Previous results with the planar robot MIT-MANUS demonstrated positive benefits in trials with over 250 stroke patients. Consistent with motor learning, the positive effects did not generalize to other muscle groups or limb segments. Therefore we are designing a new class of robots to exercise other muscle groups or limb segments. This paper presents basic engineering aspects of a novel robotic module that extends our approach to anti-gravity movements out of the horizontal plane and a pilot study with 10 outpatients. Patients were trained during the initial six-weeks with the planar module . This training was followed by six-weeks of robotic therapy that focused on performing vertical arm movements against gravity. The 12-week protocol includes three one-hour robot therapy sessions per week .Pilot study demonstrated that the protocol was safe and well tolerated with no patient presenting any adverse effect. Consistent with our past experience with persons with chronic strokes, there was a statistically significant reduction in tone measurement from admission to discharge of performance-based planar robot therapy and we have not observed increases in muscle tone or spasticity during the anti-gravity training protocol. Pilot results showed also a reduction in shoulder-elbow impairment following planar horizontal training. Furthermore, it suggested an additional reduction in shoulder-elbow impairment following the anti-gravity training.Our clinical experiments have focused on a fundamental question of whether task specific robotic training influences brain recovery. To date several studies demonstrate that in mature and damaged nervous systems, nurture indeed has an effect on nature. The improved recovery is most pronounced in the trained limb segments. We have now embarked on experiments that test whether we can continue to influence recovery, long after the acute insult, with a novel class of spatial robotic devices. This pilot results support the pursuit of further clinical trials to test efficacy and the pursuit of optimal therapy following brain injury. Rather than using robotics as an assistive technology for a disabled individual, our research focus is on the development and application of robotics as a therapy aid, and in particular a tool for therapists. We foresee robots and computers as supporting and enhancing the productivity of clinicians in their efforts to facilitate a disabled individual's functional motor recovery. To that end, we deployed our first robot, MIT-MANUS  that nurture has a significant impact in speeding motor recovery of the paretic shoulder and elbow, and that robot therapy is effective in delivering the necessary exercise. This recovery is most pronounced in the trained muscle groups and limb segments. Encouraged by these positive results, we have expanded our project to develop a family of novel, modular robots, designed to be used independently or together to rehabilitate other muscle groups and limb segments. This paper describes two different implementations of a new module developed at MIT that expands the capabilities of MIT-MANUS to include motion in a three-dimensional workspace. We will present both implementations, the basic engineering differences between these modules, and pilot clinical results from their use with stroke patients.Volpe  reportedModularity. From the outset we believed that modularity is essential to success in robotic therapy, particularly in extending the approach to patients suffering from distinct afflictions. Consider, for example, patients undergoing surgery at the wrist  who might not require a device that manipulates the payload of all the degrees-of-freedom (DOF) of the arm. Therapy for these patients requires only the wrist robot [MIT's experience with well over 250 stroke patients has reinforced the importance of one of our core design specifications: st robot -13. ConvAll of our robot modules are specifically designed and built for clinical rehabilitation applications. Unlike most industrial robots, they are configured for safe, stable, and compliant operation in close physical contact with humans. This is achieved using backdrivable hardware and impedance control, a key feature of the robot control system. Each active module can move, guide or perturb movements of a patient's limb and can record motions and mechanical quantities such as the position, velocity, and forces applied.The most profound engineering challenge specific to this family of robots is achieving the dual goals of high force production capability and backdrivability. Each module must be capable of generating sufficient force to move a patient's limb, but it must also itself be easily movable by an elderly or frail patient. Backdrivability is essential in keeping the patient engaged in the task and in allowing him to observe his successful and unsuccessful attempts at motion. Backdrivable hardware also improves the performance of systems controlled by impedance controllers. Achieving backdrivability and high force production, together in a single machine, is often difficult and becomes more so when the robot geometry is more complex.The robot control system is an impedance controller that modulates the way the robot reacts to mechanical perturbation from a patient or clinician and ensures a gentle compliant behavior. Impedance control refers to using a control system  to impose a desired behavior at a specified port of interaction with a robot, in this case the attachment of the robot to the patient's hand. Conceived in the early 1980's by one of the co-authors , it has The MIT-MANUS project was initiated in 1989 with support from the National Science Foundation. MIT-MANUS has been in daily operation since 1994 delivering therapy to stroke patients at the Burke Rehabilitation Hospital. This robot has been extensively described in the literature [The selected design created a highly backdrivable robot capable of delivering therapy in a workspace of 15" by 18" with an end-point anisotropy of 2:1 ratio  and achievable impedances between 0 and 8 N/mm. Note that the static friction is significantly below the just noticeable difference (JNF) for force, which is 7% of the reference force. The robot maximum achievable impedance is above the human perception of 4.2 N/mm for a "virtual wall." The robot is capable of delivering forces up to 45 N although the robot target design aimed at a force of 28 N, which corresponds to the arm strength during elbow extension for a weak woman in seated position .Following the successful clinical trials of MIT-MANUS, a 1-dof module to provide vertical motion and force was conceived and built. The primary goal of this module is to bring the benefits of planar therapy on MIT-MANUS to spatial arm movements, including movement against gravity. The module can be used independently or mounted to MIT-MANUS for movement in a limited spatial workspace. The module can permit free motion of the patient's arm, or can provide partial or full assistance or resistance as the patient moves against gravity. Because the vertical module moves with the endpoint of the planar module when the two are integrated, overall module mass is an important design concern in addition to on-axis mass and friction. Two embodiments are described below. is used to hold the patient's hand in place.One prototype of the 1-dof module was completed at MIT late 2000 and is shown alongside a test stand in Figure This prototype has been fully characterized at MIT ,18 Figu and 5. IThe screw-driven prototype has proven very successful both in standalone operation and mounted at the end of the planar module enabling spatial movement therapy in the clinic with compliant and stable behavior. However recent changes in linear motor technology have created the potential to achieve similar outcomes with effective vertical endpoint inertia comparable to the planar MIT-MANUS and much lower friction, without the need for force feedback control. The main change is complete enclosure of the magnets within the motor forcer. While this does not increase the magnetic field strength, it dramatically increases the line integral and concatenates magnetic lines.The practical advantages of converting the spatial system to direct-drive linear motors would be a significant reduction of friction and elimination of backlash. This simplification would also carry through to the control system and controller, as well as affording a reduction in the system's overall dimensions and weight. To determine if the expected friction levels are realistic, we tested Copley Control ThrustTube TB2504. Figures To test the novel vertical module we conducted a pilot study to analyze whether additional anti-gravity training further improves motor outcomes for "graduates" of the planar robot-assisted protocol.Outpatients were included in the study if they met the following criteria: a) first single focal unilateral lesion with diagnosis verified by brain imaging (MRI or CT scans) that occurred at least 6 months prior; b) cognitive function sufficient to understand the experiments and follow instructions ; c) Motor Power score ≥1/5 or ≤3/5 (neither hemiplegic nor fully recovered motor function in the 14 muscles of the shoulder and elbow); d) informed written consent to participate in the study. Patients were excluded from the study if they have a fixed contracture deformity in the affected limb that limited pain-free range of motion. We have found severe tendon contractures around the rotator cuff particularly, in patients with complete hemiplegia for longer than 6 months after stroke. It is reasonable to expect that robotic training for the upper limb would not have an impact on a fixed contracture deformity. Trials commenced only after baseline assessment across three consecutive evaluations, 2 weeks apart, shows a stable condition in three motor impairment scales . Our rationale for administering multiple baseline evaluations is based on an interesting "Hawthorne effect" that we observed in previous subjects ,20. BetwTen (10) community dwelling volunteers who have suffered a single stroke at least 6 months prior to enrollment were enrolled in the pilot protocol. The mean group age was 62 ± 4.3 years old (mean ± sem) with the onset of the stroke occurring 50 ± 8.9 months (mean ± sem) prior to enrollment. Table Patients were trained during the initial six-weeks with the planar module . This training was followed by six-weeks of robotic therapy that focused on performing vertical arm movements against gravity. The 12-week protocol includes three one-hour robot therapy sessions per week  Figure .For shoulder-and-elbow planar therapy, the center of the workspace was located in front of the subject at the body midline with the shoulder elevation at 30° with the elbow slighted flexed. The point-to-point movements started at the workspace center and extended in eight different directions of the compass Figure . A one-hJust as in the planar study , the antIn this pilot, standard clinical evaluations included the upper extremity sub-test of the Fugl-Meyer Assessment  from which we derived a Fugl-Meyer score for shoulder/elbow coordination ; a more comprehensive evaluation of motor power or strength in 14 different muscle groups of the shoulder and elbow, using the MRC Motor Power score ; and the Motor Status Score which is divided into two subscales, one for shoulder and elbow movements , and a second for wrist and hand abilities  ,3,24,6,7We have completed the study with ten (10) outpatients. Nine (9) outpatients who completed the planar training protocol at the Burke Rehabilitation Hospital were enrolled for an additional six-weeks with training three sessions per week on the vertical module. One (1) naïve outpatient completed only the six-week training on the vertical module (no previous exposure to the planar training). Table Results of this pilot suggested that the vertical protocol was safe and well tolerated by the patients with no patient presenting any adverse effect . Furthermore, pilot results suggested an additional reduction in shoulder-elbow impairment following the anti-gravity vertical training. In fact, for these 9 patients the reduction in impairment during the vertical training phase was comparable to the reduction during the planar phase . As mentioned earlier, we reliably incorporated therapists' feedback during the design phase of the protocol and have not observed increases in muscle tone or spasticity, as indicated by the Modified Ashworth scale, during the anti-gravity training protocol . Note also that consistent with our past experience with the persons with chronic strokes, there was a statistically significant reduction in tone measurement from admission to discharge of performance-based planar robot therapy.While the benefits from the additional anti-gravity therapy did not achieve statistical significance, this is likely due to the small sample size (9 outpatients). We anticipate that a modest increase of sample size will demonstrate statistical improvement for the shoulder and elbow of the anti-gravity training across the three clinical scales and we plan to commence trials shortly . We have now embarked on experiments that will test whether we can continue to influence recovery, long after the acute insult, with a novel class of robotic devices.It is broadly accepted that outcome measures do not significantly change in persons with chronic motor impairments more than six months from stroke onset. However, recent task specific training programs have resulted in improved motor performance in persons with chronic stroke. For example, our trials with persons with chronic stroke-related impairments showed that planar robotic training contributed to statistically significant improvements in motor abilities -10. If sOur pilot results and novel robotic modules provide a proof of concept that supports our engineering efforts, as well as further clinical trials to test efficacy. We found that the protocol was safe and well tolerated by the patients with no patient presenting any adverse effect . Furthermore, the pilot results suggested an additional reduction in shoulder-elbow impairment following the anti-gravity vertical training without detrimental changes in muscle tone or spasticity. Therefore we plan to investigate in detail the effect of training each movement component in isolation versus integrated spatial movement, and study its impact on disability. We expect that this will bring us closer to our ultimate goal, efficient delivery of optimal therapy, personalized to serve the individual's needs.


Hyperlipidemia secondary to protease inhibitors (PI) may abate by switching to anti-HIV medications without lipid effects.An open-label, randomized pilot study compared changes in fasting lipids and HIV-1 RNA in 104 HIV-infected adults with PI-associated hyperlipidemia  who were randomized either to a regimen in which their PI was replaced by abacavir 300 mg twice daily (n = 52) or a regimen in which their PI was continued (n = 52) for 28 weeks. All patients had undetectable viral loads (HIV-1 RNA <50 copies/mL) at baseline and were naïve to abacavir and non-nucleoside reverse transcriptase inhibitors.3 in the abacavir-switch and PI-continuation arms, respectively. At week 28, the abacavir-switch arm had significantly greater least square mean reduction from baseline in total cholesterol , LDL-cholesterol , and triglycerides  than the PI-continuation arm, with no differences in HDL-cholesterol . A higher proportion of patients in the abacavir-switch arm had decreases in protocol-defined total cholesterol and triglyceride toxicity grades, whereas a smaller proportion had increases in these toxicity grades. At week 28, an intent-to treat: missing = failure analysis showed that the abacavir-switch and PI-continuation arms did not differ significantly with respect to proportion of patients maintaining HIV-1 RNA <400 or <50 copies/mL or adjusted mean change from baseline in CD4+ cell count. Two possible abacavir-related hypersensitivity reactions were reported. No significant changes in glucose, insulin, insulin resistance, C-peptide, or waist-to-hip ratios were observed in either treatment arm, nor were differences in these parameters noted between treatments.At baseline, the mean total cholesterol was 243 mg/dL, low density lipoprotein (LDL)-cholesterol 149 mg/dL, high density lipoprotein (HDL)-cholesterol 41 mg/dL, and triglycerides 310 mg/dL. Mean CD4+ cell counts were 551 and 531 cells/mm3, substituting abacavir for hyperlipidemia-associated PIs in combination antiretroviral regimens improves lipid profiles and maintains virologic suppression over a 28-week period, and it simplifies treatment.In hyperlipidemic, antiretroviral-experienced patients with HIV-1 RNA levels <50 copies/mL and CD4+ cell counts >500 cells/mm Protease inhibitors (PIs) used as components of highly active antiretroviral therapy (HAART) have been well documented to reduce both the morbidity and mortality associated with HIV infection ,2. Howevn = 46), 46 mg/dL with nelfinavir (n = 21), 31 mg/dL with indinavir (n = 26), and 4 mg/dL with non-PI-containing antiretroviral regimens (n = 28)  400 copies/mL) and the Roche PCR assay Amplicor HIV-1 MONITOR UltraSensitive Version 1.0 (LLOQ 50 copies/mL) . Virologic failure was defined as HIV-1 RNA >1000 copies/mL on two occasions at least 1 week apart. CD4+ cell counts were determined by flow cytometry at baseline, and at weeks 4, 12, 20, and 28.Frequency and severity of all clinical and laboratory adverse experiences were assessed at each visit. A cardiovascular disease risk factor assessment was conducted at baseline only. Body mass index (BMI) and waist-to-hip ratio (WHR) were measured at baseline and at weeks 4, 8, 12, 20 and 28. Three types of waist measurements were conducted: mid, minimal, and umbilicus. Insulin resistance measures (C-peptide and fasting insulin to glucose ratio) were assessed at baseline and at weeks 4, 8, 12, 20, and 28. Leptin and lactate were measured at baseline and week 28.Diagnosis of possible abacavir-related hypersensitivity reaction was to be considered if the following multi-organ signs and symptoms appeared in a patient following initiation of abacavir: fever, rash, gastrointestinal symptoms , lethargy, or malaise with or without concomitant respiratory symptoms , musculoskeletal symptoms , headache, paresthesia, and edema. No rechallenge of abacavir was permitted in patients developing this syndrome. The definition and usual clinical presentation of abacavir-related hypersensitivity has been defined previously ,25.Adherence was assessed by the Patient Medication Adherence Questionnaire-7 (PMAQ-7) . The PMAHealth-related quality of life (QOL) was evaluated using the Medical Outcomes Study 36-item Short Form Health Survey (SF-36) , which pt-test with a two-sided significance level of 0.05, assuming a dropout rate of 20%. This sample size calculation is based on data from NZTA4002, in which the standard deviation of total cholesterol in the nelfinavir/zidovudine/lamivudine group at baseline was 39.5 mg/dL.A sample size of 80 patients per treatment arm was deemed necessary for 80% power to detect a difference of 20 mg/dL in fasting total cholesterol between groups using a two-group The primary efficacy population was the intent-to-treat (ITT) population, which consisted of all patients who were randomized into the study. The safety population consisted of all randomized patients who received at least one dose of study drug. Analysis of covariance (ANCOVA) was used to compare the least squares means (LSM) of the two treatment groups. The LSM represented the mean value adjusted for the average value of the covariate from both treatment groups. LSMs of change from baseline in serum lipids , apolipoprotein B, apolipoprotein E, leptin, C-peptide, glucose, insulin, and lactate were reported. The ANCOVA model included terms for treatment group, baseline value of the variable of interest, PI strata and gender.Two types of analyses were performed with HIV-1 RNA data: the ITT: observed analysis and the ITT missing equals failure analysis (ITT: M = F). In the ITT: observed analysis, only available assessments were used , regardless of whether the patient was still receiving their original therapy. In the ITT: M = F analysis, all missing values were considered as failure. Proportions of patients with HIV-1 RNA <400 copies/mL and <50 copies/mL were compared between treatment groups with a 95% confidence interval (CI) on the difference between proportions.P value of 0.05 was considered statistically significant.Differences in domain scores to the SF-36 and the PMAQ-7 were compared using the Wilcoxon rank sum test. Differences between treatment arms in the incidence of treatment-related adverse events by body system were evaluated by Fisher's Exact test. A 3 in the abacavir-switch and PI-continuation arms, respectively. About two-thirds of the patients were HIV Category A. The abacavir-switch and PI-continuation arms were well matched with respect to mean baseline HIV-1 RNA levels , total cholesterol (244 vs 241 mg/dL), LDL-cholesterol (149 vs 149 mg/dL), VLDL-cholesterol (68 vs 56 mg/dL), HDL-cholesterol (39 vs 42 mg/dL), triglycerides (340 vs 280 mg/dL), CAD risk factors , stavudine (36% vs 42%), lamivudine (32% vs 40%), and zidovudine (6% vs 2%).Of 104 patients enrolled in the study, 52 were randomized to the abacavir-switch arm and 52 to the PI-continuation arm . No patient required efavirenz intensification of their treatment regimen.A similar number of patients withdrew prematurely from the study for the reasons delineated in Table P <0.001), LDL-cholesterol , LDL direct-cholesterol , VLDL-cholesterol , triglycerides , apolipoprotein B , and apolipoprotein E  versus the PI-continuation arm. Over the study period, no significant differences were observed between the abacavir-switch or PI-continuation arms with respect to LDL subfractions (particle size A and major class) or HDL-cholesterol . The abacavir-switch arm showed a trend for greater LSM reduction from baseline in free fatty acids .In the ITT analysis, the abacavir-switch arm experienced greater reductions in total cholesterol Figure  and LDL-P = 0.016). A higher proportion of patients in the abacavir-switch arm had shifts to lower toxicity grades as compared to the PI-continuation arm, whereas a lower proportion in the abacavir-switch arm had shifts from baseline in cholesterol to more severe toxicity grades  goal value (<200 mg/dL) in the abacavir-switch arm (198 mg/dL); however, it remained above this value in the PI-continuation arm (230 mg/dL). The LSM LDL-cholesterol level at week 28 was 133 mg/dL in the abacavir-switch arm and 153 mg/dL in the PI-continuation arm   Figure . A higheP = 0.508; 95% CI ), and <50 copies/mL in 62% [32/52] and 75% [39/52] of patients in these respective groups .In the ITT: M = F analysis, no differences were observed between the abacavir-switch and PI-continuation treatment arms at any time during the study regarding proportions of patients achieving undetectable HIV-1 RNA. At week 28, HIV-1 RNA was <400 copies/mL in 69% [36/52] and 77% [40/52] of patients in the abacavir-switch and PI-continuation arms, respectively  . Three patients (6%) in the abacavir-switch arm were protocol-defined virologic failures. However, one of these patients remained on a 2-NRTI/abacavir regimen after withdrawing from the study and his viral load declined. The second of these patients was noncompliant and discontinued his antiretroviral treatment prior to the confirmation blood draw for virologic failure. The third virologic failure in the abacavir-switch arm had a history of prior dual nucleoside therapy for >1 year.No differences were observed regarding time to virologic breakthrough .At baseline, the mean CD4+ cell counts were 551 and 531 cells/mmP ≤ 0.027 at each week) and in the Physical Effects domain for all visits (P < 0.05) except those at weeks 4 and 28. In answer to the PMAQ-7 questions "it is easy for me to take my medicines at the time I am supposed to" and "my medicines are convenient to take", more patients in the abacavir-switch arm answered "definitely true" at week 28 . In the SF-36, the week 28 QOL scores did not differ between treatment arms for most domains, except Vitality  and Health . There were no differences between treatment arms with respect to any health care utilization parameter.PMAQ-7 results showed no apparent differences between the abacavir-switch and PI-continuation arms in overall adherence at week 28 (90% vs 94%) or in the Social Support, Adaptability, Knowledge and Attitudes, and Memory and Recall domains. However, the abacavir-switch arm had significantly higher scores in the Scheduling and Timing domain for all study visits (P = 0.002) and non-site specific adverse events (P = 0.003) were observed in the abacavir-switch arm. Treatment-related adverse events reported by >5% of patients in the abacavir-switch arm included nausea (12/50 [14%]), fatigue (6/50 [12%]), diarrhea (4/50 [8%]), and depressive disorders (3/50 [6%]). Two patients (4%) in the abacavir-switch arm experienced possible abacavir-related hypersensitivity reactions. Adverse events leading to treatment withdrawal in 7 patients in the abacavir-switch arm included possible abacavir-related hypersensitivity reaction (2), mild nausea (2), mild shortness of breath/tachycardia (1), mild disorientation (1), and combination of mild diarrhea, facial edema/numbness and malaise (1). Hyperlipidemia led to treatment withdrawal in 1 patient in the PI-continuation arm. No differences between the two treatments were observed in body weight, waist-to-hip ratio, BMI, lactate, leptin, glucose, insulin, or C-peptide.Substitution of PIs with abacavir in HAART regimens was generally well tolerated. No significant differences between treatment arms were observed for cardiovascular, endocrine/metabolic, hepatobiliary tract/pancreatic events, lower respiratory events, musculoskeletal, neurology, psychiatric, or skin adverse events, although more gastrointestinal (3), substituting abacavir for hyperlipidemia-associated PIs in HAART regimens improves lipid profiles and maintains virologic suppression over a 28-week period. The lipid findings are consistent with those of other studies in which abacavir was substituted for PIs in HAART regimens [fasting lipids rather than lipids in a non-fasted state (which may confound results). Thus, true differences between abacavir and PI-containing regimens could be determined, and lipid criteria established by the NCEP ATP III could be applied [The results of this study indicate that in hyperlipidemic, virologically suppressed, immunocompetent antiretroviral-experienced patients . A similar phenomenon was observed over 48 weeks in another PI-to-abacavir switch study, CNA30017 . As no cMaintenance of virologic suppression and increases in CD4+ cell count over the 24-week period following the switch to abacavir were expected in view of the lack of change in these surrogate markers previously observed in abacavir-switch studies conducted over 48 weeks to 1 year ,29.The abacavir-containing regimen was generally well-tolerated, and the type and incidence of adverse events (notably fatigue and nausea) and rare occurrence of suspected abacavir-related hypersensitivity reactions were consistent with what has been reported in other clinical trials . New advResults of the PMAQ-7 indicated significantly better Scheduling and Timing scores in the abacavir-switch arm. Significant improvement in this PMAQ-7 dimension was similarly reported at 24 weeks in the PI-to-abacavir switch study, COL30305 . ImproveSwitching to abacavir is just one of several switch strategies that have been investigated to date in an attempt to remedy hyperlipidemia in patients receiving PI-containing HAART. It is acknowledged that the ideal candidate for this switch strategy is a patient started on triple therapy, where pre-existing abacavir resistance is unlikely ,32. Anot3, they were highly immunocompetent and may not be representative of typical HIV-infected patients presenting to their physician with PI-associated hyperlipidemia. The study did not evaluate the influence of NRTI background drugs on lipid changes because the patients remained on the same baseline NRTIs. This could have affected the results because stavudine is known to elevate cholesterol and triglyceride levels [This study had several limitations. As patients in this study had mean CD4+ cell counts >500 cells/mme levels , wherease levels . This sh3 at 12 months). However, the abacavir-switch arm had a significantly lower incidence of adverse events than the efavirenz-switch and nevirapine-switch arms , a lower incidence of neuropsychiatric adverse events than the efavirenz group , and significantly fewer cases of discontinuation of study drug due to adverse events . Overall, NEFA, like our study, indicated that abacavir was an appropriate drug to substitute for PIs in HAART regimens as long as patients were virologically suppressed pre-switch and minimally antiretroviral-experienced. Our study differed from NEFA in that all participants in ESS40003 had PI-associated hyperlipidemia at baseline  and because our study used a less stringent definition of virologic failure .Further valuable clinical information could have been acquired from this study had switch agents in addition to abacavir been used as comparators. In the only study of this type that has been conducted to date – the Nevirapine-Efavirenz-Abacavir (NEFA) Study – a significantly lower proportion of stabilized HAART recipients (HIV-1 RNA <50 copies/mL for = 6 months) switching from a PI to abacavir developed fasting plasma triglycerides >400 mg/dL and plasma cholesterol >240 mg/dL at 12 months compared to treatment groups in which patients switched from a PI to efavirenz or nevirapine . Kaplan-3, substituting abacavir for hyperlipidemia-associated PIs in HAART regimens improves lipid profiles and maintains virologic suppression over a 28-week period, and it simplifies treatment.In conclusion, in antiretroviral-experienced patients with HIV-1 RNA <50 copies/mL and CD4+ counts >500 cells/mmThe author(s) declare that they have no competing interests.JEH, PHK and VCW conceived the study design, PHK, MGS, EDJ, AR, JFO, VCW, JWS reviewed and approved study design, VCW provided the statistical methods for the study and performed the statistical analysis of the results, EH, VCW, JHW, JWS wrote, reviewed and edited the protocol, GEP drafted manuscript and evaluated lipid data previously published in antiretroviral studies described in Background and Discussion, JWF, JEH, PHK, MGS, EDJ, AR, JFO, JEH, JWF, JHW, JWS, ADS-C, VCW reviewed and edited the manuscript, ADS-C, JWF, PHK, MGS, EDJ, AR, JFO enrolled study subjects, ADS-C, JWF, JEH, JHW monitored the study, ADS-C, JWF, JEH, JWS, VCW evaluated the clinical data from the study, ADS-C set up the study at study sites and JEH contributed to secure funding.The pre-publication history for this paper can be accessed here:


The lack of facilities to measure CD4 counts in poor countries impedes instituting rational and effective antiretroviral therapy in these countries. Can a new microchip counting technique help to solve the problem? Measurement of peripheral blood CD4 T lymphocytes is probably the most important laboratory assay for evaluation and monitoring of patients with HIV. The CD4 count is critical for determining the clinical stage of HIV infection, for deciding when to start antiretroviral therapy (ART), for evaluating the efficacy of treatment, and for changing the medications when necessary. Most HIV treatment decisions are therefore based upon the CD4 count .The most common technique for measuring CD4 counts in developed country settings is flow cytometry. Flow cytometers use lasers to excite fluorescent antibody probes specific for various cell surface markers, such as CD3, CD4, and CD8, which distinguish one type of lymphocyte from another.PLoS Medicine . TheThere are, however, a number of unresolved issues in this study that need further clarification before the assay can meet the expectations for becoming a widely used tool in resource-poor settings. Firstly, the study was performed with a prototype apparatus, tailored to meet the requirements of the study, but not yet representing a commercially established and viable production line. Secondly, though the authors state that the price of each CD4 determination will become much cheaper, it is not clear how much each assay will cost in the end, and whether the final cost is realistic in the context of developing countries. It is clear, though, that the actual price of the assay will change once it is widely and consistently used on a large scale. Thirdly, although a few children were tested , the results in this small group remain questionable, and therefore the application of the test to pediatric populations needs further testing. It may well be that application to pediatric patients will require an improved apparatus or improved handling.Despite these reservations, the authors of this study should be commended for addressing an extremely important issue and developing this novel approach for counting CD4 in patients with HIV. Their study may lead to further development of such an apparatus, which is sorely needed for the global fight against AIDS. Such efforts will hopefully be noticed by public funding agencies, leading to the improvement of tools for measuring CD4 counts.


Both recombinant FSH (r-FSH) and highly-purified, urinary FSH (HP-uFSH) are frequently used in ovulation induction associated with timed sexual intercourse. Their effectiveness is reported to be similar, and therefore the costs of treatment represent a major issue to be considered. Although several studies about costs in IVF have been published, data obtained in low-technology infertility treatments are still scarce.Two hundred and sixty infertile women  at their first treatment cycle were randomized and included in the study. Ovulation induction was accomplished by daily administration of rFSH or HP-uFSH according to a low-dose, step-up regimen aimed to obtain a monofollicular ovulation. A bi- or tri-follicular ovulation was anyway accepted, whereas hCG was withdrawn and the cycle cancelled when more than three follicles greater than or equal to 18 mm diameter were seen at ultrasound. The primary outcome measure was the cost of therapy per delivered baby, estimated according to a cost-minimization analysis. Secondary outcomes were the following: monofollicular ovulation rate, total FSH dose, cycle cancellation rate, length of the follicular phase, number of developing follicles (>12 mm diameter), endometrial thickness at hCG, incidence of twinning and ovarian hyperstimulation syndrome, delivery rate.The overall FSH dose needed to achieve ovulation was significantly lower with r-FSH, whereas all the other studied variables did not significantly differ with either treatments. However, a trend toward a higher delivery rate with r-FSH was observed in the whole group and also when results were considered subgrouping patients according to the indication to treatment.Considering the significantly lower number of vials/patient and the slight  increase in the delivery rate with r-FSH, the cost-minimization analysis showed a 9.4% reduction in the overall therapy cost per born baby in favor of r-FSH. In the last decades, follicle-stimulating hormone (FSH) has assumed a central role in ovulation induction and has been shown to be highly effective in achieving ovulation in anovulatory infertile woman as well as in ovulation induction protocols for subfertile, ovulatory women and in superovulation for IVF . VariousRecombinant FSH (r-FSH) and urinary FSH (u-FSH) have been repeatedly compared in trials dealing with superovulation induction for IVF. In some of this studies, r-FSH was reported to yield a better ovarian response with a higher number of retrieved oocytes -5, and aOn the other hand, only a few trials have compared r-FSH and uFSH in subfertile patients undergoing ovulation induction associated with intrauterine insemination (IUI) or timed sexual intercourse -14. The From the economical perspective, two studies calculated the cost-effectiveness of r-FSH and u-FSH treatment in infertile patients undergoing induction of ovulation associated with IUI, and concluded that the urinary preparation was more cost-effective ,18. EvenThe present trial was designed to compare the cost of the therapy with either r-FSH or highly-purified, urinary FSH (HP-uFSH) in patients submitted to ovulation induction. Both normoovulatory women with unexplained infertility and women with CC-resistant polycystic ovary syndrome (PCOS) were randomized in this prospective study. Assuming that the effectiveness of the two drugs in terms of pregnancy rate is similar -18, a coTwo hundred and sixty women belonging to subfertile couples undergoing ovulation induction associated with timed intercourse at the Reproductive Medicine and IVF Unit of the University of Turin were enrolled in this study.2 (mean ± SD 21.5 ± 3.4); all were in good physical and mental health with no history of alcohol and/or drug abuse within the 24 months preceding the treatment. These patients had a history of couple's infertility from at least one year (mean ± SD 2.6 ± 1.5 yrs), primary infertility in about 75% of cases, secundary in about 25%. At a routinary diagnostic workout, patent tubes and normal uterine cavity were documented in all patients by a recent (within 1 year) hysterosalpingography or laparoscopy. A recent (within 2 months) semen analysis of the partner revealed normal semen parameters according to the World Health Organization standards [Their age ranged between 28 and 38 years (mean ± SD 32.7 ± 4.3) and their body mass index (BMI) ranged between 19 and 26 Kg/mBoth normoovulatory patients with unexplained infertility (n = 184) and CC-resistant PCOS (n = 76) were included in the study. In the latter patients, CC resistance was defined as consistent failure to ovulate with incremental dose of CC up to 200 mg/day for 5 days in three previous treatment cycles.® Serono, Switzerland) or HP-uFSH  in women undergoing ovulation induction associated with timed intercourse.This prospective, randomized study aimed to compare the cost of therapy with either r-FSH  as follows. Assuming from evidence of previous studies ,14,17,18The following clinical parameters were used to accomplish a cost-minimization analysis (for details see paragraph below), and were registered as secondary end-points: monofollicular response rate, total FSH dose, cycle cancellation rate, length of the follicular phase; number of follicles between 12 and 17 mm the day of hCG administration; diameter of the leading follicle and endometrial thickness the day of hCG administration, twinning rate, incidence of ovarian hyperstimulation syndrome and delivery rate.The ovulation induction outcome was defined as monofollicular when only one follicle ≥ 18 mm without other follicles ≥ 12 mm diameter was observed the day of hCG administration. When a bi- or tri-follicular development was recorded (two or three follicles ≥ 18 mm without other follicles ≥ 12 mm diameter the day of hCG administration), the treatment was continued and hCG was administered, whereas the treatment cycle was cancelled when more then three follicles developed up to 18 mm diameter or, conversely, no follicle developed to more than 12 mm diameter within 28 days of FSH administration.Ovulation induction was aimed to obtain a monofollicular development and was accomplished using the low dose, step-up protocol starting on day 3 of a spontaneous or progesterone-induced withdrawal bleeding by means of subcutaneous or intramuscular injections of 75 IU/day of either r-FSH or HP-uFSH. If no ovarian response was detected after two weeks, the daily dose was increased to 112.5 IU for one week, and then to 150 IU. The ovarian stimulation was stopped in case of no ovarian response after 28 days of FSH administration.Ovulation induction was monitored by vaginal ultrasound every second-third day starting on day 7–9 of the cycle; in case of no ovarian response with daily dose increase, ultrasound evaluation was postponed by one week.®, Serono, Switzerland) when the leading follicle reached 20 mm diameter. Free sexual intercourses were encouraged from the day of hCG administration onward only in cases when no more than three follicles bigger than 17 mm were observed; otherwise, hCG was not administered and protected sexual intercourse was recommended in order to avoid high-order multiple conception.Follicle rupture was induced by intramuscular injection of 10,000 IU human chorionic gonadotropin  at a daily dose of 200 mg for 12 days starting on day +2 from hCG administration.The luteal phase was supported in every case by vaginal progesterone , as IHS sustains the cost of FSH for these treatments and it is delivered free of charge to patients.The cost of each treatment was calculated considering the cost per vial (75 IU) at the time of the clinical trial, that was equal to 23.40 € and 15.55 € for r-FSH and HP-uFSH, respectively. The economic impact of the other health resources on IHS in this trial (e.g. ultrasound monitoring) was the same for both study groups, and thus was not considered.The final outcome of the cost-minimization analysis was to compare the cost of FSH treatment per delivered baby. Cost-minimization analysis was preferred to cost-effectiveness analysis because it fits better to comparative studies in which the effectiveness of compared treatments is assumed to be similar, as previously reported in this case -18. Senst test for independent data and the χ2 test, setting the significance level at p ≤ 0.05.The JMP software  was used to perform statistical analysis. The parameters calculated for either HP-uFSH and r-FSH-treated patients were compared using the two-tailed Student's A total number of 260 patients were randomized and included in the study, 130 in the HP-uFSH group and 130 in the r-FSH group; the clinical and endocrine characteristics of the patients in the two study groups were homogeneous  were cancelled, whereas 210 were concluded with hCG administration, among which 178 (68.5%) were monofollicular and 32 (12.3%) were bi-/trifollicular  occurred.Results of cost-minimization analysis are shown in Table The technology used to obtain pharmacologically available human FSH has traditionally been extraction and purification from the urine of postmenopausal women. The purification process has been progressively improved, finally yielding a highly-purified urinary FSH (HP-uFSH) with less than 0.001 IU of LH per FSH ampoule and a low amount of proteinic contamination. The increasing spread of assisted reproduction techniques that has taken place all over the world in the last years has rapidly increased the need of bulk amounts of FSH for therapeutic use, and a recombinant technology to get theoretically unlimited amounts of recombinant human FSH (r-FSH) from cultured cells has been successfully developed .In comparison to uFSH or HP-uFSH, r-FSH has an absolute purity , a higher batch-to-batch consistency, and no risk of transmission of infectious diseases . HoweverAlthough r-FSH has been claimed to achieve some advantage in case of IVF treatment  ,8,22,23,In the present study, an FSH low dose, step-up protocol was adopted in order to get preferably monofollicular ovulation (with a few cases having two/three preovulatory follicles); all patients having more than three preovulatory follicles were discouraged to conceive: this minimized the risk of multiple pregnancy and ovarian hyperstimulation syndrome even in high-risk patients (e.g. young CC-resistant PCOS patients). Overall, a high proportion of ovulatory cycles and a high rate of monofollicular ovulation was obtained with either FSH formulation, very frequently (about 90% of cases) in normoovulatory women with unexplained infertility, in about 50% of cases in patients with CC-resistant PCOS, which are known to have a well defined trend toward multifollicular recruitment. In contrast with some of the reports comparing r-FSH to uFSH ,13, we dConsistently to previous reports ,14,17,18Consistently with previously published reports ,13,17,18Since both r-FSH and HP-uFSH allow to achieve comparable results in low-technology cycles, the cost of therapy is a major issue to be considered. In IVF, the overall cost per successful pregnancy resulted to be significantly lower with r-FSH, due to its higher effectiveness . HoweverIn our prospective, randomized trial the total number of FSH vials per delivered baby was about 40% lower among patients treated with r-FSH. For this reason and for the slightly higher delivery rate in the r-FSH group, even considering the higher cost per vial of r-FSH, the final cost per delivery was estimated 9.4% lower with r-FSH. The economical advantage of r-FSH was confirmed even when the confidence interval of delivery rate was considered in a sensitivity analysis; in fact, even after correction, the cost reduction per delivered baby was still ranging from 2.7% to 21.9% in favor of r-FSH.It must be remarked that differently from the previously published cost-effectiveness studies ,18, in oIn conclusion, the present study shows that both HP-uFSH and r-FSH can be safely and effectively used to induce ovulation induction both in normoovulatory patients with unexplained infertility and in CC-resistant PCOS patients. The slightly higher effectiveness of r-FSH in terms of delivered babies seems to compensate for the higher cost per IU, leading to lower final economical costs per delivered baby.


To compare glucose control over 18 months between rosiglitazone oral combination therapy and combination metformin and sulphonylurea in people with Type 2 diabetes.1c of 7.1–9.0% on maximum doses of metformin or sulphonylurea. If on metformin they were randomized to add-on rosiglitazone or sulphonylurea (open label) and if on sulphonylurea to rosiglitazone or metformin. HbA1c was managed to ≤ 7.0% by dose titration. A prospectively defined analysis of glycaemic control on the first 1122 participants is reported here, with a primary outcome assessed against a non-inferiority margin for HbA1c of 0.4%.RECORD, a multicentre, parallel-group study of cardiovascular outcomes, enrolled people with an HbA1c reduction on background metformin was similar with rosiglitazone and sulphonylurea [difference 0.07 %], as was the change when rosiglitazone or metformin was added to sulphonylurea [0.06 %]. At 6 months, the effect on HbA1c was greater with add-on sulphonylurea, but was similar whether sulphonylurea was added to rosiglitazone or metformin. Differences in fasting plasma glucose were not statistically significant at 18 months . Increased homeostasis model assessment insulin sensitivity and reduced C-reactive protein were greater with rosiglitazone than metformin or sulphonylurea . Body weight was significantly increased with rosiglitazone compared with sulphonylurea  and metformin .At 18 months, HbA1c over 18 months, and produces greater improvements in C-reactive protein and basal insulin sensitivity but is also associated with greater weight gain.In people with diabetes, rosiglitazone in combination with metformin or sulphonylurea was demonstrated to be non-inferior to the standard combination of metformin + sulphonylurea in lowering HbA The major therapeutic management goals for people with Type 2 diabetes are achieving optimal control of glycaemia, lipid levels and blood pressure to reduce the risk of long-term vascular complications. Treatment algorithms for the management of glycaemia have advocated a step-wise approach with diet and exercise as first-line management, followed by the addition of oral glucose-lowering agents . Drug mo1c targets, means that increasing numbers of people with diabetes are taking combination therapies. However, whilst clinical trials comparing glucose-lowering drugs in monotherapy are relatively common, longer-term comparisons of new dual-agent combinations with the de facto standard of metformin + sulphonylurea are rare.The progressive nature of Type 2 diabetes, coupled with rising prevalence  and bettRosiglitazone Evaluated for Cardiac Outcomes and Regulation of glycaemia in Diabetes (RECORD), a 6-year, multicentre, randomized, open-label, parallel-group study of cardiovascular outcomes . Allocated drugs were non-blinded owing to the difficulties of arranging six preparations with different dosing schedules, and the impossibility of maintaining blinded allocation with asymmetric timing of insulin therapy initiation.1c ≤ 7.0%. The randomized starting dose for rosiglitazone was 4 mg/day and those for metformin and sulphonylurea were in accordance with local clinical practice. If HbA1c rose to > 7.0% after 8 weeks of randomized treatment, the dose of randomized study medication was increased towards a maximum of 4 mg rosiglitazone twice daily, 2550 mg metformin, 15 mg glyburide , 240 mg gliclazide, or 4 mg glimepiride. If HbA1c was ≥ 8.5% (confirmed by a second measurement at least 1 month later) on the maximum tolerated dose for at least 8 weeks, a third glucose-lowering agent could be added if on rosiglitazone, or insulin initiated if on metformin + sulphonylurea.Throughout the study, participants were managed to target HbA1c after 18 months’ randomized treatment. Secondary efficacy analyses included fasting plasma glucose (FPG), serum lipids, homeostasis model assessment (HOMA) basal insulin sensitivity and islet B-cell function (HOMA %B) by the equation method . Intact proinsulin was measured using a two-site fluoroimmunometric assay  cholesterol were analysed by Olympus cholesterol esterase assays , and low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation. Triglycerides were measured by enzymatic determination of glycerol using Olympus reagents. Non-esterified fatty acids (NEFA) were measured by enzymatic colorimetric assay . PAI-1 antigen was quantified using a Biopool TintElize  enzyme immunoassay kit. Highly sensitive CRP was measured by fixed-time nephelometry  and fibrinogen by photo-optical clot detection in plasma on adding thrombin.A central laboratory was used for all routine laboratory assessments . HbA1c for the intention-to-treat (ITT) population  with rosiglitazone oral combination therapy was at least as good as the respective controls receiving metformin + sulphonylurea. The non-inferiority criterion (upper bound 95% CI of difference) was preset at 0.4 % units. With 260 participants per arm and change from baseline sd of 1.4% , the study was estimated to have 90% power.The primary objective was to test whether the 18-month mean change from baseline HbA1c) or during the 18-month treatment period  was also performed. Participants who stopped dual-combination therapy, including those who progressed to triple-combination therapy or started insulin, had their data censored from the time dual-combination therapy was stopped. Analyses of FPG and body weight data used the same methodology as for HbA1c.The primary ITT analysis of treatment difference at 18 months was by repeated-measures analysis  using a model including terms for treatment and baseline by visit interaction, using an unstructured covariance matrix to model the within-patient variability for each treatment group. A supportive per-protocol analysis excluding participants with prospectively defined major protocol deviations at baseline .Baseline characteristics were well matched between randomized groups within the background metformin and background sulphonylurea strata . HoweverAll but three participants began their randomized medication; 202 were withdrawn before 18 months. More participants in the metformin + rosiglitazone group (20%) ceased dual-combination therapy than in the metformin + sulphonylurea group 8%; . More pa1c in the rosiglitazone groups were similar to those achieved in the respective control groups  in favour of metformin + sulphonylurea at 6 months [difference 0.37 %]. However, this superiority was not sustained beyond 8–12 months  0.62 , P = 0.012] and 45 and 37% of background sulphonylurea participants using rosiglitazone and metformin . An HbA1c ≤ 7.0% at 18 months was achieved by 35 and 39% of participants comparing rosiglitazone with sulphonylurea, and 37 and 31% comparing rosiglitazone with metformin (both comparisons NS).A reduction in HbAAt 18 months, the apparently greater reductions in FPG in the rosiglitazone groups did not reach statistical significance . On backP < 0.001; P < 0.001; In both background treatment strata, 18-month HOMA-estimated basal insulin sensitivity was substantially increased in the rosiglitazone groups compared with the respective controls  vs. sulphonylurea 4.4  pmol/l; sulphonylurea strata: rosiglitazone −15.4  vs. metformin −5.9  pmol/l]. Similar differences were obtained for proinsulin for rosiglitazone vs. sulphonylurea [−4.8  vs. 1.8  pmol/l], but with overlap for the rosiglitazone vs. metformin groups [−6.4  vs. −3.5  pmol/l]. Rosiglitazone resulted in a greater reduction in proinsulin:insulin ratio than sulphonylurea [−22.3  vs. 0.9 %], whereas similar decreases were observed when rosiglitazone was contrasted with metformin [−15.0  vs. −17.1 %].P = 0.003; P < 0.001).Increases in body weight were observed in both arms of the metformin stratum; however, this increase was greater with rosiglitazone than sulphonylurea , but not with metformin , from baseline levels of 7.8–8.0%, explains the small proportion of participants reaching the conservative glucose control target by 18 months. If current guideline targets from International Diabetes Federation (IDF) and American College of Endocrinologists (ACE)/American Association of Clinical Endocrinologists (AACE) are to be met [1c, differences were observed in responder rates, with more rosiglitazone than metformin participants achieving a reduction of ≥ 0.7% in the sulphonylurea stratum, while fewer so responded on rosiglitazone than sulphonylurea in the metformin stratum. The apparent inconsistency with mean HbA1c may in part be explained by some non-normality in all groups, and the conservative assumptions made about those with missing data in the responder analysis. The non-normality may have arisen from there being subgroups of people who respond better to one drug or the other, but confirmation of this by other large trials would be needed.Reductions in FPG observed with rosiglitazone were generally greater than would be predicted from the change in HbAo be met , this em1c (< 9.0%) were above the threshold for discontinuation (8.5%). Additionally, this was an open asymmetric study [The number of participants moving off dual-combination treatment was higher for the rosiglitazone groups than the respective control groups. This was particularly noticeable for the sulphonylurea comparison, and may reflect the early and rapid effect of these drugs noted above, particularly as the entry criteria for HbAic study , where tic study ,15.The improvements observed with rosiglitazone in insulin sensitivity have previously been reported in other smaller, shorter studies –19, and Although there were no restrictions on the initiation or continued use of lipid-lowering therapy in RECORD, this does not appear to have substantially confounded the assessment of 18-month lipid end points, as they confirmed the previously reported effects of rosiglitazone in increasing total, LDL and HDL cholesterol . The sigThe improvements in CRP, PAI-1 antigen, and fibrinogen observed with rosiglitazone combination therapy are also consistent with previous monotherapy data ,24, and 1c in people with Type 2 diabetes as the standard combination of metformin + sulphonylurea, and produces greater improvements in CRP and insulin sensitivity but is also associated with greater weight gain.In conclusion, after 18 months’ treatment, rosiglitazone in combination with metformin or sulphonylurea, is as effective in lowering HbAThe RECORD study sponsor is GlaxoSmithKline, the manufacturer of rosiglitazone. Members of the Steering Committee, Data Safety and Monitoring Board, and Clinical Endpoints Committee, or their institutions, are remunerated for time and expenses in connection with this and other activities relating to the company and product. Local investigators and/or their institutions are paid fees per participant for study activities; some have other relationships with the company. PDH, SJP, HBN, RG, MH, and MK are members of the RECORD steering committee. PC and NPJ are employees of GlaxoSmithKline.


The aim of this study was to compare the efficacy of different HIV drug resistance test reports  in patients who were changing their antiretroviral therapy (ART).Randomised, open-label trial with 48-week followup.The study was conducted in a network of primary healthcare sites in Australia and New Zealand.Patients failing current ART with plasma HIV RNA > 2000 copies/mL who wished to change their current ART were eligible. Subjects were required to be > 18 years of age, previously treated with ART, have no intercurrent illnesses requiring active therapy, and to have provided written informed consent.Eligible subjects were randomly assigned to receive a genotype (group A) or genotype plus virtual phenotype (group B) prior to selection of their new antiretroviral regimen.Patient groups were compared for patterns of ART selection and surrogate outcomes  on an intention-to-treat basis over a 48-week period.> one month of followup were included in these analyses. Resistance tests were the primary means by which ART regimens were selected . At 48 weeks, there were no significant differences between the groups for mean change from baseline plasma HIV RNA  and mean change from baseline CD4+ cell count .Three hundred and twenty seven patients completing In the absence of clear demonstrated benefits arising from the use of the virtual phenotype interpretation, this study suggests resistance testing using genotyping linked to a reliable interpretive algorithm is adequate for the management of HIV infection. Background: Antiretroviral drugs are used to treat patients with HIV infection, with good evidence that they improve prognosis. However, mutations develop in the HIV genome that allow it to evade successful treatment—known as drug resistance—and such mutations are known against every class of antiretroviral drug. Resistance can cause treatment failure and limit the treatment options available. Different types of tests are often used to detect resistance and to work out whether patients should switch to a different drug regimen. Currently, the different types of tests include genotype testing (direct sequencing of genes from virus samples infecting a patient); phenotype testing (a test that assesses the sensitivity of a patient's HIV sample to different drugs), and virtual phenotype testing . The researchers of this study did a trial to find out whether providing an additional virtual phenotype report would be beneficial to patients, as compared with a genotype report alone. The main outcome was HIV viral load after 12 months of treatment, but the researchers also looked at differences in drug regimens prescribed, number of treatment changes in the study, and changes in CD4+ (the type of white blood cell infected by HIV) counts.What this trial shows: The researchers found that the main endpoint of the trial  was no different in patients whose clinicians had received a virtual phenotype report as well as a genotype report, compared with those who had received a genotype report alone. In addition, the average number of drugs prescribed was no different between patients in the two different arms of the trial, and there was no difference in number of drug regimen changes, and no change in immune response (measured using CD4+ cell levels). However, more drugs predicted to be sensitive were prescribed by clinicians who got both a genotype and virtual phenotype report, as compared with clinicians who received only the genotype report.Strengths and limitations: The size of the trial (338 patients recruited) was large enough to properly test the hypothesis that providing a virtual phenotype report as well as a genotype report would result in lower HIV viral loads. Randomization of patients to either intervention ensured that the comparison groups were well-balanced, and the researchers also tested whether selection bias had affected the results . They found no evidence for selection bias occurring within the trial. However, interpreting the results is difficult because the trial did not directly compare the two different testing platforms, but rather looked at whether providing a virtual phenotype report as well as a genotype report was better than providing a genotype report alone. The investigators also acknowledge that since the trial was conducted, the cutoffs for interpreting genotype information as resistant have been lowered. The findings may therefore not translate precisely to the current situation.Contribution to the evidence: Other cohort studies and clinical trials have shown that patients offered resistance testing respond better to antiretroviral therapy compared with those who were not, but the clinical effectiveness of different resistance testing methods is not known. This study provides additional data on the respective benefits of genotype testing versus genotype plus provision of virtual phenotype. Another trial comparing genotype versus virtual phenotype has also found that the different interpretation methods perform similarly. HIV drug resistance was first described three years after the introduction of zidovudine for treatment of HIV infection . ResistaHIV drug resistance testing is a recommended component of several international ART treatment guidelines ,6. ThreeNone of the current methods for resistance testing is ideal. Genotypic testing may not correlate completely with phenotype, requires expert interpretation, and relies heavily on the availability of unvalidated, rules-based algorithms. The technique requires a minimum plasma HIV-1 viral load of approximately 1,000 RNA copies/mL. Phenotypic testing is slow and only available in highly specialised laboratories. Moreover, the relationship between phenotypic inhibitory concentrations  and virologic response under continuing drug pressure  requires constant adjustment as more laboratory and clinical information becomes available. Both types of resistance testing may fail to detect minor quasi-species that may have clinical significance. Quality control of genotypic testing for sequence quality, the ability to detect nucleotide mixtures, the interpretation of sequences obtained, and clinician familiarity with the report generated, so that informed treatment choices are made, remain issues of concern.In general, both genotypic and phenotypic resistance testing has been shown to provide virologic benefit  compared with control arms –11. ClinWhile phenotypic and virtual phenotypic testing have been compared ,17, the HIV-infected patients taking combination ART, with plasma HIV RNA viral load > 2000 copies/mL, who were willing to change therapy and who were more than 18 years of age were eligible to enter the study. Patients were ineligible if they were ART naive, experiencing an acute illness warranting therapeutic management, or judged by the investigator as being unable to understand or comply with the protocol. All patients were required to give written, informed consent prior to entering the study. Institutional ethics approval was granted for all sites involved in the study. Subjects were recruited from a network comprising 41 clinical sites  in Australia and New Zealand.Patients were randomly assigned to receive a genotypic resistance test (group A) or a genotypic resistance test plus a virtual phenotype (group B).Each clinical site selected one laboratory to undertake genotype testing for their study participants.n = 1; ViroSeq HIV-1 Genotyping System Version 2 , n = 2; six laboratories used in-house assays with primers that enabled amplification and sequencing of the PR and RT genes. All laboratories participated in an ongoing quality assurance program to ensure that the differing technologies yielded consistent results [Blood was collected from eligible patients into tubes containing ACD or EDTA anticoagulant at the randomisation visit and sent to the designated laboratory for genotyping of HIV reverse transcriptase (RT) and protease (PR) genes. The laboratories used a variety of platforms to generate the HIV genotype; TRUGENE HIV-1 assay ,  results ,19. GenoThe viral RT and PR sequences generated for patients in group B were sent electronically to Virco  for generation of a virtual phenotype report. When there were insufficient matches in the database, a Virco rules-based interpretation of the sequence was made. At the time the study was performed, the VircoGEN II platform employed arbitrary clinical cutoffs for each class of ART . VircoGEN II was the only format of virtual phenotype used for the duration of the study.Our primary hypothesis was that provision of the virtual phenotype report would result in significant clinical benefit relative to provision of an HIV genotype report alone in terms of changes in plasma HIV RNA over 48 weeks.We were also interested in patterns of antiretroviral selection and use following receipt of the study resistance test results, changes in CD4+ cell count, and any associations between clinical outcome and a range of baseline factors.The primary endpoint was mean change in log plasma HIV–RNA load between the baseline measurement and that obtained 48 weeks later. Secondary endpoints were: the differences between the planned medication and the regimen selected at baseline following provision of the resistance test result; the proportion of patients with undetectable (<400 copies/mL) plasma HIV-RNA load at 48 weeks; the time weighted average change in log plasma HIV–RNA load from baseline; the number of ART treatment changes over duration of study; the change in CD4+ cell count from baseline to 48 weeks; the time-weighted average change in CD4+ cell count from baseline and time to first ART change.10, 300 patients were required to detect a difference between groups of 0.35 log10 with 80% power using a two-sided significance level of 5%.Based on an estimate of variability in mean change in log plasma HIV-RNA load between baseline and 48 weeks, corresponding to a standard deviation of 1.0 logRandomization was performed using a central telephone randomization office located at NCHECR. The randomization was stratified by site, first or subsequent combination antiretroviral treatment change, and baseline plasma HIV-RNA load . Computer-generated stratified randomization lists were generated using a blocking factor of two. The block size was known only to key staff at the coordinating centre. It was not known by any of the sites. Eligible patients were randomised in equal proportions to receive either an HIV genotype report (group A) or both an HIV genotype report and a virtual phenotype report (VircoGen II—group B).Investigators recorded the ARV regimen they would next prescribe based on their clinical judgment prior to receiving the resistance report. Demographic, CD4+ cell count, HIV RNA load, CDC classification, ART history, and ART intolerance data were also collected at baseline.Patients who failed treatment on the study  could receive an additional HIV resistance test consistent with their original randomisation.p-values are presented in addition to MITT results.Primary treatment comparisons were performed using a modified intention to treat (MITT) approach, using all randomised patients with baseline and at least one followup visit. Continuous endpoints were analysed using nonparametric rank-sum tests; binary variables using Fisher's Exact tests. Time to event endpoints were analysed using survival analysis methods. Secondary treatment comparisons were also performed using a strict intention to treat (SITT) approach, including all patients without data as failures, joint worst outcomes, or events at time zero, as appropriate. SITT Two prespecified subgroup analyses were performed—according to number of previous antiretroviral combinations received (≤7 versus 8+ combinations), and by number of drugs resistant according to genotype (≤6 versus 7+ resistant drugs). Evidence for subgroup effects were formally assessed by testing for statistically significant interactions between subgroup and randomized treatment group. At the request of the journal, we also performed one further subgroup analysis according to whether patients were the first or second randomized within each randomization block . The aimBaseline resistance profiles were analysed according to randomisation group using the genotype results only. Additionally, a formal comparison of genotype interpretation according to the Crest Algorithm  and virtThe results of resistance testing for each ART were cross-tabulated against the genotype or virtual phenotype. The proportion of genotype or virtual phenotype results that were derived from a rules-based assessment was calculated. The number of drugs against which the virus was classified as sensitive or intermediate by genotype but resistant by virtual phenotype and vice versa was assessed. A score of the overall difference between genotype and virtual phenotype was then calculated.A total of 338 patients were randomised into the study between October 2001 and April 2002. Eleven (3.3%) did not have any followup data and were excluded from all analyses. The remaining 327 patients composed the intent-to treat population. Twenty-five patients (7.6%) were lost to followup before 48 weeks, 12 from group A and 13 from group B. These patients were included in analyses using their available data. For participant disposition, please refer to 3 in group A and 318 cells/mm3 in group B. All patients had previously received nucleoside RT inhibitors (NRTIs), 80% of group A and 79% of group B, had previously received a non-nucleoside RT inhibitor (NNRTI), and 90% in both groups had previously received a protease inhibitor (PI). The mean total number of ARV drugs ever received was eight and the mean duration of ARV treatment was six years in both groups. The mean number of combinations of ARVs used was eight for group A and seven for group B. On average, patients in both treatment groups were known to be intolerant to one ARV drug. The median turnaround times for generation of resistance reports were 33 days (range 16–118 days) for group A and 33 days (range 7–127 days) for group B.Baseline patient characteristics were summarised according to randomisation group and are shown in p = 0.230, SITT p = 0.685) .After 48 weeks there were no significant differences between the groups for mean change from baseline plasma HIV RNA  . The timp = 0.553, SITT p = 0.502). Mean change from baseline CD4+ cell count at 48 weeks was not significantly different between study groups . The mean CD4+ weighted change from baseline at week 48 was +24 cells/mm3 in group A patients and +39 cells/mm3 in group B . Nine (5.5%) patients in group A and seven in group B (4.3%) experienced an AIDS-defining illness during the study. The time to developing an AIDS defining illness was not significantly different between the arms . Eight patients died during the study, six from group A and two from group B.A total of 67 patients (46%) in group A and 60 (42%) in group B had undetectable viral loads at week 48 (MITT p = 0.574). In patients randomized second, the mean log decreases were −0.65 (SD = 0.94) and −0.51 (SD = 1.09), respectively, (p = 0.144). There was no statistical evidence of an interaction between treatment group and randomization order (p = 0.535).We performed two prespecified subgroup analyses, in which we examined our study endpoints in patients with or without extensive prior treatment histories, and with multiple drug resistance point mutations or not. We did not observe any evidence of benefit for either resistance test platform in any of these analyses (unpublished data). The analysis according to randomization order also did not reveal any evidence of treatment effects. In patients randomized first within each block, mean log viral load decreases at 48 weeks were −0.70 logs (SD = 1.14) and −0.62 (SD = 0.97) in genotype and virtual phenotype patients, respectively (The average number of ARVs in regimens prior to randomisation was three in both groups. The mean duration of the current ARV regimen was 18 months in group A and 20 months in group B. The ARV drug class prescribed at study entry prior to resistance testing is shown in p < 0.001). Furthermore, on average clinicians prescribed significantly more sensitive drugs to patients in arm B than in arm A (MITT and SITT and p <0.001). There were no apparent differences between study groups for discrepancies between the planned and actual regimen employed. This is despite clear evidence that the overwhelming majority of patients in both groups started a regimen of therapy different from the one planned following receipt and interpretation of either resistance test result.Data summarised in p = 0.311, SITT p = 0.342). Thirty two patients (19%) in group A and 22 (14%) in group B were actually prescribed the ARV regimen that had been planned based on best clinical judgment . The reasons cited as being most important for selection of the prescribed ARV regimen were: resistance test result , and ARV history . The median number of ARV drugs against which plasma virus was sensitive was six in group A and eight in group B. The median number of drugs prescribed against which the virus was sensitive in the resistance test was one in group A and two in group B.The planned ARV regimen was compared with the prescribed ARV regimen following resistance testing. The mean number of ARVs planned and then prescribed in both study groups was three . Neither the time to first ARV change nor time to treatment failure were significantly different between the groups . The number of protocol-defined treatment failures was 11 (6.7%) in group A and 16 (9.8%) in group B.The mean number of changes to prescribed ARV drugs for any reason was three in group A patients and 2.7 in group B . NNRTI susceptibility was reported with greater concordance, and only delavirdine resistance was reported more often by genotype than virtual phenotype (p = 0.02). There was no significant difference in test concordance for indinavir and nelfinavir, but genotype results reported significantly more clinical isolates resistant to saquinavir (p = <0.001) and amprenavir (p = <0.001) and virtual phenotype reported significantly more resistance to ritonavir (p = 0.001).Concordance between the results from genotyping and virtual phenotyping for each drug class were determined –5. ThereThe CREST study was designed to identify whether there was any benefit in providing a virtual phenotype report in addition to a rules-based genotype to assist in the selection of ARV therapy. A no-resistance test control arm was not used because at the time of conducting the trial neither investigators nor patient advocate groups felt there was sufficient clinical equipoise for a control group. Approximately 40% of study patients had viral loads below the level of detection at week 48. Previous studies have also shown the benefit of resistance data when selecting a new ARV regimen . The choIn this heavily pretreated group of patients, we found no virological or immunological differences at week 48 between these two resistance test platforms.Subsequent to this study, the clinical cutoffs for resistance employed by virtual phenotyping were lowered from >10-fold to 2.0, 2.0, and 1.8 for didanosine, zalcitabine, and stavudine, respectively. As such it is tempting to dismiss the relevance of the findings. We would argue that the impact of the new cutoffs would be to reduce the number of drugs that are reported back to clinicians as being sensitive and in so doing further reduce the apparent differences between the two virtual phenotype and the genotypic test platforms. This view cannot be informed by data from our trial because the necessary analyses are not possible.It is reasonably well-established that extensive prior use of ART results in multidrug resistant virus and that the utility of drug resistance testing in heavily pretreated patients is questionable because their options are very limited ,10. HoweOur randomised comparison provided clinicians with either one or two forms of report and interpretation describing the scale and scope of antiretroviral drug resistance in clinical isolates taken from patients in the trial. It is not possible to ascertain with confidence which report carried more weight when clinicians reviewed the results provided for patients in group B. Consequently there may be concern that many decisions regarding future treatment in that group were made on the basis of interpretations provided by the genotype result alone. However, our data clearly show that virtual phenotype results provided clinicians and their patients with significantly more treatment options and this resulted in significantly more apparently active drugs being prescribed for patients enrolled in group B. As such, we contend that for the most part our randomised comparison maintained integrity and that the results should be viewed as valid and robust comparisons. Of some importance in this regard is the fact that it is not possible to exclude an interpretation of genotype from the virtual phenotype report since the observed point mutations for a given sample are an integral part of that report.A majority of clinical HIV isolates in each study arm had at least one ARV with either intermediate or full sensitivity. However, clinician prescribing was limited as not all of these ARVs could be used. For example, a viable combination regimen could not be constructed from three NNRTI drugs. A further limitation of this study is that the impact of low dose ritonavir, used to inhibit cytochrome P450 isoenzymes and thereby increase the plasma levels of co-administered PIs, was not examined. Further work is needed to interpret resistance results when a boosted PI regimen is planned ,23. OngoCREST resulted in all Australian and New Zealand HIV reference laboratories being able to offer HIV genotype testing and participation in a local quality assurance program. Other outcomes were sharing of expertise, collaboration between different laboratories, and the availability of a standardised genotypic interpretation and reporting format developed and maintained by Australian experts. The study was undertaken in a wide variety of settings and provided investigators with access to HIV resistance test reports. The genotype report was designed to be user friendly for both those experienced and inexperienced in interpreting the results. The provision of a resistance test result significantly impacted the choice of ARVs prescribed across all sites, both in general practice and in specialist hospitals.Overall, CREST found that HIV resistance test results affected ARV prescribing but there was no additional benefit derived from receiving a virtual phenotype report in addition to a genotype report. This may be due to factors including the quality of the genotype interpretation developed for CREST and the high biological cutoff used in the virtual phenotype analysis. Similar data have been generated in one other trial . Across CONSORT ChecklistClick here for additional data file.(56 KB DOC)Trial ProtocolClick here for additional data file.(1.0 MB DOC)


To evaluate the efficacy of triple therapy consisting of single-session photodynamic therapy (PDT), intravitreal bevacizumab (IVB) and intravitreal triamcinolone (IVT) as initial pulse therapy followed by repeat intravitreal bevacizumab injections for maintenance treatment in neovascular age-related macular degeneration (AMD).In a prospective interventional case series, patients with subfoveal choroidal neovascularization (CNV) secondary to AMD underwent pulse therapy with single-session PDT according to the standard protocol followed by 1.25 mg IVB and 2 mg IVT 48 hours later. Best corrected visual acuity (BCVA) was assessed and optical coherence tomography (OCT) and fluorescein angiography (FA) were performed prior to treatment. BCVA and OCT measurements were repeated at 6 week intervals and FA was obtained after 12 weeks and when necessary thereafter. Repeat injections of IVB were performed based on fluorescein angiographic evidence of CNV leakage.This series included 17 eyes of 17 patients with mean age of 67.6 ± 7.2 years. Mean follow up duration was 50.4 ± 15.5 weeks. Mean BCVA prior to treatment was 0.74 ± 0.33 logMAR which improved to 0.52 ± 0.36 logMAR after 12 weeks (P = 0.012) and 0.41 ± 0.38 logMAR after 24 weeks (P = 0.001). Mean pretreatment central macular thickness (CMT) was 395 ± 181μ which was significantly reduced to 217 ± 69μ (P = 0.005), 231 ± 79μ (P = 0.028) and 221 ± 87μ (P = 0.05) six, twelve and twenty-four weeks after initial treatment respectively. Visual acuity improvement and CMT reduction was maintained during the follow-up period. IVB injections were repeated once, twice and three times in 10, 7 and 2 eyes at a mean interval of 20.2 ± 10.1, 19 ± 13.7 and 15 ± 1.4 weeks after initial therapy, respectively.Initial pulse triple therapy consisting of single-session PDT combined with IVB and IVT improves vision and reduces CMT in neovascular AMD. Repeat IVB injections maintain the visual gain from the initial combination therapy. Photodynamic therapy is an FDA-approved treatment for neovascular age-related macular degeneration (AMD); however this treatment modality seems to stabilize, rather than improve vision -3. FurthCombination of PDT with intravitreal injection of an anti-angiogenesis drug and a steroid may address the multifactorial pathogenesis of AMD, improve vision and reduce the need for retreatments. Results of triple therapy with PDT, bevacizumab and dexamethasone have been reported recently . We perfThe study was a prospective, interventional case series. The treatment protocol was approved by the Ethics Committee of the Ophthalmic Research Center. Patients were informed about the risks and benefits of combined therapy and written consent was obtained. Eyes with all types of active subfoveal CNV secondary to neovascular AMD with lesions less than 4 disc areas and Snellen visual acuity of 20/30 to 20/400 at baseline were included. Exclusion criteria were glaucoma, diabetic retinopathy, macular disorders other than AMD and previous treatment for CNV.Eligible eyes were evaluated before treatment completely. Best corrected visual acuity measured by Snellen chart was recorded in logMAR. Applanation tonometry, anterior segment examination with slit lamp and fundus examination was performed. All eyes underwent color fundus photography and fluorescein angiography (FA) using a confocal scanning laser angiograph (Heidelberg Retina Angiograph II: Heidelberg engineering). Optical coherence tomography  was performed before intervention for all cases to measure the central macular thickness (CMT). Photodynamic therapy with verteporfin was performed according to the standard regimen. Forty eight hours after PDT, intravitreal injection of 1.25 mg/0.05 ml bevacizumab  and 2 mg/0.05 ml triamcinolone acetonide was carried out at two different sites under sterile condition. A topical antibiotic, every 6 hours for 3 days, was prescribed and patients were instructed to return in case of ocular pain or redness or any deterioration of vision. Patients were examined one day and one week after injection. Topical antiglaucoma medication was prescribed if intraocular pressure (IOP) was more than 21 mmHg.Visual acuity and optical coherence tomography (OCT) were evaluated at 6 week intervals. Fluorescein angiography was repeated at week 12 and when considered necessary thereafter. CNV activity was defined as presence of leakage from the CNV on fluorescein angiography. Eyes with active CNV underwent repeat intravitreal injection of 1.25 mg bevacizumab. Repeat PDT and intravitreal triamcinolone injections were not considered in the study protocol.Paired T-test was used to compare findings before and after treatment.Seventeen eyes of 17 patients  with the mean age of 67.6 ± 7.2 years were included in this study. Lesion types were predominantly classic in 5 eyes, minimally classic in 3 eyes, occult in 8 eyes and retinal angiomatous proliferation (RAP) in one eye. Mean follow-up was 50.4 ± 15.5 weeks and all 17 patients completed the 24 week follow up (Table Mean BCVA was 0.74 ± 0.33 logMAR range: 1.20 to 0.22) before treatment (Table 0 to 0.22Seven eyes (41.2%) remained stable following the initial therapy. Follow up period for these 7 eyes ranged from 24 to 73 weeks (mean: 42.6 ± 17.6 weeks). A second intravitreal injection of bevacizumab was required in 10 eyes (58.8%), a third injection was performed in 7 eyes (41.2%) and a fourth injection was needed in 2 eyes (11.8%). Mean interval between the first and second injections and between the second and third injections were 20.2 ± 10.12 and 19 ± 13.7 weeks respectively. The interval between the third and fourth injections had a range of 14 to 16 weeks. Mean follow up for eyes with 2, 3 and 4 injections were 56 ± 11.6 weeks, 60.1 ± 5.5 weeks and 58.5 ± 9.2 weeks respectively. No adverse ocular or systemic events were observed during the follow up period.Mean IOP was 14 ± 1.9 mmHg before treatment and increased to 17.3 ± 6.3 mmHg at week 6 and returned to pretreatment levels 12 weeks after intervention. In one patient IOP was increased to 34 mmHg after one week. Intraocular pressure was controlled in this patient with a combination of timolol eye drop bid and dorzolamide eye drop every 8 hours, which were continued for 5 weeks.In this small case series, combination of single-session PDT with intravitreal bevacizumab and triamcinolone was used as pulse therapy for neovascular AMD. Our goal was to increase the efficacy of treatment and reduce side effects of monotherapy. Triple therapy resulted in significant visual improvement and CMT reduction irrespective of lesion type and also reduced the need for retreatments. Visual acuity improved significantly in the majority of cases in our series and remained unchanged in others. This treatment effect persisted during the follow up period. More than one third of our patients remained stable after the initial pulse therapy. The visual gain could be maintained in the remaining cases with few retreatments. Intravitreal bevacizumab was used for maintenance therapy to avoid the side effects of repeat PDT and repeat intravitreal triamcinolone. The average interval between the first and second treatment sessions was about 20 weeks which may reflect less need for retreatment following the initial pulse therapy.Augustin et al recently published the results of triple therapy with PDT, bevacizumab and dexamethasone for neovascular AMD . They coThe rationale for single-session PDT emanates from the present knowledge regarding the possible adverse effects of repeat PDT on physiological choroid and even RPE and neurosensory retina ,25. In aPDT may cause phototoxicity-induced VEGF expression and increase vascular permeability by triggering generation of free radicals and lipid peroxides . TriamciWe used intravitreal bevacizumab as one of the components of pulse triple therapy. It was also used as a maintenance therapy in our series. Use of an antiangiogenic factor can counteract the PDT induced over-expression of VEGF and reduce its adverse effects. On the other hand, PDT may disrupt the architecture of CNV and help the antiangiogenic factor to affect the lesion more potently . Aggio eThe optimal time for intravitreal injection of bevacizumab with or without a steroid after PDT is not known. It has ranged from 16 hours to 2 weeOur preliminary results suggest that intravitreal bevacizumab has a synergistic effect with PDT and intravitreal triamcinolone for treatment of neovascular AMD. Using this triple therapy as an initial pulse can improve or stabilize visual acuity. In case of recurrent CNV, repeat intravitreal injection of bevacizumab may help maintain the visual gain. Further studies are required to confirm the results of this pilot study.LogMAR: Logarithm of the Minimum Angle of ResolutionVEGF: Vascular Endothelial Growth FactorRPE: Retinal Pigment EpitheliumThe author(s) declare that they have no competing interests.HA participated in the design of the study, contributed in acquisition and analysis and interpretation of data, was involved in drafting the manuscript and its revising. RT participated in the design of the study, contributed in acquisition and analysis and interpretation of data, was involved in drafting the manuscript and its revising. MS participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. MR participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. RK participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. AL participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. MA participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. MD participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version. SM participated in the design of the study, contributed in acquisition and analysis and interpretation of data, gave final approval of the version.The pre-publication history for this paper can be accessed here: