Mycobacterium tuberculosis isolates sharing identical DNA fingerprint patterns can be epidemiologically linked. However, municipal health services in the Netherlands are able to confirm an epidemiological link in only around 23% of the patients with isolates clustered by the conventional variable number of tandem repeat (VNTR) genotyping. This research aims to investigate whether whole genome sequencing (WGS) is a more reliable predictor of epidemiological links between tuberculosis patients than VNTR genotyping.Patients with Mycobacterium tuberculosis complex isolates received at the Netherlands National Institute for Public Health and the Environment in 2016. Isolates were clustered by VNTR when they shared identical 24-loci VNTR patterns; isolates were assigned to a WGS cluster when the pair-wise genetic distance was ≤ 12 single nucleotide polymorphisms (SNPs). Cluster investigation was performed by municipal health services on all isolates clustered by VNTR in 2016. The proportion of epidemiological links identified among patients clustered by either method was calculated.VNTR genotyping and WGS were performed in parallel on all In total, 535 isolates were genotyped, of which 25% (134/535) were clustered by VNTR and 14% (76/535) by WGS; the concordance between both typing methods was 86%. The proportion of epidemiological links among WGS clustered cases (57%) was twice as common than among VNTR clustered cases (31%).When WGS was applied, the number of clustered isolates was halved, while all epidemiologically linked cases remained clustered. WGS is therefore a more reliable tool to predict epidemiological links between tuberculosis cases than VNTR genotyping and will allow more efficient transmission tracing, as epidemiological investigations based on false clustering can be avoided. The Netherlands is a low incidence country with 5.2 tuberculosis cases per 100,000 inhabitants. In 2016, 889 tuberculosis patients were notified, which was an increase of 3% compared to 2015 due to the high influx of asylum seekers from high tuberculosis incidence countries, i.e. Eritrea, Ethiopia, and Somalia . Whole gM. tuberculosis complex isolates in the Netherlands are sent to the National Tuberculosis Reference Laboratory for genotyping. Variable number of tandem repeat (VNTR) genotyping is the current DNA fingerprinting method and has been routinely applied in the Netherlands since 2009 [M. tuberculosis, rather than insufficient epidemiological investigations. This might be especially true for isolates from patients originating from high-prevalence geographic areas such as the Horn of Africa, where transmission is less efficiently interrupted by tuberculosis control efforts and strains may be genetically highly conserved [All nce 2009 , 7. Isolnce 2009 . This loonserved .M. tuberculosis isolates potentially has a higher resolution than VNTR genotyping as a much larger fraction of the genome, more than four mega base pairs of DNA, is analysed for diversity [M. tuberculosis is rapid enough to study the transmission of tuberculosis efficiently. In 2016, a four-year nationwide WGS project was initiated in the Netherlands, which has been to date applied for research purposes only. In this project, WGS runs simultaneously with the conventional VNTR genotyping, while cluster investigation by municipal health services remains to be performed based on VNTR genotyping, until WGS is implemented in the Netherlands. In this study, we aim to 1) compare clustering on basis of VNTR versus WGS, and 2) compare the degree of confirmed epidemiological links by municipal health services on basis of information from both typing methods.WGS of iversity , 12. HowM. tuberculosis complex isolates cultured in the Netherlands between January 1, 2016 and December 31, 2016, were included in this study. VNTR genotyping and WGS were simultaneously performed for all included samples. Laboratory cross contaminations , M. bovis Bacillus Calmette-Guérin (BCG), as well as multiple isolates from the same patient were excluded.All DNA used for both typing methods was isolated from a positive Mycobacteria Growth Indicator Tube and purified with the QIAamp DNA mini kit method  under BSL-3 laboratory conditions. VNTR genotyping was performed as described earlier , 7 and VM. tuberculosis complex DNA samples were also sequenced on an Illumina HiSeq2500 sequencer that generated paired-end reads of 125-bp. A minimum sample yield of 350 Mb was required to achieve an average sequencing coverage of 80 reads for M. tuberculosis samples considering the 4.4 Mb genome size. Reads were mapped unpaired against the H37Rv reference genome version 3.0 (GenBank accession number AL123456.3) using Bowtie2 in Breseq version 0.28.1 [In parallel with VNTR genotyping, n 0.28.1 . Single Tuberculosis is a notifiable disease and cases are reported to the Netherlands Tuberculosis Register. Data on patient characteristics, i.e. pulmonary tuberculosis (PTB)/ extra pulmonary tuberculosis (ETB), geographical region of residence in the Netherlands, age, gender, ethnicity, rural/urban living, risk group, resistance, and co-morbidities were collected from this register. The register also includes data on epidemiological links as established by municipal health services by interviews with tuberculosis patients. However, since these data are entered into the Netherlands Tuberculosis Register at a relatively late stage, municipal health services were in this study actively contacted by phone to obtain results of cluster investigations on patients that had an isolate with an identical VNTR pattern with another patient in 2016. Public health nurses in the Netherlands perform VNTR cluster investigations according to a standardized questionnaire. In this study, only confirmed epidemiological links were included, which is in the Netherlands Tuberculosis Register defined as A) patients know each other by name and were present on the same time and place, or B) patients do not know each other by name, but the patients within the same VNTR cluster were present in the same period on the same address/location .R statistics version 3.3.2  was applM. tuberculosis complex isolates were subjected to both WGS and VNTR genotyping; data from the Netherlands Tuberculosis Register was missing for eight patients. The median age of patients was 35 years (range 0–102) and 60% were men. The majority of patients were first generation migrants (78.4%) and more than half (62.3%) of the patients had PTB or the combination with ETB  M. bovis, 0.2% (1/535) M. caprae, and 0.2% (1/535) M. orygis. Twenty-four percent (127/520) of M. tuberculosis isolates belonged to the EAS lineage, followed by 22.5% belonging to the Delhi/CAS lineage  were  lineage . Ten isoThe median age of the WGS clustered cases was 23 years (range 0–89) compared to 37 years (range 13–102) of non-WGS clustered cases. WGS clustered cases were more likely to be male , contact of a tuberculosis patient , and asylum seekers  with associated Delhi/CAS lineage . The Haarlem  and S-type  lineages were also more frequently observed among isolates of the WGS clustered cases .Using VNTR genotyping, 46% (246/535) had a unique pattern, 29% (155/535) were clustered with isolates from before 2016, and 25% (134/535) were clustered with another isolate in 2016. The 134 isolates clustering with another isolate in 2016 belonged to 41 different VNTR clusters with cluster sizes ranging from 2–21 isolates; 25/41 clusters consisted of two isolates.WGS clustered 14.2% (76/535) of the isolates. Sixty-eight of these isolates were also clustered by VNTR and an additional three clusters comprising a total of eight cases were clustered by WGS only; two clusters of three cases and one of two cases . The VNTAs described earlier, cluster investigation by municipal health services is currently only performed on VNTR clustered cases, as WGS is not yet routinely implemented in the Netherlands. In 2016, 134 patients were clustered by VNTR; cluster investigation resulted in 41 patients to be epidemiologically linked and for the remaining 93 patients an epidemiological link could not be identified. The proportion of confirmed epidemiological links identified in WGS clustered isolates was 57.4% (39/68) compared to 30.6% (41/134) among VNTR clustered cases. Among the 66 patients that were not clustered by WGS, two were epidemiologically linked, but their isolates showed a genetic distance of 27 SNPs . The genetic distance for events between patients that could not be epidemiologically linked was up to 201 SNPs; 2–12 SNPs for 29 of the 93 non-epidemiologically linked patients that did cluster by WGS, and 14–201 SNPs for 64 of the 93 non-epidemiologically linked patients that were not clustered by WGS .The confirmation of epidemiological links was also compared with the information on the geographic spread and infectiousness (PTB or ETB) of the patients. All 41 patients with a confirmed epidemiological link were from the same geographic area and were diagnosed with either PTB or the combination of PTB/ETB. Non-epidemiologically linked cases were in general geographically more distant from each other  Tables.Cluster investigation was not performed on the three additional clusters containing in total eight patients identified by WGS only, as they were not clustered by VNTR. Based on the geographic proximity and infectiousness of tuberculosis, for two of the three additional clusters comprising five patients, an epidemiological link appears likely. Thus, with VNTR 134 epidemiological investigations were required to identify 41 epidemiologically linked patients, which is a yield of 30.6%, whereas WGS would have required 76 (68+8) epidemiological investigations to identify 44 (39+5) epidemiologically linked cases, a yield of 57.9% .M. tuberculosis complex isolates. Typing by WGS increased efficiency by reducing the number of cases requiring cluster investigations by half, while the degree of confirmed epidemiologically linked cases doubled. Furthermore, WGS clustered an additional eight cases that were not clustered by VNTR genotyping, five of which based on geographical proximity could possibly belong to chains of transmission.This is the first prospective, population-based study to quantify the benefit of WGS over the current standard VNTR genotyping on a nation-wide collection of more than 500 Previous studies already indicated that VNTR can be misleading and some VNTR clusters can be identified as false when WGS is applied –23. In fA limitation of our study is that cluster investigation by municipal health services was only included for patients with isolates clustering on basis of VNTR with other isolates in 2016, which can lead to an underestimation of transmission. Of the 93 patients clustered by VNTR for which no epidemiological link could be confirmed, 29 also clustered by WGS. Almost all of these 29 patients were asylum seekers from Eritrea/Ethiopia. A recent study from the Netherlands showed that transmission among asylum seekers from the Horn of Africa most likely occurred during the escape route, but that a proportion of these patients might been infected after arrival in the destination country . This isM. tuberculosis and ten M. tuberculosis isolates had a relatively low mean coverage. Re-analysing the data by excluding these samples did not significantly affect the results (data not shown) as only one of the low coverage isolates was part of the 134 isolates clustering by VNTR in 2016. This low coverage isolate belonged to the epidemiologically linked patient with 27 SNPs genetic distance with its pair, meaning this link was missed by WGS due to bad sequence quality.Furthermore, 15 of the 535 (2.8%) isolates had WGS data originating from subspecies other than M. tuberculosis complex isolates in the Netherlands are genotyped at one national tuberculosis reference laboratory, making the results of this study generalizable to the entire patient population. Also, all municipal health services perform extensive source case tracing and contact investigation and support patients on a regular basis. The results of these investigations together with a wide spectrum of patient characteristics are reported to the Netherlands Tuberculosis Register, increasing the validity of this study. However, information bias remains possible due to language/cultural barriers when interviewing patients, which include many immigrants and asylum seekers.The main strength of this study is that all Currently there is no international standard for the SNPs distance cut-off to rule in a possible transmission, and various cut-offs have been applied in studies in different countries, which limits the ability to compare data , 28–30. In conclusion, the clustering of tuberculosis cases in 2016 was reduced by half on the basis of WGS compared to VNTR, while retaining the number of epidemiological links. This observation confirms that VNTR genotyping leads to a considerable proportion of false clustering, as was already suggested by the low confirmation of epidemiological links identified by municipal health services in the Netherlands. The lower degree of clustering by WGS will prevent unnecessary cluster investigations. Moreover, the much higher degree of confirmed epidemiological links within WGS clusters will contribute to improved understanding of tuberculosis transmission. Therefore, basing epidemiological investigations on WGS typing can support tuberculosis elimination in a low incidence country such as the Netherlands.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Sudden cardiac death (SCD) and pump failure death (PFD) are common endpoints in chronic heart failure (CHF) patients, but prevention strategies are different. Currently used tools to specifically predict these endpoints are limited. We developed risk models to specifically assess SCD and PFD risk in CHF by combining ECG markers and clinical variables.αTpe) and T-wave morphology restitution (TMR), both reflecting changes in dispersion of repolarization due to heart rate changes. Standard clinical indices were also included.The relation of clinical and ECG markers with SCD and PFD risk was assessed in 597 patients enrolled in the MUSIC (MUerte Súbita en Insuficiencia Cardiaca) study. ECG indices included: turbulence slope (TS), reflecting autonomic dysfunction; T-wave alternans (TWA), reflecting ventricular repolarization instability; and T-peak-to-end restitution  for SCD and PFD for patients in the high-risk group (fifth quintile of risk score) with respect to patients in the low-risk group (first and second quintiles of risk score) were both greater than 4. HRs for SCD and PFD increased to 9 and 11 when using a model including only ECG markers, and to 14 and 13, when combining clinical and ECG markers.The indices with the greatest SCD prognostic impact were gender, New York Heart Association (NYHA) class, left ventricular ejection fraction, TWA, ΔThe inclusion of ECG markers capturing complementary pro-arrhythmic and pump failure mechanisms into risk models based only on standard clinical variables substantially improves prediction of SCD and PFD in CHF patients. Sudden cardiac death (SCD) and pump failure death (PFD) are both common endpoints in patients with chronic heart failure (CHF) . PreventChronic heart failure is a complex clinical syndrome that can result from a number of functional or structural cardiac disorders, impairing the ventricle’s ability to fill with or eject blood . A commoRisk models based on clinical variables have been proposed for risk stratifying CHF patients at risk of SCD or PFD , 13, 14 The original study population consisted of 992 consecutive patients with symptomatic CHF corresponding to NYHA classes II and III enrolled in the MUSIC (MUerte Súbita en Insuficiencia Cardiaca) study, a prospective, multicenter study designed to assess risk predictors for cardiovascular mortality in ambulatory CHF patients . PatientFollow-up visits were conducted on an outpatient basis every 6 months, for a median of 44 months. Primary endpoints were SCD and PFD. Cardiac death (CD) was divided into SCD and PFD. Death was defined as SCD if it was: (i) a witnessed death occurring within 60 min from the onset of new symptoms, unless a cause other than cardiac was obvious; (ii) an unwitnessed death (<24h) in the absence of preexisting progressive circulatory failure or other causes of death; or (iii) a death during attempted resuscitation. Deaths occurring in hospitals as a result of refractory progressive end-stage CHF, or CHF patients undergoing heart transplantation, were defined as PFD. Endpoints were reviewed and classified by the MUSIC Study Endpoint Committee .QT, ΔαTpe, TMR) and sympathovagal balance (TS) were computed and used to develop the risk models, together with other commonly used ECG variables , an ECG-based model (including ECG markers only) and a combined model . Finally, a point scoring system was constructed in which points were assigned to each marker using beta-coefficients from each of the final multivariable Cox regression models , 23, 24.The area under the receiver operating curve (ROC), denoted by AUC, was calculated by considering the Sensitivity and Specificity of the three risk models  in identifying patients associated with a specific outcome (SCD or PFD) at any time during the follow-up.There were 425 men and 172 women in the sample population . The majority of patients (83%) were in NYHA class II, while the remaining 17% were in NYHA class III and LVEF was 37% ± 14%. The detailed characteristics of the study population are shown in SCD victims were more frequently men (p = 0.048), were in NYHA class III (p = 0.047) and had LVEF≤35% (p = 0.010), while PFD victims were more likely older than the rest of patients (p = 0.013), were more frequently diabetic (p = 0.009), in NYHA class III (p = 0.001), were not treated with beta-blockers (p = 0.012) and also had low LVEF (p = 0.044). Ischemic etiology was not associated with SCD or PFD .Tpe (p = 0.002), ΔαQT (p = 0.041), IAA (p = 0.008) and TMR (p = 0.001) and lower TS (p = 0.004). PFD victims were associated with lower RR range (p<0.001), ΔαTpe (p = 0.003), TS (p<0.001) and a higher rate of a complex index of arrhythmia (CIA) composed of non-sustained ventricular tachycardia (NSVT) and more than 240 ventricular premature beats (VPBs) in 24 h (p = 0.014). QRS duration was not associated with SCD or PFD.ECG analysis showed that SCD victims were associated with higher ΔαTpe≥0.028, ΔαQT≥0.228, IAA≥3.7μV, TS≤2.5 ms/RR and TMR≥0.04 . In the HFrEF group, the number of PFD victims was also significantly higher than in the HFpEF group .Tpe (p = 0.008) and TMR (p = 0.001) but lower TS (p = 0.025), while PFD victims were more frequently diabetic (p = 0.001), in NYHA class III (p = 0.001) and showed lower RR range (p = 0.003), ΔαTpe (p = 0.005) and TS values (p<0.001) but a higher rate of CIA (p = 0.030). Univariable Cox analyses showed that the only clinical variable significantly associated with SCD in the HFrEF group was administration of ARB or ACE inhibitors  and were associated with higher Δαhibitors . Hence, TMR≥0.04 , as in tTMR≥0.04  , while PFD victims were characterized by higher age (p = 0.012) and lower values of TS (p = 0.005). Univariable Cox analyses showed that no clinical variable was significantly associated with SCD or PFD (QT≥0.228 (AUC of 0.72 (0.55–0.89), p = 0.008). The ECG model for PFD included only TS≤2.5 ms/RR, as this was the only variable significantly associated with PFD along follow-up. For SCD, no multivariable combined model could be calculated. The multivariable combined model for PFD included age and TS  and higher values of ΔαD or PFD  Tables. D or PFD . The mule and TS  Tables , p = 0.009), with no significant association with PFD. Also, the high PFD risk group in the combined model was significantly associated with PFD (HAR 2.578 (1.453–4.573), p = 0.001), with no significant association with SCD.The main result of this study is that in mild-to-moderate CHF patients the combination of clinical and ECG markers significantly improves prediction of both SCD and PFD, as compared to the use of clinical variables only. This indicates a possible new strategy to identify CHF patients specifically at risk of SCD or PFD.In a combined model, the clinical variables that predicted SCD were male gender, NYHA class III and LVEF≤35%. Previous studies have also shown that men have higher SCD risk than women , 26, whiDiabetes, NYHA class III, absence of treatment with beta-blockers and LVEF≤35% predicted PFD in a multivariable clinical model. These results confirm previous findings reporting the relation between end-stage CHF and low cardiac output and LVEF, diabetes due to increased congestion as well as advanced stages of NYHA class . Also, tTpe, IAA, TS and TMR. Our results confirm that SCD risk is associated with increased dispersion of repolarization restitution [The ECG variables that independently predicted SCD in the ECG model were Δαtitution , 33, inctitution  and barotitution , 35. MorTpe, TS and the range of RR. This indicates that PFD is also characterized by baroreceptor-heart rate reflex sensitivity [Tpe indicative of higher PFD risk suggest that PFD victims experience a reduction in the ability of the ventricles to adapt to changes in heart rate, as opposed to SCD victims [The ECG markers that independently predicted PFD in the ECG model were Δαsitivity , 35. How victims .The ECG models for SCD and PFD showed better prognostic value than the clinical models. The combination of clinical and ECG markers synergistically improved the prognostic value for both SCD and PFD. For PFD prediction the improvement achieved by combining clinical and ECG markers was only marginal with respect to the results of the ECG model. This suggests that clinical variables do not add complementary information to ECG markers for PFD risk prediction. More importantly, the ECG and combined risk models demonstrated high sensitivity (association with the designated mode of death) and specificity  for SCD and PFD prediction, while the clinical risk model for SCD prediction lacked specificity and predicted PFD in addition to SCD.αQT) demonstrated specific SCD predictive value in the HFpEF group. These results support previous studies where ECG-derived arrhythmic markers were found to be predictive of SCD in patients with preserved LVEF [Separately considering HFrEF and HFpEF subpopulations, the number of SCD and PFD victims was significantly higher in the HFrEF group than in the HFpEF group, supporting previous studies . A largeved LVEF . For PFDved LVEF , 40. Oveved LVEF .Due to the low number of SCD and PFD victims, a division of the sample population into training and test groups for validation of the results was not performed. Moreover, the proposed risk scores were calculated in patients in sinus rhythm, which limits its applicability, especially in patients with reduced LVEF, where the incidence of AF is significant. Further studies may consider modifications of ECG markers to assess the proposed models in CHF patients with other rhythms. The assessment of clinical and combined models integrating additional variables deserves further investigation. The results obtained in this work are not directly comparable with those of a previous publication reporting clinical scores on the MUSIC study , becauseThis study demonstrates that two risk prediction models combining clinical and ECG markers of electrophysiological and autonomic abnormalities specifically predict SCD and PFD. For SCD, the combination of clinical and ECG markers substantially improved risk prediction as compared to the use of only clinical or ECG markers. For PFD, the use of only ECG markers demonstrated its superiority over the use of clinical markers. The combination of clinical and ECG variables only marginally improved the PFD predictive value of the ECG model.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Background: The temporal pattern of ventricular repolarization is of critical importance in arrhythmogenesis. Enhanced beat-to-beat variability (BBV) of ventricular action potential duration (APD) is pro-arrhythmic and is increased during sympathetic provocation. Since sympathetic nerve activity characteristically exhibits burst patterning in the low frequency range, we hypothesized that physiologically enhanced sympathetic activity may not only increase BBV of left ventricular APD but also impose a low frequency oscillation which further increases repolarization instability in humans.Methods and Results: Heart failure patients with cardiac resynchronization therapy defibrillator devices (n = 11) had activation recovery intervals  recorded from left ventricular epicardial electrodes alongside simultaneous non-invasive blood pressure and respiratory recordings. Fixed cycle length was achieved by right ventricular pacing. Recordings took place during resting conditions and following an autonomic stimulus . The variability of ARI and the normalized variability of ARI showed significant increases post Valsalva when compared to control . The oscillatory behavior was quantified by spectral analysis. Significant increases in low frequency (LF) power (p = 0.002) and normalized LF power (p = 0.019) of ARI were seen following Valsalva. The Valsalva did not induce changes in conduction variability nor the LF oscillatory behavior of conduction. However, increases in the LF power of ARI were accompanied by increases in the LF power of systolic blood pressure (SBP) and the rate of systolic pressure increase (dP/dtmax). Positive correlations were found between LF-SBP and LF-dP/dtmax , LF-ARI and LF-SBP  and between LF-ARI and LF-dP/dtmax . There was a strong positive correlation between the variability of ARI and LF power of ARI .Conclusions: In heart failure patients, physiological sympathetic provocation induced low frequency oscillation (~0.1 Hz) of left ventricular APD with a strong positive correlation between the LF power of APD and the BBV of APD. These findings may be of importance in mechanisms underlying stability/instability of repolarization and arrhythmogenesis in humans. Factors which modulate or destabilize ventricular repolarization are of fundamental importance in arrhythmogenesis. Spontaneous beat-to-beat fluctuation in repolarization is an inherent property of ventricular myocardium . Informed consent was obtained in writing from all subjects.Studies were performed in 11 ambulatory heart failure patients  who were recipients of a cardiac resynchronization therapy defibrillator (CRT-D) device . Patient characteristics are shown in Table The implanted CRT-D device was used to record unipolar electrograms (UEGs) from the left ventricular epicardial lead, sampled at 512 Hz Figure . The devFollowing recruitment to the study, beta-adrenergic blocking agents (bisoprolol) were discontinued for 5 days prior to the start of the protocol to allow for a sufficient wash-out period  for each beat using a script written in MATLAB . Ectopic beats  were removed from analysis together with the successive beat.Raw digital UEG traces were analyzed off-line using custom built MATLAB software  as described previously , and the normalized variability values of SBP (nSD-SBP) and dP/dtmax (nSD-dP/dtmax) were computed with the same formula as for ARI. The same formula was applied to AT-AT intervals to assess for any evidence of a change in conduction variability generated by the Valsalva.Beat-to-beat variability of ARI was computed over the entire 60 s period as per established QT variability measures  were also calculated by dividing the LF and HF variability by the total power in the range above 0.04 Hz , as recommended by Malik . A P < 0.05 was considered to be statistically significant for all tests.Results are presented as mean ± standard deviation. Continuous variables were compared using the Wilcoxon signed-rank test or Mann–Whitney All UEG recordings and respiratory recordings were analysable. Three blood pressure recordings were inadequate to allow analysis throughout the entire 60 s . A total of 22 UEG, 22 respiratory recordings, and 19 blood pressure recordings were analyzed. Throughout the protocol the pacing cycle length remained constant for all patients. The mean pacing cycle length used was 703 ± 80 ms.max measurements during rest and following the Valsalva are reported in Table p = 0.019). Mean nSDARI also increased significantly (p = 0.032). Figure p = 0.147). There was no evidence of an increase in conduction variability following the Valsalva as assessed by AT-AT interval variability: AT-AT interval variability at rest (4.42 ± 0.41 ms) and following the Valsalva (4.52 ± 0.57 ms) was similar (p = 0.7).Mean ARI, SBP, and dP/dtp = 0.004). Mean nSD-SBP also significantly increased (p = 0.004). Figure max increased following the Valsalva (p = 0.004). Mean nSD-dP/dtmax also significantly increased (p = 0.004). Figure max and nSD-dP/dtmax during control and following the Valsalva. As per the behavior observed in mean ARI, there was no change in mean SBP (p = 0.426) nor mean dP/dtmax (p = 0.652).Mean SD-SBP increased following the Valsalva .Evaluation of the heart rate response for assessment of individuals autonomic response to the Valsalva was not possible due to the fixed paced cycle length. It has also been documented that heart failure patients may show an altered blood pressure response to the Valsalva without a fall in blood pressure during the strain phase known as a “square wave” blood pressure response  vs. non-ischaemic cardiomyopathy (NICM) (n = 5) was similar. There was no observed difference in resting SDARI , nor SDARI post Valsalva  between patients with ICM vs. NICM.The behavior of SDARI in patients with ischaemic cardiomyopathy (ICM) (p = 0.473). This excluded the possibility of low frequency respiration producing associated low frequency changes in blood pressure and ARI at rest and post Valsalva.The respiratory frequency at rest (0.28 ± 0.08 Hz) and following termination of the Valsalva (0.26 ± 0.11 Hz) was similar . When normalized the mean LF power of ARI continued to demonstrate a significant increase during the 60 s following the Valsalva when compared to control (p = 0.019). Figure p = 0.365), nor in the normalized LF power of AT-AT intervals .Mean spectral energy measurements of ARI, SBP, and dP/dtmax significantly increased following the Valsalva (p = 0.004). Figures max during control and following the Valsalva.The Valsalva produced the same modulation of spectral energy measurements of blood pressure with that seen in ARI. Mean LF and nLF power of SBP and dP/dt2, p = 0.247), nor LF-ARI post Valsalva  between patients with ICM vs. NICM.The behavior of LF-ARI in patients with ICM vs. NICM was similar. There was no observed difference in resting LF-ARI   and between LF-ARI and LF-dP/dtmax . There was a strong positive correlation between SDARI and LF-ARI  .A very strong positive correlation was found between LF-SBP and LF-dP/dt) Figure . A positmax of systolic pressure, (2) the low frequency power of APD increased also associated with an increase in the low frequency power of SBP and dP/dtmax, (3) the increase in beat-to-beat APD variability correlated with increasing low frequency power of APD.We have investigated the oscillatory behavior of ventricular repolarization in the low frequency range (0.04–0.15 Hz) in response to a standard autonomic challenge . ARIs as a conventional surrogate for APD were recorded from the left ventricular epicardial lead of an implanted biventricular pacing device while pacing from the right ventricular lead. The main findings were: (1) BBV of APD (ARI) increased associated with an increase in BBV of SBP and dP/dtSympathetic nerve activity is organized in bursts in a range of frequencies including the so called low frequency (LF) range in the region of 0.04–0.15 Hz, i.e., approximately one every 10 s in humans . Coherence analysis suggested an interaction between the low frequency oscillatory behavior of APD and the BBV. These observations provide insight at the level of the ventricular action potential into mechanisms underlying low frequency oscillation of repolarization derived from the ECG T-wave which have been shown to be exaggerated by sympathetic stimulation and are strongly predictive of sudden cardiac death in post MI patients. Further work aimed at unraveling mechanisms at the cellular level may help to elucidate the link to arrhythmogenesis and possibly point to therapeutic targets.BP, SvD, MB, JG, and PT conceived and designed the experiments. All authors took responsibility in collecting, analyzing, and interpreting the data. All authors contributed to drafting the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine the accuracy of peripheral  arterial access as compared to central  arterial access for measurement of invasive blood pressure (IBP) in critically ill patients after cardiopulmonary bypass.nd attempt were included in this study. The duration of this study was from June 2015 to August 2016. Radial and femoral arterial access was achieved in all patients for simultaneous measurement of blood pressure. Arterial pressures were noted after 5, 15 and 30 minutes of weaning from cardiopulmonary bypass for both radial and femoral artery simultaneously.Sixty patients (60) who required high inotropic/vasopressor support on weaning from cardio-pulmonary bypass and weaned off in 2Mean age of study patients was 56.48±11.17 years. 85% patients were male. There was significant difference in systolic blood pressure, diastolic blood pressure and mean arterial pressures between the radial artery and femoral artery cannulation. Mean arterial pressures were significantly high in femoral artery as compared to the radial artery. The mean arterial pressures after five minutes of weaning using central access were 76.28±10.21 mmHg versus 64.15±6.76 mmHg in peripheral arterial access . Similarly we also found significant difference in mean arterial pressures after 15 minutes of weaning from cardiopulmonary bypass 78.70±10.12 mmHg in central access versus 72.03±6.76 mmHg using peripheral arterial access . The difference in arterial pressures were less marked as compared to the previous differences after 30 minutes of weaning from cardiopulmonary bypass as compared to the earlier readings .Peripheral arterial pressures are unreliable in critically ill patients after cardiopulmonary bypass receiving high dose of inotropic drugs. Central arterial access should be used in these patients to get accurate estimates of patients’ blood pressure in early periods after cardiopulmonary bypass. Monitoring of arterial blood pressure is very important for the evaluation of hemodynamic measurements as it gives a mandatory information about cardiovascular performance and hence tissue perfusion.nd most commonly used artery.Radial artery cannulation is used in about 92.0% cases and femoral artery cannulation is the 2nd attempt from CPB were included in this study. The duration of this study was from June 2015 to August 2016. The following criteria was used for high inotropic support; epinephrine or norepinephrine >0.1 μg/kg/minute or dobutamine >10 μg/kg/minute. Patients requiring mild to moderate or no inotropic support on weaning from cardiopulmonary bypass and patients who required inotropic support before cardiopulmonary bypass were excluded. Ethical approval was taken before starting the research work.This prospective study was conducted in Cardiac surgery unit of CPE Institute of Cardiology Multan. Sixty patients (60) who required high inotropic/vasopressor support on weaning from cardio-pulmonary bypass (CPB) and weaned off in 2st performing the Allen’s test. On weaning from cardiopulmonary bypass if patient required high inotropic support then femoral artery of the opposite leg of radial artery before weaning the patient from cardiopulmonary bypass. Right femoral artery was cannulated using 16 G femoral arterial catheter . Separate transducers were used for measurement of radial and femoral artery pressures simultaneously. The position of both transducers were set at the level of the right atrium and zeroed to atmospheric pressure before connecting with the arterial line. The whole system was flushed with normal saline to remove air and to check any leaks at connections. Rapid flush test was used to check damping co-efficient, kinking of arterial line, and mal-positioning of the catheters. The transducers were connected to the monitor for continuous monitoring of the arterial pressures.In all patients radial artery was cannulated using a standard 20 G radial artery catheter  for measurement of Invasive blood pressure during surgery. Radial artery of non-dominant hand was selected by 1Arterial blood pressure was noted for both radial and femoral artery immediately after 5, 15 and 30 minutes of weaning from cardiopulmonary bypass. Systolic arterial pressure, diastolic arterial pressure and mean arterial pressures were noted at these time intervals.Data were analyzed using SPSS v23 software. Independent sample t-test statistics were used for comparison of arterial pressures taking p-value <0.05 as significant difference. Repeated measurement ANOVA was used to compare difference in mean arterial pressures in femoral and radial line for different time intervals.Sixty (60) patients who required high inotropic support on weaning from cardiopulmonary bypass were selected. Mean age of study patients was 56.48±11.17 years and 85% patients were male. There were 90% patients who underwent coronary artery bypass grafting and only 10% patients underwent valvular heart surgery. Mean cardiopulmonary bypass time was 124.6±22.7 minutes .There was a significant difference in systolic blood pressure, diastolic blood pressure and mean arterial pressures between the radial artery and femoral artery cannulation after 5, 15 and 30 minutes of weaning from CPB. The mean arterial pressures after five minutes of weaning using femoral access were 76.28±10.21 mmHg versus 64.15±6.76 mmHg in radial arterial access . Similarly we also found significant difference in mean arterial pressures after 15 minutes of weaning from cardiopulmonary bypass 78.70±10.12 mmHg in femoral access versus 72.03±6.76 mmHg using peripheral arterial access . There was also a significant difference between mean arterial pressures in radial and femoral artery, but this difference was less marked as compared to the previous ones .Similar trends were seen in systolic blood pressure of patients at femoral and radial artery, SBP were significantly high in femoral artery as compared to the radial artery . The difWe concluded from the results of our study that femoral artery cannulation gives more accurate pressures recordings as compared to the radial artery cannulation in patients who become critically ill after cardiac surgery and require high inotropic/vasopressor support on weaning from cardiopulmonary bypass. On the contrary, O’Rouke et al. concluded that there is no difference in MAPs measured simultaneously in radial artery and the aorta in healthy volunteers.14Chauhan and colleagues recommended that femoral arterial pressures are more accurate and reliable during cardiopulmonary bypass and monitoring of femoral arterial pressures results in lower doses of vasoactive drugs during CPB.15Kim et al.19Fuda et al.Some other studies have concluded that arterial pressure gradients after cardiac surgery are associated with body temperature, bypass time and catecholamine levels.27In our study, there was significant difference in mean arterial pressures between the peripheral and central arterial access. Similar trends were seen in systolic blood pressure and diastolic blood pressure in central versus peripheral access. There was a significant reduction in difference in mean arterial pressures between the femoral and radial artery with the passage of time and after 30 minutes of CPB this difference was only of 5 mmHg. So we concluded that peripheral arterial pressures are unreliable in critically sick patients after cardiopulmonary bypass receiving high dose of inotropic drugs. Central arterial access  should be used in these patients to get accurate estimates of patients’ blood pressure in early period after cardiopulmonary bypass.Peripheral arterial pressures are unreliable in critically sick patients after cardiopulmonary bypass receiving high dose of inotropic drugs. Central arterial access should be used in these patients to get accurate estimates of patients’ blood pressure in early period after cardiopulmonary bypass.RAA: Conceived, designed the research methodology and supervion of research work, and is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. SA: Did data collection, helped in drafting and review the manuscript. ANJ: Drafted the manuscript and gave final approval of the manuscript to be published. MARB: Did data collection, analysis and interpretetion of results.


The NLR resulted the best discriminating variable for the occurrence of the adverse outcome . The NLR predicted ND after acute ICH and can aid in the risk stratification of patients.Immunity plays key roles in pathophysiology of intracerebral hemorrhage (ICH). The aim of the study was to determine whether the peripheral leukocyte count and neutrophil-to-lymphocyte ratio (NLR) predicted neurological deterioration (ND) after ICH. We identified consecutive patients with ICH who had blood sampling performed within 24 hours from symptom's onset. Total white blood cells (WBC), absolute neutrophil count (ANC) and absolute lymphocyte count (ALC) were retrieved, and the NLR computed as the ratio of the ANC to ALC values. The study endpoint was the occurrence of neurological deterioration (ND) within 7 days after ICH. One hundred ninety-two subjects were enrolled, whose 54 (28.1%) presented ND. At multivariate analysis, the WBC (adjusted odd ratio [ Spontaneous intracerebral haemorrhage (ICH) represents approximately 10% to 15% of all strokes and affects over 1 million people per year worldwide. It is characterized by high rates of mortality and residual disability among survivors, and currently no therapeutic strategies have demonstrated definitive benefit ; notwithImmune reaction is a major feature of ICH pathology and influences its course; the response to cerebral hematoma is not bounded to the brain and results in systemic effects, and inflammatory markers on admission, such as fever, elevated leukocyte count, interleukin-6 and C-reactive protein are associated with worse prognosis –5. The np < 0.001), higher ANC , lower ALC , and higher NLR  compared to those who did not experience ND ; the WBC, ANC and ALC were weakly to moderately correlated to the baseline ICH volume  and initial NIHSS score . After logistic regression analysis, the WBC, ANC, ALC and NLR were all significantly associated (p≤0.001) with ND  presented ND during the first week after ICH onset; univariate comparisons of baseline characteristics between the outcome patient groups are summarized in Table The main results of our study are that higher neutrophils and lower lymphocytes upon admission independently predicted ND during the initial week after ICH onset, and the NLR represented a reliable predictive biomarker.Inflammation takes place soon after the ICH and in its early stage is mainly sustained by polimorphonuclear activation. Neutrophils represent the first leukocytes to actively migrate since the first hours after ICH from peripheral blood into the brain, and promote secondary injury. The infiltrating leukocytes release inflammatory and cytotoxic mediators which enhance the peri-lesional edema by favoring capillary permeability, cell swelling and blood-brain barrier damage , 9. The Lymphocytes are major regulators of immunity and their reduction and functional deactivation are signatures of brain damage which occur as early as 12 hours after stroke as an effect of hyperactivity of sympathetic nervous system and hypothalamic-pituitary adrenal axis , 14. In-The NLR was independently associated to the occurrence of ND and outperformed the leukocytes counts as predictors of clinical worsening. The NLR is a composite inflammatory index that integrates information on the innate and adaptive pathways. In acute ICH, it could represent a surrogate systemic bio-marker of degree and direction of the immune response to hematoma. Despite obvious limits for any time-based classification, the drivers of ND are heterogeneous and both intra-cerebral and systemic conditions play different roles according to the worsening time-point . Blood aThe study should be interpreted in the light of some limitations. The retrospective design and the performance of all testing as for real-word practice and physician's judgment allowed to describe associations and raise working hypotheses. The relationships between immune response and ND can be definitively understood only through prospective studies based upon pre-specified time points for blood sampling and imaging. Further, findings could not be extended to hyper-acute clinical worsening according to study protocol and inclusion of patients up to 24 hours post stroke. The main study strengths allowed generalizability of results to everyday clinical setting and included the enrollment of patients regardless of ICH location, the wide availability of all laboratory variables, and the cost-effectiveness of the NLR as a readily accessible biomarker.In conclusion, our findings suggested a simple, inexpensive and easily available tool to early identify patients at increased risk of clinical deterioration. Neurological stability across the initial week after stroke onset translates into low risks of long-term morbidity and mortality , and theWe retrospectively identified consecutive patients hospitalized at the Stroke Unit of the Marche Polytechnic University, Ancona, Italy from January 2008 to July 2016 for stroke syndrome due to acute spontaneous ICH who underwent admission routine blood sampling and cranial CT neuroimaging within 24 hours from symptom onset. Demographics, medical history, Glasgow Coma Scale (GCS) and National Institutes of Health Stroke Scale (NIHSS)  scores aThe outcome measure was the occurrence of neurological deterioration (ND) defined as a 4 point or greater increase in the NIHSS score or 2 point or greater decrease in the GCS or death from the time of admission to 7 days post-hemorrhage .Patients presenting with isolated intraventricular hemorrhage, hemorrhage secondary to brain tumor, dural venous sinus thrombosis, ruptured arteriovenous malformation or aneurysm and patients receiving immunomodulatory treatment  before admission were not considered for the study.p values < 0.05 from comparison of baseline characteristics and selected variables  ) for continuous variables and as the number (percent) of subjects for categorical variables. Comparisons were made through the Student t test, Mann-Whitney test or Chi-squared test as appropriate. Spearman correlation was used to correlate continuous variables. The associations between the WBC, ANC, ALC, NLR and the study endpoint were determined using logistic regression models; the variables with  of ICH)  were for


Intravenous administration of FDG is sometimes halted due to troubles. In such cases, estimations of the FDG dosage injected prior to halting administration may be helpful. We have established a method of estimating the activity of FDG to patients on the basis of the dose equivalent rate on the surface of the right temporal region of the head. The correlation of actual administered dosage with independent variables, such as the dose equivalent rate on the right temporal region of the head, age, sex, and body weight, was analyzed using multiple regression analysis to obtain linear, quadratic, and cubic regression equations.Positron emission tomography (PET) with When entering independent variables, the cubic regression equation could be used to estimate an administered dosage with an accuracy of ±10 % for 62 % of all patients and ±20 % for 90 % of all patients.We conclude that this method is useful for estimating the administered dosage from the dose equivalent rate on the temporal region of the head. Intravepatients –6. Thus,patients . In thisSeven hundred six patients underwent PET examination in our site during the period between the 9th of March 2015 and the 23rd of May 2015. Of these, 520 patients were prospectively enrolled in this study with Institutional Review Board approval. The requirement to obtain informed consent was waived. The remaining 180 patients were not included in this study because of refusal to be involved, unfavorable physical condition, or incomplete records.Dosage calculation in our site:where Int is the function of “round down to the nearest decimal.” For example, for a 69-kg patient, 180 MBq is administered using an FDG automated injector . After FDG administration, using an FDG automated injector, the dose equivalent rate on the right temporal lobe (DER) was measured with the patient sitting upright using an ionization chamber type survey meter . No patients voided their bladders between injection and dose-rate measurement. The right temporal lobe was chosen based on patient comfort and the ability to conduct stable measurements. DER, administered dosage, and elapsed time following FDG administration were recorded, as well as age, sex, height, weight, and blood sugar. There are two reasons for having adopted the elapsed time following FDG administration as a dependent variable, the first is to reflect the physical decay and the second is to follow time-dependent uptake of the tracer in the brain. All data were recorded by six nurses working in our PET institution.This study was performed on a presupposition that DER should be in proportion to AD. However, DER/AD  is not completely a constant value. DER/AD is influenced by patients’ characteristics, such as sex, elapsed time following administration, patient age, height, weight, and blood sugar. Therefore, to achieve the presuppositions, DER had to be converted to corrected DER (CDER), which was unaffected by the influence of patients’ characteristics. The process of obtaining CDER is demonstrated as follows. IBM SPSS statistics  was employed for analysis and calculation of multiple regression analysis.Figure And, corrected DER (CDER) is given by the equation:In Fig. Here, it should be noted that weight, height, and BMI exhibit multicollinearity. Therefore, the most appropriate variable, of the three, was determined using the Pearson test. The variable with the highest Pearson correlation coefficient was determined to be the most important independent variable.Next, an estimation of the administered dosage was calculated using CDER by linear, quadratic, and cubic regression analyses.L(1), Q(1), Q(2), C(1), C(2), and C(3) are regression coefficients, and the intercept is zero.where Finally, the accuracy  of the regression equations was calculated.p values for DER, DER/AD, height, and others variables. Because the p values for DER and DER/AD were higher than 0.05, the null hypothesis was not rejected, indicating that the DER and DER/AD data were normally distributed and that the independent variables were appropriate for calculation of CDER with stepwise multiple regression analysis.There were no significant differences in DER/AD among the nurses (data now shown). Descriptive statistics and tests of normality, with the Shapiro-Wilk test results for each of the dependent and independent variables, are shown in Tables The bivariate Pearson correlation coefficients for height, weight, and BMI  are shown in Table R-square.Table Figure X-axis and administered dosage on the Y-axis. Table R-square for linear, quadratic, and cubic equations is 0.664 (moderate correlation), 0.744 (strong correlation), and 0.747 (strong correlation), respectively.Figure Figure We have described a method to estimate the actual dosage of FDG administered using corrected DER and four independent variables of sex, weight, blood sugar, and elapsed time following administration in a cubic regression equation obtained by analyzing 520 patients. The ratio of DER over actual administered dosage becomes practically constant when we correct DER using the independent variables, as indicated, by stepwise multiple regression analysis. This method is useful for calculating the radioactivity of FDG administered to patients when administration must be halted for any reason. However, it does not necessarily give a precise value of administered dosage but just an estimate. Care should be taken when an administered dosage is estimated by our method and it may affect the diagnostic interpretation or quantification of FDG accumulation. Of the four independent variables, the influence of blood sugar was less than weight and elapsed time following administration in stepwise multiple regression analysis. This is interesting because usually blood sugar affects brain uptake of FDG , 9. One Because the proposed estimation method makes use of dose equivalent rates on the surface of the right temporal lobe, other factors, that may affect brain uptake of FDG and, consequently, the accuracy of the method, should be considered. Diversion of FDG to large tumors, leading to decreased brain uptake, has already been reported in malignancies including lymphoma . In thisOne limitation of this study is that incomplete injection of FDG, in patients, was not examined, which means that the in vivo predictive power of this estimation method has not been verified. Of particular concern is the case of extravasation, where measurement of the actual radioactivity of FDG injected into the circulation is difficult to determine because FDG could be absorbed by subcutaneous tissue or vascular tissue. DER is most likely associated with radioactivity injected into the circulation. In time, the accuracy of our proposed estimation method can be verified using data accumulated from cases in which DER, the independent variables, and quantitative image data are all known. Another limitation is that this study was conducted at only one facility. To establish such regression approaches, including populations from multiple sites may be crucial to enhance generic applicability and wide clinical adoption. Further, multi-center studies are needed to determine the viability of our estimation method in PET facilities with various clinical circumstances.The actual administered dosage of FDG in patients was successfully estimated on the basis of dose the equivalent rate using multiple regression analysis with four independent variables of sex, weight, blood sugar, and elapsed time following administration. We propose using this method to determine the radioactivity of FDG administered to patients in cases of incomplete injection.


In this study, we determined the relationship between the ambulatory arterial stiffness index (AASI) and clinical and laboratory parameters in patients with acromegaly.Sixty-five patients with acromegaly, who visited to Dicle University Medical Faculty Department of Endocrinology , were included in this study. The study control group consisted of 65 subjects. Demographic and clinical data were recorded. Laboratory data  performed over the last year were evaluated. The AASI was obtained from 24-hour ambulatory blood pressure monitoring records of all patients. This study was completed in 15 months from 2013 to 2015.Twelve patients (18.4%) had diabetes and 21 patients (32%) had hypertension. The mean AASI value was 0.41 ± 0.14. The mean AASI value in the control group was 0.25 ± 0.09. Growth hormone (GH) levels were positively correlated with the AASI values. AASI values tended to be higher in hypertensive subjects than that in normotensive individuals.Our results show that the AASI value increased in patients with acromegaly, independent of the increase in blood pressure. The AASI was strongly dependent on the degree of the GH increase in patients with acromegaly and may have an important role predicting cardiovascular risk in patients with acromegaly. Acromegaly is a clinical syndrome that develops due to excess release of growth hormone (GH) with an annual incidence of 3–4/1,000,000. The majority of cases (95%) are due to excessive GH secretion from the anterior pituitary.2Interest in the cardiovascular complications of patients with acromegaly has increased. Cardiac complications include decreased diastolic function with exercise at early and late stages; and cardiomyopathy characterised by cardiac output and advanced congestive heart failure along with dilated cardiomyopathy at later stages.5Over the last decade, cardiovascular research has focused on “arteriosclerosis”, which is a common state of artery hardening in contrast with atherosclerosis, which involves local activity. Influenced by various factors, such as age and high blood pressure, an intense restructuring process takes place in the viscoelastic properties of the large vessel walls, resulting in arteriosclerosis characterised by the formation of hardened arteries, i.e., reduced arterial elasticity or compliance.6As the arteries harden, their width increases and the arterial wall thickens, which is manifested as increased systolic blood pressure (SBP), decreased diastolic blood pressure (DBP), and expanded pulse pressure. These three changes are the main determinants of cardiovascular morbidity and mortality.6Although several measurement methods are currently used to determine arterial stiffness, which is accepted as a strong indication of cardiovascular complications, the challenges and limitations of this method remain controversial.8A new easy-to-use index has recently been recommended as an indicator of arterial stiffness , which can be obtained from arterial blood pressure records and is easy to calculate. The ambulatory arterial stiffness index (AASI) is strongly correlated with conventional measurement methods of arterial stiffness, such as pulse wave velocity (PWV) and the augmentation index (Aix), and provides prognostic information on cardiovascular mortality.10All patients diagnosed with acromegaly and under supervision of the Endocrinology and Metabolic Diseases Clinic at Dicle University Medical Faculty Hospital were included in this study. The patients provided written informed consent. This study was approved by the ethics committee of medical research at the university. None of the patients enrolled in the study had advanced cardiovascular or metabolic diseases that could adversely affect the course of the study or the results. The study groups consisted of patients with acromegaly who were hypertensive and normotensive, patients with acromegaly with and without diabetes, patients in and out of remission with acromegaly, and controls. Age, gender, and any previous chronic diseases were recorded. In concordance with the number of patients, 65 subjects were included in the study as controls. The control group was selected among those whose age and gender distribution and metabolic status were close to the enrolled patients without any advanced chronic disease or any history of drug usage that could adversely affect the present study.In the endocrinology clinic, the patients were subjected to treatment and follow-up at regular intervals of 1–3 months depending on disease severity. During these check-ups, the patients were requested to provide the relevant examinations, such as GH, insulin-like growth factor-1 (IGF-1), and the 75-g of oral glucose tolerance test (OGTT), a blood glucose-loading test for disease progression and regression, plasma glucose, liver and kidney function tests, and glycated haemoglobin in patients with diabetes.Blood pressure was measured in all patients in an office setting before the start of the ambulatory blood pressure monitoring (ABPM). The ABPM was carried out using a non-invasive blood pressure monitoring device based on an oscillometric method for 24 hour . After fitting the device, the patients were allowed to return to their daily routine. The device was programmed to take a blood pressure measurement at 60 minutes intervals. Thus, SBP, DBP, and heart rate were obtained an average of 24 times/day in all patients.A technically valid reading of at least 80% was required during all registration periods for the operation to be considered successful and this was achieved in all patients. The individual ambulatory AASI values of the patients were calculated from ambulatory blood pressure levels over 24 h using statistical methods. During the index calculations, SBP and DBP from the patients were entered on an individual basis and measured separately.The slope of the regression curve obtained from marking the SBP and DBP values from 24- hour blood pressure records of each patient on a  plane was calculated. The AASI was calculated by the formula: 1 − regression slope. As the stiffness of the arterial system increased, the slope of regression gets closer to zero and the AASI gets closer to 1.t-test and the Mann–Whitney U-test were used to compare variables with and without a normal distribution, respectively. The relationship between continuous variables was tested by Pearson's correlation analysis, while Spearman's correlation analysis was used to test the relationship between non-parametric variables. This study was completed in 15 months from 2013 to 2015.The statistical evaluation was performed using SPSS 16.0 software . A p-value < 0.05 was considered significant. The results are expressed as the mean ± standard deviation and percentage (%). Student's The study included 65 patients, of which 33 were women (50.8%) and 32 were men (49.2%). The mean age of the patients was 43.10 ± 12.2 years. Twelve patients had (18.4%) diabetes mellitus, and 21 patients (32%) had hypertension. The mean age of the control group was 45.14 ± 16.30 years .The mean and median AASI values of patients were 0.41 ± 0.17 and 0, respectively. The mean AASI value of the control group was 0.24 ± 0.09. The mean AASI value of the patient group (0.41 ± 0.14) was significantly higher than that of the control group (0.24 ± 0.09) (p < 0.001).A significant positive correlation was detected between the basal GH level and AASI in patients with acromegaly . A significant relationship was observed between the AASI and GH level at 90 and 120 minutes of the OGTT .No significant difference was found between the genders in patients with acromegaly in terms of the AASI . No correlation was observed between the AASI and age in patients with acromegaly .The AASI value tended to increase with the increase in IGF-1 level . The AASI values were not different between hypertensive patients (0.42 ± 0.16) and normotensive patients (0.40 ± 0.140) with acromegaly (p = 0.016).Of the 65 patients with acromegaly enrolled in this study, 12 had diabetes mellitus with an AASI value of 0.34 ± 0.11. No significant difference was found with the non-diabetic group.Of the 65 patients, acromegaly was under control in eight patients; however, 57 patients with acromegaly were actively being treated. The AASI values were 0.34 ± 0.07 and 0.42 ± 0.16 in the non-cured group and the group with active disease, respectively (p = 0.28).Acromegaly is associated with an increased risk of premature mortality. Cardiovascular diseases are the most important cause of the increased mortality in this patient group. Nearly all patients with cardiac disease at the acromegaly diagnosis die within 15 years.14The prevalence of hypertension was 30% in two large series. Variability in the results may depend on differences in patient selection or technical and environmental factors affecting the blood pressure values during clinical measurements. In a study that investigated the prevalence of hypertension in 40 patients with acromegaly using 24-hour ABPM, the prevalence of hypertension was 42.5% by clinical measurement and 17.5% by the ABPM technique.Some studies have evaluated arterial stiffness using the AASI, a new index obtained from 24-hour blood pressure records, and it was deemed to be more practical than other measuring methods, which require specialised equipment, and the results were similar and consistent.18Regression curves for blood pressure have been produced by marking DBP corresponding to SBP values of each individual. Accordingly, they obtained AASI values by extracting the slope of the regression curve from 1.The mean AASI value obtained from the 24-hour ABPM in our patient group was 0.41 ± 0.14. This value was similar to that reported [0.41 (0.15–0.68)] from a study conducted in 11,291 subjects, which included patients with hypertension, diabetes, and cardiovascular disease.Aortic and carotid artery stiffness measured by PWV increases by approximately 10–15% per decade with age across this population. It has also been suggested that arterial stiffness in women is 5–10% lower compared with that of men of the same age.In a study with 11,291 subjects, both the AASI and pulse pressure were higher in women than men, which could have been due to the shorter average stature of women.Dassia et al. reported an assessment of cardiovascular risk factors and 24-hour ABPM in 96 patients with active acromegaly . Based on the ABPM measurements, the patients with acromegaly were categorised as normotensive (64 patients) and hypertensive (32 people). Moreover, they were compared to 69 controls consisting of 35 normotensive and 34 hypertensive subjects. Consequently, the AASI values from the patients with acromegaly were higher than those of the controls (p < 0.001). The AASI values of the patients with acromegaly and hypertension were higher compared with those of normotensive subjects (p = 0.01).A multiple logistic regression analysis demonstrated a significant correlation between the highest AASI value and serum IGF-1 (p = 0.034) across all acromegaly groups.25In our study, the mean AASI in patients with acromegaly (0.41 ± 0.14) was significantly higher than that in the control group (0.24 ± 0.09) (p < 0.001). The AASI values of patients with acromegaly with (21 patients) and without (44 patients) a history of hypertension were 0.42 ± 0.16 and 0.40 ± 0.14, respectively. However, no significant difference (p = 0.016) was found. The AASI values tended to increase with the increase in IGF-1. GH levels increased significantly along with the AASI values.A significant correlation was found between the AASI values and GH levels measured 90 and 120 minutes after performing the 75-g OGTT in patients with acromegaly. As a result, the values obtained at 90 and 120 minutes could be more significant for predicting the risk of cardiovascular disease.Similarly, in our study, an increase in the AASI was observed corresponding to an increase in basal GH levels in patients with acromegaly, suggesting the likelihood of increased cardiovascular morbidity and mortality.Dolan et al. demonstrated in their prospective study that AASI strongly predicts cardiovascular mortality in both hypertensive and normotensive patients. They indicated that the AASI could vary considerably, similar to 24-hour ABPM, but could provide additional hemodynamic information. They asserted that the AASI was a significant indicator of fatal stroke, particularly in young normotensive subjects. Thus, higher AASI values can also be used to predict cerebrovascular mortality.19In their subsequent study, they suggested that the AASI was highly correlated with aortic PWV as well as with central and peripheral Aix, suggesting a novel method for measuring arterial stiffness. Again, this study demonstrated that AASI value was directly proportional to age and mean arterial pressure, but inversely proportional to height, and that AASI values in both women and hypertensive subjects were significantly higher than those in men and normotensive subjects, respectively, if the other variables were held constant.Our study shows that the AASI value increased in patients with acromegaly, independent of the increase in blood pressure. The AASI was strongly dependent on the degree of increase of GH in patients with acromegaly and may have an important role predicting cardiovascular risk in this patient population.Author's Contribution:FK and AKT: Designed and performed the study.FK and ZP: Did data collection and writing of manuscript.FD and NG: Did statistical analysis and editing of manuscript.


The INVEST  study, a very large international study of hypertensive patients, has shown that patients with type 2 diabetes and coronary artery disease do not benefit from tightening systolic blood pressure levels to below 130 mmHg, and this may in some cases be harmful.INVEST, initiated in the mid-nineties, continues to contribute to the improved management of patients with CAD and this latest evaluation of the 6 400 patients with diabetes and CAD included in the INVEST cohort is the first study to critically evaluate the effects of systolic blood pressure lowering in patients with both diabetes and documented CAD.These results are likely to require guideline committees, including the local SEMDSA’s guideline committee, to include a warning that blood pressure need not be driven below 130 mmHg in diabetic patients as this does not result in any further cardiovascular benefit.This finding is particularly pertinent to drug selection and the use of verapamil SR/trandolapril, which was successful with more than 70% of INVEST patients reaching the target blood pressure of less than 140/90 mmHg. The verapamil SR/ trandolapril therapy group also experienced significantly fewer cases of newonset diabetes than those patients treated with atenolol/hydrochlorothiazide.‘Current guidelines suggest “lower is better” with regard to blood pressure’, said Rhonda M Cooper-DeHoff, PharmD, MS, and associate professor of pharmacy and medicine at the University of Florida, Gainesville. ‘The INVEST data suggest that in patients with both diabetes and coronary artery disease, there is a blood pressure threshold below which cardiovascular risk increases’.For the study, INVEST randomly assigned 6 400 patients with diabetes and CAD to blood pressure-lowering therapy based on either a calcium channel blocker or a beta-blocker, plus an angiotensin converting enzyme (ACE) inhibitor and/ or a thiazide diuretic. The target was a blood pressure of < 130/< 85 mmHg.For the analysis, patients were categorised according to the degree of blood pressure control actually achieved. Patients with a systolic blood pressure of 140 mmHg or higher, almost onethird of patients, were classified as ‘not controlled’. Those with a systolic blood pressure below 130 mmHg were classified as ‘tight control’ and those with a systolic blood pressure in between  were classified as ‘usual control’.During a follow-up period equivalent to more than 16 893 patient-years, researchers found that patients in the notcontrolled group had nearly a 50% higher combined risk of death, heart attack or stroke when compared with the usual-care group. However, those in the tight-control group had a similar risk to those in the usual-control group.Further analysis showed that lowering systolic blood pressure below 130 mmHg significantly increased the risk of allcause death when compared to usual care, an increase that became apparent about 30 months into the study and persisted for an additional five years of follow up. When researchers then analysed blood pressure in 5-mmHg increments in the tight-control group, they discovered that a systolic blood pressure below 115 mmHg was associated with increased mortality.‘Diabetic patients with CAD in whom blood pressure is not controlled have increased risk for unfavourable cardiovascular outcomes, so the message to lower systolic blood pressure below 140 mmHg is still important’, Cooper-DeHoff said. ‘However, it is not necessary to lower systolic blood pressure below 130 mmHg to reduce that risk. Most importantly, reducing systolic blood pressure below 115 mmHg may be associated with increased mortality.’


Markers of imaging, structure, and function reflecting vascular damage, integrating a long time accumulation effect of traditional and unrecognized cardiovascular risk factors, can be regarded as surrogate endpoints of target organ damage before the occurrence of clinical events. Prevention of cardiovascular disease requires risk stratification and treatment of traditional risk factors, such as smoking, hypertension, hyperlipidemia, and diabetes. However, traditional risk stratification is not sufficient to provide accurate assessment of future cardiovascular events. Therefore, vascular injury related parameters obtained by ultrasound or other noninvasive devices, as a surrogate parameter of subclinical cardiovascular disease, can improve cardiovascular risk assessment and optimize the preventive treatment strategy. Thus, we will summarize the research progress and clinical application of early assessment technology of vascular diseases in the present review. In the year of 2010, a concept of “ideal cardiovascular health” was put forward, including ideal healthy behavior, such as status of smoking, body mass index, physical activity and diet, and ideal healthy factors, such as blood pressure, blood glucose, and blood lipids . TherefoVascular endothelial cells as a layer of single cell interface between blood flow and vessel wall, responding to the stimulation of body fluid, the nerve, especially the hemodynamics, can regulate vascular tone by the synthesis and release of vasoactive substances, regulate platelet function, inflammatory response, and vascular smooth muscle cell growth and migration, and play an important role in the pathogenesis of vascular diseases , 5. VascAcetylcholine stimulation by coronary angiography is the traditional method of vascular endothelial function detection . It is tDoppler ultrasound equipment was used to measure the baseline and cuff released diameter of brachial artery, and FMD was calculated as the rate of brachial artery diameter changes. It reflects the flow-mediated endothelial nitric oxide (NO) release function and is considered as a commonly used noninvasive method. The method is simple, noninvasive, economical and accurate, reproducible, and clinically beneficial for the promotion of early vascular lesion detection . FMD as FMD reflects NO-mediated function of conduit brachial artery and is measured by Doppler ultrasound apparatus. Measurement of digital pulse amplitude tonometry (PAT) is based on a technique similar to that of FMD. It uses photoplethysmography to measure pulse wave amplitude (PWA) in the fingers reflecting small vessel microcirculation function and gets the reactive hyperemia index (RHI). RHI is defined as the ratio of postdeflation to baseline pulse amplitude in the hyperemic finger divided by that in the contra-lateral finger . StudiesBoth brachial FMD and RHI are independent predictors of cardiovascular events and all-cause mortality and both with similar prognostic magnitude , 18. RHITherefore, whether the prognostic values of these 2 methods are independent of each other and whether an endothelial function-guided strategy can provide benefit in improving cardiovascular outcomes need further research to determine .Arterial stiffness is mainly determined by larger arteries, because the thoracic aorta and abdominal aorta contribute the greatest to arterial buffering function. Elastic properties of conduit arteries vary along the arterial tree, with proximal arteries being more elastic and distal ones stiffer. In addition, aging and blood pressure are the major determinants of arterial stiffness, which causes a decrease in elastic protein synthesis and an increase in degradation, while the synthesis of type 3 and type 1 collagen is increased and the degradation is reduced . StudiesPWV is a common indicator of arterial stiffness and can be obtained by measurement of distance and pulse wave transit time between two points of vessels . It can β and can be calculated by the electrocardiogram, phonocardiogram, and brachial ankle artery pulse waveform and pulse waveform records and is mainly related to aortic stiffness and compliance [CAVI is a new evaluation index of arterial stiffness in recent years and can also be measured by means of arteriosclerosis detection device. Compared with PWV, CAVI does not depend on the immediate effect of blood pressure. CAVI comes from the stiffness coefficient mpliance . A seriempliance –35. In ampliance , and CAVmpliance . In addimpliance .Central aortic pressure (CAP) can be measured by invasive cardiac catheterization and noninvasive applanation tonometry of the radial or carotid artery , 40. CAPAugmentation index (AI) was obtained from the synthesized aortic pressure waveform . AI, meaPulse pressure (PP), with simple and convenient measurement, is widely used as roughly reflection of arterial stiffness, and increased PP showed increased arterial stiffness .The large artery elasticity index (C1) and small artery elasticity index (C2) obtained by the pulse waveform analysis of the radial artery are two of the parameters of arterial elasticity, getting through the analysis of the radial artery diastolic blood pressure pulse waveform. Studies suggested that C1 and C2 were better than the traditional blood pressure detection for clinical application of early recognition of the cardiovascular disease , and C1 Most importantly, the common limitation of CAP, AI, C1, or C2 is the lack of reference values and adequate evidence to date, therefore limiting the current widely clinical application.ABI concept was proposed by Winsor in the 50s in twentieth Century, which was originally proposed as a noninvasive diagnostic technique for peripheral arterial disease (PAD) . In 1982Ultrasound imaging technique as a noninvasive detection for clinical evaluation of carotid plaque (CP), carotid stenosis, and carotid artery intima-media thickness carotid (CIMT) is widely used. Ultrasound detection of carotid atherosclerotic lesions can be used as the observation window of the whole vascular bed atherosclerosis and can observe early atherosclerotic lesions dynamically. Its operating convenience, low cost, and good reproducibility made it a noninvasive screening of some high-risk and asymptomatic vascular disease patients . AlthougWhether CIMT and CP are distinct phenotypes or represent a different stage of atherosclerotic development is unclear. Some studies indicated that common CIMT can independently predict CP occurrence and suggested that increased CIMT might occur in an earlier phase of the atherosclerotic process , 69. HowAn article summarizing studies on CIMT and CP showed that CP was a better marker of CVD risk than CIMT and combined CIMT with CP assessment appear better than either measure alone . Meta-anCACS can be used for early diagnosis of coronary heart disease, review, and follow-up after treatment, screening for cardiovascular disease and high-risk population, and coronary artery CT angiography is the main noninvasive method for quantitative coronary artery calcified plaque at present . CACS isThe ambulatory blood pressure recording instrument is composed of a transducer, a miniature recording box, and a recovery system and can automatically store data within 24 hours of the day and night, including systolic blood pressure, diastolic blood pressure, mean arterial pressure, heart rate, and the highest and lowest values. The 2013 European updated guidelines on hypertension stressed the importance of ABPM for the prognosis of clinical cardiovascular events, especially for clinic blood pressure and family pressure in apparent inconsistency, night hypertension, and blood pressure variability assessment . 24-hourThe study of dynamic electrocardiogram was initiated in 1957 by Holter and first applied in the monitoring of cardiac electrical activity, thus also called Holter ECG. It can continuously record the daily activity under the condition of 24 hours of ECG changes, is noninvasive and easy to operate, and is widely used in clinical practice. The results of Holter ECG include heart rate, arrhythmia analysis, ST-T segment analysis, and analysis of heart rate variability. Statement of clinical application of ECG and dynamic ECG in the United States made dynamic ECG monitoring an important indication .About early assessment of vascular disease, in addition to direct or indirect imaging techniques or noninvasive evaluation techniques for arterial stiffness, there are also many risk biomarkers of vascular disease, including traditional biomarkers and some new markers found in recent years. Basic research showed that vascular endothelial cells are an important paracrine organ and play a variety of physiological roles through the secretion or release of a series of biologically active medium, and vascular endothelial dysfunction can lead to some abnormal indicators. Therefore, the detection of biomarkers can be used as the evaluation index of endothelial dysfunction . In addiArteriosclerosis is not the patent of the modern society, its existence precedes the modern civilized society and the present risk factor , and traHyperhomocysteinemia (HHcy) is characterized by elevated plasma total homocysteine levels and associated with increased risk of cardiovascular disease and stroke . StudiesAll the technologies of vascular early detection mentioned above were summarized within a table see , includiBased on the preliminary research foundation, in 2006, our research team released the first report of Chinese vascular disease early detection technology application guide . The guiAt present, there are a variety of cardiovascular risk prediction models , to pred


Tacrolimus (FK506) and cyclosporine A (CsA) are widely used to protect graft function after renal transplantation. The aim of the present study is to determine whether the single nucleotide polymorphism of CYP3A5 is a predictive index of FK506 dose requirement, and also the selection yardstick of FK506 or CsA treatment.We tested archival peripheral blood of 218 kidney recipients for CYP3A5 genotyping with PCR-SSP. Meanwhile, the dose of FK506 and CsA was recorded, blood concentration of the drugs was measured, and graft outcome was monitored.These results indicate that CYP3A5*AA/AG carriers need higher FK506 dose than CYP3A5*GG homozygote to achieve the target blood concentration. For CYP3A5*GG carriers, taking FK506 or CsA are both advisable. CYP3A5*AA/AG carriers preferred to CsA treatment depending on the graft outcomes and drug costs. CYP3A5 genotyping is a new approach to detecting FK506 dose requirement and a predictive index for the FK506 or CsA treatment selection in kidney recipients. Tacrolimus (FK506) and cyclosporine A (CsA), two kinds of immunosuppressive drugs, have been widely used in the field of renal transplantation. As triple-immunosuppressive regimen, FK506-Mycophenolate Mofetil (MMF)- Prednisone (Pred) or CsA-MMF-Pred was applied mainly depended on doctors’ experience and drug economics. However, there was no any detail treatment guidelines for the drug selection.FK506 could protect transplant function. Underdosing of immunosuppressive drugs could cause immunological graft rejection and influence the recipients’ survival quality. However, once the recipients accepted FK506 as their immunosuppressive drug, most of them would have to take it all life long. Furthermore, it could cause a series of side effects, such as toxicity to graft, infection, and even malignant tumors. Therapeutic FK506 monitoring has been carried out routinely, since target blood concentrations was expected to achieve with the lowest dose of drug given.There were several reasons for the marked heterogeneity among individuals in blood concentrations of FK506, which were achieved by a standard body-weight-based dosing. According to recent studies, the variation was determined by genetic factors to some extent , and alsIn this present study, we intend to determine CYP3A5*A and CYP3A5*G allele frequencies in Chinese kidney transplanted recipients and its association with FK506 or CsA dose requirement. We also intend to figure out whether the single nucleotide polymorphism of CYP3A5 could be a selection yardstick of FK506 or CsA treatment.p = 0.0011, Table A cohort of 218 kidney recipients was recruited. The basic data of the patients were shown in Table p = 0.0012, Figure p < 0.0001, Figure FK506 blood concentrations were measured on day 7, 1st month, 3rd month, 6th month and 12th month for each recipient after renal transplantation. CYP3A5*GG carriers taking FK506 treatment get the highest blood concentration (8.85±3.97 ng/ml) at day 7 post-transplantation with the standard dose(0.10±0.04mg/kg) Figure . They weThe influence of CYP3A5 genotype on dose-adjusted concentration was also analyzed for the patients who took CsA treatment. There was little if any difference in CsA blood concentration or CsA standard dose at any time point during the first year after kidney transplantation Figure . ConsequThe relationship between CYP3A5 genotype and liver/renal injury after kidney transplantation was analyzed for each patient. There were no significant differences in liver injury biomarkers alanine transaminase (ALT) or aspartate transaminase (AST), as well as renal function biomarkers serum creatinine (Scr) or bloodureanitrogen (BUN) in different CYP3A5 genotypes regardless of the therapy regime during the first year, as shown in Figure P = 0.030, Table P = 0.982, Table There were 218 patients in this cohort, and 21 of them had got acute rejection. For CYP3A5*GG carriers, those taking FK506 subjected to greater rejection rate than those taking CsA .Univariate analysis showed that neither genotype no calcineurin inhibitor was associated with renal acute rejection. Induction therapy, however, influenced renal acute rejection rate. Multivariate Logistic regression analysis revealed similar results Table . ImportaP = 0.127, Table P = 0.654, Table Among 95 initial CsA treated patients, 20 CYP3A5*GG carriers and 16 CYP3A5*AA/AG carriers shifted CsA to FK506 treatment after several years for some reasons. 7 CYP3A5*GG carriers shifted therapy regime when they encountered acute rejection, and never suffered second acute rejection. Another 2 CYP3A5*GG carriers, however, encountered acute rejection after therapy shift. The acute rejection rate didn't change much before or after shifting to FK506 treatment in CYP3A5*GG subgroup .There was only one participant suffered graft loss in each group  during follow-up periods. There was no statistical significance in the graft survival rate among these groups (p = 0.371), drug-induced liver disease , postoperative diabetes mellitus  and postoperative Leukocytopenia (p = 0.513) among these groups , MMF or azathioprine, and Prednisone (in tapering doses from 80 to 10 mg/day) within the first month after transplantation for Chinese people It was reported that different ethnic groups require different doses of FK506 to achieve the target concentration –14. The Previous researches have suggested that the CYP3A5 genotype could predict the FK506 dose requirement to achieve target concentration in transplant recipients . EvidentOn the other hand, we found some CYP3A5*GG carriers still needed higher FK506 doses. And some CYP3A5*AA/AG carriers just needed lower FK506 to keep the target blood concentration. Further research is necessary to explore the factors which induce the heterogeneity.Previous studies also suggested the CYP3A5 genotype could be an important risk factor for the transplant rejection –18 and CIn our study, there was no significant difference in the 3-year graft survival rate between different CYP3A5 genotypes (data not shown) regardless of the immunosuppressive agents regime, which was consistent to previous reports . Short fThe liver injury  and kidney functions (Scr and BUN) exhibited no significant difference among the different genotype groups during the first year after the kidney transplantation. It seems that even high dose of FK506 taken by the CYP3A5*AA/AG groups did not cause serious burden on liver or kidney, as they were supervised strictly and judged by the professional doctors carefully.22-26, spending more however. In China, the average cost of tacrolimus in a stable renal-transplant recipient every day is approximately 1.8 times as high as CsA. In our study, for the FK506 treated patients, CYP3A5*GG carriers can sustain effective therapeutic concentration with lower FK506 dose and thus cost less than CYP3A5*AA/AG carriers. For the CsA treated patients, there was no significant difference of CsA expense between the two genotype groups. For CYP3A5*GG carriers, FK506 treatment had to afford higher expense than CsA treatment after half a year. For CYP3A5*AA/AG carriers, it cost even more to take FK506 treatment.It was accepted that FK506 was associated with lower rejection rates and longer graft life spans compared to CsAIn conclusion, CYP3A5 polymorphism profoundly influenced pharmacokinetics of FK506 but not CsA. Taking all these into consideration, if a recipient is a CYP3A5*GG carrier, taking FK506 or CsA are both advisable. However, for a CYP3A5*AA/AG carrier, FK506 should not be the first choice due to the crushing costs and similar outcome. CYP3A5 genotyping is a reasonable approach to detecting FK506 dose requirement and a predictive index for FK506 or CsA treatment selection in kidney recipients.The studies on human samples were approved by Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University. And Experiments conducted was in accordance with Declaration of Helsinki. Written informed consent was obtained from all participants. 218 live-donor renal transplant recipients transplanted in our center between Jun. 2008 and Oct. 2010, and treated with FK506 or CsA, were invited to participate in the study. All the patients were treated with standard triple-drug maintenance immunosuppression included calcineurin inhibitor (FK506: trough level 5-10 ng/ml) or CsA, either MMF or azathioprine and prednisone which tapered doses from 80 to 10 mg/day within the first month after transplant. The FK506 and CsA dose requirement was assessed after transplantation. All demographic characteristics or clinical data of patients were blind to the researchers that were responsible for the drug blood concentration detection or CYP3A5 genotyping. All patients were followed up until Aug. 2014.Blood samples of 2 to 3 milliliters were drawn from each patient in a vacuum tube containing ethylene-diaminetetracetic acid. Genomic DNA was extracted from 250μl whole blood by Blood Genomic DNA Miniprep kit (Axygen). Polymerase chain reaction using sequence-specific primers (PCR-SSP) was performed to identify CYP3A5 genotype . 112μL reaction mixture consisting of 12μL genomic DNA, 0.8μL Taq DNA polymerase (Promega) and 100μL dNTP-Buffer that was provided in the kit was prepared and 10 μL reaction mixture was added into each well of the 96-well plates that have contained sequence-specific primers. Amplification involved 1 cycles of 96°C for 2 min, 5 cycles of 96°C for 20 seconds and 68°C for 60 seconds, 10 cycles of 96°C for 20 seconds,65°C for 50 seconds and 72°C for 45 seconds, 15 cycles of 96°C for 20 seconds, 62°C for 50 seconds and 72°C for 45 seconds followed by final extension at 72°C for 5 min. The PCR product was obtained and aliquots of 10μL were analyzed by electrophoresis on a 2.5% agarose gel containing ethidium bromide to check the sizes and amounts of the amplicons. Finally, the band patterns were visualized using an ultraviolet, and the CYP3A5 genotypes were identified according to the typing table.TM II FK506 ELISA kit (DiaSorin Inc.), which were performed on a clinical analyzer.The dose and blood concentrations of FK506 and CsA for recipients were measured at day 7, 1st month, 3rd month, 6th month and 12th month after renal transplantation, as well as hepatic and renal function. The FK506 blood concentration was measured using a PRO-TracP < 0.05 were considered statistically significant.Mean and standard deviation (mean ± SD or mean ± SEM) were used to describe continuous variables, while percentage was used to describe categorical variables. Continuous variables were compared via analysis of variance (ANOVA). Comparison of frequencies was performed by chi-square test. Graft survival was analyzed with Kaplan- Meier method and differences between survival curves were calculated by the log-rank test. Logistic regression was conducted to reveal the influence factors of renal acute rejection. Values of