To document the diurnal intraocular pressure (IOP) profile with rebound tonometry performed by primary glaucoma patients in non-clinic environment.Fifty-three medically-treated eyes of 31 primary angle closure glaucoma (PACG) and 22 primary open angle glaucoma (POAG) patients with no previous eye surgery were recruited. Diurnal IOP was measured 5 times per day at four-hourly intervals from 08:00 to 24:00 for 1 week in patients’ study eye using rebound tonometry in a non-clinic environment. The diurnal IOP profiles were compared between PACG and POAG eyes.p < 0.001). The diurnal IOP fluctuation ± 1 standard deviation (SD), as documented by SD in daily IOP values, was lower in PACG group (1.6 ± 1.1 mmHg) than in POAG group . The mean trough IOP ± 1 SD was higher in PACG group (12.9 ± 2.8 mmHg), compared to POAG group . The mean IOP level at midnight ± 1 SD in PACG group (14.0 ± 3.2 mmHg) was higher than that in POAG group .For both PACG and POAG eyes, mean patient-measured IOP was highest in the morning, gradually decreased over the course of a day, and was lowest by midnight (IOP in primary glaucoma patients was highest in the morning, and decreased over the course of a day in non-clinic environment. Treated diurnal IOP fluctuation seemed to be greater in POAG than PACG eyes. Glaucoma is the second leading cause of irreversible visual impairment and blindness worldwide. More thaIOP has been shown to fluctuate over time. Recent e® tonometer  is one of the newer generation of RBT devices that allows glaucoma patients, after adequate training, to measure their own IOP safely and accurately outside the clinic environment.[For accurate IOP measurement, Goldmann applanation tonometry (GAT) is the gold standard in clinical practice. GAT has ironment. Good corironment.,14–18 ThIn this study, the diurnal IOPs were self-measured by PACG and POAG patients using the RBT device (iCare ONE tonometer) in their ambient environments. The diurnal IOP profiles were compared between PACG and POAG eyes.Patients with PACG or POAG were prospectively recruited at the eye clinics of the Chinese University of Hong Kong and Hong Kong Eye Hospital between April 2012 and December 2013. Written informed consents were obtained from all study subjects. The study protocol and consent procedure was approved by the Ethics Committee for Human Research at the Chinese University of Hong Kong. The study adhered to the tenets of the Declaration of Helsinki. Authors do not have access to information that could identify individual participants during or after data collection.Clinic IOP was measured by Goldmann applanation tonometry on a slit-lamp biomicroscope. The reading in mm Hg was rounded to the next higher integer. Two IOP measurements were taken but if the readings differed by greater than 2 mmHg, a third measurement would be taken. Clinic IOP was defined as the mean of two IOP measurements, or the median of three measurements. Angle status was determined with darkroom gonioscopy. Shaffer system was used for the grading of angle width. Grade 0 was defined as closed, and indentation will distinguish appositional from synechial angle closure. The diagnosis of glaucoma was based on characteristic glaucomatous optic nerve head morphology, and confirmed by Humphrey automated perimetry . The criteria for glaucomatous visual field defect were adopted from a published report. All eyesWe randomly selected one eye of a patient using a random number table if the patient had bilateral disease. If the patient had unilateral glaucomatous optic neuropathy, the diseased eye would be included. The patients received topical IOP-lowering eye drops, as indicated clinically.http://www.youtube.com/watch?v=9Ov4VZXAZN4) with commentary by a study investigator who is experienced in using the self-rebound tonometry. Subjects also had hands-on training in using the tonometer under guidance and supervision. By the end of the training session, all subjects were assessed for their performance of the self-rebound tonometry to ensure that they followed a standard operating procedure. A hard copy of Icare ONE Quick Guide was distributed to every patient after the training session. All subjects were clearly instructed to remain in their usual living environment, e.g. at home, office or school, during the 1-week IOP documentation. All subjects were instructed to continue their usual activities of daily living, and to stop such activities only when they had to measure their IOP. Subjects were taught to measure their IOP in the upright sitting position, and eye drops were taken after the IOP measurements. The iCare ONE tonometer automatically acquires a mean IOP reading with every 6 consecutive automated contacts with the central cornea. Five IOP measurements were conducted every day at four-hourly intervals, from 08:00 to 24:00  for a week. At least three valid IOP readings were obtained at each time point. The readings and times of IOP measurements were automatically stored in the tonometer, and transferred to a computer for further analysis by the Icare® LINK software. The median of all valid readings at each time point was used for statistical analyses. A new sterilized and disposable probe was used for each IOP measurement to avoid damaged probes affecting accuracy of measurement, and cross infection.Before using the iCare ONE tonometer, all recruited patients participated in a 2-hour training session on the correct method for self-measurement of IOP using rebound tonometry. The training included watching a standard training video . All statistical analyses were performed using SPSS software .Based on previous sample size calculation, we aimed to recruit 16 patients in the two groups. Subject would be excluded from the database if found missing data. The demographic characteristics of the study subjects were collected. The IOP value at each time point was calculated by averaging (mean) the IOPs measured at the same time point of every day during the whole study week. The diurnal IOP values at the 5 time points of the day constituted the ‘individual diurnal IOP profile’. The ‘group diurnal IOP profiles’ were acquired by the medians of the IOP values at the study time points of all recruited subjects within the group. Differences were compared by independent t-test  or Mann-Whitney U test . Normality of the data was tested by Shapiro-Wilk test. Categorical data were compared using the Pearson Chi-Square test or Fisher’s test. IOP differences among 5 time points were tested by Friedman test, and post-hoc test was further evaluated by Wilcoxon Signed Ranks test. A p < 0.001). The mean vertical cup-to-disc ratio ± 1 SD was 0.64 ± 0.20  in PACG, and 0.79 ± 0.24  in POAG (p = 0.003). The mean ± 1 SD, and median  extent of synechial angle closure in PACG group was 262.7 ± 126.5 degrees, and 325.0  degrees, respectively. The corresponding values in POAG group were 2.7 ± 10.9 degrees, and 0  degree (p < 0.001). The average mean deviation (MD) and pattern standard deviation (PSD) ± 1 SD, on Humphrey automated perimetry were -10.41 ± 7.72 dB  and 4.55 ± 2.75 dB  in PACG group, and -14.69 ± 7.75 dB  and 8.45 ± 3.66 dB  in POAG group, respectively   .p = 0.358). Forty-four patients (25 PACG and 19 POAG) (83.0%) completed all 5 measurements each day in the study, while 9 patients (6 PACG and 3 POAG) (17.0%) had missed some time points. The percentage of IOP measurements missed in the 9 study patients ranged from 11.4% to 31.4%. Data from these 9 patients were included in the calculation of mean diurnal IOP and IOP fluctuation, and were therefore included in the diurnal IOP profile of the two groups. Data from these 9 patients were not included in the calculation of peak and trough IOP frequencies, because of their incomplete measurements during the study week.Overall, the mean number ± 1 SD of IOP-lowering drugs received by PACG eyes was 1.8 ± 1.6 , whereas that of POAG eyes was 2.5 ± 1.4  (p = 0.041).In order to study the characteristics of diurnal IOP profile in primary glaucoma patients, the average IOP, peak IOP, trough IOP, and IOP fluctuation, were calculated in both PACG and POAG groups . The difth percentile, 75th percentile; range) of IOP fluctuation  was 1.6 ± 1.1 mmHg  mmHg; 0.3 to 5.1 mmHg) in PACG group, and 2.0 ± 1.2 mmHg  mmHg; 0.8 to 6.3 mmHg) in POAG group (p = 0.049) (The mean ± 1 SD (median (25= 0.049) . The POAth and 75th percentiles of diurnal IOP profiles for PACG and POAG groups, respectively. In PACG group, the median IOP was 16.4  mmHg, 14.6  mmHg, 14.1  mmHg, 14.7  mmHg and 14.4  mmHg at 08:00, 12:00, 16:00 20:00 and 24:00, respectively (p < 0.001). Wilcoxon Signed Ranks test further showed that the IOP at 08:00 was significantly higher than that at 20:00 (p = 0.001) and 24:00 (p = 0.001) in PACG eyes (p < 0.001). The IOP at 08:00 was significantly higher than that at 16:00 (p = 0.004), 20:00 (p = 0.004) and 24:00 (p < 0.001). In addition, IOP at 24:00 was significantly lower than other time points (12:00 (p < 0.001), 16:00 (p = 0.004) and 20:00 (p = 0.002)) in POAG group (ectively . There wACG eyes . In POAGACG eyes . The diuAG group .p = 0.013).There was also statistically significant difference in mean IOP at midnight (24:00) between the PACG group (14.0 ± 3.2 mmHg) and the POAG group (12.1 ± 3.7 mmHg) , followed by 16:00 . The percentage of occurrence of peak IOP was significantly different among the 5 time points (p = 0.012), the occurrence at 08:00 was higher than that at 20:00 (p = 0.003) and 24:00 , and least frequently documented at 08:00 . However, the frequencies of trough IOP did not show any statistically significant differences among the 5 time points . The percentage of occurrence throughout the course of a day is presented in e points .th percentile = 23.2%, 75th percentile = 70.8%). The percentage of peak IOP occurring among the 5 time points also showed a statistically significant difference (p < 0.001), the frequency at 08:00 being significantly higher than those at 16:00 (p = 0.003) and 24:00  and least frequently at 08:00 . Statistically significant difference was found among the 5 time points (p = 0.020); however, individual differences did not reach statistical significance after Bonferroni corrections  and POAG (p < 0.001) eyes, and the peak IOP was most frequently documented at 08:00 in both PACG and POAG groups. However, the range of diurnal IOP in our cohort was smaller, with a median IOP range of 2.3 mmHg in PACG and 3.2 mmHg in POAG, whereas previous studies showed a range of approximately 4 to 5 mmHg in glaucoma eyes.[Previous studies have documented the diurnal IOP fluctuation in POAG and PACG eyes, and alsooma eyes. The possThe mean IOP appeared to be higher in the POAG group than in the PACG group at all time points, but only the difference in mean IOP at midnight between the two groups reached statistical significance. In terms of IOP fluctuation parameters, the SD of diurnal IOP was lower and the trough IOP was higher in PACG eyes compared to POAG eyes. This might be explained by the differences of number and type of medicine between PACG and POAG patients taking. It also raised a possibility that a greater influence of light on the dilatation status of the pupil for PACG eyes as compared to POAG eyes. Slower op = 0.682) between Goldmann applanation tonometry and rebound tonometry. The between-measurement ICC of these two tonometries was 0.72.[The current study has a few limitations. First, the patients themselves conducted all of the IOP measurements. The accuracy of the IOP measurements depended on the training and skills of the patients. A total of 6 readings were collected for each IOP measurement. The rebound tonometer is able to recognize wrong positioning or large variations, and automatically delete possibly unreliable readings. Only the more consistent readings would be stored, allowing us to ensure a certain degree of compliance with the measurement protocol. In addition, we provided a 2-hour standard training program in using the rebound tonometer to all study patients to minimize the skill effect, with an evaluation at the end of training, to ensure accuracy and consistency of the IOP measurements. Three IOP readings were documented by both Goldmann applanation tonometry and self- conducted rebound tonometry on all of the participants after the training program. The within measurement intra-class correlation coefficient (ICC) of Goldmann applanation tonometry and rebound tonometry were 0.98 : 0.97–0.99) and 0.94 (95% CI: 0.90–0.96), respectively. Although rebound tonometer was showed may underestimate the IOP compared to Goldmann applanation tonometry measurements in previous studies, We evaluwas 0.72. Second, was 0.72.–38 oculawas 0.72. and PACGwas 0.72. than norwas 0.72. A study In summary, mean patient-measured IOP in the home environment was highest at 08:00, tended to drop over the course of a day, and was lowest by midnight, in both PACG and POAG eyes in this study. The PACG eyes had lower diurnal IOP fluctuation than POAG eyes, and higher midnight IOP. This study demonstrated the feasibility of diurnal IOP documentation by patients themselves using rebound tonometry in their ambient environments. Such measurements may potentially be of clinical relevance in patient management.S1 File(XLSX)Click here for additional data file.


Streptococcus pneumoniae serotype 12F (Sp12F) invasive pneumococcal disease (IPD) dynamics in the 7 years after vaccine introduction. Of 4,573 IPD episodes during July 2009–June 2016, a total of 434 (9.5%) were caused by Sp12F. Sp12F IPD rates  increased in children <5 years of age, from 1.44 in 2009–2010 to >3.9 since 2011–2012, followed by an increase in all ages. During 2011–2016, Sp12F was the most prevalent IPD serotype. Sp12F isolates were mostly penicillin nonsusceptible  and predominantly of sequence type 3774), a clone exclusively found in Israel (constituting ≈90% of isolates in 2000–2009). The sharp increase, long duration, and predominance of Sp12F IPD after vaccine implementation reflect a single clone expansion and may represent more than a transient outbreak.Israel implemented use of 7- and 13-valent pneumococcal vaccine in 2009 and 2010, respectively. We describe results of prospective, population-based, nationwide active surveillance of  Streptococcus pneumoniae is a leading cause of illness and death worldwide; the highest incidence occurrs in children <2 years of age and in the elderly (Streptococcus pneumoniae serotype 12F (Sp12F) , a substantial increase in nasopharyngeal carriage of nonvaccine serotypes (NVT) has been observed in surveillance studies  was introduced to the national immunization plan (NIP) in July 2009, with a catch-up plan for children <2 years of age, and was gradually replaced with the 13-valent PCV (PCV13) in November 2010, without further catch-up. Uptake rates were high (>90%)   and incubated them at 37°C for 24 hours in a 5% enriched CO2 atmosphere. Identification of S. pneumoniae was done as previously described . Serotype 6A was differentiated from serotype 6C by PCR  according to Clinical and Laboratory Standards Institute (CLSI) guidelines  with dice coefficients, a 1% position tolerance, and optimization values. We performed cluster analysis by the unweighted pair-group mean analysis. All isolates in the same cluster, defined as clonal, were assigned a letter, A to T, for analysis purposes.aroE, ddl, gdh, gki, recP, spi, and xpt) were amplified according to the S. pneumoniae PubMLST (We characterized selected isolates representing pulsed-field gel electrolysis (PFGE) cluster and isolate years by multilocus sequence typing (MLST). The 7 housekeeping loci . We retrieved MLST allelic profiles and STs from the We performed statistical analysis using SPSS version 14.0 software for Windows . A p value <0.05 was considered statistically significant.>30 days for the same serotype or by any interval for different serotypes. Rate reductions and ratios were calculated. The following age groups were defined: <5 years, 5–17 years, 18–64 years, and ≥65 years of age.The data from the active surveillance during 2009–2016 are presented according to epidemiologic years, July through June. Each isolate was counted only once per episode; episodes were separated by During the study, we identified 4,573 IPD episodes (93% bacteremia and 7% meningitis). Of those, 434 (9.5%) were caused by serotype 12F .Patients <5 years of age accounted for 27% of the overall study population; those 5–17 years of age, 8%; those 18–64 years of age, 31%; and those ≥65 years of age, 34%. Overall, 56.3% of all episodes occurred in male patients, and 86.4% of all episodes occurred among the Jewish population.We ompared the first (2009–2010) and the last (2015–2016) study years and found that overall IPD rates  declined by 34% , from 10.2 to 6.7 . IPD ratRates of IPD caused by PCV13 serotypes and proportions of all IPD episodes declined substantially throughout the study period. Rates  of IPD caused by PCV7 serotypes declined by 90%, from 2.6 to 0.5, from the first to the last study years. Similarly, rates of IPD caused by PCV13 serotypes declined by 80%, from 7.4 to 1.5, during the study period.Proportions of all IPD episodes caused by PCV7 serotypes substantially declined by 74%, from 24.8% in 2009–2010 to 6.4% in 2015–2016. Proportions of all IPD episodes caused by PCV13 serotypes decreased by 68%, from 69.4% to 22.4%.IPD caused by non-PCV13 serotypes increased by 93%. Rates  increased from 2.8 in 2009–2010 to 5.4 in 2015–2016.We observed a steady increase (except for the last study year) in the proportion of Sp12F out of all IPD episodes throughout the study: 2.7% in 2009–2010, 4.9% in 2010–2011, 10.1% in 2011–2012, 12.6% in 2012–2013, 13.7% in 2013–2014, 16.1% in 2014–2015, and 12.7% in 2015–2016 ; Table 1To assess the association between PCV7/PCV13 introduction and the dynamics of Sp12F rates, we analyzed strains isolated during 2000–2015. Overall, we analyzed 445 Sp12F strains by PFGE . The isoWe further analyzed representative Sp12F isolates by MLST. Among 25 isolates with the predominant PFGE pulsotype, 24 were typed as ST3774 and 1 as the closely related ST3524. Four isolates from other PFGE pulsotypes were typed as ST989 and 1 isolate from another PFGE pulsotype was typed as ST3377.We compared the MLST type with isolates reported as Sp12F in the global PubMLST database . ST3774 50 0.12 μg/mL, range 0.02–1.2 μg/mL). However, most strains were fully susceptible to ceftriaxone , clindamycin (95%), rifampin (100%), and vancomycin (100%).Among all Sp12F isolates, 89% were penicillin nonsusceptible (MIC >0.06); nonsusceptibility rates varied by year from 70% to 99% (MICS. pneumoniae serotype 12F, a non-PCV13 serotype, is currently the most prevalent pneumococcal serotype causing IPD in Israel. This previously rare serotype caused 434 IPD cases during July 2009–June 2016 and became prevalent in all age groups, most distinctly in young children. The population of Sp12F during 2000–2016 was >90% of the same clonal complex according to PFGE and MLST analyses, indicating the expansion of the ST3774 sequence type, which is currently unique to Israel. The predominant global Sp12F outbreak clones ST218 and ST989 (Sp12F was previously reported to have high invasive disease potential and is sometimes called hyperinvasive (In a recent study from France that evaluated the invasive potential of specific serotypes and compared nasopharyngeal carriage in healthy children to carriage in children infected with IPD, Sp12F was shown to be a major cause of IPD but was not found in nasopharyngeal cultures (The emergence of IPD caused by Sp12F in Israel, mainly by clonal expansion, may be attributed, at least in part, to the observed more general serotype replacement after PCV implementation. The emergence of this serotype in the youngest age groups after PCV7/PCV13 introduction supports this hypothesis. However, the previously reported low carriage rate of Sp12F may counter this possibility, and current data on Sp12F carriage in Israel are needed to support this theory. Alternatively, a large-scale clonal outbreak, of long duration and high invasiveness rather than true replacement, may be at the origin of this serotype emergence.Our results emphasize the role of Sp12F as a serious current public health challenge. Continued monitoring of IPD and specific serotype distribution are needed to enable the development of future vaccination strategies. The evolutionary origin of this emerging clone will be further studied by whole genome sequencing, which may offer insights into the virulence of Sp12F strains.


Staphylococcus aureus is primarily related to target-site modification through acquisition of an erm gene. In the present study the prevalence of both phenotypic and genotypic tylosin resistance in S. aureus isolates (n = 103) from subclinical mastitis in nine dairy farms belonging to three different province of Iran were investigated. Overall, ermA, ermB and ermC was found in 7.80%, 32.00%, and 20.40% of S.aureus isolates, respectively. A very high percent of isolates (56.90%) were resistant to tylosin. MIC90 and MIC50 values were 64 and 32 µg mL-1, respectively. Most of tylosin resistant isolates did not harbour any erm gene but ermB was dominant gene among 58 tylosin resistant isolates of S. aureus. In overall, tylosin resistance was prevalent in S. aureus isolates obtained from bovine mastitis in Iran. The macrolides appear to have considerable effects for treatment of bovine mastitis because of excellent diffusion into the mammary gland, long half-life, low protein binding, high intracellular concentration and lipid solubility. Acquired resistance to macrolides in  Effective control of S. aureus mastitis relies on antibiotic treatment of intramammary infections at drying-off and treatment of clinical and sub clinical mastitis during lactation.2Bovine mastitis is the most prevalent production disease affecting dairy farms worldwide, accounting for 38.00% of direct costs incurred by the dairy industry.S. aureus mastitis by the intramammary or systemic administration route. Given high bioavailability from the injection site, lipid solubility, long half-life, low protein binding and intracellular concentration in phagocytes, macrolides are of considerable interest for parenteral mastitis therapy.1 Tylosin and tilmicosin are routinely used for dry cow therapy in most of dairy farms in Iran.Beta-lactams, aminoglycosides, lincosamides, fluoroquinolones and macrolides are common antimicrobial compounds for treatment of Staphylococcus species occurs by three primary mechanisms: Through target-site modification, through efflux of the antibiotics, and by drug inactivation. Target modification is predominant mechanism that is dependent on the erm genes.3 Because of extraordinary usage of macrolides for treatment of S. aureus mastitis in Iranian dairy farms for a long time, it is not surprising that resistance is prevalent among Staphylococcus species. In this study, we determined the prevalence of both phenotypic and genotypic tylosin resistance in S. aureus isolates from bovine subclinical mastitis.Macrolide resistance in Bacterial isolates. The present study was performed in nine dairy farms belonging to three different province of Iran including Tehran , Khorasan Razavi  and Alborz  from 2014 to 2015. The California mastitis test (CMT) was done and milk samples were taken from quarters with score 1 or more in CMT and cultured. A total number of 103 bovine S. aureus isolates from more than 500 individual quarter milk with subclinical mastitis were collected. Collection of milk samples and microbiological procedures were performed according to the National Mastitis Council procedures.4 Isolation and identification of these presumptive S. aureus colonies were based on conventional methods, including Gram staining, colony morphology, production of coagulase, catalase, DNase and fermentation of mannitol. To confirm the identity of the isolate as S. aureus, the nucA gene was amplified by a PCR-based method using primers listed in S. aureus isolates were stored at –70 ˚C in brain heart broth plus 20% glycerol.DNA isolation and polymerase chain reaction (PCR) amplification. For detecting macrolide resistance genes, the whole genomic DNA from cultured strains was prepared using DNA extraction kit . The PCR was carried out on the three related genes, including ermA, ermB, ermC, using specific oligonucleotide primers listed in 2, Tris-HCl, (NH4)2S04, 0.20% Tween 20, 0.40 mM dNTPs, 0.20 units per μL ampliqon Taq DNA polymerase inert red dye and stabilizer , 0.50 mg mL-1 of each primer and 3µL of template DNA. The PCR conditions consisted of a pre-denaturation step at 94 ˚C for 5 min, followed by 30 cycles of 45 sec at 94 ˚C, 50 sec at 50 ˚C (for ermA and ermC genes) or 54 ˚C (for ermB gene) or 55 ˚C (for nucA) and 75 sec at 72 ˚C. A final extension step was performed at 72 ˚C for 5 min. After amplification, DNA bands were analyzed by staining with ethidium bromide and electrophoresis  on 1% agarose gel.Antibiotic susceptibility testing. The broth micro-dilution method was used to determine minimum inhibitory concentrations (MIC), in accordance with the Clinical and Laboratory Standards Institute (CLSI).5 Mueller-Hinton broth was used as the test medium. Plates were incubated at 35 ˚C for 18 hr. Staphylococcus aureus ATCC29213 was used as reference strain for MIC quality controls. MIC were tested in the concentration range of 0.125 to 128.00 µg mL-1. Tylosin breakpoint 20 µg mL-1 was also considered in our test. 6Statistical analysis. SPSS software , was used for statistical analysis. Differences in the prevalence of macrolides resistant genes and distribution of MIC values in different provinces were calculated using the chi-square test. A p-value of 0.05 was considered as statistically significant. S. aureus in milk samples obtained from cows with subclinical mastitis were 22.00%, 23.50% and 25.00% in Tehran, Khorasan Razavi and Alborz, respectively. Among 103 S. aureus isolates 58 (56.86%) and 31 (30.09%) isolates were resistant and intermediately resistant to tylosin, respectively. The MIC values ranges for the all strains were from 0.50 to 128.00 µg mL-1. The MIC90 values in our test were 64.00 µg mL-1, while the MIC50 values were 32.00 µg mL-1. The distribution of MIC values in different provinces showed no significant differences (p > 0.05). The MIC distribution data of tylosin for the 103 S. aureus isolates and macrolide resistant genes are summarized in ermA, ermB and ermC genes, respectively . Among 58 tylosin resistant S. aureus, ermB gene was dominant and 27.60% of isolates harboured this gene (Fifty-six isolates (54.36%) of 103 had at least one his gene . S. aureus isolates from subclinical mastitis. The prevalence of S. aureus isolated obtained from subclinical mastitis was 23.00%. This prevalence varies in different countries. Prevalence of S. aureus in China is reported to be as high as 25.20%,10 others reported a prevalence of 10.20% and 30.60% in Finland and Kenya, respectively.11,12 The same prevalence rate (25.00%) was found among nine farms in Alborz, Iran.13We have determined the prevalence of macrolides resistant genes and the distribution of tylosin MICs in S. aureus was high (56.86%). These results were completely in agreement with other investigation who reported 56% resistance in S. aureus isolated from cow milk samples with clinical and subclinical mastitis in Romania.14 Other studies from different geographical locations reported lower resistance to tylosin ranged from 4.40% to 40.30%.1,10,15In the present study, frequency of acquired resistance to tylosin in et al. determined antimicrobial resistance patterns of S. aureus isolated from bovine subclinical mastitis in Alborz province and showed that resistance to tylosin was 28.80% of isolates.13Pourtaghi 50 value of tylosin for S. aureus isolates in our study was higher than those measured by previous investigators.1,10,14 The overall resistance of S. aureus isolated from bovine mastitis to macrolides in different countries is low or scarce.16,17 Result of present study showed higher frequency of resistance to macrolides. High resistance of S. aureus isolates to tylosin might be associated with the increase in highly resistant strains and rapid transfer of cloned resistance which could result from introducing and extraordinary usage of macrolides in Iranian dairy farms. When a single resistance determinant was considered, ermB was the most common. These observations are contrary to most other findings especially in human research that reported ermA was more frequent genes and ermB was present in only a minority of strains.18,19,20 Others showed that ermC was the most frequently encountered gene responsible for macrolide resistance among S.aureus isolates.3 Interestingly, in our investigation most of tylosin resistant isolates did not harbour any erm gene. It seems that other resistant genes are more important in tylosin resistance isolates obtained from bovine mastitis. Other resistant genes like ermF, ermY and lunA have also been detected in S. aureus isolates that were macrolide-lincosamides and streptogramin resistant.3,21 Also, it may suggest that one or several new resistance mechanisms for macrolides may be widespread among S. aureus isolates.Furthermore, the MICS. aureus isolates obtained from bovine mastitis in Iran. The most frequent determinant of macrolides resistance was ermB and with ermC the next most common.In conclusion, tylosin resistance was prevalent in


CDH1), SLUG (SNAI2), and NCAM (NCAM1) by real-time PCR. The samples were fixed in formalin and embedded in paraffin for immunohistochemical analysis of E-cadherin and NCAM. Thirty-five patients with acromegaly were included in the study. Of these, 65.7% had invasive tumors. Immunohistochemically, E-cadherin was expressed in 96.7% of patients, and NCAM in 80% of patients. There was no statistically significant relationship between tumor grade or invasiveness and immunohistochemical expression of these markers. Regarding gene expression, 50% of cases expressed CDH1, none expressed SNAI2, and 53.3% expressed NCAM1. There was no statistically significant relationship between tumor grade or invasiveness and gene expression of CDH1, SNAI2, and NCAM1. The absence of Slug overexpression and of E-cadherin and NCAM suppression suggests that expression of these markers is not associated with tumor invasiveness in GH-secreting pituitary adenomas.Pituitary adenomas account for 10–15% of primary intracranial tumors. Growth hormone (GH)-secreting adenomas account for 13% of all pituitary adenomas and cause acromegaly. These tumors can be aggressive, invade surrounding structures and are highly recurrent. The objective of this study was to evaluate E-cadherin, Slug and neural cell adhesion molecule (NCAM) expression in GH-secreting pituitary adenomas and its relationship to tumor invasiveness. A cross–sectional study of patients who underwent hypophysectomy due to GH-secreting pituitary adenoma from April 2007 to December 2014 was carried out. The medical records were reviewed to collect clinical data. Immediately after surgery, tumor samples were frozen in liquid nitrogen and stored in a biofreezer at –80°C for assessment of E-cadherin 1 ( Pituitary adenomas are common tumors, with an estimated prevalence of 16.7%, and autopsy studies have reported their presence in 20 to 25% of cases, accounting for 10 to 15% of primary intracranial tumors .TP53 and KRAS, rarely show mutations. In addition, the mitosis index is generally low and Ki67 and PCNA, markers of cell proliferation, have little relevance in predicting tumor behavior  have tumor recurrence after surgical treatment. The mechanisms underlying the pathogenesis of these tumors are unclear and genes classically involved in neoplastic development, such as behavior . The absSome studies have shown that loss of adhesion protein expression may be involved in the pathogenesis of pituitary adenomas and contribute to tumor aggressiveness and invasiveness –7. CurreThe factors involved in the growth and invasiveness of these tumors are not fully understood, but once identified, they will aid in the identification of invasion and recurrence markers and of potential therapeutic targets . The aimThis cross-sectional study consisted of 35 consecutive patients with a clinical and laboratory diagnosis of acromegaly who underwent neurosurgery performed by a single surgeon (NPF) at Hospital São José, Complexo Hospitalar Santa Casa de Porto Alegre, Southern Brazil, from April 2007 to December 2014. All patients undergoing the procedure during this period were included in the study. Written informed consent was obtained from all individual participants prior to their inclusion in the study.The diagnosis of pituitary adenoma was confirmed by anatomopathological examination. The histopathological diagnosis was made by the neuropathologists of the Department of Pathology of the institution in accordance with the World Health Organization (WHO) guidelines. Hematoxylin and eosin (HE)-stained slides were available for all cases to confirm the presence of tumors. The HE-stained slides were used to guide sampling of the tissue of interest. Each adenoma was immunohistochemically stained for six pituitary hormones  using commercially available antibodies. Immunohistochemical analysis was performed using chromogranin A antibody for all cases in order to characterize the pituitary tissue. The patients' medical records were then reviewed to collect data on gender, age, preoperative images and immunohistochemistry. Tumor grade and invasiveness were defined based on magnetic resonance images (MRI 1.5T) or X-ray computed tomography (CT) scans obtained preoperatively and classified according to the criteria proposed by Hardy : grade IThe study was approved by the Research Ethics Committee of Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), protocol No. 512/230, and conducted in accordance with the provisions of the Declaration of Helsinki.2O2) 30 V in methanol. Nonspecific protein binding was blocked using 1% BSA for 30 min. Incubation with primary antibody was performed overnight at 4°C. Incubation with secondary antibody and tertiary antibody was performed for 40 min at room temperature. The primary antibody was replaced with saline to serve as a negative control. Human tonsil was used as a positive control for ECAD and glioma for NCAM. The antigen-antibody binding was visualized with the diaminobenzidine (DAB) chromogen.Tumor tissue samples were fixed in 10% buffered formalin for 24 h and embedded in paraffin. Serial 4-µm thick sections were cut for immunohistochemical analysis. To detect protein expression, sections were incubated with the monoclonal anti-ECAD antibody G-10 , at a 1:50 dilution, and anti-NCAM antibody , at a 1:300 dilution. The labeled streptavidin-biotin method  was used for detection. Due to technical problems related to the primary antibody, the analysis of SLUG by means of immunohistochemistry was not possible. Endogenous peroxidase activity was blocked using three 10-min baths in 5% hydrogen peroxide  and SLUG (SNAI2), and of 100 ng/µL for qRT-PCR of NCAM (NCAM1). The samples were amplified using Sybr Green  in a total reaction volume of 15 µL under the following conditions: initial denaturation at 50°C for 2 min and at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The following primers were used: CDH1 , SNAI2 , and NCAM1 . All reactions were run in duplicate using a StepOnePlus system . Samples without cDNA were used as negative controls. As an endogenous control, GAPDH was used as a reference for normalization . GAPDH was amplified by qRT-PCR for each sample and for each RT-negative control, with the same specified conditions for gene analysis. A cycle threshold (Ct) <40 was classified as showing good quality cDNA. Commercially available pooled RNA , consisting of 39 healthy pituitary glands from adult males and females, was used for calibration of qRT-PCR. Data were converted to normalize expression ratios using the Applied Biosystems-recommended 2^(−ΔΔCt) method. Relative expression  was given by the 2^(−ΔΔCt) formula, where: ΔΔCt = [(Ct target gene – Ct GAPDH gene in samples) – ]. Data from tumor tissues are reported as equal to 1 (reference level). An expression level < 1 was defined as lower expression compared to normal pituitary gland pool and ≥1 as presence of equal or higher expression compared to normal pituitary gland pool , according to the manufacturer's instructions. RNA was then reverse transcribed into cDNA at a final volume of 21 µL using the SuperScript III First-Strand Synthesis System , according to the manufacturer's instructions. The resulting cDNA samples were diluted to a final concentration of 250 ng/µL for qRT-PCR of ECAD (CDH1), SLUG (SNAI2), and NCAM (NCAM1) was presented as median and interquartile range due to their asymmetrical distribution. Immunohistochemical expression was presented as frequencies and percentages. Noninvasive and invasive adenomas were compared using the chi-square and Mann-Whitney tests. The level of significance was set at 5% and data were analyzed using SPSS (USA), version 23.0.Descriptive statistical analysis and normality testing of the data (Shapiro-Wilk test) were performed to determine the distribution of the data. Quantitative variables are reported as means±SD or medians according to their distribution. Age was symmetrically distributed and reported as mean±SD. The gene expression of ECAD  were women. Mean patient age was 47.1±13.4 SD years, ranging from 18 to 74 years. Immunohistochemically, 15 (42.9%) adenomas expressed GH alone, 14 (40%) expressed both GH and prolactin, and the remaining adenomas expressed GH and other hormones.Regarding tumor grade based on preoperative images, 3 (8.6%) patients had grade I adenomas, 9 (25.7%) had grade II adenomas, 10 (28.6%) had grade III adenomas, and 13 (37.1%) had grade IV adenomas. Of these, 12 (34.3%) were noninvasive and 23 (65.7%) were invasive tumors. There was no statistically significant difference in gender or age between the noninvasive and invasive groups .Due to material availability, the samples of 30 patients were subjected to immunohistochemical analysis of protein expression. Of these, 11 (36.7%) were noninvasive tumors (3 grade I and 8 grade II tumors) and 19 (63.3%) were invasive tumors (8 grade III and 11 grade IV tumors).The samples of 29 (96.7%) patients expressed ECAD. Of these, 26 (86.7%) showed strong expression (+++), 2 (6.7%) showed moderate expression (++), 1 (3.3%) showed weak expression (+), and 1 (3.3%) showed no expression .When analyzed according to tumor grade, ECAD was strongly expressed in 100% of grade I and II adenomas, 62.5% of grade III adenomas, and 90.9% of grade IV adenomas. Strong expression of ECAD was observed in 100% of noninvasive tumors and 78.9% of invasive tumors .The samples of 24 (80%) patients expressed NCAM. Of these, 3 (10%) showed strong expression (+++), 8 (26.7%) showed moderate expression (++), 13 (43.3%) showed weak expression (+), and 6 (20%) showed no expression .When NCAM expression was analyzed according to tumor grade, grade I adenomas showed moderate expression in 100% of cases, grade II adenomas showed weak and strong expression in 75% of cases, grade III adenomas showed weak and moderate expression in 87.5% of cases, and grade IV adenomas showed weak, moderate and strong expression in 72.7% of cases . Weak, mCDH1 gene expression. Of these, 10 (50%) showed expression equal or higher compared to normal pituitary gland pool . Of 12 samples analyzed for SNAI2, none showed gene expression. Of 15 samples analyzed for NCAM1, 8 showed gene expression   .CDH1, none expressed SNAI2, and 3 (42.9%) expressed NCAM1. Among invasive tumors, 5 (45.5%) expressed CDH1, none expressed SNAI2, and 5 (62.5%) expressed NCAM1  expressed ed NCAM1 .CDH1 (P=0.295), SNAI2 (P=0.485), and NCAM1 (P=0.463).There was no statistically significant relationship between tumor grade or invasiveness and gene expression of Epithelial-mesenchymal transition is an important process in embryonic development that has been associated with cancer progression and metastasis, and loss of ECAD is considered a key initial step in the transdifferentiation of epithelial cells into a mesenchymal phenotype –19. InSeveral studies of pituitary adenomas have analyzed ECAD and reported positivity in most cases, albeit at varying levels. Yamada et al.  analyzedCDH1 gene expression. Elston et al.  transcripts, which may influence the prognosis of patients  and the Wnt (wingless) signaling pathway, although the exact mechanisms of molecular regulation of SNAI2 are unclear (CDH1 gene is also responsible for gene silencing in certain pituitary tumors (There are few studies in the literature that report on SLUG expression in pituitary adenomas. In a study of 41 nonfunctioning adenomas, the expression of SLUG by immunohistochemistry, PCR and western blotting was significantly increased in invasive tumors  investig unclear ,31. Seve unclear . In addi unclear . Likewisy tumors ,34.NCAM is a membrane glycoprotein that belongs to the immunoglobulin superfamily and plays a role in the proliferation, growth, differentiation and survival of different cell types . It is kNCAM1 gene expression, Rubinek et al. (Regarding invasiveness, Kleinschmidt-DeMasters et al.  analyzedk et al.  showed, SNAI2 overexpression and of CDH1 and NCAM1 suppression suggests that they are not associated with tumor invasiveness in GH-secreting pituitary adenomas. Further studies with a larger sample size are needed to better demonstrate the interrelationship of ECAD, SLUG, and NCAM and to determine the influence of these and other markers on the pathogenesis of acromegaly and tumor invasiveness.In conclusion, the characterization of GH-secreting pituitary adenomas allows the early identification of patients with tumors with a high risk of recurrence or resistant to conventional therapy, thus providing a basis for the development of patient-tailored treatment strategies and follow-up. The absence of


Overall, influenza viruses when detected in patients with ILI, SARI, or SCRI are likely attributable to illness. This finding is particularly likely among children and the elderly irrespective of HIV serostatus and among HIV-infected persons irrespective of age.The attributable fraction (AF) of influenza virus detection to illness has not been described for patients in different age groups or with different HIV infection statuses. We compared the age group–specific prevalence of influenza virus infection among patients with influenza-like illness (ILI) or severe acute or chronic respiratory illness  with that among controls, stratified by HIV serostatus. The overall AF for influenza virus detection to illness was 92.6% for ILI, 87.4% for SARI, and 86.2% for SCRI. Among HIV-uninfected patients, the AF for all syndromes was highest among persons <1 and  We defined a case of SARI as illness in a hospitalized patient who had symptom onset within 10 days before admission and who met age-specific clinical inclusion criteria. Cases in infants 2 days to <3 months of age included any hospitalized patients in this age group with a diagnosis of suspected sepsis or physician-diagnosed acute lower respiratory tract infection, irrespective of signs and symptoms. Cases in children 3 months to <5 years of age included any hospitalized patients in this age group with physician-diagnosed acute lower respiratory tract infection  or pleural effusion. Cases in children >38°C or a history of fever and cough of <10 days’ duration. We defined controls as persons of any age who sought medical care at 1 of the sentinel clinics for reasons other than fever or respiratory or gastrointestinal symptoms during the 14 days preceding the visit. Most controls visited the clinics for dental procedures, family planning, well baby visits, voluntary HIV counseling and testing, or acute care for nonfebrile illnesses. We aimed to enroll 1 HIV-infected and 1 HIV-uninfected control every week in each clinic within each of the following age categories: <1 year, 1–4 years, 5–24 years, 25–44 years, 45–64 years, and >65 years.During May 2012–April 2016, we also conducted prospective surveillance for persons with ILI and for asymptomatic persons (controls) at 2 outpatient clinics located in the catchment areas of the hospitals where we conducted SARI and SCRI surveillance: the Edendale Gateway Clinic in KwaZulu-Natal Province and the Jouberton Clinic in North West Province. We defined a case of ILI as illness in an outpatient of any age who sought medical care at 1 of the sentinel clinics and had a recorded temperature The procedures for these surveillance programs have been previously described ; specimens consisted of nasopharyngeal aspirates for children <5 years of age and nasopharyngeal and oropharyngeal swabs for persons >18 months of age, and we used PCR to test samples from children <18 months of age if the ELISA was reactive.We obtained HIV status for study participants from 2 sources: patient clinical records, when available; and, for consenting study participants, by testing dried blood spots at the National Institute for Communicable Diseases. We used an HIV ELISA to test samples from all patients We used unconditional logistic regression or exact logistic regression to estimate the AF of influenza-associated hospitalizations and outpatient consultations by comparing the influenza virus detection rate among patients with ILI, SARI, or SCRI with that among controls. The attributable fraction was estimated from the odds ratio (OR) obtained from the regression models as follows:DetectRateIllInfl) from the observed detection rate (DetectRateObsInfl) as follows:We subsequently estimated the influenza virus detection rate associated with illness among patients with ILI, SARI, or SCRI  were also included as covariates in the non-HIV–stratified overall model and in the models implemented among children <5 year of age  and persons >5 years of age . We also evaluated the AF of influenza virus types and subtypes overall and by HIV serostatus, using the same approach. For this analysis we also adjusted for co-circulating influenza virus types and subtypes.We implemented this analysis overall and by HIV serostatus within the following age categories: <1 year, 1–4 years, 5–24 years, 25–44 years, 45–64 years, In addition, we implemented the described analysis after reclassifying the SARI and SCRI cases by using a duration of symptoms cutoff of 7 days. This classification was in accordance with practices previously recommended by WHO for global influenza surveillance  or first-order and second-order  polynomial terms for age categories treated as a continuous numerical variable. We used STATA version 14.1  to implement the statistical analyses.The protocol for patients with SARI and SCRI was approved by the University of the Witwatersrand Human Research Ethics Committee  and the University of KwaZulu-Natal Human Biomedical Research Ethics Committee . The protocol for controls and patients with ILI was approved by HREC (protocol no. M120133) and BREC (protocol no. BF080/12). The surveillance was deemed nonresearch by the Centers for Disease Control and Prevention.During May 2012–April 2016, we enrolled 14,431 persons, of whom 13,335 (92.4%) had known age and available influenza and HIV results and were thus included for further analyses. Of these 13,335 persons, 2,504 (18.8%) were controls; 4,797 (36.0%) had ILI; 3,755 (28.2%) had SARI; and 2,279 (17.1%) had SCRI.Children <5 years of age accounted for 34.9%  of controls; 32.6%  of patients with ILI; 53.1%  of patients with SARI; and 6.7%  of patients with SCRI. Overall, the HIV prevalence was 41.0%  among controls (owing to enrollment criteria for controls); 27.0%  among patients with ILI; 38.7%  among patients with SARI; and 73.1%  among patients with SCRI (p<0.001). The HIV prevalence among patients with ILI, SARI, or SCRI was lowest among infants <1 year of age: 2.0% (11/554) among those with ILI; 9.1%  among those with SARI; and 20.2% (20/99) among those with SCRI. The HIV prevalence among these patients was highest among persons 25–44 years of age: 60.4%  among those with ILI; 89.0% (745/837) among those with SARI; and 91.6%  among those with SCRI. The median duration of illness was 2 days among patients with ILI, 3 days among patients with SARI, and 16 days among patients with SCRI.We detected influenza viruses in 1,082 (8.1%) of 13,335 specimens. Of these, 466 (43.1%) were influenza A(H3N2) viruses; 227 (21.0%) were influenza A(H1N1)pdm09 viruses; 20 (1.8%) were influenza A viruses that were not subtyped; and 369 (34.1%) were influenza B viruses Figure 1.>5 years of age with severe illness (SARI or SCRI), 46.6% (117/251) had symptoms for >10 days, compared with 12.0% (16/133) among children <5 years of age (p<0.001). Among controls, after we adjusted by age, the influenza virus detection rate was statistically significantly higher among HIV-uninfected compared with HIV-infected persons  .During the study period, we detected influenza viruses in 32 (1.3%) of the 2,504 controls; 666 (13.9%) of the 4,797 patients with ILI; 251 (6.7%) of the 3,755 patients with SARI; and 133 (5.8%) of the 2,279 patients with SCRI (p<0.001) . Among iThe overall influenza virus AF was 92.6% (95% CI 89.3%–94.8%) among patients with ILI; 87.4% (95% CI 81.3%–91.5%) among patients with SARI; and 86.2% (95% CI 77.7%–91.5%) among patients with SCRI . Our fin>65 years of age and lowest among persons 25–44 years of age  was 12.9% among patients with ILI, 5.9% among those with SARI, and 5.0% among those with SCRI . Using a>65 years of age and lowest among persons 25–44 years of age for all syndromes. This trend was not observed among HIV-infected patients, for whom the AF was similar across age groups for all syndromes.We provide estimates of the influenza virus AF among HIV-infected and HIV-uninfected patients of different age groups with mild or severe respiratory illness. Overall, influenza virus detection was statistically significantly associated with illness across syndromes, age groups, and HIV serostatus. Given the elevated influenza virus AF that we found in this study, the difference between the observed influenza virus detection rate and that attributable to illness was minimal. Nonetheless, among HIV-uninfected patients, the influenza virus AF was highest among persons <1 and >5 years of age with severe illness had symptoms for >10 days, compared with 12.0% of children <5 years of age. Chronic lung diseases are a known risk factor for influenza-associated severe illness  virus was consistently the highest and that of influenza A(H1N1)pdm09 virus was consistently the lowest across syndromes and HIV serostatus. A higher severity of influenza A(H3N2) compared with influenza A(H1N1)pdm09 and influenza B infections has been found in some studies . In addition, a differential influenza virus AF may also occur among persons with conditions associated with increased risk of influenza-associated severe illness other than age and HIV infection, and this possibility should be investigated. WARNING: Content in this section is NOT in the correct location. Check paragraph styling and re-export Detailed results for a study showing the proportion of influenza virus–positive cases attributable to mild and severe respiratory illnesses in patients with and without HIV, South Africa, 2012–2016.


Background: Several studies indicate contribution of hypothalamus-pituitary-adrenal (HPA) axis in multiple sclerosis (MS) disease. This study was designed to determine whether there is an effective difference in pituitary height, shape, and anterior-posterior diameter (APD) between patients with MS and the control group.Methods: In this study, sagittal pituitary height and APD of 134 men and women  were measured by T1 sequence magnetic resonance imaging (MRI). All the subjects were free of sellar or parasellar pathology without a history of surgical intervention or prolactin affecting drugs like bromocriptine and cabergoline or corticosteroid consumption.Results: Mean height of pituitary gland was 6.62 ± 1.43 and 5.78 ± 1.15 mm for patients and the control group, respectively, and the difference between the two groups was statistically significant (P = 0.001). Mean APD was 10.40 ± 1.29 mm for the group of patients and 10.25 ± 1.41 mm for the control group, respectively, without significant differences. 46.9%, 37.5%, and 15.6% of patients had flat, convex, and concave hypophyseal surfaces, respectively. This rate was 50%, 30%, and 20% among the control group, respectively. There was no significant difference between our measurements among patients on whom imaging study was performed at time of disease onset with others.Conclusion: Mean height of pituitary gland among patients with MS was significantly greater than the control group (P = 0.001). So can we consider the same etiology for pituitary hypertrophy among patients with MS as a hypothesis? It’s a complex neurological condition with demyelination and axonal loss.3 and 31.5 individuals per 100000 in Kerman Province, Iran.4MS incidence rate has been estimated as 2.5 million individuals worldwide. The incidence of MS in Iran is estimated to be 54.51 per 100000 population,5The stress system has peripheral and central parts: the hypothalamus-pituitary-adrenal (HPA) axis, and the sympathetic and adrenomedullary systems are the peripheral limbs, however, central components are in the hypothalamus and the brain stem; the main function of this system is to maintain basal and stress-related homeostasis.6 This study was designed in order to evaluate these responses on anatomy of pituitary gland and to determine whether there are differences in height, shape and anterior-posterior diameter (APD) of pituitary between patients with MS and the control group.Several studies indicate the contribution of HPA axis among patients with MS, increasing activity of HPA.In this case-control study, 64 definite patients with MS (57 women and 7 men) with the age range of 15-40 years old and 70 healthy, age and sex–matched controls were selected according to revised Mc-Donald's criteria. All the patients and control group had no history of sellar or parasellar pathology or cranial surgical intervention. During last 3 months prior to study, they had no history of consumption of drugs like bromocriptine, corticosteroids, and cabergoline, which may affect HPA.Pregnancy and lactation were considered as criteria of excluding from the study. Imaging was performed using GE Signa Excite, 1.5 Tesla Scanner.For 28 patients (43.75%), imaging was performed at disease onset (before MS treatment).The mean age was 27.06 and 27.54 years for patients and the control group, respectively, without statistically significant difference (P = 0.580).The mean height of pituitary gland was respectively 6.62 ± 1.43 and 5.78 ± 1.15 mm among patients with MS and among the control group, being higher for patients than the control group (P = 0.001).The mean APD diameter was 10.40 ± 1.29 mm for the group of patients and 10.25 ± 1.40 mm for the control group, respectively (P = 0.520). 46.9%, 37.5%, and 15.6% of patients had flat, convex, and concave hypophyseal surfaces, respectively. These rates were respectively 50%, 30%, and 20% among the control group. There was no difference in shape distribution percentage between the two groups (P = 0.610). It can be observed that the convex shape has increased among the patients with MS, however the concave shape has decreased among them.There is no significant difference between the patient individuals and control group members in our measurements  versus . The imaging study among the patients was performed at the time of disease onset (P < 0.050).All the patients and controls were divided into 3 groups according to their ages.The mean height of pituitary gland for men in the group of patients was respectively 6.60 ± 1.66 mm in the age range of 15 to 20 years old, 7.06 ± 1.19 mm in the age range of 21 to 30 years old, and 6.16 ± 1.60 mm in the age range of 31 to 40 years old. There was no significant difference with P value equal to 0.11. Mean AP diameter of hypophysis in above mentioned groups was 9.98 ± 1.30, 9.99 ± 1.36 and 10.37 ± 1.52 mm respectively.7 These findings are in agreement with other studies.8Our results showed higher pituitary gland height among patients with MS compared to healthy controls. The height and AP diameter values of hypophysis among normal individuals in Kerman Province are 5.78 and 10.25 mm, respectively.To our knowledge, we didn’t find any report about measurements of pituitary gland among patients with MS in the researches published in English language.8Clinical and experimental studies show that HPA axis has abnormality among patients with MS undergoing severe levels of MS. It might be associated with the pathogenesis of the disease. Hyperactivity of HPA axis is seen among 50% of patients with MS.9 MS disease is more frequent among women in almost the same age group.Pituitary height was significantly more among 15-to 20-year-old controls. We know that pituitary height is significantly greater among women than men, especially among the adolescent and young individuals; this problem is known as physiologic hypertrophy.10Among individuals undergoing MS with fatigue, HPA axis is more active compared to patients without fatigue. Adrenocorticotropic hormone (ACTH) concentration is significantly high in patients with fatigue. Proinflammatory cytokines are increased among patients with MS, which might be the reason for fatigue and changes of HPA axis.11In this study, 28 cases (43.75%) of patients with MS did not receive interferon-beta (IFN-), however, 36 cases (56.25%) received IFN-. There is no significant difference in height and AP diameter of hypophysis . Acute IThe mean height of pituitary gland among patients with MS was significantly greater than that of the control group (P = 0.001). So can we consider the same etiology for pituitary hypertrophy among patients with MS as a hypothesis?


Elevated serum ferritin concentration is associated with insulin resistance and diabetes. Recently it has also been described in gestational diabetes mellitus (GDM).A prospective study was done to determine whether there was a relationship between serum ferritin concentration in early pregnancy and the risk of GDM.A study was performed on 1,384 pregnant women with gestational age of 12-16 weeks. A blood sample was obtained for measurement of ferritin in the first trimester. Diagnosis of GDM was done by 75 gr oral glucose tolerance test between 24-28 wk.th percentile for healthy pregnant women. Considering this level 32% in the GDM group and 25.2%of normal subjects exhibited high ferritin levels (p=0.01). The risk of GDM with these high levels of ferritin was 1.4-fold higher than that for subjects with lower concentrations. The Odds Ratio was 1.4 (95% CI= 1-1.87) (p=0.01). After adjusted for age Odds Ratio was 1.38 (95% CI=1.02-1.86) (p=0.03) and after adjustment for pre-pregnancy Body Mass index, the adjusted odds ratio was 1.31 (CI= 0.96-1.79) (p=0.08). After multivariable adjustment (age and body mass index), the adjusted odds ratio was 1.3 (0.95-1.8) (p=0.09). Women who developed GDM had a higher concentration of serum ferritin than women who did not develop GDM (p=0.01). A ferritin concentration of 45 ng/ml was calculated to be the 75High serum ferritin can be regarded as a significant risk factor for the development of gestational diabetes. Ferritin, the major iron storage protein, has a function in iron metabolism , 2. SeruSome studies revealed a significant association between higher serum ferritin and risk of type 2 diabetes , 15. NevThe goal of this cohort study was to determine the relationship between serum ferritin concentration and risk of GDM.A prospective study was done on pregnant women with 12-16 wk gestational age. These subjects were recruited from two prenatal clinics  in Yazd, Iran between Jan 2010 and Feb 2013. Pregnant women were excluded from the study because of serious non-obstetric problems such as type 1 or 2 diabetes, malignancies, acute or chronic inflammatory or infective diseases, acute or chronic liver disease and iron deficiency anemia. General information including maternal age, the level of education, reproductive medical histories was obtained from the subjects’ medical records; Height was measured at entry to prenatal clinic, and pre-pregnancy BMI was obtained by re-calling pre-pregnancy weight in Kilogram divided by the square of height in meters. oC up to assayed. Prenatal iron supplementation was continued by all the participants based on the national policy, and all women received the same dosage  after the fourth month of pregnancy. Assessment of gestational diabetes was done by 75 gr oral glucose tolerance test between 24-28 wk of pregnancy. The oral glucose tolerance test was performed after an overnight fast of 8-14 hr while the subjects were on an unrestricted diet with unlimited physical activity for at least three days. Plasma glucose measured at fasting and 1 and 2 hr after 75 gr glucose load. Women were diagnosed with GDM if at least 1/3 diagnostic criteria were met  . Blood samples were stored at -70ctively) . GlucoseSerum ferritin was measured by ELISA method using two high-affinity monoclonal antibodies in an immune metric assay system . The Intra-assay and inter-assay coefficient variation of ferritin were 5.7% and 6.6% respectively. Iron was measured by the spectrophotometry method using the Bio system Iron Ferrozin Kit code 11509 . The Intra-assay and inter-assay coefficient variation were 2.2% and 2.9%, respectively. Iron binding capacity was measured by the spectrophotometry method using the Bio system Kit . Hb was assayed by the ELISA method , and hematocrit (HCT) was measured by micro hematocrit capillaries.Ethical considerationThis study was approved by Shahid Sadoughi University of Medical Science Ethical Committees. All subjects gave written Informed consent for participation in the study.Statistical analysis2 test was used for categorical variables. Data of continuous variables were expressed as mean±SD, and because ferritin levels were not normally distributed, the results were shown as medians (interquartile range). For the assessment of correlation between ferritin and risk of GDM the Spearman correlation was used. Multiple linear regression analyses were used to determine the relations between elevated serum ferritin  and pre-pregnancy BMI and age that were the dependent variables. Univariate statistics were considered for continuous variables, and a The logistic regression analyses were performed to find the effect of high serum ferritin on the risk of GDM. Odds ratios (ORs), adjusted odds ratios (AORs) and their 95% CIs from the logistic regression coefficients and corresponding covariance matrices, and the P for trend were computed. Potential confounding variables known to be associated with GDM were included in multivariable models  were tested in separate models because they correlated with serum ferritin. All statistical analyses were performed using statistical package for the social sciences, version 17.0, spss Inc Chicago, Illinois, USA. In this study from 1,384 subjects, 1,358 subjects performed OGTT at 24-28 weeks of pregnancy, and 26 women (1.6%) did not follow our study (OGTT was not done or referred to another center). The characteristics of participants are shown in two groups . Women wThe mean serum iron concentration was 111.5±58.3 µg/dl in the GDM and 98.6±41.7 µg/dl in the normal group; no significant difference was found. Total iron binding capacity was 344±72.5 µg/dl in the normal and 334±56 µg/dl in the GDM group, which was not statistically significant. The serum level of hemoglobin was not significantly different between the two groups. In pregnant women with gestational diabetes, the serum ferritin level was found to be higher in comparison with healthy pregnant women and the difference was statistically significant (p=0.001). The linear relationship between serum ferritin with maternal age was 0.05 (p=0.06) and with pre-pregnancy BMI was 0.1 (p=0.001). th percentile for healthy pregnant women. Considering this level as the cut-off point to define high ferritin, 32% in the GDM group and 25.2% of normal subjects exhibited high ferritin levels (p=0.01). The risk of having GDM with these high levels of ferritin was calculated to be 1.4-fold higher than that in subjects with lower ferritin concentrations (OR 1.4 95% CI= 1-1.87) (p=0.01) (th percentile), and risk of GDM with OR=1.19 (95% CI 0.88-1.6) (p=0.27). The sample size of our study was sufficient because it had 90% power to detect a 40% increase in the risk of GDM in pregnant women who had ferritin concentrations more than 45 ng/ml in the first trimester.A ferritin concentration of 45 was calculated to be the 75(p=0.01) . We furt(p=0.01) . After a(p=0.01) . Our stuOur study demonstrated that pregnant women with elevated serum ferritin concentration in early pregnancy had a higher rate of gestational diabetes. Our results were in concordance with Chen which showed that women with the highest quintile of serum ferritin had a twofold increased risk of GDM . Shimin and colleagues showed that among pregnant Chinese women, the mean serum ferritin concentrations at 28-30 weeks of gestation increased significantly in women with impaired glucose tolerance and patients with GDM compared with control subjects  (p=0.08). In addition, Jiang revealed that risk of type 2 diabetes is increased when the level of ferritin is elevated and this association is independent of other diabetes risk factors in healthy women . Chen shet al found that hemoglobin more than 13 g/dl) in pregnant women was an independent risk for GDM and that women with iron deficiency anemia had a reduced risk of GDM (Lao k of GDM , 25. We k of GDM , 25, 26.k of GDM , 28. Extra iron and oxidative stress affect the pathogenesis and increase the risk of type II diabetes and other associated diseases. Recently, it has been suggested that iron has impacts on glucose metabolism even if there is no excess iron. The studies revealed that body iron stores are involved in impaired glucose tolerance and gestational diabetes since iron compounds can change insulin synthesis and secretion, increase lipid oxidation, decrease in glucose transport into the muscle and elevation in gluconeogenesis, therefore insulin resistance in tissues developed , 23, 29.Our study has some limitations. This study measured ferritin once during pregnancy and did not assess the effect of inflammatory markers on GDM.In this cohort study, we showed associations between elevations in serum ferritin levels during early gestation and the risk of GDM in pregnant women. Therefore, high ferritin can be regarded as a significant risk factor for the development of gestational diabetes.


To investigate the intra-cameral level of ganciclovir following topical application of ganciclovir gel, 0.15% for cytomegalovirus (CMV) anterior segment infection.Non-randomized, prospective, interventional clinical study.Patients with active CMV anterior segment infection seen at Singapore National Eye Centre, confirmed by positive CMV real time PCR (RT-PCR) of the aqueous humor, that had not been treated with any form of ganciclovir in the preceding 1 month were recruited. They were treated with ganciclovir gel, 0.15% 1cc 5 times a day. Following 6 weeks of treatment, CMV load in the aqueous humor was measured using CMV RT-PCR and the ganciclovir drug levels in tears and aqueous humor were measured using high-performance liquid chromatography-mass spectrometry. The clinical features of the disease activity and the central corneal thickness (CCT) were recorded at the baseline and post-treatment.There were 29 eyes of 29 patients, of which 23 eyes had CMV anterior uveitis and 6 eyes had CMV endotheliitis. At the end of week 6, 26 eyes had undetectable CMV titre in the aqueous humor and no anterior chamber (AC) activity. Two patients had an increased CMV titre and increased AC inflammation. Both of these patients were non-compliant with the treatment. One patient had a reduced CMV titre in the aqueous humor with minimal AC inflammation. The mean ganciclovir concentration in the aqueous humor and the tears were 17.4 ± 30.6 ng/ml and 20,420.9 ± 33,120.8 ng/ml respectively. Mean CCT was 552.2 ± 42.3 microns. There was a weak correlation between the ganciclovir concentration in the aqueous humor and CCT . There was no significant correlation between the ganiclovir concentration in the tears and CCT .Ganciclovir levels in the aqueous humor was below the 50% inhibitory dose (ID50) for CMV replication, following topical application of the ganciclovir gel, 0.15%.NCT01647529.SingHealth Centralized Institutional Review Board, Singapore; R733/17/2010, ClinicalTrials.gov;  Chee heliitis  were posheliitis ,3.Various regimes have been used to manage CMV AU and endotheliitis, including systemic treatment with intravenous and oral ganciclovir, local therapy using repeated intravitreal ganciclovir injections or implants and topical therapy with ganciclovir gel, resulting in varying rates of success –9. ClincCurrently, ganciclovir ophthalmic gel, 0.15%  which is intended for the treatment of herpes simplex keratitis, is being used off-label in Singapore to treat CMV anterior segment infection. It is considered an inexpensive alternative with good tolerence and less relapses when compared to other forms of ganciclovir treatment . The recPre-clinical studies on rabbit eyes had shown good intra-cameral penetration following topical application of ganciclovir ophthalmic gel in varying concentrations, 0.05%, 0.15% and 0.2% –13. ThisWe recruited consecutive patients diagnosed with CMV anterior segment infection, either uveitis or endotheliitis at the Singapore National Eye Centre from July 2012 to July 2013. There was no sample size calculation as this was a pilot study.This was a non-randomized, single group, prospective interventional study with no comparison group.Written informed consent was obtained from all the participating subjects. The study was approved by the SingHealth Centralized Institutional Review Board (Protocol number: R733/17/2010). This trial was carried out in accordance with the tenets of the Helsinki declaration.We included the subjects aged between 21 and 99 years, diagnosed with CMV anterior segment infection, either AU or endotheliitis based on clinical manifestations and positive CMV real time PCR (RT-PCR)  of the aWe excluded those with other causes of hypertensive AU and endotheliitis such as Herpes simplex virus (HSV) and Varicella-zoster virus (VZV), confirmed by simultaneous PCR analysis  of the aAfter written informed consent, eligible patients were prescribed topical application of Virgan® ganciclovir ophthalmic gel, 0.15% 1cc 5 times a day, which worked out to be once every 3.5 hours, for 6 weeks. This is the only form of topical ganciclovir that is commercially available in Singapore. All the patients were instructed how to use the medication and asked to keep a drug diary to monitor their compliance.Primary outcomes were mean ganciclovir concentration in the aqueous humor and tears and its correlation to the central corneal thickness (CCT), following 6 weeks of topical application of the ganciclovir gel, 0.15%. CCT and the clinical features of the disease activity such as state of the cornea, presence of KPs and AC cells were recorded both at the base line and 6 weeks post treatment. In addition, intraocular pressure (IOP) using Goldmann applanation tonometry and cup to disc ratio (C/D ratio) were also recorded. CCT was measured using Sonogage Corneo-gage™ Plus pachymeter, and an average of five readings were taken. The AC cells and AC activity were graded according to the SUN criteria .At the end of week 6, the patients were reviewed in the clinic, 3.0 hours following the last application of the ganciclovir gel. As the half-life of ganciclovir is 3.5 to 4 hours ,18, we sAdverse events (AE) were monitored by the study team throughout the study period. Treatment-related AEs were defined as the AEs for which the causal relationship with the investigational drug could not be ruled out definitively, including the AEs assessed as ‘unlikely related’ to ‘definitely related’ . These iThe concentration of ganciclovir in the aqueous humor and tears was determined by HPLC-MS/MS. Ganciclovir pure standard and HPLC grade ammonium formate were purchased from Sigma-Aldrich . Acetonitrile and formic acid were purchased from Merck Chemicals . Chromatographic separation was performed by a Waters 2695 Separations Module  with a ZIC HILIC column, 2.1 × 100 mm, 3.5 μm, 200 Å . The autosampler and column heater temperatures were maintained at 10°C and 30°C, respectively. The mobile phase was: A, 20 mM ammonium formate in 9:1 water/acetonitrile and B, 0.1% formic acid in 9:1 acetonitrile/water. The gradient profile was: 95% B at 0 min to 85% B at 8 min, 40% B from 8.5 to 10 min and 95% B at 10.5 min. The flow rate was 0.2 ml/min. Detection was performed by an API 2000 triple quadruple mass spectrometer  with an ESI source operating in positive ionization mode. The MRM transition 256.1/152.1 was used for quantitation. The retention time of ganciclovir was 4.7 min.Aqueous humor samples (25 μL) were dried in a vacuum concentrator and reconstituted in 2:1 acetonitrile/water (50 μL). Samples were vortexed, centrifuged and the supernatants similarly analyzed by LC-MS/MS. Tear samples were diluted with 2:1 acetonitrile/water (10 to 1000-fold) and vortexed briefly. Samples were then centrifuged for 10 min at 14000 g (4°C) and the supernatants were analyzed by LC-MS/MS. In both cases, 20 μL of supernatant was injected for LC-MS/MS analysis. The limit of quantitation (LoQ) was estimated at 1 ng/ml (S/N = 10).Statistical significance was set at p≤0.05. As the data were not normally distributed, non-parametric Spearman rank correlation test was used to evaluate the correlation between CCT and the ganciclovir concentration in aqueous humor and tears. SPSS ver. 17  was used for analysis.Twenty nine eyes of 29 patients  aged between 32 and 82 years (Mean age 60.4 ± 12.5 years) were recruited . Twenty The mean concentrations of ganciclovir in the aqueous humor and tears were 17.4 ± 30.6 ng/ml and 20,420.9 ± 33,120.8 ng/ml respectively. The ganciclovir drug levels in the aqueous humor and tear samples are shown in Figs None of the patients developed any side-effects nor adverse events from using the medication. Neither were there any serious adverse events reported in this study. We did not perform subgroup analysis to compare the ganciclovir concentrations in the aqueous humor and tear samples between AU and endotheliitis eyes due to small sample sizes in each subgroup.In this study, topical application of ganciclovir ophthalmic gel, 0.15% exhibited a low intra-cameral drug level with a mean aqueous humor concentration of 17.4 ± 30.6 ng/ml, falling severely short of the 50% inhibitory dose (ID50) for CMV replication. ID50 for CMV replication is 250 ng/ml . As seenGanciclovir drug levels in both aqueous humor and tear samples were variable. There was only a weak correlation between the ganciclovir concentration in the aqueous humor and the CCT. Despite these findings, there was clearance of CMV from the aqueous humor following continuous application of ganciclovir gel for 6 weeks with good tolerance.Ocular pharmacokinetics studies in rabbit eyes have shown a rapid and relevant penetration of ganciclovir into the cornea and the anterior segment of the eye. Intra-cameral levels of ganciclovir in rabbit eyes reached a concentration of 394 ± 419 ng/ml following topical application of ganciclovir ophthalmic gel, 0.2% 4 times a day for 12 days –13. GancGanciclovir has a poor ocular bioavailability as a result of its relatively low partition coefficient . The difA recent study on seven patients with CMV endothellitis reported a mean ganciclovir concentration of 162.0±202.4 ng/ml in the aqueous humor following topical application of ganciclovir gel, 0.15% six times a day for 12 weeks . The lonIn rabbits, ganciclovir concentration was markedly higher in the solid ocular tissues than that in the ocular fluids . A histoIn a previously published study on healthy human eyes, there was a wide range of inter-individual and intra-individual variation in the ganciclovir concentration in tears, ranging from 0.92 to 6.86 μg/ml. These concentrations were measured 2 hours and 45 minutes after 4 instillations of ganciclovir gel, 0.15% . In our Similar to other studies ,29,35–37Ganciclovir concentrations in the aqueous humor were below the ID50 for CMV replication following topical application of the ganciclovir gel, 0.15%. There was a weak correlation between the aqueous humor drug levels and CCT. The preferential concentration of the drug in the solid ocular tissue in CMV infected eyes ,30–33 maHence, the topical ganciclovir gel, 0.15% can be considered a well-tolerated and inexpensive alternative to other forms of ganciclovir therapy for CMV anterior segment infection. Further research into improving the pharmacokinetics of ganciclovir is needed to improve the efficacy of the topical therapy which carries significant advantages over oral administration of the drug.S1 File(PDF)Click here for additional data file.S2 File(DOC)Click here for additional data file.


Vaginal intraepithelial neoplasia (VAIN) is a pre-malignant lesion, potentially leading to vaginal cancer. It is a rare disease, representing less than 1% of all intraepithelial neoplasia of the female genital tract. Similar to cervical intraepithelial neoplasia (CIN), there are three different grades of VAIN. VAIN 1 is also known as a low-grade squamous intraepithelial lesion (LSIL), whereas VAIN 2 and VAIN 3 both represent high-grade squamous intraepithelial lesions (HSIL). Risk factors for the development of VAIN are similar to those for cervical neoplasia, i.e. promiscuity, starting sexual activity at an early age, tobacco consumption and infection with human papillomavirus (HPV). However, compared to other intraepithelial neoplasia such as CIN or VIN , there still is little understanding about the natural course of VAIN and its capacity for pro- or regression. Furthermore, there is controversial data about the HPV detection rate in VAIN lesions.67 patients with histologically confirmed VAIN, who were diagnosed between 2003 and 2011 at the University Women´s Hospital of Heidelberg Germany, were included in this study. The biopsies of all participating patients were subjected to HPV genotyping. GP-E6/E7 Nested Multiplex PCR (NMPCR) was used to identify and genotype HPV. Eighteen pairs of type-specific nested PCR primers were assessed to detect the following "high-risk" HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, as well as the "low-risk" genotypes 6/11, 42, 43 and 44. The data was analyzed with the software SAS .All 67 cases were eligible for DNA analysis. The median age was 53 years. The largest group with 53% (n = 36) was formed by women, who were first diagnosed with VAIN between the age of 41 to 60 years. 50% (n = 37) of the patients presented a VAIN in the upper 1/3 of the vagina. 58 (87%) were diagnosed with HSIL (VAIN). The median age in patients with LSIL (VAIN) was 53 years and in patients with HSIL (VAIN) 53.5 years. 12 women (18%) had an immunosuppression. HPV positivity was confirmed in 37 patients (55%). Except for a single patient, who had a triple infection with HPV types 6/11, 16 and 68, only infections with one single HPV genotype were detected. An infection with the HPV genotypes 31, 39, 45, 51, 58, 59, 66, 42, 43 and 44 couldn’t be found in any of the patients. In 28 patients with diagnosed VAIN, an infection with HPV 16 could be shown, 24 (86%) of them were diagnosed with a HSIL (VAIN). 16 (24%) women presented condylomata and 13 of them (81%) had a positive HPV status. However, only 47% of the women without condylomata presented a positive HPV status, resulting in a significant correlation (p = 0.0164) between condylomata and HPV infection. In 28 of all 67 patients (42%), recurrence of the neoplasia occurred.HPV 16 is the main virus-type to be associated with the development of a VAIN. Also, HPV 16 infection, VIN or condylomata acuminata in the past medical history seemed to be significant factors for early relapse. Vaginal intraepithelial neoplasia (VAIN) is a pre-malignant lesion, potentially leading to vaginal cancer. VAIN is rare, representing less than 1% of all intraepithelial neoplasia of the female genital tract .Similar to CIN, there are three grades of VAIN according to the depth of epithelial involvement. Whilst VAIN 1 only affects the lower one-third of epithelium, VAIN 2 involves the lower two-thirds and VAIN 3 more than two-thirds or the complete epithelium. VAIN 1 is also known as a low-grade squamous intraepithelial lesion (LSIL), whereas VAIN 2 and VAIN 3 both represent a high-grade squamous intraepithelial lesion (HSIL) [VAIN can affect women of any age, but it is more common in women above 50. Interestingly, age and grade of disease correlate in the sense that women of a higher age suffer from higher grades of VAIN . Risk faFurthermore, there is controversial data about HPV detection rate in VAIN lesions. Chao et al reported a 69.3% detection rate of HPV in VAIN lesions , whereas2 laser vaporization as a primary therapy. The treatment was done consistently between providers . We defined recurrence as detection of new lesions 8–12 weeks after initial therapy at earliest. In all cases the recurrence was histological confirmed by vaginal biopsy. All patients gave their written consent for analysis of their tissue. The study was approved by the ethical committee of the university of Heidelberg (S-277/2015).67 patients with histologically confirmed vaginal intraepithelial neoplasia (VAIN) and clinical examination between 2003 and 2011 at the University Women´s Hospital of Heidelberg, Germany, were included in this study. All patients were referred by the gynecologist to University Hospital due to a suspicious vaginal finding. VAIN was diagnosed according to guidelines, using colposcopy, followed by biopsy of the suspicious areas, which were revealed through application of acetic acid and Schiller's Iodine test. All parts of the vagina are thoroughly examined according to an examination scheme viewing the vagina clockwise starting at 3 o'clock. Hematoxylin-eosin sections were made from the biopsies and reviewed by an experienced pathologist. In uncertain cases, immunohistochemical analysis of p16 (INK4a) was additionally performed. We included only patients who were free of any suspicious area 8–12 weeks after initial diagnose or treatment. According to the WHO classification preinvasive lesions of the vaginal squamous epithelium are differentiated into LSIL (VAIN 1) and HSIL (VAIN 2 and 3) [Before HPV-testing all biopsies were reviewed by an independent pathologist. Data regarding the patient’s history and neoplasia status were collected retrospectively from the clinical documentation system. The biopsies of all patients, who were included in this study were subjected to HPV genotyping. GP-E6/E7 Nested Multiplex PCR (NMPCR) was used to identify and genotype HPV . The pri®, NORMAPUR®, VWR, Vienna) was added and heated to 55°C. After 10 minutes, the liquid was removed and the previous step repeated. Next, xylol was replaced by 100% ethanol and the samples were left to rest for 10 minutes twice. Between each step, the tissues were centrifuged at 13300G. After removal of alcohol, the tissues were incubated at 37°C. 0,788g Tris-HCl , 0,0372g EDTA  and 0.5 ml Tween 20  were added to a vessel. 50 - 75ml distilled water was added to the mixture and adjusted to a pH of 8.5. 5μl proteinase K  was added. The samples were then incubated overnight at 56°C. The next day the product was heated to 95°C for 5 minutes. As a next step 5μl were removed from this solution and transferred to a glass cuvette. Eventually, the DNA concentration was determined photometrically (NanoDrop ND-1000 Spectro Photometer). Subsequently, the solution was diluted with nuclease-free water  to 50ng/μl, after which 2.5 μl DNA were extracted for PCR.The samples, embedded in paraffin, were cut with a layer thickness of 7μm. Xylol  and 0,75μl 6P primer (10μM)  and 12,5μl "MasterMix" consisting of deoxynucleotide, Mg2+, 10-fold concentrated polymerase buffer solution, water and Taq polymerase. The Taq activation and denaturation of the DNA took place when heated to 94°C, in the first step for 4, then repeated for 1 minute. The primer hybridization was carried out at 40°C for 1 minute, the elongation phase at 72°C for 2 minutes initially and then again for 5 minutes. A total of 40 cycles were performed.2,5μl DNA were assessed with 9,25μl water  and 12,5μl "MasterMix" were added to 0,5μl E6/E7 product. The Taq activation and denaturation of the DNA were carried out again at 94°C for an initial 4 minutes, then again for 30 seconds. The primer hybridization took place at 56°C for 30 seconds. Finally, the elongation phase was carried out at 72°C, initially for 45 seconds, and in a second step for 4 minutes. A total of 35 cycles were performed.Separation of nucleic acid strands allows determination of the amplicon length, which leads to specific identification of HPV types. The agarose powder (3g to 100ml buffer) was dissolved in 1 × Tris-borate-EDTA (TBA) buffer and heated to near-boiling point. After brief cooling, 10μl "Gel Red" per 100ml buffer was added. The solution was poured into a cast, after which a comb was placed to create wells for loading. Next, the gel was layered with 1x TBE buffer in an electrophoresis chamber and loaded. Furthermore, 5μl PCR product with 2μl 40% glycerol were added on a microtiter plate and applied to the gel. As a molecular weight marker served 50 bp. The term was 1 hour at a voltage of 100 volts. Finally, the gel was removed from the chamber and photographed under UV light.Data were analysed with the software SAS . Categorical variables were compared by chi-square test or Fisher's exact test. p-values < 0.05 were considered statistically significant. Due to the small number of cases and partially missing values a multivariate analysis was not performed .All 67 cases were eligible for DNA analysis. The median age was 53 years (range 26–84 years). Subgroup analysis revealed a median age in patients with LSIL (VAIN) of 53 (range 28–66 years) and HSIL (VAIN) of 53.5 years (range 26–84 years). The largest group with 53% (n = 36) was formed by women, who were first diagnosed with VAIN between the age of 41 to 60 years. 50% (n = 37) of the patients presented with VAIN in the upper 1/3 of the vagina. 58 (87%) were diagnosed with HSIL (VAIN). According to conventional histology, only 6 cases were unclear, which meant that additional p16 (INK4a) staining was necessary. In all 6 cases p16 (INK4a) was detectable. In two cases no HPV infection could be shown, both patients had a HSIL. Three patients were HPV16 and one patient HPV 33 positive. Overall positive HPV status was confirmed in 37 patients (55%). 6  patients with LSIL (VAIN) were HPV positive and 31  of the patients with HSIL (VAIN). The median age of the group with detected HPV infection was 53 years (range 26–79). It differs only slightly from that of the HPV negative group . Detailed patient characteristics are shown in 12 women (18%) had an immunosuppression—4 of those received chemotherapy, 2 had an HIV infection, 3 suffered from diabetes mellitus type 1 or 2, and 3 patients had an autoimmune disease or history of transplantation with immunosuppressant medication.Except for a single patient who had a triple infection with HPV types 16, 6/11 and 68, only infections with one single HPV genotype were diagnosed. The genotype distribution is shown in 17 patients of this study presented a simultaneous or previous VIN. Twelve (71%) of those developed VAIN recurrence, whilst only 16 of the 50 patients (32%) without a VIN in the medical history did (p = 0.0053). 32 patients (48%) presented a simultaneous or previous cervical intraepithelial neoplasia (CIN). 10 patients of those patients developed a recurrence. In 18 of 35 patients without CIN the VAIN recurred . In 10 out of 16 women with a VAIN recurrence a history of condylomata was known . In one patient a vaginal carcinoma could be found with first diagnose of VAIN.Patients underwent follow-up examinations with a median duration of 12.5 months . A follow-up examination was scheduled every 2–3 months. In 28 of 67 patients (42%) recurrence of the VAIN occurred .Of the 28 patients, who suffered a recurrence, 20 (71%) had an HPV infection. In comparison, only 17 of the 39 (44%) recurrence free women had a HPV infection (p = 0.0238), indicating a significant correlation between HPV infection and recurrence of disease.The median time from initial diagnosis to recurrence was 12 months (range 3.3 months to 48.1 months). Of 58 patients with HSIL (VAIN), 25 (43%) developed a recurrence. Recurrent vaginal intraepithelial neoplasia was highly related to HPV infection . Only one patient progressed to vaginal carcinoma after one year. None of the other patients progressed to a higher grade of VAIN.The only patient with a low-risk HPV type 6/11 infection remained recurrence free, as well as two patients with HPV type 56. A significant correlation between HPV 16 infection an VAIN recurrence could be found. 16 (57%) out of 28 patients with a HPV 16 infection VAIN was recurrent (p = 0.0351). Also patients with HPV types 18, 33, 35 and 52 respectively, developed a recurrence. Furthermore, the women with a triple HPV infection with types 16, 6/11 and 68 also developed a recurrence.In accordance with other studies we were able to demonstrate that the median age of women suffering from VAIN was 53 years , 14. FurOther studies have revealed that VAIN is most frequently localised in the upper third of the vagina , 15. ThiAccording to the study of Dodge et al., a strong association between VAIN and coexisting CIN or VIN was present in 65% and 10% of cases, respectively .In addition, 25% of patients were either simultaneously diagnosed with VIN or had a VIN in the past and 48% of patients were diagnosed with CIN respectively. In summary, VAIN frequently presents not as a single pathological phenomenon in 60% of patients it is accompanied with other intraepithelial neoplasia of the lower genital tract. Patients with immune deficiency show a higher risk for developing multiple lesions of the lower genital tract –19, whicThere are heterogeneous results concerning the association of VAIN and HPV infection rate. Regarding to this diversity, non-standardised and differing approaches in HPV identification seem to be the leading issue. Smith et al reported a HPV identification rate of 98,5% in 66 LSIL (VAIN) cases and 92,6% in 166 HSIL (VAIN) cases . In contThe cellular tumor suppressor gene p16 (INK4a) is an important biomarker for HPV-associated intraepithelial neoplasia. Klaes et al. found an overexpression of p16(INK4a) in all examined LSIL (CIN) lesions—except for those being caused by "low-risk" HPV types—as well as in all HSIL (CIN) lesions. There was no expression in healthy cervical epithelium or inflammatory altered epithelium . In thisSimilar to the HPV detection rate, our HPV type analysis revealed different data compared to previous reports. The most common type of HPV in VAIN lesions is generally believed to be HPV 16. De Vuyst et al described HPV 16 in 57,6% of HSIL (VAIN) cases, Smith et al even showed a rate of 65,8% [Condylomata acuminata represent the most common benign genital tract tumours, originating from infection with low risk HPV types such as HPV type 6 and 11 . HistoloThe prevalence of vaginal carcinoma varies in different areas. Whereas in Eastern and Western Asia the prevalence rate is 0,2/100000, in Southern Asia and the Caribbean seems to be a higher prevalence rate  due to high HPV infection rate , 25. IrrFor some authors a premalignant or malignant cervical lesion is an stronger indication for hysterectomy –29 than However, the closer follow-up of these patients after hysterectomy due to intraepithelial neoplasia may partially cause this difference in the mean development time of VAIN as those patients might be monitored more closely.In the past there have been several attempts to identify risk factors for relapse, such as nicotine abuse, grading of VAIN, localisation, age and many others . NeitherHPV 16 is the main virus-type to be associated with the development of VAIN. Also, repeated HPV 16 infection, VIN or condylomata acuminata in the past medical history seem to be significant risk factors for relapse.S1 Table(XLSX)Click here for additional data file.S2 Table(RTF)Click here for additional data file.S3 Table(RTF)Click here for additional data file.S4 Table(RTF)Click here for additional data file.S5 Table(RTF)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.