Background: Pathological complete response (pCR) to neoadjuvant chemoradiotherapy (CRT) in locally advanced rectal cancer (LARC) is still ascribed to a minority of patients. A pathway based-approach could highlight the predictive role of germline single nucleotide polymorphisms (SNPs). The primary aim of this study was to define new predictive biomarkers considering treatment specificities. Secondary aim was to determine new potential predictive biomarkers independent from radiotherapy (RT) dosage and cotreatment with oxaliplatin. Methods: Thirty germ-line SNPs in twenty-one genes were selected according to a pathway-based approach. Genetic analyses were performed on 280 LARC patients who underwent fluoropyrimidine-based CRT. The potential predictive role of these SNPs in determining pathological tumor response was tested in Group 1 , Group 2 (73 patients treated with high RT dosage), Group 3 (113 patients treated with standard RT dosage), and in the pooled population (280 patients). Results: Nine new predictive biomarkers were identified in the three groups. The most promising one was rs3136228-MSH6 (p = 0.004) arising from Group 3. In the pooled population, rs1801133-MTHFR showed only a trend (p = 0.073). Conclusion: This exploratory study highlighted new potential predictive biomarkers of neoadjuvant CRT and underlined the importance to strictly define treatment peculiarities in pharmacogenetic analyses. The standard treatment of locally advanced rectal cancer (LARC) in Europe is represented by a multimodal approach. Patients undergo neoadjuvant radiotherapy (RT) or chemoradiotherapy (CRT) primarily based on fluoropyrimidines (5-fluorouracil (5-FU) or capecitabine), sometimes associated with platinum derivatives or monoclonal antibodies, followed by surgery and adjuvant treatment if needed.Response to neoadjuvant therapy plays a pivotal importance in rectal cancer patients. This treatment aims at reducing the tumor mass leading to a more sparing surgery choice, reducing the risk of the side effects, such as urinary and sexual dysfunction, fecal urgency and incontinence . MoreoveThe identification of reliable predictive biomarkers represents a pivotal need for optimizing cancer patients’ treatment, leading to fewer invasive surgical approaches in case of pCR, and to upfront surgery or intensified neoadjuvant treatment in patients with a lower possibility to achieve treatment response.TYMS, XRCC1, EGFR, and MTHFR [Many studies have already addressed this issue. One discipline with big potentialities in this field is represented by pharmacogenetics, which aims at defining genetic variants, called polymorphisms, that affect response to therapy in terms of both efficacy and risk to develop toxicities. The relevance of common variations in DNA sequences like nucleotides’ deletion/insertion, microsatellites, and single nucleotide polymorphisms (SNPs), is related to their possible implication in gene expression regulation or in their influence on protein activity, thus impacting pharmacokinetics and pharmacodynamics. In particular, germline variants exert a potential impact on the pharmacodynamics of drugs and on the body reaction to treatment , and thend MTHFR . As a coIn light of these considerations and currently available literature data, we adopted a pathway-based approach in order to investigate the role of thirty SNPs in twenty-one genes involved in DNA repair system, proliferation and angiogenesis, and fluoropyrimidines response and toxicity. These cellular mechanisms orchestrate indeed in a direct way cellular response to treatment and tumor aggressiveness, thus determining patients’ response to neoadjuvant treatment. We have analyzed these SNPs on 280 LARC patients who underwent fluoropyrimidines-based CRT. These patients have been classified into three subgroups according to treatment characteristics, considering radiotherapy (RT) dosage and association with oxaliplatin. The primary aim of this study was to highlight potential predictive biomarkers according to the peculiarities of neoadjuvant treatment. The secondary aim was to identify potential predictive biomarkers suitable for the entire casistic.p = 0.074) was highlighted for rs1801133-MTHFR.We have identified nine potential predictive biomarkers from the analyses of patient subgroups. None of these SNPs was significantly associated with pCR in the pooled population, only a trend , clinical nodal and metastasis stage , tumor distance from anal margin, neoadjuvant treatment parameters , date of diagnosis, surgery, end of RT, post-CRT pathologic T stage (ypT), kind of surgical intervention, intraoperative RT, recurrence, adjuvant treatment, and date of last follow-up/death. Moreover, the pathological response to neoadjuvant treatment according to Tumor Regression Grade (TRG) was collected for each patient. Patients’ characteristics are reported in Genotyping analyses were successfully performed in our samples. The average genotype call rate was 97.24%, and 29 out of 34 assays had a call rate ≥96.00% (range: 92.14%–99.29%).The association between genotypes and pCR was tested separately in the three subgroups of patients with multivariate analysis, adjusted for gender, age, RT dosage, distance of the tumor from the anal margin, concomitant platinum treatment, and interval between the end of RT and surgery . Nine SNMDM2 and rs1801133-MTHFR, resulted significantly associated (p ≤ 0.05) to pCR in Group 1 according to a dominant model  = 0.24, 95% confidence interval (CI) = 0.07–0.90; p = 0.019, Adj-OR = 3.49, 95% CI = 1.23–9.91, respectively). In addition, according to an additive model, rs1130409-APE1 was significantly associated to treatment response in Group 2 . The analyses in Group 3 highlighted six different potential predictive biomarkers. The most significant one (p < 0.01) was rs3136228-MSH6. According to the dominant model, bearing at least one variant allele reduced the risk not to obtain a pCR . Moreover, rs3213239-XRCC1, rs1799977-MLH1, and rs2010963-VEGFa played a potential strong predictive role . Specifically, according to a dominant model, bearing at least one variant allele of rs3213239-XRCC1 or of rs2010963-VEGFa increases the risk not to achieve a pCR . In addition, according to a recessive model, the rs1799977-MLH1 is a protective factor . Finally, two other SNPs arose as predictive biomarkers according to a dominant model, even if their association is significant (p < 0.05) but not particularly strong considering the quite small sample size. Specifically, bearing at least one variant allele of rs3212986-ERCC1 and rs12917-MGMT increases the risk not to achieve a pCR .Two SNPs, rs2279744-MTHFR, resulted significant in at least one group, showed a concordant genetic effect and compatible genetic models in the three subgroups. Thus, we have tested its predictive value in the pooled population. It did not maintain its significant predictive value, only a trend was observed .Only one SNP, rs1801133-Different and unpredictable treatment response to neoadjuvant CRT in LARC represents a critical clinical problem. A not negligible percentage of patients did not achieve a pCR, thus rendering this treatment less effective in improving patients’ prognosis. The identification of patients who are at risk not to achieve a pCR could be the key to treatment optimization.We aimed at determining new potential predictive biomarkers of neoadjuvant treatment. To this purpose, we considered the pCR, defined as TRG 1 , as clinical endpoint, since its prognostic value is well known and corroborated also by our previous findings . The keyWe have focused our efforts on germline DNA because it could have a great clinical impact. Germline SNPs are indeed easily analyzable thanks to a blood draw. This is a minimal invasive procedure on patients, an important factor in the oncological setting.p-values in order to underline the strength of the associations (p < 0.01), those robustly associated (p < 0.025), and those significantly associated (p < 0.05) with treatment response. Interestingly, from the analysis of Group 3 we obtained six out of nine significant predictive biomarkers, including the most significant one (rs3136228-MSH6), and two SNPs robustly associated with pathological response . This is a really interesting point considering that Group 3 represents the standard treatment, based on fluoropyrimidines and standard dose of RT (5040 cGy).The primary aim of this study was to highlight potential predictive biomarkers considering the peculiarities of the prescribed neoadjuvant treatment. For this purpose, we have analyzed thirty SNPs in twenty-one different genes involved in key pathways of response to RT and fluoropyrimidines-based chemotherapy in 280 LARC patients. In particular, we focused our attention on genetic variants located in genes involved in DNA repair pathways, proliferation and angiogenesis, and response to fluoropyrimidines. We have searched for their independent predictive role in predicting the complete pathological response to neoadjuvant therapy in three different patient subgroups defined according to treatment peculiarities. Globally, nine SNPs arose as potential predictive biomarkers of response to neoadjuvant treatment: six located in genes involved in DNA repair, two in genes controlling proliferation and angiogenesis, and one in a gene modulating fluoropyrimidines response. Considering the explorative nature of this study, we had coarsely classified the identified predictive biomarkers according to the obtained ciations . Specifip < 0.01) obtained in this study is represented by rs3136228-MSH6. The MSH6 gene codifies for a factor mainly involved in mismatch repair (MMR) and in other kind of DNA repair mechanisms such as the response to double strand breaks (DSB). Specifically, MSH6 seems to be involved in non-homologous end joining pathway, that is activated in case of DNA breaks. Interestingly, in vitro MSH6 deficiency leads to an impaired DSB repair, with a consequent higher sensitivity to cell death induced by radiation exposure [MSH6 is located in the upstream region of the gene and seems to affect the binding with the transcription factor Sp1 [MSH6 in predicting tumor response in this subgroup. The lack of significance in Group 1 is trickier to interpret due to the central role of MMR in response to oxaliplatin. Nonetheless, we can suppose that oxaliplatin, fluoropyrimidines and RT act in a synergistic effect promoting more impacting damages in DNA. However, this result claims for further analyses in order to verify our hypothesis and to confirm the predictive value of this SNP.The strongest association  are rs3213239-XRCC1, rs2010963-VEGFa, rs1801133-MTHFR, and rs1130409-APE1.Other biomarkers strongly associated with treatment response  was rs1801133-MTHFR, that arose from the analyses conducted on Group 1. This gene has been selected for its central role in fluoropyrimidines response. It codifies for an enzyme, methylentetrahydrofolate reductase, that catalyzes the conversion of 5,10-methylentetrahydrofolate  to 5-MTHF. Cellular levels of 5,10-MTHF can exacerbate the cytotoxicity exerted by 5-FU. The rs1801133-MTHFR is a missense SNP that causes a substitution of alanine to valine in position 222, thus inducing a 60% reduction in enzyme activity [MTHFR has been specifically investigated in colorectal- and rectal cancer setting [Another robust predictive biomarker . It is quite perplexing that we did not find any biomarker suitable for the entire population, but we can ascribe this to the not homogeneous treatments included in this analysis. This study addresses the potential strong association of genetic biomarkers to specific neoadjuvant treatments administered to LARC patients. The specificity of the biological response to various insults can justify the observed differences among the new potential biomarkers identified in the three patient subgroups. The biomarkers potentially characterizing Group 1 are associated with fluoropyrimidines pathway (rs1801133-MTHFR) and with cell proliferation (rs2279744-MDM2), whereas those of Groups 2 and 3 belong mainly to DNA repair mechanisms  and to angiogenesis (rs2010963-VEGFa). These data are hypothesis generating and suggest an important impact of treatment peculiarities in tumor response. This specificity is also underlined by the lack of a solid predictive biomarker that fits for the pooled population. Nonetheless, the explorative nature of this study cannot allow us to draw final conclusions. Performing the analysis on a validation group of patients is mandatory to clearly and ultimately define the potential predictive role of the identified genetic variants. It would be also of great potential clinical interest to investigate the prognostic value of these SNPs in terms of disease-free survival and overall survival in order to optimize LARC patients’ management.The secondary aim of this study was to identify potential predictive biomarkers suitable for the entire group of patients. We had introduced this secondary aim in order to highlight biomarkers with a potential easier introduction in the clinical practice due to their less specificity to treatment peculiarities. To avoid the selection of false positive biomarkers, we compared the effect of the SNPs among the three different subgroups, selecting only those resulted significant in at least one subgroup, with a concordant genetic effect (increased or reduced possibility to achieve pCR) and compatible genetic models in the three subgroups. Only rs1801133-MTHFR, a central gene in fluoropyrimidines response. Analyses performed in larger populations could for sure be the key to solve these dilemmas. In addition, a more comprehensive approach that considers the joint effect of different SNPs and also of clinical parameters could pave the way to a more holistic comprehension of treatment response that barely can be considered as a monogenic trait due to the involvement of several cellular pathways. This strategy requires for sure larger groups of patients but could suggest a more efficient model to explain and predict treatment response.We have to acknowledge that this study presents some limits. First of all, it is necessary to underline the small sample size of the analyzed subgroups, which cannot give the ultimate response to the need to identify strong biomarkers to be translated into the clinical practice. As already stated, these results are preliminary and only hypothesis generating. However, the performed multivariate analysis gave an additional value to the obtained results. In addition, a prioritization of our data according to the strength of the associations has been suggested in order to conceive them in a critical way. Moreover, as already stated, we have to underline that we did not have the possibility to validate these results in an external validation group of LARC patients, that is necessary to identify biomarkers of clinical interest. This is for sure one of the main limits of this study. Nonetheless, we believe that these preliminary results are of some interest because they are based on a numerous group of LARC patients with homogeneous clinical characteristics. Consequently, due to the explorative nature of this analysis, we hope that it could be considered as a starting point for future analyses. Furthermore, in some cases the biological function of the identified biomarkers has not been elucidated yet, thus rendering not possible the fully comprehension of the obtained results. Another potential limit is represented by the total independence of the results among the three subgroups, even if all patient underwent fluoropyrimidines-based CRT. On the one hand, this sustains our hypothesis about the central role of treatment peculiarities, such as association treatments and RT dosage. On the other hand, it is quite puzzling that none of the selected SNPs could be considered as potential predictive biomarker of response to neoadjuvant treatment suitable for the pooled population. Only a trend was observed for As previously described , from DeAll procedures were reviewed and approved by the Ethical Committee of each participating institution, and all patients signed a written informed consent for research purposes. Specifically, patients were enrolled according to Phase III study on rectal cancer INTERACT-LEADER CRO-07-2006, approved by the Ethics committee CRO-Aviano on 20 January 2006, and according to the Study RF-2011-02349645 about the use of non invasive biomarkers on rectal cancer, 3533/AO/15 approved by the Ethics committee of Padua on 21 June 2016.As already described elsewhere , patient2/day intravenous continuous infusion for 5 weeks or capecitabine 1650 mg/m2 in two daily oral administrations for 5 weeks) was prescribed with 50.4 Gy or 55.0 Gy, whereas the capecitabine (1300 mg/m2) was administered with oxaliplatin (130 mg/m2 every 19 days) and concurrently standards RT dose of 50.4 Gy.Two different RT programs were performed, according to clinical trials ongoing in the considered period time. Specifically, in the first RT program patients were treated with a standard dose of 50.4 Gy/28 fractions: a consecutive boost of 50.4 Gy/3 fractions to the tumor and mesorectum was given following the CTV dose of 45.0 Gy/25 fractions, for a total dose of 50.4 Gy. In the second program, patients underwent a dose of 55.0 Gy/25 fractions: specifically, a concomitant boost of 10 Gy/10 fractions over 5 weeks, 2 times a week , was delivered to the tumor and mesorectum during the CTV dose of 45 Gy fractions, for a total dose of 55 Gy. Fluoropyrimidines alone , in proliferation and angiogenesis , and fluoropyrimidines response . The selection of the SNPs was based on literature data analysis. We have specifically focused our attention on the most promising genetic variants according to the literature data available at the time of the study design.A set of thirty SNPs in twenty-one genes was selected. Specifically, according to literature data, we have chosen genes involved in pathways with a key role in chemoradiotherapy response . More inXRCC1, rs1052133-OGG1, and rs2279744-MDM2. PCR amplifications were performed in a Eppendorf Mastercycler gradient, with TaqGold DNA polymerase .Genomic DNA of LARC patients was extracted from peripheral blood samples using the automated extractor BioRobot EZ1, in association with the Kit “EZ1 DNA Blood Kit 350 μL”  and stored at +4 °C until the time of this study. Positive controls were included in the analyses. Pyrosequencing  was used for the analysis of rs3213239-XRCC1, rs25489-XRCC1, rs1799796-XRCC3, rs1799794-XRCC3, rs861539-XRCC3, rs3136228-MSH6, rs11136410-PARP1, rs4149963-EXO1, rs1799793-ERCC2, rs3212986-ERCC1, rs11615-ERCC1, rs1799977-MLH1, rs1130409-APEX1, rs12917-MGMT, rs1695-GSTP1, rs1138272-GTSP1, rs4880-SOD2, rs1642785-TP53, rs1801516-ATM, rs1801133-MTHFR, rs1801131-MTHFR, rs2227983-EGFR, rs4444903-EGF, rs2010963-VEGFa, and rs1570360-VEGFa. The gene copy number variations of GSTT1 (Hs00767125_cn) and GSTM1 (Hs00273142_cn) were analyzed with TaqMan technology assays.Pre-designed TaqMan SNP genotyping assays with the Applera TaqMan Universal Master mix were used on ABI 7900HT  according to the manufacturer’s instructions for the discrimination of the following SNPs: rs25487-TP53 and rs34743033-TS were analyzed by PCR coupled with agarose gel electrophoresis.Finally, rs17878362-Primers and PCR conditions are available upon request.Pathological tumor response to neo-adjuvant treatment was defined according to the TRG. This is an index that considers the amount of therapy-induced fibrosis in relation to residual tumor in surgical specimens. Complete responders  were compared to non-complete responders . We have performed this classification according to our previous analysis demonstrating the significant difference in patients with TRG 1 compared to patients with TRG 2–5 . PatientX2-test.The association between genotypes and TRG was tested separately in the three groups of patients. Odds ratio (ORs) and 95% confidence interval (95% CI) were computed through logistic regression model, adjusting for gender, age, distance of the tumor from the anal margin, platinum treatment, RT dose, and time between the end of RT and surgery. Dominant, recessive, and additive genetic models were considered for each genotype combining heterozygous and homozygous genotypes. The best fitting genetic model was selected according to the Wald Polymorphisms resulting significant in at least one group, showing a concordant genetic effect, and with a “compatible” genetic model in the three groups were further investigated in the entire population. A “compatible” genetic model was considered the combination of an additive model in one group, with either a dominant or recessive model in the other group.Logistic regression was performed using SAS 9.2 .p < 0.01) was highlighted for rs3136228-MSH6 in the patient subgroup undergoing standard treatment (fluoropyrimidines combined with a dose of 5040 cGy of radiotherapy). In the same subgroup, we identified two additionally strong biomarkers (p < 0.025), rs3213239-XRCC1 and rs2010963-VEGFa. Moreover, other strong biomarkers (p < 0.025) arose in the group also undergoing oxaliplatin treatment, rs1801133-MTHFR, and in the group treated with high dose of RT, rs1130409-APE1. We did not find any significant association between genetic variants and pathological response to neoadjuvant treatment in the pooled population, only a trend was observed for rs1801133-MTHFR.To conclude, this study highlighted the potential predictive key role of nine SNPs involved in key pathways of CRT response in three different LARC patient subgroups defined according to the peculiarities of neoadjuvant treatment. The strongest association (These data, even if only preliminary and hypothesis generating, address the great potentialities of genetic analyses performed on germline DNA and the importance of treatment peculiarities in determining new predictive biomarkers. If validated with further analyses performed in an external group of LARC patients with homogeneous clinical parameters, this study could lead to the introduction in the clinical practice of biomarkers to optimize patients’ treatment.


Specialized early intervention programs are efficient in treating patients with a first episode of psychosis (FEP) at least after 2 years. However, few studies have examined long-term outcomes, and particularly prognostic implications of the sex of FEP patients.We aimed to investigate long-term neuropsychological and functional outcomes in female and male 10 years after the first presentation of a non-affective psychotic episode. One hundred sixty-five FEP patients, 73 women and 92 men were assessed for sociodemographic, clinical and neuropsychological information.Differences in outcome between female and male based on baseline, 1-year, 3-year and 10-year follow-up information were substantial, showing women better outcomes on several variables. Schizophrenia diagnosis was significantly more frequent in men . Women were more likely than men married  and having children (41% vs. 13%); p < 0.001). Significant differences arose for social function  and processing speed . There was also some weak evidence  for negative symptoms and global neurocognitive function.Women who suffered a first episode of psychosis have better functional and neurocognitive outcomes compared to men. This differential outcome profile is important for clinicians to consider sex specific therapeutic approaches.


To examine the functional recovery differences after stroke rehabilitation in patients with uni- or bilateral hemiparesis.In this retrospective study, we included data from the medical record of all 383 patients with uni- or bilateral hemiparesis after stroke who were admitted to King Fahad Medical City-Rehabilitation Hospital between 2008 and 2014 in Riyadh, Kingdom of Saudi Arabia. According to the site of hemiparesis, we classified patients into 3 groups: right hemiparesis (n=208), left hemiparesis (n=157), and bilateral hemipareses (n=18). The patients (n=49) who did not have either site of hemiparesis were excluded. The Functional Independence Measures (FIM) instrument was used to assess the score at admission and discharge. A post hoc test was conducted to examine the functional recovery differences between groups. Multiple regression analyses were used to confirm the findings.Amongst the three groups, there were significant (p<0.05) differences in the total-FIM score as well as motor- and cognitive-FIM sub-scores between admission and discharge of stroke rehabilitation. The differences were significantly greater in the bilateral hemipareses group than in either unilateral hemiparesis group. Multiple regression analyses also confirmed that the site of hemiparesis significantly (p<0.05) differs in the total-FIM score as well as motor-FIM and cognitive-FIM sub-scores.Our results demonstrate that differences in functional recovery after stroke rehabilitation may be influenced by the site of hemiparesis after stroke. Hemiparesis described as complete or incomplete muscular weakness or paralysis affecting either side of the body after a stroke.5The brain is a highly complex organ that influences different body functions.Despite the presence of intensive stroke rehabilitation programs in Saudi Arabia, there remain limited data discussing the functional outcomes in patients with uni- or bilateral hemiparesis after stroke.This retrospective study received ethical approval by the Committee on Human Research, the Institutional Review Board at King Fahd Medical City-Rehabilitation Hospital (KFMC-RH), Riyadh, kingdom of Saudi Arabia .The electronic medical record database review was conducted by 2 of the present investigators at the KFMC-RH. The time frame between 2008-2014 was determined based on the availability of medical records by stroke diagnosed according to the International Classification of Diseases, Ninth Revision (ICD-9) codes 348-438 and 799.3. Differences of opinion regarding medical records were resolved by discussion between the investigators until consensus was reached. The KFMC-RH is the largest tertiary inpatient rehabilitation hospital in Riyadh, Kingdom of Saudi Arabia. The hospital follows the Uniform Data System for Medical Rehabilitation (UDSMR) protocol when administering the FIM scale.We included data of 383 patients with uni- or bilateral hemiparesis after stroke who were treated at the KFMC-RH stroke rehabilitation program between 2008-2014. Based on the site of hemiparesis, we classified patients into three groups: right hemiparesis (n=208), left hemiparesis (n=157), and bilateral hemipareses (n=18). The patients (n=49) who did not have either site of hemiparesis were excluded.The FIM was used to score the level of assistance required for a patient to accomplish ADL.15Frequencies were presented as percentages (%) for categorical measures, and continuous measures were presented as means (standard deviations). For group comparisons, a chi-square test was conducted for frequencies, a post hoc analysis (ANOVA) was performed for continuous variables and for analyzing the differences in FR.18Table 1 shows descriptive characteristics of all patients in the 3 groups. The mean patient age was 54.9±15.3 years, and just over half of the patients were male (56%). Patients with bilateral hemipareses were 11 years younger (age 48 years) than patients with right hemiparesis (age 59 years) and were 10 years younger than patients with left hemiparesis (age 58 years). The usual length of hospital stay was 47.5 days. The majority of patients with uni- or bilateral hemiparesis were discharged to home (98%). Among the 3 groups, functional score improved on total-FIM as well as motor-FIM and cognitive-FIM subscales after discharge from inpatient rehabilitation facility .Table 2 shows outcome measures for the 3 groups after stroke rehabilitation in terms of total-FIM as well as motor-FIM and cognitive-FIM sub-scores. Among the 3 groups after stroke rehabilitation, 3 were significant differences between admission and discharge (p<0.05) in total-FIM improvement , motor-FIM sub-scores , and cognitive-FIM sub-scores .Table 3 shows multiple regression analyses between site of hemiparesis and FR. The table confirms that site of hemiparesis signficantly differs in total-FIM score , motor-FIM sub-scores , and cognive-FIM sub-scores  after adjustment for age, gender, and LOS. The full adjusted model shows an explained variance (R2) of 0.029 for total-FIM, 0.015 for motor-FIM, and 0.034 for cognitive-FIM.The objective of this study aims to examine the FR differences after stroke rehabilitation in patients with uni- or bilateral hemiparesis. Our results reveal that there is a significant difference in FR by the site of hemiparesis after stroke rehabilitation. Patients with bilateral hemipareses had significantly greater FR after stroke than patients with unilateral hemiparesis. Additionally, patients with left hemiparesis exhibited significantly higher FR than patients with right hemiparesis. In summary, overall outcomes indicated significantly different FR after stroke rehabilitation in patients with uni- or bilateral hemiparesis due to stroke. The magnitude of change was significantly higher for patients with bilateral hemipareses.Our findings also showed significant differences between admission and discharge in total-FIM as well as motor-FIM and cognitive-FIM sub-scores in patients with uni- or bilateral hemiparesis. Compared to patients with bilateral or left hemiparesis, patients with right hemiparesis had the lowest magnitude of FIM score change. This finding was constant with the results of the similar study, which described that patients with left hemiparesis had significantly greater FIM score improvements than patients with right hemiparesis.In contrast, another study reported that patients with left hemiparesis had more severe FR than patients with right hemiparesis after stroke rehabilitation.12In the present study, patients with bilateral hemipareses were younger (by 10 years or more) than patients with unilateral hemiparesis. Additionally, patients with bilateral hemipareses had greater FIM scores. Furthermore, patients with left hemiparesis had higher FIM score improvements than patients with right hemiparesis after stroke rehabilitation. This finding was related to that of the previous study conducted in AustriaAlso, our findings indicate that differences in the total-FIM score, motor-FIM, and cognitive-FIM sub-scores all had thresholds above which they could be considered clinically significant. This limit is called the minimum clinically significant difference (MCID). A recent study in Saudi Arabia reported that the change in total-FIM was not clinically significant (the MCID was 20).In acute care, the physician has the established goal of significantly improving the blood flow or break up blood clots in the area of damaged brain tissue through medications (antithrombotics and thrombolytics) to stabilize the patient’s health status.27The strengths of our work were as follows: first, KFMC-RH is a large tertiary in-patient rehabilitation hospital in Riyadh, Saudi Arabia, and is certified to use the FIM instrument. Second, this is the first study in Saudi Arabia that examined the differences in FR after stroke rehabilitation in patients with uni- or bilateral hemiparesis. The limitations of this study include its retrospective design and small sample size; larger studies may produce more accurate results. Additionally, the results are not generalizable as the study did not include all tertiary rehabilitation hospitals in Riyadh. Additionally, we have not addressed the manner of stroke onset and stroke severity that influences the FR of stroke rehabilitation. Another limitation is the deficiency of data regarding health covariables , as well as some sociodemographic information  that may affect FR.In conclusion, this study demonstrates that differences in FR after stroke rehabilitation may be influenced by the site of hemiparesis after stroke. Patients with bilateral hemipareses may need a special intensive rehabilitation compared with patients having a right or left hemiparesis. Future large-scale studies at national level tertiary rehabilitation hospitals and studies exploring sociodemographic and health variable effects may provide better insights into FR among patients with hemiparesis. It may, in turn, help justify the establishment of more rehabilitation centers in Saudi Arabia.


Reserpine treatment led to decreased NE re-uptake at 1d and 5d compared to saline treatment. Expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of the NE synthesis, was elevated in left stellate ganglia after reserpine. Mechanistically, measurement of NET mRNA expression in left stellate ganglia and myocardial NET density revealed a post-transcriptional downregulation of the NET by reserpine. In summary, present data demonstrate that depletion of cardiac NE stores alone is sufficient to impair cardiac NE re-uptake via downregulation of the NET, independent of systemic neurohumoral activation. Knowledge about the regulation of the cardiac NE homeostasis may offer novel therapeutic strategies in HF.In heart failure (HF), a disturbed cardiac norepinephrine (NE) homeostasis is characterized by depleted cardiac NE stores, impairment of the cardiac NE re-uptake by the neuronal norepinephrine transporter (NET) and enhanced cardiac NE net release. Reduced cardiac NE content appears to be caused by enhanced cardiac NE net release from sympathetic neurons in HF, triggered by neurohumoral activation. However, it remains unclear whether reduced NE itself has an impact on cardiac NE re-uptake, independent of neurohumoral activation. Here, we evaluated whether depletion of cardiac NE stores alone can regulate cardiac NE re-uptake. Treatment of Wistar rats with reserpine (5 mg/kg/d) for one (1d) or five days (5d) resulted in markedly reduced cardiac NE content, comparable to NE stores in experimental HF due to pressure overload. In order to assess cardiac NE re-uptake, the specific cardiac [ Activation of the sympathetic nervous system is a major characteristic in chronic heart failure (HF) and an elevation of norepinephrine (NE) plasma levels is associated with a poor prognosis in HF patients –4. Under1) via the sodium-dependent, neuronal norepinephrine transporter (NET), whereas extraneuronal NE re-uptake (uptake2) plays only a minor role in the heart -NE uptake was determined as described previously [3H]-NE  was injected into the perfusion system and proportionally distributed to the hearts and blank channels. Radioactivity was measured in the effluent. To avoid artificial findings due to different experimental conditions, we only compared animal groups that were measured together in one single NE re-uptake experiment with one batch of [3H]-NE. Experiments were performed in the absence and presence of the NET inhibitor desipramine  in order to distinguish between neuronal and extraneuronal NE uptake. The amount of [3H]-NE, which was extracted by the hearts (uptake), is expressed as the percentage of radioactivity measured in the blank channels.For isolated perfusion, hearts were prepared according to a modified Langendorff technique, as previously described in detail , 24. TheTotal cellular RNA was isolated from frozen left stellate ganglia by acid guanidium thiocyanate/phenol/chloroform extraction . ReverseLeft (LV) and right (RV) ventricular NE content was determined by high-performance liquid chromatography and electrochemical detection as described previously , 26.3H]-mazindol binding assay as described previously [3H]-mazindol  as a specific ligand of the NET. Nonspecific binding was determined by measuring residual binding in the presence of desipramine (100 μmol/l). The incubation was carried out at 30°C and was terminated by rapid vacuum filtration through a MultiScreenHTS-FB filter plate . All experiments were performed in triplicate. To calculate the binding capacity, the remaining filter radioactivity was determined.Plasma membranes of LV were prepared for -NE uptake into isolated perfused hearts from TAC animals, indicating a reduced NE re-uptake into sympathetic nerve endings   . In summ3H]-NE (~40%) in the Langendorff perfusion, indicating a reduced sympathetic NE re-uptake in reserpine-treated animals  led to a slight reduction in total body weight, reflecting the systemic effects of reserpine , and to  animals . Moreove the NET .3H]-NE in animals treated with reserpine for five days (3H]-NE was entirely mediated by the NET (Prolonged treatment with reserpine over five days (5d) led to an even further reduction in total body weight , and carive days . Again,  the NET .3H]-mazindol measured a lower presence of the NET on the plasma membrane, indicating a post-transcriptional downregulation of the NET after reserpine, comparable to what is described in HF (We previously demonstrated that reduced NET function in experimental HF is mediated by a post-transcriptional mechanism , 20, 28.ed in HF  11]. Th. Th3H]-med in HF  20, 29]3H]-mazinOur data demonstrate that the depletion of cardiac NE stores leads to changes in cardiac NE that resemble the findings in HF namely, a decreased cardiac NE re-uptake mediated by post-transcriptional downregulation of the NET and, possibly as compensation, an upregulation of TH, the rate-limiting enzyme of NE synthesis. To our knowledge, this is the first study that directly connects myocardial NE content to changes in NE homeostasis independent of HF and HF-associated activation of the neurohumoral system.But is reserpine an appropriate model to study the depletion of cardiac NE stores? Reserpine irreversibly blocks the vesicular monoamine transporter that normally transports norepinephrine and other monoamines in the presynaptic nerve terminal to presynaptic vesicles for subsequent release into the synaptic cleft . It has As a sympathetic response to NE depletion, we detected an increased level of mRNA expression of TH in the stellate ganglia, the enzyme that initiates NE biosynthesis by hydroxylation of tyrosine to dopa . We inte2 carrier. This is in accordance with many other studies that have identified NET-dependent mechanisms causal for impaired NE re-uptake in cardiac disease models [3H]-mazindol binding assay. Mazindol specifically binds to the NET and this assay presents a valid measurement of NET density on the surface of cardiac sympathetic neurons when plasma membrane preparations are used [The central finding of our study is that we observe a significant reduction of cardiac NE re-uptake not only in the HF but also in the reserpine model. Reserpine was previously shown to impair NE-re-uptake in the cerebral cortex and adrenal glands . Howevere models , 11, 34.are used . This isare used . Althougare used . The autare used . Trafficare used –40. Otheare used , 41. Morare used . This stcirculus vitiosus of chronic heart disease.The concept that activation of the neurohumoral system leads to disturbed NE homeostasis in HF is well established. Given the sympathetic over-activation in HF , it has Heart failure is a pandemic disease with increasing incidence, economic importance and rising morbidity and mortality . MyocardIn summary, we demonstrate that the depletion of cardiac NE stores alone impairs cardiac NE re-uptake via post-transcriptional downregulation of NET, independent of systemic neurohumoral activation. We believe that a deeper knowledge about the regulation of the cardiac NE homeostasis may offer novel therapeutic strategies for HF in the future.


A few studies have shown that methotrexate (MTX) use exacerbates liver fibrosis and even leads to liver cirrhosis in rheumatoid arthritis (RA) patients, although the risk is low compared to psoriatics. We therefore conducted a population-based cohort study to investigate the impact of long-term MTX use on the risk of chronic hepatitis C (CHC)-related cirrhosis among RA patients. We analyzed data from the National Health Insurance Research Database in Taiwan and identified 450 incident cases of RA among CHC patients (255 MTX users and 195 MTX non-users) from January 1, 1998 to December 31, 2007. After a median follow-up of more than 5 years since the diagnosis of CHC, a total of 55 (12%) patients developed liver cirrhosis. We did not find an increased risk of liver cirrhosis among CHC patients with long-term MTX use for RA. Furthermore, there was no occurrence of liver cirrhosis among the 43 MTX users with a cumulative dose ≧3 grams after 108 months of treatment. In conclusion, our data showed that long-term MTX use is not associated with an increased risk for liver cirrhosis among RA patients with CHC. However, these results should be interpreted with caution due to potential bias in the cohort. Methotrexate (MTX) is commonly used for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and psoriasisChronic hepatitis C (CHC) is one of the main etiological factors of liver cirrhosisThis retrospective cohort was based on the NHIRD7Risk factors for liver cirrhosis, such as non-alcoholic fatty liver disease , diabetes mellitus (ICD-9-CM code 250), and dyslipidemia (ICD-9-CM code 272), as well as comorbidities were documented for each patient. Diagnosis of decompensated liver cirrhosis was made based on the inclusion in the CIPD. To be registered in the CIPD, at least one of the following criteria needs to be fulfilled for patients with liver cirrhosis: (1) intractable ascites, (2) variceal bleeding, or (3) hepatic coma or liver decompensation.Statistical analysis was conducted using SAS software version 9.3 . All quantitative data are presented as means plus the standard deviation unless specified otherwise. For numerical variables, comparisons were made using the Student’s t test. For categorical variables, comparisons were made using the Chi-squared test. Hazard ratios of developing liver cirrhosis since the diagnosis of CHC between subgroups of RA patients were calculated using the Cox proportional hazards model after adjustment for age, gender, and comorbidities. The Kaplan-Meier survival analysis and the log rank test were used to compare the probability of liver cirrhosis-free survival since the diagnosis of CHC between MTX non-users and MTX users. A p value less than 0.001 in a two-sided test was considered statistically significant.This population-based cohort study investigated the impact of long-term MTX use on the risk of liver cirrhosis among RA patients with CHC. There was no significant increase of liver cirrhosis in RA patients with CHC who received long-term MTX treatment, which was in line with our previous results in RA patients with CHBAmong RA patients with CHC in Taiwan, 255 (57%) of 455 patients (excluding those patients who had received biologics) had received MTX to treat RA, a prevalence comparable to that among RA patients with CHB11After a median follow-up of more than 5 years since the diagnosis of CHC, 55 (12%) of 450 RA patients with CHC developed liver cirrhosis. This incidence of liver cirrhosis was higher than among RA patients with CHB 1415Previous studies have demonstrated that the risk for liver cirrhosis after long-term MTX therapy is low among RA patientsHowever, it is not reasonable to infer that MTX use is protective against liver cirrhosis due to the well-known hepatoxicity of MTX21Our study has some limitations. First, as mentioned in our previous studyIn conclusion, our data showed that long-term MTX use is not associated with an increased risk for liver cirrhosis among RA patients with a comorbidity that contributes to liver cirrhosis, such as CHC. This finding is compatible with our previous observation in RA patients with CHB. However, the results should be interpreted with caution due to potential bias in the cohort.How to cite this article: Tang, K.-T. et al. Methotrexate is not associated with increased liver cirrhosis in a population-based cohort of rheumatoid arthritis patients with chronic hepatitis C. Sci. Rep.6, 33104; doi: 10.1038/srep33104 (2016).


As the first step to discover protein disease biomarkers from saliva, global analyses of the saliva proteome have been carried out since the early 2000s, and more than 3,000 proteins have been identified in human saliva. Recently, ethnic differences in the human plasma proteome have been reported, but such corresponding studies on human saliva in this aspect have not been previously reported. Thus, here, in order to determine ethnic differences in the human saliva proteome, a Korean whole saliva (WS) proteome catalogue indexing 480 proteins was built and characterized through nLC-Q-IMS-TOF analyses of WS samples collected from eleven healthy South Korean male adult volunteers for the first time. Identification of 226 distinct Korean WS proteins, not observed in the integrated human saliva protein dataset, and significant gene ontology distribution differences in the Korean WS proteome compared to the integrated human saliva proteome strongly support ethnic differences in the human saliva proteome. Additionally, the potential value of ethnicity-specific human saliva proteins as biomarkers for diseases highly prevalent in that ethnic group was confirmed by finding 35 distinct Korean WS proteins likely to be associated with the top 10 deadliest diseases in South Korea. Finally, the present Korean WS protein list can serve as the first level reference for future proteomic studies including disease biomarker studies on Korean saliva. Saliva is secreted from salivary glands, including three major glands  and minor glands. Saliva has various functions. It maintains oral cavity homeostasis, lubricates oral tissues, promotes chewing, swallowing, digestion, and speaking, and protects the oral cavity against microorganisms –4. It isAs the first step to discover protein disease biomarkers from saliva, global analyses of the saliva proteome have been carried out since the early 2000s. As a result, more than 3000 proteins have been identified in human saliva , 7–11. SRecently, ethnic differences in human plasma proteome have been reported. Jeong et al. confirmed 100 unique proteins out of 185 proteins in Korean plasma compared to 3,380 proteins in the HUPO Plasma Proteome Project dataset . These rTherefore, the Korean WS proteome catalogue indexing 480 proteins was built and characterized in this study through proteomic analyses of WS samples collected from eleven healthy Korean male adult volunteers for the first time. It was then compared to the integrated human saliva proteome including 3,449 proteins to determine ethnic differences in human saliva proteome. Confirmed differences of protein identities and GO category distributions between the two proteomes strongly support that there are ethnic differences in the human saliva proteome. In addition, some distinct proteins in the Korean WS are likely to be associated with highly prevalent diseases in South Korea, demonstrating the high diagnostic potential of ethnicity-specific human saliva proteins for diseases highly prevalent within an ethnic group. Finally, the present list of Korean WS proteins can serve as the first level reference for future proteomic studies including disease biomarker studies on Korean saliva.This study was approved by Dankook University Institutional Review Board. The participants of this study were recruited between August 1, 2014 and Augst 31, 2014 by posting its notices including the brief summary of this study around Dankook University, Cheonan, Chungnam, South Korea. A total of 15 volunteers wanted to participate this study, but 4 of them were excluded based on their history of diseases informed through their introductory screening surveys. Finally, eleven healthy South Korean male adults  were decided to be the participants and each participant signed an informed consent form. Any specific baseline demographic characteristics of the study populations were not available in this study, because only basic personal information and history of diseases were obtained through the introductory screening survey. WS (15 mL/person) was collected from volunteers at 9:30 am prior to eating and after rinsing the mouth with water. A protease inhibitor cocktail solution  was spiked  to WS samples immediately after sample collection. These protease-spiked samples were centrifuged at 12,000 rpm and 4°C for 10 min. Each supernatant was stored at -70°C until use. Prior to protein digestion, 4 mL of the thawed protease-spiked sample supernatant was applied to a 3 kDa cutoff filter unit  for buffer exchange with water. The filter unit was centrifuged at 3,500 rpm and the retentate was dried by vacuum centrifugation. The dried residue was resuspended with 500 μL of water and its total protein concentration was determined by BCA assay . An appropriate portion of the resuspended solution  was then dried by vacuum centrifugation again, and the resulting residue was applied to procedures described previously with slight modifications  20, 21]21]. A poE). Prior to fragmentation processes, IMS was carried out to separate similar precursor ions. Parameters related with mass spectrometry are listed in All nLC-Q-IMS-TOF analyses were carried out on a Waters nanoACQUITY UPLC system  and a Waters SYNAPT G2-S HDMS system. The prepared sample was injected into a Waters nanoACQUITY UPLC Symmetry C18 trap column . It was desalted with 99% mobile phase A (0.1% formic acid in water) and 1% mobile phase B (0.1% formic acid in acetonitrile) for 5 min at a flow rate of 10 μL/min. Trap column-retained peptides were eluted into a Waters nanoACQUITY UPLC BEH300 C18 column  and separated by a linear gradient of mobile phase B from 1 to 60% for 120 min at a flow rate of 250 nL/min. Peptides eluted from the analytical column were delivered into the mass spectrometer through a nanoelectrospray ionization (nESI) source operating in positive ion mode. Mass spectrometry of peptide ions was performed in resolution data-independent acquisition mode (MS2). Parameters related with mass spectrometry are listed in All nLC-Q-orbitrap analyses were carried out on a Thermo Scientific Easy-nLC 1000 system  and a Thermo Scientific Q Exactive system. The prepared sample was desalted by Top Tip  following the direction by the manufacturer and the desalted sample was separated on an in-house analytical column (0.075×250 mm), packed with C18 resin , by a linear gradient of mobile phase B from 1 to 80% for 120 min at a flow rate of 300 nL/min. Peptides eluted from the column were delivered into the mass spectrometer through a nESI source operating in positive ion mode. Mass spectrometry of peptide ions was performed in data-dependent product ion scan  v3.0.2 and Thermo Proteome Discoverer v2.1, respectively. For the identification of peptides and proteins, database search against the IPI human database v3.87 was performed and database search parameters are listed in For GO analysis of saliva proteomes, the Generic GO term mapper was used . SignifiDatabase of disease-related biomarkers was used to check probable association between distinct proteins observed in Korean WS proteome but not in the integrated human saliva proteome and diseases .Based on nLC-Q-IMS-TOF analyses of Korean WS samples, the Korean WS proteome was built successfully for the first time. In order to enhance the credibility of protein identification results, the following criteria were set: 1) any identification derived from only one unique peptide was rejected, 2) FDR was kept at no more than 1%, 3) only protein identification with at least 95% probability from PLGS results were accepted, and 4) all results which passed the above criteria were verified manually. These criteria were applied to all downstream protein identifications. As a result, a total of 480 proteins were identified . Also, tTo determine ethnic differences in the human saliva proteome, the present Korean WS proteome was compared to P. Sivadasan et al.'s updated human saliva protein list  built by the integration of their own and previously-reported five human saliva protein lists , 7–11. FFor the determination of the inter-platform variability in the nLC-Q-IMS-TOF system used in this study and the validation of the identities of proteins, especially, the distinct Korean WS proteins, results of the analyses of the pooled Korean WS sample by the nLC-Q-IMS-TOF system and a nLC-Q-orbitrap system were compared. As a result, 141 and 208 proteins were identified from the nLC-Q-TOF platform and the nLC-Q-orbitrap platform, respectively, and 98 out of 141 proteins (69.5%) from the nLC-Q-TOF platform were overlapped with those from the nLC-Q-orbitrap platform. Among proteins identified in the pooled sample, 130 proteins from the nLC-Q-TOF platform and 147 proteins from the nLC-Q-orbitrap platform were found to be within the Korean WS proteome index. Additionally, among those proteins overlapped with the Korean WS proteome, 22 out of 130 proteins (16.9%) and 29 out of 147 proteins (19.7%) were confirmed to belong to the distinct Korean WS proteins from the nLC-Q-TOF platform and the nLC-Q-orbitrap platform, respectively. Finally, the portion of the distinct proteins from the nLC-Q-IMS-TOF platform, which overlaps with those from the nLC-Q-orbitrap plarform was 68.2% .p < 0.05). GO biologic process categories also showed higher portions of proteins for response to stimulus, cell communication, protein metabolism, and transport in the Korean WS proteome than those in the integrated human saliva proteome (p < 0.05). However, the opposite tendency was observed in proteins for other primary metabolic and organization and biogenesis (p < 0.05). Finally, in the case of GO molecular function categories, over-representation of the Korean WS proteome was observed in other binding, catalytic activity, antioxidant activity, and enzyme regulatory activity with under-representation in protein binding compared to the integrated human saliva proteome were found (p < 0.05). Allocation of proteins observed in the Korean WS proteome according to their GO annotation can be found in In addition to the comparison of protein identities, GO annotation in terms of cellular component, biological process, and molecular function between the Korean WS proteome and the integrated human saliva proteome was compared . First, To evaluate the clinical applicability of ethnicity-specific human saliva proteome, 226 proteins observed in the Korean WS proteome, but not in the integrated human saliva proteome, were searched against the Database of disease-related biomarkers . As showAs the initial step to determine ethnic difference of human saliva proteome, the Korean WS proteome was constructed for the first time due to the fact that Korea is the most ethnically homogenous country in the world . A totalThe numbers of every type of proteins identified from the analyses of the pooled sample by using each platform are smaller than the counter parts of the Korean WS proteome, likely the consequence of the dilution of individual proteins by saliva pooling and the analyses of a single sample. However, the portion of the distinct proteins from the nLC-Q-IMS-TOF platform, which overlaps with those from the nLC-Q-orbitrap platform, is still close to 70% (68.2%). Thus, no significant influence of the inter-platform variability in our system is observed, lending high credibility of the protein identities in the Korean WS proteome. Interestingly, the number of proteins from the nLC-Q-orbitrap platform, which belong to distinct protein in Korean WS is larger than that from the nLC-Q-IMS-TOF platform  of proteins were unique in the Korean WS proteome. Discovering a large portion of Korean WS unique proteins from the Korean WS proteome was expected, because similar portion to that  was already reported from distinct Korean plasma proteins compared to human plasma proteome , no signWhile it was observed that the nLC-Q-IMS-TOF system of this study did not bring higher performance than other proteomics platforms due to the relatively small number of proteins identified, the identification of the distinct proteins confirmed that the nLC-Q-IMS-TOF system still has good performance for the identification of distinct Korean WS proteins. Actually, to build a global protein list, most proteomic studies have employed multi-dimensional proteomics technique to include as many as possible proteins in their lists , 30, 31.From comparison of GO annotations between the Korean WS proteome and the integrated human saliva proteome, some categories in the Korean WS proteome showed over-representation or under-representation . RegardiFor further evaluation of the clinical applicability of ethnicity-specific human saliva proteome, 226 distinct proteins observed in Korean WS, but not in other human saliva, were searched through the Database of disease-related biomarkers. As a result, 22.1% (50 out of 226) of these distinct proteins were found to be disease biomarker candidates , firmly However, this study has a couple of limitations. First, as mentioned above, it did not employ any multi-dimensional separation technique, and, as a result, a relatively small number of proteins was catalogued in the Korean WS proteome index. Interestingly, however, its limited performance must have played an important role in supporting ethnicity-related differences in human saliva, because it did not seem to produce any significant platform-specific performance, the source of the inter-platform variability. Also, since WS samples were collected from only eleven young male adult volunteers, there would be concerns of gender bias as well as a lack of representativeness in the results because of the narrow age range of the participants. Thus, the expansion of the Korean WS proteome by analyzing more samples, including female WS and a broader range of participant ages, by using the nLC-Q-IMS-TOF system or a multi-dimensional proteomics technique is expected in the near future.The Korean WS proteome catalogue indexing 480 proteins was built and characterized from nLC-Q-IMS-TOF analyses of WS samples collected from eleven healthy Korean male adult volunteers in this study for the first time. From comparison of the Korean WS proteome with the integrated human saliva proteome in terms of protein identities and GO annotations, evidences strongly support ethnic difference in human saliva proteome. Additionally, the potential value of ethnicity-specific human saliva proteins as biomarkers for diseases highly prevalent in that ethnic group was confirmed by finding 35 distinct Korean WS proteins probably associated with the top 10 deadliest diseases in South Korea. Finally, the present Korean WS protein list can serve as the first level reference for future proteomic studies including disease biomarker studies on Korean saliva.S1 TableAmong multiple results on a certain protein from different sample and replicate analyses, only one with the highest PLGS score was selected for this table.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S1 FigTotal proteins identified (A). Proteins which belong to the Korean whole saliva proteome (B). Proteins which belong to the distinct Korean whole saliva proteins (C).(PPTX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity. Current estimates for DENV infections reach 400 million worldwide, occurring in over a hundred tropical and subtropical countries, and leading to thousands of deaths per year.3 Recently, several candidate DENV-vaccines have been tested in clinical trials, and the first product was licensed in some endemic countries.5 ZIKV, which had remained unnoticed in Africa for decades, emerged in the South Pacific in 2007 and was introduced to South America in 2014, where it is currently spreading.7 ZIKV causes febrile illness, but it also appears to be linked to Guillain-Barré syndrome as well as microcephaly in newborns.8 Many of the human pathogenic flaviviruses are transmitted by the same mosquito species , and areas where different flaviviruses co-circulate are increasing in number, most importantly DENV and ZIKV in South America.10Arthropod-transmitted flaviviruses are small, enveloped RNA viruses, which are endemic to many parts of the world. They include a large number of important human pathogens, such as dengue, Zika, yellow fever, West Nile, Japanese encephalitis and tick-borne encephalitis viruses .13 Acute infections are diagnosed either by the detection of IgM or by a rising IgG antibody titer from the acute to the convalescent phases of infection. In secondary flavivirus infections, a measurable IgM response can be very low or absent 14 and IgG tests become the method of choice. However, specificity in flavivirus antibody-measurement is significantly hampered by the structural similarity of the viruses and the resulting antibody cross-reactivity.17 This is of considerable concern especially for IgG-based assays, as it leads to false-positive test results.19 The massive co-circulation of ZIKV and DENV has dramatically increased the problem. Antibodies against ZIKV are particularly cross-reactive to DENV and vice versa due to conserved parts of their proteins.2 As a consequence, currently available tests are severely compromised by cross-reacting antibodies.20 To validate serological results, virus neutralization tests have to be performed, which are time consuming and require BSL-3 facilities.21 Therefore, there is a strong need for the development of techniques that have high sensitivity and specificity to detect and differentiate flavivirus antibodies and allow a high-throughput analysis.Serological diagnosis of flavivirus infections relies on the measurement of IgM and IgG antibodies which usually appear about a week after symptoms onset.24 It was shown before that mutating residues within this domain reduces binding of cross-reactive antibodies.27 By using recombinant proteins that contain point mutations within and near the FL, we have previously generated systems to specifically diagnose WNV and DENV infections, respectively.29 Here, we have analyzed the potential of this technology to differentiate antibody responses to DENV and ZIKV infections. Our results suggest that FL-mutant E-proteins of ZIKV and DENV can be used for a specific serological diagnosis of both infections.The predominant target for cross-reacting antibodies is the fusion loop (FL) domain in the flavivirus envelope (E) protein, a short amino-acid sequence which is almost identical in many pathogenic flaviviruses.Serum samples were divided into different groups regarding their virus infections, described in n=15), ZIKV (n=14) and CHIKV (n=8) IgM and/or IgG-positive samples were obtained from persons returning from a stay in endemic areas, TBEV seropositive samples (n=24) were obtained from a seroprevalence study in forest rangers in Northeastern Italy, WNV seropositive sera (n=28) were obtained from patients with neuroinvasive disease or fever during outbreaks in Northeastern Italy. All these samples, as well as 14 negative control sera, were collected and characterized at Padova University Hospital, Italy. Ethical approval for these studies was obtained from the Padova University Hospital Ethics Committee. Another DENV (n=31), ZIKV (n=18) and JEV (n=4) IgM and/or IgG positive serum samples were obtained from returning travelers after a stay in endemic areas and were collected at the Bernhard Nocht Institute for Tropical Medicine, Hamburg, for diagnostic purposes with written informed consent from each patient. DENV PCR positive sera (n=42) were collected in Sri Lanka with the approval of Ethical Review Boards of the Medical Research Institute and Lady Ridgway Children Hospital Colombo. 13 ZIKV IgG positive sera were collected in Salvador, Brazil, from mothers having children with ZIKV infection-associated microcephaly. Sampling and testing were approved by the institutional research ethics board of the federal university of Bahia Climério de Oliveira. Another DENV (n=14) IgG and ZIKV (n=9) IgM and/or IgG positive samples were collected in Rio de Janeiro, Brazil, with ethical approval by the ethical committee from the University Hospital Clementino Fraga Filho (HUCFF). Additional DENV-positive samples (n=14), as well as one negative control, were obtained from Sera Care Life Sciences  and from Zeptometrix Corporation  (4 DENV-positive samples and two negative controls) with sample origin from Colombia, Honduras and Ecuador. The YFV (n=8) samples  were from YFV-vaccinated individuals who had participated in a randomized controlled vaccination study . The Malaria immune sera (n=6) from Ghana were obtained from the Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany. Ethical clearance was obtained from the Committee on Human Research Publication and Ethics of the Kwame Nkrumah University of Science and Technology. All participants in this study provided informed consent and all samples were analyzed anonymously.DENV , in concentrations of 300 ng and 160 ng per well, respectively, as described.29 The ZIKV Equad antigen was used in the indicated amounts (Results section).The quadruple mutant E-protein from ZIKV  bearing the point mutations T76A, Q77G, W101R and L107R was cloned into pMT/BiP/V5-His vector , expressed in 2SO4 and signals were read out at 450 nm with background reduction at 520 nm in a micro plate reader .Indicated amounts of ZIKV Equad or DENV 1–4 Equad mixtures were coated overnight on Nunc polysorb plates (Thermo Scientific) in 100 μL coating buffer  at 4 °C. The plates were washed three times with 350 μL per well of PBS-0,05% Tween and blocked with 200 μL of 5% non-fat milk powder (blocking solution) for 2 h at room temperature. After a second washing step, human sera were diluted 1:100 in 100 μL blocking solution per well and incubated for 1.5 h at room temperature. Following a third washing step, the HRP-conjugated secondary goat anti human IgG  or rabbit anti human μ-chain IgM  antibody was added for 1 h at room temperature. After a fourth washing step, 100 μL TMB substrate (Biozol) per well were incubated for 30 min at room temperature. The reaction was stopped with 50 μL 1 M HIn competition IgG ELISAs, sera (diluted 1:100 in blocking solution) were pre-incubated with indicated amounts of the competing antigen for 1 h at room temperature. Subsequently, they were added to the blocked antigens on the ELISA plate and incubated for 1.5 h at room temperature. The protocol was then continued as described above with IgG antibody detection.All antibody measurements were performed in duplicates in at least two independent experiments, except in Figure 6, where single measurements were performed due to limited amounts of serum. Graphical and descriptive statistical analysis of data was carried out using GraphPad Prism 6 . Statistical significance was determined using the Holm-Sidak method, with alpha=5.000%. Receiver operating characteristics (ROC) optimal curve calculations were performed in GraphPad Prism 6 with ELISA signals of the infected specimen and negative sera as control values. Signal cutoffs with optimal sensitivity and specificity were chosen and data were interpreted as positive with a signal/cutoff ratio higher than1.1 to ensure the best specificity. The cutoffs for the individual assay types are listed in 29To facilitate a specific serological differentiation between DENV and ZIKV infections, we inserted four amino acid point mutations in the conserved fusion loop (FL) domain of the ZIKV E-protein (Equad) and compared it to the previously described DENV 1–4 Equad mixture, which was shown to significantly reduce cross-reactivities in dengue serological diagnosis.The optimal concentration of ZIKV Equad for ELISA-based IgG and IgM tests was established through titration of the antigen with three ZIKV-positive and -negative sera each . Saturat29For the DENV 1–4 Equad mixture, the antigen amount per well yielding optimal sensitivity and specificity in IgM- and IgG- based ELISAs was determined previously.IgM antibodies of DENV-, ZIKV- and WNV-infected human sera  and negaNext, we used the DENV 1–4 Equad mixture and ZIKV Equad to measure IgG responses in serum samples from DENV-, ZIKV-, WNV-, JEV- and TBEV-infected persons . For DENTo further reduce the IgG cross-reactivity between DENV- and ZIKV- positive sera, a competition ELISA setup was chosen and tested with 3 DENV-positive sera which were known to be ZIKV-negative because they were collected in South America before the ZIKV-outbreak and 2 ZIKV-infected samples. All of them displayed similarly high signals in both the DENV- and the ZIKV- IgG ELISA. These sera were now pre-incubated with a competing antigen (either ZIKV Equad or the DENV 1–4 Equad mixture) and then measured on the coated DENV 1–4 Equad or ZIKV Equad. After having established suitable conditions for competition experiments, DENV- and ZIKV- IgG-positive as well as the negative sera were then measured on DENV 1–4 Equad after competition with 0.2 μg ZIKV Equad  and on ZZIKV- and DENV-positive sera were divided into two different groups each : Europea30 However, due to the epidemic spread of ZIKV in areas of simultaneous DENV circulation, the problem has gained another dimension. The specific diagnosis of these infections is mandatory not only for the implementation of effective control and surveillance activities, including the conduction of recently started DENV vaccine trials, but also for the timely treatment of potentially life-threatening disease symptoms. However, available DENV-tests are severely affected by ZIKV-antibodies, leading to a large number of false positive test results.32 Recently, ZIKV ELISAs based on NS1-proteins as antigens have been made commercially available, but limitations in IgM and/or IgG detection have been reported.36 Here we present a system for the specific and sensitive serological diagnosis of DENV- and ZIKV infections, based on the E-protein as antigen. The E-protein is widely used in serological flavivirus diagnosis, as virtually all infected individuals generate antibodies against it. On the other hand, it is targeted by cross-reactive antibodies, which mainly recognize the conserved FL-domain. By using mutations within and next to the FL-domain of the E-proteins of DENV and ZIKV, we found that IgM antibodies show greatly reduced cross-reactivity and bind specifically to the homologous antigen. This is in line with previous reports indicating that IgM responses against flaviviruses are more specific than IgG,19 although cross-reactivity has also been described.37 However, IgM antibodies are often produced only in low amounts, especially in secondary flavivirus infections or after a vaccination and are shortlived,38 hence IgG measurements become necessary. Using the mutant E-proteins, IgG antibodies present in the DENV- and ZIKV-positive sera did show cross-reactive binding to the ZIKV- and the DENV-antigens, respectively, although serum samples from WNV-, JEV- and TBEV-infected persons remained negative in both ELISAs, confirming previous results with the DENV antigen.29 On the basis of the sequence conservation of the E-protein ectodomains, ZIKV is more closely related to DENV than to the JEV-serocomplex, with amino acids identities of 52% and 54–57% when comparing ZIKV to WNV and DENV (four serotypes), respectively.39 This minor difference in amino acids identities is apparently enough to result in a very different binding of cross-reactive antibodies in sera from infected persons. Antibodies against non-flaviviral vector-borne pathogens  did not bind to either antigen, which underlines the usability of the mutant E-proteins in the diagnosis of flavivirus infections, whereas false-positive results with sera from malaria infected persons have been reported for an NS1-based ZIKV assay.41Cross-reactivity is a long-known complication in the serological diagnosis of flavivirus-infections, and several attempts have been made to develop systems to increase specificities of available test systems.42 which corresponds to the detection of DENV-antibodies in 18/21 samples using the mutant antigens. This result underlines the need to simultaneously test for ZIKV- and DENV- IgG antibodies in areas of co-circulation.In order to increase the specificity of the test in the differentiation of DENV- from ZIKV IgG-antibodies and vice versa, we performed competition experiments by pre-incubating the sera with one antigen before measuring them on the ELISA plate coated with the second one. This resulted in a statistically significant reduction of cross-reactivities . Now, ZI45 Therefore, tests to identify and discriminate such antibodies are an unmet need which has to be addressed urgently. Methods as the one presented here, which are able to detect DENV and ZIKV IgM- and IgG- antibodies with high specificity and sensitivity, could be very useful in this respect.Such secondary flavivirus infections represent a major challenge, not only for serological diagnosis. Pre-existing flavivirus antibodies might lead to severe complications of acute DENV- or ZIKV-infections, including severe dengue and neurological or congenital complications with ZIKV, as has been suggested by several studies.46 However, higher numbers of serum samples need to be analyzed in order to test whether this is indeed a difference between the two infectionsUsing paired serum samples we analyzed the rise of IgG antibodies upon infection with ZIKV and DENV in returning European travelers. A strong increase in the first 40 days was observed, and DENV-antibodies seemed to reach a plateau earlier than ZIKV antibodies, which might be attributed to a generally higher viral load in infections with DENV compared to ZIKV.47 and a monoclonal ZIKV NS1-antibody in a blockage-of-binding format.48 The system presented here relies on another antigen (mutant E-protein) and could, therefore, be used as an alternative approach in serology, independent of NS1 and also not dependent on only a single epitope. As the infrastructure for virus neutralization tests is only available in few specialized laboratories, confirmation and evaluation of inconclusive results from a particular serological assay can be performed best with an assay that is based on a different antigen. Therefore, resulting ELISAs for ZIKV and DENV could contribute to an accurate diagnosis and surveillance of these two virus infections, both as stand-alone tests and as means to complement existing methods.Most of the currently available ZIKV-ELISAs are based on the NS1 protein. Recently published attempts for improvement of ZIKV serological diagnosis include a microarray-based assay using ZIKV NS1 and DENV virus particles in a multiplexed format based on a plasmonic gold platform.


The aim of this 2-year cohort study (2003 to 2005) was to investigate how cariesexperience, at initial lesions (early or non-cavited lesions) and cavited stages,predicts caries increment in permanent teeth in 7-10- year-olds.The random sample of 765 children attending public schools in the city ofPiracicaba, SP, Brazil, was divided into two groups: 423 children aged 7-8 yearsand 342 children aged 9-10 years. All subjects were examined by a calibratedexaminer, using dental mirror and ball-ended probes, after tooth brushing andair-drying in an outdoor setting, based on the World Health Organization criteria.Active caries with intact surfaces were also recorded as initial lesion (IL).Univariate analysis was used for statistical analysis (Odds Ratios andChisquare).The association between the DMFT  increment andthe presence of IL was significant only for 9-10-year-old children. The childrenwith DMFT>0 at baseline were more prone to have DMFT increment, with thehighest risk for caries increment occurring in children aged 7-8 years.The predictors of caries increment were the presence (at baseline) of cariesexperience in permanent teeth for both age groups  and thepresence of the IL (at baseline) for 9-10-year-olds. In fact, detecting initial caries lesions inepidemiological studies is important to estimate the real disease prevalence and to knowthe treatment needs, targeting either invasive or non invasive procedures, to subjectsand/or groups of populations at risk.Evidence from literature has shown that the early detection of initial caries lesionsand the preventive approach are both the main aims for maintaining a good oral healthstatus7, it is also important to determine the impact of initialcaries lesions in caries risk assessment, verifying its influence as a predictor ofcaries increment, as assessed by some studies23. Therefore, the aim of this cohort study was toinvestigate how caries experience, at initial caries lesions (early lesions) andcavitated stages, predicts caries increment in permanent teeth over a two-yearperiod.Since recent scientific studies have reported that initial caries lesions aresignificantly more prevalent than cavitated caries lesionsThe study was approved by the Research Ethics Committee of Piracicaba Dental School,State University of Campinas (FOP/UNICAMP), under the protocol number 151/2003. TheSchool Principals granted permission for the study and an informed consent wasobtained from each child’s parents. All children diagnosed with treatment needs inthe epidemiological examinations were treated at FOP/UNICAMP’s preventive-restorativeprogram.This is a two-year prospective cohort study conducted between 2003 and 2005 inschoolchildren attending four different schools in the city of Piracicaba, SP,Brazil. At baseline (2003), 983 7-10-year-old schoolchildren of both genders wereexamined for dental caries and 765 children (77.8% response rate) were reexamined in2005 .16Piracicaba is a city located in the State of São Paulo in the southern regionof Brazil. Its population is about 365,000 inhabitants, and its Human DevelopmentIndex is 0.81. Fluoride has been added to the water supply system since 1971. Overthe last 3 decades, epidemiological surveys have shown that caries disease hasdecreased significantly, mainly due to the fluoridated water supply and use offluoridated dentifrice by the population.In order to calculate the sample size, the random sampling method was usedconsidering the caries experience by age group, based on previous surveys carried outin Piracicaba with a design error of 2, sample error 2%, a sampling loss of 20% and aconfidence level of 95%, that added up to 1,037 children. Considering the exclusioncriteria: having no parental consent, presence of systemic diseases or communicationand/or neuromuscular disorders, use of a fixed orthodontic appliance, presence ofsevere fluorosis or hypoplasia (n=54), the initial sample was composed of 983, 7 to10-year-old children. In 2005, 765 children aged 9 to 12 years were reexamined fordental caries.All the schools selected in this study are run by the municipality and are situatedin low-income urban communities from the outskirts of Piracicaba. The schoolchildrenwere similar concerning socioeconomic characteristics.The dental examinations carried out in 2003 (baseline) and in 2005  followed the same protocol. All subjects were examined by a calibratedexaminer, helped by a note-taking assistant, using dental mirror and ball-endedprobes with a diameter of 0.5 mm for removing debris and assessing presence offissure sealants in case of doubt and also to check the surface texture of initiallesion (IL). Before the examination each child brushed their teeth with fluoridateddentifrice for about 2 min, using the modified Bass technique, under supervision of adental hygienist. Moreover, dental drying was carried out for about 5 s per toothusing air from a dental compressor . Examinations were performed only on days with anappropriate natural luminosity with the child seated in a school chair in front ofthe examiner. No radiographs were taken in both baseline and final examinations.25 thatconsider a tooth as decayed only when cavitations are present. Active caries withintact surfaces were also recorded as ILs, following an adaptation of the criteriaproposed by Nyvad, et al.15 (1999)and Fyffe, et al.5 (2000). Thus, anIL is defined as a presumably active carious lesion which, upon visual assessment bya calibrated examiner, has an intact surface with no clinically detectable dentaltissue loss, with a whitish/yellowish area of increased opacity, roughness and lossof luster. When the probe is used, its tip should move gently across the surface. Forthe smooth surfaces, caries lesion is typically located close to gingival margin. Forthe occlusal surface, the lesion extends along the fissure walls. In this study,localized surface defects (active microcavities) restricted to enamel were alsoincluded in the IL group. IL and microcavities contiguous to sealants, restorationsand cavitations were also recorded.The criteria and codes used in this study were those based on the WHOrecommendationsTwo diagnostic thresholds were used in the study: WHO diagnostic thresholds  and WHO+IL diagnostic threshold.25 wascalibrated before baseline and final examinations by a benchmark “Gold Standard”dental examiner, who routinely uses the WHO criteria for training and calibration fororal health surveys. The benchmark examiner had also been previously trained andcalibrated in diagnosing ILs using similar criteria of other studies3. The calibration process consisted of theoretical discussions inclassrooms and clinical training sessions held in outdoor setting, under naturallight, in four periods of 4 h. Mean inter-examiner agreement (benchmark examinerversus examiner), measured by the Kappa statistics9 was K=0.88 for the WHO+IL. Reexaminations wereperformed in 10% of the sample to determine intra-examiner error. Mean weighted Kappavalue of intra-examiner agreement was K=0.89 (WHO+IL).One examiner with epidemiological experience in surveys using the World HealthOrganization criteria19 at 5% significancelevel.In data analysis the dependent variable was DMFT increment>0 over the 2-yearperiod . First the influence of ILs on caries increment was tested according to agegroups (7-8-year-olds and 910-year-olds) by the Chi-square test. Then each age groupwas divided according to caries experience as follows: DMFT and dmft=0 (Controlgroup); DMFT=0 and dmft>0 (caries experience in primary teeth); DMFT>0 anddmft>0 (caries experience in permanent and caries experience or no in primaryteeth) to test the influence of caries experience at cavitated stage on the cariesincrement. Odds Ratios (OR), their 95% confidence intervals (CI) and significancelevels were estimated. All statistical tests were performed using the softwareSASThe response rate in this 2-year cohort study was 77.8%, as from the 983 children whowere examined at baseline, 765 completed the study. The reasons for children dropoutwere: moving to another school and refusal to participate in the final examination.In this prospective cohort study investigating how caries experience predicts cariesincrement in permanent teeth, the presence of IL at baseline was associated with cariesincrement in permanent dentition after 2 years for 9-10-yearold children . This in4 (1989), teeth in eruption process are more susceptible to developdental caries because the biofilm finds favorable conditions for accumulation as theseteeth are not yet in function. Moreover, children may present poor dental cleaning,becoming a more susceptible group for dental caries in permanent dentition.Another important result is that all the children with DMFT>0 and dfmt>0 weresignificantly more prone to develop caries in permanent dentition in comparison to thosecaries-free in both dentitions . Thechi24. In general, children aged 7-8 years are in a period ofdentition transition, with many primary teeth exfoliating and permanent teeth ineruption. Especially the last condition may increase the caries risk due to somecharacteristics such as: a) a higher carbonate content in dental enamel, which causeschanges in the hydroxyapatite crystal lattice, resulting in a more acid-susceptibleenamel surface; and b) teeth in eruption have no functional occlusal contact, which mayincrease dental biofilm accumulation and hinder toothbrushing1.An unexpected result was that a child with caries experience in permanent dentition atbaseline presented statistically higher probability of developing DMFT increment , since m13 (1991), 74% of ILsdetected at baseline halted or receded during the 2-year period of the research. It isalso important to note that in a recent study also conducted in Piracicaba with 6-8year-olds, there was no association between ILs and caries increment after 7 years ofevaluation.21 However, theinclusion of ILs within epidemiological surveys is likely to establish a clearerrelation between the epidemiological estimates of dental caries prevalence and thetreatment needs.17 Moreover, in alongitudinal study, changes in behavioral characteristics, such as, improvement indental cleaning, reduction in sugar consumption, etc., may modify the oral environmentin such a way that caries risk declines over time. Therefore data collection in relationto ILs in oral health surveys should be encouraged in order to know the real prevalenceof caries and to help oral health administrators in planning prevention actions.Regarding the lack of association between the DMFT increment and the presence of ILs foryounger children , in a stIt is also important to mention the limitations of this study. The results cannot beextrapolated to all populations and regions or countries because some differences existcompared to other regions, such as availability of fluoridated dentifrice since about1990’s and water fluoridation since 1971.The predictors of caries increment were the presence (at baseline) of caries experiencein permanent teeth for both age groups  and the presence of the IL(at baseline) for 9-10-year-olds.


Accurate diagnosis and subsequent treatment of latent tuberculosis infection (LTBI) is essential for TB elimination. However, the absence of a gold standard test for diagnosing LTBI makes assessment of the true prevalence of LTBI and the accuracy of diagnostic tests challenging. Bayesian latent class models can be used to make inferences about disease prevalence and the sensitivity and specificity of diagnostic tests using data on the concordance between tests. We performed the largest meta-analysis to date aiming to evaluate the performance of tuberculin skin test (TST) and interferon-gamma release assays (IGRAs) for LTBI diagnosis in various patient populations using Bayesian latent class modelling.Systematic search of PubMeb, Embase and African Index Medicus was conducted without date and language restrictions on September 11, 2017 to identify studies that compared the performance of TST and IGRAs for LTBI diagnosis. Two IGRA methods were considered: QuantiFERON-TB Gold In Tube (QFT-GIT) and T-SPOT.TB. Studies were included if they reported 2x2 agreement data between TST and QFT-GIT or T-SPOT.TB. A Bayesian latent class model was developed to estimate the sensitivity and specificity of TST and IGRAs in various populations, including immune-competent adults, immune-compromised adults and children. A TST cut-off value of 10 mm was used for immune-competent subjects and 5 mm for immune-compromised individuals.A total of 157 studies were included in the analysis. In immune-competent adults, the sensitivity of TST and QFT-GIT were estimated to be 84%  and 52% (50–53%), respectively. The specificity of QFT-GIT was 97% (96–97%) in non-BCG-vaccinated and 93% (92–94%) in BCG-vaccinated immune-competent adults. The estimated figures for TST were 100% (99–100%) and 79% (76–82%), respectively. T-SPOT.TB has comparable specificity (97% for both tests) and better sensitivity (68% versus 52%) than QFT-GIT in immune-competent adults. In immune-compromised adults, both TST and QFT-GIT display low sensitivity but high specificity. QFT-GIT and TST are equally specific (98% for both tests) in non-BCG-vaccinated children; however, QFT-GIT is more specific than TST (98% versus 82%) in BCG-vaccinated group. TST is more sensitive than QFT-GIT (82% versus 73%) in children.This study is the first to assess the utility of TST and IGRAs for LTBI diagnosis in different population groups using all available data with Bayesian latent class modelling. Our results challenge the current beliefs about the performance of LTBI screening tests, and have important implications for LTBI screening policy and practice. We estimated that the performance of IGRAs is not as reliable as previously measured in the general population. However, IGRAs are not or minimally affected by BCG and should be the preferred tests in this setting. Adoption of IGRAs in settings where BCG is widely administered will allow for a more accurate identification and treatment of LTBI. Reliable detection of latent tuberculosis infection (LTBI) is a priority as this will help direct appropriate use of limited resources for tuberculosis (TB) control. One-third of the world’s population have LTBI with 10% of these individuals eventually developing active TB . TreatmeMycobacterium tuberculosis-specific antigens absent in BCG and most non-tuberculosis mycobacteria ) OR “TST” OR “Mantoux”) and (“interferon gamma release assay” OR “interferon gamma assay” OR “QuantiFero*” OR “IGRA” OR “T-SPO*” OR “TSPO*” OR “Elispot” OR CFP10 OR ESAT6) and (tuberculosis[mesh]). No restrictions on date, language, or type of studies were applied. The full search strategy is described in Data from each eligible study were extracted by two independent reviewers. Discrepancies between the two reviewers were resolved by consensus or by consultation with a third reviewer (DE) if consensus could not be reached. The following variables were extracted: year of publication, country of origin, population group, BCG vaccination rate, TST cut-off value, methods of IGRAs, age range and mean/median where available, proportion of participants on immunosuppressive therapy, and 2x2 test agreement data . If separate agreement tables were available for different subgroups of patients, these data were included separately . AuthorsThe primary outcome was the diagnostic performance, i.e. sensitivity, specificity, positive predictive value and negative predictive value, of TST, QFT-GIT and T-SPOT.TB in immune-competent adults aged 15 years or above. For studies to be included in this primary analysis, the prevalence of immune-compromised conditions had to be less than 5% . Subgrouπ) of LTBI in the population, and the sensitivity  and specificity  of TST  and IGRA . Let D be the unknown (latent) true disease status, the prevalence, sensitivity and specificity can be formally expressed as follows:π, S1, S2, C1 and C2 as follows:We developed a Bayesian latent class model to describe the observed 2x2 data to estimate the true prevalence  distribution for the priors of the sensitivity and specificity. Beta distribution was chosen because its region of positive density ranges from 0 to 1, matching the range of these parameters  estimated the pooled sensitivity of QFT and TST to be 70% and 77%, respectively; the specificity of QFT to be 96–99%; and the specificity of TST in non-BCG-vaccinated and BCG-vaccinated populations to be 97% and 59%, respectively. Our estimate of the sensitivity of QFT-GIT is lower than that of Pai et al. [Accurate identification and subsequent treatment of LTBI is essential to TB control and elimination. The lack of a gold standard for diagnosing LTBI means that the true prevalence of the disease is unknown, and the estimations of the sensitivity and specificity of diagnostic tests are unreliable. This study represents the most comprehensive Bayesian latent class analysis of published data on the performance of TST and IGRAs for the diagnosis of LTBI. We have confirmed that IGRAs have high specificity but that these tests have considerably lower sensitivity than TST in immune-competent populations than had previously been demonstrated ,7,183. AConventional meta-analysis of diagnostic tests simply entails pooling of data to provide pooled estimates of test sensitivity and specificity. Simple pooling of data may cause serious bias due to confounding of disease prevalence in the contributing studies . Our latImmune-compromised patients have an increased risk of LTBI reactivation . ScreeniThe limitations of our study must be considered. Our results are derived from studies where the estimates of LTBI prevalence vary widely. This is due to the heterogeneity in study settings, populations and methodology of the included studies. Bayesian analysis requires prior information on model parameters. One criticism of Bayesian latent class models is that they may be sensitive to the choice of prior information. This may particularly be the case when there are limited observed data. When the number of observed data are large, as in our study, these begin to dominate any prior information. We believe that we have used the most informative priors obtained from the literature. Furthermore, we performed sensitivity analysis and found that our results are not sensitive to choice of prior .In conclusion, our study represents the most comprehensive Bayesian latent class analysis of the diagnostic accuracy of TST and IGRAs derived from all published agreement data. Our results challenge the current beliefs about the performance of LTBI screening tests and provide important information to guide choice of tests for LTBI screening that will enhance the millennium goals for elimination of TB. Our findings show that IGRAs may be inferior to TST for diagnosing LBTI in non-BCG-vaccinated populations. For BCG-vaccinated individuals, IGRAs appear to be a more favourable choice. IGRAs will therefore allow physicians and TB controllers to better understand the background prevalence of LTBI for targeted preventive therapy in settings where BCG vaccination is widely administered. QFT-GIT and TST have suboptimal sensitivity in immune-compromised patients and results should be interpreted with caution. A combination of both tests could potentially overcome the problems of false-positives in this setting. Considerations regarding cost-effectiveness, logistics, availability for clinicians and patient acceptability should be taken into account to decide which test to use for the diagnosis of LTBI.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 Text(PDF)Click here for additional data file.S1 TableIGRA, interferon gamma release assay; N, No; Q, Question; TST, tuberculin skin test; U, Unclear; Y, Yes.(PDF)Click here for additional data file.S2 Table*Results are for immune-competent adults. BCG, Bacillus Calmette-Guérin; CrI, credible interval; QFT-GIT, QuantiFERON-TB Gold In Tube; TB, tuberculosis; TST, tuberculin skin test.(PDF)Click here for additional data file.S3 Table*Results are for immune-competent adults. BCG, Bacillus Calmette-Guérin; CrI, credible interval; QFT-GIT, QuantiFERON-TB Gold In Tube; TB, tuberculosis; TST, tuberculin skin test.(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.