Previous structural imaging studies have found evidence of brain morphometric changes in patients with major depressive disorder (MDD), but these studies rarely excluded compounding effects of certain important factors, such as medications and long duration of illnesses. Furthermore, the neurobiological mechanism of the macroscopic findings of structural alterations in MDD patients remains unclear. In this study, we utilized magnetization transfer imaging, a quantitative measure of the macromolecular structural integrity of brain tissue, to identify biophysical alterations, which are represented by a magnetization transfer ratio (MTR), in MDD patients. To ascertain whether MTR changes occur independent of volume loss, we also conduct voxel-based morphometry (VBM) analysis. The participants included 27 first-episode, drug-naive MDD patients and 28 healthy controls matched for age and gender. Whole-brain voxel-based analysis was used to compare MTR and gray matter volume across groups and to analyse correlations between MTR and age, symptom severity, and illness duration. The patients exhibited significantly lower MTR in the left superior parietal lobule and left middle occipital gyrus compared with healthy controls, which may be related to the attentional and cognitive dysfunction in MDD patients. The VBM analysis revealed significantly increased gray matter volume in right postcentral gyrus in MDD patients. These findings in first-episode, drug-naive MDD patients may reflect microstructural gray matter changes in the parietal and occipital cortices close to illness onset that existed before volume loss, and thus potentially provide important new insight into the early neurobiology of depression. In vivo studies of the brains of MDD patients serve as an important strategy for learning about the neurobiology of depression and are helpful for developing effective therapies.As one of the leading worldwide causes of disability, major depressive disorder (MDD) is characterized by persistent, pervasive feelings of sadness, guilt and worthlessness, and often results in an increased risk of suicide.4 insula,4 temporal lobes,4 anterior cingulate cortex,5 striatum6 and hippocampus.7 Larger volumes of white matter in the inferior parietal lobule8 and periventricular9 regions and deep or subcortical10 white matter hyperintensity have also been reported. However, these reports are inconsistent in detail, probably because of potential confounders associated with medications and/or long duration of illnesses. Thus, studies of first-episode, drug-naive MDD are a starting point for assessing brain structure before it is influenced by potential confounders and thereby provide core information relevant to models of pathogenesis.11In recent decades, numerous structural magnetic resonance imaging (MRI) studies have sought to identify the key brain areas involved in the pathogenesis of depressive symptoms. Voxel-based morphometry (VBM) studies have reported that MDD is associated with widespread local abnormalities in many brain regions, such as the frontal gyrus,12 MDD patients have also showed reduced density of neuronal cell bodies with large cell body size in cortical layers 2 through 5 of the orbitofrontal cortex (OFC), and in layers 2, 3 and 6 of the dorsolateral prefrontal cortex (dlPFC), concurrent with an increased density of small body size neurons in layer 3 (OFC) and layers 3 and 6 (dlPFC).14 Observations of reduced glial cell density in the dlPFC and anterior cingulate cortex in the post-mortem brain of depressed patients provide further evidence of glial changes in patients with MDD.16 However, it is difficult to characterize the histopathological changes in vivo that underlie the macroscopic abnormalities. Techniques with sufficient neuropathological sensitivity to relatively subtle macromolecules are needed.The neurobiological mechanism of the macroscopic structural alterations in MDD patients remains unclear. A post-mortem study has demonstrated reduced levels of the metabotropic glutamate receptor 5 monomer protein in the prefrontal cortex in cases of depression.in vivo to subtle or early neuropathological changes than volumetric MRI.18 By utilizing the magnetization exchange between the spins of free water and water bound to macromolecules, MTI creates a contrast between tissues, namely, magnetization transfer ratio (MTR) map. The MTR measures the efficiency of these exchange phenomena, which depends on biophysical integrity of macromolecular protein pools and their local microenvironment.19 MTI has been used to characterize the status of macromolecular proteins in several clinical brain disorders including depression,23 schizophrenia24 and multiple sclerosis, even when a conventional MRI is negative.26 Previous MTI studies of depression have reported lower MTR in the corpus callosum, caudate nucleus, putamen, and the occipital white matter in late-life depressed patients,21 and lower MTR in the head of the caudate nucleus in both MDD patients22 and MDD patients with diabetes.27 Treatment-resistant depressed patients have been reported to show reduced MTR in the anterior cingulate cortex, insula, caudate tail and amygdala-parahippocampal areas compared with healthy controls.20Magnetization transfer imaging (MTI) is a MR technique with the potential for providing more neuropathological information in vivo in first-episode, drug-naive MDD patients relative to healthy controls without the confounding effects of antidepressant therapy. To ascertain whether MTR changes occurs independent of volume loss, we also conduct VBM analysis.However, these studies included patients taking antidepressants or patients who were free of antidepressants for at least 2 weeks. Furthermore, most of these studies used only region-of-interest (ROI) methods that focused on specific brain regions. In this study, we use both whole brain and ROI MTI approaches to explore and further characterize the neuropathological abnormalities 28 All patients also met the following inclusion criteria: (1) were between 18 and 60 years; (2) were experiencing first-episode depression; and (3) had a 17-item Hamilton Rating Scale for Depression (HRSD)29 total score of 18 or higher on the day of their scan. Exclusion criteria included the following: (1) currently taking or had previously taken antidepressant or anti-psychotic drugs; (2) currently taking psychotropic medications, including benzodiazepines, hypnotics or anticonvulsant agents; (3) a history of electroconvulsive therapy; (4) contraindications to the MRI scan; (5) any psychotic disorders other than MDD; and (6) significant medical or neurological illness, including any history of significant head trauma with the loss of consciousness.We recruited patients with 27 MDD from the Department of Psychiatry at West China Hospital of Sichuan University. All patients were assessed by an experienced psychiatrist and determined to meet the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) criteria for MDD.We recruited 28 healthy comparison subjects from the local area by poster advertisement who were assessed using the Structured Clinical Interview for the DSM-IV. Exclusion criteria included the same medical and psychiatric factors used to recruit MDD patients, as well as any DSM-IV Axis I disorder or known history of significant psychiatric illness. All participants were of Chinese Han nationality.The West China Hospital Clinical Trials and Biomedical Ethics Committee of Sichuan University approved the study protocol (registration no. 84). The experiment was performed in accordance with the approved guidelines, and written informed consent was obtained from all participants.2; and data matrix=320 × 192. High-resolution, three-dimensional, T1-weighted images were acquired using a spoiled gradient recalled sequence .Images were acquired using a 3.0 Tesla General Electric magnetic resonance scanner . Participants were fitted with soft earplugs that were positioned comfortably in the coil, and they were then instructed to relax and remain still. Head motion was minimized using foam pads. Whole-brain MT images were acquired using a three-dimensional fast, low-angle shot sequence. One acquisition was performed with, and another without, the magnetization saturation pulse at 1.5 kHz off-resonance. In this way, MT- and non-MT-weighted images were generated separately. The other sequence parameters were as follows: repetition time/echo time (TR/TE)=37/5 ms; flip angle=15° 50 contiguous axial slices with slice thickness=3 mm; field of view=24 × 24 cmhttp://www.fil.ion.ucl.ac.uk/spm). For each subject, we first co-registered the MT- and non-MT-weighted images using a mutual information registration algorithm. MTR was calculated on a voxel-by-voxel basis as follows: MTR=(M0−Ms)/M0 × 100, where M0 and Ms are the signal intensities without and with, respectively, the saturation pulse applied. As the non-MT images are partially T1-weighted, we directly normalized them to the MNI T1 W template and then used the transformation parameters to normalize the co-registered MTR map. The normalized non-MT images were skull-stripped using the brain extraction tool (http://www.fmrib.ox.ac.uk/fsl/bet/)30 and were then used as masks to remove non-brain tissues on the normalized MTR maps. Finally, MTR maps were smoothed with a Gaussian kernel of 6-mm full width at half maximum.MR images of all the subjects were first reviewed by a neuro-radiologist to ensure that there were no structural abnormalities or data quality flaws. Data processing and analyses were conducted using the statistical parametric mapping software SPM8 . Significance in the resulting statistical maps was set at 0.05 and corrected for multiple comparisons. The correction standard was determined by Monte Carlo simulations, with the following parameters: individual voxel P-value=0.01, 1000 simulations, full width at half maximum=6 mm, applied using the Resting-State fMRI Data Analysis Toolkit of the AlphaSim program (http://www.restfmri.net).31 In this way, a corrected significance level of P<0.05 was obtained for a minimum cluster size of 132 voxels. To quantify changes in the affected regions, MTR values were extracted using a volume-of-interest approach in SPM. We conducted correlation analyses between the average regional values in these regions and the patients' age, HRSD score and illness duration.MTR maps of MDD patients were compared with those of healthy controls using two-tailed two-sample 34 These brain regions were selected as ROIs, and the segmentation was performed with the Freesurfer software package  and 40 subcortical structures. The T1 image of each subject was then co-registered to his/her non-MT-weighted image. The resultant transformation was used to map the segmented map to the non-MT image space. Finally, we overlaid the segmented map onto the MTR images and extracted the MTR values of bilateral ROIs using ITK-SNAP (www.itksnap.org),36 and the MTR values of MDD patients were compared with those of healthy controls using two-tailed univariate analysis of covariance controlling for age and gender.We were also interested in determining the MTR changes of specific brain regions, such as the bilateral medial OFC, hippocampus, caudate nucleus, globus pallidus and thalamus, as they have been frequently reported in studies of depression.37 as implemented in SPM8. Two-tailed two-sample t-tests controlling for age and gender were performed to compare group differences of gray matter volume between first-episode, drug-naive MDD patients and healthy controls. Significance in the resulting statistical maps was set at 0.05 and corrected for multiple comparisons using the Resting-State fMRI Data Analysis Toolkit AlphaSim utility same as in voxel-based magnetization transfer analysis.To identify whether morphological abnormalities could be detected in the first-episode, drug-naive MDD patients, T1W SPGR images were used to conduct VBM analysis using Exponentiated Lie algebra (DARTEL)P>0.05).P<0.05, corrected for multiple comparisons). No marked regional MTR increase in the MDD group compared with the controls was found. t =4.244, P<0.001 for left SPL; t =3.681, P=0.001 and t =3.014, P=0.004 for the two left MOG regions, respectively). No association was found between regional MTR values and patient age, HRSD scores or illness durations .As presented in t=1.801, P=0.186; right t=0.238, P=0.628), hippocampus , caudate nucleus , globus pallidus  or thalamus  when controlling for age and gender  metrics to assess the integrity of macromolecules in the brain tissue such as myelin or cellular membranes components .43 This brain region has been consistently associated with the volitional allocation of attention to visual aspects of, and coordinated motor actions to, specific regions in extrapersonal space.45 Previous functional MRI studies have revealed that shifting the location of attention produces a rapid, transient increase in SPL activation.47 Impairments in attention may be due to the neuropsychological deficits that correspond to the cognitive criterion ‘impaired ability to think or concentrate' as stipulated for MDD in the DSM-IV.28 A structural neuroimaging study has reported a reduction in the gray matter volume in the SPL in MDD patients compared with normal subjects.48 Functional MRI studies have reported that first-episode, drug-naive MDD patients exhibited reduced resting-state activity in the parietal lobe,49 increased activation in bilateral SPL after escitalopram oxalate treatment during sad facial expression recognition50 and decreased functional connectivity between the dlPFC and parietal lobe in first-episode MDD patients.51 Furthermore, decreased glucose metabolism was also observed in the parietal cortex of depressed patients compared with that of the healthy controls.52 Therefore, our findings are generally consistent with these studies and further reveal that the microstructural gray matter changes in the parietal lobe may have already existed in the early stage of MDD because our group only included first-episode, drug-naive MDD patients. Together with previous findings on the parietal lobe in MDD patients, our result of lower MTR in the superior parietal lobe of MDD patients may improve our understanding of the cerebral mechanisms underlying attentional deficits in depression.In this study, we found the first-episode, drug-naive MDD patients had a significantly lower MTR in the left SPL than did the healthy controls. The SPL is an important brain region of the dorsal attention network, which is hypothesized to have a key role in the top-down, or goal-driven, allocation of attention.53 and is involved in the perception of facial emotion.54 Depressed patients are thought to have a negative cognitive bias in their appraisal of people or life events and are believed to be unable to carry out normal interpersonal interactions.55 Thus, abnormal facial emotion processing may give rise to some of the affective and social symptoms in major depression. Neuroimaging studies have suggested that the occipital cortex may be relevant in MDD. A study comparing patients with psychotic major depression, patients with non-psychotic major depression and a healthy group of individuals revealed greater right occipital activation only in the non-psychotic major depression group compared with healthy controls during a verbal working memory task, thus suggesting that increased effort is required for basic visual processing.56 Decreased resting-state activity in the left middle occipital gyrus has been reported in MDD patients compared with healthy controls.58 It has also been reported that regional cerebral blood flow (rCBF) in the parieto-occipital regions was decreased in MDD patients and was significantly increased following pharmacotherapy.59 A magnetic resonance spectroscopy study found that gamma-aminobutyric acid (GABA) neurotransmission was altered in the occipital cortex of patients with MDD.60 The molecular mechanism behind the neuroimaging results may be related to the reduced number of GABAergic neurons in the occipital cortex.61 Consistent with our results, these findings of microstructural gray matter changes alterations in the middle occipital gyrus indicate that the visual recognition network interference may contribute to cognitive dysregulation in MDD.We also identified a lower MTR in the left middle occipital gyrus in first-episode, drug-naive MDD patients compared with that of healthy controls. The middle occipital gyrus is within the visual recognition network62 In this study, bilateral medial OFC, hippocampus, caudate nucleus, globus pallidus and thalamus were chosen as ROIs based on previous studies and no significant difference of MTR was found between MDD patients and healthy control group that was consistent with the our whole-brain voxel-based analysis. Nonetheless, lower MTR has been demonstrated in gray matter regions in previous MTI studies of MDD.63 Two MTI studies compared the MTR value changes between MDD patients and healthy controls by manually placing ROIs in the key components of cortical–subcortical circuits—the caudate, thalamic, striatal, orbitofrontal, anterior cingulate and dorsolateral prefrontal regions, and the results showed significant group differences of MTR values in the bilateral caudate22 and thalamus.63 However, it should be noted that the MDD patients in these two studies were free of antidepressant medication at least 2 weeks, whereas patients in our study were first-episode and drug-naive. In another whole-brain MTR study, treatment-resistant depressed patients with long mean illness duration were recruited, and the results showed a reduced MTR in the anterior cingulate cortex, insula, caudate tail and amygdala-parahippocampal areas relative to healthy controls.20 The discrepancy between our results and those of previous studies may be mainly attributed to inter-study differences in sample size, the sociodemographic and clinical characteristics of the participants, such as the various medication statuses and the incompatible illness duration.Whole-brain voxel-based analysis investigates region-specific changes by spatially normalizing brain images and conducting statistical tests at each voxel, which is both reproducible and automatic. Whereas ROI analysis, usually applied in the original image space, allows one to carry out group analyses of functionally corresponding regions with high statistical power and substantially reduces the correction needed for multiple comparisons as is needed for the many thousands of voxels of a whole-brain analysis.64 A study has reported that the MDD patients showed reduced cerebral activation in the right postcentral gyrus than healthy controls during empathy for others pain.65 Similarly, another functional MRI study demonstrated that following escitalopram oxalate treatment, first-episode, treatment-naive MDD patients showed decreased brain activation in bilateral postcentral gyrus during emotion recognition.50 Although the underlying mechanism for increases in gray matter volume in our study is still unknown, one possible account for this effect may be the inflammatory response, as a compensatory effect in the early phase of depression.67 It is worth mentioning that different regions were implicated when MTI and VBM methods were separately used. MTI provides information on the integrity of macromolecules in the brain tissue,19 whereas VBM allows for the study of the gray matter morphology. These isolated findings of MTI and VBM analyses suggested that MTR changes in occipital and parietal cortices existed beyond volume loss in the early stage of MDD which needs further longitudinal studies.The VBM analysis revealed increased gray matter volume in the right postcentral gyrus in the first-episode, drug-naive MDD group compared with the healthy controls. The postcentral gyrus is the primary sensory area of the brain. The somatosensory-related cortices are considered to not only encode bodily sensations but also have a key role in using social cues to understand emotional states of others.Our study has certain limitations. First, the sample size is modest. It is possible that a larger sample size will provide further insight. However, it should be noted that our patient group was a homogeneous group of first-episode MDD patients without medication. Second, MTR is very sensitive to pulse sequences and relaxation properties. This makes it difficult to accurately address the specific neurobiological causes of the identified MTR alterations. Third, as our study uses a cross-sectional design, the question arises whether these differences are a consequence or precondition of MDD. This issue can only be resolved by longitudinal design studies.In conclusion, this study demonstrates that first-episode, drug-naive MDD patients exhibit structural abnormalities, namely, lower MTR in the left superior parietal lobule and left middle occipital gyrus and increased gray matter volume in right postcentral gyrus that largely avoided the confounding effects from medications and/or long duration of illness. The MTR changes in the parietal and occipital cortices existed beyond volume loss in the early stage of MDD. The results indicate that the attentional and cognitive dysfunction in MDD patients may be associated with microstructural gray matter changes in the parietal and occipital cortices close to the illness onset that existed before volume loss, and thus potentially provide important new insight into the early neurobiology of the depression.


Mycobacterium tuberculosis (MTB), which is resistant to at least isoniazid and rifampicin. Previous studies have documented variable prevalences of multidrug-resistant tuberculosis (MDR-TB) and its risk factors in Ethiopia. Therefore, this meta-analysis is aimed, firstly, to determine the pooled prevalence of MDR-TB among newly diagnosed and previously treated TB cases, and secondly, to measure the association between MDR-TB and a history of previous anti-TB drugs treatment.Efforts to control the global burden of tuberculosis (TB) have been jeopardized by the rapid evolution of multi-drug resistant PubMed, Embase and Google Scholar databases were searched. Studies that reported a prevalence of MDR-TB among new and previously treated TB patients were selected. Studies or surveys conducted at national or sub-national level, with reported MDR-TB prevalence or sufficient data to calculate prevalence were considered for the analysis. Two authors searched and reviewed the studies for eligibility and extracted the data in pre-defined forms. Forest plots of all prevalence estimates were performed and summary estimates were also calculated using random effects models. Associations between previous TB treatment and MDR-MTB infection were examined through subgroup analyses stratified by new and previously treated patients.P < 0.001) and there was an odds ratio of 8.1 (95% CI 7.5–8.7) for previously treated TB patients to develop a MDR-MTB infection compared to newly diagnosed cases. For the past 10 years (2006 to 2014) the overall MDR-TB prevalence showed a stable time trend.We identified 16 suitable studies and found an overall prevalence of MDR-TB among newly diagnosed and previously treated TB patients to be 2% (95% CI 1% - 2%) and 15% (95% CI 12% - 17%), respectively. The observed difference was statistically significant (The burden of MDR-TB remains high in Ethiopian settings, especially in previously treated TB cases. Previous TB treatment was the most powerful predictor for MDR-MTB infection. Strict compliance with anti-TB regimens and improving case detection rate are the necessary steps to tackle the problem in Ethiopia. Despite the recent progress of global control efforts, tuberculosis (TB) remains a major public health threat, worldwide . AccordiTB continues to be one of the major public health problems in developing countries and is particularly compounded by high burdens of MDR-TB. Approximately, 75% of TB infections occur in Africa, South-East Asia, and Western Pacific regions where HIV is fueling the epidemic . HIV is The overall burden of MDR-TB is exceptionally high in resource limited countries, where health resources, finances, and the skilled personnel required for diagnosis and management are limited, making containment and the prevention of further spread more difficult . In EthiIn Ethiopia, TB has been recognized as one of the main public health threats since half century ago. The national TB control program has adopted a standardized TB prevention and control program so called directly observed treatment, short course (DOTS). At national level, there is one TB program manager who oversees a team of 11 TB program officers who are responsible for each region and work closely with the regional health bureau to maximize appropriate implementation of national TB control and prevention strategies. Ethiopia remains one of the high TB and MDR-TB burden countries where TB remains a significant cause of morbidity and mortality .Although MDR-TB is a growing concern in resource limited countries like Ethiopia, it is largely under-reported, compromising control efforts. Information concerning the true extent of the problem of MDR-TB in the African Region is limited. Since, there are significant gaps in surveillance, and lack of standards for methodology, data sharing and coordination. The overall epidemiology of drug resistant TB is not well understood in Ethiopia –16. HencEthiopia is a highly populated country in Africa, with 101 million lives in its nine regions and two federal cities. The operation of health system has been decentralized to regional governments and district health offices. Each district has a primary hospital with multiple health centers, and every health center is administratively linked to five health posts. Besides, health posts have been structured with two health extension workers who provide a package of up to 16 basic services including TB prevention and treatment follow up. The tuberculosis case definitions and management is as per the WHO TB treatment guidelines , 12.In this meta-analysis, we have used the following terms; MDR-TB described as resistance to at least the two powerful first line anti-TB drugs (isoniazid and rifampin). According to WHO, MDR-TB among new cases is defined as resistance to isoniazid and rifampin drugs in patients that have never been treated for TB, and MDR-TB among previously treated TB patients, on the other hand, is defined as resistance to isoniazid and rifampin drugs in patients that have been treated for TB .A literature search of the PubMed, Embase and Google Scholar databases was conducted, and studies potentially relevant to our study were identified. The search was carried out by 2 authors  independently, by using the following keywords; (MDR-TB or (Drug AND Resistant AND Tuberculosis)) AND (Associated AND Risk-factors) AND (Ethiopia). Among the citations extracted, abstracts were reviewed in an attempt to retrieve the clinical studies on MDR-TB among newly and/or previously treated TB patients. Studies that were relevant by title and abstract were accessed in full text to determine those that provided sufficient information to be included in our meta-analysis. Finally, the references cited by each eligible study were examined to identify additional articles. This systematic review and meta-analysis was done in accordance with QUOROM  guidelines .We have included studies carried out in Ethiopian settings, reporting MDR-TB prevalence and have sufficient data to calculate prevalence of MDR-TB among newly and/or previously treated TB cases. Studies were also only eligible if they reported the burden of MDR-TB for the past 10 years and were conducted in Ethiopia . Studies conducted among presumptive MDR-TB cases were excluded from the analysis, to minimize bias of reporting erroneously increased prevalence of drug resistant TB. Presumptive MDR-TB can be defined as: smear positive previously treated patients who define as relapse, return after default, and failure; new smear positive pulmonary TB patients who sputum remains smear positive at month 2 or 3 of treatment; symptomatic close contacts of known MDR-TB patient, and new smear positive with Human Immunodeficiency Virus (HIV) infected patients. Only studies published in English were considered.The main outcome of interest was the prevalence of MDR-TB among newly and/or previously treated TB patients. The prevalence was calculated by dividing the number of MDR-TB cases by the total number of TB patients. The results have been stratified by geographic location, types of TB cases (new versus previously treated TB cases), type of sample (sputum versus other possible specimens), and methods of MDR detection . Secondly, we have also calculated the odds of developing MDR-TB among previously treated TB cases, compared with new TB cases.The relevant data from each selected study has been extracted independently by two authors  and summarized into an Excel spreadsheet. Discrepancies were resolved through consensus and discussion with a third author (FM). For each selected study, the following parameters were extracted: first author and reference, year of publication, study region/area, year/s/ of the study period, study patients as pulmonary tuberculosis (PTB) or extra PTB cases, types of sample utilized for MDR-TB isolation, total number of TB patients, and the method employed for MDR-TB detection . Moreover, we have also extracted the proportion of MDR-TB among new, previously treated and both cases, accordingly. To depict the time trends of the burden of MDR-TB in the country, we used the index year as the year that the study has been conducted not the year of publication.The included papers were assessed for quality using a checklist based on Newcastle-Ottawa quality assessment scale. The quality of the selected studies was examined independently by two authors  using a set of predefined criteria, namely: clear data on the research design and study population (PTB versus extra PTB cases), quality of the reported data (stratified data for both new and previously treated cases), and the appropriateness of the method used for MDR-TB detection among newly diagnosed and previously treated TB cases.Since the included studies were conducted with different study designs and populations, we used a pooled random effects analysis to calculate the combined prevalence and the 95% confidence intervals (CI’s), by using the approach of DerSimonian and Laird . We used2 and p-values [2 ≥ 50% was considered as statistically significant. Possible sources of variation were explored using subgroup analysis by stratifying studies through predetermined variables; study regions, used methods, types of patients (new versus previously treated TB cases), and study population (≥ 100 and <100). The effect of small studies in publication bias was explored by the Begg’s funnel plot [P-values <0.05 were considered as statistically significant.We assessed the heterogeneity among reported prevalence by computing values for chi-square (Q), Ip-values . I2 ≥ 50nel plot . For timOur electronic database search yielded 735 citations, of these 243 non-duplicate papers were assessed and 119 excluded after reviewing their title and study setting. The remaining 124 were examined by abstract screening of which 71 were excluded. The remaining 53 were initially considered as eligible by title and abstract for our meta-analysis, however after retrieving the full texts; only 16 studies met our inclusion criteria , 24–38 aThe included studies were published from 2008 to 2016 and reported data from 2002 to 2014. Of the 16 eligible studies, 12 reported MDR-TB data on both new and previously treated TB patients. Three studies have been done only among newly diagnosed TB cases, while 1 study has assessed drug resistant TB among previously treated TB cases (Table P = 0.016). Interestingly, the prevalence of MDR-TB among studies having study participants 100 or above was 1.9% (95% CI 1.9% - 2%), this was significantly lower than a prevalence calculated from studies with their study participants less than 100 patients, 7.9% (95% CI 5.2% - 10.6%). Regarding the microbiological assays used in studies included in our meta-analysis, 8 studies have exclusively used culture to identify MDR-MTB, 8 studies used both culture and molecular methods (PCR based tests). Of note is that culture alone yielded a prevalence of 1.9% (95% CI 1.9% - 2%), which was statistically lower from the 4.3% (95% CI 3.1% -5.5%) of MDR-MTB detection using both culture and molecular methods.Summary prevalence estimates are presented in Table P = 0.770).Additionally, among the eligible studies, 12 were selected to determine the time trend of MDR-TB prevalence since the selected studies have data for MDR-TB rate in both newly and previously treated TB cases , 28–30. 2 statistic, and it was marginally insignificant . Furthermore, we have also calculated the overall prevalence of MDR-TB among previously treated TB cases; it was 15% (95% CI 12% - 17%). The heterogeneity test indicated that no statistical significant variation among pooled estimate of MDR-TB in previously treated cases . Moreover, we explored graphically the possibility of a publication bias. We did not observe an indication of such a bias in the studies included to calculate the pooled prevalence of MDR-TB among newly diagnosed and previously treated cases, accordingly  and 15% (95% CI 12% - 17%), respectively. It is comparable with a previous meta analysis report that was documented in Sub-Saharan Africa countries, where HIV co-infection is rampant . SimilarHowever, the pooled prevalence of MDR-TB among newly diagnosed and previously treated TB cases in the present study; was comparatively higher than that reports noted in economically developed countries –54. WithAccording to subgroup analysis, we observed that the pooled estimate of MDR-TB was significantly higher among studies with sample sizes of less than 100 participants, compared to studies where 100 or more participants were recruited. It is known that variation in sample size has a great effect on estimation. And possibly, the included studies have been carried out in different geographical locations that could have also impact on the observed prevalence. The calculated estimate of MDR-TB was also significantly higher in studies other than the two national surveys. Among the selected studies, 14 have been conducted either in hospital setups or other health institutions, and possibly results high estimate. Moreover, the prevalence of MDR-TB was higher among studies that have used both culture and molecular based detection methods compared to studies used culture alone. The significant variation could be presumably the fact that molecular based assays are the most powerful and highly sensitive techniques compared to conventional methods.P < 0.001). Most importantly, TB patients who had history of anti-TB treatment exposure were 8.1 times more likely to develop MDR-TB infection compared with newly diagnosed TB cases. Previously treated patients often constitute a very heterogeneous group including those who experience relapse after receiving successful treatment, those who return after default, and those who start receiving a re-treatment regimen after having experienced previous treatment failure [A second finding that highlights the association of previous history of anti-TB treatment with MDR-TB infection was high. As indicated in the study, the observed prevalence of MDR-TB was significantly higher than that among newly diagnosed TB cases . Also, there were some methodological differences between studies.Our analysis showed that the burden of MDR-TB remains a huge concern in Ethiopian settings. Particularly, the prevalence of MDR-TB was higher among previously treated TB patients. As detailed earlier, previously treated TB patients were 8.1 times more likely to develop a MDR-TB infection compared with newly diagnosed TB patients. Moreover, in the past 10 years, the time trend of MDR-TB prevalence was stable. Therefore, this finding underscores the importance of reducing the burden of MDR-MTB infection in Ethiopian settings. Effective implementation of DOTS program, strict compliance with infection control policies, and enhancing case detection rate through strengthening the TB laboratory capacity including introduction of rapid molecular diagnostic methods and implementation of active case finding through MDR-TB contact screening, seem to be the necessary next steps in this effort.


There were no significant results in Cycle 2. In Cycle 3, global QoL (p = 0.040) and fatigue (p = 0.013) were significantly better in the intervention group compared to placebo. This trial suggests adjuvant ginger supplementation is associated with better chemotherapy-induced nausea-related quality of life and less cancer-related fatigue, with no difference in adverse effects compared to placebo.Ginger supplementation could be an effective adjuvant treatment for chemotherapy-induced nausea (CIN). The aim of this clinical trial was to address significant methodological limitations in previous trials. Patients (N = 51) were randomly allocated to receive either 1.2 g of standardised ginger extract or placebo per day, in addition to standard anti-emetic therapy, during the first three cycles of chemotherapy. The primary outcome was CIN-related quality of life (QoL) measured with the Functional Living Index- Emesis (FLIE) questionnaire. Secondary outcomes included acute and delayed nausea, vomiting, and retching as well as cancer-related fatigue, nutritional status, and CIN and vomiting-specific prognostic factors. Over three consecutive chemotherapy cycles, nausea was more prevalent than vomiting (47% vs. 12%). In chemotherapy Cycle 1, intervention participants reported significantly better QoL related to CIN ( The prevention and management of chemotherapy-induced nausea and vomiting (CINV) is a priority in the oncology setting. While the development of a range of anti-emetic medications has reduced the prevalence of CINV, vomiting and, in particular, nausea, are still experienced by up to 25% and 61% of cancer patients, respectively ,4. Furth3 receptor antagonism, which is also one of the cornerstones of modern antiemetic drug therapies such as ondansetron and granisetron [Various interventions to reduce CINV have been investigated. These include pharmaceuticals , behavioral interventions , and nutraceuticals such as ginger supplementation ,6,7. Thenisetron .Ginger has been trialed with some success for other types of nausea, including morning sickness and post-operative nausea and vomiting ,10,11. TThe primary aim of this double-blind, randomized placebo-controlled trial was to determine the effect of adjuvant time- and dose-standardized ginger on chemotherapy-induced nausea (CIN)-related QoL, compared to placebo, in chemotherapy-naïve patients over three cycles of moderately- or highly-emetogenic chemotherapy.Previously unexplored, but clinically important outcomes, were also investigated. These include cancer-related fatigue and malnutrition; both prevalent in chemotherapy cohorts, and consistently associated with CINV, and linked with significant decrements in patient QoL ,13. In aThe design of this double-blind, randomised placebo-controlled trial is based on our fully detailed protocol manuscript . The priPatients were recruited if they were chemotherapy-naïve, were due to receive a moderately- or highly-emetogenic chemotherapy regimen, were at least 18 years old, had a baseline Karnofsky score >60 , had no Patients were recruited from the Princess Alexandra Hospital, Brisbane, Australia from March 2014 to February 2015. Potentially eligible patients were identified by research staff during daily chemotherapy education sessions and through the hospital chemotherapy scheduling system within one week prior to the first cycle of chemotherapy.Participants were randomly assigned to receive either 1.2 g (4 × 300 mg) of a standardized ginger extract or placebo in conjunction with the standard antiemetic therapy prescribed by their physician. The rationale for the selected dose is based on the dose used in previous trials that have reported significant improvements, pharmacokinetic data, and concerns regarding possible contraindications at high doses, as described in our previous protocol manuscript . The ginEligible patients were randomly allocated to ginger or placebo capsules by an independent company using a computer-generated sequence. Participants received three questionnaire booklets, one for each cycle of chemotherapy, which were either mailed back to the researchers upon completion or collected during the following cycle. All staff members involved in recruitment and outcome assessment were blinded to the results of randomization. Participants were followed over three chemotherapy cycles in order to evaluate the effect of the intervention over an extended period of their chemotherapy treatment. For each cycle, outcomes were assessed 3 days prior to chemotherapy until 4 days post-chemotherapy . Participants were asked to consume the study capsules 4 times per day, with each meal, for 5 days per chemotherapy cycle, commencing on the day of chemotherapy.The primary outcome was chemotherapy-induced nausea-related (CIN) quality of life (QoL) based on the rationale that this outcome is more clinically meaningful compared to other standard measures of CINV that may not fully reflect the impact of reported CINV on all domains of daily living. For example, a patient may report a low frequency of nausea but this may significantly impact on their QoL. This was measured using the Functional Living Index Emesis 5 Day Recall (FLIE-5DR) questionnaire, a validated measure of the impact of CINV on patients’ general well-being . It compA total score for CINV as well as separate scores for nausea, retching and vomiting were elicited using the validated, 8-item self-report tool, the Rhodes Inventory of Nausea, Vomiting and Retching (INVR) . The INVThe following operational definitions defined each phase of CINV. Anticipatory CINV was defined as any symptom occurring in the 24 h prior to chemotherapy administration . Acute CThe INVR is designed to measure symptoms over a 12-h period; however, to minimize survey burden, this period was extended to 24 h so that participants would only need to complete one questionnaire per 24 h. For each 24-h period, a score  is given for each symptom and a total score is derived from each symptom score (from 8 to 40). For delayed symptoms, scores from the three 24 hour periods after the acute phase were combined. Nutritional status was assessed once per chemotherapy cycle, on the day of chemotherapy, by an appropriately-trained research dietitian using the Patient-generated Subjective Global Assessment (PG-SGA) tool . The PG-Global cancer-related QoL and cancer-related fatigue were assessed at baseline and 4-days post-chemotherapy using the Functional Assessment of Cancer Therapy-Global (FACT-G) and the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) assessment questionnaires, respectively ,23. BothA new questionnaire was developed as part of this project to assess the prevalence of prognostic factors identified to increase the risk of CINV. The rationale for implementing this questionnaire was that an uneven distribution of these prognostic factors between the intervention and placebo groups could influence the results. The questionnaire included five items that assessed the patient’s history of morning sickness and motion sickness, their average weekly alcohol intake and their history of anxiety ,28. AddiTo determine adherence to the study protocol, participants were asked to record the number of capsules consumed each day during the study period. The quality of patient blinding was assessed at the end of each chemotherapy cycle during participant interviews, in which participants were asked to state the capsule (placebo or ginger) they believed they had received.Safety concerns and adverse events were monitored via telephone interviews during each cycle, 5-day post-chemotherapy. Participants were asked about any hospitalizations or adverse events during the study period. To assess any negative effects of ginger supplementation, between-group differences in a range of symptoms were also assessed using the Edmonton Symptom Assessment Scale, which was administered at each cycle of chemotherapy, at baseline and 5-day post-chemotherapy . This is® . Descriptive analysis of baseline participant characteristics was undertaken. Bivariate outcomes were assessed using Chi-square analysis. Normally distributed continuous outcomes were assessed using independent sample t-tests. The Mann-Whitney U test was used for non-parametric outcomes. Data were analyzed on an intention-to-treat basis. A p value < 0.05 indicated statistical significance. Mean and standard deviation (SD) were reported for normally distributed data. Median with 25th percentile and 75th percentile were reported for non-parametric data. Missing data were handled using multiple imputation. In order to explore the association between ginger supplementation combined with aprepitant and worse delayed-CINV, a subgroup analysis in patients receiving aprepitant was also conducted.Statistical analysis was conducted using SPSS Version 20Sample size was calculated based on the ability to detect a clinically meaningful difference in the primary outcome of nausea-related QoL. Hence, using the standard deviations from a preliminary feasibility study of ten participants and a desired mean difference of 9 points on the nausea-related subdomain of the FLIE-5DR, a sample size of 82 was estimated to provide sufficient power to detect a statistically and clinically significant difference in nausea-related QoL with 80% power and 5% significance.p > 0.05 for all characteristics). The majority of patients (84%) were scheduled to undergo moderately emetogenic chemotherapy regimens  and better total CINV-related QoL  = 124.5  vs. 111 ; p = 0.043) compared to patients assigned the placebo. Examination of median CINV- and nausea-related QoL at Cycle 1 in the placebo and intervention groups suggests that the clinical significance of this effect was minimal in both groups  = 61.5  vs. 54 ; h groups . No otheOver the three chemotherapy cycles, acute and delayed CINV occurred in 39% and 65% of all participants . In bothn = 18) assigned to the intervention with and without being prescribed aprepitant, there were no statistically significant differences in CINV between groups at any time point.In a subgroup analysis of participants (p = 0.006) and Cycle 3  compared to placebo. There was also a statistically significant difference in cancer-related QoL at Cycle 1  and Cycle 3 .Clinically significant fatigue and malnutrition were experienced by 35% and 22% of participants over the study period. Ginger supplementation was associated with improved measures of chemotherapy-related fatigue in Cycle 1  and cancer-related fatigue (>3 point difference) were clinically important. No significant difference in nutritional status was detected between the intervention and placebo group during the study period . For participants in the intervention group who successfully identified their allocation, the most common rationale provided was a lack of nausea (60%), the smell of the capsules (20%), and ginger taste or reflux (13%). Adherence to the study intervention was moderate-to-high, with 71% of all participants (67% in ginger group and 74% in placebo group) consuming at least 3 of the 4 prescribed capsules per day.p > 0.05) between these variables and any measure of CINV.The hypothesized prognostic factors of age, gender, anticipatory CINV, and chemotherapy emetogenicity were analyzed with no significant associations detected  compared to some previous studies, which have reported an attrition rate of approximately 20% ,31. Due Another limitation was that, while this trial was sufficiently powered to detect a significant difference in the primary outcome at Cycle 1, due to timing constraints and attrition, the trial did not meet the required sample size for the second and third cycles of chemotherapy. Introducing sufficient inflation factors in sample size calculations, as well as reducing study burden, is recommended in future studies to ensure sufficient power during subsequent cycles.In summary, the results of this clinical trial suggest that compared to placebo, adjuvant ginger is associated with better chemotherapy-induced nausea-related and cancer-related quality of life, and less cancer-related fatigue. The results confirm several previous studies that report ginger supplementation to be well-tolerated and without significant side-effects. Further studies with larger sample sizes are required to confirm these results and to further explore the safety profile of ginger supplementation during chemotherapy.


To investigate the effect of laser pan-retinal photocoagulation with or without intravitreal bevacizumab injections on macular choroidal thickness parameters in eyes with high-risk proliferative diabetic retinopathy.High-risk proliferative diabetic retinopathy patients undergoing laser treatment were prospectively enrolled in this study. One eye was randomly selected for laser treatment combined with bevacizumab injections, study group, whereas the corresponding eye was subjected to laser treatment alone, control group. Spectral-domain optical coherence tomography with enhanced depth imaging was used to measure the macular choroidal thickness prior to and 1 month after treatment. Measurements in both groups were compared. Clinicaltrials.gov: NCT01389505.Nineteen patients (38 eyes) with a mean±standard deviation age of 53.4±9.3 years were evaluated, and choroidal thickness measurements for 15 patients were used for comparison. The greatest measurement before treatment was the subfoveal choroidal thickness . No significant difference between groups was found in terms of macular choroidal thickness measurements at baseline or after treatment. However, within-group comparisons revealed a significant increase in choroidal thickness parameters in 10 measurements in the study group and in only 5 temporal measurements in the control group when 1-month follow-up measurements were compared to baseline values.The macular choroidal thickness does not appear to be significantly influenced by laser treatment alone but increases significantly when associated with bevacizumab injections in patients with proliferative diabetic retinopathy and macular edema. Because bevacizumab injections reduce short-term laser pan-retinal photocoagulation-induced macular edema, our findings suggest that the choroid participates in its pathogenesis. Diabetes mellitus (DM) is an important metabolic disease that is often associated with injury to several ocular structures. Diabetic retinopathy (DR) is one of its most common chronic microvascular complications Laser pan-retinal photocoagulation (PRP) is the primary treatment modality for preventing vision loss in patients with PDR Previous studies have indicated that DM may also affect the choroid via obstruction of the choriocapillaris, vascular degeneration, and the development of choroidal aneurysms and neovascularization that may be present even before the onset of DR Most previous studies evaluating possible involvement of the choroid in patients with DM were performed using indocyanine green angiography In relation to the changes in macular CT for PDR patients treated with PRP with or without anti-VEGF, the published data are still inconsistent likely due to differences in samples and the presence or absence of DME. The only finding that has been confirmed is that PRP causes macular CT thinning in long-term follow-ups Despite the vast literature addressing the effects of PRP on the retina, previous OCT studies have not addressed the possible effects of PRP with or without IVB injections on the macular CT measurements of high-risk PDR patients. Thus, we prospectively investigated the effects of PRP alone or with the adjuvant use of IVB injections on macular CT measurements in patients with high-risk PDR.This trial applied an interventional, prospective, masked, and randomized study design. The study followed the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of the Ethics Committee of our institution. All participants signed an informed consent form. Clinicaltrials.gov: NCT01389505.The inclusion criteria for the study were best corrected visual acuity (BCVA) ≥20/200, type 2 DM, and similar high-risk PDR in both eyes with or without DME. The exclusion criteria were pretreatment of diabetic retinopathy (laser photocoagulation or intraocular surgery), vitreous hemorrhage, vitreous-retinal interface alteration , active external eye disease, systolic or diastolic blood pressures greater than 180 or 110 mm Hg, respectively, A1C levels exceeding 15%, and chronic renal failure.®Stratus-OCT, Carl Zeiss Ophthalmic System, Inc.; Dublin, California, USA) and spectral-domain  OCT scanning. Diabetic ME was diagnosed if the OCT foveal thickness (FT) was ≥250 μm on ®Stratus-OCT measurements Twenty-two patients  were enrolled in the study. All patients received a complete clinical evaluation and the following ophthalmic examinations: BCVA assessment, intraocular pressure measurements, slit-lamp examinations, fundoscopy and time-domain  at 870 nm with enhanced depth imaging (EDI) was used to measure macular CT, defined as the vertical distance between the posterior edge of the retinal pigment epithelium to the choroidal-scleral junction. CT measurements at 500-μm intervals up to 2,500 μm from the temporal and nasal to the fovea were used for comparisons. An investigator blinded to the study design performed all measurements in one afternoon. On a different occasion, another examiner independently measured the same group of OCTs. To calculate intra- and inter-observer variability, the same two observers repeated their measurements unaware of the previous results.The The fourth-generation OCT employed was equipped with a camera that can be used to monitor the stability of patient fixation. Therefore, the tomograms should occur at the same point. The images were generated using six macular radial scans, centered at the fovea at equally spaced angular orientations, with 20 raster lines spaced 200 μm apart. The machine rated the quality of the images, and only ratings greater than 25 were considered high-quality scans. Cross-sectional images were analyzed using built-in software.Both eyes of each patient were included. The first eye was randomly assigned to the study group to receive PRP with IVB injections, and the other eye was enrolled in the control group and received PRP alone. A schematic representation of the study protocol is shown in The control group received PRP once per week for 3 consecutive weeks beginning on the same day as randomization. Between 300 and 500 shots were given per episode. The retinal laser parameters were a spot size of 250 μm, an exposure time between 0.1 and 0.2 msec, and a moderate intensity (200 to 500 mW)In the study group, on the day of randomization, one eye received an IVB (1.25 mg/0.05 mL) injection following standard international guidelines The laser treatment for both eyes was performed after ophthalmic evaluation using a double-frequency Nd:YAG laser  via a 160 Mainster OMRA-PRP lens .The patients were followed for 1 month. Ophthalmic examinations at the end of a 1-month follow-up visit included intraocular pressure measurements using a Goldmann applanation tonometer, slit-lamp biomicroscopy, fundus examination, OCT scanning and fluorescein angiography.The primary outcome was a determination of macular CT measurements of the eyes in the experimental and control groups at baseline and at a 1-month follow-up; these measurements were then compared between the two groups.The secondary outcome included a longitudinal comparison of macular CT measurements within each group, comparing the measurements at baseline with those at the first-month follow-up visit.Potential injection-related local side effects, such as ocular hypertension, lens opacity progression, and anterior chamber reaction, were reported to evaluate ocular safety. Arterial thromboembolic events were reported to evaluate systemic safety.p<0.05. The analyses were performed using SPSS, version 15.0 .Descriptive statistics included the mean±standard deviation. The Shapiro–Wilk test was used to evaluate the normality assumption. Comparisons of macular CT measurements between and within groups were assessed using the rank-sum and the Wilcoxon signed-rank test, respectively. An intra-class correlation coefficient (ICC) was calculated to assess inter- and intra-rater variability for measurements of macular CT. Fisher’s exact test was applied to compare the number of significantly different measurements in the treatment and the control group. The level of statistical significance was defined as Twenty-two patients were eligible, and 19 were included in the study. Three patients were excluded before randomization, 2 because of unreliable CT measurements and one because of vitreous hemorrhage. Nineteen patients (38 eyes) were randomized and treated, but at the one-month follow-up, 4 patients were excluded (2 because of vitreous hemorrhage and 2 because of unreliable CT measurements at follow-up). The fourth excluded patient had one eye (from the control group) excluded due to unreliable CT measurements, whereas the eye subjected to PRP and IVB injections was suitable for study.p<0.001), indicating excellent inter-observer variability. The corresponding value for repeated measurements by the same reader was 0.98, indicating excellent intra-observer variability (p<0.001).The intra-class correlation coefficient for the macular CT measurements by the second observer at the fovea, 500 μm nasal and 500 μm temporal was 0.99 for all three measurements  were males. The average glycated hemoglobin level was 8.89 (range 7.5-9.7). DM had lasted 17.3±8.7 years, and systemic arterial hypertension had lasted 7.3±6.2 years. The mean initial BCVA was 0.21±0.21 and 0.21±0.21 logMAR for the study and control groups, respectively.DME was present in 11 and 10 eyes of the 15 patients from the study and control groups, respectively. p=0.024, Fisher’s exact test).Throughout the study, we did not detect ocular hypertension, lens opacity progression, anterior chamber reactions, or thromboembolic events.The relationship between the choroid and DR and the effects of PRP with or without intraocular anti-VEGF agents on macular CT are not well understood in high-risk PDR patients. There is conflicting evidence on the effects of anti-VEGF agents on macular CT, even in patients not subjected to PRP. Nevertheless, in PDR diabetic patients, because PRP can increase macular retinal thickness in the short-term, it is possible that it may also alter macular CT. Furthermore, in a prospective, non-comparative, 1-month follow-up study, Takashi et al. Zhang et al. Roohipoor et al. We believe that the finding of a greater CT increase at the end of 1 month in the group subjected to PRP + IVB injections compared to the group subjected to PRP alone may be primarily explained by four mechanisms: a) PRP temporarily increases the macular CBV and CBF We therefore believe that because laser treatment causes increased VEGF levels inside the eye leading to a more significant breakdown of the EHB, fluid may shift from the choroid to the retina, particularly in eyes with long-standing DME, which have retinal pigment epithelium that has been progressively damaged by the presence of fluid. The use of anti-VEGF bevacizumab treatment as an adjuvant may, in part, restore the hemato–retinal barrier function, somehow preventing a fluid shift and therefore contributing to an increase in choroid thickness, relative to pretreatment measurements in the study group. This reasoning may explain why the control group showed less significant increases in macular CT measurements compared to the treatment group in the current study; this may also explain why Regatieri et al. We understand that the limitations of the present study include its relatively small sample size, its short follow-up period and its inclusion of patients with and without DME. However, we believe that the results of this pilot study may stimulate further research on the choroidal contribution in the appearance of DME after PRP.In conclusion, although significant between-group differences in the macular CT measurements were not observed, our study indicates that when IVB injections were used as an adjuvant to PRP in the treatment of PDR, a more pronounced increase in the macular CT was observed over a brief follow-up. Additional larger studies are required to confirm these findings.Preti RC conceived and designed the study, was responsible for data acquisition, analysis and interpretation, and manuscript drafting. Takahashi WY conceived and designed the study, and was responsible for the manuscript critical revision. Mutti A and Ferraz DA were responsible for data acquisition. Nakashima Y was responsible for data analysis and interpretation. Zacharias LC was responsible for data analysis and interpretation, and manuscript drafting. Monteiro ML was responsible for the manuscript drafting and critical revision.


Of 11,215 exons detected in the maternal blood whole-transcriptome, a panel of 395 genes, including PPP1R15B, EXOC8, ACTB, and TPT1, were identified to comprise exons with considerably less variable expression level  than any GAPDH exon . Upon validation, the selected genes from this panel remained more stably expressed than GAPDH in maternal blood. This panel is over-represented with genes involved with the actin cytoskeleton, macromolecular complex, and integrin signaling. This groundwork provides a starting point for systematically selecting reference gene candidates for normalizing the levels of circulating RNA transcripts in maternal blood.RNA transcripts circulating in peripheral blood represent an important source of non-invasive biomarkers. To accurately quantify the levels of circulating transcripts, one needs to normalize the data with internal control reference genes, which are detected at relatively constant levels across blood samples. A few reference gene candidates have to be selected from transcriptome data before the validation of their stable expression by reverse-transcription quantitative polymerase chain reaction. However, there is a lack of transcriptome, let alone whole-transcriptome, data from maternal blood. To overcome this shortfall, we performed RNA-sequencing on blood samples from women presenting with preterm labor. The  Quantitative polymerase chain reaction (qPCR) is a standard method for the quantification of nucleic acid sequences ,2. CombiPreterm birth (delivery before 37 weeks of gestation) is a major cause of neonatal morbidity and mortality . Less thReference genes for normalizing RNA transcripts in the human circulatory system have been reported on patients with tuberculosis , schizopTo address this paucity of relevant study, we embarked on a search for reference genes suitable for the normalization of RT-qPCR data on whole blood collected from women during their presentation of preterm labor. Similar searches for reference genes often rely on a candidate gene approach or gene expression microarray data, which is sometimes referred to as transcriptome data. Such microarray-based transcriptome studies measure RNA levels based on oligonucleotide probes, which requires prior knowledge of the RNA transcript sequences, and thus are limited essentially to the more characterized genes of known sequences. To maximize the chance of finding suitable reference genes, in this study, we expanded this search to the whole-transcriptome dataset generated by RNA-sequencing (RNA-seq), which is not limited by probes of well-characterized gene sequences. We hypothesized that the whole-transcriptome of maternal blood harbors many RNA transcripts that are expressed at relatively constant levels. Based on the RNA-seq dataset, 395 genes, comprising 458 exons, were systematically identified by detecting at a low variation across all tested maternal blood samples. Subsequently, the expression stability of the selected genes was assessed using RT-qPCR in another set of maternal blood samples. Our data suggest that the whole-transcriptome harbors gene candidates of higher expression stability than the commonly used reference genes.In this study, RNA-seq data were interpreted at the finer resolution of exons, which are sub-regions within an expressed gene transcript. Different exons of the same gene were detected at considerably different levels of variation. Based on the exon-level RNA-seq data, one can readily pinpoint the exon with the least variation in the design of RT-qPCR assays for the quantification of candidate reference genes. Moreover, we explored how the list of 458 stably detected exons could be used as a starting point for the systematic identification of reference genes in other blood compartments and different groups of patients. Lastly, we discuss the pros and cons of our exon-level RNA-seq approach, and how it may be further improved for identifying reference genes.We obtained the informed consent of, and collected peripheral whole blood samples from, pregnant women during their presentation of preterm labor. Twenty blood samples were subjected to strand-specific RNA-seq (ssRNA-seq). Forty libraries  were constructed for strand-specific pair-end cDNA sequencing on the HiSeq 4000 sequencer . Low-quality sequences and adapter sequences were trimmed by Trimmomatic, v.0.33 , and aliCV = standard deviation divided by the mean) across all maternal blood samples were calculated. mean and CV of the robustly detected exons in the log10 scale.Each gene comprises one or more sub-regions called exons. At the above sequencing depth, RNA-seq allows investigators to profile RNA levels not only at gene-level but also at the higher resolution of exon-level. Since the higher resolution exon-level data are advantageous for developing RT-qPCR assays, the read counts mapped to each exon in GRCh38 were summarized. To account for technical variations, including the differing sequencing depth and the amount of RNA input for each library, read counts were normalized using the Remove Unwanted Variation from RNA-seq data (RUVSeq) method , which is based on the factor analysis of control samples, such as technical replicates . Unless median  of the mean RNA levels was 5.7 counts (3.2–13 counts), and the median of the CV (IQR) of the RNA levels was 32.5% (24.0% to 42.0%). The 10th and 90th percentiles of the CV are 17.7% and 53.8%, respectively. The Spearman rank order correlation coefficient was only −0.06  between the mean and the CV of the RNA levels of these exons. Exons with a low CV are good candidates for reference genes, because they exhibit a low variation across all maternal blood samples (CV below the 10th percentile (10log(CV%) < 1.25) are viable candidates  = 1.4; Without presuming a normal distribution of the linear-scale data on RNA levels across the 4579 exons, we describe the data as follows: the  samples . For exap-value after correction for multiple testing by the Bonferroni method. Nine cellular component PANTHER GO-Slim terms were over-represented among our list of stably detected genes, as compared with the reference list of all genes in the human genome  terms  was statn genome A. The 12n genome B. The rep, 9.7 × 10−4), intracellular , cell part , organelle , and actin cytoskeleton . Overall, this is in line with the major components expected of blood cells after lysis in the RNA extraction step.Compared with the reference list of all genes in the human genome, the list of 395 genes that are stably detected in maternal blood is over-represented with GO terms in cellular components , T cell activation , inflammation mediated by a chemokine and cytokine signaling pathway , fetal growth factor (FGF) signaling pathway , epidermal growth factor (EGF) receptor signaling pathway , and integrin signaling pathway . The B cell activation pathway, T cell activation pathway, and inflammation mediated by a chemokine and cytokine signaling pathway highlight the important roles played by immune regulation in pregnancy [Moreover, this list of 395 stably detected genes is over-represented with 12 pathways , Fc γ receptor (FCGR)-dependent phagocytosis , and clathrin-mediated endocytosis . These are consistent with the presence of white blood cells.Furthermore, the list of stably detected genes in maternal blood is over-represented with pathways in other curated databases , EXOC8 (ENSE00001442235), and PPP1R15B (ENSE00001443770) genes are 7.7%, 9.0%, and 9.4%, respectively. See CV ranked 1st, 4th, and 6th among the 4579 robustly detected exons in maternal blood. Their mean RNA levels (± standard deviation (SD)) are 85 counts ± 7 counts, 34 counts ± 3 counts, and 27 counts ± 3 counts, respectively.Ideally, the RNA levels of the reference gene and our gene of interest should not differ by more than a few orders of magnitude. A panel of 36 preterm birth-associated transcripts are reported to be aberrantly expressed in the preterm placenta and released into maternal plasma . Of thesRPL37A was found to be suitable for normalizing RT-qPCR data in whole blood from tuberculosis patients [RPL37A was also robustly detected. In a meta-analysis of microarray data on 1053 blood samples from healthy individuals and patients with cancer and other abnormalities, less than a dozen genes were found to be expressed at less than a CV of 30% [ACTB, HUWE1, and TPT1 showed the highest RNA levels. Historically, GAPDH was often used as a reference gene, despite the controversy over whether its expression is stable enough to serve this purpose [Additionally, other reference genes were shortlisted for normalizing the RT-qPCR data in human blood sample. Previously, other investigators also applied high-throughput data to systematically search for reference genes for normalizing RNA transcripts levels in blood. In a study based on microarray data, among several other genes, patients . RPL37A V of 30% . Of thes purpose .IQR of the RNA levels of DDX17, EXOC8, and PPP1R15B are small, because they were shortlisted based on their small CV in the same dataset. The IQR of the RNA levels of GAPDH is the largest among the shortlisted genes. The CV of the RNA levels for TPT1 (ENSE00001618156), ACTB (ENSE00001902558), GAPDH (ENSE00001817977), RPL37A (ENSE00001900648), and HUWE1 (ENSE00000978660) are 21%, 25%, 27%, 29%, and 43%, respectively. The highly variable expression levels of the RPL37A and HUWE1 genes, and the low RNA levels expressed by the HUWE1 gene, make them unsuitable to serve as reference genes in maternal blood samples. In fact, of these six candidates identified from the literature, the CV of five genes are larger than the lower quartile of the CV (24%), as shown by the viable reference gene candidates that may be identified from our RNA-seq dataset  guidelines . The expM), which is defined as the average pairwise variation of that particular gene compared with all other candidate genes, is calculated. In the NormFinder software , a stability value is also calculated for each gene by a model-based approach, taking into account its intra- and inter-group variations. In both softwares, the lowest stability value indicates the most stable gene.We used two common softwares, namely geNorm  and Normr = 0.94, p = 0.005), and so are those calculated using NormFinder . The 95% confidence interval of the best-fitting line between the two subsets in geNorm appears to be narrower than that in NormFinder  of a certain number (n) of genes was calculated, starting with the most stable genes. To determine the possible need or utility of including a certain number of genes for normalization, the pairwise variation nVn+1/ was calculated between nNF and nNF+1 . Its presence may affect the accuracy of the quantification of the amount of RNA input for library construction. Moreover, since the predominance of rRNA often masks the signals from other more informative mRNA in RNA-seq experiments, a common practice is to reduce the rRNA before library construction. However, such a practice contributes to a source of technical variations. Combining the quality-filtering, mapping, and normalization steps in the data analysis, each RNA-seq dataset contains a considerable amount of noise, which should be vigorously controlled for reliable gene expression profiling. Therefore, it is important to validate the findings from RNA-seq by an independent technology platform, such as RT-qPCR and cohort. This is especially true for the identification of reference genes, because the impact of technical variation in RNA-seq on a gene expression stability analysis has not been extensively studied. Furthermore, we have supplemented our RNA-seq-based selection of candidates with relevant studies which have employed another platform for profiling transcriptome.We have also been able to assess the variations of candidate reference genes in terms of r = 0.94 and 0.89, respectively) stability values between two different subsets of samples. Thus, the data suggest that both softwares generate similar stability values across different batches of samples. Although geNorm has generated slightly more correlated stability values across the two subsets, it is noted that both geNorm and NormFinder have consistently ranked PPP1R15B, EXOC8, and ACTB as the most stably detected genes , EXOC8 (9.0%), and PPP1R15B (9.4%). On the other hand, in the geNorm analysis, ACTB turned out to be one of the most stably detected genes. A closer examination revealed that the ACTB exons targeted by the commercially available RT-qPCR assay were not detected in our RNA-seq dataset. The RT-qPCR assay pre-designed by that company targeted an amplicon that is mapped to more than one genomic location. This type of exonic sequencing reads been filtered out after mapping, because they may interfere with the counting of transcripts in the RNA-seq experiment. This illustrates how a search for reference genes based on RNA-seq may benefit from relevant studies using an alternative technology.We observed that the RNA-seq data is not always predictive of the gene expression stability analysis data based on RT-qPCR. For instance, in the RNA-seq data, the RNA levels of ACTB exon targeted by the commercial RT-qPCR assay was not detected in our RNA-seq data. It has been estimated that the RNA levels of hundreds of genes in the human genome could not be quantified accurately by RNA-Seq [The major advantage of our RNA-seq approach is that reference gene candidates could be systematically identified from the whole-transcriptome, which is more comprehensive. Moreover, the exon-level data allow a more specific design of RT-qPCR assays. The downside is that certain short sequencing reads that are mapped to more than one unique location in the genome are missed. For instance, the  RNA-Seq . These g RNA-Seq . CombineIn this study, we provided a list of 458 exons (395 genes) that are most stably detected in maternal blood. Unlike many other tissues, peripheral blood is readily and non-invasively obtainable from a patient. Many RNA transcripts expressed in other tissues are often released into the blood circulation, including preterm birth-associated placental RNA and pregnancy-associated microRNA ,30. NeveTo find reference genes in blood samples from patients with other diseases, one may first shortlist about 10 stably detected exons from our RNA-seq data  with simIn addition, we noted that the stably detected genes in maternal blood are over-represented with annotation terms in macromolecular complex and actin cytoskeleton A. This iIntriguingly, terms in the FGF and EGF receptor signaling pathways and Integrin pathways are also over-represented in the stably detected genes. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning . The EGFr = 0.39, with 6135 out of 9302 mRNA–protein pairs (66.0%) correlating significantly at a false discovery rate <0.05 [Analogous to the systematic search for reference genes to normalize RNA data based on RNA-seq, investigators have also searched for reference genes to normalize protein data based on proteomics. It would be interesting to contemplate whether the genes with stable RNA expression in blood, as identified in this study, may also have stable protein expression. However, in a recent study on breast cancer, a global analysis of the correlation of mRNA-to-protein yielded a median Pearson value of te <0.05 . Hence, CV in maternal whole blood samples collected from women presenting with preterm labor. They are good reference gene candidates for normalizing expression data from these patients. Particularly, as further suggested by the results of the expression stability analysis, the RNA transcripts of PPP1R15B, ACTB, EXOC8, and TPT1 are stably expressed in the maternal whole blood samples. Hence, these four genes are useful to serve as reference genes for normalizing the concentrations of other circulating transcripts in maternal peripheral whole blood.Taken altogether, the maternal circulation harbors RNA transcripts that are stably detected. We identified a list of 395 genes that were detected at a low PPP1R15B, ACTB, EXOC8, and TPT1. Then, an expression stability analysis on the shortlisted candidates could be performed directly without going through the time- and resource-consuming screening experiments like RNA-seq or expression microarray. Since this list is based on data from human blood, we reason that the candidates proposed in this study are more suitable for normalizing RNA data in blood when compared with other lists based on tissues besides blood.Our list may also be useful as a starting point for finding reference genes for normalizing data from patients with other disease, from non-pregnant females or from pregnant women without preterm labor, or from samples of different blood compartments. A handful of candidates could first be shortlisted from the RNA-seq identified list of stably detected genes , particuWith reference genes that are more stably detected in blood, it is hoped that the hurdles of normalization could be overcome, and that more differentially expressed transcripts could be identified in the circulatory system.This study was approved by the Joint Chinese University of Hong Kong and New Territories Easter Cluster Clinical Research Ethics Committee  and the Institutional Review Board of Hallym University Kangnam Sacred Heart Hospital . Women presenting with preterm labor, and that fulfilled the inclusion and exclusion criteria, were invited to participate in this study. The inclusion criteria were women with (i) uterine contractions at least once every 10 min <34 weeks, (ii) intact membrane, (iii) singleton pregnancies, and (iv) a Chinese or Korean ethnicity. The exclusion criteria were women with pregnancies complicated with (i) preterm prelabor rupture of membrane, (ii) multiple gestation, (iii) preeclampsia, (iv) fetal growth restriction, (v) macrosomia, (vi) fetal distress, (vii) antepartum hemorrhage, (viii) fetal chromosomal or structural abnormalities, (ix) history of uterine abnormality or cervical surgery, and (x) indicated preterm births before 37 weeks , where deliveries are iatrogenic. Gestational ages were established based on menstrual date confirmed by sonographic examination <20 weeks. Delivery outcome was traced and categorized accordingly into spontaneous preterm birth  and term birth  groups. Cases with chorioamnionitis were included in the sPTB group.DNase I (Thermo Fisher Scientific) to minimize contaminating genomic DNA. The quality of the RNA preparation was assessed by spectrophotometry and RNA Pico chip on the Bioanalyzer .Blood sample (9 mL) was collected from the antecubital fossa in ethylenediaminetetraacetic acid (EDTA)-containing tubes . To minimize RNA degradation, the blood sample was mixed with RNAlater  shortly after venesection and stored at −80 °C until extraction. RNA was extracted using the RiboPure RNA Purification Kit (blood) (Thermo Fisher Scientific), and treated with CV of RNA levels were calculated at the exon-level. When multiple exons were robustly detected in the RNA-seq dataset, the data from the exon with the lowest CV were reported unless otherwise stated. After all of the quality checking and filtering procedures mentioned were performed, RNA-seq data from a total of 19 blood samples, collected from 10 women ending in sPTB and 9 women ending in TB, were analyzed.Forty libraries  were constructed for strand-specific pair-end cDNA sequencing on the HiSeq 4000 sequencing platform (Illumina) (TruSeq 3000 4000 SBS Kit v3) according to the TruSeq Stranded Total RNA Sample Prep Guide (Part # 15031048 Rev. E). To minimize the highly abundant but uninformative ribosomal RNA and globin mRNA transcripts from masking the signals from the more informative transcripts, RNA samples were subjected to pre-treatment by Ribo-Zero Globin (Illumina). The remaining ribosomal RNA reads were removed (RSeQC v2.6.2) . Low-quaCV of RNA levels for all except two genes in this study using the on-line PrimerQuest software (https://eu.idtdna.com/Primerquest/Home/Index) by Integrated DNA Technologies . The assays for the ACTB and GAPDH genes were predesigned by and purchased from Integrated DNA Technologies, Inc. To improve the accuracy of the quantification, hydrolysis probes were used. The primers and probes were validated in silico for possible secondary structures and non-specific binding by Primer-BLAST [DNase I before RT-qPCR. To monitor for environmental contamination, no template controls were run in parallel.To leverage on the exon-level transcriptome data, we designed RT-qPCR assays to target the exon with the lowest er-BLAST . The fulDNase I-treated RNA was added to each reaction. The geNorm analysis was carried out in qBase PLUS, which is MIQE-compliant [A RT-qPCR primed by random primers is linear over a narrower range than a similar reaction primed by target-specific primers . Furtherompliant . The PCRUnless mentioned above, all data analysis, including correlation tests, descriptive statistics, and graph plotting, were performed using DeduceR , which iPPP1R15B, ACTB, EXOC8 and TPT1 could be used as reference genes for normalizing the concentrations of other circulating transcripts in maternal peripheral whole blood.The RNA transcripts of 395 genes in


IntroductionPrimary central nervous system lymphoma (PCNSL) is a rare tumor without a well-defined standard of care. For immunocompetent patients, therapeutic regimens have largely evolved from treatment with whole-brain radiation therapy (WBRT) to treating initially with systemic chemotherapy regimens that include high-dose (HD) methotrexate (MTX) with or without WBRT. Looking at population-based treatment trends may help define which therapies are most effective. This study was conducted to determine treatment patterns and outcomes for patients with PCNSL in the Louisville, KY metropolitan area during the period 2000 to 2012.MethodsData were collected by retrospective chart reviews of patients identified using the International Classification of Diseases (ICD) code from three major oncology practices in the Louisville metropolitan area during the period 2000 to 2012. Patients were excluded if they were under age 18, positive for human immunodeficiency virus (HIV), had histology other than B-cell lymphoma, or had systemic lymphoma.ResultsA total of 21 patients were identified. The median age was 65 years (range: 30 to 90). All patients were Caucasian, and the median Karnofsky performance status (KPS) score was 80 (range: 50 to 100). The ratio of males to females was 1:1.3. Median overall survival (OS) for all patients was 22 months (range: 1 to 155 months). Of 21 patients, 11 (52 percent) received chemotherapy regimens that included systemic HD-MTX at their initial diagnosis with a median OS of 22 months (range: 1 to 155 months). Nine of 21 patients (42 patients) were offered other therapies, including WBRT or non-MTX-based chemotherapies; they had a median OS of 5 months (range: 2 to 150 months). The median OS for patients receiving at least four cycles of HD-MTX was 40 months (range: 4 to 155 months).ConclusionsThis population-based study shows that patients with PCNSL and the ability to undergo HD-MTX-based therapy had a superior survival rate compared to those receiving radiation alone or other non-HD-MTX-based therapies.  Primary central nervous system lymphoma (PCNSL) is a rare tumor accounting for only two percent to three percent of all adult central nervous system (CNS) tumors in the United States . The lowIn the past 25 years, there has been a shift from using whole-brain radiation therapy (WBRT) for pre-radiation high-dose (HD) systemic methotrexate (MTX) regimens and delaying or declining WBRT altogether to avoid neurotoxicity . Series The purpose of this study was to analyze a regional cohort of patients with PCNSL to assess treatment trends and outcomes in ordinary clinical practice. This may provide an important source of information for optimizing treatment in the future.Study CohortWe performed a retrospective review of the medical records of patients diagnosed with PCNSL and who were treated at three major oncology practices in the Louisville, Kentucky area from January 2000 to December 2012. The three institutions consisted of a university teaching hospital, a large metropolitan hospital, and a private practice oncology group. Patients were identified by International Classification of Diseases (ICD) code 9590/3. The date range was chosen because adequate medical records were available at all three institutions for that period.Data Collection and VariablesThe data collected included patient demographics, symptoms at presentation, the duration of the symptoms before diagnosis, age at diagnosis, the location of the tumor by imaging studies, staging results, Karnofsky performance score (KPS), cerebrospinal fluid (CSF) protein levels, serum lactate dehydrogenase (LDH) levels, treatments, initial progression-free survival , survival after first recurrence, and overall survival . Our secondary outcomes were initial progression-free survival and the time from relapse to death. The institutional review boards (IRBs) of all three institutions approved this study.Statistical AnalysisAn analysis was performed on data from all 21 patients, and the effect of MTX treatment was evaluated by comparing treatment regimens containing MTX with treatment regimens without MTX.The continuous variable (age) was summarized as a mean with standard deviation, median with associated interquartile range (IQR), and the range (minimum to maximum). Categorical variables  were summarized as a frequency count, with associated percentages. To evaluate whether the two treatment groups had statistically similar demographics, we compared age using the Wilcoxon rank sum test and categorical variables with the chi-square test.Time-to-event variables  are presented with the median survival time and associated IQR as well as the full range . OS was compared between the two treatment groups using the log-rank test. A visualization of the survival curve is presented with Kaplan-Meier curves. Data pre-processing and analysis was performed in SAS 9.4 . All tests were two-sided with a significance level of 0.05.A total of 21 patients were identified and included in the study . Surgical data were available for 17 patients. A total of 11 patients underwent biopsy, 4 had a gross total resection (GTR), and 2 had subtotal resections. One of the patients included in the GTR group had ocular lymphoma and underwent a vitrectomy. This patient was observed without any other documented therapy. The median OS for all patients was 22 months (range: 1 to 155 months) (Table Of the 21 patients (52 percent), 11 received systemic HD-MTX-based regimens Table . The medWithin the 21 patients included in this study, 14 different treatment regimens were used Table . The 21 Treatment information was available for six patients at the time of recurrence Table . The treWe collected serum LDH and CSF protein data in order to give patients a prognostic score based on the international extranodal lymphoma study group where patients are stratified based on age, performance score, serum lactate dehydrogenase (LDH), cerebrospinal fluid (CSF) protein, and the involvement of deep regions of the brain . All variables were available for four patients in this cohort. Detailed data were not available for XRT or HD-MTX dosing.Historically, treatment using WBRT for patients with PCNSL improved overall survival from 2 to 4 months to 12 to 15 months . By the In this study, we looked at a regional cohort of 21 PCNSL patients from the Louisville, KY metropolitan area that were diagnosed and treated from 2000 to 2012. We found that consistent with therapeutic recommendations after 2000, a slightly larger number of patients (11 of 21 or 52 percent) were treated with HD-MTX-based systemic chemotherapy regimens and in most cases, XRT was omitted or deferred.Highlighted by this investigation is the lack of a standard of care even in newly diagnosed patients receiving HD-MTX-based regimens. Over the 13-year interval of this study, seven different upfront HD-MTX regimens were used in 11 patients. The two used most frequently were single agent HD-MTX (three cases) and HD-MTX in combination with cytarabine (two cases). The other regimens used in this patient cohort included HD-MTX, rituxan, vincristine, and procarbazine; HD-MTX, rituxan, vincristine, procarbazine, and WBRT; HD-MTX, vincristine, procarbazine, and cytarabine; HD-MTX, cytarabine, and rituxan; HD-MTX with rituxan and cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP).In these 11 patients, upfront XRT was given only two times. In one case, WBRT was used for consolidation in a 43-year-old patient considered young enough to avoid neurotoxicity. In the second case, a patient was initially started on HD-MTX and then switched to WBRT because of toxicity from HD-MTX.Like previously published studies, we see that patients treated with HD-MTX regimens had a significantly improved survival compared to those patients that received alternative therapies (22 months vs. 5 months) Figure  11]. Ho. Ho11]. Many of the treatment regimens used on patients in this study reflect some of the combined modality trials for patients with PCNSL that were being conducted or were published during the interval of the study. In a Phase II study of 52 patients published in 2000 by Aubrey et al., patients were given HD-MTX plus procarbazine and vincristine and some patients received WBRT. The median overall survival for the 52 patients was 60 months . HoweverThis regimen was later modified to include rituxan with reduced-dose WBRT followed by a high-dose cytarabine consolidation. The initial results of this multicenter single-arm Phase II trial published in 2007 were favorable, and, in 2013, the final results showed that survival on an intent-to-treat basis was 6.6 years in 52 patients -18.Another chemotherapy combination reflected in this study was investigated in a Phase II randomized trial published by Ferreri et al. in 2009 . This grOne patient in this study group was treated with HD-MTX and R-CHOP, and although the combination chemotherapy regimen of R-CHOP is the best treatment for systemic non-Hodgkin’s lymphoma, it has not been shown to be effective for PCNSL . Since 1In the nine patients that did not receive a systemic chemotherapy regimen that included HD-MTX, the most frequent alternative treatment was WBRT . The other alternative treatments were thalidomide; R-CHOP; intrathecal MTX (IT-MTX); topotecan with IT-MTX; and cyclophosphamide, vincristine, doxorubicin, and dexamethasone with IT-MTX. Although an explanation or justification for the use of alternative therapies is beyond the scope of this analysis, published studies describe the use of most of these agents in patients with PCNSL -28.Information on treatment after recurrence was available for six patients. The treatments used at recurrence were rituxan and temozolomide; WBRT, rituxan, and temozolomide; HD-MTX, procarbazine, vincristine, and cytarabine; WBRT; cytarabine; and HD-MTX and rituxan.Not surprisingly, treatments for recurrent disease were also varied. In this sample of patients, therapies used were already established as being effective in the upfront setting. In addition, temozolomide and rituxan to treat recurrent PCNSL has been looked at in several studies and can be used to treat recurrent disease in patients that cannot tolerate HD-MTX or have failed that therapy -30. Our study’s retrospective design along with incomplete records and a small sample size does not allow for statistically significant conclusions to be drawn with regard to treatments and outcomes. Additionally, selection bias does not allow rigorous unconfounded comparisons to be performed. However, this study does highlight the lack of a standard of care for patients with PCNSL at the community level and supports the use of HD-MTX-based regimens in the upfront treatment setting for these patients.A large-population data-based study to help further the investigations of variability in treatment and mitigate selection bias issues would be valuable.