This study aimed to determine the effects of dietary MH, delivered as an extract of un-ripened avocado, on energy expenditure (EE) in healthy adult Labrador Retriever dogs . The study was a double-blind, cross-over with each dog receiving both dietary treatments, control (CON) and MH , in random order. Resting and post-prandial (10 h) EE and respiratory quotient (RQ) were determined by indirect calorimetry (d 42). The following day, body composition was assessed using dual X-ray absorptiometry. Continuous activity monitoring was conducted using an Atical® accelerometer (d 43–47). A vastus lateralis muscle biopsy was obtained prior to the morning meal (d 49) and 4 h after consumption of their meal (d 56) to determine the protein content and phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK). Diet did not affect body weight, resting EE or skeletal muscle AMPK phosphorylation. Dogs fed MH had significantly lower post-prandial RQ (p = 0.02) and ratio of fat to lean body mass (p = 0.02). Physical activity during light time periods (but not dark) was lower in dogs fed MH (p < 0.05) during weekends, but not on weekdays. These results suggest that MH affects energy balance of adult dogs, but that these effects are not dose dependent and not due to physical activity.Mannoheptulose (MH), a sugar found in avocados that inhibits glycolysis  The average lifespan of dogs has been increasing; it has been estimated that more than 50% of dogs in the United States are over the age of six . As in hEnergy restriction (ER), without malnutrition, has been shown to increase median lifespan in yeast, flies, nematodes, rodents  and dogsFood ingredients enriched with compounds thought to favourably alter metabolism or extracts of these compound may provide an alternative to ER. In humans, several plant-derived polyphenols are currently under investigation, including, but not limited to, resveratrol and epigallocatechin gallate . How theMannoheptulose (MH), a seven-carbon sugar found in avocados, has been preliminarily investigated as a novel food ingredient for dogs. Early research demonstrated that MH competitively inhibits hexokinases ,9. StudiThe primary objective of this study was to examine the effects of dietary MH  on EE in adult Labrador Retrievers. In addition, we examined the effects of MH on skeletal muscle AMPKα protein content and phosphorylation. We hypothesized MH induced inhibition of hexokinases would limit glucose as a substrate of energy production and consequently increase fat utilization. Furthermore, MH was expected to decrease post-prandial EE and this decrease would be associated with increases in the activation of AMPK in skeletal muscle.All procedures were approved by the Institutional Animal Care and Use Committee of Procter and Gamble Pet Care . A total of 12 black Labrador Retrievers  were usThis study was designed as a parallel, double-blind, cross-over with each dog receiving both dietary treatments, control (CON) and mannoheptulose (MH), in random order. The study took place January 2013 to July 2013 and included a ten week dietary adaptation period, two study periods (each 63 d), and a three week dietary washout between study periods. A summary of study measurements is presented in Both diets, CON and MH, were made from identical ingredients and were similar in terms of nutrient content . The MH Animals were individually fed their daily ration in two meals (0700 h and 1300 h) and food intake was measured daily. Beginning 10 wk prior to study initiation and in between study periods (3 wk), dogs were fed the CON diet. A 10 wk dietary adaption period was selected partly because dogs included in the present study were involved in other research trials and fed diets of varying composition prior to the initiation of this study. Furthermore, given the duration of this cross-over study, a 10 wk adaption period was necessary to accommodate scheduling of study events and support staff. During the dietary wash-in period, energy intakes were adjusted to maintain body weight and a body condition score of 3.0. The day before study initiation, dogs were randomized to dietary treatment and energy intakes  were fixed throughout the entire duration of the study (including in between treatment periods).2 levels in the chamber between 0.4 and 0.8%. Exiting chamber air was dried by passing it through columns of Drierite™ and magnesium perchlorate before reaching the O2 and CO2 analyzers .Respiratory gas exchange measurements were conducted via whole-body indirect calorimetry. The calorimetry chambers  were made of clear plexiglass and fitted with a hinged access top door for providing food to the dog. Chambers were designed as open-flow circuits with room air pulled into the chambers at a rate of 22 to 26 L/min to maintain CO2 and CO2 analyzers were calibrated with standard gases and dogs rested in the chamber for a minimum of 25 min to ensure adequate CO2 equilibration. Two fasting measurements were taken, after which dogs were fed their full daily ration of test diet as a single meal (time 0) and gas exchange measurements continued for 10 h. Animals were fed their full daily ration  in order to maximize circulating MH concentrations and presumably its biological effects. Each chamber was sampled every 3 s over a 5 min period every 25 min. O2 and CO2 exchange and respiratory quotient data were logged in real time using data acquisition software . Energy expenditure was calculated from O2 consumption and CO2 production (VO2 and VCO2) using the abbreviated Weir equation [Prior to study initiation, dogs were acclimated over an 8 wk period (1 to 8 h per week) to rest comfortably and calmly in the chamber with no excessive activity or movement. Dogs that did not acclimate were ineligible to participate in the study. Gas exchange measurements were conducted 2 d prior to the initiation of each study period (baseline) and on d 42 of each period. Prior to any measurements, the Oequation  and exprOn d 43 of each period, body composition analysis was completed using an X-Ray Bone Densitometer . Dogs were fasted overnight  and sedated using Dexmedetomidine  at a dose of 0.02 mg/kg and Carprofen  at a dose of 2 to 4 mg/kg administered i.m. Propofol  at a dose of 5 to 7 mg/kg was administered i.v. for induction. Dogs were positioned on their sternum with the cranial aspect of ante brachium placed on the table to ensure the phalanges faced caudally. The hind limbs were extended with the tail placed straight and in between the hind limbs. A whole body scan was performed of the following regions: left arm, right arm, trunk, left leg, right leg and head. Scans were done in triplicate for each dog and the median value of the three scans was recorded. Following the scan, atipamezole  was administered to each dog at a dose of 0.2 mg/kg. Dogs were placed in a heated cage until fully recovered and monitored for 1 wk for complications.Continuous physical activity measurements were made using the Actical accelerometer . The Actical device  was attached to the dog’s collar in the ventral position and all dogs were acclimated to wearing the device prior to study initiation. Raw activity values were converted to activity counts using ActiReader software .Baseline activity measurements were taken the week prior to study initiation and again the third week of the dietary washout period. Treatment measurements were taken on d 43 to 47 of each period . The average activity per minute was calculated during dark (1800 h to 0600 h) and light (0600 h to 1800 h) time periods. The percent of active time was calculated by dividing the time when dogs were active  by the total time. Weekday (0600 h Wednesday through 0600 Saturday) and weekend (0600 h Saturday through 0600 h Monday) activity were analyzed separately, as there is less human interaction with the animals on weekends.2 and stored at –80°C for later immunoblot analysis.Muscle biopsies were taken after an overnight fast  on d 49 of each treatment leg. On d 56 of both treatment legs, dogs were fed their full daily ration of test diet at their morning meal and muscle biopsies were taken 4 h post-feeding . This sampling time point coincides with peak MH concentrations in the plasma ,14. Animg. Supernatants were removed and protein concentration was determined by Bradford protein assay. Protein (25 μg) from muscle lysates was diluted in Laemmli sample buffer  and loaded onto 7.5% Tris HCL pre-cast resolving gels  and separated by SDS-PAGE. Gels were transferred onto nitrocellulose membranes  (200 mA/tank). Membranes were blocked in 5% milk for 1 h at room temperature and washed in TBST for 10 min before being incubated in primary antibody overnight at 4°C. Primary antibodies  were diluted (1:1000) in 5% BSA/TBST. Membranes were washed twice in TBST for 15 min and then incubated with anti-rabbit immunoglobulin–horseradish-peroxidase–linked secondary antibodies (1:2000 dilution in TBST) for ~1 h. The proteins of interest were visualized using enhanced chemiluminescence  and captured and quantified using AlphaView Software for FluorChem Systems. All samples from individual dogs were loaded on to the same gel, in addition to a control sample. Data were normalized to the control sample and expressed as the ratio of phosphorylated to total protein content.Muscle was homogenized in ice cold NP40 Cell Lysis Buffer (15:1 v:w) with 1 mmol/L PMSF  and protease inhibitor cocktail  and spun at 4°C for 5 min at 1500 x 1 = 480 KJ/Kg0.75·d; μ2 = 447 KJ/Kg0.75·d; SD = 26 KJ/Kg0.75·d; α = 0.05; β = 0.8 [A sample size of 12 was calculated from energy expenditure data reported by McKnight et al.  for all dogs throughout the entire study.time < 0.01) irrespective of diet (p diet = 0.27) (Body weight significantly increased throughout the study (p  = 0.27)  and ther = 0.27) , but abslean mass · d), respectively)  between dietary groups . Restingctively) . Post-prctively) . From 3 For all baseline and period activity measurements, dogs were more active in light than dark time periods , Table 5Weekday and weekend baseline activity per minute was not different between diets prior to study initiation or during the dietary washout period . SimilarPeriod was significant for weekday and weekend activity per minute and percent active time in the dark time period . For botAn inadequate amount of muscle tissue (< 20 mg) was harvested to complete the necessary analyses in some animals and as such, sample sizes were reduced across all treatments. AMPK was expressed as the ratio of phosphorylated to total AMPK protein content . There wDietary MH , delivered as an extract of the flesh of un-ripened avocados significantly decreased post-prandial RQ andvoluntary physical activity in adult Labrador Retrievers. These MH induced changes were not associated with alterations in EE or the content and phosphorylation of AMPK in skeletal muscle. Interestingly, decreased physical activity associated with MH feeding and led to a reduction in body fat to lean mass, without altering body weight . Energy expenditure as measured by indirect calorimetry includes obligatory and facultative components of REE, adaptive thermogenesis and a small amount of voluntary activity. It does not include the energy expended during voluntary physical activity. REE represents the major component of total EE (~70%) and is largely predicted by fat free mass . As leanKm for glucose compared to that of hexokinase IV found in liver, pancreas and brain. As glucose phosphorylation in skeletal muscle is saturated at low glucose concentrations, AMPK may not be as sensitive to changes in glucose supply as it would be in tissues that express glucokinase. In addition, MH was delivered in a mixed meal. Therefore, it is also likely that effectiveness of MH as competitive inhibitor of hexokinase II was overcome due to the abundance of dietary derived carbohydrate (glucose). A dose response study is necessary to better understand the role of dietary MH as a glycolytic inhibitor in skeletal muscle and other tissues, particularly adipose tissue, given the observed reduction in fat to lean body mass in the present study.As MH has been shown to inhibit hexokinases, the reduced flux through glycolysis was expected to limit glucose as a substrate of energy production and consequently increase fat utilization. In agreement, post-prandial RQ was transiently reduced in dogs fed MH, suggesting a higher proportion of fat relative to carbohydrate oxidation. However, RQ does not indicate net carbohydrate or fat oxidation, nor does it account for protein oxidation. Therefore, more sensitive measures of substrate oxidation are warranted. Inhibition of glycolysis may lead to a decrease in intracellular ATP. Lowering the ATP to ADP ratio would activate AMPK. Once activated, AMPK increases ATP production by potently promoting fat oxidation. Indeed, glucose deprivation, as imposed by another glycolytic inhibitor, 2-deoxyglucose, has been shown to activate AMPK , 23. In The final component of EE, physical activity, can account for 20–30% of EE. Dogs in this study exhibited diurnal patterns of activity; specifically dogs were more active during the light than dark periods (irrespective of diet). This finding agrees with the activity patterns displayed by laboratory Beagles . In contWith prolonged reductions in physical activity, as imposed by daily MH feeding, one may expect weight gain. However, MH decreased the ratio of body fat to lean mass, despite all dogs having equivalent energy intakes. To understand the impact of MH on EE better, other measurement techniques, including the doubly labelled water technique, should be considered. The doubly labelled water technique would enable average estimates of EE over variable time periods in the dog’s regular environment. Furthermore, other sources of energy loss including fecal and urinary losses, which were not measured in the current study, should be examined.Irrespective of MH, study period had a significant impact on several study outcomes, including body weight. Dogs in this study were fed to maintenance weight based on historical energy intake records yet still gained weight. Basing energy intakes on historical energy intake records is a common practice in companion animal nutrition, largely due to the impracticality of using more sensitive measures such as indirect calorimetry. However, the accuracy of energy intake records is debatable, especially for studies of long duration such as the present one. The primary reason for fixing energy intakes in this cross-over designed study (opposed to adjusting intakes weekly to maintain body weight) was to ensure individual dogs received an equivalent amount of MH in each study period. The body weight gain observed in this 6 month study was modest, approximately 3% of the animal’s body weight . As lean mass was not different between study periods, the gain in body mass was considered fat deposition. Fat deposition was likely influenced by reduced physical activity observed in the second study period. Seasonal changes, including differences daylight exposure and climate, between period 1 (winter) and period 2 (spring) may have influenced animal activity patterns and in turn body composition. Seasonal changes in fat deposition have been noted in ruminants –28 and rA modest but statistically significant transient reduction in post-prandial RQ was observed in dogs fed MH. The biological significance of the decrease in RQ is uncertain. It may be reflective of increased fat oxidation, as a reduction in fat to lean body mass with MH feeding was observed . However, changes in body composition with daily MH feeding (~6 mg/kg BW) were likely influenced by decreased voluntary physical activity, particularly on the weekend when there is reduced human animal contact. As physical activity encompasses a broad range of complex behaviours, a more critical analysis of physical activity (and related behaviours) is warranted. These results suggest that MH affects energy balance of adult dogs, but that these effects are not dose dependent and not due to physical activity.S1 Data(XLS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


N = 83), were matched to “referents” (N = 232), those who had not died of prostate cancer, using incidence density sampling. Diagnostic tissue was retrieved to assess expression of AZGP1, MUC1, NKX3.1, p53, and PTEN by semiquantitative immunohistochemistry (IHC). Poisson regression was used to estimate mortality rate ratios (MRRs) adjusted for age, Gleason score, and stage and to estimate survival probabilities. Expression of MUC1 and p53 was associated with increased mortality , whereas AZGP1 expression was associated with decreased mortality . Analyzing all markers under a combined model indicated that the three markers were independent predictors of prostate cancer death and survival. For men with localized disease at diagnosis, assessment of AZGP1, MUC1, and p53 expression in diagnostic tissue by IHC could potentially improve estimates of risk of dying from prostate cancer based only on Gleason score and clinical stage.Only a minority of prostate cancers lead to death. Because no tissue biomarkers of aggressiveness other than Gleason score are available at diagnosis, many nonlethal cancers are treated aggressively. We evaluated whether a panel of biomarkers, associated with a range of disease outcomes in previous studies, could predict death from prostate cancer for men with localized disease. Using a case-only design, subjects were identified from three Australian epidemiological studies. Men who had died of their disease, “cases” ( In developed countries, prostate cancer (PCa) is the most common noncutaneous cancer in males, but with the widespread use of prostate-specific antigen (PSA) testing and prostate biopsies, the majority of prostate tumors now diagnosed are localized , slow growing, and unlikely to metastasise κB and KPNA2 proteins were also found to be associated with biochemical recurrence, independently of clinical prognostic factors Numerous immunohistochemistry (IHC) studies have examined the prognostic value of biomarkers in PCa tumor tissue. While the majority of studies have involved small numbers of samples resulting in detected associations not holding up to multivariable analysis, recently there have been some promising results from a few larger, more robust studies. In an IHC analysis of tissue from 5718 radical prostatectomy specimens, Tsourlakis et al. found expression of the death-domain-associated protein (DAXX) to be independently associated with biochemical recurrence and to be a potential independent prognosticator of PCa outcomes As disease prognosis and treatment decisions are the principal challenges for those patients with clinically localized disease at diagnosis Despite these promising results, few biomarkers have been tested and validated in diagnostic tissue from localized tumors and no biomarkers have yet been established for widespread clinical usage. The aim of this study was to assess expression of a panel of tissue biomarkers including AZGP, MUC1, p53, NKX3.1, and PTEN using IHC on archival tissue collected at the time of diagnosis and to determine whether expression of these biomarkers predicts PCSM independently of Gleason score and clinical stage.Study participants were men diagnosed with histologically confirmed PCa recruited into three epidemiological studies run by the Prostate Cancer Research Program of the Cancer Council Victoria. These studies include 964 men from the Melbourne Collaborative Cohort Study (MCCS), 786 men from the Melbourne arm of the Risk Factors for Prostate Cancer case–control study (RFPCS), and 1230 men from the Early-Onset Prostate Cancer Family Study (EOPCFS). The MCCS series included all men diagnosed with PCa during follow-up of a cohort of 41,514 volunteers  recruited in Melbourne during 1990–1994 In 2007, we began a nested case–referent study within the MCCS, RFPCS, and EOPCFS to identify factors associated with PCSM. Cases were defined as those men diagnosed with localized disease (T1–T3b) and whose death was ascribed to PCa by the Australian Bureau of Statistics (cause of death codes C61 or 185) between diagnosis and 1 January 2005 for MCCS participants, or 1 January 2007 for RFPCS and EOPCFS participants. Cause of death was reviewed by an expert coder and PCSM was confirmed for 213 men (hereafter referred as “cases”). For each case, we then randomly selected two men (hereafter referred to as “referents”) using risk set  sampling with years since diagnosis as the time scale. Thus, to be eligible as a referent, each man had to have survived at least as long as the corresponding case. Cases and referents were matched for Gleason score at diagnosis as recorded by the Victorian Cancer Registry .This study was approved by the Human Research and Ethics Committee of the Cancer Council Victoria.Three micron tissue sections were taken from selected blocks following review and stored at 4°C in desiccated containers until used. IHC was performed using the Leica Bond-Max® autostainer . All primary antibody incubations were for 30 min. The detection system used was the Novocastra Novolink Max polymer  with the DAKO DAB chromogen . The antibodies used were the following: Novocastra NCL-PTEN HIER citrate 1/400 (PTEN), Novocastra NCL-MUC-1 HIER citrate 1/400 (MUC1), Novocastra-P53-D07 HIER 1/300 (p53), Zymed 35-9700 HIER citrate 1/250 , and Santa Cruz SC-11358 . Level of staining was assessed by one of the authors (J. P.), an anatomical pathologist specializing in urological cancers, who was masked to the status of the participant . For each case the tissue block corresponding with the highest Gleason score was selected, sections were cut, and the resulting slides were stained for the five biomarkers by IHC. Biomarker expression was recorded as both the proportion of stained tumor cells (0–100%) and overall staining intensity . If different Gleason patterns were present on the same slide, expression was recorded for each of them. If multiple areas with the same Gleason pattern were present on the slide, expression was averaged across them. For MUC1, AZGP1, and PTEN staining was only present in the cytoplasm while for p53 it was present only in the nucleus. For NKX3.1 we considered only cytoplasmic staining as positive as nuclear staining was ubiquitous. The data used for this study were binary, including only the proportion of stained tumor cells for the highest Gleason pattern categorized by either not expressed (<5% staining) versus expressed (≥5% staining).Reproducibility of the IHC scoring was assessed using 20 slides for each marker which were independently assessed by another pathologist (A. L.) masked to J. P.'s results. Slides for this study were selected at random and in a way to ensure an equal distribution of expression and nonexpression of each biomarker.S(t) were calculated using the delta method estimate of the variance of log(−log(S(t))) back transformed to the natural metric. All models were adjusted for tumor stage, Gleason score , and age at diagnosis. The Wald test was used to compute P-values for coefficients.Mortality rate ratios (MRR) and 95% confidence intervals (CI) for IHC biomarker expression were estimated using Poisson regression models with PCSM as the outcome In secondary analyses, we restricted the models to cases and referents that had been treated by radical prostatectomy. For sensitivity analyses, we fitted conditional logistic regression for the complete case–referent sets  to compare the odds ratios with the MRR obtained from the Poisson models . For the analyses conducted to determine whether the scoring of the staining was reproducible, we calculated separately for each marker the proportion of slides that were scored consistently by the two pathologists and estimated the Cohen's kappa statistics using expression in two categories (expressed vs. not expressed) as for the other analyses.All statistical analyses were performed using Stata 12.1 for Linux 64-bit .Due to financial limitations, the present study was restricted to a randomly selected subset of 102 cases and 254 referents for whom we attempted to retrieve diagnostic archival tissue. In most instances diagnostic tissue was in the form of “cores” obtained by a trans-rectal, ultrasound-guided (TRUS) needle biopsy, in the others it was in the form of “chips” obtained by a TURP. Diagnostic tissue specimens were not able to be retrieved for 19 cases and 22 referents, leaving a total of 83 cases and 232 referents available for analysis. This sample of cases and referents was similar to the remaining 283 cases and referents for whom we did not attempt tissue collection in terms of age at diagnosis  and time to death . The distribution of Gleason score did not differ dramatically between the two groups: of those not included in this study, 30% had Gleason score less than 7, 30% had Gleason score of 7, and 40% had Gleason score of 8–10 while the proportions were 18%, 42%, and 42%, respectively for those included in this study.The main clinical characteristics of subjects in this study are shown in Table As a sensitivity analysis we also fitted a combined conditional logistic regression model using data from the 57 matched case–referent sets with at least one matched referent . These results were broadly consistent with those of the combined Poisson model, with odds ratios (95% CIs) of 0.58 (0.25–1.35), 2.68 (0.94–7.59), and 1.96 (0.80–4.82) for AZGP1, MUC1, and p53 expression, respectively. A sensitivity analysis including only TRUS samples also provided similar results, with MRRs (95% CIs) of 0.35 (0.12–1.01), 1.79 (0.61–5.28), and 3.10 (1.24–7.74) for AZGP1, MUC1, and p53, respectively.κ-statistics being 0.76, 0.69, 0.89, and 1, respectively. For NKX3.1, the agreement was slightly lower with 78% concordant and κ statistics equal to 0.49.We found no evidence of statistical interaction between any of the assessed markers (data not shown) and, therefore, calculated predicted marginal survival probabilities for given marker expression patterns directly from the combined Poisson model including AZGP1, MUC1, and p53. In our study the overall 5-year survival probability for men with PCa was 97% (99% for Gleason score 6 prostate cancer). The predicted 5-year disease-specific survival probabilities for a man diagnosed at the median diagnosis age (63 years), with a Gleason score 8–10 and a clinical stage T1c tumor are shown in FigureOur study shows that semiquantitative IHC assessment of the expression of three proteins in diagnostic tissue, AZGP1, MUC1, and p53, taken together as a biomarker panel, predicts risk of PCSM beyond the risk predicted by “conventional” variables such as Gleason pattern and stage. Expression of AZGP1 alone was associated with a threefold decreased risk of PCSM compared with nonexpression, while MUC1 or p53 expression was associated with a twofold increased risk compared with nonexpression. Most importantly, a combined assessment of these three markers suggests that they are independent predictors of PCSM and can discriminate between cases at high risk from those at low risk of dying from PCa.The expression of p53 protein has previously been shown to be associated with PCSM in diagnostic tumor specimens AZGP1 and MUC1 were first identified as candidate markers of PCa aggressiveness by a study that used cDNA microarrays to compare gene expression in fresh-frozen radical prostatectomy specimens, fresh-frozen “normal” prostate tissue from these same surgical specimens, and lymph node metastases Deletions of the tumor suppressor gene PTEN and of the transcription factor NKX3.1 are common in PCa but evidence that expression of these proteins is associated with prognosis is limited and inconclusive Our study has several major strengths (1) the relatively large number of cases that died from PCa and therefore, the ability to use PCSM as the outcome; (2) the simultaneous evaluation and assessment of multiple biomarkers; (3) limiting the analysis to patients with localized PCa; (4) assessing formalin-fixed, paraffin-embedded diagnostic tissue, thereby expanding the data to all patients with prostate cancer, not just those who have had radical prostatectomies or those where fresh-frozen tissue is available; (5) assessment of all biomarkers by a single, masked, expert uropathologist, and review by another, general pathologist showing that these markers could be assessed consistently; and (6) our results can be rapidly translated into practice due to the use of standard prostate diagnostic tissue  and IHC procedures using commercially available antibodies. We selected IHC to assess protein expression as it is used by virtually all pathologists, including those in middle- or low-income countries and because it is simple and inexpensive .Despite the many strengths mentioned above, our study does have some limitations. First, serum PSA concentrations at diagnosis were not available for our patients, although this omission is unlikely to have confounded the observed associations. PSA at diagnosis is a strong predictor of biochemical relapse In summary, our results, if validated in an independent set of cases, show that a panel of markers, including AZGP1, MUC1, and p53, could be assessed using routine IHC at the time of diagnosis of clinically localized PCa. This panel may help clinicians and patients make more informed decisions around treatment options and therefore decrease unnecessary treatment for men at low risk of dying from the disease and improve our ability to identify men that might benefit from aggressive treatment.


The pneumococcus is a diverse pathogen whose primary niche is the nasopharynx. Over 90 different serotypes exist, and nasopharyngeal carriage of multiple serotypes is common. Understanding pneumococcal carriage is essential for evaluating the impact of pneumococcal vaccines. Traditional serotyping methods are cumbersome and insufficient for detecting multiple serotype carriage, and there are few data comparing the new methods that have been developed over the past decade. We established the PneuCarriage project, a large, international multi-centre study dedicated to the identification of the best pneumococcal serotyping methods for carriage studies.Reference sample sets were distributed to 15 research groups for blinded testing. Twenty pneumococcal serotyping methods were used to test 81 laboratory-prepared (spiked) samples. The five top-performing methods were used to test 260 nasopharyngeal (field) samples collected from children in six high-burden countries. Sensitivity and positive predictive value (PPV) were determined for the test methods and the reference method .For the alternate serotyping methods, the overall sensitivity ranged from 1% to 99% (reference method 98%), and PPV from 8% to 100% (reference method 100%), when testing the spiked samples. Fifteen methods had ≥70% sensitivity to detect the dominant (major) serotype, whilst only eight methods had ≥70% sensitivity to detect minor serotypes. For the field samples, the overall sensitivity ranged from 74.2% to 95.8% (reference method 93.8%), and PPV from 82.2% to 96.4% (reference method 99.6%). The microarray had the highest sensitivity (95.8%) and high PPV (93.7%). The major limitation of this study is that not all of the available alternative serotyping methods were included.Most methods were able to detect the dominant serotype in a sample, but many performed poorly in detecting the minor serotype populations. Microarray with a culture amplification step was the top-performing method. Results from this comprehensive evaluation will inform future vaccine evaluation and impact studies, particularly in low-income settings, where pneumococcal disease burden remains high. Catherine Satzke and collaborators exploit a panel of spiked and field pneumococcal samples to compare serotyping methods used for nasopharyngeal carriage studies. Streptococcus pneumoniae bacterium. S. pneumoniae is transmitted through contact with infected respiratory secretions and harmlessly colonizes the nose and throat of many healthy children . Occasionally, however, S. pneumoniae spreads into the lungs, the blood stream, or the covering of the brain, where it causes pneumonia, septicemia, and meningitis, respectively. These potentially fatal invasive pneumococcal diseases can be treated with antibiotics but can also be prevented by vaccination. Vaccination primes the immune system to attack disease-causing organisms (pathogens) by exposing it to weakened or dead pathogens or to pathogen molecules that it recognizes as foreign (antigens). Because there are more than 90 S. pneumoniae variants, or “serotypes,” each characterized by an immunologically distinct complex sugar coat, S. pneumoniae vaccines have to include multiple serotypes. “Pneumococcal conjugate vaccines” (PCVs) effectively prevent invasive pneumococcal diseases caused by common serotypes in resource-rich countries and are now being introduced into resource-poor countries.About 800,000 young children, mostly living in low-income countries, die annually from pneumococcal diseases, illnesses caused by the Although vaccination with PCVs reduces the carriage of the pneumococcal serotypes contained in them (vaccine serotypes), in the absence of these serotypes, non-vaccine serotypes can rapidly colonize the nasopharynx and become more common in both carriage and disease. Thus, “serotype replacement,” which may be more pronounced in low-income settings, threatens the global control of pneumococcal disease through vaccination; thus, when evaluating the impact of pneumococcal vaccines, it is important to analyze the carriage of multiple serotypes. Unfortunately, the traditional serotyping method—in which bacterial colonies are grown and a small number of colonies are typed using tests called the Quellung reaction and latex agglutination—is cumbersome and frequently misses the carriage of multiple serotypes. Several new serotyping methods have been developed over the past decade, and, here, in a multi-center comparative study (the PneuCarriage project), the researchers investigate which of these new methods is best for the examination of pneumococcal carriage in vaccine evaluation studies.The researchers used 15 clinical isolates containing a mixture of pneumococcal serotypes to prepare 81 “spiked” samples, which they distributed to 15 laboratories for testing with 20 serotyping methods. They determined the sensitivity (the percentage of serotypes in the samples that were correctly identified) and the positive predictive value  for each method and used the five top-performing methods  to test 260 nasopharyngeal (field) samples collected from children in six high-burden countries. When testing the spiked samples, traditional serotyping of over 100 colonies per sample had a sensitivity of 98% and a PPV of 100% overall, whereas the sensitivity of the alternative methods ranged from 1% to 99%, and their PPV ranged from 8% to 100%. Fifteen methods detected the major serotype in the spiked samples with ≥70% sensitivity, but only eight detected the minor serotypes with the same sensitivity. For the field samples, the sensitivity and PPV of the top-performing tests ranged from 74.2% to 95.8% and from 82.2% to 96.4%, respectively ; a culture microarray method had the best overall performance (95.8% sensitivity and 93.7% PPV).A pneumococcal serotyping method for use in carriage studies needs to have high sensitivity, to detect multiple serotypes in individual samples, and to detect most or all serotypes. These findings show that although most of the recently developed serotyping methods detected the dominant serotype in a sample, many failed to detect minor serotypes. Moreover, the performance of similar methods varied markedly, and methods optimized for testing pure isolates did not necessarily work well when testing more complex samples. These findings identified microarray with a culture amplification step as the top-performing method, but, importantly, this study did not test all the available serotyping methods. Also, because it assessed each method in only a single laboratory, no conclusions can be reached about the reproducibility of these methods or their suitability for use in less-experienced laboratories or in resource-limited settings. Nevertheless, these findings should help to guide future vaccine evaluation and impact studies, particularly in low-income settings, where the burden of pneumococcal disease remains high.http://dx.doi.org/10.1371/journal.pmed.1001903.This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at pneumococcal disease and pneumococcal vaccination, including personal stories (including some information in Spanish)The US Centers for Disease Control and Prevention provides information on all aspects of pneumococcal diseaseThe UK National Health Service Choices website provides information about pneumococcal vaccination (in English and Spanish)Kidshealth, a website provided by the US-based not-for-profit Nemours Foundation, includes information on pneumococcal disease and vaccinationPublic Health England provides guidance on pneumococcal disease, including personal storiesThe not-for-profit Immunization Action Coalition has information on pneumococcal vaccination in resource-poor countriesGavi, a not-for-profit global vaccine alliance, is helping to roll out PneuCarriage project is availableMore information about the pneumococcal infections (in English and Spanish)MedlinePlus provides links to other resources about pneumococcal vaccines.The World Health Organization provides information about pneumococcal disease and  Streptococcus pneumoniae (the pneumococcus) is a dominant cause of childhood illness and death worldwide. It has been estimated to cause ~800,000 deaths in children aged under 5 y annually, most due to pneumonia and most in low-income countries [ountries . Pneumocountries . PneumocPneumococcal conjugate vaccines (PCVs) are effective at preventing pneumonia and invasive pneumococcal disease, and are currently being introduced in many resource-poor countries . PCVs reduce nasopharyngeal carriage of the pneumococcal serotypes they contain (vaccine types). Although transmission and disease due to vaccine types is reduced, non-vaccine types fill the ecological niche, becoming more common in carriage and disease –5. This Carriage studies offer a practical approach for monitoring serotype replacement and can help in assessing the impact of PCVs and other pneumococcal vaccines ,10. PneuA pneumococcal serotyping method suitable for use in carriage studies should have high sensitivity (including the ability to detect multiple serotypes), detect most or all serotypes, be suitable to scale up for large projects, and be practical for resource-poor countries. We established the PneuCarriage project, a large, international multi-centre study, with the aim of identifying the best pneumococcal serotyping methods for carriage studies.We compared 20 different pneumococcal serotyping methods from 15 research groups using 81 laboratory-prepared samples. We then tested the five top-performing methods using 260 nasopharyngeal samples collected from children in six high-burden low- and middle-income countries. All samples were subjected to comprehensive conventional microbiology to thoroughly characterise the pneumococcal content, and the results were used to assess the performance of the new methods to identify the best pneumococcal serotyping methods. Sensitivity and positive predictive value (PPV) were selected as the key parameters for method evaluation, as these measures best convey the overall ability of a method to correctly detect pneumococcal serotypes present in a sample.All research involving human participants was approved by the relevant institutional review board or an equivalent committee, specifically the Bangladesh Institute of Child Health Ethics Review Committee; the Fiji National Research Ethics Review Committee; the Gambian Government/Medical Research Council Laboratory Joint Ethics Committee and the Medical Research Council (UK) Gambia Scientific Coordinating Committee; the Kenya Medical Research Institute National Ethical Review Committee and Scientific Steering Committee; the Government of Papua New Guinea Medical Research Advisory Committee and the Papua New Guinea Institute of Medical Research Institutional Review Board; the Human Research Ethics Committee of the University of the Witwatersrand, the Ethics Committee of Stellenbosch University, and the Medicine Control Council of South Africa; and the Clinical Science Review Committee of the Division of AIDS, US National Institutes for Health. Written informed consent was obtained from the parents/guardians of all participants.Fifteen pneumococcal isolates were used to prepare 81 laboratory-prepared (“spiked”) samples in skim milk-tryptone-glucose-glycerol (STGG) medium ,15. The Viable counts were performed in saline to assess the concentration of the inocula, and the associated load (and proportion) of each serotype in the spiked samples was determined. Samples containing one serotype were inoculated with low numbers of pneumococci ; however, the serotype contained within them represented 100% of the pneumococcal content. As such, the viable count data from samples with one serotype were included when calculating the mean load of the minor type, but excluded when calculating the mean percent of the minor serotypes.n = 4) or contained one, two, three, or four serotypes . Samples inoculated with pneumococci contained a mean of 2.4 serotypes , with a mean load of 2.11 × 105 colony-forming units (CFU)/ml . Samples inoculated with more than one serotype contained a “major type” (the serotype with the highest percent abundance in each sample) and one or more “minor types” (the other serotypes present in each sample). Major types ranged from 67.78% to 98.71%  of the total pneumococcal content in each sample, with a load ranging from 3.08 × 104 to 5.70 × 105 CFU/ml . The minor types ranged from 1.29% to 14.49%  of the total pneumococcal content in each sample, with the load ranging from 6.95 × 102 to 3.89 × 104 CFU/ml .Spiked samples consisted of medium alone  in the project laboratory at the Murdoch Childrens Research Institute.The 260 nasopharyngeal (“field”) samples were from 260 children aged ≤24 mo in six low- and middle-income countries . Samples2, up to 120 well-separated alpha-haemolytic colonies were randomly selected. To do this, the culture plate was evenly divided into eight sections, and a section was chosen from a previously prepared random list. The outermost colony in the designated sector was then picked and designated the “first” colony. The remaining colonies on the whole plate were then picked. If not all of these colonies were required, sections were prioritised from a list of randomly generated numbers. If not all the colonies in a particular section were required, the operator selected colonies from the outer edge of the plate working towards the centre. Throughout the random colony selection process, only well-separated alpha-haemolytic colonies were chosen. An example of each morphological variant was also subcultured if not chosen during random selection. Selected colonies were then subcultured onto HBA plates. Subcultured colonies underwent pneumococcal identification using optochin, and then bile and Phadebact Pneumococcus (MKL Diagnostics) tests, as appropriate. Pneumococcal isolates were serotyped by Quellung reaction and/or latex agglutination (see below). Latex agglutination was conducted using the Denka Seiken kit [Conventional methods were used to identify and serotype the pneumococci carried in the samples. Sample aliquots were thawed and mixed, and 50 μl was used to conduct a viable count by serial dilution and plating on selective medium (HBA plates containing 5 μg/ml gentamicin). After 36–44 h of incubation at 35–37°C in 5% COiken kit  accordiniken kit , except iken kit ,26 usingiken kit ,28 usinghttp://www.rcpaqap.com.au).The primary colony  was serotyped with both the Quellung reaction and latex agglutination. Subsequent colonies were serotyped with latex agglutination, using the minimum number of reactions needed to confirm whether the serotype was the same. Any different serotypes detected were fully serotyped by latex agglutination and Quellung reaction. Laboratory staff were fully blinded during sample processing and participated in an external quality assurance program during the course of the project , all occurring when the target was present as a minor serotype.n = 1).To identify serotyping methods for testing, we conducted literature reviews and contacted pneumococcal researchers, gave presentations at international meetings to explain the project, and circulated a project fact sheet. We identified and contacted 29 research groups. Fifteen research groups joined the study. Of the remaining 14 groups, 11 had methods that were no longer in use, had been superseded by other approaches, or were considered by the investigators to be unsuitable for detecting multiple serotype carriage. Three research groups were unable to participate because of funding constraints, existing work commitments, or an inability to reach agreement on the terms of the material transfer agreement  added, and the pellet resuspended. DNA was extracted using the QuickGene DNA tissue kit S with the QuickGene Mini 80 apparatus (Fuji Film Corporation) and eluted in 200 μl of buffer CDT. Then, 10 μl of extracted DNA was used as a template for multiplex PCR (mPCR) reactions. Each reaction was performed in a total volume of 50 μl using AmpliTaq Gold DNA Polymerase with GeneAmp (Applied Biosystems), and amplified in a Veriti Thermo Cycler (Applied Biosystems) for 40 cycles. PCR products were detected using a Beckman CEQ 8000 genetic analyser system. Primers were labelled for recognition of the product in the CEQ 8000 analyser as green or blue peaks and by size (141 to 515 base pairs). LytA PCR was used to detect pneumococcal DNA in the sample as previously described [A subset of 22 spiked samples was processed with this method. Initially, 25 μl of sample was spun in a microcentrifuge at 5,220escribed , except Taq polymerase produced in house. If the DNA was positive for cpsA, the cps primers were omitted and the reaction repeated to avoid competition of these primers with primers for minor serotypes that may be present. A similar approach was taken for any positive serotyping reactions.In this method, 50 μl of sample was plated onto a selective blood agar plate (containing 5% sheep blood and 5 μg/ml gentamicin) and incubated overnight. DNA was extracted from the entire culture plate using a DNeasy Blood & Tissue Kit (Qiagen). Approximately 500 ng of extracted DNA was then used for each of seven subsequent mPCR reactions  using TaInitially, 50 μl of sample was plated onto Columbia III agar with 5% sheep blood (BD) and incubated overnight. DNA was then extracted from all the plate growth using a DNeasy Blood & Tissue Kit (Qiagen). All seven mPCR reactions were then applied to each sample, using 5 ng of DNA as template. To detect the minor serotypes, the seven mPCR reactions were repeated, using 100 ng of DNA. PCR reactions and primer sequences were as previously described –32.lytA PCR [LytA-positive samples were then tested by mPCR/reverse line blot (RLB) assay to identify serotypes, as previously described [lytA, but non-typeable by mPCR/RLB, were tested by cpsA-B PCR and sequencing [DNA was extracted from 50 μl of sample using the NucliSENS easyMAG total nucleic acid extractor kit (bioMérieux) or the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). DNA was eluted in 110 μl, and 10 μl used for each subsequent test. Initially, lytA PCR  was perfescribed ,34. All escribed  was condquencing .plyNCR was performed using 31.4 μl of extracted DNA. To identify co-colonisation, restriction fragment length polymorphism (RFLP) analysis of the plyNCR PCR amplicon was performed on the extracted DNA, as described previously [In this method, 50 μl of sample was streaked onto a Columbia sheep blood agar plate and incubated overnight. DNA was extracted from a loopful of the bacterial lawn using the QIAamp DNA Mini Kit (Qiagen). The entire lawn was then harvested from the primary culture agar plate and stored at −80°C. PCR amplification of eviously . Latex aeviously  except tIn this method, 50 μl of sample was diluted 1:4, lysed by bead beating, and treated with proteinase K at 56°C for 15 min, and DNA was extracted using the KingFisher DNA extraction instrument (Thermo Scientific), as previously described . Then, 5In this method, 1 μl of sample was cultured on a sheep blood agar plate and incubated for 48–72 h. Growth was then harvested using a needle, and boiled for 15 min at 95°C. Twenty-nine of the 81 spiked samples tested did not grow even when 20 μl was cultured, so DNA was directly extracted from ~30 μl of original sample by boiling lysis. Extracted DNA was then subjected to PCR and ESI-MS as outlined for method 19.Initially, 50 μl of sample was cultured on a fresh rabbit blood agar plate for 24 h. Growth was harvested for DNA extraction and analysis, as previously described . In brie2O and quantified using a NanoDrop spectrophotometer. Approximately 300 ng of the DNA was fluorescently labelled with either ULS-Cy3 or ULS-Cy5 using the Agilent Technologies Genomic DNA ULS Labeling Kit. The fluorescently labelled samples were then hybridised overnight to the BμG@S SP-CPS v1.4.0 microarray, according to the Agilent Array CGH protocol. Following hybridisation, the microarrays were washed and scanned, and intensity data acquired using an Agilent microarray scanner and feature extraction software. The statistical calls of which serotypes, or combination of serotypes, were present in the sample, together with the relative abundance of each serotype, were determined using Bayesian-based algorithms [In this method, 45 μl of the neat sample, or of two 10-fold serial dilutions, was spread on selective agar plates  and incubated overnight. For the plate with the highest density of distinct non-confluent colony growth, all colonies were scraped into 1 ml of sterile PBS. DNA extractions were performed using the QIAamp DNA Mini Kit (Qiagen), including lysis buffer  and RNase treatment as previously described . DNA wasgorithms .2O. Whole genome amplification was performed on 10 μl of extracted DNA using the GenomePlex Whole Genome Amplification Kit (Sigma). The amplified DNA was cleaned up using an Amicon Ultra 0.5 ml (Millipore) centrifugal filter and quantified using a NanoDrop spectrophotometer. Note that a subset of 16 spiked samples was processed with this method.This method was performed as per method 4, except that DNA was extracted from 50 μl of sample using a NucleoSpin Tissue XS DNA isolation kit (Macherey-Nagel) and eluted in 20 μl of Hcps regions and the lytA gene. Then, 5 μl of extracted DNA was added to each tube and reactions were performed using the Bio-Rad CFX96 real-time PCR system.DNA was extracted from 50 μl of sample using the easyMAG automatic extraction system (bioMérieux) and eluted in 100 μl. The multiplex real-time PCR  was perfThis method was performed as per method 7, except that the 50 μl of sample was inoculated into 8 ml of Todd Hewitt broth (Sigma-Aldrich) (with 0.5% yeast extract) and incubated overnight. Then, 0.5 ml of the overnight culture was used for DNA extractions as in method 7.lytA by quantitative real-time PCR were included in serotyping analysis. Real-time PCR was performed as previously described using 6 μl of extracted DNA for each reaction using 33 sets of primers/probes [DNA was extracted from 20 μl of sample using the QIAmp DNeasy Blood & Tissue Kit (Qiagen). Only samples positive for s/probes . If no iIn this method, 10 μl of sample was cultured in 3 ml of Todd Hewitt broth  overnight, and DNA from 200 μl of broth culture was extracted using the QIAmp DNeasy Blood & Tissue Kit (Qiagen). Testing was then conducted as in method 14.cpsB gene was amplified by PCR (using 2 μl of extracted DNA per reaction), the amplicon purified, and the nucleotide sequence determined. The nucleotide sequences were used to interrogate a publically available gene database (GenBank), and the match with the greatest nucleotide homology indicated the serotype. If co-colonisation was detected, the amplicons were subcloned into a plasmid vector, cloned, and re-sequenced. If a sample was cpsB negative, the PCR was repeated using 10 μl of template. A subset of 29 spiked samples was tested with this method.DNA from 50 μl of sample was extracted by heat lysis , the deb2O, heat lysed, and processed as above.This method was conducted as per method 12, except that the 50 μl of sample was inoculated into 10 ml of Brain Heart Infusion (Oxoid) broth. After 18–24 h of incubation, cells from 8 ml of broth were pelleted by centrifugation, resuspended in 50 μl of HIn this method, 25 μl of sample was added to 3 ml of serum broth (SSI Diagnostica) and incubated overnight. The culture was then serogrouped and/or typed using the commercially available Pneumotest-Latex kit as recommended by the manufacturer (SSI Diagnostica) . Each cuInitially, 10 μl of sample was cultured on a selective agar plate (Columbia CNA agar with 5% sheep blood) overnight. A suspension of all the colonies was made and tested as described previously . In brieR-phycoerythrin conjugate was added to the plate. After another 20 min, the plate was washed as before and read on a Bio-Plex reader (Bio-Rad) to generate a mean fluorescent intensity (MFI) reading for each antigen. Positive samples were indicated by a 30% or greater reduction in sample MFI compared to the positive control MFI.Samples were heat-killed by incubation at 60°C for 45 min, and assayed as described previously . In brieg for 2 min and the supernatant diluted 1:2 in PBS. Following this, 25 μl was applied in duplicate to the Luminex immunoassay, performed as previously described [R-phycoerythrin fluorescent conjugate.In this method, 50 μl of sample was processed by centrifugation at 16,000escribed . In brieFor both the spiked and field sample testing, our primary analyses were the percent sensitivity and the PPV of each method. Sensitivity was defined as the percentage of serotypes present in samples that were correctly identified by each method. For spiked samples, sensitivity was calculated for the major serotype, minor serotype(s), and overall. For field samples, sensitivity was calculated for samples containing a single serotype, for samples with multiple serotypes (as a proxy for major and minor serotypes), and overall. The PPV was defined as the percentage of serotypes identified that were actually present in the samples, i.e., the proportion of identified positives that were true positives.Sensitivity and PPV were calculated according to the level of discrimination provided by the method. For example, if a method claimed to detect 6A/B, and the sample contained 6B, results reported as 6A/B would be deemed correct. However, if a method claimed to detect 6A and 6B as individual serotypes, and the sample contained 6B, then results reported as 6A would be incorrect. Note that for microarray (methods 4 and 15), some closely related serotypes were reported as a group, with the individual serotype call in brackets . In this case, results were analysed both to the level of the group and to the level of the individual serotype call. For simplicity of analysis, if a method did not claim to detect a serotype  but the sample contained that serotype, this result was deemed incorrect. Pneumococci identified as non-typeable were excluded from all analyses. Serotypes 15B and 15C were analysed as 15B/C, as these serotypes can interconvert .For the spiked samples, results were compared with the inocula. The result 19A or 19F was deemed correct for strain PMP843, which types as 19F by phenotypic methods but has genetic characteristics of 19A . We alsSelection of methods for field sample testing was primarily based on scientific performance in the testing of the spiked samples, using a cutoff of ≥70% sensitivity to detect minor serotypes and ≥90% PPV. Methods that met these criteria were also assessed on other aspects (see below), specifically the number of serotypes that they were currently capable of detecting  and the perceived ease of incorporating additional serotypes. This approach was used to ensure that methods were able to serotype carriage isolates (which may be more diverse than invasive isolates) and were also capable of detecting the emergence of non-vaccine serotypes in the post-PCV era. Methods were also assessed for their potential for quantification. Using these criteria, five methods progressed to testing of field samples.For field samples it was more challenging to differentiate false from true positives, because the exact sample contents were unknown, and a highly sensitive method would likely detect serotypes that other methods did not. Results from the reference serotyping method, research group methods, and additional testing of discrepant results were used to create a “study gold standard”, against which the performance of the alternate serotyping methods was assessed.To determine the study gold standard, we examined results for each sample, and considered a serotype to be a true positive if it was found by (1) the reference method and one or more alternate methods or (2) three or more alternate methods or (3) two methods that covered both phenotypic and genotypic approaches . Note that methods 14 and 21  were considered to be one method for these analyses. Where a serotype was found by only one method or by two methods of similar approach , this was deemed a discrepant result. Field samples with discrepant results were further investigated by testing of sample aliquots by single-plex quantitative real-time PCR  and/or sStatistical tests were performed using GraphPad Prism version 5.04 for Windows, (GraphPad Software). Quantitative data were analysed with the D’Agostino and Pearson omnibus normality test, and reported as mean (95% CI) or median (IQR) accordingly. Spearman’s correlation was used to examine the relationship between variables.The reference serotyping method  had 100%, 96%, and 98% sensitivity to detect the major, minor, and overall serotype populations, respectively. No false positives were identified (PPV 100%).The 20 alternate serotyping methods had overall sensitivities ranging from 1% to 99%, with similar ranges for the major and minor serotype populations. The PPV ranged from 8% to 100% ; S1 DataWhen spiked samples were tested with method 4 (culture microarray), only one serogroup 6 serotype was reported  for three samples containing both serotypes 6A and 6B, so the sensitivity and PPV were slightly lower when results were analysed to the level of the individual serotype call . The samSeven methods met our initial criteria of ≥70% sensitivity to detect minor serotypes and ≥90% PPV when testing the spiked samples . Method 5 CFU/ml . The mean relative abundance of minor serotypes  was 18.7% . When testing the 260 field samples, the reference serotyping method had 96.3% sensitivity for samples containing one serotype, 90.9% sensitivity for samples containing multiple serotypes, and 93.8% sensitivity overall. The PPV was 99.6% .The 260 field samples contained 307 serotypeable pneumococci. Forty-nine of the known 97 serotypes, including twelve of the PCV13 types, were identified at least once. PCV7, PCV10, and PCV13 types represented 27.1%, 29.0%, and 45.6% of the 307 serotypeable pneumococci, respectively. Serotypes 19F, 23F, 6A, 15B/C, 19A, 14, 16F, 6C, 11A, 13, and 6B were the most common ; S3 DataFor the five methods used to test field samples, the resultant sensitivities for samples containing one serotype, samples containing multiple serotypes, and overall ranged from 75.5% to 97.5%, 65.7% to 93.7%, and 74.2% to 95.8%, respectively. The PPVs ranged from 82.2% to 96.4% ; S2 Datan = 27) that contained multiple serotypes, had consistent serotyping results for both methods, and did not contain any non-typeables  and field samples .For methods 4 (culture microarray), 10 (culture RFLP), 14 , and 21 , we conducted secondary analyses. We evaluated the ability of method 4 (culture microarray) to provide accurate serotype-specific relative abundance for each serotype present within a sample ; S4 Dataypeables ; S4 DataMethod 10 (culture RFLP) contains a screen for co-colonisation that indicates whether multiple isolates of pneumococci are present, which might be useful as a screening test for multiple serotype carriage. For spiked samples, the ability of this method to detect co-colonisation was assessed against the inocula and had 90% sensitivity and 100% specificity. For field samples, the co-colonisation screen was assessed against the study gold standard and had 44.3% sensitivity and 65.8% specificity. Specificity results should be interpreted with caution, as samples that contain a single serotype may contain multiple strains of that serotype with different RFLP profiles. The ability of the quantitative real-time PCR method 14 to quantify pneumococcal loads was determined using the spiked samples, with the finding that the estimated loads were higher than, but significantly correlated with, the inocula ; S4 DataThe PneuCarriage project was a multi-centre comparative study designed to identify the best pneumococcal serotyping methods in order to support future carriage studies and to facilitate monitoring of pneumococcal vaccine impact in resource-poor settings. Five methods were selected for testing nasopharyngeal samples based upon their performance serotyping laboratory-prepared samples. Method 4 (culture microarray) had the best performance overall.The performance of individual pneumococcal serotyping methods was highly variable. When 20 serotyping methods were evaluated using 81 laboratory-prepared (“spiked”) samples, 13 failed to meet our performance criteria of ≥70% sensitivity to detect minor serotypes and ≥90% PPV. Although this raises concerns about the performance of these methods and the validity of some previous studies, it is important to note that many of these methods performed well when testing pure cultures and/or identifying the major serotype, often reflecting the original purpose of the assays. Although some of the methods we investigated may be appropriate for diagnostic use, we did not test their suitability for such a purpose.Some methods  performed well but were not selected as they were capable of detecting only a smaller subset of the 97 known serotypes. This is of concern for monitoring serotype replacement and PCV impact as rare serotypes may emerge and become more common, particularly with the introduction of higher valency vaccines. Some methods had particular technical issues. For example, it is likely that the heat-kill step in method 8 (direct multiplex immunoassay) greatly diminished its sensitivity. Although we did not fully unblind the research groups (to enable future use of the reference samples), details on “problem” serotypes were provided to facilitate optimisation of the methods.Given that performance in spiked sample testing was a critical component in assessing methods in this study, spiked samples were constructed to reflect nasopharyngeal samples in terms of their overall pneumococcal load, as well as in having a range of serotypes, including representatives of serotypes that are common or rare in carriage. Conventional serotyping was used to underpin development of the “study gold standard”, as the Quellung reaction and latex agglutination methods employed are recommended by the World Health Organization . To thorBased on scientific performance and technical aspects of the methods, we selected five methods to test the 260 nasopharyngeal (“field”) samples. Although this study was not designed to survey the serotypes carried in children, the serotype diversity and distribution results from the field samples  are geneThe reasons for the differences between the spiked and field sample results were not explored, but may include the increased biological complexity of the field samples, such as the presence of cells and nucleic acids from other microorganisms and the host. This finding is important as it indicates that spiked samples alone are insufficient to properly assess method performance. Previous studies have found that using a culture amplification step increases sensitivity of detection ,56, and The performance of individual pneumococcal serotyping methods was highly variable, and there was also considerable variation within a particular type of technology . As such, it is important that establishment of any pneumococcal serotyping method is supported by appropriate training and a rigorous quality system framework. As exemplified by the “19A-like” 19F isolate used in the spiked samples, serotyping results may occasionally differ depending on whether genotypic or phenotypic methods are employed ,58. ThislytA quantitative real-time PCR [The microarray (method 4) is able to detect all known serotypes and accurately measure their percent relative abundance and is suitable for high-throughput testing. Furthermore, the method can determine the genetic relatedness of isolates, and these results could be deposited in a global database . Howevertime PCR ) to deteAlthough this study is, to our knowledge, the most comprehensive of its kind to date, not all published pneumococcal serotyping assays were tested ,63. SomeAnother limitation of our study is that we did not include detection of non-typeable pneumococci, which are commonly carried in some populations ,67, in oThe aim of this study was to identify the best pneumococcal serotyping methods for carriage studies. Since the commencement of this study, there has been a growing realisation of the importance of pneumococcal carriage studies as a means of monitoring the impact of existing pneumococcal vaccines, evaluating community (herd) immunity to pneumococci, and evaluating new pneumococcal vaccines. Studies are now in place to use pneumococcal carriage as a proxy for herd immunity, providing a means by which the impact of different PCV schedules can be evaluated. Developers of new pneumococcal vaccines are relying on demonstrated impact on carriage as an important step in vaccine development. Our findings can now be applied to many of the open questions in the field—including whether carriage data (including detection of minor serotype populations) are useful in predicting serotype replacement—or to understanding the emergence of epidemic serotypes. Recent studies have explored whether nasopharyngeal carriage samples can be used to diagnose pneumococcal pneumonia (which is particularly difficult in children) or examine the severity of pneumonia –71. EmplThe effect of PCVs on multiple serotype carriage is unclear ,73, and S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S1 FiglytA quantitative real-time PCR  were compared with the loads of the inoculum (CFU/ml). The correlation between the inocula and pneumococcal loads determined by real-time PCR was significant (p < 0.001): Spearman’s r = 0.800 . Four spiked samples that did not contain pneumococci were excluded from this analysis.The pneumococcal loads of 77 spiked samples determined by (TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Chronic Otitis Media (COM) is characterized by middle ear effusion (MEE) and conductive hearing loss. MEE reflect mucus hypersecretion, but global proteomic profiling of the mucosal components are limited.This study aimed at characterizing the proteome of MEEs from children with COM with the goal of elucidating important innate immune responses.MEEs were collected from children (n = 49) with COM undergoing myringotomy. Mass spectrometry was employed for proteomic profiling in nine samples. Independent samples were further analyzed by cytokine multiplex assay, immunoblotting, neutrophil elastase activity, next generation DNA sequencing, and/or immunofluorescence analysis.109 unique and common proteins were identified by MS. A majority were innate immune molecules, along with typically intracellular proteins such as histones and actin. 19.5% percent of all mapped peptide counts were from proteins known to be released by neutrophils. Immunofluorescence and immunoblotting demonstrated the presence of neutrophil extracellular traps (NETs) in every MEE, along with MUC5B colocalization. DNA found in effusions revealed unfragmented DNA of human origin.Proteomic analysis of MEEs revealed a predominantly neutrophilic innate mucosal response in which MUC5B is associated with NET DNA. NETs are a primary macromolecular constituent of human COM middle ear effusions. Otitis Media (OM) is one of the most common conditions of early childhood accounting for a very high proportion of all pediatric office visits and surgeries annually,2 at a nLittle is known about the biological mechanisms in OM that fully explain the progression in OM from acute OM (AOM) to COM. It has been well-demonstrated that during this process, the healthy single layered middle ear epithelium remodels into a pseudo stratified epithelium in COM able to potently produce mucins,16,17. TRecently, separate studies of MEE from children with either recurrent AOM or COMdemonstrated the presence of neutrophil extracellular traps (NETs) in the middle ear fluid and showed that the extensive DNA stranding evident within the MEE is largely NET derived ,22 NETs For this study we posited that, an unbiased proteomics approach would reveal neutrophilic markers and NETs would be abundant in COM fluid. We aimed to demonstrate that NETs were present in the MEE of children with COM and characterize the COM MEE NET and proteome focusing the analysis on mediators associated with innate immune responses.The Institutional Review Board Committee of Children’s National Health System approved this study. After obtaining written informed consent from the legal guardian, effusions from children aged 0–35 months with COM, undergoing myringotomy with tube placement irrespective of race/ethnicity/gender were collected. For sample analysis, effusions from each ear were pooled into one sample per patient. Exclusion criteria included: cleft palate or other craniofacial dysmorphic syndromes, immunosuppressive states or conditions, cystic fibrosis, immotile cilia syndrome, or prior history of skull base radiation therapy or skull base malignancy. A total of 49 samples of MEE from x pediatric patients with COM were collected fresh, aliqouted, and if not immediately used for experiments, frozen at -80°C as previously described. ImportaProtein concentration was determined from MEE aliquots using Bicinchoninic Acid Microtiter Plate Assays . As some of the effusion samples had blood contamination, a serum sample was also collected as a control for protein presence for the proteomic analysis.Proteins were separated by SDS-PAGE electrophoresis and analyzed by proteomics as previously described. BrieflyMS was carried out as previously described. BrieflyBecause proteins less than 20kDa in size are typically too small to be detected by one-dimensional gel mass spectrometry proteomics approaches, we also analyzed MEE samples (n = 49) for quantitative levels of IL10, macrophage derived chemokine (MDC), IL13, IL17A, IL1β, IL6, IL8, RANTES, TNFα, and VEGF using a commercially available multiplex magnetic bead immunoassay  according to the manufacturers’ instructions using provided standards and quality controls as previously described. For thiFreshly obtained MEE samples were gently spread directly over a glass slide and allowed to dry for 2 to 4 hours on ice, before fixation in paraformaldehyde 3% for 20 min and storage at 4°C in PBS 1X until immunostaining was performed. Cells were permeabilized with PBS/Tween 20 0.05% for 5 min; non-specific sites were saturated using 0.01% PBS/Tween-20/3% Bovine Serum Albumin for 30 min. This solution was also used to incubate primary anti-human antibodies for -neutrophil elastase , Citrullinated H3 , MUC5B , Lysozyme , Lactoferrin , Myeloperoxidase , S100A8/9 , S100A9 , SPLUNC  at 1/500 dilution and the secondary antibodies anti-goat Alexa 488 A11055 oranti-rabbit Alexa A10042 at 1/500 . Three washes of 30 min were done in order to reduce the non-specific signal of the antibodies. 4',6-diamidino-2-phenylindole(DAPI) at 1:8000 in PBS was finally used to stain DNA. The chambers were removed and the slide was mounted using Permount mounting medium  with a coverslip. Slides were stored at 4°C in dark before immunofluorescent analysis using an Olympus FV1000 confocal microscope .® West Dura Extended Duration Substrate kit  according to the manufacturer’s instructions.A previously described protocol was used for the western blotting of non-m® West Dura Extended Duration Substrate kit  according to the manufacturer’s instructions.For mucins, western blot analysis was performed as previously described. BrieflyNeutrophil elastase protein activity was assayed with the Elastase Substrate I, Colorimetric . The substrate was reconstituted in 0.1M Hepes, 0.5M NaCl and 10% dimethylsulfoxide at 1 mg/ml as performed previously. 200μl oDNA from 200 uL of middle ear effusions was purified using the QiaAmp mini kit  prior to electrophoresis in 1% Agarose Gels run at 100 V in 1X TAE buffer containing ethidium bromide for visualization under ultraviolet lighting. Quantification of relative DNA concentrations in each effusion sample was performed by the Qubit double stranded DNA kit .Quality control was performed on 49 DNA samples from middle ear effusion using the Qubit 2.0 fluorometer system  and the nanodrop. These samples were further processed for shotgun sequencing using a Truseq Nano prep kit . Genomic libraries were validated (concentration and size) with the Qubit and 2100 Bioanalyzer. Forty-four samples passed the validation and were sequenced on the Nextseq 500 using the 2x151 basepair paired end protocol, after an initial loading titration trial sequencing run. Raw reads (FASTQ format) were preprocessed (QC) using PRINSEQ-lite 0.20.4 . FiltereThe entire middle ear proteome from 6 middle ear effusions was previously reported by out group. This prDue to their very high abundance in COM samples, immunoglobulins as well as complement proteins are summarized in Surprisingly, five actin and three histones were detected in the high abundance list, but only in the MEE samples and not in serum. Additionally, the three neutrophil/leukocyte proteins in the highly abundant list were present only in MEE and not in serum.We hypothesized the presence of NETs as an explanation for the high abundance of actin, histones and neutrophil markers in MEE. Given the above mentioned results and findings we postulated that visualization of fresh, independent MEE samples would reveal a copious amount of NETs. Through DAPI staining of DNA and antibody staining of specific proteins, IF unequivocally revealed abundant extravasation of DNA from cellular structures resulting in variable stranding of DNA in MEE specimens. Validation with NE, LYZ, and LTF confirmed these cells to be neutrophils. Colocalization of extracellular DNA strands with citrullinated histone-3 (CitH3) confirmed these networks of extravasated DNA and extracellular proteins in MEE fluid are indeed NETs, as the citrullination of histones is characteristic of NET DNA release,33 Fig  Notably,Immunoblotting was subsequently used to further evaluate the presence or absence of NET markers in independent samples. Western blot analyses demonstrated the presence of NET markers lactotransferrin, CitH3, Histone H4, lysozyme, and neutrophil elastase, as well as the innate immune mediators LPLUNC, α-antitrypsin, and proteins S100A9 and A8 in a majority of the samples evaluated. . As expeIn order to determine whether the DNA present in MEE from COME patients  was more-18 and p = 4.29x10-14 respectively)   was then used to analyze the relationships between the proteins of the MEE proteomic dataset. Notably, abundant MEE proteins were predicted to be directly linked to the action of IL-8, which given these relationships, was determined to most likely be involved in MEE for the process of neutrophil chemotaxis as well as for the movement of phagocytes/myeloid cells (p = 8.33x10ctively) . Almost Because proteomic techniques are limited in their ability to detect small molecules such as cytokines, we performed a multiplex assay to quantify the levels of a group of representative Th1, Th2 and other cytokines that have previously been associated with COM,36 Fig . As predThis study comprehensively validates the fact that NETs are present in the MEE of pediatric patients and reports for that proteins derived from NETS are the predominant innate immune response mediators in COM, providing a detailed proteomic analysis of COM MEEs.ex vivo and airway epithelial cell secretions in vitro. Joo et al. compared their proteomic analysis of normal human airway gland secretions with proteomic studies of of bronchial alveolar lavage fluid, apical fluid of primary human tracheobronchial epithelial cell cultures, and nasal lavage fluid and demonstrated an overall similarity in the dataset profiles from each of the sample sources[Analysis of the MEEs proteome revealed a majority of proteins implicated in mucosal innate immune responses as observed in proteomic analysis of airway secretions e sources. Our MEEe sources,38,39,40e sources and thate sources,17,42.Our MEE dataset was further characterized by the abundance of proteins produced by neutrophils, such as neutrophil elastase , myeloperoxidase (MPO), Cathepsin G (CTSG), Lactotransferrin (LTF) but also intracellular proteins typically not expected in such abundance in secretions: actin and histone H4. This finding led us to investigate the presence of NETs in the MEE of patients with COME, as recently reported ,22. In 2in vitro NETosis in neutrophils from healthy subjects [S. Aureus (MRSA) challenge,  with a parallel reduced ability for neutrophils from aged mice to form NETs kill MRSA[Of particular note, IL-8 was found to be by far the most abundant cytokine detected by multiplex assay in out MEE samples. This is of major relevance to the finding of NETosis in MEE, because not only is IL-8 a well known chemoattractant for neutrophils, its presence has been shown to positively correlate with the abundance of NETs in bronchial fluids. Furthersubjects . Aged mikill MRSA. Finallykill MRSA, suggestIt thus appears that rapid clearance of NETs from mucosal surfaces the airways is critical in preventing their deleterious effects. In the lung, the effective clearance of NET structures is a critical process in the maintenance of healthy airways. The lack of appropriate NET homeostasis, with accumulation of bacteria, extracellular DNA and NET-associated enzymes such as MPO and elastases, worsening of lung inflammation and tissue damage has been demonstrated in diseases such as CF and asthOur work confirms and establishes the presence of abundant NETs in COM fluids using immunofluorescence analysis of fresh MEEs. This is important in order to avoid sample degradation and spontaneous neutrophil activation due to temperature among other factors. All the mucoid MEEs were characterized by the presence of cells releasing high amounts of DNA in the extracellular space, which co-localized with CitH3 and other antibacterial proteins as lysozyme, lactotransferrin, neutrophil elastase which are proteins known to be characteristic of NETs along wiBased on our results we hypothesize a model for OM progression whereby acute bacterial infection leads to middle ear epithelial secretion of mediators , which sClearly our report is limited though by the fact that our characterization of mediators in middle ear fluid only represent a “snapshot” in time, and as such the proposed model is only an extrapolation of the potential role of the identified mediators in OM progression over time, and cannot necessarily be directly surmised from the data on hand. Another limitation of our work includes the fact that not every single middle ear fluid specimen could be profiled by each experimental technique. However, we believe the clear uniformity of NET presence in all specimens (stained fresh and immediately to avoid spontaneous production of NETs) by each technique employed demonstrates the importance of NETs in COM. Notably, detection by WB in every single specimen proved to be difficult given the fact that proteins might be degraded by the high abundance of proteases likely coming from neutrophils. As such mass spectrometry techniques, which recognize peptides and not specific epitopes, are more sensitive for protein identification than western blotting and less affected by natural protein degradation during sample procurement and processing. A final limitation of our work is the fact that we did not measure viscosity of the effusions, as such although we surmise that the noted association between NETs and MUC5B is likely to significantly contribute to increased MEE viscosity, we cannot demonstrably conclude as much.In conclusion, an unbiased proteomic analysis of MEE revealed that innate immune responses in COME are typically neutrophilic in nature, with NETs being a primary macromolecular constituent of human mucoid middle ear effusions.S1 TableThis table shows the peptide counts of the serum sample and the average peptide count of 9 MEEs for each protein. The cutoff peptide count was set at 2 per MEE.(DOC)Click here for additional data file.


Measures of visual cortical development in children demonstrate high variability and inconsistency throughout the literature. This is partly due to the specificity of the visual system in processing certain features. It may then be advantageous to activate multiple cortical pathways in order to observe maturation of coinciding networks. Visual stimuli eliciting the percept of apparent motion and shape change is designed to simultaneously activate both dorsal and ventral visual streams. However, research has shown that such stimuli also elicit variable visual evoked potential (VEP) morphology in children. The aim of this study was to describe developmental changes in VEPs, including morphological patterns, and underlying visual cortical generators, elicited by apparent motion and shape change in school-aged children. Forty-one typically developing children underwent high-density EEG recordings in response to a continuously morphing, radially modulated, circle-star grating. VEPs were then compared across the age groups of 5–7, 8–10, and 11–15 years according to latency and amplitude. Current density reconstructions (CDR) were performed on VEP data in order to observe activated cortical regions. It was found that two distinct VEP morphological patterns occurred in each age group. However, there were no major developmental differences between the age groups according to each pattern. CDR further demonstrated consistent visual generators across age and pattern. These results describe two novel VEP morphological patterns in typically developing children, but with similar underlying cortical sources. The importance of these morphological patterns is discussed in terms of future studies and the investigation of a relationship to visual cognitive performance. The visual system at the level of the cortex has been described in depth, from single cell recordings demonstrating encoding of object orientation to peripheral deprivation and sensitive periods in visual development , 1970. Oboth the dorsal and ventral visual networks may be responsive to motion and form  via EEG in response to such visual stimuli in children has described developmental changes in peak latency and amplitude, but also identified group and individual differences in the overall morphology of the waveform. This variability in morphology may indicate that visual cortical development, as reflected by changes in the VEP response, should be evaluated not only in terms of peak latency and amplitude, but VEP morphology as well.In the aforementioned study, The overview of the literature above suggests that the latency and amplitude of the distinct morphological changes in VEP response will show systematic age-related changes in school-aged children. These changes should mainly be observed specifically in the N1 and P2 peak components, as reported by Thus, with the goal of examining VEP peak components and identifying underlying visual cortical generators responsive to apparent motion and shape change across the school age years, we recorded high-density EEG while presenting continually morphing, radially modulated, visual gratings in 41 typically developing children over the age range of 5–15 years. We aimed to replicate the morphological patterns described in the aforementioned studies, and if these patterns were indeed observed, to better understand the development of the underlying visual cortical generators.n = 10), 8–10 year olds (n = 13), and 11–15 year olds (n = 18) consistent with previous VEP developmental studies  on a 26-inch flatscreen LCD television at a viewing distance of approximately 42 inches. The circle and star figures were presented 150 times each. The star grating remained on the screen for 600 ms and was immediately followed by the circle grating, also lasting for 600 ms. Observation of the circle-star pattern provided the percept of transformational apparent motion and change in form to the viewer. A total of 300 stimulus presentations  were presented, for a testing time of 3 min. The VEP was time-locked to the onset of each star and circle grating. Participants were instructed to direct their gaze to the center of the pattern at a black dot and to not shift their gaze during the 3 min. All participants reported no difficulty following the instructions.All children were shown a high contrast sinusoidal concentric grating that transitioned into a radially modulated grating or circle-star pattern . Ocular electrodes were designated to record vertical and horizontal eye movements for rejection of ocular artifact. The sampling rate for the EEG recordings was 1 kHz, with an online band-pass filter set at 0.1–200 Hz.A high-density 128-channel EEG electrode recording net  was fitted to the scalp of each participant while they were seated in a comfortable reclining chair in an electro-magnetically shielded sound booth. All stimuli were presented via E-Prime® . The data were baseline-corrected to the pre-stimulus time of -100 to 0 ms, and noisy channels were removed from the recording. Epochs greater than ±100 μV were rejected as artifacts. Data were down-sampled to 250 Hz to reduce processing time, altering the post-stimulus time to 492 ms. Remaining data were re-referenced using common average reference and averaged, and removed channels replaced with interpolated data via a spherical interpolation algorithm.Individual participant EEG data were high-pass filtered oﬄine at 1 Hz and segmented around each stimulus presentation, with 100 ms pre-stimulus and 495 ms post-stimulus time, then exported from Net Station into EEGLAB  using MaA central occipital region of interest (ROI) was created for each individual participant by grand-averaging the average VEP response at seven electrodes: 70 or O1, 71, 74, 75 or Oz, 76, 82, and 83 or O2 , 2016. V® Scan 7 Neuroimaging Suite (Compumedics NeuroscanTM) for source modeling. In CURRY, the selected components were averaged into the appropriate age groups according to each VEP peak , and an additional ICA was run for each group for verification of relevant independent components.Independent component analysis (ICA) was applied to individual participant concatenated EEG data in EEGLAB following artifact rejection and common average referencing , 2014. ICurrent density reconstructions (CDR) were then performed for each VEP component of each age group, using sLORETA (standardized low-resolution brain electromagnetic tomography). sLORETA is a specific statistical method that takes into account variance of the cortical source itself as well as variance arising from EEG measurement noise . Head moTable 1; Figure 1). Children with a VEP response consisting of multiple peaks  were classified as pattern B . Peak components for pattern A were identified as follows: P1 as the first positive peak occurring at approximately 100 ms, N1 as the first negative peak at approximately 270 ms, and P2 as the second positive peak occurring at approximately 360 ms. Peak components for pattern B were identified as follows: P1 as the first positive peak occurring at approximately 100 ms, N1a as the first negative peak occurring at approximately 200 ms, P2a as the second positive peak occurring at approximately 250 ms, N1b as the second negative peak occurring at approximately 300 ms, and P2b as the third positive peak at approximately 360 ms. Figures 1 and 2 show the average VEP waveforms for the 5–7, 8–10, and 11–15 year olds that were categorized as pattern A and B, respectively. For the 5–7 years old group, five children (50%) demonstrated pattern A morphology, and 5 children (50%) showed pattern B morphology. In the 8–10 years old group, six children (46%) showed pattern A morphology, while seven children (54%) exhibited pattern B morphology. Finally, five children (28%) in the 11–15 years old group showed pattern A morphology, while the majority of 13 children (72%) showed pattern B morphology. There was no significant difference in ages of children exhibiting pattern A  vs. pattern B  . Comparable morphological patterns have been observed in response to a similar visual stimulus  were classified as pattern A , two significant developmental differences were observed. First, the 5–7 years old group showed significantly larger P1 amplitude than the 8–10 years old group . This finding is consistent with previous reports of age-related decreases in VEP amplitude (2(2) = -2.594, p = 0.028]. This finding is atypical, as cortical evoked potential latency normally decreases with cortical maturation or increasing age  did not show any significant latency or amplitude differences across age. This result is consistent with other developmental findings in vision that have observed relatively stable VEP latency across age . Statistical comparisons were made using the Kruskal–Wallis test. The Bonferroni correction for multiple comparisons was applied for post hoc pairwise comparisons. As seen in Figure 3, P1 amplitude was significantly greater in pattern A compared to pattern B , while both N1a and P2a peak components occurred at significantly earlier latencies in pattern B in comparison to N1 and P2 peak components in pattern A . In contrast, there were no significant differences between P2b and P2 and N1b and N1 (p > 0.05). Thus, the data in Figure 3 clearly illustrate that N1a and P2a appear as additional independent components in pattern B.Due to the lack of major developmental differences between the age groups for each pattern, the average VEP waveforms were collapsed across age groups in order to provide a grand average waveform comparison between pattern A and pattern B  and B , as well as the collapsed pattern A and B comparison . The resulting activations were superimposed on an average MRI  with the MNI co-ordinates shown beneath each slice. The scale of the F distribution, indicating the strength of the activations, is also shown. Activated cortical regions in approximate order of response strength are listed in the table to the right of the CDR images.Current density reconstructions were calculated using the sLORETA algorithm provided by CURRYFigures 4A,B depicts the CDR images for VEP P1, N1, and P2 components in the 5–7, 8–10, and 11–15 years old groups for pattern A. Overall, visual cortical generators were comparable between the age groups, with expected activation of cerebellar areas, striate, and extrastriate visual cortex including Brodmann Areas 18, 19, fusiform gyrus, and lingual gyrus , and pattern B collapsed across all age groups. This comparison allowed for observation of possible different activations of visual cortex between the two patterns. Consistent with the previously described CDR results for both patterns, cortical activation was present in areas comprising Brodmann Areas 18 and 19, fusiform gyrus, lingual gyrus, and cerebellum. The P1 and P2 components in Pattern A, as well as the P1 and P2b components in pattern B, showed more fusiform and cerebellar activation, approaching that of the adult response to similar stimuli . We recorded high-density EEG in 41 typically developing children and compared VEP peak components and morphological patterns across children 5–7, 8–10, and 11–15 years of age . Pattern B included additional peak components . Interestingly, there were no major developmental differences across age for either pattern, which allowed for the averaging of patterns A and B across age for the CDR analyses. (ii) CDR imagery illustrated the active cortical generators underlying each VEP peak component, consistently showing responses in lingual gyrus, cerebellum, and Broadmann Areas 18 and 19. Visual generators were robust and stable across age and components for both patterns A  and B . When all age groups were combined according to pattern , visual cortical activation remained consistent, though pattern B clearly demonstrated redundant or additional activations of visual cortex.The main findings of this study are as follows: (i) Two VEP morphological patterns were identified in each age group. Pattern A consisted of the classic VEP response morphology, comprised of three obligatory peaks . In pattern B, no significant amplitude or latency differences were found . The lack of major developmental differences in P1 peak amplitude and latency is expected and consistent with other studies that have shown stability of this component as early as age 3 years, and as it is considered to arise from primary visual cortex  and when patterns A and B were compared , with consistent responses in lingual gyrus, cerebellum, and Brodmann Areas 18 and 19. CDR images for patterns A and B illustrated the lack of difference in major visual network activation across age . There was a slight developmental shift in cortical response for pattern A, as the response of the 5–7 year olds was oriented medially and superiorly in comparison to the stronger ventral response of the 8–10 and 11–15 year olds . In contrast, the visual generators illustrated in pattern B were comparable for each age group, across components .Visual cortical activation in children in response to radially modulated gratings was quite similar across patterns when evaluated according to age groups . Though feedforward and feedback networks between frontal and occipital cortices have been identified for various visual functions . This slight shift in the visual response may represent feedback and feedforward communication between primary and higher-order visual areas underlying processing and awareness of complex visual input (Because of the similarities in activated cortical regions, age groups within each pattern were combined and CDRs calculated for the VEP components. When age groups were combined within each morphological pattern, these similarities remained robust, with all components showing activation in striate and extrastriate regions (unctions , we did al input . In addial input . Overallal input .Altogether, in our study, the responsive visual cortical regions in children appeared to be different from those reported in adult studies. For instance, we did not observe activation of typical visual motion generators, such as hMT+/V5 in the dorsal pathway . HoweverOverall, there were no major differences across age for VEP morphology, component latency or amplitude, or in activated visual cortical regions as illustrated by CDR imagery. However, two distinct VEP morphological patterns were observed, which to our knowledge have not been previously described to such an extent . The undThe findings described in this study provide new data regarding developmental aspects of visual motion/form processing in children. We have identified two distinct patterns in the processing of continuously morphing, radially modulated, visual grating stimuli in children aged 5–15 years . VEP comTaken together, these results demonstrate consistent and robust VEP waveforms and visual source generators in response to apparent motion and shape change across age, VEP component, and VEP pattern. Though the visual cortical activation did not demonstrate major developmental differences, it will be of interest in the future to determine the functional significance of these two morphological patterns, including possible relationships with literacy and similar higher-order visual skills in typically developing and clinical populations.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.