellular) predominance), endocrinology (attenuated HPA axis), skeletal muscle function  and cognition (impaired memory and information processing). Finally, we propose a causal link between sustained arousal and the experience of fatigue. The model of sustained arousal embraces all main findings concerning CFS disease mechanisms within one theoretical framework.We present an integrative model of disease mechanisms in the Chronic Fatigue Syndrome (CFS), unifying empirical findings from different research traditions. Based upon the Cognitive activation theory of stress (CATS), we argue that new data on cardiovascular and thermoregulatory regulation indicate a state of permanent arousal responses –  Chronic Fatigue Syndrome (CFS) is characterized by unexplained and disabling fatigue, accompanied by symptoms such as musculoskeletal pain, impaired memory and concentration, headache and sleep problems [Coxiella burnetii [problems . Researcproblems . Recent problems . Personaproblems , as do iproblems . In manyburnetii ,7. In adburnetii ,9.Regarding perpetuating and associated factors, hemodynamic disturbances characterized by increased sympathetic and attenuated parasympathetic cardiovascular neurotransmission have been documented ,11. Immusustained arousal.A coherent and integrative model of CFS disease mechanisms combining these findings is lacking. In this article, we propose such a model, based upon the Cognitive activation theory of stress (CATS) . SpecifiBelow, we first present CATS – the stress theory upon which our model is based. Then we outline empirical indications of an association between sustained arousal and CFS. From this point of departure, we apply CATS to hypothesize the potential mechanisms leading to sustained arousal in CFS, and finally we substantiate our model by discussing sustained arousal consequences in other organ systems in relation to CFS research evidence.Among various definitions of the term "stress", a common denominator is that stress denotes any condition being a threat to homeostasis in a broad sense ,21. Strearousal response, involving the somatic and autonomic nervous system as well as several endocrine axes [Stress normally elicits a quite non-specific ine axes   [stimulus expectancies), and instrumental or operant conditioning as the acquisition of expectancies of the results of available responses (response outcome expectancies). Arousal response intensity increases if the stimulus expectancy has high affective value or if the response outcome expectancy is inadequate. Furthermore, experimental studies have demonstrated that arousal response can be modified by sensitization, the enhanced response to repeated stimulation [Although quite uniform in gross, the details and dynamics of the arousal response vary among individuals. Genetic variability has some impact , but cogs (CATS) . The thes (CATS) ,26, whermulation . This phmulation , and is mulation ,30. Thusmulation , irritabmulation , chemicasustained arousal – may contribute to disease [The arousal response is primarily adaptive and health-promoting. However, alterations of the response dynamics – in particular a state of maintenance, in CATS denoted  disease ,21.The first author investigated cardiovascular and thermoregulatory homeostasis in adolescent CFS patients and healthy controls. During supine rest, CFS patients had increased sympathetic nerve activity to the heart, the skeletal muscle arterioles and the adrenals; the latter causing increased plasma levels of epinephrine. There was also evidence of increased arterial blood pressure and body temperature -35. DuriSimilar findings have previously been reported in other studies ,11, and According to stress theory, set-point changes of homeostatic control circuits are hallmarks of the arousal response, as is increased level of epinephrine , demonstThe mechanism leading to sustained arousal in CFS might be hypothesized from stress theory Figure . InfectiHowever, this arousal response might be insufficient in solving the initial problem. An attempt of compensation would be to generate a stronger one. As there is no apparent solution to the individual, such attempts might be perceived as inadequate, resulting in negative stimulus and response outcome expectancy. Thus, a vicious circle is established, as the evaluation of the arousal response depends upon expectancies: negative expectations reinforce the arousal response . This inTherefore, when certain prerequisites are met, the arousal response might be strongly and paradoxically reinforced. This, in turn, may counteract homeostasis rather than restoring it, resulting in another vicious circle. Similar phenomena are demonstrated in hyperventilation, where an unpleasant experience triggers a mutual amplifying cascade of arousal response and unstable respiratory homeostasis, resulting in grossly abnormal blood gas levels . A paralWhen the initial triggering factor subsides, classical conditioning may lead to associations between the arousal response and common neutral stimuli , like moWe suggest that these mechanisms altogether can elicit a state of sustained arousal.Sustained arousal might have detrimental effects, as evident from stress research experiments Figure . Such coA broad range of distressing events, including psychosocial challenges, have an impact on the immune system, such as attenuated cellular immunity and a tendency towards reactivation of latent virus infections . Some ofAn arousal response has profound endocrine consequences, mainly influencing plasma levels of adrenal hormones . WhereasArousal responses also include behavioral changes, which seem to be mediated – at least in part – by catecholaminergic neurotransmission to brain motor areas . AccordiProlonged arousal has a negative influence on memory and information processing . SimilarYet the question remains to explain CFS patients' experience of overwhelming fatigue as an effect of sustained arousal. The processes underlying physiologic as well as pathological fatigue are still largely unknown. However, there are several indications of a potential association between fatigue and sustained arousal. Painful and inescapable stimuli increase the serotonin (5-HT) neurotransmission in different brain stem and limbic areas in rats, such as the raphe nucleus  and the In addition, more indirect relation involving the immune system might be important. As outlined above, sustained arousal can explain increased production of IL-6 and other Th2 cytokines, which in turn might promote the experience of fatigue through a direct action on the brain stem ,68. FinaThe model we have presented corresponds with other, recently presented models of CFS disease mechanisms. Altered sympathetic nervous activity at rest and during different challenges has been regarded a key feature by some researchers, but not interpreted within the frame of stress theory ,70. Althall main findings concerning CFS disease mechanisms within a unifying theoretical framework. Thus, it represents a synthesis where several important features are included. First, complying with recent cognitive behavioral and self-regulation models [The CFS sustained arousal model holds the capacity to explain n models ,72, the n models .In principle, the arousal response is general and quite non-specific. However, genetic factors, early experiences, personality traits, attributions, and beliefs may all contribute to the specificity of complaints developed by each individual . LikewisAlthough the assumptions of the sustained arousal model cannot be proved per se, it allows for deduction of testable hypotheses. Below, we suggest a selected sample:Certain polymorphisms of genes whose products are involved in arousal responses are more common among CFS patients than healthy controls. This could explain the hereditary predisposition for CFS. A search for polymorphisms should be governed by updated knowledge of the genetic basis for normal arousal responses.• Infections associated with CFS elicit a more comprehensive and long-lasting arousal response than other infections of similar clinical presentation. This is to be expected if certain infections are more prone to establish a conditioned arousal response. For instance, patients with verified acute EBV infection might be compared with patients suffering from acute viral pharyngitis of other origin. Autonomic and endocrine reactions to different experimental challenges could be used as markers of an arousal response.• When exposed to physical or mental challenges, perfusion, metabolism and transmitter activity in brain areas responsible for arousal responses differs among CFS patients and controls. Of particular interest are the serotoninergic pathways in the brain stem and limbic structures. Modern neuroimaging technology makes such studies feasible.• The characteristics of autonomic reflexes differ among CFS patients and controls during rest and challenges due to set-point alterations. Exploring further the characteristics of the homeostatic control circuits in CFS patients might add to the evidence supporting a sustained arousal model. A possible analytic tool would be the mathematical technique of transfer function analyses.• Cognitive behavioral therapy (CBT) specifically designed to abolish sustained arousal is more effective in the treatment of CFS than unspecific cognitive behavioral therapy. CBT is of proven value in CFS [• e in CFS ; to our Pharmaceutical inhibition of brain centers eliciting the arousal response will improve the functional abilities of CFS patients and normalize the hemodynamics in distressing situations. Clonidine, an agonist to the inhibitory alfa2-adrenoceptor, attenuates central sympathetic outflow by an effect on brain stem centers which also constitutes fundamental elements of the arousal response [• response . Thus, tIn this article, we have applied CATS and related stress theories to propose the concept of sustained arousal as a coherent and integrative model of disease mechanisms in CFS. This model opposes the reductionist approach where explanations from psychology, neurology immunology and other areas are seen as competing rather than complementary. As stated by Manu , page 17CFS: Chronic fatigue syndrome; CATS: Cognitive activation theory of stress; HPA-axis: Hypothalamus-pituitary-adrenal-axis; CNS: Central nervous system; CRH: Corticotropin-releasing hormone; CBT: Cognitive behavioral therapy.The authors declare that they have no competing interests.VBW conceived of the theory and drafted the manuscript. HRE and KM elaborated on the theory and helped to draft the manuscript. All authors have read and approved the final manuscript.VBW has conducted research projects regarding autonomic control in CFS, and is currently head of the Unit for Chronic fatigue syndrome at Division of Paediatrics, Rikshospitalet University Hospital, Oslo, Norway. HRE has published extensively on the Cognitive activation theory of stress, and is professor at the Dept. of Psychology, University of Bergen, Norway. KM has conducted clinical research on CFS and heads the national research network 'CFS in theory and practice'.


The diagnosis of respiratory virus infections has evolved substantially in recent years, with the emergence of new pathogens and the development of novel detection methods. While recent advances have improved the sensitivity and turn-around time of diagnostic tests for respiratory viruses, they have also raised important issues such as cost, and the clinical significance of detecting multiple viruses in a single specimen by molecular methods. This article reviews recent advances in specimen collection and detection methods for diagnosis of respiratory virus infections, and discusses the performance characteristics and limitations of these methods. Respiratory virus infections have a major impact on health. Acute respiratory illnesses, mostly caused by viruses, are the most common illness experienced by otherwise healthy children and adults worldwide. Upper respiratory tract infections (URIs) such as common cold are exceedingly prevalent in infants and young children and continue to be common in older children and adults. Infants and young children may have 3–8 episodes of cold per year; those who attend daycare centers may have many more episodes per year –4. URI cCommon respiratory viruses include influenza A and B, respiratory syncytial virus A and B, parainfluenza virus types 1–3, adenovirus, rhinovirus, human metapneumovirus, and coronavirus types OC43 and 229E. Less common respiratory viruses include parainfluenza virus type 4, influenza virus C, and specific types of enteroviruses. The significance of more recently discovered viruses such as human bocavirus, coronavirus NL63, and HKU1 has yet to be elucidated , 8.Clinical presentations of respiratory virus infections overlap among those caused by various viruses. In addition, clinical manifestations may mimic those of diseases caused by bacteria. Therefore, antibiotics are most often used in these infections, most of them unnecessarily. Furthermore, LRIs often require hospitalization for management such as intravenous antibiotics and symptomatic and supportive treatment. Specific antiviral treatment for respiratory virus infections is only available for influenza. Respiratory viral diagnosis is an integral part of patient management. Accurate diagnosis of specific respiratory virus infection not only improves the knowledge of disease the patient has but also can affect patient management and help prevent secondary spread of the infection. Rapid viral diagnosis may result in discontinuation of unnecessary antibiotics, initiation of antiviral drug for influenza, reduction of costs related to reduction of unnecessary investigations, and shortened hospital stay –11. This paper describes up-to-date information on laboratory methods presently available in the diagnostic virology laboratories and those upcoming for detection of respiratory viruses. Perhaps one of the most significant advances in the detection of respiratory viruses is the recent introduction of novel swab types such as flocked swabs. Flocked swabs consist of nylon fibers perpendicular to the swab shaft. Unlike standard woven rayon swabs which trap sample material, the fibers of flocked swabs act like a brush to collect respiratory epithelial cells and efficiently release them in transport medium . StudiesTraditionally, nasal washes and aspirates have been considered to be superior to swab specimens for isolation of respiratory viruses in cell culture and detection of viral antigens in immunoassays –18. HoweParent-collected respiratory swabs have been utilized in research studies, where they have been shown to yield sensitivity equivalent to that of healthcare worker-collected swabs when combined with PCR testing . A practProviding a result at the point of care in less than 30 minutes, rapid immunoassays are widely used for the detection of influenza viruses and RSV. Rapid immunoassay formats include membrane-based enzyme immunoassay, lateral flow immunochromatography, and optical immunoassay. Most test results are read manually by eye although one test includes an automated reader. The sensitivity of rapid RSV immunoassays in pediatric patients is generally higher than that of cell culture due to the lability of RSV . The senDirect fluorescent antibody (DFA) staining of viral antigens in patient specimens is generally considered to be more sensitive than rapid immunoassays . The speLong considered the gold standard for detection of respiratory viruses, the turn-around time of conventional cell culture in tubes for respiratory viruses is generally too long to be clinically relevant . When peThe diagnostic utility of serology generally is limited by the need to collect both acute and convalescent sera to identify either seroconversion or a fourfold rise in antibody titer. As such, serologic methods that detect IgG responses usually have little impact on patient management. Tests that detect IgM antibodies can detect acute infection, but sensitivity is reduced because serum IgM levels are often low due to repeated exposure to vaccine or circulating virus. Recent advances in serologic assays for respiratory viruses are largely limited to novel microagglutination assays that detect and measure antibody titers to novel influenza A virus subtypes. These assays utilize engineered reporter viruses—a lentiviral vector pseudotyped to contain the influenza hemagglutinin protein that eliminates the need for enhanced biocontainment facilities . The pseNucleic acid amplification methods such as RT-PCR and nucleic acid sequence-based amplification (NASBA) are becoming more commonly used for detection of influenza virus and other respiratory viruses. Using realtime, fluorescent detection of amplified product, laboratories are able to perform molecular tests in less than 3 hours. High specificity requires judicious selection of primers and probes, optimization of amplification conditions, and interpretation of results. Continuous adherence to laboratory protocol is essential to avoid false positives due to carryover contamination. Due to the limited number and cost of FDA-cleared nucleic acid amplification tests, many laboratories perform tests developed and verified in house (referred to as laboratory-developed tests (LDTs)). LDTs  are viable options for the detection of common respiratory viruses such as RSV , as wellIn real-time PCR, amplification (as detected by an increase in fluorescence) and analysis occur simultaneously. Real-time PCR is well suited for quantitative analysis, but currently available commercially kits for detection of respiratory virus nucleic acids have only qualitative claims. Most real-time PCR instruments are limited in the number of emission channels available for multiplexing. As a result, commercially available multiplex real-time PCR kits are generally limited to the detection of four or fewer nucleic acid targets. One of the targets is usually an internal control. FDA-cleared real-time RT-PCR assays are currently available for the detection of influenza A (including subtypes) and influenza B viruses, RSV, parainfluenza viruses 1, 2, and 3, and human metapneumovirus. Studies have demonstrated that molecular amplifications methods including real-time RT-PCR provide the most sensitive detection of respiratory viruses , 43.Conventional PCR requires the postamplification detection of PCR product . Either format allows for highly multiplexed assays that are able to detect more pathogens in a single PCR reaction than real-time PCR. An FDA-cleared assay utilizing conventional RT-PCR and a microarray of sequence-specific beads can detect RSV, influenza A (including subtypes) and influenza B viruses, parainfluenza viruses 1, 2, and 3, human metapneumovirus, rhinoviruses, and adenoviruses from a single RT-PCR reaction. This PCR assay format provides sensitive detection of influenza viruses, as well as the ability to detect a broad scope of respiratory pathogens .The high sensitivity of molecular amplification tests has made the interpretation of positive results challenging. These assays detect respiratory virus nucleic acid in the absence of symptoms –46 and aDuring the past several years, we have witnessed an explosion of improvements in respiratory virus diagnostics, from novel specimen collection instruments to highly sensitive and multiplexed nucleic acid amplification tests. With the expanding list of antigen and molecular-based tests, it is now possible for laboratories to offer comprehensive testing for respiratory viruses without even performing virus isolation. However, cell culture still remains the gold standard in many laboratories. Recent advances have also been made to this traditional approach to improve the turn-around time by using a combination of shell vial cultures and immunostaining.Because of the sensitivity and rapid turn-around time, nucleic acid amplification technologies such as PCR will likely be the focus of further advances in respiratory virus diagnostics. The future will likely promise additional commercial test kits for molecular detection of respiratory viruses, including multiplexed assays. Because of their exquisite sensitivity, nucleic acid amplification tests can create diagnostic conundrums. Additional research is needed to elucidate the clinical significance of positive PCR results that are persistent in a patient, and when two or more respiratory viruses are detected in a single specimen. Future research on respiratory virus diagnostics should aim towards the ability to accurately detect a spectrum of clinically significant viruses rapidly enough to affect patient management and initiation of infection control measures while keeping the costs affordable. Commercial kits provide standardization not achievable with laboratory-developed tests, but at a substantially higher cost. Ultimately, the utilization of molecular testing, particularly highly multiplexed tests in routine patient management will depend on the cost/benefit ratio. Current and future diagnostic options will include antigen, molecular, and culture-based methods. The performance characteristics and limitations of these methods will vary greatly with the new generations of assays. It is important that diagnostic virologists and clinicians understand these characteristics and limitations.


The concept of risk dominates the HIV/AIDS literature pertaining to People Who Use Injection Drugs (PWUID). In contrast the associated concept of worry is infrequently applied, even though it can produce important perspectives of PWUID's lives. This study asked a sample (n = 105) of PWUID enrolled in a Victoria, British Columbia needle exchange program to evaluate their degree of worry about fourteen factors they may encounter in their daily lives.Exploratory factor analysis was used to analyze their responses.Factor analysis delineated three factors: 1) overall personal security, 2) injection drug use-specific risks including overdosing and vein collapse and, 3) contracting infectious diseases associated with injection drug use (e.g. HIV/AIDS and hepatitis C).PWUID in this study not only worry about HIV/AIDS but also about stressful factors in their daily life which have been linked to both increased HIV/AIDS risk behaviour and decreased anti-retroviral treatment adherence. The importance PWUID give to this broad range of worry/concerns emphasizes the need to place HIV/AIDS intervention, education, and treatment programs within a broader harm-reduction framework that incorporates their perspectives on both worry and risk. Injection drug use is a driving force in historic HIV epidemics in North America and emerging epidemics in Asia and Eastern Europe , and is In contrast to this long-standing concern with the concept of risk there has been relatively little development of a related concept, that of worry, in the HIV/AIDS literature. A notable exception is Smith and Watkins' ,11 substet al. ), our results again emphasize the need to address larger structural problems which form risks and worries for PWUID. Unfortunately in the present case the closure of the AVI-SOS fixed site needle exchange facility limits the organization's ability to address these broader programs. Throughout its existence the fixed-site provided a suite of services, ranging from providing hot meals through access to street nurses, referrals to housing/shelter organizations, personal counsellors and HIV/HCV testing to simply providing a safe, dry, warm place. With its closure AVI-SOS must attempt to provide these services via newly established outreach services which cannot individually offer the array of services provided by the now defunct fixed-site; which this analysis reveals their clientele want and need.In conclusion, combined with the historically more frequently applied concept of risk, consideration and inclusion of PWUID's panoply of everyday worries into broader-based harm reduction interventions could provide important insights or "windows" into their lives and yield effective programs featuring the convergence of PWUID perspectives and public health goals.The authors declare that they have no competing interests.HE, EKG, JL, RS and EAR designed the study questionnaire, constructed the research design, collected the data upon which this analysis is based, and interpreted analysis results. LC, RS and EAR completed the statistical analysis, and wrote the manuscript draft. All authors read and approved the final manuscript.


The approaches for shotgun-based sequencing of vertebrate genomes are now well-established, and have resulted in the generation of numerous draft whole-genome sequence assemblies. In contrast, the process of refining those assemblies to improve contiguity and increase accuracy (known as 'sequence finishing') remains tedious, labor-intensive, and expensive. As a result, the vast majority of vertebrate genome sequences generated to date remain at a draft stage.To date, our genome sequencing efforts have focused on comparative studies of targeted genomic regions, requiring sequence finishing of large blocks of orthologous sequence (average size 0.5-2 Mb) from various subsets of 75 vertebrates. This experience has provided a unique opportunity to compare the relative effort required to finish shotgun-generated genome sequence assemblies from different species, which we report here. Importantly, we found that the sequence assemblies generated for the same orthologous regions from various vertebrates show substantial variation with respect to misassemblies and, in particular, the frequency and characteristics of sequence gaps. As a consequence, the work required to finish different species' sequences varied greatly. Application of the same standardized methods for finishing provided a novel opportunity to "assay" characteristics of genome sequences among many vertebrate species. It is important to note that many of the problems we have encountered during sequence finishing reflect unique architectural features of a particular vertebrate's genome, which in some cases may have important functional and/or evolutionary implications. Finally, based on our analyses, we have been able to improve our procedures to overcome some of these problems and to increase the overall efficiency of the sequence-finishing process, although significant challenges still remain.Our findings have important implications for the eventual finishing of the draft whole-genome sequences that have now been generated for a large number of vertebrates. Currently, the standard and most efficient approach for the de novo generation of sequence from complex genomes  is 'shotgun sequencing' ,2, althoThe NISC Comparative Sequencing Program  has beenIn generating these BAC sequences, we have paid considerable attention to the sequence-finishing process. We routinely refine all BAC-derived sequence assemblies to produce 'comparative-grade' finished sequence , the quaOur efforts in sequencing the same orthologous genomic regions in many different species and in producing two 'grades' of finished sequence for a large subset of BACs provided us a unique opportunity to examine results from application of the same standardized process to a variety of genome sequences. Here, we report a series of studies that reveal substantial differences in the effort required to finish shotgun-generated genome sequences from different vertebrates, reflecting significant architectural differences between orthologous regions of these vertebrate genomes.The nature of our comparative genome mapping and sequencing program ,11 has rCFTR); this genomic segment corresponds to ENCODE pilot project region ENm001  contains a stretch of 2,100 bp with a GC content of 83%; an embedded gap of ~ 250 bp  was refractory to most routine gap-filling efforts and ultimately required an extraordinary finishing effort to close. Several other dog BAC sequences contain at least one gap that presented similar challenges during finishing. Interestingly, such high GC-content regions in dog sequence gaps often reside near CpG islands.For many species, the GC content of the gap sequence is only marginally different from the GC content of the total sequence Figure . For somPreviously, we found that sequence within gaps is disproportionately more repetitive than non-gap sequence; specifically, total repetitive sequences identified by the program RepeatMasker  account An early step in sequence finishing involves ordering and orienting contigs within the initial sequence assembly. This process extensively utilizes information about the paired forward- and reverse-primed sequence reads generated from each plasmid template during the shotgun phase. Two contig ends are considered adjacent to each other if two or more sequence reads in one contig connect to their plasmid 'mates' in the other contig; in such cases, the intervening gap is considered 'captured' by the spanning subclones. Furthermore, the calculated minimum distance between read pairs should not greatly exceed the average insert size of the subclone library. Sequence gaps not spanned by at least two appropriately spaced sequence read-pairs are deemed 'uncaptured' and add no spatial information to the sequence contig map. An assembly containing several uncaptured gaps can thus be quite challenging to finish, with other data required to deduce contig order and orientation; substantial additional effort is frequently involved during the finishing process, particularly in cases where the auxiliary data are weak or limited. Furthermore, generating the sequence of regions within captured gaps  is most readily achieved by sequencing the spanning plasmid subclones; in contrast, generating the sequence of regions within uncaptured gaps requires sequencing alternative templates , which is more costly and labor-intensive.We analyzed the comparative-grade finished sequence generated for the 2,031 BACs in the second data set for the presence of captured and uncaptured gaps. In aggregate, these sequences contain roughly 30 gaps per Mb, with approximately two-thirds of these gaps being captured. This result is generally expected for shotgun sequence assemblies with at least eightfold sequence redundancy. However, the characteristics of the sequence gaps vary among genomic regions and among species Figure . None ofThe effort required for establishing the order and orientation of sequence contigs--a key part of the sequence-finishing process--directly relates to the number of uncaptured gaps in the sequence assembly. While total gaps per Mb is an important metric to monitor, in practice, it is the presence of two or more uncaptured gaps in a given BAC sequence assembly that presents a significant challenge during sequence finishing. Within the second data set, the fraction of BAC sequence assemblies containing two or more uncaptured gaps ranged from 16% (ENr213) to 47% (ENm003) among the different ENCODE pilot project regions and 12% (vervet) to 60% (platypus) among the different species. BAC sequences from ENCODE regions ENm003, ENm005, ENr111, ENr211 and ENr312 (as well as ENr333 and ENr231) have routinely required extra finishing effort, mostly because over 40% of the sequence assemblies contained two or more uncaptured gaps. Meanwhile, in the case of vervet and squirrel BACs, nearly all sequence assemblies contained fully ordered and oriented contigs, since few of these BAC sequences had two or more uncaptured gaps. In contrast, platypus BAC sequence assemblies had an average of over eight gaps per BAC, with more than 44% of those gaps being uncaptured; this characteristic (in addition to other features of platypus sequence mentioned above) has made the finishing of platypus sequence more challenging than that of any other species' sequence encountered to date.In recent years, we have implemented various steps to reduce the number of gaps in our BAC sequence assemblies. For example, in the case of sequence assemblies containing two or more uncaptured gaps, we often will construct fosmid shotgun libraries from the starting BAC DNA, and then generate a modest number  of sequence read-pairs from fosmid insert-ends. The resulting fosmid clones often capture the DNA residing in previously uncaptured gaps, thereby helping to order and orient the sequence contigs. While effective, such a fosmid-based approach is labor-intensive and expensive; it also introduces delays in finishing a BAC sequence because of the manual nature of fosmid-library construction and subclone-DNA purification. Of note, this approach has not been particularly successful for some species . In these cases, the sequence assemblies frequently contain too many contigs that cannot be unequivocally ordered even with fosmid-derived read-pairs . As an alternative, we found that generating read pairs with a 10- kb insert plasmid library was more productive.Another approach that we have implemented involves the use of an alternate bacterial host strain for constructing the initial shotgun plasmid library. We reasoned that uncaptured gaps might reflect regions of foreign DNA that are 'poisonous' to the bacterial host, with subclones propagating such DNA in high copy-number plasmids either failing to grow or growing poorly -32. ThusE. coli DH10B tonA). Aliquots of the ligation reactions, which earlier produced the initial shotgun libraries, were used to separately transform competent cells of copy-control E. coli strain EPI400 (Epicentre Biotechnologies); BAC sequencing was then performed per our usual routine, after which we compared assemblies from sequence reads obtained with each host strain. Representative results from those comparisons are shown in Figure To test this idea, we selected three previously generated BAC sequences  associated with the use of EPI400 cells, we only utilize copy-control host strains in special cases. For example, for species  whose initial sequence assemblies regularly have large numbers of gaps, we now routinely use copy-control EPI400 cells during the initial shotgun phase; for other species, these cells are used only after the BAC sequence assembly is reviewed and deemed to require such an approach {with the key determinant being a large variation in sequence-read redundancy . The purified DNA from each BAC was sheared and then ligated with a unique 'indexing' linker. All 12 samples were then mixed together, and the resulting pool then used to make a single shotgun sequencing library using the Genome Analyzer instrument (Illumina) . The resCombining the Sanger- and Genome Analyzer-derived shotgun sequence data simplified some of the sequence-finishing process, but did not solve the most vexing finishing problems. For example, in some cases, the use of Velvet-assembled contigs helped to extend sequence from contig ends, provided complementary strand data, and resolved miscellaneous ambiguities . In fact, adding Genome Analyzer-derived data to the Sanger-derived assemblies reduced the total number of contigs for the 10 problematic BACs; for five BACs, a few of the uncaptured gaps were closed. However, new problems sometimes arose with the addition of Velvet-assembled contigs; for example, in several instances, new misassemblies formed where none existed in the Sanger-derived assembly. Most disappointing was the fact that many troublesome gaps in the Sanger-derived assemblies, which had been extremely problematic (and expensive) to close during human-grade sequence finishing, remained unchanged following the incorporation of Velvet-assembled contigs. Although some small contigs were found to partially fill these gaps, much of the missing sequence had no matching Genome Analyzer-derived sequence. These results point out the biases and limitations of both sequencing methods that utilize DNA polymerase.The refinement of sequence assemblies generated from shotgun reads  is a multi-step procedure that is influenced by various features of the starting template. Some of these features can present major problems during the sequence-finishing process; the most common being gaps (missing sequence) of various types and characteristics, and sequence misassemblies. Here, we present some representative findings based on our experience in finishing in a standardized fashion the sequence of over 11,000 BACs from 75 different vertebrate species. This unique data resource allows for a rigorous assessment of the requirements for finishing sequence of diverse origins; the current study reveals substantial differences in the effort needed to finish the sequence from different genomic regions and, in particular, from different species.While our experience provides the opportunity to generate quantitative summaries about the comparative grade-finished assemblies of shotgun sequence from various sources  can recover some of the sequence that is otherwise missing in initial assemblies. We are investigating further alternative approaches for employing these new platforms in generating sequence data that are more complete, reducing the effort required for finishing (unpublished data).However, our experience also confirms the continued labor-intensive nature of sequence finishing and the absence of any 'magic bullet' that resolves all major sequence-finishing problems. Some of the most challenging sequences to finish are from species for which a draft genome sequence has already been generated and currently sits at a draft stage, such as for dog  and platE. coli DH10B tonA host cells (Invitrogen). Plasmid DNA was purified from individual subclones by an automated magnetic bead process (Agencourt Bioscience). Paired sequence reads were generated from randomly selected subclones by universal forward- and reverse-primed fluorescent dideoxy sequencing reactions [xl sequencing instruments (Applied Biosystems). Typically, 2,500-2,800 sequence reads were generated for each BAC (which averaged ~ 165 kb in size), with the resulting Phrap-based [Purified BAC DNA was sheared and 'shotgun subcloned' into plasmids containing 3- to 5- kb inserts using eactions  using ABap-based ,26 sequeet al. [Sequence assemblies were refined to produce comparative-grade finished sequence; this process, described in detail by Blakesley et al. , includiet al. ,39. The All sequencing reactions for the shotgun phase and most reactions for the finishing phase employed Big-Dye Terminator chemistry (Applied Biosystems) as described by the manufacturer. Certain other sequence finishing reactions used alternative chemistries, where dGTP BigDye Terminator Mix (Applied Biosystems) or an 80:20 mixture of BigDye:dGTP Big Dye substituted for BigDye Terminator Mix in the sequencing reaction mixture. In certain cases, one-tenth volume of SeqRxA (Invitrogen) was added to the reaction.RWB, NFH, JCM, BM, GGB, and EDG conceived the study. NFH led the bioinformatics analyses, with contributions from JCM, BM, GGB, and RWB. JG, BBB, SYB, HC, PH, S-LH, KS, SS, JLV, PJT and members of the NISC Comparative Sequencing Program generated the data and performed some of the bioinformatic analyses. RWB, NFH, and EDG wrote the manuscript.