Atrial fibrillation (AF) is the most common arrhythmia seen in adults. Atrialstunning is defined as the temporary mechanical dysfunction of the atrialappendage developing after AF has returned to sinus rhythm (SR).We aimed to evaluate atrial contractile functions by strain and strain ratein patients with AF, following pharmacological and electrical cardioversionand to compare it with conventional methods.This study included 41 patients with persistent AF and 35 age-matched controlcases with SR. All the AF patients included in the study had transthoracicand transesophageal echocardiography performed before and after. Septum(SEPsSR), left atrium (LAsSR) and right atrium peak systolic strain rate(RAsSR) were defined as the maximum negative value during atrial contractionand septum (SEPε), left atrium (LAε) and right atrium peaksystolic strain (RAε) was defined as the percentage of change.Parameters of two groups were compared.1, 0.75±0.04s-1 vs1.35±0.04s-1, p<0.0001; RAsSR:1.13±0.06s-1, 1.23±0.07s-1 vs2.10±0.08s- 1, p<0.0001; SEPsSR: 0.76±0.04s-1, 0.78±0.04s- 1 vs 1.42±0.06 s-1, p<0.0001).In the AF group, 1st hour and 24th hour LAε, RAε, SEPε,LAsSR, RAsSR, SEPsSR found to be significantly lower than in the controlgroup (LAε: 2.61%±0.13, 3.06%±0.19 vs6.45%±0.27, p<0.0001; RAε: 4.03%±0.38,4.50%±0.47 vs 10.12%±0.64, p<0.0001; SEPε:3.0%±0.22, 3.19%±0.15 vs 6.23%±0.49, p<0.0001;LAsSR: 0.61±0.04s-Atrial strain and strain rate parameters are superior to conventionalechocardiographic parameters for the evaluation of atrial stunning in AFcases where SR has been achieved. In our study, we aimed to evaluate atrial contractile functions bystrain and strain rate, which is a new echocardiographic evaluation method inpatients with AF, following pharmacological and electrical cardioversion and comparethem with conventional methods.Atrial fibrillation (AF) is the most common arrhythmia seen in adults. Its incidenceincreases with age, and the total length of hospitalization is the longest of allthe arrhythmias.st hour, 24thhour and 1st month controls, and strain and strain rate examinations wereevaluated. The Vivid 7 Pro was used for echocardiographic evaluations, the 3.5-MHzprobe for TTE and the multiplane 6-MHz probe was used for TEE. Left ventricleejection fraction was measured by the modified Simpson method. The sampling volumewas placed parallel to the ventricle filling flow at the apical four-chamber planbetween the mitral and tricuspid valve endings, the peak early diastolic flowvelocity (E), the peak late diastolic flow velocity (A), the peak early and latediastolic flow velocity ratios (E/A) for pulsed Doppler evaluation and velocity-timeintegrals of peak early and late diastolic flow velocities, the left atrial ejectionforce [0.5×1.06×mitral valve area×(peak A velocity)2Kdyne] and the right atrial ejection force [0.5×1.06×tricuspid valvearea×(peak A velocity)2 Kdyne] were calculated and atrialfunctions were evaluated by conventional methods.We performed 45 elective echocardiographic evaluations. This study consisted of 41 ofthose 45 participants , whose SR was achieved with persistentAF and who applied to the cardiology department, and 35 control group cases  with SR and no severe valve disease on echocardiography. The studyprotocol was approved by the local ethics committee. Written informed consent wasobtained before enrollment. The cases with pre-procedure contraindications for oralanticoagulation, New York Heart Association class III-IV congestive heart failure,severe native valve and prosthetic valve disease, left atrial thrombus detected inTEE, sick sinus syndrome and hyperthyroidism, and left atrial diameter larger than5.5 cm measured with TTE were excluded from the study. All the AF patients includedin the study had TTE and TEE performed before cardioversion, as recommended by theAmerican Society of Echocardiography. After SR was provided, they had conventionalechocardiographic evaluations with TTE at the 1In the strain rate echocardiographic apical four-chamber evaluation, in an average158 number of frames, the peak systolic strain and peak systolic strain rates weremeasured from the left atrium, the right atrium and the inter atrial septum 5mmabove the atrioventricular junction. All values were found based on the average offour consecutive cycles. Peak septum systolic strain rate (SEPsSR), peak left atriumsystolic strain rate (LAsSR) and peak right atrium systolic strain rate (RAsSR) weredefined as the maximum negative value during atrial contraction and peak septumsystolic strain (SEPε), peak left atrium systolic strain (LAε) andpeak right atrium systolic strain (RAε) were defined as the percentage ofchange. After being recorded on digital media, the images were analyzed offline inthe EchoPac PC (GE Vingmed Ultrasound) program. Before cardioversion, AF cases weregiven aspirin and intravenous standard heparin infusion (17 u/kg) and the activatedpartial thromboplastin time was adjusted to remain at 1.5-2.0 times its value. AfterTTE and TEE, the cases without intracardiac thrombus were randomly divided into twogroups to receive either medical cardioversion with amiodarone infusion  or transthoracicdirect current cardioversion. For cases where SR was achieved, anticoagulation withwarfarin was provided, keeping international normalized ratio (INR) values at2.0-3.0. All other antiarrhythmic medications and digoxin were stopped beforecardioversion, while other medications were continued. Intravenous midazolam wasused for sedation. Transthoracic electrical direct current cardioversion wasperformed by using synchronized monophasic direct current with the cardioverterdefibrillator device. For cardioversion, in order, 200 joules, 300 joules and 360joules of energy were used. SR was achieved with electrical cardioversion in caseswhere SR could not be medically provided.t test or Mann-Whitney U test. For allevaluations, a p value < 0.05 was accepted as statistically significant.Was performed with the SPSS 10.0 program. Continuous variables are presented asmean ± standard deviation, whereas categorical variables are presented aspercentages. Categorical variables were evaluated by the Pearson chi-square orFisher's exact chi-square test, and continuous variables were evaluated by theunpaired Student The results from the 41 patients, among the performed 45 elective echocardiographicevaluations, where SR was achieved, were compared to those of the control group. Thecomparison of demographic characteristics of the two groups is demonstrated in The duration of AF was evaluated by patient history. The AF duration was estimatedaccording to the first diagnosed AF ECG date. The mean AF duration in SR achievedcases was 207.76±47.72 days. The mean heart rate in the AF group before theprocedure (94.29±2.21 bpm) was significantly higher than in the control group .Baselinst hour (2.61%±0.13) and 24th hour(3.06%±0.19) LA systolic strain were found to be significantly lower than inthe control group (6.45%±0.27) (p<0.0001). At the end of the first month,there was no significant difference between two groups. In the AF group,1st hour (0.61±0.04s-1) and 24th hour(0.75±0.04s-1) LA peak systolic strain rates were found to besignificantly lower than in the control group (1.35±0.04s-1)(p<0.0001). There was no statistically significant difference at the end of thefirst month. In the AF group, SEP systolic strains of the 1st hour(3.0%±0.22) and of the 24th hour (3.19%±0.15) were found tobe significantly lower than in the control group (6.23%±0.49) (p<0.0001).Similarly, there was no statistically significant difference at the end of firstmonth. In the AF group, the SEP peak systolic strain rates at the 1sthour (0.76±0.04s -1) and 24th hour(0.78±0.04s-1) were found to be significantly lower than inthe control group (1.42±0.06s-1) (p<0.0001). At the end of thefirst month, there was no statistically significant difference between two groups.In the AF group, RA systolic strains of the 1st hour (4.03%±0.38)and of the 24th hour (4.50%±0.47) were found to be significantlylower than in the control group (10.12%±0.64) (p<0.0001). In the AF group,the RA peak systolic strain rates at the 1st hour(1.13±0.06s-1) and 24th hour (1.23±0.07s-1) were found to be significantly lower than in the control group(2.10±0.08s-1) (p<0.0001). At the end of the first month,there was no significant difference in the RA peak systolic strain rate of the AFgroup (2.07±0.10s-1) when compared to that of the control group(2.10±0.08s-1).Sinus rhythm was obtained in 41 patients (91.1%) following cardioversion. AFrecurrence was observed in one patient 7 hours after the procedure, and in onepatient 10 hours after the procedure. At the end of the first month, a total of 32patients (71.1%) had SR. The atrial strain and atrial strain rate values of patientswho had achieved and remained in SR were compared to the control group. In the AFgroup, 1The flow rates through the mitral and tricuspid valve were evaluated in the AF groupand compared with the control group. The findings are shown in In the subgroup analysis of the study, patients who underwent cardioversion due to AFwere divided into two groups according to AF durations as less than 1 year (Group A)and more than 1 year (Group B). These groups were compared amongst themselves andthe control group (Group C). There were 24 patients in Group A with a mean age of57.63±2.13 years, 17 patients in Group B with a mean age of 61.38±1.70years, and there were 35 patients in Group C with a mean age of 55.83±1.65years. There was no statistically significant difference between the groups in termsof risk factors, body surface area, left atrial diameter, left ventricle size, leftventricle volumes, left ventricle ejection fraction and fractional shortening andvalve insufficiencies.st and 24th hour, they were significantlylower in Group B at the end of the 1st month . There was no significant difference between the two groups withrespects to SEP systolic strain and SEP peak systolic strain rate. When the valuesof Group B were compared to Group C, the left atrial systolic strain and rightatrial systolic strain, left atrial peak systolic strain rates and right atrial peaksystolic strain rates of Group B were found to be significantly lower. There was no statistically significantdifference found in Group A and Group C at the end of the 1st month interms of strain and strain rate parameters (When the patients in Groups A and B, who achieved SR after a successful cardioversionwere compared, while left atrial systolic strain and right atrial systolic strain,left atrial peak systolic strain rate and right atrial peak systolic strain ratewere similar in the 1rameters .st month for mitral and tricuspid valves A wave flow velocities,velocity-time integrals, diastolic E/A ratios and atrial ejection force measurements(When the conventional echocardiographic parameters were evaluated, there was nostatistically significant difference between the three groups at the end of the1urements.One of the primary results in our study was that in the early period, the contractilefunctions were depressed in both right and left atriums when cases with persistentAF, who had SR achieved following medical or electrical cardioversion, were comparedwith the control group. With conventional echocardiographic parameters, strain andstrain rate examinations, atrial functions were observed to recover at the end ofthe first month. The second, duration of AF before cardioversion is the mostimportant factor related to the severity of atrial stunning.2Atrial stunning is defined as the transient mechanic dysfunction that develops in theatrium and atrial appendage after SR is achieved by cardioversion in AF cases. Theincidence of atrial stunning is reported as being 38%-80%.4 Another factor that determines theduration and severity of atrial mechanical dysfunction is the size of the atrium. Inconducted studies, associations have been found between left atrial size and atrialstunning. When Mattioli et al.5compared cases with large left atriums with those of normal left atriums, they havedemonstrated that in large atriums, atrial stunning was more severe and prolonged.When left atrial appendage flow rates were evaluated with TEE in the study by Omranet al.,6 they found that, as theleft atrial size increased, the flow rates decreased. In our research, the aim wasto evaluate atrial contractile functions and atrial stunning with the newechocardiographic parameters of strain and strain echocardiography in patients whohad returned to SR after cardioversion and compare them to conventionalechocardiographic parameters.Atrial stunning is important in SR achieved patients following cardioversion becauseit can lead to thrombus formation and prepares a basis for thromboembolic events.The factors determining the duration and severity of atrial stunning include theduration of AF, size of the atrium and presence of underlying structural heartdisease. The duration of AF before cardioversion is the most important factordetermining the duration and severity of atrial stunning. As the duration of AFincreases, the rate of returning back from atrial stunning delays.11 In the study by Manning et al.,8 they have demonstrated that atrialstunning recovered immediately in cases where the duration of AF was shorter thantwo weeks before cardioversion, recovered within 24 hours in cases where duration ofAF was between two and six weeks and recovered in one week in cases longer than sixweeks. With patients having received pharmacologic and electrical cardioversion,Shapiro et al.11 have found nodifference in atrial stunning between cases with a less than one week AF durationand the control group in their study of AF. However, they did find significantlylower transmitral flow rates in AF cases with a duration longer than one week whencompared to the control group and the less than one week duration AF group.The duration of AF before cardioversion is the most important factor determining theduration and severity of atrial stunning. As the duration of AF increases, it takeslonger to recover from atrial stunning.st hour and 24thhour evaluations in terms of conventional echocardiographic parameters for atrialmechanical dysfunction, there was no significant difference found statistically whencompared with both control and AF cases of less than one year at the end of thefirst month. In all cases where SR was achieved, the "p" waves were visualized onECG after cardioversion, which reflects atrial electrical activity. However, atrialmechanical functions were found to be depressed with conventional echocardiographicparameters, strain and strain rate evaluations.In the subgroup analysis of our study, we compared the cases of AF duration longerthan one year, with the cases with duration less than one year and the controlgroup. In cases where the AF was longer than one year, at the end of the firstmonth, lower strain and strain rate values for the right and left atriums were foundcompared to cases where AF was shorter than one year and the control group. Whilesignificant results have been found in the 112 hasdemonstrated that before the left atrium expands, passive stretching of the atrialwall is compromised in cases with paroxysmal AF, since this pathology cannot bedemonstrated with methods of conventional echocardiography, they did demonstrate itin the early period of the disease with strain rate echocardiography. Modesto etal.13 have comparedconventional echocardiographic parameters with strain and strain rateechocardiography to determine the prevalence of atrial dysfunction in patients withcardiac amyloidosis. As a result of that study, while atrial functions were found tobe normal with conventional echocardiographic parameters in patients with cardiacamyloidosis, atrial dysfunction was demonstrated quantitatively in those patientswith strain and strain rate echocardiography. The results obtained in our studysupport the results of those two studies. Furthermore, mitral and tricuspid valvepeak A flow rates are affected by various factors, such as ventricle compliance,heart rate, site of the sample taken, and preload and afterload.15 The other conventional parameters, including E/A ratio, Awave rate-time interval and atrial ejection force, are used to calculate the peak Aflow passing the valves. In clinical and experimental studies, structural remodelingleading to fibrosis and atrial cardiomyopathy has been demonstrated in the atrium asa response to atrial arrhythmia. This structural remodeling is associated with theduration of the AF and is a progressive process. Sometimes structural remodeling maynot completely return to normal, especially due to the development of atrialfibrosis and atrial cardiomyopathy in cases with long duration of AF.16 In one study, atrial mechanicalfunction was deteriorated more severely in cases where AF lasted longer than threeyears and recovery of mechanical dysfunction lasted longer aftercardioversion.17 In ourstudy, with strain and strain rate echocardiography, it was demonstrated that atrialstunning continued at the end of the first month in cases with an AF duration longerthan one year. Due to the long duration of AF in these cases, the progressiveprocess of structural remodeling affects atrial myocytes more severely and preparesa basis for the development of atrial cardiomyopathy.The "A" wave, which reflects atrial contraction, was demonstrated on the Dopplerechocardiography of the mitral and tricuspid flows in all cases in the early periodfollowing cardioversion. However, atrial contractile dysfunction was detected withatrial strain and strain rate evaluation. Demonstration of the presence of an "A"wave is not adequate for the evaluation of atrial contractile functions with Dopplerechocardiography in cases where SR was achieved following cardioversion. Thesuperiority of strain and strain rate examination over conventional methods can beexplained by the direct and quantitative lengthening and shortening of atrialmyofibrils. There are limited numbers of studies in literature that evaluate atrialsystolic functions with strain and strain rate echocardiographic examination. Thestudy by Inaba et al.Atrial strain and strain rate echocardiographic evaluation is a useful method forthe evaluation of atrial stunning in AF cases where SR has been achieved. Incases having had cardioversion due to persistent AF, this new method is superiorto the A wave measured from the mitral and tricuspid valve with pulse Dopplerand the dependent parameters of A wave speed-time integral, E/A rate and atrialejection force in demonstrating the continuation of atrial stunning. Atrialstrain and strain rate echocardiographic examination reflects atrial systolicfunctions quantitatively, and can be used safely both in the diagnosis phase andin planning optimal medical treatment, including anticoagulant therapy in caseswith persistent atrial stunning.The main limitations of this study were the relatively small number of patientsand the short duration of follow-up. The limited number of patients in thisstudy may not represent the whole population. So, the inclusion of a larger andmore representative group of patients would provide a more comprehensivepicture. We did not evaluate the diastolic and systolic functions by hemodynamicand biochemical tests. Assessment of the relationship between strain, strainrate and diastolic and systolic functions by hemodynamic and biochemical testsmight be more illuminating.The results of this pilot study on atrial stunning showed atrial strain and strainrate parameters to be better than conventional echocardiographic parameters in AFcases where SR has been achieved. This result needs to be confirmed with a largergroup of patients and long-term follow-up studies.


The incidence of oropharyngeal cancer (OPC) in New Zealand (NZ) has more than doubled over the last 14 years with 126 cases in 2010. Overseas studies have shown that human papillomavirus (HPV) plays a significant role in the development of these cancers. However, the role of HPV in OPC and the burden on the NZ health system is unclear.The aim of the study was to determine the prevalence and the genotypes of HPV associated with OPC in New Zealand.In this study, 621 OPC were identified from cancer registry data from 1996–98, 2003–05, and 2010–12. Biopsies of 267 cases were then retrieved from laboratories throughout New Zealand. p16 immunohistochemistry and a human beta globin PCR were performed on all specimens. HPV genotyping was performed on all beta globin positive specimens using real-time PCR with melt analysis.Using a p16/PCR algorithm, 77.9% (95% CI: 71.1–83.5%) of cases were attributable to HPV. Of these, 98.5% were HPV 16 positive. There was also one case each of HPV 33 and 35. The percentage of HPV positive cases increased from 61.9% (95% CI: 40.9%– 79.2%) in 1996–98 to 87.5% (95% CI: 79.8%– 92.5%) in 2010–12. Results from the multivariable model, adjusted for sex and ethnicity found statistically significant associations between HPV positivity and timeframe , and between HPV positivity and patient age .® 9.This data is consistent with data from other developed countries showing an increase in cases of HPV positive OPC in New Zealand, and the majority of cases being attributable to HPV 16. These results support the recent inclusion of males into the nationally funded immunization schedule for Gardasil Head and neck squamous cell carcinomas (HNSCC) comprise cancers of the oral cavity, larynx, hypopharynx, and oropharynx . The incIn the past, the majority of HNSCC was seen in the larynx and oral cavity and was associated with smoking and heavy alcohol consumption . The incThe number of cases of OPC caused by HPV in New Zealand is unknown, however, when looking at anatomical site alone there has been an approximately threefold increase in the number of OPC cases from 35 cases in 1996 to 126 cases in 2010 . In contrast, the incidence of oral cavity cancers has remained steady during the same time period . CurrentEthics approval was obtained from the Health and Disabilities Ethics Committee (reference: 14/STH/128). Participant privacy was preserved by de-identification of samples.Cases were identified by a New Zealand cancer registry search of all cases of oropharyngeal cancer of ICD-10 codes C01, C05.1–5.2, C09.0–9.9, and C10.0–10.9 from 1996–98, 2003–05, and 2010–12. The New Zealand cancer registry is a population based register of all primary malignant diseases diagnosed in New Zealand. The information available from the registry comprises: patient demographics  and tumour specific information ). Data was cross checked with National Health Index (NHI) data to identify patients that were deceased. Study information sheets and consent forms were sent to all living patients whose samples were stored in laboratories with five or greater specimens in storage as per the laboratory code from the cancer registry data, and with current and complete address details. In addition the whānau (extended family) of deceased participants identified as Māori from the Auckland region were requested to give consent for these specimens to be used, as per local Iwi (tribe) wishes. All participants provided written informed consent prior to specimens being requested from storage. After specimen requests were made, laboratories sent the specimens (n = 267), a copy of the pathology report, and any existing p16 slides (n = 57) to Massey University.Samples with a historic p16 slide had the existing slide used in this study. Samples were recoded and read blinded. All other samples had p16 IHC performed at a diagnostic medical laboratory . Formalin fixed paraffin embedded (FFPE) tissue was used for IHC. Sections were cut at four microns and baked onto Superfrost Plus slides at 60°C for 60 minutes. Staining was performed on the Ventana Benchmark Ultra , with antigen retrieval performed on board, using Ventana CC1 buffer for 32 minutes at 100°C. A 1:150 dilution of the p16 (INK4a) antibody  , was incubated on the slide for eight minutes at 36°C. The detection of p16 was then visualised using the Ventana Optiview DAB kit . A multi-tumour block positive control containing a serous ovarian carcinoma was included with each run of samples. All slides were read independently by two pathologists (BL and UV), and discordant cases reviewed to reach a consensus. Samples showing strong diffuse staining in the nucleus and cytoplasm of greater than 75% of tumour cells were considered positive. Samples showing staining in 10% to 75% of tumour cells were considered focally positive, and samples with staining in less than 10% of tumour cells were considered negative.® Blood and Tissue kit  as per manufacturer’s instructions for tissue. A control extraction was included with each set of samples. The blank extractions were then used as negative controls in the beta globin and HPV 16 qPCRs. A pre-treatment for paraffin embedded samples was included, and a final elution volume of 100 μL was used. The quality of extracted DNA was assessed using a Nanodrop™ Spectrophotometer .A 10 μm slice of representative FFPE tumour block from each case with sufficient material (n = 224) was cut for DNA extraction using a new blade between each sample to avoid cross contamination of samples. DNA extractions were performed using the DNeasyTm of 81.0°C (±1.0°C). Samples showing low amplification on the qPCR were visualised using gel electrophoresis through a 1.0% agarose gel containing 1X SYBR® Safe (Thermo Fisher Scientific) to confirm product amplification.The presence of amplifiable DNA was assessed on all cases with extracted DNA by qPCR targeting the human beta-globin gene using the PC03 and PC04 primers . All priTm of 77.6°C (±1.0°C).The presence of HPV 16 was assessed on all cases with a positive beta globin qPCR result. A qPCR with melt curve analysis was developed using previously described primers . Each re®-T Easy kit with JM109 High Efficiency Competent Cells , according to the manufacturer's instructions with blue/white selection. The cloned plasmid contained an approximately 1.8 kb fragment of the HPV 16 genome that included the E6 and E7 gene sequence amplified using the primers detailed in A positive control was made from cloned DNA of a HPV 16 positive clinical sample provided by MedLab Central Ltd . Cloning was performed using a pGEM7 copies of HPV 16 were used to determine the limit of detection of the HPV 16 qPCR.Duplicate standard curves from eight, 10-fold serial dilutions starting from 9.56 x 102, 1X PCR buffer, 0.3 mM each dNTP, 1 unit Platinum™Taq , 1.5 μM Syto® 9 (Invitrogen). Up to 50 ng extracted DNA was used as template. Cycling conditions were 95°C for 10 minutes, followed by 40 cycles of: 95°C for 15 seconds, 49°C for 30 seconds and 72°C for 30 seconds. PCR was followed by a melt curve from 70–85°C with 0.2°C increments and a five second hold. Known HPV 16, 18, 33, and 52 positive samples were included in each run. Samples with a Tm in the range of 76.0–81.0°C were considered positive for HPV DNA. The GP5+/6+ PCR could not reliably discriminate HPV genotypes therefore, the product of samples positive for the GP5+/6+ PCR were purified using the PureLink® PCR purification kit . The HPV type present was then confirmed by sequencing as previously described.Samples that were p16 positive / focally positive, and HPV 16 negative were subject to qPCR using the GP5+/6+ primers . Each reThe continuous variable age was categorised into a binary variable: aged 60 years or younger, and 61 years or older. Ethnicity was classified into NZ European, NZ Māori, and other.To investigate the association between HPV positive OPC and putative risk or confounding factors , each factor was tested individually for significance at p < 0.2 in a logistic regression model using the software package R version 3.2.0 . A multivariable model was built by a stepwise selection process, retaining all variables significant at p < 0.05 and any confounding variables. Once a main effects model was built two-way interaction terms were introduced to the model and retained if significant at p < 0.05.The national dataset from the cancer registry comprised a total of 621 cases from the 1996–98 (n = 113), 2003–05 (n = 185), and 2010–12 (n = 323) time periods. Diagnosis was based on histology in 94.8% (n = 589) of cases, cytology in 4.6% (n = 29) of cases and other non-specified tests in 0.6% (n = 3) of cases. The proportion of cases diagnosed on histology versus cytology was consistent over all time frames. The mean age of an OPC patient was 59.2 years. NZ Europeans made up 68.8% of the national cases, and NZ Māori a further 10.5%. There was an approximate 4:1 ratio of males to females.Of the 344 living patients identified from the national dataset, 303 were able to be contacted, of which 52% (157/303) gave consent. Of the 157 cases, 109 specimens could be retrieved. There were 277 deceased patients identified in the national dataset. Of the 277 cases, 158 specimens were retrieved for use in the study.Thus, the total study population comprised 267 cases with retrievable specimens, and represented all major centres. Between four and 61 cases were retrieved from each of the 15 laboratories involved. The demographic factors of the national dataset and study population were comparable as shown by the high p-values for age, sex, ethnicity and timeframe . Therefop16 results were categorised as positive, focally positive, or negative . ConsensThere were 57 cases with a historic p16 slide. When comparing study p16 results to original results from cases with historic p16 slides, concordance was seen in 49/49 positives, 1/2 focally positives, and 5/5 negatives. The discordant focally positive case was identified as negative by our study. The remaining case had a slide sent for the study but no p16 result in the original pathology report.DNA could be extracted from 224 samples. Following the beta-globin qPCR  a furtheResults of the GP5+/6+ qPCR (n = 17) were 82% (14/17) negative, and 18% (3/17) positive . Of the For the purpose of further analysis, a sample is considered positive if positive by p16, and positive for either the HPV 16 qPCR, or a high risk HPV type was confirmed by sequencing. A negative sample is negative by p16, and HPV 16 qPCR negative. This approach resulted in an overall study prevalence of HPV positive OPC of 77.9% (95% CI: 71.1–83.5%). HPV 16 accounted for 98.5% of HPV positive OPC.The proportion of cases attributable to HPV increased from 61.9% (95% CI: 40.9%– 79.2%) in 1996–98 to 63.8% (95% CI: 49.5%– 76.0%) in 2003–05, and finally to 87.5% (95% CI: 79.8%– 92.5%) in 2010–12.The mean age of an HPV negative patient was 66.2 years (95% CI: 65.8–66.4), compared to 56.8 years (95% CI: 56.6–56.9) for an HPV positive patient. The univariate analysis of putative risk factors showed age (p < 0.01) and timeframe (p < 0.01) to be significant . In the The increase in the proportion of HPV positive cases between 1996–98 and 2010–12 seen in this study augments Ministry of Health data showing over a three-fold increase in OPC case numbers, regardless of HPV status between 1996 and 2010. Overall, the results of this study showed that the majority of OPC cases in New Zealand are caused by HPV. An HPV positive patient is more likely to be under 60. The younger age for HPV positive patients and increase in prevalence from this study are consistent with international data ,12,21. OThere was a higher proportion of deceased patients in our population than the national dataset (59% versus 45%) and this is likely due to the different consenting requirements. Due to the poorer prognosis of HPV negative tumours , the incWe requested 389 specimens from laboratories throughout New Zealand to capture our final study population of 267 cases. There were several reasons why cases were not retrieved, such as 1) there were logistical difficulties identifying cases , 2) cases were diagnosed on cytology only and had no biopsy / cell block, 3), specimens had been destroyed in the 2011 Christchurch Earthquake, and 4) specimens had been removed from storage and could not be located. Given the many reasons for samples not being included and the final comparisons of our study population to the national dataset, there was no reason to suspect any systematic bias in our sample.In New Zealand, laboratories are required by law to report any new diagnosis of cancer to the cancer registry. This generates complete national incidence data. Our study compared only risk factors with data available from the cancer registry . It is important to note these are not the only risk factors for HPV positive OPC and other significant factors such as alcohol consumption, tobacco smoking and sexual behaviours ,11,12 coIt should be noted, that our final criteria for a positive result was both p16 positive and either HPV 16 qPCR positive, or a high risk HPV type confirmed by sequencing. These criteria resulted in 172 results from 267 cases for analysis. This approach excluded focally positive cases and those cases that were p16 negative but HPV 16 positive by PCR. The exclusion of focally positive cases, and use of the p16 positive cut-off at 75% of tumour cells stained, aligned our definition of a p16 positive result with that used in many other studies ,12,21,25Although p16 is routinely used alone in the diagnostic setting , the use2 and 1.34 x 107 copies per 50 ng of DNA [et al [et al [The viral load in tonsillar cancer has been reported to be between 1.54 x 10g of DNA . Our HPVg of DNA ,28. JunoA [et al  reportedet al [et al [et al [While multi-levelled result categories or scoring systems for p16 have been reported –34, moreet al  and Lewil [et al ,34. Apprl [et al ,34. It il [et al , howeveret al, with HPV 6 the most commonly detected with a prevalence of 3.1% [Worldwide HPV is responsible for over 90% of HPV positive OPC . Co-infe of 3.1% . It is aet al, focused on tumour site alone without assessing HPV status, the results support the apparent increase seen in our study.Based on our results, a HPV positive patient is more likely to be under 60, and diagnosed recently (2010–12). The prevalence of HPV positive OPC in females appears to be increasing at a faster rate than in males based on our study data. However, interpretation of this apparent trend is limited due to the low number of cases in females in this study. There were no HPV positive cases in females in 1996–98, although there was one case that was p16 positive that could not have DNA extracted. This is compared to 20% of p16/HPV DNA positive being in females in the 2010–12 time period. A statistically significant increase in New Zealand OPC cases in females of 2.1% per year between 1982 and 2010 has been previously reported [et al [®9 in New Zealand. HPV types 16, 18, 6, 11, 31, 33, 45, 52, and 58 are included in Gardasil®9 [Chaturvedi et al  estimaterdasil®9 . TherefoS1 File(XLSX)Click here for additional data file.


Background. Date palm's pollen has been identified as a source of allergy; the rate of sensitization of this pollen is between 6 and 29%. Objective. To determine the prevalence of sensitization to date palm in Marrakesh and to identify the clinical profile. Patients and Methods. This study is based on a questionnaire and the prick test on 7 allergens, in population aged 5 years and above with clinical symptoms suggesting allergic diseases, from November 2012 to February 2013 in Marrakech. Results. We included 468 patients (women: 79.5%). The prick tests were considered interpretable in 467 cases. The prevalence of skin sensitization to pollen of date palm was 6.6%. The 31 cases of sensitization to date palm involved 7 men and 24 women with an average age of 37.5 years. Cutaneous monosensitization to date palm's pollen was observed in 2 cases. Asthma, rhinitis, and conjunctivitis were recorded, respectively, in 48.4%, 93.5%, and 67.7%. Conclusion. Skin sensitization to pollen of date palm does not seem unusual among allergic patients in Marrakech and is comparable to that found in Casablanca (7%), Barcelona (6.6%), and Cartagena (6.1%).  Marrakesh palm groves are considered as a historical monument  . Date pa Dermatophagoides pteronyssinus (DP), 314P3-ST for Dermatophagoides farinae (DF), 688P3-ST for pollen of the 5 grasses, 651P3 for olive tree pollen, 626P3 for cypress tree pollen, 668P3 for date palm pollen, and 301P3 for German cockroach. This study was conducted over four-month period from 1 November 2012 until the end of February 2013 at Marrakesh in six health centers, located near date palms implantation. We included in the study any patient aged 5 years and more, with clinical symptoms suggesting asthma and/or rhinitis and/or conjunctivitis and/or cutaneous manifestations such as eczema or urticaria. We used the histamine dihydrochloride (10 mg/ml) as a positive control to ensure skin reactivity and glycerol containing the diluent of the prick test solution as a negative control to eliminate dermographism. We considered a test positive if the wheal diameter obtained was 3 mm greater than the negative control and/or 50% greater than the diameter of the positive control. Data analysis was performed by the software Epi Info version 6, and statistical comparisons were made by the Chi-2 test.This is a descriptive cross-sectional study based on a questionnaire and the realization of a prick test (PT) on 7 allergens provided by Stallergenes Laboratory whose references are 315P3-ST for Parietaria in (10.1%), and cypress tree (9%) and before the 5 grasses (6%). Skin sensitization to different allergens is grouped in p = 0.018). In fact there is no female predominance because 7.3% of women sensitized to pollen of date palm against 6.5% men (p = 0.8). 16 patients of the 31 (51.6%) sensitized to the date palm reported the presence of date palm tree in their immediate surroundings and only 3 patients were directly exposed to this tree. Cutaneous monosensitization to date palm was noted in only 2 cases, among 29 other patients; skin sensitization was most commonly associated with the cockroach sensitization in 13 cases/29 which corresponds to 44.8% of cases, with cypress sensitization in 11 cases/29 (37.9%), with the olive tree sensitization in 9 cases/29 (31.0%), with the Parietaria sensitization in 8 cases/29 (27.6%), with the 5-grass sensitization in 8 cases/29 (27.6%), and with the mite in 7 cases/29 (24.1%). In terms of the clinical history and the physical examination, allergic manifestations were diverse. But it appears that rhinitis was an almost constant symptom and it was found globally in 29 cases/31 (93.5%) (p = 0.01). Asthma was found globally in 15 cases/31 (48.4%) (p = 0.6). Conjunctivitis was found in 21 cases/31 (67.7%) (p = 0.3). p = 0.04); this rate was at 23.3% for rhinitis (p = 0.02) and at 23.8% for conjunctivitis (p = 0.1). The average size of the wheal due to the date palm PT was 4.9 mm (range from 3 to 7 mm) against 7.6 mm +/− 2 mm for others allergens, in the general population studied.In our study, we included 468 patients, 372 women and 96 men with female predominance of 79.5%. The average age was 34.9 +/− 16.5 years with extremes ranging from 5 to 83 years. Asthma, rhinitis, and conjunctivitis were noted, respectively, in 53.4%, 73.7%, and 57.1%. The prick tests were performed in 468 cases and considered interpretable in 467 cases. They are considered positive in 190 cases or 40.7% of the total studied population. Allergy to pollen of date palm (6.6%) came in the sixth skin sensitization after house mite dust (17.9%), olive tree (12.4%), German cockroach (10.5%),Different types of studies have focused on determining the prevalence of allergy to pollen of date palm, but the populations were heterogeneous and not well defined. Furthermore, the used allergen extracts were not standardized; we used various allergens and various concentrations. All these factors make it difficult to compare the results of all the work developed significantly. In Spain, five studies have investigated the prevalence of skin sensitization to pollen of date palm in patients with respiratory allergic symptoms or pollinosis. In these patients, the prevalence of skin sensitization was between 6.11% and 29.41% . In BrusAllergy to pollen of date palm does not seem to be rare around the Mediterranean basin. Patients sensitized to pollen constitute a homogeneous group of patients characterized by frequent polysensitization and a clinical feature dominated by allergic rhinitis which is the most common clinical manifestation. Therefore, we recommend including the date palm allergen among the routine allergic skin prick tests.


To characterize hepatitis C virus (HCV) epidemiology in Pakistan and estimate the pooled mean HCV antibody prevalence in different risk populations, we systematically reviewed all available records of HCV incidence and/or prevalence from 1989 to 2016, as informed by the Cochrane Collaboration Handbook. This systematic review was reported following the PRISMA guidelines. Populations were classified into six categories based on the risk of exposure to HCV infection. Meta-analyses were performed using DerSimonian and Laird random-effects models with inverse variance weighting. The search identified one HCV incidence study and 341 prevalence measures/strata. Meta-analyses estimated the pooled mean HCV prevalence at 6.2% among the general population, 34.5% among high-risk clinical populations, 12.8% among populations at intermediate risk, 16.9% among special clinical populations, 55.9% among populations with liver-related conditions and 53.6% among people who inject drugs. Most reported risk factors in analytical epidemiologic studies related to healthcare procedures. Pakistan is enduring an HCV epidemic of historical proportions—one in every 20 Pakistanis is infected. HCV plays a major role in liver disease burden in this country, and HCV prevalence is high in all-risk populations. Most transmission appears to be driven by healthcare procedures. HCV treatment and prevention must become a national priority. FollowiThe Middle East and North Africa (MENA) region is the most affected region by HCV infection, with approximately 15 million individuals chronically infected . HCV is The objective of this systematic review is to characterize HCV epidemiology in Pakistan by: (i) systematically reviewing and synthesizing available published data of HCV incidence and prevalence in six population categories defined according to risk of exposure and (ii) pooling available HCV prevalence measures in each of the six pre-defined risk population categories to estimate population-specific pooled mean HCV prevalence.This work was conducted as part of the MENA HCV Epidemiology Synthesis Project, which aims to characterize HCV epidemiology in MENA to inform key public health research, policy, programming and resource allocation priorities –24.2.The methodology used in this study follows that used in previous systematic reviews of the MENA HCV Epidemiology Synthesis Project –17. The 2.1.All available records reporting HCV incidence and/or prevalence measures in Pakistan were systematically reviewed, as informed by the Cochrane Collaboration Handbook . Results2.2.Similar to our previous systematic reviews –17, all 2.3.The inclusion and exclusion criteria used in this study were adapted from our previous systematic reviews –17. BrieIn this work, for clarity, a ‘report’ refers to a document  including one or several outcome measures of those included in our systematic review, while a ‘study’ refers to any one specific single outcome measure. One report may contribute multiple studies , and multiple reports of the same outcome measure  were identified as duplicates and deemed as one study.2.4.Data from relevant reports were extracted by Z.A.K., of which 20% were double extracted by S.P.K. to ensure consistency. Nature of extracted data followed our previous systematic reviews –17. HCV Risk factors that were found to be significantly associated with HCV infection through multivariable regression analyses were extracted. HCV ribonucleic acid (RNA) prevalence among HCV antibody-positive individuals (that is HCV viraemic rate ) was extGeneral population (populations at low risk): these included blood donors, pregnant women, children, refugees, household-based survey participants and national army recruits, among others.High-risk clinical populations: these included populations exposed to frequent medical injections and/or blood transfusions, such as haemodialysis, thalassaemia, haemophilia and multi-transfused patients, among others.Populations at intermediate risk: these included populations whose risk of exposure is higher than the general population but lower than populations at high risk, such as healthcare workers (HCWs), household contacts of HCV-infected patients, patients with diabetes and prisoners, among others.Special clinical populations: these included clinical populations whose risk of exposure to HCV infection is difficult to ascertain, such as patients with non-liver-related malignancies, dermatological manifestations and rheumatological disorders, among others.Populations with liver-related conditions: these included patients with liver-related conditions of an epidemiological significance to HCV infection such as patients with chronic liver disease, acute viral hepatitis, hepatocellular carcinoma and liver cirrhosis, among others.People who inject drugs (PWID).The extracted data were synthesized by risk population in six distinct categories defined according to the risk of exposure to HCV infection as follows:2.5.The quantitative analysis approach was similar to that in our previous HCV systematic reviews –17. HCV The variance of the prevalence measures was stabilized using the Freeman–Tukey type arcsine square-root transformation . EstimatWith a recently identified potential issue with the Freeman–Tukey type arcsine square-root transformation , we condMeta-analysis of RNA HCV prevalence measures among HCV antibody-positive individuals (that is HCV viraemic rate) was also conducted to estimate the pooled mean of this prevalence measure.A sensitivity analysis was further performed to examine whether the advent of more specific and sensitive diagnostic tools (third or fourth generation assays) could have affected the prevalence estimates in the general population. Meta-analyses were performed on the general population prior to and after 2005, since after this year the vast majority of studies were likely to have been conducted using third of fourth generation assays. The results of the meta-analyses were assessed to determine whether the estimated pooled mean HCV prevalence was significantly different prior to 2005.meta [Meta-analyses were conducted in R v. 3.1.2. , using tmeta .2.6.The quality of HCV prevalence measures was assessed for each study as informed by the risk of bias (ROB) Cochrane approach , as wellStudies were considered as having high precision if the number of HCV tested individuals was at least 100 participants, as informed by previous studies –17.3.3.1.3.2.Our search identified one HCV incidence study, which reported seroconversion risk. This study included (as its baseline) HCV-negative HCWs who reported a needle stick injury from documented HCV-positive patients. After six weeks follow-up, investigators reported a seroconversion risk of 4.8% .3.3.3.3.1.n = 57), HCV prevalence ranged from 0.4 to 20.8%, with a median of 3.5%. Among pregnant women (n = 12), HCV prevalence ranged from 0.7 to 20.7%, with a median of 6.0%. Among outpatients (n = 9), HCV prevalence ranged from 4.4 to 51.0%, with a median of 9.0%. Among other general populations (n = 65), HCV prevalence ranged from 0.4 to 35.9%, with a median of 6.8%.Among the general population , our sea3.3.2.n = 12), HCV prevalence ranged from 7.7 to 60.0%, with a median of 42.2%. Among haemodialysis patients (n = 7), HCV prevalence ranged from 16.4 to 68.0%, with a median of 28.0%. Only one study was conducted for each of haemophilia patients  and multi-transfused patients .Among high-risk clinical populations , our sea3.3.3.n = 25), HCV prevalence ranged from 2.5 to 71.0%, with a median of 13.2%. Among HCWs (n = 11), HCV prevalence ranged from 0.0 to 5.6%, with a median of 3.2%. Among prisoners and/or volunteer prisoner blood donors (n = 9), HCV prevalence ranged from 8.7 to 18.2%, with a median of 13.1%. Among diabetics (n = 6), HCV prevalence ranged from 5.1 to 43.0%, with a median of 15.5%. Among household contacts of HCV index patients (n = 4), HCV prevalence ranged from 4.4 to 38.0%, with a median of 18.3%. A study conducted in Karachi among men who use roadside barbers measured HCV prevalence at 38.0% [Among intermediate risk populations , our search identified 64 prevalence measures/strata, ranging from 0.0 to 70.9%, with a median of 12.9%. Among hospitalized populations (at 38.0% .3.3.4.n = 4), HCV prevalence ranged from 3.0 to 23.4%, with a median of 7.7%. Among patients with urological conditions (n = 4), HCV prevalence ranged from 1.0 to 25.9%, with a median of 9.6%.Among special clinical populations , our search identified 18 prevalence measures/strata, ranging from 1.0 to 81.0%, with a median of 15.5%. Among patients with skin disorders (3.3.5.n = 20), HCV prevalence ranged from 4.9 to 78.4%, with a median of 41.1%. Among cirrhosis patients (n = 21), HCV prevalence ranged from 28.0 to 100.0%, with a median of 68.0%. Among hepatocellular carcinoma patients (n = 18), HCV prevalence ranged from 33.3 to 92.0%, with a median of 70.1%. Among acute viral hepatitis patients (n = 6), HCV prevalence ranged from 6.4 to 57.1%, with a median of 20.9%.Among populations with liver-related conditions , our search identified 73 prevalence measures/strata, ranging from 3.0 to 100.0%, with a median of 63.5%. Among chronic liver disease patients . The details of these measures can be found in the electronic supplementary material, table S6. HCV viraemic rate ranged from 44.4 to 98.0%, with a median of 74.2%.3.5.Pooled mean estimates for HCV prevalence for the six risk populations are summarized in Of note, the GLMM meta-analyses produced similar pooled mean estimates for all risk populations. For example, the pooled mean HCV prevalence for special clinical populations, that showed the largest difference between the fixed effects result and the random-effects result, was 13.1% (95% CI: 6.9–31.3) using the GLMM method versus 16.9% (95% CI: 6.2–31.3%) using the Freeman–Tukey type arcsine square-root transformation method.Q-statistic's p-value was always less than 0.0001; I2 > 93.7%). The prediction intervals were generally very wide. The totality of these heterogeneity measures indicates high heterogeneity in HCV prevalence measures in each risk population category.Statistically significant heterogeneity in effect size  was observed in all meta-analyses  was estimated at 74.1% (95% CI: 59.5–86.5%).The meta-analyses performed prior to and after 2005 among the general population, as part of our sensitivity analysis, estimated a pooled mean HCV prevalence of 5.0% (95% CI: 4.0–6.0%), and 6.5% (95% CI: 5.9–7.0%), respectively.3.6.Risk factors for HCV seropositivity were assessed in 11 studies using multivariable regression analyses. Healthcare-related risk factors were most commonly reported, including history of blood transfusions ,71,146, Injecting drug-use-related risk factors were also commonly reported, including history of injecting drug use ,146,148,3.7.Findings of the quality assessment are summarized in the electronic supplementary material, table S5. Only one study was identified for HCV incidence   was based on samples with greater than or equal to 100 participants, and were therefore classified as having high precision. Most studies (67.7%) reported specific details about HCV ascertainment, but nearly 70% did not report the assay generation. When information was provided, 94.2% of studies reported use of third or fourth generation assays.A sensitivity analysis was performed to assess whether HCV prevalence in the general population differed prior to and after 2005, because the vast majority of studies after this year were likely to have been conducted using third or fourth generation assays. The confidence intervals of the estimated pooled mean HCV prevalence prior to and after 2010 overlapped, indicating HCV prevalence was not significantly different between these two time durations.The majority of HCV prevalence studies (92.3%) used convenience, non-probability-based sampling approach. Nearly half of studies had low ROB in the response rate domain and 48.8% had missing information—only 1.6% of studies had high ROB in this domain.To summarize, 78.6% of studies had low ROB based on at least one domain, and 41.1% had low ROB based on at least two domains. Furthermore, 1.2% of studies had high ROB based on two domains, and no study had high ROB based on three domains. The totality of the quality assessment measures indicates reasonable study quality.4.We presented a systematic review and synthesis of HCV incidence and prevalence in Pakistan. Our results affirm that Pakistan has one of the highest HCV infection levels in both MENA –17 and wOur results collectively indicate a major role for healthcare in HCV transmission. High HCV prevalence was observed in the populations exposed to healthcare in one form or another. In high-risk clinical populations, the pooled mean HCV prevalence was high at 34.5% (95% CI: 27.0–42.3%) , with HCrelative (as opposed to absolute) role is probably small compared with healthcare procedures [Our assessment of HCV risk factors further indicates that HCV transmission appears to be primarily driven by healthcare-related exposures, such as therapeutic injections, intravenous infusions and poor sterilization of medical equipment ,54,71,87ocedures . These focedures ,21.The apparent major role for healthcare in HCV transmission distinguishes Pakistan from most other countries. Though healthcare plays a role in both developing and developed countries ,153–155,Of healthcare exposures, unnecessary therapeutic injections and reuse of syringes and needles were highlighted often as key factors ,158. Pakrelative contribution of injecting drug use to HCV incidence is probably substantially smaller than that of healthcare, although the exact quantitative contribution remains uncertain.The regional context of Pakistan and drug trafficking routes  support Pooled mean HCV prevalence estimates), are chronically infected with HCV and can transmit the infection further. In spite of heavily discounted prices for DAAs in Pakistan [Our results highlight the urgent and immediate need for expansion of HCV treatment and prevention programmes in Pakistan. High HCV prevalence was observed among all risk populations , with abPakistan , treatmePakistan . To reacPakistan . To addrPakistan .Our study has identified key gaps and weaknesses in HCV epidemiological evidence in Pakistan. Despite the large epidemic, only one (now outdated) nationally representative and probability-based population-based survey was conducted in this country . RepeatiDespite the major role for healthcare, a relatively small number of studies have been conducted among clinical populations, or investigated healthcare-related exposures. This is to be contrasted, for example, with Iran where a large number of studies investigated the role of healthcare—despite the relatively small role of this mode of exposure in this country . Hardly Our study is limited by the quantity and quality of reviewed studies, as well as their representativeness of the different risk populations—most studies used convenience sampling as opposed to probability-based population-based sampling. Only PubMed and Embase databases were searched, but other HCV data may exist in unpublished (grey literature) form, or are published in non-indexed journals. There was extensive heterogeneity in HCV prevalence measures in each risk population—possibly because of variability within the specific studied subpopulation, geographical location, sex and age-group representation in the sample, sampling technique and participant recruitment, year of study and study quality.Despite these limitations, the main strength of our study is that we identified a large number of studies that covered different risk populations, and that facilitated a comprehensive synthesis of evidence and identification of gaps and weaknesses that preclude a satisfactory understanding of HCV epidemiology in Pakistan.5.Pakistan is enduring an HCV epidemic of historical proportions—one in every 20 Pakistanis has been already infected with this infection playing a major role in liver disease burden in this country. HCV prevalence is high in all risk populations with most transmission apparently driven by healthcare procedures. Though our knowledge of the specific modes of exposure that drive transmission is improving, our understanding is still hampered by key gaps and weaknesses in available evidence. Conduct of repeated and comprehensive nationally representative and probability-based population-based surveys is critical to assess HCV prevalence and trends, identify risk factors and modes of exposure, examine the spatial variability in prevalence, and assess HCV knowledge and attitudes.HCV treatment and prevention must become a national priority in Pakistan. Although Pakistan has made efforts to increase coverage of safe injection and blood screening and to improve infection control ,182–184,


Streptococcus suis is a zoonotic cause of severe meningitis and sepsis in humans. We aimed to assess the long-term outcomes in patients who survived S. suis infection, in particular the progress and impact of vestibulocochlear sequelae.S. suis infection at discharge and 3 and 9 months post-discharge for 47 prospectively enrolled cases and at 11–34 months for 31 retrospectively enrolled cases. Outcomes in patients were compared to 270 controls matched for age, sex and residency.This case-control study evaluated outcomes of The prevalence ratio (PR) of moderate-to-complete hearing loss was 5.0(95%CI 3.6–7.1) in cases at discharge, 3.7(2.5–5.4) at 3 months, 3.2(2.2–4.7) at 9 months, and 3.1(2.1–4.4) in retrospective cases compared to controls. Hearing improvement occurred mostly within the first 3 months with a change in hearing level of 11.1%(95%CI 7.0–15.1%) compared to discharge. The PR of vestibular dysfunction was 2.4(95%CI 1.7–3.3) at discharge, 2.2(1.4–3.1) at 3 months, 1.8(1.1–2.5) at 9 months, and 1.8(1.1–2.6) for retrospective cases compared to controls. Cases also indicated more problems with mobility, self-care and usual activities.Both hearing and vestibular impairment were common and persist in cases. Appropriate patient management strategies are needed to reduce the incidence and impact of these sequelae. Streptococcus suis is a commensal bacterium in the respiratory, genital and digestive tract of pigs, which can infect humans through penetrating injuries or consumption.S. suis infection is common in East and Southeast Asia.4S. suis infection, less is understood about the frequency, persistence and impact of clinical sequelae. Reported rates of hearing loss vary greatly between studies, from 6% to 100%, with the weighted pooled estimate at 39% in a recent review and meta-analysis.S. suis meningitis prospectively to quantify the prevalence of sequelae. This clinical trial of adjuvant dexamethasone found 26/46 (56%) of cases with hearing loss in the placebo group and 15/41 (37%) in the intervention group at 6 months, but did not report on vestibular dysfunction.S. suis infection.Despite the growing literature on the epidemiological, microbiological and clinical aspects of S. suis infection at the National Hospital for Tropical Diseases (NHTD) in Hanoi, Viet Nam. Patients were assessed at discharge and after 3 and 9 months since discharge. To assess the long-term health outcomes beyond 9 months we retrospectively enrolled patients admitted to NHTD between January 2013 and October 2014 with laboratory confirmed S. suis infection who were assessed once using the same assessment tools.This case-control study includes prospectively and retrospectively enrolled patients, and controls. We followed up prospectively enrolled patients admitted between November 2014 and October 2015 with laboratory confirmed S. suis infection or meningitis. We assumed that these controls represent the source population from which the cases were derived, and that, except for the studied exposure, both patients and controls were similar in terms of the baseline risk of hearing and vestibular dysfunction. All cases and controls gave written consent to participate in the study. The study was approved by the institutional review board of NHTD and the University of Oxford Tropical Research Ethics Committee (OxTREC).Four non-patient visitors at NHTD were matched for sex, age (±5 years) and location  with cases recruited between November 2014 and August 2016 and enrolled as controls. Exclusion criteria included a history of Baseline characteristics were collected from the hospital charts (cases) and through interviews (both cases and controls). Main outcomes including hearing, balance, history of vestibular symptoms, impact of hearing impairment and dizziness, visual acuity, cognitive function and overall health status were assessed for all cases and controls as described below .Air conduction audiometry was conducted according to a standardized protocol by trained staff in a sound-proof booth  or in a quiet place (for patients who were visited at home), following a modified Hughson-Westlake automated procedure . The hearing threshold in each ear was tested for frequencies from 0.5 to 8 kHz with the first threshold tested at 1 kHz. Pure tone average (PTA) is defined as the average of hearing thresholds measured over four frequencies 0.5, 1, 2 and 4 kHz in the better ear. Impairment severities are categorized as normal (PTA < 20 dB), mild (20–34 dB), moderate (35–49 dB), moderately severe (50–64 dB), severe (65–79 dB), profound (80–94 dB), and complete (≥95 dB),The Modified Clinical Test of Sensory Interaction and Balance (m-CTSIB) was used to assess vestibular function.The Vertigo Symptoms Scale (VSS) was used to assess the subjects' recall of experiencing 34 items in the previous 12 months, a score between 0 (never experienced) to 4 (quite often) is given to each symptom.We used the Hearing Handicap Inventory for Adults (HHIA)We compared the prevalence of disabling outcomes among cases and controls, and the change in the main outcomes over time. We determined the minimum sample size to detect a difference of 25% in the prevalence of vestibular dysfunction between cases and controls (assuming a similar rate of 25% among Vietnamese controls as reported among adults aged 40–60 years in the U.S.).To compare outcomes between cases and controls, we used log-binomial models and calculated adjusted prevalence ratios (PR), the ratio between the probability of an outcome for cases and for controls after considering potential confounders.nlme package.To evaluate the change in hearing thresholds among cases over time, we excluded patients showing no response to sound stimuli at the four frequencies used to calculate the PTA and ran a linear mixed model controlling for between-patient variations (random effect) over the main effect of interest (assessment time-points) using the S. suis infection at NHTD between November 2014 and October 2015, 47 were enrolled as cases prospectively (From 76 patients admitted with laboratory-confirmed ectively . Thirty ectively . Almost Profound-to-complete and moderate-to-severe hearing impairments were found in 14/44 (32%) and 23/44 (52%) of patients at discharge; this proportion fell to 13/45 (29%) and 13/45 (29%) at 9 months, respectively . ImpairmAfter discharge, hearing improved in 18/29 (62%) and 29/42 (69%), while deteriorated in 3/29 (10%) and 5/42 (12%) cases at 3 months and 9 months, respectively . Most imBalance disorder was observed in 37/44 (82%) of cases at discharge, and in 20/30 (67%) and 30/45 (65%) at 3 and 9 months, respectively . ImproveCases also reported more symptoms with a mean VSS score of 18.7 (95%CI12.0-25.5) at 3 months, 12.7 (8.6–16.7) at 9 months, and 22.1 (16.1–28.1) for retrospective cases, compared to 10.4 (9.1–11.7) in controls. Dizziness and unsteadiness, but not vertigo, were significantly more common in cases at 9 months and retrospectively enrolled cases . All thrProportion of cases experiencing problems on the five health domains is between 13–33% for cases at 3 months, 13–22% at 9 months and 3–32% for retrospectively enrolled cases . Cases rS. suis infection during 9 months post hospital discharge, and we evaluate the same outcomes in a group of retrospectively enrolled cases 11–34 months after discharge. We found significantly increased rates of hearing and vestibular impairments among cases. Profound/complete hearing loss (PTA ≥ 80 dB) was common among cases, which did not or hardly improve after 9 months. Likely, severe hearing loss post S. suis infection is irreversible given the similar rates between prospectively (at 9 months) and retrospectively (at 11–34 months) enrolled cases. Improvement within the first three months was observed among cases with non-severe hearing loss (<80 dB), consistent with current literature on hearing recovery after bacterial meningitisS. suis patients in the placebo group (33.3% at discharge and 21.7% after 6 months).S. suis infection may improve in the first three months post-discharge whereas severe hearing loss may not. Regardless of severity, the hearing status after 3 months is likely irreversible. We also explored the effect of corticosteroid treatment on vestibulocochlear function over time, but found no significant impact .To measure vestibular outcome, we used the postural metric m-CTSIB, which has been reported to have 90% sensitivity and 95% specificity in patients with vestibular disordersS. suis infection have a significantly lower health status than controls. The prevalence of experiencing problems across the five EQ-5D domains among patients in this study (aged between 28–79 years) are comparable to a sample from the general population aged 60 years and older in a rural setting in Viet Nam (10–39%).Survivors of One limitation of this study was that age matching was not fully achieved, as a result we did not reach the ratio of 1:4 for cases and controls. Therefore we included all recruited controls in the analyses for both case groups, and age was included as a potential confounder in our analyses. Secondly, 9-month audiometry was done at the home environment for some patients, which may have led to an overestimation of hearing loss prevalence and severity among cases. Nonetheless, cases evaluated at home had a smaller PR than those evaluated in the sound-proof booth as shown in the analysis stratified by location of audiometry at 9 months, despite having overlapping CIs (see notes in S. suis infection suffer from serious hearing and vestibular impairment. Hearing mostly improves in the first 3 months after discharge among the less severely affected cases, while vestibular dysfunction shows little recovery. Patients also experience multiple symptoms, which aggravate the impact of disease sequelae on their health and quality of life. Further research is required to identify appropriate treatment strategies to reduce the incidence and severity of hearing and vestibular impairment in survivors of S. suis infection.In conclusion, this study is the first to provide strong evidence that a large proportion of patients who survive All authors declare that they have no conflicts of interest in relation to the present manuscript.The funder had no role in the design, collection, analysis, interpretation of the data, writing the report, or in the decision to submit the manuscript.


NCR and FPR were determined in 254 paired urine/blood samples collected simultaneously from 254 women at median gestational week (GW) 24  without hypertension or significant proteinuria in pregnancy (SPIP). Fifteen (5.9%) developed SPIP and hypertension at GW 34.0 (26.0–38.6) and 35.3 (27.6–38.6), respectively, and were diagnosed with PE at GW 35.7 (27.6–38.6). The 90th percentile level determined in 239 women normotensive throughout pregnancy gave NCR (139) sensitivity and positive predictive values (PPV) of 60% (9/15) and 27% (9/33), while those for serum FPR (4.85) were 40% (6/15) and 20% (6/30), respectively. Relative risks (95%CI) of later PE were 10.0  and 4.98  for NCR-positive and FPR-positive women, respectively. Cut-offs suggested by ROC gave NCR (86.6) sensitivity and PPV of 87% (13/15) and 17% (13/79), and FPR (8.8) values of 40% (6/15) and 40% (6/15), respectively. Thus, 2nd trimester NCR was superior to FPR for PE prediction.This prospective observational study compare urine nephrin:creatinine ratio  with serum soluble fms-like tyrosine kinase-1:placental growth factor ratio  for preeclampsia (PE) prediction among unselected asymptomatic pregnant women in 2 Nephrin is a podocyte-specific transmembrane protein that is predominantly localized at the glomerular slit diaphragm of podocytes2345935nd trimester cut-off level of PCR < 0.27 can differentiate between women with higher and lower risks of later developing PEProteinuria increases gradually with advancing gestation even in normotensive women612Circulating maternal serum levels of soluble fms-like tyrosine kinase 1 (sFlt-1) are increased and placental growth factor (PlGF) levels are decreased in women with PE1416171819202122231617181920212223Thus, all three variables, i.e., nephrinuria corrected by urine creatinine (NCR), PCR, and FPR, can be used as biomarkers of PE. To our knowledge, however, there have been no studies comparing NCR with FPR with regard to prediction of later development of PE. The present study was performed to compare NCR with FPR as well as PCR with regard to prediction of later PE among unselected asymptomatic women.Of the 254 women included in this study, 15 (5.9%) later developed SPIP at gestational week (GW) 34.0 (26.0–38.6) and hypertension at GW 35.3 (27.6–38.6) and were diagnosed with PE at GW 35.7 (27.6–38.6), while the remaining 239 women developed neither SPIP nor hypertension . In the Although all 254 women were normotensive and not proteinuric at blood/urine sampling, all biomarkers except PlGF differed significantly between women that did and did not develop PE later .We were concerned with degradation of urine nephrin detectable by our method with increasing number of days after specimen sampling. However, time intervals until assay of nephrin after sampling appeared not to affect the NCR level .th  or 10th percentile (for PlGF) levels in the 239 women without later PE development as shown by red lines in th percentile NCR level was superior to the corresponding FPR level with regard to both sensitivity  and PPV   . The relctively) . The cutctively) .NCR gradually increased significantly toward onset of SPIP, but not toward hypertension . FPR incr values were within the range of −2.49 to 2.49  of women with positive results on both NCR and FPR tests later developed PE, while only 2.0% (4/197) of women with negative results on both NCR and FPR tests showed later development of PE r values To our knowledge, this is the first study to compare NCR with FPR with regard to ability to detect women at higher risk of PE among asymptomatic women.th percentile value had RR of PE = 4.98 (1.91–13.0) compared to women with FPR <90th percentile value.The antiangiogenic factor, sFlit-1, and proangiogenic factor, PlGF, have been implicated in the pathogenesis of PE67891011121314151617181920212223et al.et al.et al.et al.et al.The median values were 1414 vs. 813 pg/mL for sFlit-1 levels and 336 vs. 497 pg/mL for PlGF levels determined at around GW 24 in 15 vs. 239 women that did and did not later develop PE, respectively, in this study. These levels were similar to those determined in women with similar clinical conditions in a previous study by Levine nd trimester NCR around GW 24 appeared to be superior to simultaneous FPR with regard to the detection rate of women with later development of PE in our setting. Women with NCR > 90th percentile value had RR of PE = 10.0 (3.82–26.4) compared to women with NCR < 90th percentile value; the corresponding RR was 4.98 (1.91–13.0) for FPR in this study. In addition, AUC of ROC was greater for NCR than for FPR, and the NCR cut-off suggested by the ROC yielded high sensitivity of 87%. In our previous study using longitudinal urine samples from women that did and did not develop PE later324The 2.0 3.82–2.4 compar.0 3.82–2.4 compar0.0 3.82–.4 comparnd trimester NCR levels were independent of the 2nd trimester sFlit-1, PlGR, and FPR  in this study was somewhat higher than the prevalence rate of 2.3% in the general Japanese population2728In conclusion, non-invasive urine test of NCR around GW 25 was compared with simultaneous serum FPR with regard to predictability of later development of PE. Results were promising and larger prospective studies are warranted regarding the ability of NCR for prediction of PE.This study was conducted in accordance with the principles of the Declaration of Helsinki and with the approval of the Institutional Review Board of Hokkaido University Hospital , a tertiary teaching hospital managing mainly high-risk pregnant women.st or 3rd trimester. Then, from the 443 women with availability of 2nd trimester urine specimens but without chronic hypertension, the following 189 women were excluded: one woman that exhibited hypertension before reaching GW 20, three women that exhibited new onset of hypertension and or SPIP at the time of urine collection, and 185 women whose blood specimen collected simultaneously with urine collection was not available. Thus, the remaining 254 women fulfilling the following two criteria were finally enrolled in this study: (1) no known pre-existing hypertension, and (2) no hypertension or SPIP at the simultaneous urine and blood sampling during the 2nd trimester. Thirty-three of the 254 women (13%) were also enrolled in our previous studyDuring a study period between from May 2014 to June 2016, 827 women received antenatal care and gave birth at Hokkaido University Hospital. Among them, 540 (65%) women gave written informed consent to participate in this study and provided at least one spot urine specimen with or without simultaneously collected blood specimen during pregnancy. From these 540 women, 97 women were first excluded from the present analyses: 15 women with known hypertension (pre-existing hypertension) at the establishment of the current pregnancy and 82 women that provided urine samples only collected during the 1SPIP was defined as PCR (mg/mg) >0.27 (corresponding to 30 mg/mmol) in spot urine specimens. Hypertension was diagnosed in women with systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg on at least two occasions recorded more than 12 hours apart. The GW at new onset of hypertension and SPIP were specified in each subject. PE was diagnosed in women that showed both hypertension and SPIP on and after GW 20.g for 5 minutes. Urinary supernatant was stored at –20 °C until measurement of protein, creatinine, and nephrin levels. Protein and creatinine concentrations were measured using a Protein Assay Rapid Kit Wako and Laboassay Creatinine , respectively. Nephrin concentrations of all 254 urine samples were measured using a commercial ELISA kit  that was different from the kit used in our previous studyAll spot urine samples were coded and processed within 2 hours of collection. Urine samples were transferred into tubes and centrifuged at 700 × © statistical software package . The Mann–Whitney U test was used to compare median or mean values between two groups. The Kruskal–Wallis test was used for comparison of medians or means of three groups. Receiver operating characteristic curves (ROC) were constructed for the biomarkers to assess their ability to differentiate women with PE onset later. The Spearman’s rank-order correlation was used to test associations between two variables. In all analyses, P < 0.05 was taken to indicate statistical significance. However, a significant finding regarding a linear correlation between two variables was defined as that meeting both P < 0.05 and correlation coefficient (r) > 0.25 or <–0.25.Data are presented as the median (range) and or mean ± SD. Statistical analyses were performed using the JMP10How to cite this article: Zhai, T. et al. Second-trimester urine nephrin:creatinine ratio versus soluble fms-like tyrosine kinase-1:placental growth factor ratio for prediction of preeclampsia among asymptomatic women. Sci. Rep.6, 37442; doi: 10.1038/srep37442 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


HADHA and HADHB), both located on chromosome 2p23, cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by decreased activity of MTP. The most common MTP mutation is long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency caused by the c.1528G>C  substitution in exon 15 of the HADHA gene.The mitochondrial β-oxidation of fatty acids is a complex catabolic pathway. One of the enzymes of this pathway is the heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and β-subunits. Mutations in MTP genes  carriers among Kashubians (1/57) compared to subjects from other regions of Poland (1/187). We found higher frequency of c.652G>C  polymorphism in the HADHA gene within population of Silesia, southern Poland (1/107) compared to other regions.HADHA gene in Kashubian population, suggesting the founder effect. For the first time we have found high frequency of rs71441018 in the South Poland Silesian population.Our study indicate described high frequency of c.1528G>C variant of  HADHA and HADHB), both located on chromosome 2p23 CTCACCCGCATTCTCCGAT-3') and HADHARev199 (5'-ACAGCCCCTTACCTTAACCACA-3') were used. The PCR amplification was performed using a 50 μL reaction volume containing 2 μL of genomic DNA, 2x PCR Master Mix Plus (A&A Biotechnology), and 1 μL of each primer (10 μM). The PCR used the following steps: 94°C for 3 min, followed by 40 cycles with 94°C for 15 s, 58°C for 30 s and 72°C for 30 s.; the final extension was at 72°C for 5 min. The sequencing primer was 5'-GTTTTCTCGGTCGTGATAA-3', and the nucleotide dispensation order was TCTSCAGC. Sequencing was carried out as instructed by the protocol using the PSQ™ 96MA pyrosequencing aparatus and the PyroMark® Gold Q96 kit (Qiagen).The subjects . To amplHADHA gene region (NC_000002.11 (26413504..26467665)) which gave 30 SNPs for the analysis. All sequence coordinates were compared to the GRCh37/hg19 reference sequence and were obtained from GenBank (http://www.ncbi.nlm.nih.gov). These results were exported from GenomeStudio using the PLINK Input Report Plug-in v2.1.3 by forward strand [The 5901 DNA samples were genotyped for 551,945 SNPs using the 24x1 Infinium HTS Human Core Exome PLUS  microarrays according to the protocol provided by the manufacturer. Briefly, DNA samples were amplified, then enzymatically fragmented and hybridized to the BeadChips. Afterwards, the BeadChips underwent an extension and X-staining processes. Next the BeadChips were scanned using iScan . Raw fluorescence intensities were imported to the GenomeStudio V2011.1 with the Genotyping Module v1.9.4 . All data first underwent stringent quality control including sample exclusion if call rate was below 0.94 and if the 10% GenCall parameter was below 0.4. We filtered the data from the d strand .http://www.ncbi.nlm.nih.gov) and were used in this paper as the nomenclature of variants.For each of the polymorphisms detected in this study the following parameters were assigned: dbSNP IDs (rs numbers), nucleotide position within or relative to the coding sequence based on the NM_000182.4, and amino acid position in the protein for SNPs in the coding sequence based on the reference sequence NP_000173.2. These data were obtained from GenBank , rs7593175 , rs146406360  and rs71441018 .Among 30 analyzed SNPs we have detected genetic variation for only four variants of The observed genotype distribution was determined for all the detected polymorphisms by performing the Hardy-Weinberg equilibrium exact test assuming consistency for a P-value higher than 0.05. Differences in observed MAF values were examined with chi-square test for alleles  and shown as their P-values, assumed significant if <0.05. The prediction of the effect of aminoacid and nucleotide substitution on protein function or gene were evaluated with the PredictSNP tool .HADHA gene. We did not find individuals with abnormal homozygous CC genotype and 5,841 (99.39%) were homozygous for the GG genotype. The carriers ratio of this variant in the studied population of Poland was 1/163. After exclusion of individuals from the Pomeranian region the ratio was 1/171. The carriers frequency in the Pomeranian province was 1/103  were carriers of variants of the as 1/103 .HADHA gene. We have not found individuals with abnormal homozygous CC genotype and 1,005 (98.25%) were homozygous for the GG genotype. The carriers ratio of this variant in the Kashubian population of Poland was 1/57  were carriers of variants of the was 1/57 .HADHA gene. There were no individuals with abnormal homozygous GG genotype and 9,284 (99.47%) were homozygous—GG genotype. The carriers ratio of this variant in the studied population of Poland was 1/187. In the individuals from the Pomeranian region the ratio was 1/75 (without well-defined Kashubians)  were carriers of variants of the hubians) , Fig 3.Based on pairwise chi-square test for alleles (with Yates correction if any group <10), any significant differences in c.1528G>C variant frequency between particular regions of Poland (excluding the Pomeranian region from this comparison) were detected. Thus, we were able to put together populations from all the regions and compare these pooled data to the Pomeranian population.By using this methodology, we found that population from the Pomeranian region and Kashubian population on its own significantly differ from the rest of the country in terms of frequency of this variant  .Frequency of c.1528G>C was described previously for several populations: Finland, Holland, Estonia and Poland, summarized in For rs146406360  polymorphism from 5,879 analyzed people (POPULOUS collection) we found 21 (0.41%) persons with heterozygous GA genotype and no one with AA genotype. The frequency of heterozygotes was 1/280. We did not find any differences in the frequency between all compared provinces of Poland.For rs7593175 (c.572+32T>C) polymorphism from 5,877 analyzed persons, we found 3,097 with CC (52.70%) genotype, 2,338 with TC (39.78%) genotype and 442 (7.52%) with TT genotype. We did not find any differences in the frequency between different regions of Poland.Interesting data were uncovered for rs71441018  polymorphism. From 5,878 analyzed persons, we found 11 (0.19%) individuals with heterozygous GC genotype. The frequency of heterozygotes within the Polish population was 1/534. Nine of all 11 (82%) detected heterozygotes were found in the Silesian region. The frequency for this region was 1/107, statistically higher than in other regions of Poland (1/2933) .In silico analysis of SNP effect on protein functionality revealed that all exonic variants, such as rs71441018 , rs137852769 , rs146406360 , have deleterious effects  [Some studies, recently conducted in Poland, employed this isolated population, which is particularly attractive from a genetic point of view. For instance, Siemińska et al. showed, that the variant allele A of rs169969968 of the alpha-5 nicotinic receptor subunit gene (CHRNA5), a polymorphism which is strongly associated with nicotine dependence was significantly less frequent in comparison to the HapMap CEU reference population (map.org) , 35–37. map.org) –43.Rebala et al studied the frequencies of Y chromosome haplotypes (7 STR) have found that Kashubian are closely related to similar populations from nearby regions , and also related to other Polish populations, but were different from other Slavics  and German populations  Danish, and Got (Sweden) populations .Y-chromosome DNA haplogroup among the Kashubians who live in Kashubia, is haplogroup R1a, which is carried by 61.8% of Kashubian males. It is followed in frequency by I1 (13.2%), R1b (9.3%), I2 (4.4%) and E1b1b) (3.4%). Altogether these account for over 9/10 of the total Kashubian Y-DNA diversity. A study from January 2010 discovered similar proportions of most haplogroups , but also found Q1a in 3.1% of Kashubians. This study reported no significant differences between Kashubians from Poland and other Poles in terms of Y chromosome polymorphism. When it comes to mitochondrial DNA haplogroups, according to a January 2013 study, the most common major lineages among the Kashubians, each carried by at least 2.5% of their population, include J1 (12.3%), H1 (11.8%), H* (8.9%), T* (5.9%), T2 (5.4%), U5a (5.4%), U5b (5.4%), U4a (3.9%), H10 (3.9%), H11 (3.0%), H4 (3.0%), K (3.0%), V (3.0%), H2a (2.5%) and W (2.5%). Altogether they account for almost 8/10 of the total Kashubian mtDNA diversity [According to a study published in September 2015, by far the most common iversity –41.NPHS2 gene mutations causing steroid-resistant nephrotic syndrome in Polish patients and reported that the carriers of the c.1032delT allele were exclusively found in the Pomeranian (Kashubian) region, suggesting a founder effect origin [LDLR gene is frequent in this population) [In another study, Lipska et al explored the spectrum of t origin . Studiesulation) , 42, 43.In a 2008 study of Polish newborns a carrier frequency of 1/217 was found, whereas it was significantly higher in the Pomeranian region at 1/73. Because the majority of carriers was detected in children living in the Kashubian region, the authors suggested a probable Kashubian origin (the Kashubian origin of children and parents was not confirmed in that study) of the prevalent c.1528G>C variant. The estimated frequency of disease was 1/16,900 whereas in the rest of Poland it was 1/118,336 .HADHA gene within the Pomeranian region (Our study confirms the high frequency of carriers (1/70) of the c.1528G>C variant in the n region , and wasn region . The higHADHA gene variants was the Lubusz province. This result could be explained by the low number of analyzed persons, and must be verified in enlarged population. When we analyzed the data collected as part of our study together with data from Piekutowska-Abramczuk, the frequency of variant in this region was 1/88 i.e. lower than in Kashubians  variant consistent with its previously described clinical significance. Although the effect of the SNP variant c.2113G>A  was also classified as deleterious, amino acid substitution which is vital for protein functionality, was not predicted as negative effect.HADHA gene in the Pomeranian region of North Poland region with the highest frequency in individuals of Kashubian origin, which could confirm founder effect in this population. For the first time we have found a high frequency of rs71441018 polymorphism in the HADHA gene within the Silesian population of Southern Poland.In the summary, the results of our study confirm higher frequency of c.1528G>C variant in


Deaths from unintentional injuries are the seventh leading cause of death among older adults . The largest AAPC in mortality rates from falls (11.0% per year) occurred in Maine, followed by Oklahoma (10.9%) and West Virginia (7.8%). A significant increase in the rate from 2007 to 2016 occurred in 30 states  and DC. No significant change in fall mortality rates occurred in 11 states . After an initial increase, rates stabilized in three states . Arizona, Nevada, and Wisconsin had initial periods of stability followed by a significant increase in fall death rates. The death rate from falls decreased in Missouri during 2007–2012, followed by an increase during 2012–2016, and increased in Utah during 2007–2012, followed by a decrease during 2012–2016.The overall rate of older adult deaths from falls increased 31% from 2007 to 2016 (3.0% per year) . NationwIn 2016, death rates from falls were higher among adults aged ≥85 years (257.9), men (72.3), and whites (68.7) than among corresponding groups . From 20Approximately 30,000 adults aged ≥65 years died as the result of a fall in 2016, and state-specific rates for deaths from falls ranged from 24.4 per 100,000 in Alabama to 142.7 in Wisconsin. The rate of deaths from falls among older adults increased steadily from 2007 to 2016 in 30 states and DC. The 31% increase in the national rate of deaths from falls from 2007 to 2016 is consistent with findings from a 2010 study that estimated a 42% increase from 2000 to 2006  to 142.7 (Wisconsin). The fastest-growing rate was among persons aged ≥85 years (3.9% per year).As the U.S. population aged ≥65 years increases, health care providers can address the rising number of deaths from falls in this age group by asking about fall occurrences, assessing gait and balance, reviewing medications, and prescribing interventions such as strength and balance exercises or physical therapy.