Pseudomonas aeruginosa is a nosocomial pathogen colonizing patients with chronic infectious diseases and has gained resistance to all the known broad spectrum antibiotics available today. The present study showcases the antibiofilm potential of an essential oil (EO) from an underexplored Cinnamomum species namely, C. tamala, against P. aeruginosa biofilms. Furthermore, the synergistic effects of the EO along with a commercially available DNase (DNaseI) and a DNase (MBD) isolated from a marine bacterium were explored for its antibiofilm activity. The results showed that the synergized action has maximum efficacy in inhibiting young and preformed biofilms. The synergized effect of EO and DNaseI showed 70% inhibition against matured biofilms of P. aeruginosa. The EO from C. tamala also showed quorum sensing inhibitory potential as it could inhibit the swarming motility behavior of P. aeruginosa. The synergistic action of EO and DNases offers a novel alternate therapeutic strategy for combating P. aeruginosa biofilm associated infections. Essential oils (EOs) are natural plant secondary metabolites which constitute a complex mixture of volatile components found to have multiple antimicrobial properties . EOs conPseudomonas aeruginosa is a notorious nosocomial pathogen which is ubiquitous in health care settings where it causes persistent and chronic infection in immunocompromised individuals  and Pseudomonas quinolone signaling (PQS) molecule  T. Nees and Eberm (Indian bay leaf/Indian cassia) and DNases to disrupt biofilms. The literature scrutiny reveals that till date, the antibiofilm potential of only the commercial cinnamon/Ceylon cinnamon (Cinnamomum zeylanicum) has so far been reported  wild-type strain and two clinical P. aeruginosa isolates AU07 (GenBank Accession No: JN871909) and AU09 (GenBank Accession No: JN871910)   which wee assays .Vibrio alginolyticus (GenBank Accession Number: KX458518).Bovine pancreatic DNaseI was purchased from HiMedia and Marine Bacterial DNase (MBD) was isolated and purified from the marine bacterium C. tamala were collected from Almora, Uttarakhand, India. The leaves were partially shade dried and used for extraction of EO. On hydro-distillation, the partially shade dried leaves yielded 1.2% pale yellow color EO. After distillation, the EO was dried over by adding a pinch of anhydrous sodium sulfate and stored at 4°C until further analysis and experiments. The EO was quantitative and qualitatively analyzed by GC-FID and GC-MS following the methods and conditions used by The leaves of The Minimum Inhibitory Concentration (MIC) of the EOs were determined as per P. aeruginosa planktonic cells, growth curve analysis was done. LB broth containing 1% overnight culture of P. aeruginosa was supplemented with Biofilm Inhibitory Concentration (BIC) of the EO and the flask was incubated for 24 h at 37°C. The flask containing overnight culture without EO served as control. The optical density was read spectrophotometrically at every 1 h interval up to 24 h  and EO at its sub-MIC (5% v/v). The well without EO and only DMSO (1.25%) served as a control. Likely, the effect of commercial and MBDs and synergism of EO and the respective DNases were assessed as above to determine the maximum efficacy in inhibiting the biofilm.The effect of EO to inhibit the young biofilms was assessed using the crystal violet assay . The assTo determine the potency of the EO to inhibit mature biofilms, the BIC of EO was added to the preformed biofilms and incubated further for 24 h at 37°C. After incubation, the spent media was discarded and the wells were washed with sterile distilled water. After air drying the wells were stained with 0.4% crystal violet and the absorbance was quantified spectrophotometrically at 595 nm . SimilarBiofilms were allowed to form on 1 cm × 1 cm glass slides which were placed into the wells of the 24-well titer plates. The BIC of EO (5%v/v) was added to the wells and the plates were incubated at 37°C for 24 h. After incubation, the biofilm formed on the glass slides was stained using crystal violet dye for 5 min. It was then gently washed with de-ionized water and allowed to dry for 5 min. Then, the slides were viewed under light microscope at a magnification of 40×  .Confocal Laser scanning microscopy (CLSM) was used to determine the action of EO on mature biofilms. The biofilms were allowed to form on 1 cm × 1 cm glass slide placed in a 24-well titer plates. After 24 h incubation, the BIC of EO was added to the preformed biofilms and the plates were further incubated at 37°C for 24 h. The three dimensional architecture of the preformed biofilm treated with BIC of EO was assessed by CLSM . Images P. aeruginosa biofilm, SEM analysis was performed. Briefly, P. aeruginosa biofilms were grown in the presence and absence of enzymes (DNaseI and MBD) on the glass slides. The biofilms on the glass slides were fixed with 2.5% glutaraldehyde were washed in 0.1M sodium acetate buffer (pH 7.3). Finally, the dehydrated samples were dried, gold sputtered and examined with VEGA3 TESCAN  (Sigma), which stains the DNA present within a biofilm. Accumulation of eDNA were visualized under a fluorescence microscope  at excitation and emission wavelengths of 540 and 525 nm for PI and photographed digitally  as well as the clinical P. aeruginosa isolates. In addition, cinnamaldehyde and linalool which were the major components of EO were also assayed individually against the QS mediated virulence factors of PA01 and the clinical isolates.All the QS mediated virulence assays were performed with the EO against 2SO4 was also added. The mixture was incubated for 1 h in the dark and centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at 490 nm.Bacterial biofilm consists of self-generated extra polymeric substances which plays a distinct role during biofilm formation. EPS from EO treated and untreated test pathogen was quantified by the method of P. aeruginosa biofilms which aids in the adhesion of EPS that promote cell attachment and shields the cell surface structure. Alginate was quantified from the EO treated and untreated cell suspension. 600 μl of boric acid – H2SO4 (4: 1) was added to 70 μl cell suspension and the cell suspension was vortexed for 10 s. Further, 20 μl of 0.2% carbazole solution was added to the cell suspension which was incubated for 30 min at 55°C. After incubation, absorbance was measured spectrophotometrically at 530 nm . The suspension was boiled for 10 min and then diluted with the same buffer to an OD of 0.8 at 595 nm. To 900 μl of diluted S. aureus suspension, 100 μl of cell free culture supernatant of the strains cultured with or without EO was added. OD was measured at 595 nm after every 15 min interval for 60 min using a UV–visible spectrophotometer. Similarly, the LasA staphylolytic assay was carried out using cinnamaldehyde and linalool against P. aeruginosa and the clinical isolates.activity . A 30 mlPseudomonas aeruginosa uses rhl QS system that consists of rhlI which in conjunction with RhlR activates the expression of pyocyanin pigment production. Cell free supernatants of PA01 and the clinical isolates cultured with the presence and absence of EO at their respective sub-MIC concentrations (5 and 7% v/v) was extracted for pyocyanin quantification  was inoculated in the middle of the agar and incubated for 16 h at 37°C  and (E)-cinnamaldehyde (31.2%) were the major compounds of the oil (Table 1).GC-FID and GC-MS analysis of leaf EO of Cinnamomum sp. The major components of the EO obtained from the bark of Ceylon type C. zeylanicum were eugenol and (E)-cinnamaldehyde which represent 82.5% of the total composition . The MIC of cinnamaldehyde against PAO1 was found to be 0.36 mg/ml and for clinical isolates it showed MIC of about 2 mg/ml. Likely, the MIC of linalool against PAO1 was found to be 0.36 mg/ml and for Clinical isolates it showed MIC of about 2 mg/ml. It was observed that chemical composition of volatile oils varied significantly among the different position . Linalootiveness .Figure 1).Growth curve analysis was performed to ensure that the EO did not show any antibacterial activity at its sub-MIC (5%) concentration. The results no reduction in optical density of cells between control and treated even after 24 h of incubation . The results were in similar lines with matured biofilms. A maximum inhibition of about 70% ± 1.09 was seen in synergism of EO and DNases. EO alone on the other hand could inhibit only 50% ± 2.07 of the mature biofilm and 65% ± 2.8 inhibition was noticed for DNases alone .The results showed that difference was observed in reduction of the biofilms treated with the combination of EO and DNases than EO and DNases treated individually. Biofilm inhibition of about 75% ± 1.09 was seen when EO and DNases were used in combination. On the other hand, EO showed only 58% ± 2.9 of inhibition when used individually .To clearly demonstrate the synergized effect of EO and DNases on young biofilms, microscopic examinations were performed in the presence of sub-MIC (5% v/v) of EO. The microscopy analysis revealed maximum reduction of young biofilm upon treatment with EO and DNases. There was also reduction in EO treated biofilm but to a lesser extent on comparison with combination of EO and DNases , it is evident that there was a disintegration of biofilms in the treated samples on comparing with the control, which showed the presence of a dense biofilm. COMSTAT analysis was carried to determine the three dimensional features like biomass, average thickness and surface volume ratio of matured biofilms before and after treatment. The results show that there was a substantial reduction in different parameters of biofilm architecture such as biomass and average thickness, whereas there was an increase in surface to volume ratio which indicates the detachment of cells from biofilm matrix upon treatment (Table 2). From the Table 2, it is evident that the increase in surface to volume ratio (μm2/μm3) of around 0.043 and decrease in biomass (μm) (32.22) and average thickness (μm) (30.08) is due to the synergistic effect of both EO and DNase.Confocal Laser scanning microscopy was further done to confirm that synergized effect has maximum ability to disrupt matured biofilm than EO and DNases alone and to study the architecture of the biofilm before and after treatment. From the CLSM images  has a thick layer of EPS, whereas there is a disruption in the EPS matrix of biofilm treated with DNaseI and MBD .To analyze the antibiofilm potential of DNaseI and MBD on P. aeruginosa biofilms were stained with PI  and was visualized under a fluorescent microscope. From the fluorescent micrograph results it is evident that untreated biofilm has high accumulation of eDNA  whereas treated has lesser eDNA accumulation .To detect the presence of eDNA, P. aeruginosa treated with sub-MIC (5% v/v) . Similarly, EO exhibited a concentration dependent inhibitory effect in the production of alginate by PAO1. At sub-MIC (5% v/v) the alginate production was reduced to the maximum of 71% ± 4.38 whereas 1% and 2.5% v/v had lesser inhibitory effect on alginate production . The EO at its sub-MIC (7% v/v) inhibited the alginate production of clinical isolate AU07 by 65% ± 3.49 and 61% ± 3.58 for AU09 . Likely, cinnamaldehyde inhibits the alginate production at its sub-MIC (0.18 mg/ml) of about 69% ± 2.75 for P. aeruginosa, 63.5% ± 4.63 for AU07 PACI-01 at its sub-MIC (1 mg/ml) and 59.5% ± 2.43 for AU09 PACI-02 at its sub-MIC (1 mg/ml) . The EO at its sub-MIC (7% v/v) reduces the LasA staphylolytic activity for AU07 by 68% ± 4.95 and 75% ± 7.93 for AU09 . The reduction of LasA staphylolytic activity was found to be 70% ± 5.26 for P. aeruginosa at its sub-MIC (0.18 mg/ml), 62.5% ± 4.22 for AU07 PACI-01 and 60% ± 3.99 for AU09 PACI-02 at its sub-MIC (1 mg/ml)  treated (P. aeruginosa is regulated by QS system. P. aeruginosa biofilm treated with EO showed a maximum reduction of pyocyanin production of about 72% ± 4.65 at its sub-MIC (5%v/v) when compared to the control . The EO at its sub-MIC (7% v/v) reduces the pyocyanin production by 68% ± 3.95 for AU07 and 62% ± 3.90 for AU09 . Pyocyanin was reduced significantly of about 78% ± 4.70 at its sub-MIC (0.18 mg/ml) for P. aeruginosa treated with cinnamaldehyde and 67% ± 0.87 for AU07 PACI-01 at its sub-MIC (1 mg/ml) and 63% ± 2.01 for AU09 PACI-02 at its sub-MIC (1 mg/ml)  concentration . From the results, it is envisaged that EO at its BIC (5% v/v) has the ability to interfere in the Las and Rhl mediated QS system. The individual component of EO such as cinnamaldehyde  as its major component followed by cinnamaldehyde (31%). So, we fully concur with the view that the exhibited QSI activity might be due to the synergistic action of the major components present in the EO  alone showed very low efficacy in reducing P. aeruginosa biofilms  mediates a neutrophil dominated host immune response. During this process a very high concentration of e-DNA gets released as neutrophils disintegrate which leads to increased sputum accumulation  and rhl which coordinates several extracellular virulence factors . Swarming motility is a key virulent trait in P. aeruginosa which is mediated by Las and Rhl QS system (C. tamala suppressed the QS-based virulence factors in P. aeruginosa PAO1 as well as the clinical isolates. Cinnamaldehyde is already reported as a QS inhibitor in V. harveyi probably by inhibiting/degrading the QS molecule 3-hydroxy-butanoyl homoserine lactone. On the other hand 3-oxo-C12HSL and C4HSL are the major QS molecules in P. aeruginosa (P. aeruginosa.Of late, EOs of different plants has been documented to have QS inhibitory activity . In the  factors . From thS system  which plruginosa . So it iC. tamala and the synergistic combination of EO of C. tamala and DNases has increased efficacy in disrupting biofilms. The synergistic use of EO with a DNase of marine bacterial origin might result in a cost effective therapy and its use as a potential antibiofilm agent for the treatment of biofilm device associated infections warrants further investigations.Though several EOs have been explored for their antibiofilm property, to best of our knowledge, the present study forms a first report about the QS and biofilm inhibitory potential of an underexplored EO of All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. PN designed overall research and wrote the paper.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.


We applied multivariate logistic regression analysis to build a model based on the three biomarkers for BCa diagnosis. The diagnostic accuracy of the three biomarkers and the model were assessed and compared by the area under the curve (AUC) of the receiver operating characteristic. We validated the diagnostic accuracy of these biomarkers and the model in an independent validation cohort of 210 patients. In the training set, urinary concentrations of AIB1, EIF5A2, and NMP22 were significantly elevated in BCa. The AUCs of AIB1, EIF5A2, NMP22, and the model were 0.846, 0.761, 0.794, and 0.919, respectively. The model had the highest diagnostic accuracy when compared with AIB1, EIF5A2, or NMP22 . The model had 92% sensitivity and 92% specificity. We obtained similar results in the independent validation cohort. AIB1 and EIF5A2 show promise for the noninvasive detection of BCa. The model based on AIB1, EIF5A2, and NMP22 outperformed each of the three individual biomarkers for detecting BCa.We previously demonstrated that amplified in breast cancer 1 (AIB1) and eukaryotic initiation factor 2 (EIF5A2) overexpression was an independent predictor of poor clinical outcomes for patients with bladder cancer (BCa). In this study, we evaluated the usefulness of AIB1 and EIF5A2 alone and in combination with nuclear matrix protein 22 (NMP22) as noninvasive diagnostic tests for BCa. Using urine samples from 135 patients (training set, controls [ Bladder cancer (BCa) is responsible for approximately 250,000 deaths per year worldwide and is diagnosed in approximately 900,000 men and women each year . To dateConsidering variation among individual subjects, crosstalk between molecular pathways, and solid tumor heterogeneity, molecular diagnostic panels composed of multiple biomarkers are likely to be far more accurate than single markers. Recently, several studies embraced the multiplex approach and identified the signatures of diagnostic biomarkers. These studies showed that integrating multiple biomarkers into a single model would substantially improve diagnostic value as compared with a single biomarker –9. HowevNumerous studies have identified amplified in breast cancer 1 (AIB1) –12 and eThe aim of this study was to evaluate AIB1 and EIF5A2 as single biomarkers for detecting BCa, and in comparison and in combination with NMP22. We used multivariate binary logistic regression analysis to develop a model integrating AIB1, EIF5A2, and NMP22 to yield a prediction rule. We then assessed the diagnostic accuracy of this model in a training set, and validated it in an independent validation cohort.p > 0.05 for all). In the training set, 64.7% of patients had non-muscle invasive BCa (NMIBC) and 43.5% had low-grade disease. The independent validation set was comprised of 134 urine samples from subjects with BCa and from 76 non-cancer controls. As in the training set, there were no significant differences in sex, age, and smoking habits between the patients and the controls . In the independent validation set, 67.2% patients had NMIBC and 41.8% had low-grade disease. There were no significant differences in sex, age, smoking habits, pathological T stage, and grade between the patients in the training and independent validation sets .Table Initially, we aimed to verify that protein measurements could be reliably obtained from urine samples. For this purpose, replicate measurements of AIB1, EIF5A2, and NMP22 in urine samples from the training and independent validation sets were performed. Scatter plots were constructed to display the repeatability of three consecutive measurements across all urine samples from the training and independent validation sets. In this study, all intraclass correlation coefficients (ICCs) were >0.98, indicating that repeatability was reliable across the entire cohort  were significantly higher in the subjects with BCa than in the controls. In the independent validation set, the mean urinary levels of AIB1 (1.31 vs. 0.39 ng/ml), EIF5A2 (5.74 vs. 4.60 ng/ml), and NMP22  were significantly higher in the subjects with BCa than in the controls.Figure p < 0.05 for all) and in patients with high-grade disease as compared to patients with low-grade disease . Interestingly, we obtained similar results in the independent validation set . However, in the independent validation cohort, the AIB1 AUC was significantly greater than that of NMP22 (p = 0.0264).We compared the AIB1 or EIF5A2 AUCs with that of NMP22, and found that they were not statistically different for both comparisons in the training set  and 0.708 (95% CI 0.611–0.793), respectively. Urinary AIB1 had 73% (95% CI 59%–84%) sensitivity, 88% (95% CI 76%–96%) specificity. Urinary EIF5A2 had 64% (95% CI 50%–76%) sensitivity, 78% (95% CI 64%–89%) specificity. In the independent validation set, Urinary AIB1 had 73% (95% CI 63%–82%) sensitivity, 86% (95% CI 76%–93%) specificity, and an AUC of 0.792 (95% CI 0.722–0.850). Urinary EIF5A2 had 63% (95% CI 52%–73%) sensitivity, 74% (95% CI 62%–83%) specificity, and an AUC of 0.684 (95% CI 0.608–0.753). .p < 0.0001 for all; Table p < 0.0001 for all; Table In univariable logistic regression analyses for the training set, increased AIB1, EIF5A2, and NMP22 were all associated with increased risk of BCa  +  + . The cut-off probability score in the combination detection assay was 10.08. The distribution of probability scores revealed that the combination assay had better diagnostic accuracy Figure . The comp < 0.0001; Table p < 0.0001; Table To confirm that the combination assay had similar diagnostic value in different populations, we applied it to the independent validation set comprising 134 patients from another center and the 76 control subjects. The combination assay yielded similar diagnostic performances, as well as in the training set Figure . Figure p = 0.02) and EIF5A2 (p < 0.0001). The combination assay had 87% (95% CI 76%–95%) sensitivity and 92% (95% CI 81%–98%) specificity. In the independent validation cohort, AUC of the combination assay was 0.872 (95% CI 0.812–0.919), diagnostic performance of the combination assay was significantly greater than both of the urinary AIB1 (p = 0.0001) and EIF5A2 (p < 0.0001). The combination assay had 84% (95% CI 75%–91%) sensitivity and 91% (95% CI 82%–96%) specificity.  for NMIBC detection, outperformed both of the urinary AIBI , and negative predictive value (NPV) in the two sample sets. As a standalone assay, the 3-biomarker panel had an AUC of 0.919 for BCa detection. Similar results were obtained for the independent validation cohort. Several studies have shown that multiple protein biomarkers could increase the diagnostic performance to higher levels. For example, in a cohort of 127 patients (64 tumor-bearing subjects), Goodison et al. demonstrated that a panel based on two biomarkers (VEGF and apolipoprotein E [APOE]) had 81% sensitivity and 97% specificity for detecting BCa. By combining three biomarkers , a logistic prediction model was derived that had 90% sensitivity and 97% specificity [in vitro and in vivo [Previous reports have implicated AIB1 in bladder tumor biology, and a few studies have investigated its use as a potential biomarker of BCa –12. Prev in vivo . We also in vivo . These fEIF5A2 is overexpressed in many human malignancies and is critically associated with tumor progression, lymph node metastasis, and poor prognosis –19, 23. The present study is limited because the study was performed in a southern Chinese population from two clinical centers, and the distribution of clinical characteristics might differ in other regions, rendering it susceptible to the inherent biases of such a study format. Therefore, our results should be further validated by multicenter clinical trials and patients from different areas and races.In summary, our findings show that higher levels of urinary AIB1 and EIF5A2 are associated with the presence of BCa. Moreover, our study demonstrates that the model integrating urinary AIB1, EIF5A2, and NMP22 detection can be a useful tool for diagnosing patients with BCa.The study was performed after we had obtained approval from the local institutional review board. Informed consent was obtained from each subject. Voided urine samples from 345 subjects were collected prospectively in the morning and frozen at −20°C within 30 min until analyzed. For the training set, urine samples were obtained from 85 patients from the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, between November 25, 2013, and October 28, 2014. Fifty control subjects, comprising six patients with renal calculi, five with benign bladder lesions, seven with benign prostatic enlargement, seven with hematuria, six with urinary tract infection, and 19 healthy volunteers, were enrolled. The five patients with benign bladder lesions and the seven patients with benign prostatic enlargement had undergone TUR-Bt and transurethral resection of the prostate, respectively. Another 134 urine samples were obtained from the Cancer Center of Sun Yat-sen University, Guangzhou, China, between November 1, 2013, and November 28, 2014, and from 76 control subjects, comprising seven patients with renal calculi, eight with benign bladder lesions, 10 with benign prostatic enlargement, nine with hematuria, eight with urinary tract infection, and 34 healthy volunteers were enrolled in the independent validation set. The patients with benign bladder lesions and the patients with benign prostatic enlargement had also undergone TUR-Bt and transurethral resection of the prostate, respectively. The histological grade and stage were recorded according to the 2004 World Health Organization grading system and the sixth edition of the tumor-nodes-metastasis (TNM) classification system, respectively. All subjects were confirmed without any symptom of kidney failure. Office cystoscopy was performed in all control subjects with hematuria. According to the International Consensus Panel on Bladder Tumor Markers , this cog 4°C for 10 min. Supernatant was frozen at −20°C until analyzed.Prior to any therapeutic intervention, approximately 50 ml clean-catch, first-morning midstream-voided urine was collected prospectively from each subject. The sample was immediately centrifuged at 1000 ×ELISA kits of human AIB1 (SEE798Hu) and human NMP22 (NUMA1: SEC332Hu) were purchased from USCN Life Science . ELISA paired antibody (H00056648-AP11) and recombinant protein (H00056648-P01) for detecting human urinary EIF5A2 were purchased from Abnova . Secondary streptavidin–horseradish peroxidase-conjugated anti-mouse immunoglobulin G antibody (ab6789) was purchased from Abcam .An ELISA kit was used to quantify the urinary AIB1 and NMP22 concentrations. Assays were performed in triplicate according to the manufacturer's instructions. The minimum detection concentrations were 0.124 ng/ml for AIB1 and 0.063 ng/ml for NMP22.Sandwich ELISA was used to quantify the urinary EIF5A2 concentrations. Assays were performed in triplicate according to the manufacturer's instructions. The minimum detection concentration was 3 ng/ml.The association between categorical data and differences of categorical data between the cases and controls were tested using the chi-square test. The nonparametric Mann–Whitney sum rank U test was used for comparing the variables between groups. The precision of the repeated measurements was determined using ICC.We investigated the diagnostic performance of the three biomarkers for BCa detection. The US National Cancer Institute recommends evaluating the performance of potential markers for cancer detection using ROC curves . Accordip < 0.05 and all reported p-values were 2-sided. Statistical analyses were done using SPSS 12.0  and using MedCalc version 8.0  for ROC curve analysis.We used multivariate binary logistic regression analysis to construct a model combining AIB1, EIF5A2, and NMP22 to derive a prediction rule in the training set. We determined the Youden index cut-off probability score to maximize the sum of sensitivity, specificity, the PPV and NPV for the combination assay . We applied the model to the independent validation cohort. We generated nonparametric ROC curves for the model using the same method described above. We used univariable and multivariable binary logistic regression to calculate odds ratios and 95% CI. Statistical significance in this study was set at


To describe the incidence and progression of retinopathy in people with diabetes in Southern Malawi over 5 years. To document visual loss in a setting where laser treatment is not available.Subjects from a cohort sampled from a hospital-based, primary-care diabetes clinic in 2007 were traced in 2012. Laser treatment was not available. Modified Wisconsin grading of retinopathy was performed using slit lamp biomicroscopy by a single ophthalmologist in 2007 and using four-field mydriatic fundus photographs at an accredited reading centre in 2012. Visual acuity was measured by Snellen chart in 2007 and by ‘Early Treatment of Diabetic Retinopathy Study’ chart in 2012. HbA1c, blood pressure, HIV status, urine albumin–creatinine ratio, haemoglobin and lipids were measured.Of 281 subjects recruited in 2007, 135 (48%) were traced and assessed, 15 were confirmed dead. At follow-up (median 5.3 years) ≥2 step retinopathy progression was observed in 48 subjects . Incidence of sight threatening diabetic retinopathy for those with level 10 (no retinopathy) and level 20 (background) retinopathy at baseline, was 19.4% (11.3–27.4) and 81.3% (62.1–100), respectively. In multivariate analysis 2 step progression was associated with HbA1c (OR 1.2495%CI 1.04–1.48), and haemoglobin level . 25 subjects (18.8%) lost ≥5 letters, 7 (5.3%) lost ≥15 letters.Progression to sight threatening diabetic retinopathy from no retinopathy and background retinopathy was approximately 5 and 3 times that reported in recent European studies, respectively. Incidence of visual loss was high in a location where treatment was not available. Sub-Saharan Africa faces an epidemic of diabetes . In contQECH is a large teaching hospital. QECH provides primary and secondary care to the people of greater Blantyre (approximately 1 million). Tertiary care is provided to the Southern Region of Malawi. At the time of this study (2012) the QECH diabetes clinic was the only public sector diabetes clinic in Blantyre. In the period 2007 to 2012 the clinic underwent a number of changes. The number of registered patients increased from approximately 800 to 2000. A vibrant nurse-led patient education programme supported by the World Diabetes Foundation commenced in 2008. Its aims were improving compliance with diet and medications and educating patients on the complications of diabetes. An electronic records system  was installed in early 2010. In 2007 medications regularly available free of charge were glibenclamide and insulin (lente and soluble). Metformin was available from private pharmacies but rarely from the hospital pharmacy. By 2012 metformin was more frequently available free of charge. However, supplies of all drugs remained intermittent. Tests available at the clinic were the same in 2007 as 2012: glycaemic control measured by fasting blood glucose (FBG), blood pressure (BP), height and weight. Measurement of lipids, glycosylated haemoglobin (HbA1c) and urine test sticks for microalbuminuria were not available routinely.ad hoc (i.e. not consecutive): subjects had slit lamp examination if the ophthalmologist was present at the particular clinic at which they were recruited. At this time laser treatment was not available in the public sector. The 2007 study was not planned as a cohort study therefore no contact details were recorded. Tracing of subjects between December 2011 and November 2012 was systematic. The QECH diabetes clinic electronic patient record system was searched by subject name by a research nurse. Identified persons were then contacted by phone or home visit. The study team attended the diabetes clinic weekly between May and November 2012 to approach patients in the clinic waiting room. The majority of deaths in Malawi are not registered. The relatives of deceased subjects were visited at home by a study nurse in order to confirm the death. Death was recorded if confirmed by a first degree relative or ‘Traditional Authority’ .Patient selection in the 2007 cross sectional study has been described elsewhere ,13. BrieClinical assessment in the 2007 study has been described elsewhere ,13. BrieClinical assessment of subjects in 2012 was the same as in our 24 month cohort study described in detail elsewhere . BrieflyAs in 2007 retinopathy and maculopathy were classified by feature-specific grading using the LDES scale . In contor 1 step progression in both eyes). A multiple logistic regression model  was constructed to determine the odds ratio and 95% confidence intervals for the primary end point at 5 years. For the majority of variables the baseline value was used in the analysis. For those variables only measured at the 2012 visit this value was used . An initial 11 variables were studied: HbA1c, duration of diabetes, baseline grade of DR, type of diabetes, sBP, haemoglobin level (2012), urine albumin creatinine ratio (uACR)(2012), triglycerides (2012), HIV status, age and sex. uACR did not demonstrate a linear association with probability of 2 step progression; a logarithmic transformation (base 10) was used in analysis. All tests were two-sided and a p value <0.05 was taken to indicate statistical significance. All calculations were performed using STATA version 12 . The study was approved by the University of Malawi, College of Medicine Research Ethics Committee and the University of Liverpool Research Ethics Committee. All participants gave written informed consent.Retinopathy grades were analysed by patient according to the worse or only gradeable eye. Visual acuity scores were analysed by patient according to the better eye. For the purposes of analysis visual acuity scores from 2007 (corrected Snellen acuities in the better eye) were converted to ETDRS letter scores using a standard conversion table . Compari2 test for trend). A total of 135 subjects were assessed between December 2011 and December 2012. Median time to follow up was 5.3 years (IQR 5.1–5.4). 15 subjects were confirmed dead.  . BaselinOf the 135 subjects seen in the 2007 study and subsequently traced and assessed in 2012, 15 (11.1%) were HIV positive at baseline. By 2012 17 (12.6%) were reactive: 13 taking ART; 3 known HIV+ but not taking ART; and 1 new diagnosis (WHO stage 3) . 21 subjBetween baseline and 5 years prevalence of any retinopathy, STDR and PDR increased from 29.6% (95% CI 21.9–37.3) to 63.0% (54.8–71.1), 12.6% (7.0–18.2) to 38.5% (30.3–46.7) and from 2.2% (0–4.7) to 5.9% (1.9–9.9), respectively (n = 135). Two (or more) step progression (from baseline) was observed in 48 subjects ; three (or more) step progression in 30 subjects . Of 115 subjects without STDR at baseline (2007) 34  had developed the condition at five years  Tables. Over the follow-up period 25 subjects (18.8%) lost 5 or more ETDRS letters of which 7 subjects (5.3%) lost 15 or more letters. Of these 25 subjects 2 (1.5%) progressed to moderate visual impairment (50–59 letters) and 3 (2.3%) became ‘severely visually impaired or blind’ (<50 letters). The most common primary causes of visual loss for the 25 subjects who lost five or more letters were DR (36%) both DR and cataract (20%) and cataract alone (28%). Therefore in 56% of cases DR was the sole or equal contributing cause of visual loss. In univariate analysis loss of 5 or more ETDRS letters was associated with presence of STDR at follow up visit  but not age  or duration of diabetes .Of the 135 subjects from the 2007 cohort 41 were recruited (by systematic random sampling from the QECH diabetes clinic) into our 24 month cohort study. Of the 41 subjects; 38 were seen at 12 months and 36 at 24 months providing 6 and 7 year longitudinal data . Of 34 sWe report the progression of grades of retinopathy and visual impairment over 5 years in a cohort of people with diabetes from Southern Malawi who had no access to laser treatment. Five year incidence of any DR in those without evidence of retinopathy at baseline was 48.4% (38.2–58.5). The five year incidence of STDR for those with level 10 and level 20 retinopathy at baseline was 19.4% (11.3–27.4) and 81.3% (62.1–100), respectively. The five year incidence of PDR for those with level 10, level 20, level 30 and level 40 retinopathy at baseline was 0%, 4.5%, 22% and 40%, respectively. Higher glycosylated haemoglobin (HbA1c) and lower haemoglobin level were risk factors for progression of retinopathy in multivariate analysis. Over the follow-up period 25 subjects (18.8%) lost 5 or more ETDRS letters of which 7 subjects (5.3%) lost 15 or more ETDRS letters. 2 subjects (1.5%) progressed to moderate visual impairment (50–59 letters) and 3 (2.3%) became ‘severely visually impaired or blind’ (<50 letters). In 56% of cases DR was the sole or equal contributing cause of visual loss.High quality cohort studies of DR from Africa or Asia are scarce. In our group’s separate prospective cohort study performed in Malawi between 2012 and 2014, 2 year incidence of STDR for subjects with level 10 and level 20 retinopathy at baseline was 2.7% (95% CI 0.1–5.3) and 27.3% (16.4–38.2), respectively . A subseDR progression at 5 years was associated with lower haemoglobin level. In our group’s prospective, 24 month cohort study lower haemoglobin was associated with presence of STDR at baseline (reported elsewhere ) but notTo our knowledge, ours is the first study to report longitudinal visual acuity data in African subjects with diabetes. Procedures for assessing vision differed between the baseline and final assessments. ETDRS measurements yield better VA than Snellen measurements and differences are more marked in persons with low vision –35. The This clinic-based study has limitations. Barriers to attendance include lack of knowledge of available health services, transportation costs, and competing tasks such as planting and harvesting staple crops. Patients living in rural locations endure long journeys to clinic and may represent a selected sub-group of the rural diabetes population. Few subjects with diet controlled diabetes attend the QECH diabetes clinic ,13 and tS1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(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.


To assess the influence of central obesity on the magnitude of the error of estimate of maximal oxygen uptake in maximal cycling exercise testing.-1] was estimated by the attained maximal workload and body weight using gender- and population-specific equations. The error of estimate [mL.(kg.min)-1] and percent error between measured and estimated maximal oxygen uptake values were compared among obesity quartile ranges.A total of 1,715 adults (68% men) between 18-91 years of age underwent cardiopulmonary exercise testing using a progressive protocol to volitional fatigue. Subjects were stratified by central obesity into three quartile ranges: Q1, Q2-3 and Q4. Maximal oxygen uptake [mL.(kg.min)p<0.05)) and for women ).The error of estimate and percent error differed (mean ± SD) for men (Q1=1.3±3.7 and 2.0±10.4; Q2-3=0.5±3.1 and -0.5±13.0; and Q4=-0.3±2.8 and -4.5±15.8 (Central obesity directly influences the magnitude of the error of estimate of maximal oxygen uptake and should be considered when direct expired gas analysis is unavailable. However, due to the limited availability of metabolic testing resources, VO2max is more commonly estimated, rather than directly measured, by applying equations that take into consideration the maximal workload achieved or the exercise duration 2max are strongly associated, the error of estimate (EE) for a given subject tends to be substantial, averaging 10-20%, which far exceeds the normal error of other clinical and laboratory measurements There is strong evidence that cardiorespiratory fitness is inversely associated with cardiovascular and all-cause mortality 2max, it is generally assumed that all subjects have the same mechanical efficiency, which is biologically incorrect and likely explains most of the EE of VO2max.The ability to utilize oxygen to perform work is related to an individual’s mechanical efficiency 2max in overweight and obese subjects. Currently, there is very little data available regarding the role of central obesity as a modulator of the EE of VO2max.Among the factors that likely influence mechanical efficiency, one of the most clinically relevant is body composition, primarily due to the increased prevalence of obesity in recent decades. Obesity and, more particularly, central obesity may hinder the mobilization of the lower limbs, possibly reducing the mechanical efficiency for activities such as cycling or walking 2max.Therefore, the current study was performed to determine the influence of central obesity, based on the waist to height ratio (WHtR), in the EE of VOOur study population included volunteer adult subjects in an exercise medicine clinic who initially underwent CPET using a progressive cycle ergometer protocol between January 2008 and June 2014. All patients were evaluated for exercise prescription purposes and provided informed consent. All patients authorized the de-identified use of their collected data for research purposes. The study and retrospective data analysis were approved by the institutional ethics committee. Subjects were excluded based on the following criteria: those who had previously been tested in our clinic, those who were younger than 18 years of age, those who had undergone treadmill testing, and those who did not fulfill the criteria for a maximal CPET. An additional 100 subjects who had incomplete data were excluded. The final population sample included 1,715 subjects.Body weight was measured to the nearest 0.1 kg while the subject was barefoot and wearing light clothing. Height and waist circumference were obtained to the nearest 0.1 cm. The latter was measured in the upright position, at the umbilicus level Maximal CPET was conducted using an electromagnetically braked cycle ergometer  and an individualized ramp protocol designed to achieve voluntary exhaustion between 8 and 12 minutes. All CPET was performed under direct medical supervision in a properly equipped laboratory. Subject seat height and body position were carefully adjusted on the cycle ergometer to provide a comfortable cycling movement. The pedaling rate was maintained between 65 and 75 revolutions per minute.One lead CM5 or CC5 digital ECG continuous monitoring  was obtained at rest, during exercise and at 5-minute recovery periods. Resting, exercise and post-exercise measurements of heart rate (HR) (bpm) and blood pressure (mmHg) were obtained from ECG recordings and by auscultation of the right brachial artery, respectively.2max represents the highest oxygen uptake value obtained, expressed as mL.(kg.min)-1, during CPET.During CPET, subjects expired through a mouthpiece and a pneumotachograph Prevent  with the nose occluded. Ventilatory analysis was accomplished using a metabolic analyzer VO2000  that was calibrated daily. During exercise, expired gases were continuously collected, and the results were recorded at 10-second intervals. The data from six consecutive 10-second intervals were averaged and reported for each minute during CPET. VOA CPET session was defined as maximum if it was not prematurely terminated due to adverse signs/symptoms and if it fulfilled physiological criteria 2max [mL.(kg.min)-1] values were predicted by gender-specific equations To better characterize the study sample, VO2max [mL.(kg.min)-1] was estimated by the gender-specific equations that were previously validated for a similar population in our exercise lab as follows: C-MEN = [maximal workload (watts)/weight (kg)] x 10.79 + 7 and C-WOMEN = [maximal workload (watts)/weight (kg)] x 9.82 + 7 VO2max, subjects were categorized by gender  and then divided into three quartile ranges according to WHtR: Q1, Q2-3 (combining Q2 and Q3) and Q4. For each of these quartile ranges, VO2max per kg of body weight, EE (measured VO2max - estimated VO2max) in mL.(kg.min)-1 and percent error /measured VO2max] x 100) were calculated for both men and women. Negative values of EE and %E signified that the estimated VO2max was higher than the measured VO2max, that is, the equation overestimated VO2max.To compare measured and estimated VO2max in men and women. One-way ANOVA was used to compare EE and %E among the quartile ranges for each gender. Statistical analysis was performed using Prism 6 , with 5% probability as the criterion for statistical significance.Descriptive statistics are presented as mean and standard deviation or as percent. Demographic characteristics and CPET variables were compared by t-test or chi-square test for each gender. Pearson product-moment correlation was also calculated for measured and estimated VOThe average age of our subjects  was 52±15 years, with men representing 68.3% of the total sample. Considering the entire study population, 20.8% were apparently healthy, 17.2% had known coronary artery disease, and the remaining subjects exhibited diverse clinical conditions, including arterial hypertension, diabetes mellitus, obesity, pulmonary disease, or combinations thereof. Based on body mass index (BMI), 41.5% and 23.2% of the subjects were classified as overweight and obese, respectively, including 1.4% of the total sample that was morbidly obese. Regarding prescribed medications, 23.0% of the subjects were taking β-blockers, 34.9% were on angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists use, and 39.8% were using cholesterol-lowering medications.vs. 0.526±0.082, p<0.01). The WHtR results for each of the quartile ranges for men were as follows: Q1=0.378 to 0.515, Q2-3=0.515 to 0.612, and Q4=0.612 to 0.900. The WHtR results for each one of the quartile ranges for women were as follows: Q1=0.332 to 0.467, Q2-3=0.467 to 0.571, and Q4=0.572 to 0.813. Additional demographic and CPET data, with specific reference to progressive WHtR ranges, are presented in As estimated by the WHtR, central obesity was higher in men was higher than in women . The maximal attained workload was higher for men than for women . As a group, men achieved a higher VO2max than women (29.4±10.5 vs. 24.1±9.0 mL.(kg.min)-1, respectively, p<0.01). Measured VO2max ranged from 5.8 to 73.3 mL.(kg.min)-1 in men and from 4.1 to 60.1 mL.(kg.min)-1 in women. For both genders, these values were consistently lower for those classified in the Q4 cohort of WHtR (21.9±7.0 the expression “for men” and 16.6±5.1 mL.(kg.min)-1 the expression “for women”, p<0.01). Measured and estimated values of VO2max were strongly correlated in both sexes . For the Q4 subset, the measured versus estimated VO2max relationship remained highly significant   .2max tended to be lower than age-predicted VO2max, corresponding to 96.6±26.9% and 81.7±24.5% for men and women, respectively. Considering only Q4 subjects, measured VO2max corresponded to an even lower percent of age-predicted VO2max in men and women, 80.0±20.3% and 64.5±14.4%, respectively.Measured VO-1 for men and 0.4±2.9 mL.(kg.min)-1 for women (p=0.54). The values of %E were 0.9±13.4% for men and -1.8±15.8% for women (p=0.20). An analysis of %E for the three WHtR quartile ranges revealed a clear trend, with Q1 showing a slight tendency to underestimate VO2max and Q4 showing a strong tendency to overestimate VO2max for both sexes. For Q4 men, the %E was -4.5±15.8%, which was significantly larger than the %E for Q1 and Q2-3 . For women, an even larger %E was identified in Q4 , and as observed in men, comparably lower %E values were seen in Q1 and Q2-3 . When comparing EE and %E by sex in each of the three quartile ranges, no differences were observed between men and women (p>0.05). Nevertheless, by analyzing the magnitude of the variability of the EE and %E results, as reflected by the standard deviation of the means, it is apparent that the EE of VO2max remained high for any given subject, especially among those with a higher WHtR.The values of EE were 0.5±3.2 mL.(kg.min)2max is estimated rather than directly measured 2max. This may be especially relevant in the current era of obesity 2max, these equations should be population-specific to account for the specific features of different populations, including body habitus.Although cardiorespiratory fitness is most accurately measured during CPET 2max by considering separate population-specific equations for men and women. The merit of this approach was substantiated by noting that the magnitude of EE of VO2max was similar for men and women and that there were no other significant gender differences related to VO2max. After having previously developed these equations 2max. This was based on the assumption that this variable may adversely decrease mechanical efficiency and, consequently, increase the EE. In the current analysis, rather than proposing specific equations that could correct for central obesity, we focused on analyzing the consistency of these influences and potential gender differences.Our study was uniquely focused on reducing the magnitude of error in estimating VO2max from the peak workload achieved during maximal exercise testing, some variables may influence the EE, including body composition. First, the relationship between workload and oxygen consumption during cycling, that is, the net mechanical efficiency, has been shown to vary among lean and obese individuals. Hulens et al. When estimating VO2. As previously described 2max assume that all individuals consume 3.5 mL of oxygen per kg of body weight per minute at rest. However, subjects with increased fat mass may have a higher absolute resting VO22 and, therefore, for its use in routine clinical practice Another possible explanation for our results is a difference in resting VOPrevious studies have addressed other variables in obese individuals that may influence the cardiovascular response to exercise. Gondoni et al. 2max determination. The retrospective analysis was undertaken by only one investigator who was not directly involved in data collection. The sample size was considerably large and most likely reflected the clinical profiles typically seen in a medically based office practice.This study has some methodologic features that strengthen the relevance of our findings. All CPET data were collected by four physicians in the same clinic, using the same equipment, under controlled environmental conditions and having followed standardized criteria for VO2max even when adjusted for age-predicted values, which suggests that age was not the only variable that contributed to the difference in mechanical efficiency. Second, subjects were not specifically evaluated based on the use of different medications , which may affect mitochondrial function 2max difference. Third, there are several techniques for categorizing obesity, ranging from more sophisticated approaches, such as computer tomography and DEXA, to simpler methods, such as bioimpedance or anthropometric measurements There are several limitations of this study that should be acknowledged. First, age is a potential confounding variable when considering mechanical efficiency 2max may lead to modest errors in some subjects and to significant errors in others. In our study, the application of a sex-specific equation identified a distinct magnitude of error when subjects with higher and lower WHtR were compared. In subjects with higher central obesity, VO2max values were systematically overestimated, while the opposite occurred among subjects with less central obesity, although to a lesser degree.Therefore, in agreement with our previous reports Although the aforementioned studies used BMI to categorize obesity and its relationship to cardiorespiratory fitness and mechanical efficiency, it is widely recognized that BMI has numerous limitations in assessing body composition, because it fails to differentiate body fat from lean mass, and in providing indices of overall adiposity, fat distribution and metabolic risk 2max was based on recent studies that have demonstrated that fat distribution has important clinical implications. Because central obesity is strongly related to visceral fat and increased cardiovascular risk In the present study, the WHtR provided a simple, non-invasive, anthropometric index to quantify central obesity. Moreover, unlike BMI, WHtR values are not significantly influenced by ethnic differences, age and sex, making it possible to compare central obesity among a heterogeneous cohort of men and women. Our selection of central obesity rather than overall obesity as the variable that may influence mechanical efficiency and the EE of VO2max, with a higher EE in those at the upper end of the WHtR distribution. For subjects classified in the upper WHtR quartile (men > 0.612 and women > 0.572), the use of currently available equations for estimating VO2max may lead to excessive errors and inaccuracies in clinical practice, especially when using functional capacity for risk stratification or in surgical clearance algorithms.In conclusion, central obesity as estimated by the WHtR influences the magnitude of the EE of VOAll three authors contributed sufficiently to this work to take public responsibility for appropriate portions of the content. de Souza e Silva CG and Araujo CG participated in the conception and design of the study and in the acquisition, analysis and interpretation of the data. Franklin BA participated in the data analysis and critically revised the manuscript for important intellectual content.


Acinetobacter baumannii bacteremia are grave because of the multi-resistance of the organism to antibiotics. This study aimed to determine the prevalence of A. baumannii isolated from blood cultures and to describe their antibiotic resistance patterns.A. baumannii were performed according to standard techniques of bacteriology and susceptibility testing as recommended by the CLSI. PCR was used to detect β-Lactamase genes, blaOXA-51, blaOXA-23.A retrospective longitudinal study was conducted on blood cultures between 2010 and 2014 from all Ibn Rochd University Hospital intensive care units; it was based on the exploitation of microbiology laboratory database (duplicates were excluded). Isolation and identification of Staphylococcus was isolated in 21.6% of cases followed by A. baumannii (9.2%), and K. pneumoniae (9.1%). A. baumannii strains were resistant to most antibiotics tested: imipenem (75.7%), ceftazidim (85.4%), cefotaxim (98.6%), gentamicin (78.1%), amikacin (63.5%) and ciprofloxacin (88.2%). All A. baumannii strains, resistant to carbapenem, tested were positive for blaOXA-51 genes and 87.5% expressed the blaOXA-23 genes.Among the 4232 samples received at the laboratory, 2402 (56.8%) were positive. Negative coagulase A. baumannii was the second germ frequently isolated from blood cultures in intensive care units. It was multi-resistant to antibiotics. The strengthening of hospital hygiene measures and surveillance of antibiotic resistance is needed to limit the spread of germs and to optimize the management of antibiotics. Acinetobacter baumannii is a Gram-negative pathogen. It is frequently associated with nosocomial infections . It was also recognized worldwide as an emerging cause of nosocomial outbreaks and listed by the American Society of Infectious Diseases (IDSA) as one of the six most hazardous microorganisms (A. baumannii bacteremia in intensive care units (ICUs) are responsible of a significant morbidity and mortality and create a therapeutic problem due to the multidrug resistance of the organism to antibiotics  and to describe the evolution of the organism’s resistance to antibiotics. Then, to detect the presence of Oxacillinases: OXA-23 and OXA-51 among the isolates showing resistance to imipenem.The aim of this study was to determine the prevalence of A. baumannii identified by the standard bacteriological methods in the routine diagnosis of bacteremia from all ICUs were examined. PCR was used to target the blaOXA-23 and blaOXA-51 among the imipenem resistant isolates A. baumannii.A retrospective longitudinal study was carried out on blood cultures from all ICUs in Ibn Rochd University Hospital between 2010 and 2014. It was based on the exploitation of the Microbiology laboratory database (duplicates of the same patient were excluded). All bacteria including A. baumannii. Disk diffusion test was used to determine the susceptibity of isolates to ceftazidim (30 μg), 30 μg cefotaxim (30 μg), 30 μg amikacin (30 μg), 10 μg gentamicin (10 μg), ciprofloxacin (5 μg), ampicillin/sulbactam (10 μg), piperacillin/tazobactam (85 μg), netilmicin (10 μg), Tobramycin (10 μg), trimethoprim/sulfametoxazol (25 μg), tetracycline (30 μg), imipenem (10 μg). E-test was used to determine the MICs for colistin (Biomérieux Marcy l’Etoile France). E. coli ATCC 25922 was used as a quality control strain. Results were interpreted according to the breakpoints recommended by the Clinical and Laboratory Standards Institute .Antimicrobial susceptibility testing was done for all A. baumannii resistant to imipenem were grown on Mueller–Hinton (MH) agar plates  for 18–24 hours at 37°C, bacterial cells were suspended in 500 μl of ultrapure water. Suspension was heated at 100°C for 10 min and immediately frozen at 0°C for 5 min. 300 μl of supernatant were then recovered after centrifugation of 14000 g for 10 min. Supernatant containing DNA was stored at −20°C until further use  and Microsoft Excel. The chi square test or Fisher’s exact test was performed to compare proportions. Differences were considered significant if the p-value is <0.05.A. baumannii (9.2%) and K. pneumoniae (9.1%)  were positive. Negative coagulase Staphylococcus was isolated in 21.6% of cases followed by e (9.1%) .A. baumannii strains increased from 7% in 2010 to 10% in 2011, 2012 and 2013, and then decreased slightly in 2014 (9%).The prevalence of A. baumannii strains were resistant to: imipenem (75.7%), ceftazidime (85.4%), cefotaxime (98.6%), gentamicin (78.1%), ciprofloxacin (88.2 %), the netilmicin (14%) and colistin (1%) (tin (1%) .p=0.0003) and then decreased slightly in 2014 (75%) (p=0.35)  .A. baumannii strains resistant to imipenem, determined by PCR, all A. baumannii strains were positive for blaOXA-51 genes and 87.5% (n=147) expressed the blaOXA-23 genes (As for the genotypic characterization of 23 genes .A. baumannii was the second germ frequently isolated from blood cultures in ICUs between 2010 and 2014 (9.2%). It was characterized by a multi-resistance to the antibiotics tested . It was however, sensitive to colistin in 99% of cases.In our study, A. baumannii, resistant to carbapenem, were positive for blaOXA-51 genes and 87.5% were positive for blaOXA-23.The evolution of antibiotic resistance during the study period was increasing especially for imipenem (from 50% in 2010 to 75% in 2014). All isolates of A. baumannii is considered as an opportunistic pathogen that can survive in austere conditions. It is responsible of an increasing rate of severe nosocomial infections. They affect especially immunocompromised patients, exposed to prolonged stays in ICUs and having a previous exposure to antibiotics; carbapenems and 3rd generation cephalosporins are the most involved, followed by fluoroquinolones, aminoglyco-sides and metronidazole , E. coli (15.4%) and S. epidermidis (14.4%) accounted for almost a half (48.3%) of blood cultures isolated microorganisms; Candida albicans represented 2.5%. A. baumannii was rarely isolated   .rd generation cephalosporins and ciprofloxacin, in Algeria, Tunisia and France, is over than 50% (Resistance to the 3than 50% –14 as itA. baumannii infections) is variable: 3.3% in France, 60% to 89.9% in Tunisia and 48% in Algeria  (A. baumannii carbapenem resistance (A. baumannii strains were positive for blaOXA-51 genes and 87.5% (n=147) for blaOXA-23.The acquired carbapenem resistance in (OXA-51) . OXA-23 sistance . In our A. baumannii harbouring OXA-23 enzymes have been identified in Brazil, Argentina and Colombia  and the use of new antibiotics such as tigecycline to overcome therapeutic impasses .A. baumannii was the second germ that was frequently isolated in blood cultures of intensive care units in the last five years in Ibn Rochd University Hospital. It was multi-resistant to antibiotics with a particular increasing resistance to imipenem. The strengthening of hospital hygiene measures and antibiotic resistance surveillance is needed to limit the spread of multi resistant bacteria and to optimize the management of antibiotic therapy.


Shigella to invade, colonize, and eventually kill host cells is influenced by many virulence factors. However, there is no analysis of related genes in Jiangsu Province of China so far. Shigella flexneri was collected from 13 cities of Jiangsu Province through the provincial Centers for Disease Control (CDC) for analysis of distribution of major virulence genes  detected by PCR technology.The ability of S. flexneri which belongs to 11 serotypes of S. flexneri, among which serotype 2a was the most predominant . All isolates were positive for ipaH gene, followed by sat (94.1%), sigA (78.9%), set1B (78.0%), pic (77.6%), set1A (74.5%), virF (64.8%), sepA (63.5%), sen (56.9%), ipaBCD (50.5%), ial (47.0%) and virB (47.0%). The presence of virulence genes in different serotypes was distinct. The existence of virulence genes of serotype 1b was generally lower than other serotype-the positive rate for virulence genes was between 0.0 and 14.1% except for ipaH and sat. In addition, virulence genes also fluctuated in different regions and at different times in Jiangsu province. The result of analysis on the relationship between virulence genes of S. flexneri showed that the existence of virulence genes of Shigella could be well represented by multiplex PCR combination ipaH + ial + set1A, which had a high clinical value.A total of 545 isolates received were confirmed as S. flexneri-associated virulence genes. The data showed high diversity of virulence genes with regard to periods, regions and serotypes in Jiangsu Province of China.The present study was designed to explore the prevalence of 12 The online version of this article (10.1186/s13099-017-0222-9) contains supplementary material, which is available to authorized users. Shigella species . Different serotypes for these species exist including more than 30 serotypes of S. flexneri which are categorized based on their O antigens, [Shigella-related infections led to 14,000 deaths per year. There are some factors contributing to the high prevalence of human Shigella infection. One reason for the high infection rate in some developing countries is the low sanitary conditions, knowing that Shigella spp. is transmitted via the fecal–oral route. Another important factor is that S. flexneri possess protective mechanisms that help it to survive even at high levels of acid in the stomach, which makes it highly infectious with only 10–100 microorganisms required to cause a disease [Shigellosis is major health burden in many parts of the world. It is an acute invasive enteric infection caused by four members of ntigens, . Althougntigens, . In a syShigella spp.—associated virulence factors, invasion plasmid antigen (ipa) B, C, D, and H as well as invasion-associated locus  facilitates its penetration into intestinal cells [flexneri because they are components of the type III secretion system (T3SS) which is important for S. flexneri and other gram-negative pathogenic or symbiotic bacteria in manipulating the host cell processes and establish a successful infection [Shigella enterotoxin 1 (ShET-1), Shigella enterotoxin 2 (ShET-2) and shiga toxin (stx) are among virulence genes encoding Shigella enterotoxins. A group of genes mostly found in S. flexneri serotype 2 clinical samples encode ShET1, a 55 kDa protein complex [Shigella [S. dysenteriae 1, but this species is rare in China [Shigella spp. harbors toxic factors like serine protease autotransporters of enterobacteriaceae (SPATE) of which there are two phylogenetic classes [Shigella IgA-like protease homologue (sigA) and secreted autotransporter toxin (sat) belong to class 1 which are toxic to epithelial cells, while non-toxic SPATE class 2 toxins includes sepA, which facilitates intestinal inflammation and pic, a mucinase associated with colonization.In children, main symptoms of shigellosis vary from mild to severe which include: diarrhoea characterized by presence of blood in stool, abdominal cramping, fever, among other gastrointestinal complications. Its clinical phenotypes are determined by different virulence genes and the activity of immune system of the host. Among the many al cells . As withnfection . Shigell complex , 7. ShETin China . The train China . Finally classes . ShigellS. flexneri virulence genes in some regions in China, investigations to dozen virulence genes of Shigella spp. mentioned above are still rare throughout the world, and to the best of our knowledge there is no report in China. To develop effective control strategies, it is important to conduct an epidemic study about Shigella in terms of its drug resistance and genetic features [Shigella-related virulence genes obtained from patients with diarrhea in Jiangsu Province of China, and discussed the genetic diversity and clinical applications of these genes.Although some studies reported the prevalence and distribution of features . For thiShigella spp. isolates from different patients with either diarrhea or dysentery in different hospitals in 13 cities by using routine biochemical techniques. Shigella is a class B infectious disease in China. The bacteria detected in any local hospital must be reported to the provincial CDC by the city’s CDC. The study was conducted in collaboration with the Provincial CDC, so the collection of Shigella was the most comprehensive.CDC-based real-time surveillance program in 13 cities of Jiangsu Province from 2010 to 2015  and automated biochemical analyzer , the collected samples were processed and screened. O and H antigens were examined using hyperimmune sera through slide agglutination test , and thereafter, the serotypes were grouped according to the Kauffmann–White scheme.Qiagen DNA mini kit was used for the extraction of DNA in line with the manufacturer’s instructions. The polymerase chain reaction (PCR) assays were performed targeting virulence genes using previously reported primers listed in Table Statistical analyses were performed using SPSS 16.0 database software. Distribution of different virulence genes in serotypes, periods and regions were analyzed by the Chi square test, and statistical differences between groups were considered to be significant for p < 0.05.S. flexneri during the 6-year period of investigation. These 545 strains were distributed in 13 cities of Jiangsu province. The prevalence ranked as follows: Taizhou , Suzhou , and Lianyungang . What’s more, only 20 (3.7%) strains were from Nanjing, the capital of Jiangsu Province  compared with the other serotypes. 2a, 2b, 1a, 1b, x and 4c were the six most frequently isolated serotypes, accounting for 96.5% of all S. flexneri, while other serotypes accounted for less than 1.5%. Six major serotypes had an obvious fluctuation over time (Table All the 545 isolates of S. flexneri showed that ipaH had the highest frequency (100%) followed by followed by ipaBCD (50.5%) and ial (47.0%)  Table . There wvirF genes while 256 (47.0%) isolates were positive for virB genes , S. flexneri 2b (46.2%), S. flexneri 1a (34.1%) and S. flexneri 1b (0.0%). virF gene was found in 93.3% of S. flexneri x, followed by S. flexneri 4c (77.8%), S. flexneri 2a (75.8%), S. flexneri 2b (73.6%), S. flexneri 1a (52.9%), S. flexneri 1b (1.6%).A total of 353 (64.8%) isolates tested positive for S. flexneri strains tested, 99.3% contained genes that encode SPATE proteins, such as class II  and/or class I  (Table S. flexneri strains was sat (94.1% of strains), following by sigA (78.9%), pic (77.6%), sepA (63.5%). Regarding differences between serotypes, a total of 86.7% S. flexneri x had two class I SPATE genes and two class II SPATE genes, following by 72.2% S. flexneri 4c, 69.1% S. flexneri 2a, 57.5% S. flexneri 2b, 38.8% S. flexneri 1a, and 0.0% S. flexneri 1b.Of the 545 set1A gene was present in 406(74.5%) S. flexneri isolates , and set1B was present in 425 (78.0%) S. flexneri isolates  (Table set1A and set1B were detected in 375 (68.8%) of S. flexneri isolates. The sen was present in 310 (56.9%) S. flexneri isolates .The c) Table . Both seS. flexneri was the lowest in 2012. There was no regular change in virulence genes between regions, such as the positive rate of invasion-associated genes was the highest in Yangzhou, followed by Lianyungang, but the highest positive rate of ShET-1 existed in Lianyungang, followed by Xuzhou  which are encoded by large virulence plasmids [ial gene has been identified in invasion processes and on inv plasmid [ial and ipaBCD on invasiveness of S. flexneri are not known. Numerous studies have shown that there is a link between the ability of the Shigella spp. strains to cause diarrhea and the presence of invasive genes in the bacteria. Mokhtari et al. [ial and/or ipaBCD. A study by Phantouamath et al. [S. flexneri’ isolates were positive for ial gene, and 50.5% S. flexneri’ isolates were positive for ipaBCD gene. Comparison with other similar studies, 78.9% S. flexneri’ isolates were positive for ial gene in Iran [ipaBCD gene, our result is similar to that of a study in Peru (49%) [S. flexneri in Jiangsu Province is not strong compared with other areas. Moreover, prevalence of virulence genes showed obvious serotype characteristics, such as none S. flexneri 1b expressed both ial and ipaBCD strains. But it should be noted that the pathogenicity of S. flexneri is also related to both the number of infected bacteria and the immunity of infected people.Since the information on the variety of  studies . The preplasmids , 27. ial plasmid . Many pr plasmid –29, whici et al.  showed th et al. , showed  in Iran , and eve in Iran . For theru (49%) , but lowru (49%) . in thisShigella virulence genes is regulated by heat-stable nucleoid structural protein (H-NS) which downregulates their transcription during unfavorable conditions for invasion. In response to favorable environmental signals, transcription of a series of genes is activated starting from AraC-like protein gene virF, which subsequently turns on transcription of virB regulatory genes. Thereafter, virB protein reverses the H-NS-induced inhibition on transcription which eventually turns on the virulence genes on the plasmid [virF and virB were found in 45.0% S. flexneri isolates, indicating that there might be other pathways for regulating gene expression. In addition, virF but not virB was found in 19.8% S. flexneri isolates, suggesting that virF regulated virulence genes not only through virB pathway. Interestingly, of the 545 S. flexneri, 11 strains had only virB, which may be due to loss of the virF gene. On the other hand, because of the importance of virF in regulating virulence genes, potential novel antibiotics targeting virF have gained increasing attention [Expression of  plasmid , 34.In tttention , 36. HowS. flexneri. One is called Shigella enterotoxin 1 (ShET-1), which is encoded in the set1 chromosomal gene. It has been suggested that in its active form, the ShET-1 toxin is composed of a subunit A (encoded by set1A) and five B subunits (encoded by set1B) [set1 genes were only detected in S. flexneri serotype 2 (2a and 2b) isolates and less so in other serotypes. In contrast, in the current study, many S. flexneri serotypes tested positive for set1 genes [set1 (set1A and/or set1B) was significantly lower than in other serotypes, such as S. flexneri 1b, S. flexneri 3b  . Other iy set1B) , which it1 genes , 12, 38.Shigella collections. A similar study in Iran found that the sat gene was present in all S. flexneri isolates, and the presence of sigA, pic and sepA genes simultaneously were existed in 35.5% of S. flexneri [Shigella was sat in our study, but the positive rate of the other three genes of SPATEs was significantly higher than that of Iran. Interestingly, sat is now recognized as a pathogenic E. coli, although it was initially studied in uropathogenic E. coli strains. In comparison with previous studies on the frequencies of sat gene in E. coli [sat gene in Shigella was found to be higher. It should be noted that except for sat gene, SPATEs of serotype 1b was significantly less than that of the other serotypes.Another factor that possess virulence activities is the Serine protease autotransporters of Enterobacteriaceae (SPATEs), which are toxins secreted from gram-negative bacteria. Nevertheless, only a few studies have searched for the presence of their encoding genes in large flexneri . Compari E. coli , 40, howShigella, which had been confirmed by previous studies. Some studies [Shigella by a PCR assay targeting the ipaH gene was higher than that by the traditional culture method. The disadvantage of this method is that it can only identify one virulence gene at a time, though this disadvantage could probably be overcome by multiple PCR techniques by screening the amplified genes in view of the difficulty of multiple PCR and the restriction of the number of amplified genes. IpaH can be used as a marker gene of Shigella to detect the Shigella. Four genes  are located on the chromosome SHI-1 Island, and the pic gene overlaps with set1A and set1B. When Shigella flexneri set1A gene was positive for Shigella flexneri, 94.1% Shigella set1B was positive, and 92.4% Shigella isolates were positive for pic and sigA. set1A positive Shigella had a stronger representation of the integrity of this segment of the gene. Because of the high expression of sat in Shigella, the clinical value of its amplification is not significant. Other virulence genes include ial, ipaBCD, virF, virB, sen and sepA, all of which are located on the large virulence plasmid (140 MDa). To reflect these virulence genes of Shigella, we chose the lowest existent ial gene as a marker and found that the positive rate of ial positive S. flexneri, ipaBCD was 98.8%, the positive rate of virF was 96.1%, the positive rate of virB was 92.6%, and the positive rate of sen and sepA was 94.5%. To sum up, multiplex PCR combination ipaH + set1A + ial can comprehensively reflect the virulence of Shigella.The virulence gene can be used to identify  studies , 42 repoS. flexneri in Jiangsu Province in China. It was found that the prevalence of these virulence genes varied greatly, leading to different severities of the disease. The profile of these virulence genes correlated with serotype, period and region. We found a low pathogenicity serotype (1b) and combination between those genes. These findings may help better control and identify Shigella strains.In the present study, we provided some baseline information about the distribution of some virulence genes in clinical strains of Additional file 1. Emporal variation of virulence genes in different serotypes of S. flexneri.Additional file 2. Regional variation of virulence genes in different serotypes of S. flexneri.


The widespread use of antiretroviral therapies (ART) has increased life expectancy in HIV patients, predisposing them to chronic non-communicable diseases including Chronic Kidney Disease (CKD). We performed a systematic review and meta-analysis (PROSPERO registration number CRD42016036246) to determine the global and regional prevalence of CKD in HIV patients.We searched PubMed, Web of Science, EBSCO and AJOL for articles published between January 1982 and May 2016. CKD was defined as estimated glomerular filtration rate (eGFR) <60ml/min using the MDRD, Cockcroft-Gault or CKD-EPI equations. Random effects model was used to combine prevalence estimates from across studies after variance stabilization via Freeman–Tukey transformation.Sixty-one eligible articles  in 60 countries were selected. The overall CKD prevalence was 6.4% (95%CI 5.2–7.7%) with MDRD, 4.8% (95%CI 2.9–7.1%) with CKD-EPI and 12.3% (95%CI 8.4–16.7%) with Cockcroft–Gault; p = 0.003 for difference across estimators. Sub-group analysis identified differences in prevalence by WHO region with Africa having the highest MDRD-based prevalence at 7.9% (95%CI 5.2–11.1%). Within Africa, the pooled MDRD-based prevalence was highest in West Africa [14.6% (95%CI 9.9–20.0%)] and lowest in Southern Africa . The heterogeneity observed could be explained by WHO region, comorbid hypertension and diabetes mellitus, but not by gender, hepatitis B or C coinfection, CD4 count or antiretroviral status.CKD is common in HIV-infected people, particularly in Africa. HIV treatment programs need to intensify screening for CKD with added need to introduce global guidelines for CKD identification and treatment in HIV positive patients. As a consequence, the spectrum of kidney diseases in HIV patients has broadened, ranging from asymptomatic changes in renal function like proteinuria, , 3 electIn this analysis, we synthesized available data on CKD prevalence in the adult HIV population at both regional and global levels. The overarching goal was to provide an essential basis to guide contextualized effective prevention and control strategies to tackle the burden of CKD in this population.2 irrespective of proteinuria status. Studies that reported CKD as eGFR <60ml/min/1.73m2 and/or persistent proteinuria were only included if we could compute the frequency of those with eGFR <60 ml/min/1.73m2 from available data in the article. We also included studies that reported CKD prevalence using a single estimated eGFR in order to accommodate studies from low-income countries where repeated serum creatinine measurement might not have been performed. A comparison of the pooled prevalence from studies with a single eGFR estimate and that with multiple estimates was also undertaken. We excluded studies with small sample size (<100 participants) and those including both adult and pediatric populations in which it was not possible to disaggregate data for adults. For studies published in more than one report (duplicates), the most comprehensive reporting the largest sample size was considered.The Preferred Reporting Items for Systematic Reviews and Meta–Analysis (PRISMA) 2009 guidelines  served aWe searched PubMed/MEDLINE, EBSCO, Web of Science and African Journals Online to identify all relevant articles reporting data on the prevalence of CKD in HIV-infected adults published from January 1, 1982 (when the HIV epidemic started) to September 30, 2016. We conceived and applied a search strategy based on the combination of relevant terms relating to HIV and CKD. The search strategy for Pubmed, web of science, EBSCO and AJOL is shown in The methodological quality of included studies was evaluated using the 9-point rating system developed by Stanifer et al  and modiTwo investigators (UEE and IGO) independently screened the titles and abstracts of articles retrieved from literature search, and the full-texts of articles found potentially eligible were obtained and further assessed for final inclusion . Disagreχ2 test on Cochrane’s Q statistic [2 [metaprop [package in STATA version 14.0 for Windows .A meta-analysis was used to summarize prevalence data. We pooled the study-specific estimates using a random-effects meta-analysis model (DerSimonian-Laird) to obtain an overall summary estimate of the prevalence of CKD according to the different eGFR equations across studies , after statistic  and quanectively). Subgrouectively). We asseectively). A p-valectively) and the metaprop  package The initial literature search retrieved 1220 articles of which 99 were selected after title and abstract screening for full-text review. Finally, 61 articles , 32–91 wIncluded studies reported on 209,078 HIV-infected adults from 60 countries. There were 46,295 participants (26 studies) from Africa; 52,785 (9 studies) from Europe; 52,305 (11 studies) from North America; 3,661 (4 studies) from South America; 49,147 (9 studies) from Western Pacific and 248 (1 study) from the Eastern Mediterranean. One study  from mulThe component studies had a sample size range of 163  to 41,862 = 98.9%, heterogeneity-p<0.001) using the MDRD equation, 4.8%  with CKD-EPI and 12.3%  with the CG equation (p = 0.003 for difference across GFR estimators)  . There wimators)  all p≥0.Using the MDRD equation, the African region had the highest prevalence estimate at 7.9% (95%CI 5.2%-11.2%) while the European region had the lowest estimate at 3.7% (95%CI 2.5–5.1%); p = 0.004 for difference across regions. Summaries of pooled prevalence by region and GFR estimators are presented in CKD prevalence was also high in Africa using the CKD-EPI equation: 7.0% (95%CI 2.8–12.9%). Studies using CG equation were mostly from Africa (84% of the studies), precluding sound regional analysis. The pooled prevalence of CKD in Africa from CG estimator was 13.7% (95%CI 9.1–19.0%); Of the studies performed in Africa, studies originating from West Africa had the highest pooled prevalence estimate using the MDRD equation: 14.6% (95%CI 9.9–20.0%) while the estimates from Southern Africa  were the lowest; p<0.001 for difference across African sub-regions, , Table 2One study  reportedOlder (≥median age 38.5 years) compared with younger participants (<38.5 years) had lower but non-significant difference in CKD prevalence: MDRD: 6.1% (95%CI 4.6–7.9%) vs. 6.9% (95%CI 5.2–8.8%), p = 0.54; CKD-EPI: 4.8% (95%CI 2.6–7.5%) vs. 4.8% (95%CI 2.3–8.2%), p = 0.98; CG: 8.5% (95%CI 4.5–13.6%) vs. 14.2% (95%CI 7.9–21.9%), p = 0.17. Substantial heterogeneity was apparent within age-groups regardless of the criteria . The CKD prevalence rates for patients with co-infection with hepatitis B and C, by level of CD4 count, by ART status and for those with comorbid systemic hypertension and diabetes mellitus are summarized in Twenty-one studies , 90, 91 Using the MDRD equation, the pooled prevalence of CKD reported from African studies that used two eGFR measurements was 4.2% (95%CI 1.4–8.3%) versus 8.9% (95%CI 5.3–13.3)  for those that used one eGFR measurement. For studies from North America, the pooled CKD prevalence for studies with at least 2 eGFR measures was 6.1% (95%CI 4.5–7.9%) compared to the pooled prevalence of studies with one eGFR measure of 7.6% (95%CI 5.2–10.4%), p = 0.34. The forest plot showing pooled prevalence for studies with two or more MDRD-based eGFR estimates across the WHO regions is shown in To our knowledge, this is the first attempt to provide prevalence estimates for CKD in HIV populations across various WHO regions. Prevalence was highest in Africa and lowest in Europe although the data shows substantial heterogeneity. Despite this, sociodemographic and clinical factors such as gender, age, coinfections with HBV and HCV did not significantly affect the estimates while coincident hypertension and diabetes mellitus had significant effect on the estimates. Paradoxically, our analysis did not reveal significant contribution to CKD prevalence of HIV related factors such as CD4 counts and ART status.2 in the general population is not clear but Stanifer et al[2 of 1.6%[The overall prevalence of CKD in HIV populations is high, regardless of estimator used. This is more so in Africa where the prevalence of CKD in the general population is already high . The higfer et al reported2 of 1.6% to 8.0%[2 of 1.6% using th2 of 1.6%, 96 in cConsistently, there was significant difference in the prevalence reported across the three estimators. Prevalence estimates obtained using the CG equation were generally higher than those obtained from MDRD and CKD-EPI with CKD-EPI being the most conservative of the three. In the general population, the CKD-EPI equation appears to outperform the MDRD and CG equations –99; howe2 for at least 3 months [Although not statistically significant in most of the comparisons, there was clearly a trend towards lower CKD prevalence estimates in the studies with more than one GFR estimate compared with those with only one estimate. This validates the KDIGO position of demonstration of GFR <60ml/min/1.73m3 months  before a3 months .Hypertension and diabetes mellitus remains significant risk factors for CKD in the HIV population as seen in this analysis when head-to-head comparison was performed between HIV only cohorts and HIV/hypertension or DM co-morbidities. Both hypertension and diabetes mellitus are age-related conditions and with the increasing age of HIV patients, a higher prevalence of CKD might be predicted in future in HIV positive patients. Both conditions, however, did not appear to explain some of the heterogeneities in CKD prevalence estimates, when comparison was made based on median hypertension or DM prevalence . This maThe effect of hepatitis B and/or hepatitis C on CKD occurrence in HIV patients has not been consistent. In this study, we found no significant difference in the pooled prevalence of studies with high hepatitis B or C co-infection compared with those with low prevalence of these viral co-infections. Some observational studies have found a higher risk of CKD , 90, 110One possible reason for the relatively high prevalence of CKD in African patients is late presentation to HIV care clinics at advanced stages of disease. This is evidenced by the significantly lower CD4 counts in African patients compared to the other regions. This may be compounded by late initiation of anti-retroviral medications giving adequate time for HIV–induced or related damage to the kidneys. In North America, ARTs are given to all HIV–infected individuals regardless of CD4 count to reduce morbidity and mortality associated with HIV infection . This isFurthermore, CKD in HIV patients may occur because of repeated episodes of undocumented AKI. AKI is common among HIV patients and is an important cause of morbidity and mortality in this patient group with sepsis and hypovolemia from diarrhea being the commonest causes –121. AKIThe higher prevalence of HIV–related kidney disease in African Americans compared to Caucasian Americans  and veryThe global HIV population is quite heterogeneous; male preponderance in North America and Europe whereas females constitute 60–70% of the HIV patients in the African studies reviewed. The influence, if any, of gender difference on the CKD prevalence remains unclear. The prevalence of traditional risk factors for CKD like hypertension, diabetes mellitus and Hepatitis C is also higher in North America and Europe than in Africa. The high prevalence of these modifiable CKD risk factors present a window of opportunity for sustaining therapies that may ultimately slow down CKD progression. The experience garnered from chronic care management of HIV could be leveraged as a platform for integration of non-communicable disease services into HIV populations. The different dimensions of HIV care–prevention, diagnosis, enrollment into care, disease management and palliative care—could also be useful for NCDs. The integrated care model appears to have achieved good results in parts of SSA  and emph2 and persistent proteinuria in this study. If the definition of CKD was made to include persistent proteinuria, then the prevalence of CKD among HIV patients may be much higher than reported in this study. The lack of information on specific antiretroviral drugs and their potential contribution to the burden of CKD in this work is a limitation.There is still inadequate information about the best creatinine–based eGFR formula for Africa in general  and the The burden of CKD in HIV positive patients is high globally, particularly in African patients. HIV treatment programs need to intensify routine screening for CKD at baseline and ART follow up clinics using relatively cheap and simple test for urinary proteins. There is now a great need to produce global guidelines for CKD identification and treatment in HIV patients and integrate treatment for chronic non-communicable disease with HIV patient care.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.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Background. The ABCD2 score is now identified as a useful clinical prediction rule to determine the risk for stroke in the days following brain ischemic attacks. Aim. The present study aimed to introduce a new scoring system named “ABCD2F” and compare its value with the previous ABCD2 system to predict recurrent ischemic stroke within 90 days of the initial cerebrovascular accident (CVA). Methods. 138 consecutive patients with the final diagnosis of ischemic CVA or TIAs who referred to emergency ward of Rasoul-e-Akram general hospital in Tehran from September 2012 to December 2013 were eligible. By adding a new score in the presence of atrial fibrillation to ABCD2 system, the new scoring system as ABCD2F was introduced and the risk stratification was done again on this new system. Results. The area under the curve for ABCD2 was 0.434 and for ABCD2F it was 0.452 indicating low value of both systems for assessing recurrence of stroke within 90 days of primary event. Multivariable logistic regression analysis showed that none of the baseline factors could predict 90-day recurrent stroke. Conclusion. ABCD2 and/or atrial fibrillation are not good scoring candidates for assessing the risk of recurrent stroke within first 90 days. Cerebrovascular events still remain as one of the major leading causes of death and disability all over the world. Only in the United States, the annual incidence of these events has been estimated to be 780.000 strokes, 600.000 as the primary strokes and others as recurrent attacks . The manIn this study, 138 consecutive patients with the final diagnosis of ischemic cerebrovascular accidents (CVA) or TIAs who referred to emergency ward of Rasoul-e-Akram Hospital in Tehran, Iran, from September 2012 to December 2013 were eligible for the study. The patients with hemorrhagic CVA or those who died within 90 days of discharge from the hospital were excluded . The pri t-test or Mann-Whitney U test. A receiver operating characteristic (ROC) curve was used to determine the value of scoring systems to distinguish occurring from nonoccurring recurrent stroke and also to determine the sensitivity and specificity. For the statistical analysis, the statistical software SPSS version 16.0 for Windows  was used. p values of 0.05 or less were considered statistically significant.Results were presented as mean ± standard deviation (SD) for quantitative variables and were summarized by absolute frequencies and percentages for categorical variables. Categorical variables were compared using Chi-square test or Fisher's exact test when more than 20% of cells with an expected count of less than 5 were observed. Quantitative variables were also compared withIn total, 138 patients were included within the study with the mean age of 65.48 ± 12.03 years (ranged from 35 to 88 years) and 64.5% of them were males. Regarding the type of stroke, 76.8% had an ischemic stroke, and others suffered from TIAs. In electrocardiographic assessment, 64.5% had the normal pattern, while atrial fibrillation was revealed in 6.5%; furthermore, 13.0% had evidence of cardiac ischemic events, and 5.1% had cardiac blocks. With regard to initial symptoms on admission, 71.7% had weakness plus speech abnormality, 12.3% had speech abnormality alone, and 15.9% had symptoms related to posterior circulation. The duration of symptoms in patients who suffered from TIA was less than 10 minutes in 4.3%, while the symptoms lasted more than 60 minutes in 11.6% of them. In total, only 3.6% were admitted to ICUs on admission time. Regarding hemodynamic indices, 52.9% had first systolic blood pressure higher than 120 mmHg, 30.4% had first diastolic blood pressure higher than 90 mmHg, and 29.7% had first pulse rate higher than 90 beats per minute. Assessing cardiovascular risk factors showed the history of CVA in 13.0%, history of TIAs in 1.4%, history of ischemic heart disease in 24.6%, and history of valvular heart disease in 5.8%. Also, previous atrial fibrillation was found in 2.2%. Moreover, 28.3% were diabetic, 56.5% were hypertensive, 15.2% were hyperlipidemic, and 23.2% were smokers. With respect to oral medications, aspirin was used in 26.1%, clopidogrel in 2.9%, and warfarin in 5.8%. The mean ejection fraction of left ventricle was 50.46 ± 9.46 percent and, in 20.3% of cases, the ejection fraction of left ventricle was less than 35%. Left heart clot was only observed in 1.4% and left ventricle aneurism was observed in 4.3%, whereas wall motion abnormality in echocardiography was found in 18.8%. The mean of total initial NIHSS was 8.38 ± 5.26 and 1.06 ± 1.98 in patients with stroke and TIA, respectively. Based on NIHSS, in patients with ischemic stroke, 23.8% had mild stroke, 61.9% had the moderate stroke, 13.3% had moderate-to-severe stroke, and only 1.0% had severe stroke.The follow-up of the patients for 90 days showed recurrent stroke, as TIAs or ischemic stroke was revealed in 9.4%.p = 0.406) and ABCD2F score . In total, a strong correlation was observed between the two scoring systems .No difference was found between the patients with and without 90-day clinical recurrent stroke in both ABCD2 score  and for ABCD2F it was 0.452 , indicating low value of both systems for assessing recurrence of stroke within 90 days of the primary event (Using the ROC curve analysis for determining the value of scoring systems to predict recurrent stroke, the area under the curve for ABCD2 was 0.434 (95% CI: 0.245–0.623; ry event .Multivariable logistic regression analysis  showed tThe current study attempted to assess ABCD2 scoring system of two aspects. First, we aimed to assess the value of this tool to predict occurring recurrent brain stroke within 90 days of first attacks. Second, we would like to examine whether adding occurrence of atrial fibrillation to this scoring system could enhance its value to predict recurrent brain stroke within this follow-up time. In this regard, the study had some important points. First, we showed that the ABCD2 scoring system was not valuable enough for predicting 3-month recurrent brain stroke. In other words, by using two statistical tools, including multivariable regression modeling and calculating area under the ROC curve, we could not demonstrate its high power to distinguish recurrent stroke. As previously noted, the ABCD2 scoring system was introduced to assess the risk for recurrent stroke during the first week of initial attack and thus its value to predict long-term stroke events had been already questioned. According to our results, it seems that the pure ABCD2 cannot be applied to assess mid-term or long-term recurrent cerebrovascular events, at least in our patients' population. Second, we also showed that even adding the parameter of atrial fibrillation could not improve the predicting value of ABCD2. In total, the low power of ABCD2 or its new version as ABCD2F can be explained by some reasons. First, it seems that, for improving its predictive value, we have to consider some other potential risk factors for delayed recurrent brain stroke admission etiologic stroke subtype, prior history of TIA/stroke, and topography, age, and distribution of brain infarcts. As also shown by Ay et al. , the newIt should be noted that the risk factors for recurrence of stroke may be more complex and confounding. One of these major confounding factors is the type of the treatment following first cerebrovascular accident. From this point of view, the origin of stroke is very important and choosing of the type of treatment depends on it. As noted before, atrial fibrillation plays an important role in cardioembolic strokes. Also, Ois et al. showed that severe symptomatic cerebral arterial diseases have approximately five times risk for recurrence of stroke within 90 days . The modIn conclusion, it seems that either ABCD2 scoring system or ABCD2F scoring system cannot effectively predict 90-day recurrent brain stroke in our population. In other words, ABCD2 and/or atrial fibrillation are not good scoring candidates for assessing the risk of recurrent stroke within first 90 days. In this regard, it is better to examine the value of other confirmed scoring tools such as the RRE-90 system or to discover other potential risk profiles to structure the new scoring systems to predict recurrent brain stroke.The ABCD2 and/or atrial fibrillation are not good scoring candidates for assessing the risk of recurrent stroke within first 90 days.