One of the challenges in the interpretation of studies showing associations between environmental and genotypic data with disease outcomes such as neovascular age-related macular degeneration (AMD) is understanding the phenotypic heterogeneity within a patient population with regard to any risk factor associated with the condition. This is critical when considering the potential therapeutic response of patients to any drug developed to treat the condition. In the present study, we identify patient subtypes or clusters which could represent several different targets for treatment development, based on genetic pathways in AMD and cardiovascular pathology.CFH) on chromosome 1q25 and variants in the ARMS2/HtrA serine peptidase 1 (HTRA1) gene(s) on chromosome 10q26. We conducted a multivariate segmentation analysis of 253 of these patients utilizing available epidemiologic and genetic data.We identified a sample of patients with neovascular AMD, that in previous studies had been shown to be at elevated risk for the disease through environmental factors such as cigarette smoking and genetic variants including the complement factor H gene , and the alleles of These results have significant personalized medicine implications for drug developers attempting to determine the effective size of the treatable neovascular AMD population. Patient subtypes or clusters may represent different targets for therapeutic development based on genetic pathways in AMD and cardiovascular pathology, and treatments developed that may elevate CV risk, may be ill advised for certain of the clusters identified. The current medical literature is increasing weekly with studies identifying DNA variants and their possible interaction with environmental factors that may have impact on risk of disease. The growth of such studies has been spurred by the promise of understanding the genetic and environmental basis of complex diseases, and the possibility of identifying therapeutically responsive targets for drug development. Enormous numbers of DNA variants have been associated with diseases and traits and this number will only grow as it becomes economically feasible to sequence an individual patient's entire genome.within the affected patient population. Even in situations where the association between a risk factor and disease is highly significant, there are individuals with the disease who do not manifest all risk factors and those with risk factors who manifest no disease[One key data interpretation challenge lies in how best to assess the phenotypic heterogeneity and risk factor heterogeneity o disease. The preAge-related macular degeneration (AMD) is an example of a complex disease that has been shown to have clear genetic and environmental antecedents. The leading cause of visual loss in the aging population, neovascular AMD is characterized by the growth of abnormal new blood vessels underlying the retina which can cause severe and rapid vision loss due to hemorrhage and exudation .The general population harbors both modifiable and non-modifiable characteristics associated with AMD, however, the current study examines the afflicted subsample of population rather than at the entire population. Prior epidemiologic characteristics shown to be associated with the risk of AMD include age, gender, elevated body mass index (BMI), hyperlipidemia, hypertension, and cigarette smoking-12. ThesCholesterol and lipid metabolism have been implicated in the pathogenesis of AMD,-32 and tCFH) gene on chromosome 1q25. The CFH gene is known to play a role in the immune/inflammatory system[ARMS2/ HTRA1 genes on chromosome 10q26[Several genes have been associated with all subtypes of AMD, including the advanced stages, with the most strongly associated variants seen within the complement factor H , with many times greater magnitude of effect than important non-genetic factors such as smoking[Recognizing that any patient population with the same disease phenotype will be heterogeneous to some degree for any single risk factor or collection of factors, it is critical that a multivariate or multifactor approach is used to consider risk. Another important consideration in interpreting measures of association is that although the association may be statistically significant, not all cases with the disease will have the risk factor. For example, our group has shown that having two copies of the risk allele (TT) at s smoking. HoweverConsequently, it is reasonable to hypothesize that appropriately designed studies may be able to identify meaningfully distinct subtypes or clusters of patients within the neovascular AMD population on the basis of genetic or environmental characteristics predictive of the risk of disease. If, for example, a pharmaceutical company was developing a drug specifically targeting neovascular AMD that focused on specific genetic and cardiovascular risk characteristics, the actual patient population that might be responsive or benefit from such an agent would actually be a subset of the total, comprising only those patients with that particular risk profile. There may well be overlapping pathophysiological antecedents between risk of cardiovascular disease and neovascular AMD-12,53,54In the present study we examine the genetic and cardiovascular risk characteristics of patients with neovascular AMD in a multivariate segmentation analysis to identify clusters of patients with distinct epidemiologic and genetic risk profiles. To do this, we leverage a clustering analytic approach, a multivariate method that yields groups of individuals who have underlying similarities across a number of different behavioral, attitudinal, and/or demographic characteristics. In the public health sector, standard clustering methods have been leveraged to identify relevant subgroups of individuals with a particular disorder. For example, three distinct subgroups of individuals with obsessive compulsive disorder have been identified. Each group was characterized by pathophysiologic mechanisms and different treatment outcomes, which may have significance in classifying and treating these patients. Other clustering studies have been conducted with suicidal psychiatric patients, substancThe protocol was reviewed and approved by the Institutional Review Boards at the Massachusetts Eye & Ear Infirmary (MEEI), Boston, Massachusetts and conforms to the tenets of the Declaration of Helsinki. Eligible patients were enrolled in this study after they gave informed consent either in person, over the phone, or through the mail, before answering questions to a standardized questionnaire and donating 10 to 50 ml of venous blood.In this study of unrelated neovascular AMD, recruited patients all had a sibling with normal maculae. This is similar to what has been done in prior studies. DetailsPatients were administered a standardized questionnaire in person or via telephone to ascertain smoking exposure, with the age of the patient at the time entry into the study as the cutoff reference age for smoking exposure. Data captured included the age when they started smoking, the age when they quit smoking (if they did quit), and the number of packs of cigarettes smoked per day, on average. From these data the number of pack-years of cigarettes smoked was calculated for each smoker. A pack-year was defined as one pack of cigarettes per day for one year, with one pack defined as twenty cigarettes. To reduce the impact of any extreme outlying observations, a single patient's data were truncated to 140 pack-years.Self reported alcohol consumption was measured as grams of alcohol consumed per week, with 1 can, glass, or bottle of beer considered equal to 12.8 g of ethanol, one 4oz glass of wine equal to 11.0 g of ethanol, and one drink or shot of liquor equal to 14.0 g of ethanol,17. AlcoSelf reported weight in pounds was recorded decade by decade and then converted to kilograms, excluding years of pregnancy from the 20's until the decade of the patient's reference age. BMI was calculated as the current weight divided by the square of the self-reported height in meters at age 25 years. To reduce the impact of any extreme outlying observations in analyses, two patients' data were truncated to a BMI of 40.To classify patients has having a history of either condition, self-reported medication use was captured. Patients were classified as having treated hypertension or hypercholesterolemia if they had any period of at least six months of regular use (at least twice per week) of an anti-hypertensive, or statin or other cholesterol lowering agent.CFH and ARMS2/HTRA1), known independently to have the greatest influences on neovascular AMD risk overall. Of the several significant single nucleotide polymorphisms in each gene that have previously examined in this cohort,[CFH, the marker rs1061170 (Y402H) was selected, with genotype frequencies in the study population of CC 37.2%, CT 46.2%, and TT 16.6%. In prior research, the CC genotype has been shown to be strongly associated with neovascular AMD[ARMS2/HTRA1, the marker rs1049331 was selected, with genotype frequencies in the study population of CC 30.8%, TC 36.0%, and TT 33.2%. The TT genotype has also been shown to be strongly associated with elevated risk of neovascular AMD[For the present analyses, two consistently associated AMD-risk genetic markers (SNPs) were selected for analysis. Their statistical association through family based association testing and conditional logistic regression with neovascular AMD risk has been described in detail previously,59. We fs cohort, we seleccular AMD,60. For The analytic approach used in the present study utilizes techniques that are standard to the pharmaceutical industry in the segmentation of physician and patient populations. In brief, cluster analysis groups data objects together based only on information describing their characteristics or relationships. The goal is that the objects within a group be similar (or related) to one another and different from (or unrelated to) the objects in other groups. Cluster analytic methods have a long history of use in the life sciences. Some forty years ago these methods were used to identify approximately similar subtypes in complex populations ,62. WithTraditional clustering methods fall into two broad categories: relocation and hierarchical. Relocation clustering methods -- such as k-means and EM (expectation-maximization) -- move records iteratively from one cluster to another, starting from an initial partition until an optimal set of clusters is identified. Hierarchical clustering methods proceed in steps -- producing a sequence of partitions in which each one nests into the next partition in the sequence. Hierarchical clustering can be either agglomerative or divisive. Agglomerative clustering starts with singleton clusters (clusters that contain only one record) and proceeds by successively merging the two "nearest neighbor" clusters at each stage. In contrast, divisive clustering methods begin with one single massive cluster that contains all records and then proceeds by successively separating the cluster into smaller ones.In the present case, an industry standard two stage analytic process was used to identify meaningfully distinct clusters of patients. First, a variant of Zhang's BIRCH  algorithm was used to create a preliminary group of clusters. BIRCH iTo be included in these analyses, all patients had to have complete data on all the variables under examination, resulting in a final N for analysis of 253 patients . The group of 253 neovascular AMD patients had a mean age of 73.1 (SD = 7.4) years, and had a female majority (58.1%). The overall mean of total smoking in pack-years for the patient group was 26.7 (SD = 32.3). The overall patient mean decades of alcohol use was 3.3 (SD = 2.2). The overall patient mean for BMI was 26.5 (SD = 4.47). Across all patients, 63.6% were classified as having a history of hypertension and 48.2% were considered to have a history of hyperlipidemia.The results of the segmentation modeling are presented in Table ARMS2/HTRA1, with marked differentiation most evident for the risk genotype TT , though the CC and TC genotypes also significantly varied across clusters. The CFH marker rs1061170 (Y402H) was less sensitive in discriminating between segments. The risk genotype for this marker was only modestly differentiating  relative to the non-risk genotypes.In terms of the genetic markers, the distribution of genotypes for both genes of interest did significantly differentiate patients. The stronger differentiator was clearly the marker rs1049331 in ARMS2/HTRA1 TT risk genotype, and a third (32.4) also carrying the CFH CC genotype , is characterized as a group of patients where the clear majority have a history of both treated high blood pressure and hypercholesterolemia, who tend to be more overweight but slightly younger than the other clusters. Their genetic profile is mixed, with around half (52.1%) carrying the ARMS2/HTRA1 rs1049331 TT risk genotype at all (0.0%), and less than half (41.7%) carry the CFH Y402H CC risk genotype.Similarly, Cluster 2 patients (33.2%) are highly likely to have a history of treatment for hypertension (100%), though just under half have co-morbid hypercholesterolemia treatment histories (45.2%). They tend to be the oldest group of patients and are leaner than two of the other clusters. In terms of the genetic factors, they do not carry the ARMS2/HTRA1 rs1049331 TT risk genotype (83.9%), and a fifth carry the CFH Y402H CC genotype (21.4%).Cluster 3 patients (22.1%) have much better cardiovascular profiles than those in Clusters 1 and 2, and when they have accompanying pathology it is more likely to be hypertension. Specifically, while just less than half have a history of high blood pressure treatment (42.9%), patients in cluster 3 are very unlikely to have been treated for hypercholesterolemia (1.8%). Patients in cluster 3 are highly likely to carry the ARMS2/HTRA1 rs1049331 TT risk genotype (0.0%) but they are the group most likely to carry the CFH Y402H CC genotype (57.1%).The final group, Cluster 4 (16.6%), have the healthiest cardiovascular profile. They differ from Cluster 3 by having a tendency toward hypercholesterolemia rather than hypertension when there is accompanying pathology. None of the neovascular patients in Cluster 4 have a history of treated high blood pressure (0.0%), and only around a quarter (28.6%) have a history of hypercholesterolemia (28.6%). No one in this group carries the ARMS2/HTRA1). This has implications for the design of clinical trials, which may increasingly focus on the inclusion of genetic markers in their data collection protocols.In this analysis, various subtypes of patients with neovascular AMD were identified and the resulting segmentation was driven by both cardiovascular and genetic risk profiles. However, not all factors shared equal weight in creating the patient clusters. Importantly, several risk factors associated with developing neovascular AMD or its progression failed to differentiate clusters of patients with the disease itself. Clearly, modifiable risk factors such as alcohol consumption, body mass index, and cigarette smoking should continue to be a focus of preventive intervention efforts. However, our analysis suggests that once patients have end stage neovascular AMD, patient variability in cardiovascular health, specifically hypertension and hypercholesterolemia, tends to segregate with genotypic risk profiles .This heterogeneity may provide insights to eventual treatment development, or at the very least indicate subpopulations who may be more (or less) responsive to any potential agent targeting a vascular pathology and any genetic networks/pathways that include ARMS2/HTRA1 and/or the pathway it functions in (currently the pathway it functions in is unknown) became a focus of drug development, Cluster 2 patients represent a low-potential group that have poor cardiovascular health, but do not carry the ARMS2/HTRA1 homozygous risk TT genotype. However, Clusters 1 and 3 would represent a higher opportunity target sub-population of AMD patients, as they in total represent 50.2% of the neovascular AMD population, and tend to have high blood pressure in combination with a high likelihood of carrying the ARMS2/HTRA1 TT genotype.Similarly, if ARMS2/HTRA1) may be only suitable for a smaller sample of the population. If, for example, a new treatment carried some risk of elevated cardiovascular events, then it may well be contraindicated for Clusters 1 and 2, and the real market potential may thus only be for Cluster 3.Currently, anti-VEGF therapies delivered via injection  are the best treatments for neovascular AMD. However, it has been proposed that anti-VEGF therapies be contraindicated in those patients with cardiovascular risk factors - particularly high blood pressure . One couIn the future, it may important that efforts to identify druggable targets for potential AMD treatments look beyond bivariate tests of risk associations and take a multi-factorial approach, taking into consideration the fact that patients with the disease are very heterogeneous in their likelihood to have any genetic or cardiovascular profile of characteristics shown to elevate the risk of disease. The patient clusters identified here could reflect differential potential therapeutic targets for pharmaceuticals. Consideration of their profiles would allow drug developers to better design trials to reflect the heterogeneity of the AMD population, recognizing that subtypes of patients identified on the basis of genetic and epidemiological factors may be differentially responsive (or non-responsive or even adversely responsive) to any potential therapeutic agents in development.The authors declare that they have no competing interests.MF, JH, and RD conceived of the study, participated in its design and coordination, participated in the statistical analysis of data and helped to draft the manuscript. MAM participated in the study design and coordination, participated in the statistical analysis of data and helped to draft the manuscript. JWM and IKK participated in the study design and coordination, and helped to draft the manuscript. MMD conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/83/prepub


At birth, lung fluid is rapidly cleared to allow gas exchange. As pulmonary sonography discriminates between liquid and air content, we have used it to monitor extrauterine fluid clearance and respiratory adaptation in term and late preterm neonates. Ultrasound data were also related to the need for respiratory support.Consecutive infants at 60 to 120 minutes after birth underwent lung echography. Images were classified using a standardized protocol of adult emergency medicine with minor modifications. Neonates were assigned to type 1 (white lung image), type 2  or type 3 profiles . Scans were repeated at 12, 24 and 36 hours. The primary endpoint was the number of infants admitted to the neonatal ICU (NICU) by attending staff who were unaware of the ultrasound. Mode of respiratory support was also recorded.A total of 154 infants were enrolled in the study. Fourteen neonates were assigned to the type 1, 46 to the type 2 and 94 to the type 3 profile. Within 36 hours there was a gradual shift from types 1 and 2 to type 3. All 14 type 1 and 4 type 2 neonates were admitted to the NICU. Sensitivity was 77.7%, specificity was 100%, positive predictive value was 100%, negative predictive value was 97%. Four type 1 infants were mechanically ventilated.In the late preterm and term neonate, the lung ultrasound scan follows a reproducible pattern that parallels the respiratory status and can be used as a predictor of respiratory support. The fetal lung is filled with fluid actively secreted by the pulmonary epithelium on a chloride ion gradient . At birtAt present, there are no good techniques to follow the gradual passage to an air filled lung of a newly born infant and much relies on clinical and radiological signs.et al. found a close correlation between the dry/wet ratio in the minipig lung and the number of ultrasound lung comets also known as B-lines [In an experimental animal model, Jambrik  B-lines .In adult medicine, lung ultrasound scan has been successfully used to monitor the reverse change, that is, from a dry to a wet lung . This stThe present study explores the potential of ultrasound as a monitoring tool of fluid to air passage in the neonatal lung. Ultrasound data suggesting a deranged changeover are also correlated with the need for respiratory support. Our approach, then, differs from previous studies performed mostly on preterm babies looking at the sonographic diagnosis of specific causes of respiratory distress -10.Normal lung tissue scanned with ultrasound yields the superficial image of the pleural line rhythmically moving (the lung sliding sign) and horizontal repetition artifacts known as 'A lines'. Fluid accumulation in the alveolar interstitial space generates 'B lines', that is, comet-like, vertical artifacts . LichtenIn the present study, we have modified the previous classification into three profiles:Type 1 - full hyperechoic image of the lung fields or 'white lung' Figure ; Type 2 The investigation was carried out at the well baby nursery and neonatal ICU (NICU) of the University "Federico II" of Naples, the largest regional delivery center with 2,400 live births/year. The study was approved by the local ethics committee (AOU "Federico II") and parental consent was obtained. As a general policy, newly born babies were brought to the Nursery. Infants were admitted to the NICU if signs and symptoms of RD  were present beyond the normal observation time of four hours; it was the responsibility of the attending physician, who was unaware of infants being scanned, to set the indication for respiratory support.The purpose of the study was to evaluate the relative distribution of the three profiles and document the eventual transition from types 1 and 2 to type 3 with serial scans; to correlate the persistence of significant fluid as assessed by ultrasound with clinical outcome expressed as NICU admission rate, use of supplemental oxygen, nasal continuous positive airways pressure (N-CPAP) and mechanical ventilation. Clinical decisions were taken by a separate team of attending physicians, blinded to the study, operating according to the local NICU protocols. All live neonates with gestational age ≥34 weeks were included in the study: 112 (72.8%) full-term neonates (range 37 to 42 weeks) and 42 (27.2%) pre-term neonates (range 34 + 1 to 36 + 6 weeks); 120 (55%) had a birth weight greater than 2,500 grams, while 34 (22%) weighed less; 106 (68.8%) infants were delivered by cesarean-section while 48 (31.1%) were born vaginally; 107 (69.5%) babies were not given antenatal steroids whereas 40 (26%) received the treatment. Newborns with major congenital malformations and/or intrauterine growth retardation (IUGR) were excluded. Scans on the anterior and lateral chest walls of both lungs in supine infants were acquired in the Nursery by a single neonatologist (AS or FM) who then sent the acquired image to the Radiology Department in a separate building. The pediatric radiologist (GV) reviewed and classified the scans according to the study protocol. After completion of the study, concordance was assessed between the evaluation of the initial operator, who could not avoid observing the infant, and the pediatric radiologist who was fully masked to the infants' clinical conditions. Any clinical decision (NICU admission and type of treatment for respiratory distress) was taken by a third party physician, unaware of the present study. In our hospital, the Nursery and the NICU are separate environments attended by different medical staff. The infants were scanned in the absence of the physician in charge, taking extra care not to report the results of our investigation.A broadband linear transducer  which encompasses the superior and inferior lung fields in the same image was used. All infants had the first scan performed no sooner than one hour and no later than two hours after birth. Scans were then repeated at 12, 24 and 36 hours.The study was conducted in a level III hospital with 2,400 total births per year. The population of term and late preterm infants was 2,200 with 11% admitted to the NICU for RD in 2010. We calculated that a cohort of 150 neonates would be representative of the population given a sampling error <5% with a confidence interval of 95%. When lung ultrasound images had been classified by the neonatologists and by the pediatric radiologist who was blind to the infants' clinical conditions, we investigated the accuracy of the sonographic profile with maximal echodensity (type 1 or white lung) to predict admission to the NICU for respiratory support.We defined true positive (TP) as type 1 and admitted to the NICU; true negative (TN) as type 2 or 3 and not admitted; false positive (FP) as type 1 and not admitted; false negative (FN) as type 2 or 3 and admitted. The specificity of the test was defined TN/(TN + FP); sensitivity was TP/(TP + FN); positive predictive value (PPV) was TP/(TP + FP); negative predictive value (NPV) was TN/(TN + FN). Kappa coefficient was calculated to assess the interobserver variability.One hundred fifty nine neonates were enrolled in the study. Two were excluded for congenital malformations  and three for IUGR. Population characteristics are described in Table Sequential scans in Table The four infants in the type 2 group (8.7%) who were admitted to the NICU with clinical RD and unremarkable chest X-ray received support with N-CPAP and supplemental oxygen. No baby in the type 3 group needed NICU admission or additional respiratory support. Given the above pattern distribution, we calculated the performance of the Type 1 profile in predicting NICU admission: sensitivity was 77.7%, specificity was 100%, PPV was 100% and NPV was 97%. Likewise, we calculated the accuracy of the Type 1 profile in identifying the four HMD/RDS cases . The remaining 14 infants admitted to the NICU were affected by transient tachypnea of the neonate (TTN) and the accuracy of the Type 1 profile was as follows: sensitivity = 71.4%; specificity = 97.1%; PPV = 71.4%; NPV = 97.1%.This study shows that in the term and late preterm infant the early lung ultrasound profile can be classified in distinct patterns correlated to the lung fluid content. We document an evolution to the least hyperechogenic profile that is consistent with a clinically stable baby.et al. described a specific sign of TTN comparing a single sonographic scan with the corresponding chest X-ray [We found no previous approach using serial ultrasound scans to examine the extra-uterine respiratory adaptation in this neonatal population. Most other studies were focused on the preterm baby lung. In an unmasked investigation of 32 preterm infants, Copetti st X-ray . Using tst X-ray . In the st X-ray .Studying less immature neonates, we focused our attention on the clinical outcomes in our series and the practical implications for the clinical neonatologist regardless of nosologic classifications. When compared with neonates at 39 to 40 weeks of gestation, the risk of respiratory failure almost triples at 37 weeks and increases more than tenfold at 34 weeks. Similarly, there is a significant increase in the risk of developing symptomatic TTN  or hyaline membrane disease  .As the regional organization of deliveries is often based on a 'hub and spoke' model, the timely identification of those infants in need of moderate to advanced respiratory support is crucial. Our results can help in this task. Likewise, an early type 3 profile  was always confirmed at 12 hours and never associated with RD; this is also of clinical significance to those clinicians operating in birth centers where additional support equipment is not readily available.We acknowledge some limitations of the present study. First, although an early allocation in the Type 1 group was effective in predicting the need for respiratory support, it did not discriminate between a milder and a more severe condition requiring mechanical ventilation. Second, the neonatologists acquiring the scans could not avoid observing the infant. This, however, was not critical in classifying the images, as assessed by the full agreement with the pediatric radiologist who was masked to the clinical conditions of the neonates.In the late preterm and term neonate, lung ultrasound scan follows a reproducible pattern that parallels the respiratory status and can be used as a predictor of the need for respiratory support. Health care givers working in a low technology setting can use lung ultrasound for early screening of neonates in need of respiratory support.• At birth, lung fluid content can be assessed non-invasively by ultrasound.• Over the first 36 hours, pulmonary adaptation can be monitored with serial ultrasound scans.• Lung ultrasound permits the early and reliable identification of those newborns who fail to adapt and show signs and symptoms of respiratory distress.• Health care givers working in a low technology setting can use lung ultrasound for an early screening of neonates in need of respiratory support.HMD: hyaline membrane disease; IUGR: intrauterine growth retardation; NICU: neonatal intensive care unit; N-CPAP: nasal continuous positive airways pressure; NPV: negative predictive value; PPV: positive predictive value; RD: respiratory distress; TTN: transient tachypnea of the neonate.The authors declare that they have no competing interests.FR conceived the study design and wrote the manuscript. FM and AS performed the ultrasound scans while GV reviewed them as a third, masked observer. AU and AR helped with data management and statistics. LC critically reviewed the manuscript. All authors read and approved the final manuscript.


We describe two patients who did not experience a viral rebound after cessation of HAART which was initiated for progressive disease. CD4 T-cell count remained stable in one patient and progressively declined in the other, despite apparent viral control. We failed to identify any immune activation or genetic markers that could offer an explanation for this unusual "secondary controller" status. But their viruses are clearly less fit compared to viruses from rebounders. Highly active antiretroviral treatment (HAART) has improved life expectancy and quality of life of HIV infected patients . HoweverIn 2008 we identified a patient who, after stopping HAART, had a VL below the detection limit of 1,6log for more than 2 years. After searching the data base of 1,700 HIV infected patients under follow-up at the clinic of the Institute of Tropical Medicine, another patient was identified, who spontaneously controlled VL after cessation of HAART for at least 6 months. We labeled these two patients "secondary controllers" (SC) as opposed to "elite controllers" (EC) who spontaneously control viral replication but are treatment naive.In order to identify correlates of SC status, we compared clinical characteristics, viral factors, genetic traits and immune activation markers in the two SC and patients who rebound after treatment cessation. We used the following criteria to define SC and rebounders: (1) being HIV-1 infected for at least 3 years; (2) HAART started in the chronic progressive phase; (3) HAART discontinued for the first time; and (4) VL below the detection limit of 1.6log at treatment interruption. Pregnant women taking HAART to prevent mother-to-child transmission and patients who started treatment in the acute phase of infection were excluded. SC were defined as patients who met the above criteria and who kept their VL below 3log for at least 6 months after treatment cessation. In order to make sure that the SC were not taking antiretroviral drugs, plasma samples were tested for the presence of protease inhibitors (PI) and non nucleoside reverse transcriptase inhibitors at the Bioanalytical Laboratory of the Catholic University of Louvain.The following parameters were assessed: HIV subtype, CD4 count, VL and number of days on HAART and were compared in SC and rebounders. In addition, we evaluated plasma markers of immune activation, including: neopterin; beta-2 microglobulin (B2M) ; soluble CD14 (sCD14) ; and lipopolysaccharide (LPS) . These measurements were done on stored plasma samples taken before starting HAART (T1), during HAART (T2) and 6 months after stopping HAART (T3). Soluble markers in plasma were measured instead of activation markers on cells (Roberts AIDS 2010), because at our clinic only plasma samples of patients are stored.In order to assess viral factors, the SC provided 100 ml of blood for purification of CD4+ T-cells. One part was used to extract DNA, as described elsewhere , for seq-3 with the viruses, IIIB and BAL as reference strains , and with virus isolated from SC patient 1 and from 4 different rebounders. P24 concentrations were measured by ELISA over time.In addition, we evaluated the relative replication capacity of P1. To assess viral fitness phytohemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) from 3 healthy donors were infected at multiplicity of infection (MOI) 10This study was approved by the Institutional Review Board of ITM and the Ethics Committee of the University Teaching Hospital in Antwerp. Informed consent was obtained from all patients.When searching our database on 1700 patients in regular follow-up, we found that a total of 160 patients had stopped HAART for the first time, after a mean of 25 months of successful viral suppression. Treatment was stopped under medical guidance, mostly because of toxicity and/or because the patient wanted a drug holiday. At least two VL measurements after treatment cessation were available for 124 patients, which enabled us to classify them as "rebounders" or "SC". Median time to rebound (VL > 3log) was 7 weeks and median time to re-starting treatment was 4.5 months. Sixty-eight patients remained off treatment for at least 6 months. By that time all but 2 patients (the SC) had a VL that exceeded 3log. Both SC were negative on the drug assays.Patient 1 was diagnosed with HIV infection in 1985. In 1993 he started treatment with AZT, when his CD4 count was 228/μL. VL testing was unavailable at that time and no plasma samples from 1993 were available at the clinic in Antwerp. The first VL measurement dated from 1999 when the patient was already taking AZT monotherapy. It was 3.4 log and a PI based HAART regimen was started and continued till 2001, when the patient was switched to Nevirapine and 2 nucleoside reverse transcriptase inhibitors (NRTI's). He took this combination till treatment interruption in April 2005, when his CD4 count was 458/μl. In July 2005 the VL was 3.9log but from October 2005 until now it remained below the detection limit. The CD4 count, however, has been decreasing to 205/μL in February 2010 but the patient refuses to be restarted on treatment.Patient 2 was found to be HIV positive in 1997, but was probably infected since 1991. In 1998 her CD4 count was 377/μL and her VL 4.6log. She was started on HAART with a PI based regimen. In 1999 she switched to Nevirapine and 2 NRTIs. Treatment was stopped in March 2006. The VL remained undetectable after treatment interruption, though in April 2009 there was a blip in the plasma VL of 2,9log, which was detected with the Cobas ampliprep/cobas Taqman . The patient is still off treatment with a stable CD4 count above 1000/μL.Table Virus measured as p24 in culture supernatant of purified CD4+ T-cells from patients under HAART, typically emerges after 3 to 6 days. In contrast, in patient 1 it emerged on day 21, while in patient 2 virus culture failed despite three attempts. In 2009, however, the latter patient had a blip in plasma virus suggesting that the virus is not completely defective. Moreover the sequences of Gag, Pol and Env in the two SC did not show any obvious defects, and no CD8 T-cell associated escape mutations could be detected. Remarkably, phylogenetic analysis of env from patient 1 revealed the presence of two related but distinct viruses. Both viruses were lacking the lentivirus lytic peptides (LLP) 1 region, but could still be cultured.Autologous viruses from patient 1 could be cultured making it possible to investigate whether the virus of this patient was less fit in comparison to reference strains or virus of 4 different rebounders. Figure We identified two HIV-1 infected patients with an exceptional clinical course after stopping HAART. One patient kept stable and high CD4 T-cell counts for more than 4 years after treatment cessation, while the other patient had progressive disease with decreasing CD4 T-cell counts. Such discrepancy of evolution has also been described in elite controllers -9.To our knowledge there is only one previous study that had identified patients who maintained an undetectable viral load for several months after treatment cessation. Skiest et al mentioned 2 patients who had an undetectable viral load for 6 months after treatment was stopped as part of a study on the effects of treatment interruption. However, they just mention them without comparing them with patients whose viral load rebounded . We are One possible explanation for the SC status is that deleterious mutations have been induced in the virus by HAART. However, sequencing of the major structural genes of the 2 available viruses did not reveal any obvious defects. Virus could be cultivated in patient 1 only, but patient 2 had a spontaneous "in vivo" blip. We hypothesize that these replication competent viruses are less fit and/or are kept under control by the patient's immune system. Indeed, as shown in Figure Markers of immune activation are more disturbed in patients with rapid progression and closer to normal in EC ,13. NeopResearch on EC is very active because these patients may give clues for novel approaches to the development of a preventive HIV vaccine. Research on immune function in the secondary controllers in this study is ongoing; as we believe that they might provide new insights about correlates of protection and be useful for the development of therapeutic vaccines.Written informed consent was obtained from the patients for publication of this case report. A copy of the written consent is available for review by the Editor-in -chief of this journal.The authors declare that they have no competing interests.EVG performed all culture experiments and ELISAs for immune activation, contributed to the study design, data analysis and interpretation of the data; and wrote the paper. LH was responsible for the molecular biological aspects of this study and performed all sequence analyses. LB: performed the experiments to investigate the replicative fitness.SC performed all DNA isolations and amplifications. EF was responsible for the clinical aspects of the study. He identified the patients, abstracted the clinical information and recruited the patients in the study. AB contributed to the study concept, and the writing of the project proposal and the paper. PL performed all the statistical data analyses. GV supervisor and principal investigator of the project, contributed to the study design, data analysis and interpretation of the data. All authors have read and approved the final manuscript.EVG is a postdoc at the Institute of Tropical medicine. During her PhD she was trying to develop a therapeutic vaccination against HIV based on dendritic cells electroporated with mRNA encoding HIV Gag. In this studies patients on HAART were vaccinated with these DC. After vaccination it is the goal that they stop treatment and control the virus. Therefore these patients are so special because they can do it without vaccination. If we can find out why than we know to which parameter we have to look for protection in a therapeutic vaccination. AB, EF, GV are clinicians. EF is really working in the clinic and seeing patients. AB and GV are more interested in research. For EF is it also important if we can unravel why these patients control the virus after treatment interruption, maybe more patients can go on drug holiday.


Risk factors such as blood pressure and serum cholesterol are used, with age, in screening for future cardiovascular disease (CVD) events. The value of using these risk factors with age compared with using age alone is not known. We compared screening for future CVD events using age alone with screening using age and multiple risk factors based on regular Framingham risk assessments.Ten-year CVD risk was estimated using Framingham risk equations in a hypothetical sample population of 500,000 people aged 0–89 years. Risk estimates were used to identify individuals who did and did not have a CVD event over a ten-year period. For screening using age alone (age screening) and screening using multiple risk factors and age (Framingham screening) we estimated the (i) detection rate (sensitivity); (ii) false–positive rate; (iii) proportion of CVD-free years of life lost in affected individuals with positive results (person-years detection rate); and (iv) cost per CVD-free life year gained from preventive treatment.Age screening using a cut-off of 55 years detected 86% of all first CVD events arising in the population every year and 72% of CVD-free years of life lost for a 24% false-positive rate; for five yearly Framingham screening the false-positive rate was 21% for the same 86% detection rate. The estimated cost per CVD-free year of life gained was £2,000 for age screening and £2,200 for Framingham screening if a Framingham screen costs £150 and the annual cost of preventive treatment is £200.Age screening for future CVD events is simpler than Framingham screening with a similar screening performance and cost-effectiveness. It avoids blood tests and medical examinations. The advantages of age screening in the prevention of heart attack and stroke warrant considering its use in preference to multiple risk factor screening. Cardiovascular disease  is the commonest cause of death and a major cause of morbidity worldwide Guidelines recommend that primary preventive treatment be based on assessment of absolute risk of cardiovascular events using multiple risk factor algorithms such as the Framingham risk equations, which include age, sex, smoking status, diabetic status, serum cholesterol, and blood pressure To investigate this we compared the efficacy of offering preventive treatment based on age alone (age screening) with Framingham risk estimation (Framingham screening). Using illustrative costs, we also compared the cost effectiveness of these methods.Screening performance was assessed by estimating the detection rate  with positive screening results) for a given false-positive rate  with positive results). For example, in age screening, a 60% detection rate for a 20% false-positive rate at a given age cut-off means that 60% of all individuals in a population with a first CVD event occurring over a specified time period, and 20% of individuals without a CVD event over the same time period, are at or above the age cut-off. Regardless of the specified time period, this is equivalent to detecting 60% of all individuals in a population who have a first CVD event in every calendar year.Estimates of detection rates and false-positive rates were used to compare the performance of age screening with multiple risk factor screening. Obtaining reliable estimates for such comparisons requires a very large population with known CVD risk factor values, and the identification over 10 years of individuals who do or do not have a first CVD event (ie. distinguishing affected from unaffected). A simulation study is the appropriate method of analysis because it generates a large complete dataset that reflects the distribution of age and risk factors in a whole population.A sample of 500,000 individuals aged from 0 to 89 was generated, having the same age and sex distributions as England and Wales (2007) Framingham estimates of the annual risk of a first CVD event  were calculated for each of the 500,000 individuals in the simulated population In our evaluation of Framingham screening, risk assessments are performed either annually or five-yearly from age 40 until an individual's 10-year CVD risk reaches a specified level , after which time the individual remains screen-positive, with no further screening assessments. In age screening, an individual becomes screen-positive when they reach a specified age.Applying these two approaches to the sample population we determined, for specified 10-year CVD risk cut-off levels and age cut-offs, the: (i) detection rate; (ii) false-positive rate; (iii) person-years detection rate, which is the proportion of all CVD-free years of life lost in affected individuals which is lost by those who are screen-positive, ie. CVD-free years of life lost in affected individuals with a positive result divided by the CVD-free years of life lost in all affected individuals. The person-years detection rate is lower than the detection rate for the same cut-off because CVD events in younger people lead to more years of life lost without a CVD event than events in older people. For example, three individuals aged 50, 60 and 70 have life expectancies without a CVD event of 21, 16 and 13 years respectively. If they have a CVD event at 50, 60 and 70, in age screening with a cut-off of age 55, two of these three events (at age 60 and 70) would be detected: a 67% (2/3) detection rate, but the person-years detection rate would be 58% because 29 (16+13) of the 50 years (21+16+13) of CVD-free life lost would be detected.The years of CVD-free life gained were calculated on the basis that a standard dose statin and three half-standard dose blood pressure-lowering drugs administered together, regardless of an individual's cholesterol concentration or blood pressure, would prevent 80% of coronary heart disease deaths and non-fatal myocardial infarctions, and 70% of strokes Cost-effectiveness was estimated by calculating the costs per CVD-free year of life gained using three illustrative costs for annual treatment  and three illustrative costs of the screening assessments . The cost of inviting people to be seen is not included because it is small and applies equally to the different methods. The cost per CVD-free year of life gained was calculated by multiplying the annual cost of treatment by the person-years of treatment plus, for Framingham screening, the cost of a Framingham risk assessment multiplied by the number of assessments, all divided by CVD free life-years gained.We tested the validity of the assumptions that the Framingham risk equations ranked risk correctly in people of all ages, including those under 35 and those 75 and over by comparing the expected performance of age screening based on the expected age-specific incidence of CVD events using the Framingham risk equation with those observed from CVD registry data in England and Wales Framingham screening with annual assessments is, as expected, less cost-effective than five-yearly screening as it involves more screening assessments. For example, with an annual treatment cost of £200 and a Framingham screening cost of £150, the cost per CVD-free life year gained at a 60% person-years detection rate is £3,200 for annual screening compared with £2,100 for screening every five years. The number of screening assessments per CVD-free year of life gained would be, respectively, 23 and 6.About 90% of individuals have concordant results from Framingham screening and age screening. For example, using the above cut-offs (age 55 and 8% 10-year risk using 5-yearly Framingham screening) among affected individuals, 3% would be missed using Framingham but detected using age, and 6% would be missed using age but detected using 5-yearly Framingham. Among unaffected individuals 6% would be classified positive using age but negative using Framingham, and 4% would be positive using Framingham but negative using age. Framingham screening does not identify people at younger ages that have CVD events. Five-yearly Framingham screening with a 20% 10 year risk cut-off would miss only 5% of people over 70 who have a CVD event, but would miss 52% of people with such events in their 50's.The age-specific risk of cardiovascular disease is higher in men than in women, so we explored the use of sex-specific age cut-offs. At a fixed detection rate the age cut-offs would be about 1–2 years younger in men and about 3–4 years older in women than the age cut-off for both sexes combined.Sensitivity analyses were performed in respect of the cost-effectiveness estimates in The simulated population took account of advancing age, but ignored changes in risk factors and habits that affect risk. Allowing other risk factors to vary over the ten year follow-up period had a negligible effect on our results. The simulated population was based on Gaussian distributions of the risk factors but results are robust to changes in these distributions, for example, the results were not altered if all the risk factors were regarded as log-Gaussian. Framingham screening was taken to have started from age 40 as widely practised. Varying the age of starting Framingham screening has only a small effect on screening performance. For example starting at age 30 the screening performance is almost identical but the costs are greater. Starting at age 50 the detection rate and the false-positive rate are both about 2 percentage points less and the costs are less, but not reduced sufficiently to make such screening materially cheaper than an equivalent age screening policy.Our results show that age screening loses little in screening performance compared with multiple risk factor measurement methods and, with appropriately priced preventive treatment, is less expensive. Offering preventive treatment to everyone above a specified age has the advantage of simplicity. It avoids needless worry that would be caused through selecting individuals on account of the results of a personal medical assessment.assessment of risk to the reduction of risk.Age screening avoids the costs and time spent in connection with the measurement and explanation of risk factor levels and avoids having to issue regular invitations for blood tests and medical examinations. With age screening people are not singled out as being at risk other than on account of their age, so those taking preventive treatment are less likely to feel “abnormal” or that they have become patients and possibly given a medical diagnosis. Age screening moves the emphasis from the In multiple risk factor screening, a 10-year CVD risk cut-off of 1 in 5 (20%) has been adopted by the UK government, and recommended by the National Institute for Health and Clinical Excellence Preventive treatment has adverse effects but these are largely minor and reversible. With age screening a higher proportion of people are treated for the same number of cardiovascular events prevented but, given the extremely low incidence of serious adverse effects, the difference is not large enough to influence which screening method to adopt.All methods of screening for CVD involve some people receiving preventive treatment without benefit because they die of another cause without having a CVD event, while others who would benefit do not receive treatment. For a given detection rate the proportions in these two categories are similar with Framingham screening and with screening using age alone. The prediction of CVD events using a Framingham risk assessment is relatively poor even though the Framingham equations were used to determine CVD events. This is because a Framingham risk only determines the probability of having a CVD event, not who actually has an event .The curves in The monetary costs we have used are illustrative and designed to provide an indication of the financial implications arising from the three methods of screening in relation to their efficacy. The costs will vary according to healthcare setting. Some costs associated with both methods have not been considered here, including the initial treatment consultation with a health professional, which does not affect the comparative costs. With Framingham screening, physicians may vary treatment according to the assessment results, and such treatment tailoring is likely to increase costs relative to age screening. Our analysis provides a reasonable indication of the relative cost-effectiveness of the two screening methods using illustrative unit costs.We used the Framingham risk algorithm published in 1991 because it provides risk equations for the three cardiovascular outcomes we specified in this analysis . Framingham risk equations published in 2008 performance was based on a population aged 0–89, but screening programmes would invite people aged about 40 or over for a risk assessment or simply for preventive treatment if about 50–55 or over. Using the whole population in estimating screening performance has several advantages. First, it standardizes the estimates of screening performance and avoids variation arising from the age range selected. Starting at age 40, as in Screening A perceived limitation of this study is that it is based on statistical modelling and not on observed measurements from a cohort of individuals. The modelling is, however, based on observed data used to define the distributions of the risk factors in the population at large. The method is therefore no different from the modelling used in estimating the screening performance of, for example, Down's syndrome in pregnancy Causal CVD risk factors, even in combination, are poor CVD screening tests European guidelines on the prevention of cardiovascular disease In summary, CVD is common and serious. To have a major impact on its incidence a proactive cost-effective public health policy is needed. This should be designed to prevent most CVD events and should simplify access to preventive treatment without making people become patients. Age screening meets these objectives and warrants serious consideration, given its advantages over current methods of cardiovascular disease screening and prevention.Appendix S1(DOC)Click here for additional data file.


Subthreshold depression is highly prevalent in the general population and causes great loss to society especially in the form of reduced productivity while at work (presenteeism). We developed a highly-structured manualized eight-session cognitive-behavioral program with a focus on subthreshold depression in the workplace and to be administered via telephone by trained psychotherapists (tCBT).We conducted a parallel-group, non-blinded randomized controlled trial of tCBT in addition to the pre-existing Employee Assistance Program (EAP) versus EAP alone among workers with subthreshold depression at a large manufacturing company in Japan. The primary outcomes were depression severity as measured with Beck Depression Inventory-II (BDI-II) and presenteeism as measured with World Health Organization Health and Work Productivity Questionnaire (HPQ). In the course of the trial the follow-up period was shortened in order to increase acceptability of the study.The planned sample size was 108 per arm but the trial was stopped early due to low accrual. Altogether 118 subjects were randomized to tCBT+EAP (n = 58) and to EAP alone (n = 60). The BDI-II scores fell from the mean of 17.3 at baseline to 11.0 in the intervention group and to 15.7 in the control group after 4 months . However, there was no statistically significant decrease in absolute and relative presenteeism .Remote CBT, including tCBT, may provide easy access to quality-assured effective psychotherapy for people in the work force who present with subthreshold depression. Further studies are needed to evaluate the effectiveness of this approach in longer terms. The study was funded by Sekisui Chemicals Co. Ltd.NCT00885014ClinicalTrials.gov  Subthreshold depression, variably defined as depressive state failing to reach the diagnostic threshold for major depression and sometimes also called subsyndromal depression or minor depression, is highly prevalent, clinically relevant and societally important.The prevalence of subthreshold depression has been estimated at around 5–20% in the community and in the primary care patients Given this high prevalence and significant morbidities, subthreshold depression represents a substantive burden on the society. Its sufferers also report more days absent from work (absenteeism) and less productivity while at work (presenteeism). Taken altogether, the cost of minor depression may be as high as 160 million US dollars per year per 1 million inhabitants, which is only marginally less than 192 million US dollars per year that is due to major depression Management of subthreshold depression is therefore a matter of clinical and society urgency. However, the effectiveness of the customary medical treatment for this condition, namely pharmacotherapy, has recently been called into serious question. A systematic review of drug treatments for minor depression identified six studies (468 patients) and concluded that there is unlikely to be a clinically important advantage for antidepressants over placebo in individuals with minor depression It is well established that psychological interventions are effective in the treatment of major depressive disorders Management of subthreshold depression is therefore in need of a greater number of high quality studies. The present study focused on subthreshold depression in the workplace, targeting both depression itself and associated decreased productivity, through the novel CBT method of delivery using the telephone. Remote CBT using telecommunication technology appears to be particularly suited to the problem of depression The study was approved by the Ethics Review Committee of Nagoya City University Graduate School of Medicine, and is registered at ClinicalTrials.gov (Identifier: NCT00885014). The protocol for this trial and supporting CONSORT checklist are available as supporting information: see After full explanation of the purpose and procedure of the study, potential participants were given two opportunities to provide written informed consent. At the initial screening, the participants were asked to provide their written informed consent to fill in the screening questionnaire  and K6. Cf. below) and to answer the telephone interview when invited for the ultimate purpose of participating in the intervention study. Those scoring 9 or higher on K6 were then invited for the telephone interview by a trained interviewer using the Composite International Diagnostic Interview, mood and substance sections, and also to fill in the Beck Depression Inventory-II (BDI-II). Finally, those without current major depression, bipolar disorder or substance dependence, not receiving any current treatment for a mental health problem, and scoring 10 or higher on BDI-II were asked to provide their final informed consent to participate in the intervention study.The participants providing their definitive consent were then randomized 1∶1 either to (1) telephone cognitive-behavior therapy (tCBT) or to (2) waiting list by an independent clinical research coordinator (CRC) at the central office. The random sequence was generated independently by a study statistician, stratified for depression severity at baseline (BDI-II score ≤19 or ≥20), presenteeism in the past month (HPQ item5 ≤5 or ≥6) and study site. The random sequence within each stratum was blocked with variable length, unknown to the CRC or to the study principal investigators. The random sequence was managed by a spreadsheet program which reveals the allocation only after a participant is registered by the CRC in order to guarantee allocation concealment.Those allocated to the tCBT started the telephone sessions immediately, and were asked to fill in the end of treatment questionnaires  within one month after completion of the program. Those allocated to the waiting list had to wait for four months, fill in the end of treatment questionnaire and, if they still desired, could receive the tCBT sessions. Those who received these additional tCBT were asked to fill in the follow-up questionnaire after the end of their treatment.Working men and women with subthreshold depression were recruited at 13 factories and offices of a large manufacturing company  in Japan. Inclusion criteria were:Age 20–57 at study entryMen and womenCurrently employed full-time (either regular or temporary)Expected to be employed full-time for 6 months after screeningK6 scores greater than or equal to 9 at screeningBDI2 scores greater than or equal to 10 at screeningThe exclusion criteria were:Major depressive episode in the past month, as ascertained by CIDI Lifetime history of bipolar disorder, as ascertained by CIDIAny substance dependence in the past 12 months, as ascertained by CIDIAny other current mental disorder if it constituted the predominant aspect of the clinical presentation and required treatment not offered in the studyCurrent treatment for a mental health problem from a mental health professionalSick leave for 6 or more days for a physical or mental condition in the past monthExpected to be on pregnancy leave, maternity leave or nursing leave within 6 months after screeningA systematic review of psychological treatments, mostly CBT, for subthreshold depression yielded a Cohen's d of 0.42 (95%CI: 0.23 to 0.60) at post-test No interim analysis was pre-planned as this was a small study and we had anticipated no serious adverse event with our psychotherapeutic intervention.The telephone CBT is a structured, manualized 8-session program adapted from a previously established manual The patient manual, to be shared both by the participant and the therapist, contains all the materials to be covered in each session, with ample space for the participant to write in his/her own examples. A separate therapist manual was prepared, which specifies the actual flow of each session, followed by the therapist's session checklist after each session and sample emails to be sent to the participant between sessions. The emails to be sent to the participant a few days before the next appointed telephone session was meant to serve both as a summary of the previous session, prompter for the agreed-upon homework and reminder of the next session. The therapist manual proved to be a valuable resource in the training of new therapists, with explanation of theoretical background of the tCBT program and pragmatic tips in the actual conduct of its sessions. The participant and the therapist also shared a booklet termed “Activity Pocketbook” which bound all the homework worksheets in a small notebook format that the participant could easily carry around with him/her and jot down his/her self-monitoring results, activity results and automatic thoughts on the go.Each session was designed for completion in 30–45 minutes but actual lengths varied according to participants' and therapists' assessment of need. Sessions were meant to occur at weekly intervals but the scheduling was flexible to accommodate the working population's work schedule, especially in the latter half of the program. Each session began with a brief structured assessment of depressive symptoms with K6, review of the previous session and the homework. The initial session included psychoeducation of the CBT model and provided the rationale of the whole program. Sessions 2–4 focused on increasing pleasant activities through personal experiments Telephone counselors were master-level, doctor-level and postdoctoral clinical psychologists, social workers or nurses with at least 1 year of clinical experiences. We initially invited Dr Evette Ludman, who was one of the original developers of the tCBT program In addition to the structured, manualized style of tCBT and the specific training as described above that the counselors had to complete, the quality of the administered CBT telephone sessions was assured by on-going supervision and consultation. All the therapists had to have at least 3 out of their 8 sessions per participant supervised on an on-going basis through auditotaped recordings throughout the study. The CRC was also to check client adherence and notify the therapist and his/her supervisor if (i) more than three weeks had elapsed between sessions or (ii) K6 was 10 or greater for any of the session #2 through #4 and 6 or greater for sessions #5 or afterwards. In addition, whenever any question or need arose, they were able to consult TAF or MH. The counselors' meeting to discuss the progress of each participant's treatment was held every two months.Participants assigned to tCBT or to the waiting list condition were free to seek assistance through the Employee Assistance Program (EAP) which was run by a contracted private company, and which included stress diagnostics and reduction program on the web, telephone consultation, and email consultation. Furthermore, they were also free to seek professional help outside the company such as professional counselors and medical doctors.The Beck Depression Inventory, originally published in 1961 We administered BDI-II at baseline, at 4-month follow-up (end of treatment), and at 8-month follow-up (for those who were on the waiting list and received the tCBT). BDI-II is the primary outcome of this study.th-99th percentile range of the general population distribution because it is known that between 5–10% of the population suffer from mental disorders at any point in time. Only items displaying constant psychometric characteristics across sociodemographic variation are included in the final model. K6 has been found to work as well as and better than some widely used screening questionnaires K6 is a recently developed very short (6-item) self-report questionnaire to screen for common mental disorders We administered K6 as the initial screening instrument. The cutoff of 8/9 was selected according to the Japanese calibration study The World Health Organization Heath and Work Performance Questionnaire (HPQ) is a self-report instrument designed to estimate the workplace costs of health problems in terms of self-reported sickness absence (absenteeism) and reduced job performance (presenteeism). Validation studies have found documented significant associations (r = 0.61 to 0.87) of HPQ work hours assessments with payroll records The HPQ measures of presenteeism are based on the following two questions: “On a scale from 0 to 10 where 0 is the worst job performance anyone could have at your job and 10 is the performance of a top worker, how would you rate the usual performance of most workers in a job similar to yours?” and “Using the same 0-to-10 scale, how would you rate your overall job performance on the days you worked during the past 4 weeks?” The absolute presenteeism score is obtained by multiplying by 10 the respondent's response to the second question. The relative presenteeism score is obtained by dividing the first response by the second response, multiplied by 100.We administered HPQ at screening, at 4-month follow-up (end of treatment) and at 8-month follow-up. Our primary outcomes included the two presenteeism scores as defined by the HPQ as well as the actual hours worked in the past 4 weeks.The CIDI is a widely used fully-structured diagnostic interview for assessing mental disorders, to be used with the general population by trained lay interviewers The participants' satisfaction with the program  was measured with a single-item question “How satisfied are you with the stress management welfare program as provided by your company?” rated between 1 = “Very unsatisfied” through 6 = “Very satisfied.” The program here referred to EAP+tCBT in the intervention group and EAP alone in the control group.We also asked a few additional ad hoc questions to those who received the tCBT, including “Were you satisfied with the telephone counseling?”, “Would you recommend this program to other people?” and “Would you like to do the program again when you feel under stress?”, on a 6-level Likert scale between 1 = “Very unsatisfied” through 6 = “Very satisfied.”We compared the means in the control and intervention groups at the 4-month follow-up using a t-test, permutation test, and a maximum likelihood mixed-effects model. The permutation test was used to get an exact distribution for the t-statistic based on a Monte-Carlo simulation with 1,000,000 replications. Permutations were done within the four strata defined by baseline BDI and baseline absenteeism. The mixed model analysis, a type of repeated measures analysis, includes all randomized individuals, including those with missing outcome data at month 4 and it accounts for the within-person correlation between baseline and follow-up. In this model we adjusted for baseline covariates including BDI-II, absenteeism, site, age and gender. The maximum likelihood mixed model accounts for missing data provided that the data are MAR (missing-at-random) conditional on the covariates and the baseline values of the outcome. We decided a priori to make the mixed model our primary analysis since it is intention-to-treat and includes all randomized individuals in the analysis.For the overall satisfaction score, which did not have the baseline measurements, we used regression (rather than mixed models) to compare month 4 treatment means adjusting for the stratification variables, age and gender.All the analyses were performed with SPSS Version 18.0 One major change made to the protocol after trial commencement is the shortening of the waiting period. Originally it was planned that the waiting period would be 15 months but the initial low participation rate prompted us to shorten the waiting period to 4 months after five participants had been enrolled, in order to increase acceptability of the study.We also had to stop recruitment after we entered 118 patients after 18 months of trial commencement. The participation rate turned out to be much lower than we had anticipated. We calculated that this sample size would still allow us to detect an effect size of 0.5 or greater at an alpha error of 0.05 and a beta error of 0.20.Altogether we screened 4916 working men and women at 13 factories and offices between June 2009 and December 2010. 3105 (63.2%) returned the screening questionnaire, of whom 961 scored 9 or higher on K6. Of the latter, 243 were found to satisfy all the eligibility criteria after telephone interviews with CIDI. After the second telephone interview explaining the study purposes and procedures, 118 participants finally gave their written informed consent to be randomized to the intervention or the control arms. There were 10 telephone counselors, each providing tCBT to a median of 6 participants (range: 1 to 10).  completed at least 4 or more sessions. Of the seven who dropped out from treatment prematurely, one had no session, two had one session only, one had two sessions only, and further three had only three sessions. The reasons for dropouts as assessed by the therapists included: two for being too busy at work, one for feeling well and feeling no more need, and four for lack/loss of interest and motivation. The mean (SD) and median number of sessions delivered was 7.0 (2.1) and 8 , and the average length of a session was 56.1 (12.1) minutes.Of the seven who prematurely stopped the treatment, three responded to the 4-month follow-up, while the remaining four did not return the questionnaire. Of the 51 who finished at least half the program, only one did not return the follow-up questionnaire. Thus the overall follow-up retention percentage for the whole experimental arm was 91.4%. Of the 60 who were placed on the waiting list, only one participant did not return the 4-month follow-up questionnaire. (Follow-up percentage 98.3%)During the parallel comparison period, in the EAP group (n = 60), three utilized the job stress diagnostics on the internet page for job-related stress, two used the stress reduction program on the internet page, and one made a telephone consultation. In the EAP+tCBT group (n = 58), five used the job stress diagnostics, and one made a telephone consultation.After the 4-month follow-up, 36 (60.0%) out of the EAP only group chose to receive the tCBT sessions. 35 of them (97.2%) received at least 4 sessions.In terms of the primary outcomes, tCBT showed superiority over its control condition, with an estimated effect size of 0.69 (95%CI: 0.32 to 1.05) for depression severity as measured with BDI-II. The secondary outcomes corroborated the primary results in that the tCBT condition provided benefit in terms of overall psychological distress as measured with K6 .The overall satisfaction score for the services was 3.57 (3.31 to 3.81) for the EAP alone group and 4.42 (4.17 to 4.67) for the tCBT +EAP group (p<0.001).The evaluation of the tCBT program was satisfactory with at least 85% showing positive responses to the questions “Were you satisfied with the telephone counseling?”, “Would you recommend this program to other people?” and “Would you like to do the program again when you feel under stress?” The positive response rate was 90%, 86% and 85%, respectively.No serious adverse event, including suicidality, was reported in either the intervention arm or the control arm.The present study examined the short-term effectiveness of remote CBT using the telephone among working men and women with subthreshold depression up to four months. It reduced depression symptomatology  and general psychological distress  but failed to demonstrate significant improvement in productivity . The program was acceptable in the workplace as the dropout from active treatment was relatively low at 12.1% and the employees who received the telephone CBT were generally very satisfied.Our study therefore added further support to the effectiveness of cognitive-behavioral intervention for subthreshold depression The present study however failed to show a statistically significant improvement in presenteeism, as measured by absolute presenteeism, relative presenteeism or total hours worked according to the HPQ. We are aware of two RCTs to date which have demonstrated significant effect on work productivity through enhanced depression treatment including telephone counseling with treatment-seeking workers The strengths of the current study include, first, its explicit screening for subthreshold depression. We have screened the potential participants with two screening questionnaires (K6 and BDI-II) to ascertain their depressive symptomatology and excluded diagnoses of major depression and bipolar disorder through structured interviews (CIDI). Secondly, the quality control of the treatments was rigorous. It followed highly manualized procedures, with a detailed textbook and a workbook to be shared by both the therapist and the participants. With the training and supervision provided for this study, the quality of the therapy was well controlled. Thirdly, the attrition from treatment (10.3%) and missing follow-ups (5.1%) was relatively low in comparison with the previous studies of subthreshold depression or remote CBT which typically ranged around 20–30%. Lastly, we employed the maximum likelihood mixed effects model and accounted for all the randomized individuals, thus adhering to the intention-to-treat principle.There are, however, several weaknesses to this study. First and foremost, the control condition  was weak. It can control for the regression towards the mean, the natural course and Hawthorne effect but not the placebo effect or the non-specific general psychotherapeutic effect. The present study has therefore established the value of adding tCBT to the usual care but not the specific effectiveness of cognitive or behavioral interventions delivered in tCBT. Secondly, due to the nature of the intervention in question, we were able to blind neither the participants nor the therapist to the allocated intervention. Moreover, all our primary outcomes (BDI-II and HPQ) depended on patients' self-report and may have led to overestimation of the treatment effect. Third, our study was relatively short term (up to 4 months) and, due to our study design using the wait list condition, was unable to examine the effect of tCBT in reducing depressive symptomatology in the long term or in preventing future major depressive episodes. The most up-to-date systematic review barely failed to find a statistically significant reduction in future depression through CBT with subtheshold depression . Such effectiveness of CBT is yet to be ascertained. Fourth, our failure to demonstrate statistically significant effect on presenteeism could be accounted for by our lack of statistical power , limited range of improvement possible among our participants , insensitivity of our outcome measure In summary, remote CBT, including telephone CBT, may be able to provide easy access to quality-assured psychotherapy for people in the work force who may present with subdiagnostic psychological distress and cannot readily visit health care services on weekdays. From the viewpoint of service providers, it also means efficient use of the scarce resource, that is well trained CBT therapists, as the remote CBT enables delivery of their CBT from anywhere to anywhere. The average length of a session in our study (56.1 minutes) was somewhat longer than we had originally anticipated but are in line with some telephone-administered CBT programs Checklist S1CONSORT 2010 checklist of information to include when reporting a randomised trial.(DOC)Click here for additional data file.Protocol S1The original protocol in Japanese.(DOCX)Click here for additional data file.Protocol S2English translation of the original Japanese protocol.(DOCX)Click here for additional data file.


In the published article, the beta-actin blot under Figure 2A (cytosolic) was duplicated unknowingly by error with other published papers ; 569, 2010; and Fig. 2B; Sharma and Katiyar, Pharm Res., 27: 1092, 2010). The authors would like to apologize to readers and to the editors for this error.The experiments were repeated in A549 cells with and without treatment with grape seed proanthocyanidins (GSPs) under identical conditions. Cytosolic fractions were subjected to Western blot analysis, and new data was generated to confirm and verify equal protein loading on the gel using antibody against β-actin and to replace the duplicated one. The data obtained confirm the results originally reported in the article. The corrected figure is available here: