Cardiovascular Magnetic Resonance (CMR) is recognised as a valuable clinical tool which in a single scan setting can assess ventricular volumes and function, myocardial fibrosis, iron loading, flow quantification, tissue characterisation and myocardial perfusion imaging.The advent of CMR using extrinsic and intrinsic contrast-enhanced protocols for tissue characterisation have dramatically changed the non-invasive work-up of patients with suspected or known cardiomyopathy.Although the technique initially focused on the in vivo identification of myocardial necrosis through the late gadolinium enhancement (LGE) technique, recent work highlighted the ability of CMR to provide more detailed in vivo tissue characterisation to help establish a differential diagnosis of the underlying aetiology, to exclude an ischaemic substrate and to provide important prognostic markers.The potential application of CMR in the clinical approach of a patient with suspected non-ischaemic cardiomyopathy is discussed in this review. Cardiomyopathies encompass a broad spectrum of myocardial conditions which can affect the heart as a primary disease process or as part of a systemic disorder, evolving toward heart failure or cardiovascular death.Progress of modern molecular biology and its introduction into clinical cardiovascular medicine has considerably changed the approach to cardiomyopathies and has led to new classification schemes. .While cardiomyopathies were initially defined as disorders that were idiopathic, expert panels classify cardiomyopathies now into: primary, acquired and mixed . This reWhilst prevalence of dilated cardiomyopathy reaches 36 cases per 100 000 population, these figures as well as those reported for other cardiomyopathies, underestimate the frequency of the disorder as a number of patients are asymptomatic until reaching advanced stages of the disease. In addition, accurate diagnostic and prognostic phenotyping of cardiomyopathy remains a challenge owing to frequent overlapping features between individual cardiomyopathies.In the investigative workup for a suspected cardiomyopathy, typically a considerable number of tests are performed, ranging from the more routine and straightforward tests including ECG, echocardiogram, and exercise treadmill, to the more invasive including coronary angiography, electrophysiological studies, and endomyocardial biopsy. It is key to identify a potentially treatable substrate and then to risk stratify patients for treatment including consideration of an implantable defibrillator (ICD) or cardiac resynchronisation device.Advances in cardiovascular magnetic resonance (CMR) provide the potential to address all these important issues in a single scan setting complementing other non-invasive tools and genetic testing. The cost-effectiveness of non-stress CMR has been highlighted recently pointing out the ability of CMR to act as a cost-reducing gatekeeper to invasive coronary angiography in specific clinical settings .In a single 45–60 minute study, CMR can provide 3-dimensional data on cardiac anatomy, function, tissue characterisation, coronary and microvascular perfusion, valvular disease in any selected plane, regardless of patient’s habitus and without ionising radiation. The relative safety of gadolinium agents and tissue characterising sequences allows for repeated imaging, follow-up, family screening and serial risk stratification.In this review, non-ischaemic cardiomyopathies (NICMP) are approached from a clinical point of view outlining the diagnostic and prognostic role of CMR as an integral part of the clinical decision making algorithm.In the imaging workup of a suspected cardiomyopathy a wide range of sequences are acquired following dedicated protocols -8. FigurDark-blood imaging using mutislice single-shot spin-echo sequence  in trans-axial, coronal and sagittal planes allows a gross anatomical assessment in under 3 min. Alternatively 10–12 s breathhold images in each of the imaging planes using bright-blood imaging with steady-state free precession sequence (SSFP) can be performed.Dynamic long-axis and short-axis views of the heart are then acquired using breath-hold cine-SSFP imaging. Cine-CMR is accepted as the gold standard for accurate and reproducible quantification of left and right ventricular volumes, biventricular ejection fraction, and left ventricular (LV) mass obtained through manual planimetry or the use of semi-automated software . PresencSpecialised sequences allow characterisation of pathological tissue without the need for a contrast agent. The T2-weighted sequence, Short-Tau Inversion Recovery (STIR) is a powerful sequence for imaging oedema suppressing signal from flowing blood and from fat enhancing sensitivity to myocardial fluid content. Long T2 relaxation times of water-bound protons are used to generate a water-specific contrast resulting in high signal intensity of oedematous tissue ,15. STIRPitfalls include low signal-to-noise ratio, surface coil intensity variation, interfering bright signal from stagnant blood, motion artifacts and the subjective nature of interpretation ,22. SomeT1-weighted and turbo-spin echo sequences are useful to assess the pericardium and to look for fat infiltration.Myocardial T2*-mapping is a unique technique, validated against in-vivo histology, allowing direct identification and quantification of myocardial iron in vivo .Myocardial fibrosis or infiltration can be assessed following administration of gadolinium. Gadolinium is an extracellular agent accumulating in areas of interstitial expansion . Three phases can be assessed after gadolinium injection: the first pass (immediate imaging at rest or during stress), early enhancement (first 5 min) and late enhancement (5 to 20 min after injection). First pass can be used for perfusion imaging and is often given during vasodilator stress to detect myocardial ischaemia. Following intravenous injection of gadolinium (0.1-0.2 mmol/kg body weight), the contrast media is followed through the LV at rest and during pharmacologic stress to assess homogeneity of perfusion ,27. UltrEarly gadoliuium enhancement (EGE) imaging (1–3 min after injection) enables the detection of hypovascular regions such as dark, unenhanced intra-cardiac thrombi, and microvascular obstruction .Late gadolinium enhancement (LGE) imaging (5–20 min after injection) detects accumulation of contrast in areas of infarction or fibrosis due to slower contrast kinetics and greater volume of distribution in extracellular matrix.This is performed using inversion recovery Turbo-FLASH gradient echo or phase-sensitive inversion recovery (PSIR) in identical planes to the cine images searching for areas of fibrosis or infiltration. PSIR provides corrected images that are virtually insensitive for pre-pulse delay timing. It allows for a faster acquisition method without adjustment of inversion time and more consistent image quality to the expense of a lower spatial resolution ,30. ThisUsing inversion recovery sequences, focal fibrosis is imaged as a localised area of high signal intensity. The extent and pattern of LGE varies according to the underlying pathological process and contributes therefore to establish the correct diagnosis in NICMP Figure .Presence of fibrosis in NICMP is often only identified visually but its extent can be quantified as a percentage of total LV mass using dedicated software . All quaLGE imaging sequences delineate fibrosis by revealing a relative difference in T1-relaxation times between areas of scar (T1 shortened by accumulation of gadolinium) and normal myocardium . T1-mapping techniques work by measuring the absolute T1-relaxation time for all areas of myocardium on a pixel by pixel basis. As the shortening of T1 relaxation time is proportional to the local concentration of gadolinium, this can reveal subtle changes in T1 times due to expansion of the interstitial space with collagen and other fibrous tissue components. Early T1 mapping techniques were very time consuming. A Look-Locker sequence consisting of a gradient echo cine sequence with a nonslice selective inversion pulse after an R wave followed by a segmented gradient echo acquisition was developed ,39. Meane, (an index of fibrosis if the extracellular space is occupied by scar tissue) to be determined [The local concentration of gadolinium will be affected by several factors including the amount of scar, the rate at which gadolinium is cleared from the body but also by the amount of extracellular fluid available in the body for the contrast to distribute into . With thtermined ,44.While T1 mapping can accurately differentiate both interstitial and replacement fibrosis from normal myocardium, it cannot distinguish one type of fibrosis from another . To dateDespite a lower temporal resolution compared to echocardiography, assessment of valvular disease is achievable using CMR velocity mapping with reproducible results .CMR myocardial tagging quantifies myocardial deformation, contraction and relaxation in the radial, circumferential and longitudinal directions adding useful information particularly prior to resynchronisation therapy Figure . This teA feature tracking method has recently been made available for the post processing of routinely acquired SSFP cines using inhomogeneities of the tissue as identifiers, which are then identified by software algorithms for analysing regional function. Following manual contouring of the LV endocardial border of the first cine frame, the software automatically identifies and tracks endocardial and epicardial myocardial boundaries through the remaining phases of the cardiac cycle. Although the method has been compared to echocardiographic speckle tracking, it may be better described as ‘boundary’ tracking when applied to CMR SSFP acquisitions. Potentially derivable parameters include circumferential, longitudinal and radially directed velocity, displacement, strain and strain rate. However, some of these derived measures even more than others require cautious interpretation with respect to both spatial and temporal resolution of cine images, and the design of the software. Feature tracking based measures of global strain of short axis cine slices – essentially the relative change of length of the tracked endocardial boundary – have shown correlation with corresponding global strain measured by CMR myocardial tagging, with reduced values found in patients with Duchenne muscular dystrophy compared with volunteers and appeared as a promising technique to detect wall motion abnormalities during Dobutamine stress CMR -50. HoweAmongst limitations of CMR, metallic implants and intracardiac Devices  represent the only absolute contra-indication, but this will be overcome in the near future by new generation of CMR compatible pacing leads currently in clinical trials. Severe claustrophobia, clinical instability and the first trimester of pregnancy are among relative contra-indications. Even though gadolinium contrast agents are relatively safe, their use is restricted in chronic renal failure (GFR <30 ml/min) due to the small but tangible risk of nephrogenic systemic fibrosis (NSF) which is enhanced by concomitant heart failure . On the Cardiomyopathy is often suspected on the basis of symptoms, an associated abnormal ECG and echocardiographic findings. Following detection of ventricular hypertrophy, dilatation (right or left ventricles) or abnormal wall motion abnormalities after acute chest pain syndromes with unobstructed coronaries, characteristic imaging features on CMR greatly assist in reaching a diagnosis and providing prognostic information. The diagnostic and prognostic abilities of CMR in these various clinical conditions are reviewed. Detection of a hypertrophied LV highlights the differential diagnosis between a physiologic response to pressure overload  or pathological conditions such as hypertrophic cardiomyopathy (HCM), wall thickening due to myocardial infiltration (amyloidosis), or a glycogen storage disease (Fabry’s). CMR provides unique complementary information assisting the diagnostic process. The characteristic LGE findings distinguishing each entity are summarised in Table Although concentric LV hypertrophy (LVH) secondary to systemic hypertension (HTN), aortic stenosis (AS), or aortic coarctation is often suspected clinically and by echocardiography, CMR assists the diagnosis and provides important prognostic information. This is of particular interest in cases with discrepancies between clinical and imaging data such as marked hypertrophy unexplained by severity and duration of HTN where another underlying associated myocardial abnormality can be detected.In hypertensive hearts, CMR allows reproducible assessment of wall thickness and LV mass with greater accuracy compared to echocardiography which is particularly important for assessing small LV mass changes over time as a consequence of treatment but also of prognostic value as it represents an independent predictor of cardiac mortality ,54. Up taortic stenosis, LV mass predicts the development of heart failure independent of the degree of AS [Similarly to hypertensive hearts, in ee of AS . In addiee of AS ,60. The LGE is a frequent finding in adaptive LVH  probably related to myocardial injury secondary to mismatch between LVH and blood supply, and depends on the severity of LV remodeling . Amount However, diffuse fibrosis can be difficult to identify on standard LGE images. Myocardial signal with diffuse fibrosis will be nulled to highlight focal scar losing all information on background interstitial expansion. A growing body of publications have addressed this issue and several groups have extensively studied a way to detect interstitial fibrosis, validating T1 mapping to derive a fibrotic index -67 (see HCM represents the most common cause of sudden cardiac death (SCD) in the young, including trained athletes, and an important substrate for heart failure disability at any age. Various genetic mutations lead to a combination of myocyte hypertrophy, disarray and fibrosis.Although an abnormal ECG associated with marked asymmetrical septal hypertrophy and a dynamic LV outflow tract (LVOT) obstruction is almost diagnostic, individuals harbouring a genetic defect for HCM do not necessarily express clinical markers of their disease at all times during life. As a result, even normal or mildly increased LV wall thickness can be consistent with the presence of an HCM-causing mutant gene. Coexistence of HTN and HCM in a proportion of patients adds to the complexity. While HCM has been characterised by substantial LV wall thickening, Maron et al reported rather a spectrum of distribution and patterns of wall thickening .CMR offers the potential for high spatial resolution images with excellent border definition allowing accurate measurement of peak wall thickness and detection of localised hypertrophy especially in the apical variant of HCM and the basal anteroseptal wall, regions more difficult to image with echocardiography. LV apical aneurysms, conferring increased risk of SCD, ventricular arrhythmias, thrombo-embolic strokes and progressive heart failure, can be identified in 2% of HCM patients using CMR . In addiTypical patterns of LGE are patchy or confluent, generally confined to segments with greatest wall thickening and at the RV-LV insertion points  Figure C4A.Aside from its diagnostic value, LGE extent appears related to occurrence of ventricular arrhythmias at rest and during exercise and increased risk of SCD potentially contributing to risk assessment in borderline or controversial cases benefiting from a prophylactic ICD ,73. EvenCMR-detected myocardial fibrosis is associated with increased incidence of atrial fibrillation (AF) and heart failure, heralding advanced LV remodeling and systolic dysfunction ,75. InteDegree of LVOT obstruction and presence of systolic anterior motion of the mitral valve can be quantified using velocity flow mapping at rest and during stress and have been correlated with more extensive hypertrophy, worse heart failure symptoms and unfavourable outcome . CMR canWhilst no specific pattern of LGE has been identified in secondary LVH due to HTN or AS, focal non-subendocardial pattern has been described in the former and patchy sub-endocardial or mid-wall LGE in basal LV segments has been observed in the latter. This contrasts with the typical pattern of LGE seen in HCM patients affecting mainly insertion points and segments with maximal wall thickness Table .This common cause of infiltrative cardiomyopathy is classified as primary, secondary (reactive), hereditary, and age-related. Myocardial involvement represents an important prognostic marker of the disease influencing treatment options . DetectiTypical CMR findings include a small LV cavity size with homogeneously thickened walls affecting inconsistently also the right ventricle (RV). Bi-atrial enlargement, as a consequence of the restrictive cardiomyopathy, impaired long-axis function with preserved radial function until end-stages of the disease, thickened interatrial septum and valve leaflets, pleural and pericardial effusions resulting from interstitial amyloid fibrils deposition are among the classical features. Around 55% of patients with cardiac amyloidosis may present with asymmetrical septal hypertrophy mimicking HCM.Wassmuth et al, observed that accumulation of amyloid fibrils can change T2 relaxation times and demonstrated that myocardium appeared hypointense on T2-weighted images in cardiac amyloidosis. In addition, a lower T2 signal intensity ratio  was independently associated with shortened survival . FurtherWhile diffuse perfusion defects indicating microvascular dysfunction can be seen, the characteristic pattern of LGE and the peculiar kinetics of gadolinium are unique and discriminate this disease from other forms of hypertrophic cardiomyopathies. LGE reflects interstitial expansion by amyloid fibrils and appears as widespread, circumferential enhancement mostly of the sub-endocardium matching the distribution of amyloid protein on histology, giving rise to a characteristic ‘zebra’ pattern  Figure B4B. ThisFaster washout of gadolinium from blood into the total amyloid load leads to a technically challenging LGE acquisition with the inability to adequately null myocardial signal. High myocardial uptake and fast blood washout result in a dark blood pool due to similar myocardial and blood T1 values. To visualise myocardial and blood pool gadolinium kinetics before setting an inversion time when suspecting cardiac amyloidosis, an inversion time scout corresponding to an inversion-time mapping sequence with images generated at 30 ms inversion time increment, can be run early following contrast injection (less than 5 minutes). LGE has a reported sensitivity and specificity of 86% for the identification of cardiac involvement in patients with systemic amyloidosis, detecting early abnormalities in patients with normal LV wall thickness, and appeared strongly related to heart failure severity -82. AddiThis treatable cause of LV hypertrophy affects 1-2% of middle aged patients suspected of HCM . ConcentThe term DCM is applied in the presence of LV or biventricular dilatation and dysfunction following systematic exclusion of all obvious or detectable causes of cardiomyopathy. In up to 50% of cases the aetiology remains unknown, with 20-30% of familial cases attributed to known genetic abnormalities ,89. MyocDCM is the second most common cause of heart failure and the most frequent cause of heart transplantation. In addition to providing accurate and reproducible assessment of ventricular volumes and LVEF, the LGE technique detects a pattern of fibrosis in DCM which is typical mid-wall and seen in one third of cases. CMR by detecting the pattern of fibrosis contributes significantly to ruling out ischaemic aetiology and providing prognostic information.The specificity of the LGE pattern in ischaemic cardiomyopathy (ICM) has been underscored by several studies in which coronary angiography and CMR were performed in patients with systolic dysfunction of unknown aetiology .In a large study comparing patients with a diagnosis of DCM with unobstructed coronaries and patients with known coronary artery disease and a group of normal controls, three LGE patterns were reported . The iscPresence and transmurality of fibrosis in DCM has been linked to increased risk of SCD and ventricular arrhythmias independent of other markers of risk such as reduced LVEF ,92.Wu et al reported an eightfold increased risk of heart failure, ventricular arrhythmias and cardiac death in DCM patients with LGE referred for an ICD .Mechanisms contributing to fibrotic changes in DCM include inflammation, neurohumoral changes and microvascular ischaemia . LimitedWhilst the patterns of LGE observed in DCM patients are distinct from those seen in ICM, mid-wall but also epicardial, diffuse and focal patterns are seen ,93. It iAssomull et al, demonstrated recently that LGE was highly effective in detecting the underlying aetiology of newly diagnosed LV dysfunction and could be used as a safe and effective gatekeeper to coronary angiography . While pPilz et al, reported previously the role of Adenosine stress CMR as a gatekeeper to coronary angiography in a different patient population demonstrating the high accuracy of CMR in detecting significant coronary artery stenoses inducing 30% reduction in the rates of coronary angiography .A dilated RV of unexplained aetiology should raise the possibility of ARVC particularly if associated with aborted SCD from arrhythmias, often the first manifestation of the disease.This inheritable condition predominantly involving the RV with progressive loss of myocytes and fibrofatty tissue replacement can affect the LV in up to 75% of patients. In spite of recent advances in genotyping, clinical diagnosis remains challenging.While the classic phenotype describes progression of regional to global RV dysfunction, followed by LV involvement and biventricular failure, recognition of phenotypes with early and predominant LV involvement have raised questions regarding its differentiation from other myocardial disorders, such as DCM. Diagnosis relies largely on the ARVC Taskforce criteria, recently revised to include quantitative parameters for imaging studies, improving sensitivity of detection of gene-carriers with limited disease expression and those with left-sided disease features .2 for male and ≥100 ml/m2 for female or RVEF ≤40%; Minor criteria : Regional wall motion abnormality and RV end-diastolic volume ≥100- <110 ml/m2 for male and ≥90- < 100 ml/m2 for female or RVEF >40% - ≤45%) have been defined on the basis of comparison with normal patient data.These published cut-off values for RVEF and end-diastolic volumes  unexplained by significant coronary lesions . In a coKey features rely on the use of T2-weighted imaging increasing the sensitivity and specificity of detection of an ischaemic insult and on the typical pattern of LGE distinguishing an ICM from an NICMP and detection of myocarditis and Tako-tsubo  Figure B and 7.Myocarditis occurs with a wide spectrum of clinical manifestations ranging from flu-like symptoms to troponin positive CPS, severe heart failure and SCD. Endomyocardial biopsy recognised as the gold standard for the diagnosis remains limited by insensitivity and sampling bias.CMR can image several aspects of myocardial injury.In the acute stage of the disease, oedema following lymphocytic infiltration and myocytolysis appears as regions of high intensity on T2-weighted images. EGE matching areas of T2-hyperintensity may reflects tissue hyperemia and increased interstitial space .LGE identifies inflammation and fibrosis acutely and areas of irreversible necrosis in later stages, typically affecting the sub-epicardium sparing the sub-endocardium of the lateral wall Figure A. Mid-waAmong patients presenting with troponin positive CPS with unobstructed coronaries, CMR was able to identify a cause in 65% of patients with the commonest being myocarditis . In patiA large consensus group provided recommendations on the use of CMR in patients with suspected myocarditis suggesting the combination of 3 CMR criteria (‘Lake Louise criteria’) to increase the diagnostic accuracy . AuthorsAcute but rapidly reversible LV systolic dysfunction in absence of coronary artery disease, triggered by profound psychological stress characterises this stress-mediated myocardial stunning. Mid and apical LV segments akinesia with ballooning and compensatory hyperkinesia of basal segments producing a dynamic obstruction is a typical finding. RV is involved in 25% of patients.Focal and transient transmural hyperintensity on T2-weighted imaging reflecting oedema matching the distribution of wall motion abnormality seems to be a feature of Tako-tsubo cardiomyopathy related to the severity of LV systolic dysfunction . Lack ofRecently, Eitel et al analysed the CMR findings in Tako-tsubo cardiomyopathy in a large multicenter prospective cohort . These oThis multisystem granulomatous disease of unknown aetiology involves the myocardium in 20-30% of cases.Clinical myocardial involvement (5% of patients) ranges from asymptomatic conduction abnormalities to heart failure or fatal ventricular arrhythmias. An early diagnosis is crucial as current therapy may effectively prevent death from arrhythmias and cardiac failure. Endomyocardial biopsy and echocardiography proved insensitive diagnostic tools detecting mostly advanced stage of the disease.Although results of prospective data are awaited, CMR appears promising for the early diagnosis and for monitoring treatment response, providing novel risk stratification criteria for SCD and heart failure. T2-weighted and STIR imaging detect localised areas of myocardial inflammation. Cines identify areas of thinning and regional wall motion abnormalities and LGE detects focal scarring, typically patchy, mid-wall or sub-epicardial, frequently involving the anteroseptal and anterolateral walls  Figure .Patel et al described a large spectrum of LGE patterns. While the majority of affected myocardial segments had non-transmural involvement, explaining normal ventricular wall motion, there was a predilection for basal and mid-ventricular septum with up to 48% of regions displaying sub-endocardial LGE similar to coronary artery disease LGE. However, 86% of affected patients had also at least one region of LGE with a non-ischaemic type pattern. Sub-endocardial LGE of the RV side of the septum was a common finding and could be associated with LGE of the RV free wall .CMR appeared more sensitive for disease detection compared to Thallium SPECT and gallium SPECT . Recent This rare congenital disorder is characterised by a “spongy” appearance of the LV (and the RV in <50% of cases) resulting from an arrest in normal embryogenesis.LVNC may be associated with neuromuscular disorders, leading to heart failure, arrhythmias and thrombo-embolic manifestations. Although echocardiography is considered as the reference for the diagnosis, CMR accurately diagnosed LVNC delineating hyper-trabeculations of the apex and the lateral wall .Hyper-trabeculation with a end-diastolic ratio of non-compacted over compacted myocardium of 2.3 distinguished pathological from non-pathological conditions with a sensitivity of 86% and specificity of 99%   were highly representative of mean global myocardial iron validating current clinical practice .Interestingly, macroscopic fibrosis detected by LGE is uncommon in thalassaemia patients despite current or prior heart failure and across a broad spectrum of myocardial iron loading .Despite significant advances in anti-rejection therapy, acute cellular allograft rejection remains a leading cause of early mortality among transplanted patients. The diagnostic gold standard technique remains repeated endomyocardial biopsy largely limited by insensitivity.CMR can assist the detection of rejection mainly through myocardial T2 relaxation time quantification with sensitivities and specificities approaching 90% except for the early peri-operative period characterised by normal post-operative oedema . A complCardiac involvement by Trypanosoma cruzi infection occurs in 30% of seropositive patients representing a major cause of death from heart failure in Latin America.LGE-CMR typically involves the apical and mid-inferolateral wall in patients with advanced disease but also in asymptomatic seropositive subjects without wall motion abnormalities . IschaemThe most common adverse effect of chemotherapy is cardiotoxicity affecting patient’s prognosis and quality of life independently of the oncological issue. A wide range of agents have been associated with cardiotoxicity, anthracyclines and related compounds being the most frequently implicated.. IncidenWhile echocardiography remains the commonest imaging modality used to follow-up LVEF in patients at risk of developing cardiotoxicity, case series assessing the role of CMR in this setting have raised interesting issues. In a population of patients receiving trastuzumab therapy for breast cancer and developing LV dysfunction, authors reported the presence of sub-epicardial LGE in the lateral wall in all patients as a sign of myocarditis induced by drug toxicity ,132. OthAn optimal nulling of the myocardium is key to LGE interpretation and detection of small areas of fibrosis or quantification of sub-endocardial scars. Although the optimal inversion time can be set based on experience, a single short axis slice can be acquired automatically at different inversion times. The optimal inversion time is then selected visually as the one nulling completely the healthy myocardial signal. According to the underlying suspected pathology, LGE should be acquired before 10 minutes such as in amyloidosis due to the faster washout of gadolinium from blood pool. In cardiomyopathy patients, gadolinium washout can also be faster, needing an earlier acquisition to obtain optimal nulling of the myocardium. This is thought to be related to structural alterations of extracellular matrix associated with reduced myocardial blood flow in DCM patients . Once thLack of ionising radiation, excellent spatial resolution, 3D anatomical assessment and ability to provide tissue characterisation with and without contrast are amongst advantages of CMR making it a valuable and complementary tool to other non-invasive modalities. CMR is recognised as a class I indication for EF, volumes and mass quantification . CMR shoLGE CMR appears highly effective in identifying the aetiology of LV dysfunction in newly diagnosed heart failure and can be used as a safe, clinically effective and potentially economical gatekeeper to coronary angiography in these patients . Safety Whilst CMR offers an excellent morphological evaluation through its high spatial resolution, echocardiography excels through its unique temporal resolution, providing a non-ionising, inexpensive bedside tool, superior for haemodynamic evaluation . Limitations are linked to its restricted lateral spatial resolution, the dependency on good acoustic windows particularly in patients with chronic lung diseases, and the limited assessment of apical segments and the RV. CMR remains partially limited by arrhythmias interfering with ECG triggering and poor ability to perform breath-holds. Metallic non-CMR safe devices and severe renal failure prevent its use.Cardiac computed-tomography (CCT) provides a fast and accurate 3D assessment of coronaries and myocardium in a 30-second time frame even in patients with pacing devices. However, the use of CT remains limited by radiation, renal failure often encountered in heart failure patients (for CT contrast agents), pregnancy and limited assessment of late enhancement still inferior to CMR. It can however be considered as an alternative in patients with pacing devices.Nuclear and CT hybrid devices are rapidly evolving to incorporate high-speed MDCT along with positron-emission tomography (PET) and single-photon emission tomography (SPECT) detector systems.This dual modality imaging presents an opportunity to use a single piece of equipment for a combined assessment of coronary anatomy, perfusion, function and metabolism or coronary calcification. However, radiation exposure as well as availability and cost remain an issue. Table CMR spectroscopy (MRS) and CMR-based molecular imaging have appeared recently as non-invasive tools for assessing myocardial metabolism using the intrinsic properties of nuclei including Phosporus-31, proton, sodium-23, oxygen-17 and 13-carbon . Phospho2)[Newly developed quantitative Oxygen-sensitive CMR techniques have been designed to directly evaluate myocardial oxygen consumption . Elevate2). MBF mea2). Interes2). These gThrough a wide range of sequences, CMR offers a unique non-invasive tool which should be integral part of the clinical work-up of a cardiomyopathy.In vivo tissue characterisation remains a major strength of CMR compared to other non-invasive techniques detecting and distinguishing oedema from fat, fibrosis and myocardial infiltration. Its excellent spatial resolution set it above other imaging techniques to analyse RV mophology and detect RV cardiomyopathies and for identification of regional ventricular hypertrophy or dilatation.Although, CMR provides precious and irreplaceable additional information to standard imaging and invasive methods, it cannot be seen as an alternative to these techniques yet and as a one stop shop but as a unique supplementary method able to characterise abnormal tissue in vivo, allowing pre-clinical detection of disease process and novel parameters for risk stratification.The authors declare that they have no competing interests.All authors contributed to the design and writing of this manuscript. All authors read and approved the final manuscript.


Patients with atrial fibrillation (AF) often present with typical angina pectoris and mildly elevated levels of cardiac troponin  during an acute episode of AF. However, in a large proportion of these patients, significant coronary artery disease is excluded by coronary angiography, which suggests that AF itself influences myocardial blood flow. The present review summarizes the effect of AF on the occurrence of ventricular oxidative stress, redox-sensitive signaling pathways and gene expression, and microcirculatory flow abnormalities in the left ventricle. Angina pectoris is a typical symptom in patients with paroxysmal atrial fibrillation (AF). In most of these patients, angina pectoris is associated with mildly elevated cardiac troponin (cTn) levels suggesting a non-ST-segment elevation myocardial infarction (NSTEMI). However, in a large proportion of these patients, significant coronary artery disease can be excluded by coronary angiography despite clinical symptoms  are generated under physiological conditions in the cardiovascular system and act as second messengers in numerous redox-sensitive signal transduction pathways Figure . HoweverOxidative stress has been implicated as playing a critical role in the pathophysiology of heart and cardiovascular diseases such as heart failure, LV hypertrophy, coronary heart disease, cardiac arrhythmia. It appears that the oxidative events initiate the disease-dependent tissue remodeling and promote its propagation. Oxidative stress is associated with microvascular flow abnormalities and occurs immediately after new-onset AF likely representing key initiator mechanisms of AF-related ventricular remodeling. This has been shown in patients with lone recurrent AF and for rapid atrial pacing models  and endothelin (ET) play important roles in the regulation of cardiovascular function as well as in the pathogenesis of heart and cardiovascular diseases. AF has been associated with increased plasma and tissue levels of angiotensin II (AngII)  have been shown to increase NO availability and it is possible that this effect is mediated by a stronger ANG-II-dependent activation of AT2+-waves via IP3-dependent Ca2+-release leading to DAD  exerts its proarrhythmogenic effects by two different ways. First, due to its strong vasoconstrictory activity, ET-1 may induce ischemia which facilitates arrhythmia. Second, upon binding to endothelin receptor A (ET-A), ET-1 affects intracellular calcium handling and, in particular, provokes intracellular CaHeart failure , the expression of six homologs  has been identified  in pigs , glycogen synthase kinase 3β (GSK-3β), muscle isoform of glycogen phosphorylase (PYGM), and acyl-coenzyme A dehydrogenase (ACADL; Bukowska et al., It is fully established that even mild ischemia is associated with compromised mitochondrial function and requires metabolic adaption to maintain adequate ATP generation and cardiac output (Shohet and Garcia, AT induces oxidative stress in the atrial and ventricular myocardium. In the ventricles, AF causes alterations in gene expression and activation of specific signal transduction pathways. As a consequence, microcirculation is impaired, troponin can be released, which is associated with causing clinical symptoms like angina pectoris and dyspnea. It remains to be determined if these alterations are also related to the increased rate of death in AF patients shown by several epidemiologic studies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography–mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours. The majority of solid cancers contain poorly oxygenated areas Hypoxia may thus affect energy metabolism both directly, by limiting the availability of oxygen to the mitochondria, and indirectly through transcription-mediated metabolic rearrangements. Therefore, it is crucial to develop reliable techniques for assessing mitochondrial functions under hypoxia. This is of particular importance when looking for general cellular metabolic changes imposed by low oxygen since the mitochondrion is a complex metabolic hub that not only provides energy for the cell but is also crucial for the generation of reducing equivalents (NADH and NADPH) and building blocks for anabolic reactions Metabolomics is a powerful tool developed to systematically analyse the metabolic fingerprint of a cell, widely used in the past to analyze microorganisms metabolism ATCC® Number: CCL-247™) cells either under normoxia (21% oxygen) or hypoxia (1% oxygen) for 36 hours. The chronic exposure to this oxygen tension recapitulates the oxygen levels found in hypoxic tumours The cellular response to hypoxia was studied by culturing HCT116   . Howeverde (KCN) .+) nicotinamide adenine dinucleotide that may affect the cytosolic levels NAD+ required for glycolysis. To assess the levels of NADH in intact cells we took advantage of the spectral characteristic of the molecule, which is fluorescent when excited by ultra-violet light, while its oxidized form, NAD+, is not. Since NADH is the major autofluorescent signal present in cells, confocal real-time imaging of NADH has been widely used to detect changes in NADH oxidation and as readout of mitochondrial respiration +/NADH ratio was observed under hypoxic conditions indicating a more reduced state of the cells under hypoxia  and oxidized  together with the mitochondrial potential-independent probe Mitotracker green (MTG). Using confocal microscopy, cumulative images of sequential focal sections were acquired and z-stacks of the whole microscopic field were generated in order to measure the volume and intensity of the red and green markers. This ratiometric (red/green) quantification was required to reduce possible artefacts arising from changes in fluorescence emission of TMRE due to changes in mitochondrial shape and volume rather than from actual changes in mitochondrial membrane potential. Representative images were obtained by flattening the z-stack into a maximum projection image . To rulechondria . Hypoxicchondria . ImportaOxygen limitation, mitochondrial dysfunction, and the genetic reprogramming initiated under hypoxia are likely to elicit global metabolic changes beyond anaerobic glycolysis. To better understand the metabolic signature of hypoxic cells, we performed an unsupervised endo-metabolomic analysis of normoxic and hypoxic cells using LC-MS. The total ion current of the different samples was used as a confirmation for normalized input . The metThe unsupervised clustering analysis identified 99 annotated metabolites that are different between hypoxia and normoxia . Among tAn important catabolic process which is known to occur under hypoxia is autophagy, the process of cell’s self-cannibalism that maintains nutrient supply under starvation The role of oxygen in the regulation of cellular metabolism has been widely investigated over more than a century. Initially, the cellular adaptation to hypoxia was mainly considered as a rearrangements of metabolite fluxes as a consequence of respiratory impairment m for oxygen of less than 1 µM (∼0.1% oxygen saturation), advocating that mitochondrial metabolism is insensitive to changes in the physiological range of oxygen tensions. However, biochemical changes such as increased lactate production, alterations in the redox state of the cells and in ATP/ADP ratio were demonstrated at oxygen levels at which respiration was unaffected (i.e. >100 µM) Mitochondria are the major oxygen consumers in the cell and are in the heart of several biochemical pathways involved both energy production and the generation of anabolic intermediates required for proliferation. Therefore, changes in mitochondrial function under hypoxia are likely to contribute to the altered metabolic profile of cells, making mitochondria important oxygen sensors. While the genetic reprogramming during hypoxia has been characterized and many players in this process have been identified, the contribution of mitochondria to the hypoxic response is still uncertain. Isolated mitochondria can respire efficiently even at very low oxygen concentration m of mitochondria for oxygen. Changes in respiration at this oxygen levels might be explained either by changes in the Vmax of cytochrome c oxidase Using phosphorescent-quenching oxymetry, respiration of normoxic and hypoxic cells was characterised. This technique allows for the measurement of oxygen consumption of intact hypoxic cells without the need to reoxygenate the sample. The sharp transition from an active respiring stage to a non-respiring stage suggests that cells reach near anoxia before respiration halts, confirming the capacity of mitochondria to respire at very low oxygen levels . Importa+) and hence supporting faster glycolytic rate. In line with that, a dramatic reduction in ATP was observed in hypoxic cells under conditions where autophagy was inhibited  found in hypoxic cell line showed extensive alterations of amino acids and fatty acid/phospholipids metabolism . Among tnhibited . InteresThis hypoxic metabolic profiling revealed a shift in balance between anabolic and catabolic processes that is likely to have important survival consequences. Therefore, unbalancing the anabolic/catabolic ratio under hypoxia, by either stimulating anabolism (e.g. by inhibiting AMPK) and/or inhibiting catabolism (e.g. blocking macroautophagy) is a possible strategy for specifically targeting hypoxic cells in tumours. In support to this hypothesis, the incubation with inhibitors of macroauthophagy such as concanamycin A and bafilomycin A caused significant cell death of hypoxic cells . Taken tDetailed protocols are available on Supporting Information online in the file Figure S1Metabolomic analysis of hypoxic cells. (A) Total ion current of cell extracts obtained from hypoxic or normoxic HCT116 submitted to LC-MS analysis. (B–C) Histograms of the distribution of m/z of metabolites before (B) and after (C) log2 transformation obtained from LC-MS analysis.(TIF)Click here for additional data file.Figure S2Metabolic signature hypoxic cells. (A) Spectral clustering of cells under normoxia or hypoxia. (B) Heatmaps of annotated metabolites with the same trend (upregulated or downregulated in hypoxia) using the indicated restrictions for fold change (f.c.) and p-value.(TIF)Click here for additional data file.Table S1Metabolomic database. Database of annotated compounds detectable by Orbitrap Exactive LC-MS by negative and positive ion mode. RT =  retention time.(XLS)Click here for additional data file.Table S2Metabolic signature of hypoxic cells. List of metabolites changed upon hypoxia. Metabolites with a cut off of fold change of >1.5 or p-value <0.05 are indicated with an asterisk. The metabolic pathways (derived from KEGG) which each metabolite belongs to are indicated. Metabolic pathway coding are as follow: A: amino acid, C: carbohydrate, E: creatine, F: fatty acid/phospholipids, G: glutathione, H: heme, N: neurotransmitter, Nu: nucleotide, O: oxidative phosphorylation, P: pentose phosphate, T: TCA cycle, V: vitamins.(XLS)Click here for additional data file.Methods S1Detailed description of the materials and methods utilized in the manuscript.(DOCX)Click here for additional data file.


In this study we examined the association of two common NRGN risk single nucleotide polymorphisms (SNPs) with functional and structural brain-based intermediate phenotypes for schizophrenia. We obtained structural, functional MRI and genotype data of 92 schizophrenia patients and 114 healthy volunteers from the multisite Mind Clinical Imaging Consortium study. Two schizophrenia-associated NRGN SNPs (rs12807809 and rs12541) were tested for association with working memory-elicited dorsolateral prefrontal cortex (DLPFC) activity and surface-wide cortical thickness. NRGN rs12541 risk allele homozygotes (TT) displayed increased working memory-related activity in several brain regions, including the left DLPFC, left insula, left somatosensory cortex and the cingulate cortex, when compared to non-risk allele carriers. NRGN rs12807809 non-risk allele (C) carriers showed reduced cortical gray matter thickness compared to risk allele homozygotes (TT) in an area comprising the right pericalcarine gyrus, the right cuneus, and the right lingual gyrus. Our study highlights the effects of schizophrenia risk variants in the NRGN gene on functional and structural brain-based intermediate phenotypes for schizophrenia. These results support recent GWAS findings and further implicate NRGN in the pathophysiology of schizophrenia by suggesting that genetic NRGN risk variants contribute to subtle changes in neural functioning and anatomy that can be quantified with neuroimaging methods.The neural mechanisms underlying genetic risk for schizophrenia, a highly heritable psychiatric condition, are still under investigation. New schizophrenia risk genes discovered through genome-wide association studies (GWAS), such as  Twin and family studies suggest a high heritability for schizophrenia , but resneurogranin (NRGN) [NRGN gene was also found to be associated with schizophrenia in three additional independent samples [Genome-wide association studies (GWAS) allow for a hypothesis-free approach to genetic investigations. A recent study of more than 300,000 single nucleotide polymorphisms (SNPs) in 12,945 schizophrenia patients and 34,591 healthy controls reported associations at genome-wide significance with rs12807809 located near NRGN risk variants on brain-based intermediate phenotypes for schizophrenia. Neuroimaging-based intermediate phenotypes are heritable, disease-associated and stable traits that may show a stronger association with risk genes than behavior or diagnosis due to their greater proximity to the underlying biology [In order to understand the role of new genetic markers in disease pathophysiology careful clinical and biological follow up studies are necessary. Studying the effects of risk variants for psychiatric disorders on brain function and structure can provide insight into disease-associated changes and mechanisms on a neuroscience systems level, but also helps to verify GWAS results. Here, we studied the effects of  biology . In fact biology .NRGN protein is an important component in the NMDA-signaling pathway, which is associated with synaptic plasticity and memory formation [NRGN knockout mice display deficits in neural and behavioural correlates of learning and memory [NRGN has been associated with working memory-elicited neural activity in healthy controls [NRGN immunohistochemical staining in working memory-associated areas such as the anterior cingulate cortex (ACC) and the DLPFC [NRGN has been implicated in neuronal cortico- and synaptogenesis during brain development [ormation . In factd memory ,12. Furtcontrols ,14 whilehe DLPFC . Furtherelopment  – both felopment ,18.NRGN with a diagnosis of schizophrenia and cortical development, as well as a reduced NRGN expression in frontal brain regions of schizophrenia patients, the aim of the present investigation was to understand the neurogenetic risk mechanisms of two common NRGN SNPs (rs12807809 and rs12541) and their haplotypes by studying intermediate phenotypes for schizophrenia: abnormal working memory-elicited DLPFC activity and reduced cortical thickness. Because rostral ACC volume reduction, especially in the left hemisphere, has been associated with both, a diagnosis of schizophrenia and NRGN risk variants [NRGN genotype effects on left rostral ACC volume in an additional analysis.Given the evidence for an association of variants ,20, we aDSM-IV) diagnosis of schizophrenia, established using a Structured Clinical Interview for DSM disorders (SCID) and a review of case files by trained clinicians. There were no exclusions based on treatment with antipsychotic drugs. For further details, see SI 1.1 in We studied a total of 206 participants  who enrolled in the multisite Mind Clinical Imaging Consortium study ,22, wereControls were matched to the patient cohort for age, gender, and parental education and were excluded if they had a history of a medical or Axis I psychiatric diagnosis. Participants were excluded if they had a history of neurological or psychiatric disease other than schizophrenia, history of a head injury, history of substance abuse or dependence within the past month, severe or disabling medical conditions, contraindication to MR scanning or an estimated verbal IQ less than 70 (based on the reading subtest from the Wide Range Achievement Test (WRAT-III)).After complete description of the study the participants provided written informed consent. The human research review committees at each of the four sites (Universities of Iowa (UI), Minnesota (UMN), and New Mexico (UNM) and Massachusetts General Hospital (MGH)) approved the study protocol. We confirm that all potential participants who declined to participate or otherwise did not participate were eligible for treatment (if applicable) and were not disadvantaged in any other way by not participating in the study. During the consent process the subjects were asked a series of questions to assure that they understood the nature of the study, that if they chose to participate it was voluntary and that they could stop at any time without affecting their care, and that they understood the risks and benefits of the study. If they stated that they wanted to participate, they were also asked the reason why they chose to participate. If there was any question as to the ability to provide informed consent  then they were not recruited for the study. In addition, if during the clinical interview it was determined that they lacked the ability to provide informed consent, then they were dropped from the study at that time.The Sternberg Item Recognition Paradigm (SIRP) is a working memory task, previously shown to consistently activate the DLPFC and parietal regions in healthy controls and schizophrenia patients . In eachStructural magnetic resonance imaging (MRI) data was acquired with either a 1.5T Siemens Sonata  or a 3T Siemens Trio (UMN). Functional MRI data was acquired with either a 1.5T Siemens Sonata (UNM) or a 3T Siemens Trio . To reduce variability due to acquisition site differences, all sites followed guidelines developed by the biomedical informatics research network (BIRN) test bed, which included standardized acquisition parameters across sites  ,26.http://surfer.nmr.mgh.harvard.edu, for more details, see SI 1.3 in http://www.fmrib.ox.ac.uk/fsl). For additional information about data acquisition and processing, see SI 1.3 in Cortical reconstruction and volumetric segmentation based on high resolution structural MRI scans was performed with the FreeSurfer surface reconstruction software . More quality control measures are given in Table S1 in rs12541=17, nrs12807809=8), C/C and C/T participants were combined into one C allele-carriers group . We hypothesized that TT allele homozygosity in both SNPs would be associated with abnormal working memory-elicited DLPFC activity and reduced cortical thickness.Blood samples were obtained from 255 participants and sent to the Harvard Partners Center for Genetics and Genomics for DNA extraction. All DNA extraction and genotyping was done blind to group assignment. Genotyping was performed at the Mind Research Network Neurogenetics Core Lab using the Illumina HumanOmni-Quad BeadChip. Quality control steps included common standard procedures  using PLBasic demographic characteristics were compared across genotype group and all four acquisition sites using a series of one-way ANOVA and subsequent Bonferroni-corrected post hoc tests. Chi-square statistics were used to examine differences in categorical variables. Alpha was set to 0.05 for all analyses.In our fMRI analyses, we used a Contrast Of Parameter Estimate (COPE) that modeled all working memory loads  during the Probe phase versus fixation. In our main higher level models (referred to as model 1 for rs12541 and model 2 for rs12807809) we tested the effects of genotype of each SNP by fitting a univariate general linear model to the fMRI time course at each voxel in the whole brain to estimate the average activation during the three loads of the probe condition in a whole brain model. Equal weight was given to all loads. All models were cluster-corrected according to FSL default settings (following random field theory) with a z-value of 2.3 and a p-value of 0.05 and controlled for scanner field strength and diagnostic group. We also modeled the diagnosis by SNP interaction effect.http://hapmap.ncbi.nlm.nih.gov) showed that individuals of African ancestry (Hapmap populations: African ancestry in Southwest USA (ASW), Luhya in Webuye, Kenya (LWK), Maasai in Kinyawa, Kenya (MKK), and Yoruban in Ibadan, Nigeria (YRI)) had a much higher rs12541 C allele frequency than all other populations  . No allele frequency differences were observed for rs12807809 . We therefore tested the effect of rs12541 genotype in an additional model (model 1a) on a sample limited to participants of non-African ancestry (n=182).In order to control for potentially confounding effects of population stratification, we checked rs12541 and rs12807809 allele frequencies across population groups. Hapmap3 data (http://surfer.nmr.mgh.harvard.edu/fswiki/FsAverage). Cortical thickness maps were smoothed using a 10mm full-width-at-half-maximum Gaussian kernel. Finally, univariate general linear models were run for each SNP separately (model 3 and 4) at all vertices  per hemisphere. We included age, gender, scanner field strength and diagnostic group into the models as covariates and also tested for the diagnosis by SNP interaction effect. All cortical thickness results were corrected for multiple comparisons using a Monte-Carlo simulation with 10,000 repeats. Vertex-wise threshold and cluster-wise probability (CWP) were set to 0.05. For details, see SI 1.6 in Entire cortex vertex-wise analyses of cortical thickness were performed contrasting rs12541 and rs12807809 C allele carriers vs. TT homozygotes. Briefly, spherical registered cortical thickness data from all subjects were mapped to an average subject  and indices of neural activity for the DLPFC in mean percent signal change (mean %Δ). Haplotype analyses were carried out in Plink. In detail, a standard E-M algorithm was used to impute the distribution of probabilistically-inferred sets of haplotypes for each individual. Then we carried out a linear regression omnibus test with three degrees of freedom, jointly testing all haplotype effects on both intermediate phenotypes (model 5 and 6), covarying for the effects of diagnosis and scanner field strength and additionally for age and gender in the structural model. In case of a significant omnibus test, we carried out haplotype-specific tests to infer the direction of the effects.NRGN in schizophrenia patients and healthy individuals in separate analyses. Statistical analyses were carried out in SPSS 17.0. Power analyses were carried using G*Power 3 [To check for potential medication effects, we used the extracted activation and thickness estimates, regressed out all relevant covariates and correlated these residuals with lifetime exposure to antipsychotic medication estimates. Furthermore, we tested the effects of *Power 3 . AdditioPatients and controls did not differ in age, parental socio-economic status (SES) or handedness, but the percentage of females and participants of European descent among the healthy controls was higher and patients had lower WRAT-IIIRT Scores. There was no effect of acquisition site on gender, WRAT-IIIRT Score or handedness, but sites differed in their participants’ age, parental SES and ancestry .NRGN SNPs, rs12541 and rs12807809, there were no differences between genotype groups with respect to diagnosis, gender, age, WRAT-IIIRT Score, parental SES, handedness, working memory performance, reaction time, and acquisition site  covarying for the effects of scanner field strength and diagnosis. Local maxima were found in areas including the left DLPFC, left insula, left somatosensory cortex and the cingulate cortex .Main effects of task on activation were observed in working memory-associated brain regions such as the DLPFC and parietal regions as described previously ,33. NRGNe cortex . ClusterNRGN rs12541 and diagnosis could be found.In an additional whole-brain model (model 1a) excluding participants of African ancestry from the analysis  and again controlling for the same covariates, the main finding from our initial model could be replicated, i.e. TT homozygotes had increased neural activity in the left DLPFC, left insula, left somatosensory cortex and the cingulate cortex when compared to C carriers  showed that DLPFC activity was higher in participants with the NRGN rs12807809 C carriers showed reduced cortical gray matter thickness compared to TT homozygotes in an area comprising the right pericalcarine gyrus, the right cuneus, and the right lingual gyrus in an entire surface model (model 4) controlling for gender, diagnosis, age and scanner field strength  and no effect of lifetime exposure to antipsychotic medication on cortical thickness in the cluster identified in our surface-wide analysis.In accordance with previous findings we observed widespread bilateral thickness reductions in schizophrenia patients . NRGN rNRGN rs12541-rs12807809 TC as the risk haplotype  supporting our main findings that identified the C allele in rs12807809 as a potential risk allele  confirmed NRGN rs12807809 C carriers displayed reduced left rostral ACC volume compared to TT homozygotes (p=0.047). The average rostral ACC volume was reduced by 6.32% in C carriers (2325.95 mm^3) when compared to TT homozygotes (2482.85 mm^3). Again, no significant interaction between NRGN rs12807809 and diagnosis was found.Additional analysis (model 4a) with a region-of-interest approach revealed that NRGN on two independent schizophrenia-related brain-based intermediate phenotypes in schizophrenia patients and healthy controls. We found that NGRN rs12541 TT homozygotes showed increased neural activity during a working memory task in the left DLPFC and other task-associated areas such as the ACC and the left insula. NRGN rs12807809 C carriers showed regional cortical thinning in the right pericalcarine gyrus, the right cuneus, and the right lingual gyrus as well as reduced left ACC volume. Haplotype analyses further supported the T allele in rs12541 and the C allele in rs12807809 as potential risk alleles.In the present study we used multimodal brain imaging to explore potential effects of the schizophrenia risk gene NRGN and schizophrenia [NRGN [These results support recent findings from a GWA study and other independent case-control studies, which found associations between ophrenia –4. Givenia [NRGN –7 - possDLPFC dysfunction during working memory is a widely acknowledged intermediate phenotype for schizophrenia . CompareAs part of a larger working memory network the ACC and the insula are also involved in many cognitive and attention-related processes ,42. AberNRGN and a functionally defined brain-based schizophrenia intermediate phenotype is in line with the aforementioned GWA and post-mortem brain tissue findings [NRGN variants on brain activation. In their study, healthy controls had to memorize neutral faces for a later recognition trial. NRGN rs12807809 risk allele TT homozygotes had increased neural activity in the ACC during the encoding phase and less deactivation in the left insula during the recognition phase. Similarly, Rose et al. [NRGN genotype effects on brain activity during a spatial working memory task in healthy controls. They reported a failure to disengage ventromedial prefrontal areas in rs12807809 risk allele TT homozygotes, but found no effect on grey or white matter volume. Despite discrepancies with respect to risk SNP and allele , our joint findings suggest that NRGN may be involved in biological pathways, which eventually affect a broader working memory network including the DLPFC.Associations between findings . A studyfindings  also foue et al.  investigNRGN risk variants? NRGN binds CaM and thereby acts as a regulator of downstream CaM-associated pathways which include AMPA receptor insertion into the postsynaptic membrane [NRGN dysfunction could be an imbalance of different glutamate channels impeding working memory processes.What are the potential molecular mechanisms of membrane –52. A dimembrane ,54. ThusNRGN was also associated with cortical thinning in three adjacent occipital areas . Many studies have reported cortical thickness reduction in occipital areas of schizophrenia patients [NRGN has been suggested to play a role in the arborization processes and synaptogenesis in early development. Studies in macaque monkeys suggest that NRGN expression levels peak in early development at around postnatal day 70 [NRGN expression occurred in primary visual areas in the occipital lobe. In adult monkeys NRGN expression levels differ substantially between occipital and other neocortical layers [NRGN disturbances during development.Furthermore, patients ,56, althpatients –60. Heripatients . Corticapatients . Postmorpatients ,18. NRGNl day 70 . This pel day 70 . Moreovel layers . It is tNRGN rs12807809 TT homozygotes and reduced gray matter volume in the ACC in a group of schizophrenia patients of Japanese ancestry but not in controls. Similarly, we found an association between ACC volume and NRGN rs12807809 in our additional analysis. However, in our study C allele carriers had reduced volumes. It is not uncommon that the risk allele structure is inconsistent when comparing different studies and possible reasons for this phenomenon might be multilocus effects, variation in local patterns of LD, population structure of study samples , and environmental exposure differences between study populations [NRGN in brain development.Interestingly, Ohi et al.  reportedulations –67. TakeNRGN gene and risk for schizophrenia, the present results suggest that the studied risk variants may contribute to disease risk via increased DLPFC activation  and decreased cortical thickness as well as brain volumes in specific brain regions. However, the results of the current study indicate that the effect of these genotypes on brain-based phenotypes is not limited to schizophrenia patients. It is possible that other common polymorphisms, rare variants, epigenetic or environmental factors interact with the investigated NRGN polymorphisms and enhance the adverse effect of these variants in vulnerable individuals. If we consider that the investigated NRGN variants were previously associated with schizophrenia and that DLPFC dysfunction and reduced cortical thickness are well validated intermediate phenotypes for schizophrenia, our imaging genetics results support a robust relationship between NRGN and schizophrenia.Given the reported relationship between the NRGN SNPs that have been previously associated with schizophrenia. However, it is also possible that these SNPs are in high linkage disequilibrium with other functional variants which represent the true underlying genetic determinants responsible for the effects described in our paper. Second, the studied SNPs were associated with different brain modalities and the presumably protective allele of rs12807809 , we are currently unable to distinguish completely between the potential effects of antipsychotic medications versus those of the underlying disease process on measures of brain function and structure. However, prefrontal dysfunction and reduced cortical gray matter thickness have been shown to occur in persons with a high risk of developing schizophrenia and among neuroleptic-naïve and very young patients with a first episode of schizophrenia [The findings of our study have to be interpreted in the light of the following limitations. First, we focused on two e et al. ) was relophrenia ,68. Furtophrenia . This imNRGN gene on brain-based intermediate phenotypes for schizophrenia – DLPFC inefficiency during a working memory task and reduced cortical thickness. These results further implicate NRGN in the pathophysiology of schizophrenia and suggest that genetic NRGN risk variants contribute to subtle changes in neural functioning and anatomy which can be quantified with neuroimaging methods. Taken together, our study highlights the effects of schizophrenia risk variants in the File S1Supporting Material and Methods, Results.Table S1. Quality control measure for rs12807809 and rs12541. Table S2, Allele frequencies across populations. Table S3, Results of functional and structural imaging models for rs12541 and rs12807809 respectively. Table S4, rs12541-rs12807809 haplotype analysis results. Figure S1, Power analysis of fMRI models. Figure S2, Additional Model.(PDF)Click here for additional data file.


Auditory verbal hallucinations (AVH) in schizophrenia are typically characterized by rich emotional content. Despite the prominent role of emotion in regulating normal perception, the neural interface between emotion-processing regions such as the amygdala and auditory regions involved in perception remains relatively unexplored in AVH. Here, we studied brain metabolism using FDG-PET in 9 remitted patients with schizophrenia that previously reported severe AVH during an acute psychotic episode and 8 matched healthy controls. Participants were scanned twice: (1) at rest and (2) during the perception of aversive auditory stimuli mimicking the content of AVH. Compared to controls, remitted patients showed an exaggerated response to the AVH-like stimuli in limbic and paralimbic regions, including the left amygdala. Furthermore, patients displayed abnormally strong connections between the amygdala and auditory regions of the cortex and thalamus, along with abnormally weak connections between the amygdala and medial prefrontal cortex. These results suggest that abnormal modulation of the auditory cortex by limbic-thalamic structures might be involved in the pathophysiology of AVH and may potentially account for the emotional features that characterize hallucinatory percepts in schizophrenia. The pathophysiology of positive symptoms in schizophrenia remains a critical and poorly understood area of psychiatry. Auditory verbal hallucinations (AVH), a core positive symptom of schizophrenia, often manifest in the form of emotionally distressing voices. While several studies have successfully corroborated AVH-related activity in brain regions responsible for normal speech perception Converging evidence indicates that the mesolimbic dopaminergic system, normally involved in reward prediction and assignment of salience to environmental stimuli, may be critically altered in individuals with schizophrenia Within the mesolimbic system, the amygdala plays a central role in recognizing emotionally meaningful events and their associations with sensory inputs, including fear conditioning Schizophrenia research has long used emotionally salient stimuli to probe for limbic dysfunction in schizophrenia during emotional challenge. Functional imaging studies using diverse visual paradigms of emotional challenge have revealed abnormal evoked responses in the amygdala of patients hypothesis one) as well as abnormal interactions between the amygdala and auditory cortex representing alterations in the neural interface between emotion and perception systems (hypothesis two).We aimed to investigate emotional and linguistic processing during hallucination-like auditory stimulation in remitted schizophrenia patients with AVH compared to healthy controls. FDG-PET measured accumulated neural activity at baseline and during the perception of aversive verbal stimuli mimicking AVH. We hypothesized that patients with schizophrenia would show an exaggerated sustained response to aversive verbal stimuli in limbic and auditory sensory regions  after 4 weeks of treatment with risperidone 4–9 mg/day in all patients. Prior to this treatment, patients were antipsychotic-naïve. The current report focuses on the remission phase of AVH; FDG-PET findings during the acute psychotic episode have been previously reported for the same patients included here Nine patients meeting DSM-IV criteria for schizophrenia and eight matched healthy controls were recruited at the Hospital Clinic of Barcelona. All participants were right-handed. Only patients who reported prominent, commenting AVH during their first psychotic episode were prospectively included in this study. Additionally, psychotic symptoms remitted  and was clearly audible. The auditory stimulation condition consisted of binaural reproduction of the 30-minute recordings through headphones. Patients were stimulated using the recording based on their own report; control participants were randomly assigned to listen to one of the recordings.We collected self-reports on content and linguistic features of AVH  for each patient. We asked patients to describe as many examples of AVHs as they could remember and transcribed the whole set of example AVHs (mostly short sentences and single words) for each patient, as they were described. Nine unique 30-minute recordings were created, each mimicking individual AVH features for one patient, with the participation of several male and female speakers who read statements (from single words to complex discourse) following each patient's descriptions. Specifically, each recording included all example AVHs, each separated by a silent inter-stimulus interval of 3 s, arranged in a pseudorandom order. Each example was repeated at least twice so as to create recordings of the intended length. AVH content, and therefore the content of the recordings, was largely aversive and involved in all cases critical/derogatory comments of high linguistic complexity in the 318F-FDG (4.7 MBq/kg of body weight). At the moment of the injection, they all had a blood glucose concentration below 130 mg/dl. From at least 10 minutes before FDG administration to 30 minutes after, all subjects were silent and rested in a dark room to avoid visual stimulation. For the auditory stimulation condition, the reproduction started five minutes before FDG injection. Image acquisition began 35 minutes after FDG injection, using a PET scanner . To minimize motion during image acquisition, the subject's head was strapped to a cushioned head rest. A 1-minute emission scan was performed to verify the correct brain position in the field of view (14.5 cm). A 20-minute emission scan and a 7-minute transmission data scan were then acquired using a two-dimensional mode and 283×336 matrix. Slice thickness was 5 mm. Image reconstruction was performed on a SUN workstation , using a 128×128×35 matrix, with a voxel size of 4.29×4.29×4.25 mm3. The ordered-subsets expectation maximization algorithm with 28 subsets and two iterations was used for reconstruction. Attenuation correction was performed using the acquired transmission data. The standardized uptake value was calculated voxelwise for each session from tissue counts on static images, injected dose, and body weight We acquired FDG-PET scans at rest (scan 1) and during auditory stimulation (scan 2) for both study groups. The two scanning sessions took place within a week apart (5±2 days). All subjects fasted for at least four hours before intravenous injection of http://www.fil.ion.ucl.ac.uk/spm). Images were visually inspected for motion artifacts by an expert in quality assurance of PET images. Preprocessing included manual reorientation, affine co-registration of scan 2 to scan 1, joint spatial normalization into MNI  space of both scans and smoothing with a Gaussian kernel .Preprocessing and statistical analysis of the PET images were performed in SPM5 . We accounted for global nuisance and gain effects by including the global covariate (in an AnCova) as one regressor per group (AnCova by Group factor) and using proportional scaling by group Stimulation - ControlStimulation] and [PatientStimulation-Baseline-ControlStimulation-Baseline].The primary aim was to establish differences in relative glucose metabolic rate (rGMR) between hallucination-prone schizophrenia patients and healthy controls by means of a voxelwise, full-factorial analysis. We generated a 2  ×2  linear model in SPM (hypothesis two model) with the same design described for the hypothesis one model except for the addition of one regressor per group corresponding to rGMR extracted from a left amygdala region-of-interest (ROI). We followed a previously described method for assessing “metabolic connectivity” using resting FDG-PET, where mean counts extracted from an ROI are used as a covariate in a regression model to find voxels where activity correlates with the ROI activity across subjects hypothesis one model . A bilateral Heschl gyri ROI was generated with WFU PickAtlas to evaluate direct effects concerning the primary auditory cortex. An exploratory aim was to assess connections between amygdala and prefrontal cortex, given importance of prefrontal-limbic connections in the pathophysiology of schizophrenia To assess group differences in amygdalar correlations with other brain regions, we generated a second factorial model in SPM  according to Gaussian Random Field Theory or voxels significant at pFDR<0.05  are reported. Reported effects for findings related to the primary auditory cortex in which we applied small-volume correction survived a more stringent cutoff of pFWE<0.05 at the voxel level. Local-maxima coordinates were transformed into Talairach space using nonlinear registration http://www.talairach.org/). All coordinates are presented in MNI space and images are presented in neurological convention (left is left).Statistical parametric t maps resulting from contrasts in the linear regression models were thresholded at a height of p15 = 0.46, p = 0.69; % women, 44% in patients, 50% in controls, p = 1, Fisher's exact test). All participants were Caucasian. Patients were maintained on the same dose of risperidone (range: 4−9 mg/day) throughout the current experiment. After a minimum of one-year follow-up in our outpatient clinic, schizophrenia diagnosis was confirmed in all patients. Before symptom remission, patients reported marked (“clear evidence of voices that occur frequently”) to severe  AVH. All patients reported commenting hallucinatory voices with critical/derogatory content. There were no significant differences in sociodemographical features between the study groups , and hypoactivity in the fusiform gyri  relative to controls. In addition, we observed a substantial overlap in the regions of the temporal cortex that responded to stimulation across the study groups : −64 −4 −4). The PatientStimulation-Baseline-ControlStimulation-Baseline contrast did not yield significant findings, nor did the contrast between the groups at rest ([PatientBaseline-ControlBaseline]).As hypothesized, patients showed greater activation in limbic and paralimbic regions, as well as in temporal cortex and other brain regions, relative to healthy controls in the stimulation condition (FWE≤0.002). However, differences between the two groups were also prominent. Group-by-amygdala interaction effects were most prominent in the medial wall of the brain, encompassing bilateral supplementary motor, medial prefrontal, and middle cingulate cortices and precuneus, but were also present in bilateral hippocampi, posterior thalami, right inferior frontal, and right middle posterior temporal cortices  and stronger correlations in patients relative to controls of the left amygdala with posterior regions of the thalamus : −20 −28 4). Moreover, stronger amygdala-auditory cortex interaction was detected in patients with schizophrenia relative to controls .Both groups shared local correlations of the left amygdala bilaterally with the hippocampus, striatum (mostly putamen), globus pallidus, and orbitofrontal cortex, as well as connections with the contralateral amygdala  and 273 (right), cluster-level phypothesis two were driven by medication , among other brain regions, during auditory stimulation per se. However, several arguments weigh against this interpretation. The stimuli in our task were similar (but not verbatim) to the AVH content as reported by the patients and participants had not listened to these particular recordings prior to the experiment. Additionally, most patients had frequent AVH that changed rapidly in content and other characteristics , so their reports, and therefore the stimuli presented during stimulation, might not have represented particularly memorable or salient instances of AVH but instead AVH that happened to occur close in time to the clinical interview. Finally, San Juan et al. Our design was most similar to Ait Bentaleb et al.'s Our analyses revealed abnormal functional connections of the amygdala in patients with AVH. Specifically, the left amygdala displayed abnormally strong connections to the auditory thalamus and the auditory cortex in patients compared to controls. The left amygdala is central to enhancing the perception of aversive verbal material in healthy individuals In addition to these notable differences between patients with schizophrenia and healthy controls, we also found meaningful similarities between the groups. First, both patients and controls engaged a similar region within the temporal cortex in response to auditory stimulation. This common response may imply that the functional organization of the auditory cortex is preserved at least to some extent in patients. Second, in addition to the excess of short-range intra-limbic connections found in patients, the two groups exhibited overlapping connectivity between the amygdala and limbic-paralimbic regions. This relatively preserved functionality of the auditory cortex in conjunction with the abnormal connectivity between the amygdala and auditory regions, may suggest that the key disturbance underlying AVH lies in amygdalo-auditory circuits that modulate activity in the temporal cortex rather than in the auditory cortex itself, consistent with current views of hallucination pathophysiology hypothesis two, our analyses assume fixed group effects of amygdala connectivity and therefore do not account for individual variability. Future studies should seek to infer causality regarding the relationship between dysfunctional limbic connectivity and AVH, and aim to overcome these methodological drawbacks to better understand the amygdalar influence on auditory-cortex function in schizophrenia.There are some relevant limitations to the current study. The small sample size could make our analyses susceptible to a type II error, thus underestimating true differences between groups. While FDG-PET imaging was suitable to achieve our goal of investigating sustained neural activation, its low temporal resolution prevented an assessment of the effects of linguistic and other time-varying features of the verbal stimuli. Additionally, our study did not include a comparison group of schizophrenic patients without AVH, and this prevents us from drawing conclusions specific to hallucination-prone patients. With regard to In sum, our study on AVH replicates previous reports of abnormally elevated neural activity in limbic regions to aversive speech stimuli. In addition, we uncovered abnormal interactions between the amygdala and auditory regions of the cortex and thalamus that persisted after symptom remission. We suggest that an abnormal modulation of the auditory cortex by a limbic-thalamic circuit might underlie the generation of emotional AVH. Further research on limbic-auditory interactions and their role in AVH is warranted.Figure S1Axial view of group effects of auditory stimulation on brain metabolism (rGMR). The t-statistic map is thresholded at p = 0.005 and 10 adjacent voxels and overlaid onto a single-subject T1 scan (MNI Colin brain). Hot colors represent increased rGMR in patients relative to controls. Cold colors represent increased rGMR in controls relative to patients.(TIF)Click here for additional data file.Figure S2Axial view of group differences in functional correlations of left amygdala. The t-statistic map is thresholded at p = 0.005 and 10 adjacent voxels and overlaid onto a single-subject T1 scan (MNI Colin brain). Hot colors represent regions of increased connectivity with the amygdala in patients relative to controls. Cold colors represent regions of increased connectivity with the amygdala in controls relative to patients.(TIF)Click here for additional data file.Figure S3Orthogonal view of group differences in functional correlations of left amygdala. Compared to controls, patients had stronger correlations (orange) between the amygdala and the thalamus-hippocampus (same notation as in (TIF)Click here for additional data file.


Updated World Health Organization guidelines have amplified debate about how resource constraints should impact monitoring strategies for HIV-infected persons on combination antiretroviral therapy (cART). We estimated the incremental benefit and cost effectiveness of alternative monitoring strategies for east Africans with known HIV infection.3 rather than 200 cells/mm3).Using a validated HIV computer simulation based on resource-limited data (USAID and AMPATH) and circumstances (east Africa), we compared alternative monitoring strategies for HIV-infected persons newly started on cART. We evaluated clinical, immunologic and virologic monitoring strategies, including combinations and conditional logic . We calculated incremental cost-effectiveness ratios (ICER) in units of cost per quality-adjusted life year (QALY), using a societal perspective and a lifetime horizon. Costs were measured in 2008 US dollars, and costs and benefits were discounted at 3%. We compared the ICER of monitoring strategies with those of other resource-constrained decisions, in particular earlier cART initiation . Monitoring strategies employing routine virologic testing alone only maximized health benefits at willingness-to-pay levels (> $4400/QALY) that greatly exceeded the ICER of earlier cART initiation.CD4 testing alone never maximized health benefits regardless of resource limitations. Programmes routinely performing virologic testing but deferring cART initiation may increase health benefits by reallocating monitoring resources towards earlier cART initiation. Considerable debate exists about how resource constraints should impact laboratory monitoring for HIV-infected patients on combination antiretroviral therapy (cART) -6. This Published decision models have broadly suggested that laboratory monitoring delivers less favourable value than alternative resource allocations ,10,11. HWe have previously developed and validated a computer simulation model of HIV progression in resource-rich settings -15. Our We used a computer model to simulate alternative laboratory monitoring strategies for HIV-infected patients on cART in east Africa, and to compare the value of these strategies with alternative resource allocation options, such as earlier cART initiation. This model has been previously validated by demonstrating its ability to predict clinical data describing survival, time until cART failure, and accumulation of resistance mutations in distinct observational cohorts -15.This simulation has been revised: 1) to allow specification of a wide variety of possible monitoring strategies; (2) to allow calibration using data from resource-limited settings; and (3) to consider a specifiable number of cART regimens or a specifiable number of drugs within each cART category . The simulation is a stochastic, second-order Monte Carlo progression model that explicitly represents the two main determinates of treatment failure: accumulation of genotypic resistance and cART non-adherence and/or intolerance  of each monitoring strategy. ICERs measure the additive benefit of each strategy compared with its next best alternative, and interpret this benefit together with its additive cost. The ICER compares different choices in a systematic, quantitative manner, placing them "on a level playing field", and providing a widely used quantitative measure of value. Higher ICERs  are less favourable, corresponding to lower value. Lower ICERs  are more favourable, corresponding to higher value. ICERs are useful for informing resource allocation decisions because reallocating resources from a numerically higher (less favourable) ICER towards a numerically lower (more favourable) ICER can increase health benefits without requiring additional resources. We performed all analyses from a societal perspective using a lifetime time horizon. All costs and benefits were measured in US dollar (USD) values for 2008 and were discounted at an annual rate of 3%. In all cases, we followed recommendations of the US Panel on Cost-Effectiveness in Health . We simu3 versus 200 cells/mm3, and whether to make second- and third-line cART regimens routinely available).Considerable debate exists over acceptable value "thresholds" and their appropriate variation with resource constraints . To aid We compared the downstream effects of alternative monitoring strategies on HIV-positive patients newly started on cART. In accord with the USAID-AMPATH experience, we assumed that the first cART regimen was nevirapine in combination with two nucleoside reverse transcriptase inhibitors. Distributions of age, sex and CD4 count at cART initiation were based on characteristics of patients enrolled in USAID-AMPATH Table . We did We evaluated a matrix of different monitoring strategies: type of monitoring ; viral load threshold for switching ; and frequency of monitoring . Clinical monitoring was defined as evaluation by a health professional for signs and symptoms of AIDS . We deliBecause space limitations preclude the presentation of the numerous strategies that we evaluated, we focus on results from the subset of strategies on the efficient frontier. We first identified efficient frontiers for scenarios with two and three available cART regimens. We then identified the efficient frontier for a scenario that does not specify a fixed number of cART regimens, but rather allows the number of available cART regimens to vary.We estimated outcomes of life years, quality-adjusted life years (QALY), and costs (USD). QALYs are a preference-weighted metric that incorporate both quantity and quality of life, and reflect the idea that a year of poor-quality life is valued less than a year of high-quality life .Because some strategies may be sufficiently close to an efficient frontier that their exclusion is solely due to statistical uncertainty (from random variation in the model), we performed sensitivity analyses in which the cost and effectiveness estimates for each strategy were varied over their 95% interpercentile range. In separate sensitivity analyses, to assess the impact of biased inputs, we varied all inputs to the model across their plausible ranges, seeking to identify whether changes in model input assumptions would lead to different strategies on the efficient frontier.3, and we sought to identify strategies on the "efficient frontier" . Monitoring strategies lying outside this "efficient frontier" cannot deliver the greatest benefit regardless of willingness to pay, and therefore should not be preferred choices.We evaluated alternative monitoring strategies: first for a treatment scenario with two available cART regimens, and then a treatment scenario with three available cART regimens. In addition, we considered a scenario that does not specify a fixed number of cART regimens, but rather allows their number to vary. For all scenarios, we assumed that cART would be started at a CD4 count of 200 cells/mmWhen we explored a scenario in which two cART regimens were available Table , no labo3 rather than 350 cells/mm3, the efficient frontier was mostly comprised of monitoring strategies that were structured conditionally, in which CD4 was obtained routinely and a follow-up viral load was only obtained if the CD4 count met WHO criteria for immunologic failure. Conservative monitoring frequencies (12 or six months rather than three months) and switching thresholds  were preferred. In sensitivity analyses, only one strategy employing routine CD4 monitoring alone (every 12 months) was close to the efficient frontier.When willingness to pay remained limited to initiating cART at a CD4 count of 200 cells/mm3 rather than 200 cells/mm3, the efficient frontier continued to be mostly comprised of monitoring strategies that were structured conditionally; however, these strategies now employed less conservative monitoring frequencies and switching thresholds. Only one strategy employing routine CD4 monitoring alone (every six months) was close to the efficient frontier.As willingness to pay rose beyond initiating cART at a CD4 count of 350 cells/mmAs willingness to pay greatly exceeded the value of earlier cART initiation, the efficient frontier became comprised of monitoring strategies that used routine viral load monitoring, with progressively greater frequencies and less conservative switching thresholds.3 rather than 200 cells/mm3 (ICER $2600/QALY), the efficient frontier was comprised of monitoring strategies that were structured conditionally, where CD4 was obtained routinely and a follow-up viral load was only obtained if the CD4 count met WHO immunologic criteria. As willingness to pay greatly exceeded the value of earlier cART initiation, the efficient frontier was comprised of monitoring strategies that used routine viral load monitoring. In sensitivity analyses, no strategy employing routine CD4 monitoring alone was close to the efficient frontier.When we explored a scenario in which three cART regimens were available Table , strategWhen we considered a scenario that does not specify a fixed number of cART regimens, but rather allows the number of available cART regimens to vary Table , strategConditional strategies were no longer on the efficient frontier because the ICER of routinely offering multiple cART regimens (compared with providing one cART regimen only) was fairly high (ICER > $5000/QALY), and because the ICER of any laboratory testing strategy would only be favourable if multiple cART regimens were routinely offered. As willingness to pay exceeded the ICER of offering multiple cART regimens, they were also high enough to support the ICER of routine use of viral load testing.Sensitivity analyses suggested that efficient frontiers were robust to alternative assumptions ".Second, routine viral load testing alone is only a preferred strategy at levels of willingness to pay that far exceed those of earlier cART initiation. In other words, our results suggest that a programme routinely monitoring viral loads, but starting cART at a CD4 of 200 cells/mmThird, when monitoring includes viral load in resource-limited settings, the switching threshold conferring the greatest value is more likely to be 10,000 copies/mL than lower thresholds, and raises the question of whether the recent change in threshold advocated by WHO  is a step in the right direction, especially when the downstream cost burdens of switching first-line regimens to far more expensive, second-line regimens might make it more difficult to simultaneously adhere to other costly changes in its recommendations.Fourth, if programmes are considering alternative monitoring strategies at the same time that they are weighing how many cART regimen options to offer, our results suggest that they can save more high-quality years of life by routinely offering fewer regimens with less intense monitoring strategies, and by reallocating saved resources on earlier initiation of cART.Fifth, the bulk of expenditures from routine viral load testing did not arise from the cost of the viral load test itself, but rather originated from the downstream costs of more frequent switches to expensive second- and third-line regimens. When later cART regimens were assumed to be no more expensive than first-line cART regimens, monitoring strategies that involved routine viral load testing became more favourable. These results suggest that even if viral load tests become cheaper, they may not offer favourable value if there is no change in the relative pricing of different regimens. In contrast, if later cART regimens become less expensive relative to first-line regimens, viral load tests may offer favourable value even if the tests themselves remain expensive.Our estimates for the ICER of cART ($600/QALY) were very similar to other published analyses  ,11. LikeOur analyses have notable limitations. Our simulation is not a transmission model, and therefore does not consider how more conservative monitoring strategies might lead to: (1) delayed detection of antiretroviral resistance and its spread; and 2) higher viral loads in treated patients, which have been associated with increased transmission rates. However, this consideration is unlikely to alter inferences for decision making because the increase in resistance accumulation is likely to be modest (less than one resistance mutation over a five-year period)  because people who are treated earlier will have low viral loads for a longer portion of the time they are infected. In addition, while the simulation is sufficiently detailed to represent clinical differences among subtypes of nucleoside reverse transcriptase inhibitors , it is not sufficiently detailed to represent clinical differences among individual drugs within each subtype .A distinctive strength of our work is that we evaluated a broad matrix of monitoring options, including some strategies that are not guideline recommended at the current time, but which might seem like plausible alternatives to some decision makers . Indeed, the ability to simultaneously evaluate a broad range of monitoring options beyond those currently employed is one of the key methodological strengths of using mathematical modelling in general, and of the current report in particular.In conclusion, our computer simulation suggests that shifting resources away from routine laboratory monitoring and toward earlier initiation of cART has the potential to increase the number of lives saved with HIV treatment in a resource-constrained environment.This work is supported by National Institute of Allergy and Infectious Disease Award UO1AI069911-01 (IeDEA East Africa), US National Institutes of Health. MCB is employed by the US NIH, which provided funding for this study through a grant. The study sponsor had no role in the study design, interpretation of data, the writing of the paper, or the decision to submit the paper for publication.The authors declare that they have no competing interests.RSB designed the study concept. RSB and KN developed the model and KN programmed the model. LD, CTY, BM, SK, MCB and KWK acquired and/or interpreted the data. RSB analyzed results and wrote the manuscript. RSB, CTY, LD, MCB and KWK revised the manuscript. All authors read and approved the final manuscript.Appendix. The Appendix describes in detail the methods underlying the revision of the model and its calibration. The Appendix figures show results of model calibration and results of sensitivity analyses [analyses -29.Click here for file