Previous research has sought to quantify head impact exposure using wearable kinematic sensors. However, many sensors suffer from poor accuracy in estimating impact kinematics and count, motivating the need for additional independent impact exposure quantification for comparison. Here, we equipped seven collegiate American football players with instrumented mouthguards, and video recorded practices and games to compare video-based and sensor-based exposure rates and impact location distributions. Over 50 player-hours, we identified 271 helmet contact periods in video, while the instrumented mouthguard sensor recorded 2,032 discrete head impacts. Matching video and mouthguard real-time stamps yielded 193 video-identified helmet contact periods and 217 sensor-recorded impacts. To compare impact locations, we binned matched impacts into frontal, rear, side, oblique, and top locations based on video observations and sensor kinematics. While both video-based and sensor-based methods found similar location distributions, our best method utilizing integrated linear and angular position only correctly predicted 81 of 217 impacts. Finally, based on the activity timeline from video assessment, we also developed a new exposure metric unique to American football quantifying number of cross-verified sensor impacts per player-play. We found significantly higher exposure during games  than practices  (p<0.05). In the traditional impacts per player-hour metric, we observed higher exposure during practices (4.7) than games (3.7) due to increased player activity in practices. Thus, our exposure metric accounts for variability in on-field participation. While both video-based and sensor-based exposure datasets have limitations, they can complement one another to provide more confidence in exposure statistics. Concussions are a common injury in contact sports, with an estimated 300,000 sports-related concussions in the United States annually . ConcussTo quantify head impact exposure to players on the field, previous studies have focused on instrumenting American football players with wearable sensors ,9–19 Ta. These sHowever, recent studies have shown that HITS, and many other wearable sensor systems, may suffer from poor accuracy in both classifying head impacts (impact count) –25, and Video assessment represents an option for collecting an independent dataset of head impact exposure. Video assessment has been used extensively to review plays and sports injuries by coaches and medical professionals –33. SeveIn this work, we introduce a video assessment protocol to collect an independent video-based head impact exposure dataset from seven collegiate American football players for a single season. We also equipped players with an instrumented mouthguard ,27,34,35In addition, we combine sensor and video information to introduce a new exposure metric for quantifying head impact exposure unique to American football, which measures the number of head impacts sustained over a single player-play. While traditionally, exposure is quantified as the number of impacts per player exposure (defined as a player participating in a practice or game) or per hour of play time, American football is a unique sport where every player is expected to spend varying amounts of time on the field and in play. As an example, statistics on National Football League games report an average of 133.6 plays per game, of which 10.3 involve punting. Thus, specialized players such as punters are on the field, and thus exposed, far less often than players such as linemen. Our exposure metric accounts for variability in on-field participation, and normalizes exposure by the base unit for American football: a single play. This metric is also a prime example for the complementary analysis of video and sensor exposure because head impact kinematic sensors cannot measure non-impact activities and video does not have sufficient resolution to identify discrete head impacts.To collect a video-based head impact exposure dataset, we relied on a tiered video assessment protocol. Impact exposure and kinematics were also independently measured using the instrumented mouthguard.Data were collected from the Stanford University American football team during the 2015 fall season. Seven players were consented for study participation through the Stanford Internal Review Board IRB #34943. We obtained informed consent from players in written form. Players represented a variety of positions .Players wore instrumented mouthguards ,35 Fig  for the The electronic boards contained a tri-axial linear accelerometer (H3LIS331) and tri-axial gyroscope ITG3701A), both sampling at 1000Hz. The linear acceleration data were filtered at CFC180 (300Hz fourth order Butterworth low pass filter)  while th01A, bothnist.time.gov) prior to an event. A unique “time-sync” mouthguard not built for any players was used to synchronize mouthguard times with the camera times for impact correlation. This was accomplished by having an athletic trainer apply known impacts on the “time-sync” mouthguard in view of all cameras. The real-time stamps on the “time-sync” mouthguard were matched with those on the cameras after the event.Impacts collected on the instrumented mouthguards were time stamped based on an internal real-time clock to within one second. Internal clocks were synchronized to the National Institute of Standards and Technology time  and had IR readings indicating instrumented mouthguards were properly worn. We then cross-verified helmet contact periods and discrete impact events between video-based and sensor-based exposure sets respectively by comparing the synced time-stamps. This differs from previous video confirmation protocols in that we identified helmet contact periods in video independently ,25.Using the independent video-based helmet contact periods, sensor-based impacts, and cross-verified impacts, we computed a traditional exposure metric: the number of helmet contact periods or head impacts per player-hour . We then computed our new exposure metric: the number of cross-verified sensor-based head impacts per player-play. Plays were discretized using the video-based player activity timeline combined from all videos during an event. Helmet Contact, Body Contact, and No Contact activities were associated with players in play, and the Idle and Not in Video activities were associated with players not in play. Sequences of Helmet Contact, Body Contact, and No Contact activities were represented by a single play. Finally, we also computed the number of plays per player-hour.Next, we compared the impact location distributions from cross-verified video-based helmet contact periods and sensor-based head impacts. We defined location vectors representing front , rear , side , oblique , and top  impacts . During First, we integrated sensor linear acceleration and angular velocity data to obtain linear velocity, linear position, and angular position (defined using XYZ Euler angles) during each impact. We also differentiated angular velocity data to obtain angular acceleration ,38. We tTo utilize the peak angular data, we note that if we treat the head and neck as a simple pendulum, the linear and angular motion vectors should be orthogonal. Thus, we define a corrected motion vector by first taking the cross product of the peak linear acceleration, velocity, or position vector with the respective angular acceleration, velocity, or position vector. We then cross the result with the respective angular acceleration, velocity, or position vector to obtain a corrected motion vector, whose location we bin by again finding the location vector with the smallest angle.The cross-verified discrete impacts from the instrumented mouthguard were also used to assess impact severity exposure to compare against previously reported values. We defined severity based on the peak linear acceleration, angular velocity, and angular acceleration magnitude from each impact.Fourteen raters reviewed over 163 hours of video for the seven players spanning 11 practices and 6 games during the 2015 fall season. This represented over 62 hours of mouthguard recordings with over 50 hours of actual in-game or in-practice time. In that time, players participated in 927 plays. Raters identified 1004 Helmet Contact activities after first round video assessment, of which 271 were confirmed following second round video assessment. The instrumented mouthguard recorded a total of 13,034 impacts, of which 10,949 occurred during practice or game periods. 2,032 of these were determined to be in-mouth impacts based on IR readings. When matching time stamps of video-based helmet contact periods and sensor-based head impact events, we cross-verified 193 video-based helmet contact periods and 217 sensor-based impact events .Treating sensor-based and video-based exposure data separately, we observe a large discrepancy in exposure rates . The insNext, we further delineate exposure based on event type (games or practices). When considering sensor-based results independently, the instrumented mouthguard had double the head impact rate in games than in practices. For independent video-based exposure and cross-verified exposure, exposure during practices was higher. Even for cross-verified mouthguard impacts, we found greater head impacts per player-hour exposure in practices  than in games , though the difference is not significant (p = 0.105). When considering the number of cross-verified sensor-based impacts per player-play however, we found that most players sustained more impacts per player-play during games than during practices. Indeed overall, players experienced significantly fewer impacts per player-play during practices  than during games  (p<0.05). This discrepancy is likely due to a difference in amount of play seen, where all players participated in significantly more player-plays per player-hour in practices  than in games  (p<0.05).We quantified the distribution of impact locations in our cross-verified video-based helmet contact periods and sensor-based head impacts . In bothWhen comparing the location determined through video with the location determined used the processed mouthguard kinematics, we found that our best processing technique (corrected linear position) only predicted impact location correctly in 81 of 217 impacts (37.3%). The traditional peak linear acceleration process, however, was least accurate with only 61 of 217 impacts (28.1%) correctly predicted. In general, correctly identifying impact location improved with integration, and improved when assuming orthogonality with angular measures.We also quantified head impacts based on the opposing impact object , which can only be determined from video assessment. We found 64% were to an opponent’s helmet, 27% were to an opponent’s body, 5% were to an opponent’s upper or lower limbs, 3% were to the ground, and 1% were to other objects such as practice pads.2 (95% CI: 1109.1rad/s2-1475.9rad/s2) respectively during practices, and 23.2g (95% CI: 18.5g-26.1g), 12.5rad/s (95% CI: 10.8rad/s-13.9rad/s), and 1435.0rad/s2 (95% CI: 1288.4rad/s2-1723.3rad/s2) respectively during games. While the medians were larger during games, only peak angular velocity magnitude had a significant difference (p<0.05).Impact severity was only quantified in the 217 impacts recorded by the instrumented mouthguard with high IR and cross-verified with video. We delineate impact severity based on event (games or practices). Over all players, the median peak linear acceleration, angular velocity, and angular acceleration magnitude was 18.4g (95% CI: 15.2g-19.8g), 9.4rad/s (95% CI: 8.5rad/s-10.2rad/s), and 1240.5rad/sIn this work, we present a tiered video assessment protocol for obtaining an independent video-based dataset for head impact exposure in American football to compare against sensor-based impact exposure and impact location distributions. This work differs from previous head impact exposure datasets that rely primarily on imperfect head impact sensors, with a few datasets further confirming true positive vs. false positive head impacts in video. Instead, we treat the video assessment as an independent exposure dataset. Exposure datasets collected with impact sensors alone are limited by a high occurrence of false positives due to the sensitivity of motion sensors, and known innaccuracies of many sensor systems. Video assessment of exposure data are limited by the qualitative nature of analyzing video and the inability to quantify impact kinematics. However, in this study, we demonstrate that the two methods can complement one another and reveal directions for improvement in sensor-based and video-based data collection. Combining these information, we also introduce a new American football specific exposure metric based on the number of sensor-based discrete head impacts per video-observed player-play, which account for players varied involvement during events.To demonstrate the limitations of independent video-based and sensor-based head impact exposure, we highlight key differences between between the independent exposure sets and the exposure set determined with impacts cross-verified in both video and sensors.First, we note that the instrumented mouthguard only detected 71.2% of the video classified Helmet Contact events, missing 76 of 271 events. One possible reason for this is that the instrumented mouthguard was set to record an impact event when the linear acceleration magnitude exceeded 10g, which is similar to impact thresholds employed in previous work . In videPreviously, the 10g sensor threshold was chosen to differentiate head impacts from other events resulting in head motion, such as jumping or running . But, asSecond, the instrumented mouthguard alone detected 2,032 impacts, which was an order of magnitude greater than the number of cross-verified impacts. This demonstrates the relatively high rate of false positives that have plagued similar impact sensors in the past, and are commonly mitigated using video confirmation . On the In addition to differences in exposure, we also discuss discrepancies in impact location distributions between video-based and sensor-based data. In video, we found that 93.8% of impacts occurred to the front, front oblique, and side of the head after the second round of video assessment. Processing instrumented mouthguard kinematics found similar trends for impact location with an average of 85.8% of impacts occurring in the front, front oblique, and side locations; however, the mouthguard impact location matched video identified location in only 37.3% of the impacts using the best corrected position process. Current methods for predicting impact location from impact kinematics are relatively primitive, with peak linear acceleration being the most common method . Peak liDespite some improvements using integrated signals and angular kinematics, matching between video-based and sensor-based impact location was still relatively poor. This could be the result of decoupling between helmet impact location observed in video, and head motion as measured by the mouthguard. In a direct impact, the impact location is expected to correspond with the direction of motion, however we note that in some cases, our raters observed impacts in which helmets “slid past one another”, resulting in impact locations that do not necessarily correspond to the measured direction of motion. Furthermore, identifying impact locations in video is subjective in nature. When relaxing the criteria for matching video-based and sensor-based impact locations to allow for neighboring impact locations , all sensor processing methods were able to predict similar video-based impact locations in 140–150 of 271 impacts.In addition to discrepancies in head impact location between video-based and sensor-based datasets, our observed distribution of head impact locations differs with previously reported impact location distributions, which report up to 31.9% of impacts to the rear of the head . The disTo take advantage of both our sensor-based and video-based exposure data, we present a novel head impact exposure metric specific to American football. For our exposure metric, we look specifically at the number of sensor-based head impacts normalized by the number of video-based player-plays, to account for variability in on field activity between different player positions. Schnebel et. al. alluded to head impact exposure per player-play, stating that linemen tend to have head impacts every play whereas skill players have head impacts more rarely . We haveIn the second example, we observe two different linemen participating in the same game. The first lineman is a starting lineman, whereas the second lineman is only used in special packages. We see that while both players were recorded for 75–95 minutes, the starting lineman sustained many more head impacts. In terms of head impacts per player-hour, the starting lineman had a more than eight-fold increase compared with the second lineman. Again, the per play exposure is more consistent across the two linemen. We believe this metric of defining head impact exposure in American football prevents skewing from variable on-field participation. We note that while plays are well-defined during games, they are not as clear during practices. Delineating plays in the video activity timeline was done automatically based on video assessment datasheets and may over-estimate the number of plays in practice due to the use of fast and short individual drills which were commonly counted as a single play. The metric can be further refined to properly count practice drills, though we note that practice drill time is currently counted in per-hour or per-event exposure statistics. Similar metrics accounting for player participation may also be useful for sports like ice hockey and basketball where players also spend varying amounts of time in play.2 (95% CI: 1109.1rad/s2-1475.9rad/s2) respectively during practices, and 23.2g (95% CI: 18.5–26.1g), 12.5rad/s (95% CI: 10.8rad/s-13.9rad/s), and 1435.0rad/s2 (95% CI: 1288.4rad/s2-1723.3rad/s2) respectively during games. Together, these results suggest that head impacts are both more frequent and more severe in games than in practices, which agrees with previous exposure data.With this exposure metric, we found that there were significantly more head impacts per player-play during games  than during practices . From instrumented mouthguard kinematic measurements, we found median head impact linear acceleration, angular velocity, and angular acceleration magnitude was 18.4g (95% CI: 15.2g-19.8g), 9.4rad/s (95% CI: 8.5rad/s-10.2rad/s), and 1240.5rad/sWhile we successfully collected an independent video-based exposure dataset utilizing a tiered video assessment protocol, this study has several limitations. First, video assessment takes a substantial amount of time. Analyzing 160 hours of video required 1000 man-hours, primarily due to the rigor and detail required. This prevents manual video assessment from being viable for everyday use. However, this protocol can be used to collect datasets with which to improve sensor processing algorithms for head impact detection and location identification, and automated video assessment based on these protocols can be developed for a more streamlined post-event analysis ,43. In fSecond, video quality (1080p at 30fps) and quantity (two views per field) also affected the subjectivity of video classification. To account for uncertainty due to poor video quality, we had instructed raters to be sensitive to any potential instance of Helmet Contact, and labeled any helmet overlap with another object as a Contact event. Multiple views were only considered in second round of video review, which accounted for the substantial loss of Helmet Contact events between the first and second round of video assessment . Improved video quality and quantity would increase certainty in video labels. While some institutions can afford superior video quality and quantity, we believe our equipment is a reasonable representation of what many collegiate and lower-level institutions currently use.Third, we have shown previously that the instrumented mouthguard design can be susceptible to lower mandible disturbances, resulting in overestimation of the measured kinematics . DespiteFourth, we note that our sample population of seven players from a single team during a subset of practices and games in a single season is relatively small. Thus, the purpose of this study was not to generalize exposure data but describe differences in video-based and sensor-based datasets. Despite the small sample size, exposure rates and kinematic magnitudes fall within the range of previously reported American football exposure. Because of the small sample population, we did not include delineation of exposure by player position in the main manuscript, but have included it as supporting information  Figs witFinally, we do not consider Obstructed View activities as head impacts. In some activities labeled as Obstructed View, the tracked player is taking part in a large tackle on the field and is hidden beneath other players. This is particularly true of linemen. It is possible that we miss head impacts during these plays, and it is possible that these plays produce more impacts in under-represented locations . In our analysis, there were 29 observed instances of an Obstructed View.In this paper, we have demonstrated through a small field study, the comparison and combination of independent sensor-based and video-based measurements of head impact exposure. Currently in head injury research, many sensor-based approaches are being developed for impact exposure studies. While many sensors have demonstrated promise in enabling on-field measurements of head kinematics, some remaining limitations in kinematic accuracy and impact detection accuracy lower the confidence in estimating impact exposure using a single sensor. As such, many researchers have begun using additional information, such as video verification, to improve the quality of sensor-based measurements, and a working group in the National Institutes of Health are recommending video verification as a component in field deployments . As showIn conclusion, we have developed a tiered video assessment protocol for collecting an independent impact exposure dataset in American football. This video assessment protocol can be implemented in other contact sports. We have also developed a new head impact exposure metric for American football based on the number of head impacts occurring per player-play. We have found players sustain significantly more impacts per player-play in practices (0.20) than in games (0.35) (p<0.05). This method can provide additional independent exposure information to verify exposure data gathered using wearable sensors that are not fully validated. The verified exposure information can be used to better manage players so that they are not over-exposed to potentially dangerous head impact events.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Stanford University Internal Review Board IRB #34943. Informed consent was obtained from all individual participants included in the study in written form.S1 FigFirst, we compare exposure across player positions. We compared the exposure data defined by (A) cross-verified sensor-based head impacts per hour and (B) cross-verified sensor-based head impacts per play. We use cross-verified sensor-based head impacts as we believe sensor-based head impacts are the best representation of discrete head impacts and cross-verification gives us high confidence for these impacts. We note that linemen experienced higher exposure than other positions. We also note that, as with the aggregated statistics presented in the main manuscript, players generally experienced more impacts per hour in practices, but more impacts per play in games. (C) This is likely because players participated in more plays per hour during practices than games .(TIF)Click here for additional data file.S2 FigWe delineated impact location distributions by player position for both cross-verified (A) video-based helmet contact periods and (B) sensor-based head impacts. We used the most common peak linear acceleration vector method for sensor-based head impact location distribution for comparison to previously reported distributions. Players generally have similar impact location distributions in both datasets, though we note that all positions except the linemen are represented by a single player, and some positions observed relatively low exposure.(TIF)Click here for additional data file.S3 FigFinally, we delineated impact severity by player position. Impact severity were determined from cross-verified sensor-based impacts and were defined with the impact kinematics: (A) peak linear acceleration magnitude, (B) peak angular velocity magnitude, and (C) peak angular acceleration magnitude. The violin plots represent the kinematics distribution observed and were fit using a log-normal distribution. In general, impact severities were higher during games than during practices for all players. Furthermore, while linemen experienced higher exposure rates, they experienced relatively low impact severities.(TIF)Click here for additional data file.


Pneumocystis jirovecii pneumonia (PJP). This study aimed to evaluate the risk factors for PJP among patients with lung cancer. This retrospective case-control study compared patients who had lung cancer with PJP (n = 112) or without PJP (n = 336) matched according to age, sex, histopathology, and stage. PJP definition was based on (i) positive PCR or direct immunofluorescence results for pneumocystis, (ii) clinical symptoms and radiological abnormalities that were consistent with a pneumonic process, and (iii) received targeted PJP treatment. The development of PJP was associated with radiotherapy (RTx), concurrent chemoradiotherapy (CCRTx), lymphopenia, and prolonged high-dose steroid therapy . Multivariate analysis revealed independent associations with prolonged high-dose steroid therapy  and CCRTx . Steroid use was frequently related to RTx pneumonitis or esophagitis . Prolonged high-dose steroid therapy and CCRTx were risk factors for PJP development among patients with lung cancer. As these patients had a poor prognosis, clinicians should consider PJP prophylaxis for high-risk patients with lung cancer.Solid malignancies are associated with the development of  Pneumocystis jirovecii pneumonia (PJP) is an opportunistic infection caused by the fungus Pneumocystis jirovecii1. During 1980–2000, most PJP cases involved patients with acquired immunodeficiency syndrome (AIDS), although PJP has become common in non-human immunodeficiency virus (HIV)-infected cases because of improvements in AIDS treatment and more patients receiving immunosuppressive therapy2. However, non-HIV-infected patients with PJP have an overall mortality rate of approximately 35–55%5, compared to 10–20% among patients with AIDS6.Hughes et al.7 reported that the incidence of PJP in acute lymphoblastic leukemia (ALL) ranged from 22% to 45%, depending on the chemotherapy used and the stage of leukemia. Another study also reported that without prophylaxis, PJP rates may approach 25% among patients with non-Hodgkin’s lymphoma8. In a recent study, PJP incidence of acute leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma was estimated to be >45 cases per 100,000 patient year, whereas PJP incidence of solid tumors was <25 cases per 100,000 patient-year9. Possible explanations of higher incidence of PJP in hematologic malignancies are related to high intensity of chemotherapy7 and severely impaired immune function10. After routine performance of prophylaxis using trimethoprim-sulfamethoxazole (TMP-SMX), incidence of PJP among patients with hematologic malignancies has shown remarkable decrease11.12. However, little is known about the risk factors for PJP development and who would benefit from PJP prophylaxis in lung cancer patients. Therefore, this study aimed to evaluate the risk factors for PJP among patients with lung cancer.Although the exact incidence of PJP in solid tumors still remains unknown, recent reports have reported PJP cases in lung cancer, which is the most common cause of solid malignancy-related death14.Electronic medical records from the Severance Hospital  were searched to identify 3,877 patients with lung cancer who underwent surgery, chemotherapy (CTx), and/or radiotherapy (RTx) between January 2013 and December 2016. Among these patients, 311 patients had received treatment for PJP using intravenous and/or oral TMP-SMX . However, cases were excluded if they had an unsatisfactory definition of PJP (n = 188), other cancers (n = 7), organ transplantation (n = 3), or HIV infection (n = 1). Thus, records from 112 patients with confirmed lung cancer and PJP were matched 1:3 with control patients  according to age, sex, histopathology, and stage using exact match modeling , or tissue samples. The real-time PCR assay was performed with the AmpliSens Pneumocystis jirovecii-FRT PCR kit , which is a qualitative test based on the endogenous control, the β-globin gene. Ten-microliter DNA samples were added in each reaction tubes. PCRs were performed on the Bio-Rad CFX-96 real-time PCR system  according to the manufacturer’s instruction. The second condition was the presence of lung infiltration during chest radiography or computed tomography (CT)16. The third condition was clinical symptoms of PJP and PJP treatment during their hospitalization. We defined the prophylaxis group as those without a PJP diagnosis who received medication to prevent PJP.PJP was considered present when the patient fulfilled three conditions. The first condition was a positive result for 17. The daily dosage of steroids was expressed in prednisolone equivalents, and the continuous maximum use of steroids was recorded as the cumulative steroid dose. We did not consider intermittent steroid use , or steroid use that was stopped at >1 month before the PCP diagnosis, in order to more accurately account for the effect of steroid treatment on PJP development18. Steroid dosage during the last 2 weeks before the PJP diagnosis was compared to the dosage during the preceding weeks, and changes during the 2 last weeks were categorized as maintained, increased, tapered, or stopped17. Adjunctive steroid use during PJP treatment was defined a high-dose , low-dose , or absent19. The 60-mg cut-off was selected because it was the lowest effective dose in controlled trials for HIV-infected patients with PJP20. RTx-induced pneumonitis was diagnosed based on the patients’ symptoms  and radiological manifestations22. Radiation-induced esophagitis was also defined based on clinical symptoms 23. Data from chest radiography and CT were evaluated independently by one radiologist at the PJP diagnosis, and then independently confirmed by two of the authors . Medical records related to risk factors for PJP development were studied until PJP development in PJP patients, whereas those in controls were studied until death, transfer to another hospital, or December 2016.Demographic and clinical data were collected through a chart review. The lowest total lymphocyte count was selected from the 2 weeks before the PJP diagnosis (PJP group) or during the follow-up period (control group), and lymphopenia was defined as <1,000 cells/µLNormally distributed continuous variables were reported as mean ± standard deviation, while non-normally distributed variables were reported as median (range). Continuous variables were analyzed using the Student t-test or the Mann-Whitney test. Categorical variables were reported as number and percentage, and compared using the chi-square test or Fisher’s exact test. The independent risk factors for PJP development were evaluated using conditional logistic regression modeling based on the matched paired data, and the results were reported as odds ratios (ORs) and 95% confidence intervals (CIs). A two-tailed P-value of <0.05 was considered statistically significant. All analyses were performed using SAS software .The research protocol was approved by the Institutional Review Board (IRB) of Severance Hospital (IRB No. 4-2017-0431). The need for informed consent was waived due to the retrospective nature of the study. All methods were carried out in accordance with relevant guidelines and regulations.The most common symptom of PJP was dyspnea 71.4%), followed by fever (27.7%) and cough (23.2%) . Surgery was more common in the control group . The median daily steroid dose and proportion of steroid use were similar in the two groups . The median cumulative steroid dose tended to be higher in the PJP group  and the duration of steroid use was significantly longer in the PJP group . Patients with prolonged high-dose steroid use  were more common in the PJP group, compared to the control group . The mean lymphocyte counts were 425/µL  in the PJP group and 540/µL  in the control group. The PJP group had a higher proportion of patients with lymphopenia . Only 30 of the 448 cases and controls (6.7%) received PJP prophylaxis, which was less frequent in the PJP group, compared to the control group .Table p = 0.001), CCRTx , lymphopenia , and prolonged high-dose steroid therapy ; p = 0.002). PJP development was inversely but non-significantly associated with TMP-SMX prophylaxis . In the multivariate analyses, PJP development was independently associated with prolonged high-dose steroid therapy  and CCRTx . Surgery also was inversely associated with PJP development in the multivariate analysis , although this is likely because patients who were able to undergo surgery would be less likely to receive CTx or RTx for their early-stage cancer.In the univariate analyses Table , PJP dev24. Disease progression itself may have influenced the prognosis and patient’s survival, infection and pneumonia are typically associated with poor prognoses among these immunocompromised patients25. Bacterial pneumonia is relatively common in this setting, compared to PJP, although recent studies have also indicated that PJP occurs among patients with solid malignancies, such as lung cancer28. Thus, the present study compared patients who had lung cancer with or without PJP, and the results revealed that prolonged high-dose steroid use and CCRTx were independent risk factors for developing PJP in this setting.Lung cancer is the most common cause of cancer death worldwide although the survival rate has improved due to the use of novel targeted therapies plus conventional therapies30, which is likely related to their effects on T-cell immunity8. However, the present study detected a relatively small proportion of patients with steroid use and relatively low cumulative steroid dose in the PJP group, compared to the findings from previous studies18. This discrepancy may be related to differences in the definition of steroid use and other risk factors that might influence PJP development. For example, the present study excluded cases from the steroid use group if the steroid treatment was stopped at >1 month before the PJP diagnosis. In addition, we excluded cases with intermittent steroid use, and compared the maximum cumulative continuous steroid dose in the PJP and control groups. As a result, we did not detect a significant inter-group difference in daily steroid dose or cumulative maximum dose. However, when we categorized the patients according to daily steroid dose and duration, we found that a specific cut-off value  was independently associated with PJP development, although the area under the curve was low (0.568).Long-term corticosteroid use is a known risk factor for developing PJP among non-HIV-infected patients31. In this study, univariate analysis revealed the RTx was associated with PJP development, this association was statistically insignificant in the multivariate analysis. Other reports have indicated that specific chemotherapeutic agents, such as fluorouracil, bleomycin, asparaginase, fludarabine, and gemcitabine, were associated with the risk of PJP development32. Patients who receive high-dose steroids with cytotoxic CTx or RTx could be even more vulnerable to PJP development28. In the current study, specific chemotherapeutic agents did not increase the risk of PJP, while CCRTx was significantly associated with PJP development in multivariate analysis (p = 0.004).Previous studies have indicated that RTx could be a risk factor for PJP developmentet al. have suggested that a cut-off value of 300 CD4+ cells/µL would include 91% of PJP cases among HIV-negative patients, which is a high-risk group, although 39–46% of individuals who received long-term corticosteroid therapy also had CD4+ counts of <300/µL33. Moreover, the accuracy of this biomarker is poorly characterized for non-HIV-infected patients, which limits its use in that population, although it is an accurate method for assessing PJP risk among patients with AIDS34.The present study revealed that most patients who developed PJP had decreased lymphocyte counts , which was a significant risk factor in the univariate analysis but not in the multivariate analysis. Mansharamani 35. However, the frequencies of fever and cough were relatively low in our study, compared to previous studies35. This discrepancy may be related to reduced immunity caused by CTx and/or RTx, which could mask signs of infection. The reported prognosis of PJP is poorer among non-HIV-infected patients, compared to HIV-infected patients36, although we observed a much higher mortality rate during PJP treatment (approximately 60%), compared to other studies conducted in non-HIV-infected patients35. In this current study, most patients received first-line TMP-SMX treatment and approximately 67% of the patients with PJP also received high-dose adjunctive steroids during their PJP treatment19. Thus, the poor prognosis may be related to the advanced lung cancer and/or the weakened general condition of patients who receive CTx and/or RTx before developing PJP. It is also possible that the large proportion of patients with do-not-resuscitate or do-not-intubate statuses was responsible for the relatively high mortality rate among our patients with lung cancer and PJP.The characteristics of our patients with PJP and lung cancer were different from those of non-HIV-infected patients in other studies. The most common clinical symptom was dyspnea in the present study, and our patients’ respiratory symptoms tended to manifest quickly, which was also observed in previous studies34. A recent meta-analysis also indicated that, among solid organ transplant recipients and patients with hematological malignancies, TMP-SMX treatment was associated with reductions in the occurrence of PJP  and PJP-related mortality , which suggests that PJP prophylaxis is warranted when adult patients have a >3.5% risk of PJP11. Several studies have indicated that solid malignancies are associated with an increased risk of PJP37, although there are no clinical data supporting the routine use of PJP prophylaxis for patients with solid tumors. The American Thoracic Society recommends considering prophylaxis when immunocompromised patients (including patients with solid tumors) receive prolonged high-dose corticosteroid treatment , although it acknowledges that this recommendation is based on a low level of evidence38. The present study also confirmed that PJP prophylaxis is not routine for patients with lung cancer, even for patients who are receiving prolonged high-dose steroid therapy. As PJP prophylaxis can significantly reduce mortality among patients with other diseases, it may be beneficial to provide PJP prophylaxis to high-risk patients with lung cancer. For example, prolonged high-dose steroid therapy and CCRTx were possible risk factors for PJP among patients with lung cancer, and the most common reason for steroid use was radiation-induced pneumonitis, which is relatively common after CCRTx (vs. RTx)21. Therefore, it seems reasonable to use PJP prophylaxis for patients who are receiving prolonged high-dose steroid treatment and CCRTx.Among HIV-infected patients, TMP-SMX prophylaxis is highly effective for preventing PJP39. However, non-HIV-infected patients have a lower overall organism burden40 compared to HIV-infected patients, and conventional staining methods might have provided lower sensitivity and false-negative results. A meta-analysis of PCR-based PJP diagnosis revealed a sensitivity of 99% and specificity of 92% among non-HIV-infected patients, and that 31.8% of patients with false-positive results had or subsequently developed PJP41. Thus, patients with a positive PCR result require careful follow-up and/or PJP treatment. Other reports have also suggested that PCR assays for PJP are sensitive and increase the diagnostic yield among non-HIV-infected immunocompromised patients43. Therefore, to reduce the likelihood of false-positive results, we only included patients with symptoms of acute pulmonary infiltrates, such as cough, dyspnea, and radiological abnormalities.The present study has several limitations. First, the single-center retrospective design is associated with known risks of bias, and we were unable to collect exact information regarding symptom presentation or duration before the PJP diagnosis. Thus, our findings may not be generalized to other centers or patient populations. Second, proportion of surgery in PJP cases and controls are somewhat different, suggesting the possibility of inadequate cancer stage matching. Third, we used PCR and immunofluorescence data to diagnose PJP, and our data may include false-positive results, as a confirmed PJP diagnosis generally requires special microbiological staining of respiratory specimensDespite its limitations, the present study also has several strengths. To the best of our knowledge, this is the first case-control study of PJP among patients with lung cancer. In addition, we matched the patients with PJP to a relatively large number of control patients, which provides a clearer perspective regarding the risk factors for PJP development among patients with lung cancer. Furthermore, our findings may be useful for identifying high-risk patients with lung cancer who should receive PJP prophylaxis. Nevertheless, multicenter randomized controlled trials are needed to examine the value of PJP prophylaxis in this setting.In conclusion, the present study revealed that prolonged high-dose steroid therapy  and CCRTx were risk factors for PJP development among patients with lung cancer. These patients had a very poor prognosis. Furthermore, the most common reasons for the steroid treatment were CCRTx or RTx-induced pneumonitis. Therefore, clinicians should consider PJP prophylaxis for high-risk patients with lung cancer.


Supplemental Digital Content is Available in the Text.Direct clinicopathologic correlation of an eye with geographic atrophy secondary to age-related macular degeneration provides histologic correlates of features commonly seen by optical coherence tomography, such as end-stages of drusen, subretinal drusenoid deposit, plaques near the Bruch membrane, and hyporeflective wedge. In an eye with geographic atrophy (GA) secondary to age-related macular degeneration, we correlated ex vivo histologic features with findings recorded in vivo using optical coherence tomography (OCT), near-infrared reflectance imaging, and fundus autofluorescence.In the left eye of an 86-year-old white woman, in vivo near-infrared reflectance and eye-tracked OCT B-scans at each of 6 clinic visits and a baseline fundus autofluorescence image were correlated with high-resolution histologic images of the preserved donor eye.Clinical imaging showed a small parafoveal multilobular area of GA, subfoveal soft drusen, refractile drusen, hyperreflective lines near the Bruch membrane, subretinal drusenoid deposit (reticular pseudodrusen), and absence of hyperautofluorescent foci at the GA margin. By histology, soft drusen end-stages included avascular fibrosis with highly reflective cholesterol crystals. These accounted for hyperreflective lines near the Bruch membrane in OCT and plaques in near-infrared reflectance imaging. Subretinal drusenoid deposit was thick, continuous, extracellular, extensive outside the fovea, and associated with distinctive retinal pigment epithelium dysmorphia and photoreceptor degeneration. A hyporeflective wedge corresponded to ordered Henle fibers without cellular infiltration. The external limiting membrane descent, which delimits GA, was best visualized in high-quality OCT B-scans. Retinal pigment epithelium and photoreceptor changes at the external limiting membrane descent were consistent with our recent histologic survey of donor eyes.This case informs on the extent, topography, and lifecycle of extracellular deposits. High-quality OCT scans are required to reveal all tissue features relevant to age-related macular degeneration progression to GA, especially the external limiting membrane descent. Histologically validated signatures of structural OCT B-scans can serve as references for other imaging modalities. Treatment exists for neovascular AMD, but not for the atrophic end-stage, geographic atrophy (GA). Geographic atrophy is variably defined, depending on the imaging modality used. Based on optical coherence tomography (OCT), a new term proposed for GA is complete retinal pigment epithelium (RPE) and outer retinal atrophy (cRORA).5 Based on histology,6 we proposed as the border of atrophy in the photoreceptor layer a curve of the external limiting membrane (ELM) toward the Bruch membrane .8 A progressive RPE dysmorphia toward this border thickened the reflective RPE band and was suggested as an OCT biomarker8 and a correlate to hyperautofluorescence at the GA margin.9 With longitudinal OCT, we showed that during GA enlargement, Müller cells scrolled up the outer retina at the ELM descent, in advance of RPE degeneration.11Remarkably, subcellular-level detail is available in commercially available OCT, thanks to signal averaging and point-of-capture quality control, eye-tracking, and integration with multiple imaging techniques.12 and associates with progression from intermediate AMD to GA and Type 3 neovascularization .16 Basal laminar deposit (BLamD), a thickened basement membrane material between the RPE and its native basal lamina (BL), is considered diagnostic for AMD when continuous under the fovea.20 Soft drusen and basal linear deposit (BLinD) are lump-and-layer forms of the same AMD-specific lipid-rich material, located between the RPE-BL and the inner collagenous layer of BrM.22 Deposits are dynamic, showing phases of growth, regression, and persistence visible by longitudinal in vivo eye-tracked OCT.26 Furthermore, the topographies of SDD and BLinD follow those of rods and cones, respectively, suggesting dysregulation of previously unknown pathways specific to each photoreceptor type.27Recent high-resolution histology of newly described and better-defined extracellular lesions, deployed in precise layers Figure , has inf28 our recent histologic surveys of GA in donor eyes,8 and a clinicopathologic correlation of macular atrophy due to mixed causes in AMD.29 Our current results demonstrate the subcellular detail available for clinical decision-making today through OCT-anchored imaging.Clinicopathologic correlation, i.e., histology of eyes imaged during life, can both validate diagnostic technology with anatomical ground truth and probe disease pathogenesis. In this study, we describe the first clinicopathologic correlation of an eye with GA secondary to AMD for which serial OCT and near-infrared reflectance (NIR) are available, along with baseline fundus autofluorescence (FAF). The index eye can be compared with the fellow eye that progressed to Type 3 neovascularization,Studies were approved by the institutional review board at the University of Alabama at Birmingham and adhered to the Tenets of the Declaration of Helsinki.1 for cRORA, i.e., atrophy ≥250-µm diameter involving outer retinal layers and RPE. We use consensus OCT terminology30 with the addition of the RPE-BL–BrM (RBB) band.31 The presence of choroidal hypertransmission defined the presence of cRORA/GA on structural cross-sectional OCT scans. To delineate the areas of GA, these areas were then mapped on the NIR image. As recommended,33 AMD pathology is most easily described if BrM is considered as a subendothelial space with three layers , and the RPE-BL or BLamD is considered with the RPE. Between the RPE-BL (or BLamD) and the inner collagenous layer is the sub–RPE-BL space. We refer to the combination of SDD, RPE, BLamD, drusen/BLinD, and BrM as the RPE-deposit complex.34 For macular regions, we refer to the (rod-free) fovea, parafovea, and perifovea .35The index eye met proposed criteria28 she initiated anti–vascular endothelial growth factor therapy. At this time, the left eye was found to have GA without neovascularization, and it was not treated. From November 2012 to July 2013, she received in the right eye a total of 6 intravitreal injections of bevacizumab (1.25 mg/0.05 mL) with optimal response and resolution of the exudation. Available for review were an FAF baseline image from the left eye, eye-tracked OCT B-scans, and NIR for both eyes at each of the six examinations. The last clinical evaluation, on September 2013, lacked OCT imaging.An 86-year-old white woman with GA in her left eye and Type 3 neovascularization in her right eye, both secondary to AMD, was referred to a private retina practice for evaluation and treatment. She had undergone chemotherapy, radiotherapy, and upper lobectomy for lung cancer in 2002 but was not a smoker. Her ocular history included open-angle glaucoma stable under acetazolamide, dorzolamide, and timolol therapy with 20/20 visual acuity in both eyes. In the left eye, pigment changes were noted 3 years prior and soft drusen 2 years before referral to a retina specialist, which was prompted by suddenly decreased visual acuity in her right eye (20/70) and progressive vision loss in her left eye (20/200). Because of a diagnosis of Type 3 neovascular AMD in the right eye,28 The clinical diagnosis of GA was supported histologically by a 1,127-µm-wide absence of continuous RPE, in the presence of BLinD and drusen, and the absence of neovascularization.Four months after the July 2013 visit with OCT, the patient died of a cerebrovascular accident. Both eyes were recovered for research 7.5 hours after death by personnel of the Alabama Eye Bank.µm at the nonatrophic and atrophic side of the ELM descent) as described.29 Measurements were taken in consecutive visits . For comparison with histology, thicknesses were measured for outer retina (inner boundary of the outer plexiform layer [OPL] to BrM), OPL to ELM, outer nuclear layer (ONL), RBB, and subfoveal choroid  with the manual caliper in the OCT device software . The RBB thickness was also measured in the perifovea at 2,000 µm nasal and temporal to the fovea (17 different locations per side). Visibility of the ELM descent was assessed in all available OCT volumes . We previously showed29 that the number of images averaged for each B-scan in the automatic real time (ART) function and the quality value in decibels (dB) impact visibility of small features, and thus, these values were recorded.A quantitative analysis using in vivo eye-tracked OCT scans was performed at four anatomical points . Although our previous studies used eye-tracking to align in vivo and ex vivo OCT scans, for this eye, such tracking was not possible, and we correlated histology to in vivo scans as follows. An 8-mm-diameter full-thickness tissue punch centered on the fovea was post-fixed with osmium tannic acid paraphenylenediamine to preserve extracellular lipids37 and embedded in epoxy resin. The tissue punch was oriented in the embedding mold assisted by a drawing to ensure sectioning in a strictly superior-to-inferior direction. The polymerized epoxy block was reviewed under a dissecting microscope. A reference line drawn across the block face between the optic nerve and fovea was used to align the block parallel to the plane of sectioning in an ultramicrotome . The ex vivo OCT volume was reviewed to identify retinal contours, pathologies of interest, and superior-to-inferior sequence of occurrence within the volume. Cumulative distances were calculated using this OCT volume and a digital counter on the ultramicrotome. Three-micrometer sections were removed and reviewed with a 10× microscope objective to seek features expected from the OCT volume and initiate the collection of serial 0.8-µm-thickness sections that were then stained with toluidine blue. A total of 45 sections were obtained . The location of each histologic section was aligned to the ex vivo OCT, thus allowing for point-by-point in vivo/ex vivo correlation.The preserved globe underwent ex vivo OCT imaging before histologic processing, as described39 then viewed on a monitor at magnifications up to 1,240× using ImageJ software (https://imagej.nih.gov/ij/download.html). Photomicrographs were composited for figures with adjustments for exposure, contrast, and background color correction .To provide seamless zoom-in for details and zoom-out for context without switching between objectives, sections were scanned in their entirety using a 60× oil-immersion objective , a robotic microscope stage, and slide scanning software . Scanned images were scaled for tissue units and centered,39 we determined the frequency of cellular and laminar phenotypes and measured layer thicknesses in seven regions , choriocapillaris (ChC) density, and associated phagocytic and inflammatory cells, as described.7 Near the GA border, where retina was attached, we also measured the thickness of OPL–ELM, ONL, and inner segment myoid. When an assessment location had concurrently applicable phenotypes in one given layer, the most advanced one was recorded.As described,s Figure : two perAtrophic retina remained attached to the underlying support tissues in one area. Although torsion (twisting) around this attachment is possible, we reduced the impact of torsion on alignment by considering the spatial ordering of tissue features  across scans and sections. We accounted for shrinkage of detached retina. Inner retina layers were not measured because they exhibited postmortem edema.28 to 13 donor eyes with histologically diagnosed GA7 , and to an eye with macular atrophy due to mixed causes.29Data were compared with similar data from the fellow eye of this same patient, which had macular atrophy meeting criteria for cRORA in association with Type 3 neovascularization,P values <0.05 were considered significant. For clinical imaging, Student's t-test was used to compare mean ART function values and quality values between OCT scans with visible versus invisible ELM descent. Binary logistic regression was used to assess the influence of ART function and quality in visualizing the ELM descent. For histology, linear regression models were used to compare layer thicknesses and ChC density across the locations. Tissue samples were assumed to be independent of each other.40 42  and merged . These areas exhibited loss of outer retinal bands , discontinuation of the RPE-BL band that uncovered BrM, and then choroidal hypertransmission. Also, RBB thickened at the GA edge  and 31.77 ± 14.22 . At the GA border  and significantly lower-quality values .In the 6 available OCT volumes, the ELM descent was detectable in 8 to 9 of 10 B-scans crossing GA in 3 volumes, 2 to 3 of 10 scans in 2 volumes, and in no scans in one volume. Automatic real time function and quality values ranged from 7 to 11 and 3 to 27, respectively. Relative to B-scans with visible ELM descents, B-scans with invisible ELM descents had similar ART values  show cellular phenotype frequencies and layer thicknesses, for RPE–deposit complex, BrM–ChC, and outer retina, in order. To clarify relations with both photoreceptor topography and GA borders, Table Geographic atrophy corresponded to a small, multilobular atrophic area in the nasal parafovea, 408 to 882 46 SDD comprised mounds of extracellular material consisting of a dispersed phase of small, regularly spaced, and gray-staining particles within a flocculent and light-staining continuous phase. Some deposits had caps of outer segment fragments . A histologic druse with nodules . Within fibrosis and not within soft drusen/BLinD were found clefts created by the extraction of cholesterol crystals by solvents during histological processing . In the sub–RPE-BL space of the atrophic area (26/45 sections) were processes  and extending beyond it under the fovea was a fibrotic material of undulating thickness, with few cells and no apparent vessels  across the nonatrophic area toward the ELM descent, then declined on the atrophic side , and no intraretinal RPE cells were detected.Considering BLamD and RPE, BLamD was thick and present in early  and late  forms in the nonatrophic area Figures C. Basal de Table . Near GAµm) and thinned across the ELM descent into GA . Figures Table 3, Supplemental Digital Content 7, http://links.lww.com/IAE/A960). Corresponding to the OCT B-scans, the choroid was found to have relatively preserved thickness .BrM was notable for the absence of refractile unstained patches that signify calcification. As shown in Table 48 on the atrophic sides of each ELM descent . Ectopic photoreceptor nuclei in OPL/HFL were common at the nonatrophic side of the ELM descent, as were ectopic photoreceptor nuclei in the inner segment. Five measures of photoreceptor abundance and health declined significantly (P < 0.0001) to zero on the atrophic side of the ELM descent . Absence of continuous ONL, ELM, and RPE layers  apolipoprotein B, E-containing lipoprotein particles rich in esterified cholesterol that fill BrM throughout adulthood to form BLinD and soft drusen, as egress across the RPE–BrM–ChC complex is impaired, age-dependently.52 Very large drusen have a lifecycle of growth due to underclearance of these normally secreted RPE products and collapse after RPE migration and death terminates their production.46Mound-shaped RPE elevations with homogeneous and mildly hyperreflective contents seen in OCT B-scans correlated with histologically identified soft drusen containing a homogeneous lipid-rich material–lacking cells and cellular fragments.19 , the index case exhibited the latter four, with calcification and fibrotic change visible by OCT. Within numerous RPE elevations were nodules, i.e., multilobed refractile structures 5 to 100 µm in diameter that are rich in a molecularly distinct hydroxyapatite. Nodules were recently confirmed as correlating to a heterogeneous internal reflectivity of drusen associated with 6-fold increased risk of progression to advanced disease.54 As seen by the Sarks, avascular fibrosis distinct from both fibrin deposition and exudation-associated fibrosis replaced soft drusen/BLinD while maintaining the shapes of drusenoid RPE elevations.56 We saw histologic evidence without imaging correlates for sub–RPE-BL cells potentially clearing drusen .58Of soft druse end-stages described by the Sarks et al49 and others59 attributed reflective horizontally oriented plaques to “densification” of BrM .19 Querques et al60 proposed that hyperreflective, variably oriented lines in regressing drusen resulted from either BrM splitting and bowing inward or a process “similar to the onion sign.”61 We subsequently correlated the onion sign in eyes with sub–RPE-BL hemorrhage and fluid to groups of cholesterol crystals,36 suggesting that an aqueous environment is required for super-saturation and precipitation. Conversion of the sub–RPE-BL environment from primarily lipid (soft drusen/BLinD) to nonlipidic  may promote cholesterol crystallization in the available fluid, e.g., the hydration water of collagen fibrils. Our data thus support crystallization as a biomarker for the replacement of soft drusen contents, while also not supporting BrM as a source of mirror-like reflectivity, because BrM in the index case lacked calcification. The Fleckenstein plaques49 may thus represent cholesterol crystals that are too close to BrM (<1.8–11.8 µm) to be resolved with present technology. Conversely, RPE elevations over avascular fibrosis can be distinguished from dome-shaped soft drusen by the slight irregularity of contour and content of reflective crystals , suggesting that a relatively recent onset of atrophy could underlie these differences. Like the fellow eye that progressed to Type 3 neovascularization,28 the index eye also had abundant extracellular deposits, relatively intact ChC, cells in the sub–RPE-BL space, and progression to advanced AMD at the same  eccentricity, supporting a causal relationship with the topography of outer retinal cells.65 However, although the index eye had sub–RPE-BL avascular fibrosis, in the fellow eye, Müller cell processes accompanied a neovascular stalk from the retina that implanted in this compartment.Compared to similarly analyzed donor eyes with GA,66 and autofluorescence,67 and eyes with thick choroids, like the index eye, show low NIR within areas of GA. Although BLamD was >11-µm thick under the fovea (Table 44 because caverns are best seen in en face reconstruction of dense OCT B-scan raster patterns.We provide new information about device-independent and device-dependent visibility of specific pathologies. Both the atrophic zone and drusen were poorly visible in the NIR locator image. Light reflected from the sclera impacts the intensity of retinal reflectivityea Table , it coul39 Descents were visible in a scan with ART = 29 and quality = 24 dB (Figures 29 The invisibility of the ELM descent in color fundus photography and FAF imaging, in concert with inaccurate histologic quantification of outer retinal cells in GA,68 can lead to an overestimate of viable retina available for therapeutic rescue in the atrophic zone. The loss of relevant detail at quality ≤15 dB when ART is below 9 to 10 encourages heightened attention to these settings for accurate assessment of GA and its precursors.Our data help define imaging parameters necessary to consistently visualize the ELM descent, proposed as a histologically meaningful border of atrophy.Strengths of this study include serial eye-tracked OCT with the last clinic visit 4 months before tissue recovery, baseline FAF imaging, a detailed and comprehensive histology technique, multiscale viewing of both digital sections and OCT scans, and current nomenclature for both OCT and AMD pathology. Limitations include insufficient image quality in OCT volumes to reveal all relevant features, poor preservation of druse contents, and postmortem detachment that impeded comprehensive quantification of retinal morphology. Nevertheless, this is the first clinicopathologic correlation of GA with in vivo OCT, NIR, and FAF imaging, with the most complete accounting of currently recognized AMD layers since the Sarks' monumental description. Age-related macular degeneration's notorious complexity has been exacerbated by limitations of imaging technologies that could not reveal major pathology within and beyond the RPE layer. The catalog of histologically validated OCT signatures, expanded by this report, can serve as references for other modalities. A timeline from AMD precursors to end-stages visible clinically at the subcellular level is a holy grail for multifactorial diseases of aging. Recent trial imaging data can be interpreted to inform preventive measures and direct new therapies to disease stages before irreversible tissue damage.


The Sub-Saharan Africa region still remains the epicentre of the global HIV/AIDS epidemic. With regards to new paediatric HIV infections, almost 90% of new HIV infections are among children (aged 0–14 years), largely through mother to child transmission. Male Partner Involvement in Prevention of Mother to Child Transmission programmes is now strongly advocated as being key in improving infant outcomes. This study describes the role of Male Partner Involvement on infant HIV infection and mortality survival in the first year among HIV-exposed infants born from HIV positive mothers.This study was a two-phase, two condition (intervention or control) longitudinal study as part of a clinic-randomized Prevention of Mother to Child Transmission controlled trial. For Phase 1, female participants were recruited without their male partners. In Phase 2, both female and male participants were enrolled in the study as couples in order to encourage active Male Partner Involvement during pregnancy. Participants had two assessments prenatally (8–24 weeks and 32 weeks) and three assessments postnatally About 1424 women were eligible for recruitment into the study and 18 eligible women declined to participate. All women had a partner; 54% were unmarried, 26% were cohabiting, and 20% were married. Just over half (55%) of the women had been diagnosed with HIV during the current pregnancy. Phase 1 had significantly more HIV-infected infants than Phase 2 at 12-months postpartum . Increased depressive symptoms were associated with infant HIV infection at 12-months . Phase 1 also had a significantly greater proportion of dead and HIV-infected infants than Phase 2 at 12-months .Male partner involvement in antenatal care is critical in ensuring infant survival and HIV infection among children born to HIV-positive mothers. This study highlights the high risk of ante-and-post natal depression and underscores the need of screening for depression during pregnancy.NCT02085356.ClinicalTrials.Gov; Trial Number  The Sub-Saharan Africa (SSA) region still remains the epicentre of the global HIV/AIDS epidemic, with Eastern and Southern Africa being the most affected. Among the 1.8 million new HIV infections in 2017, the SSA region accounted for more than half .Earlier PMTCT programmes tended to focus mainly on women and children with little emphasis being placed on male partner involvement (MPI) in antenatal care . MPI in Even though there has been a recent realisation and enthusiasm towards MPI in maternal health care, the actual rates of male participation in antenatal care among indigenous African communities in SA remain relatively low.There have been few studies reporting on the development and implementation of targeted, contextually relevant behavioural interventions that are aimed at increasing MPI in PMTCT programmes and antenatal care,17, and This study was a two-phase, two condition (intervention or control) longitudinal study as part of a clinic-randomised PMTCT controlled trial. Eligible Participants were HIV-infected pregnant women at least 18 years of age, having a male partner, and were recruited between April 10, 2014 and January 30, 2017.The trial aimed at increasing PMTCT uptake, family planning and male partner participation in the antenatal and postnatal process in 12 randomly selected community health centres in Gert Sibande and Nkangala districts in Mpumalanga province, South Africa . ParticiEligible participants were HIV-seropositive pregnant women, between 8 and 24 weeks pregnant , with male partners and aged 18 years or older. In Phase 1, their partners were not enrolled and did not participate in the study. In Phase 2, the women’s partners were also enrolled and participated in study procedures. Those agreeing to participate were enrolled following provision of informed consent.Subsequent to enrolment, all participants (both men and women) completed a baseline survey in their preferred language  using Questionnaire Development System Audio Computer-Assisted Self-Interview (ACASI) software in order to enhance disclosure, accommodate all levels of literacy and reduce interviewer bias. To familiarize participants with the software, assessors completed the demographic component of the questionnaire with participants prior to completion of all other assessments. In addition, an on-site assessor was available at all times to assist where necessary and answer any questions.Ethics approval for this study was granted by the Human Sciences Research Council (HSRC) Research Ethics Committee (REC), protocol approval number REC4/21/08/13. Furthermore, additional approval was also obtained from the Department of Health and Social Welfare, Mpumalanga Provincial Government, and the University of Miami Miller School of Medicine Institutional Review Board (IRB ID: 20130238). Written informed consent was obtained from all participants.Twelve CHCs were matched in a 1:1 ratio according to patient census and average antenatal clinic patient load, and one clinic in each pair of clinics was randomly assigned to intervention or control using a computer program written by the data manager. The matched clinics were then assigned to the opposite condition. The randomization process was carried out by four people. The first conducted the computer-generated randomization assignments stratified by clinic size . The second implemented the assignments generated by the program, providing a table of all clinic site assignments to study personnel. The third activated each intervention site individually, and the fourth activated each control site individually.Only the Human Sciences Research Council (HSRC) study staff activating and overseeing the sites were aware of site assignment. All assessments were conducted using an audio computer-assisted self-interview (ACASI) program. As such, participants enter their data themselves and are blind to their assignment. Following randomization, clinic sites were activated individually, and clinic staff were blinded to the condition. Training for clinic study staff was conducted by condition, and clinic study staff conducting the study were also blind to clinic randomization status. Finally, data analysis to evaluate study outcomes was blinded to the clinic’s status as an intervention or control intervention arm.Prenatally, participants attended three group intervention  sessions, along with one individual or couples session; postnatally, participants attended two individual or couples sessions. In both conditions, mothers who had not disclosed their HIV status were invited to (1) disclose their status to their partner during a one-on-one couple session, or (2) not have a male partner attend the one-on-one session. Both the control and intervention groups also received the standard of care National Department of Health antenatal PMTCT education sessions.Intervention. Intervention participants received the PMTCT standard of care plus the ‘Protect Your Family’ intervention led by study-trained clinic staff. The intervention consisted of three prenatal weekly two-hour (between five and seven participants) gender-concordant group sessions and one prenatal individual or couples counselling session, along with two postpartum (6 weeks and 3 months) individual or couples counselling sessions. The ‘Protect Your Family’ intervention is a manualized, closed, structured behavioural risk-reduction programme. The intervention targeted prevention of vertical transmission, adherence to PMTCT and medication use, HIV testing of family members, prevention of HIV transmission and stigma, serostatus disclosure, partner communication, intimate partner violence (IPV), safe infant feeding, safer conception, family planning and dual method sexual barrier use. The intervention sessions for men were designed to be appropriate for partners of both HIV-positive and HIV-negative women, addressing men’s issues, PMTCT, positive family relationships, communication, and child health issues. More details on the intervention are provided in the published research protocol [protocol .Control condition. Control condition participants received the PMTCT standard of care plus three time-equivalent, group-administered video presentations prenatally. These video sessions covered various topics related to child health, specifically: 1) diarrhea management, dehydration and exclusive breastfeeding, 2) infant nutrition, and 3) immunization and sexual abuse. In addition to the group-administered presentations, control participants attended three further health promotion video presentations administered in an individual or couples setting: one prenatally on 4) fevers, plus two postnatally at 6 weeks and 3 months postpartum on 5) burns and 6) alcohol use.Infant HIV status and survival. Infant HIV status at 12 months was assessed via DNA polymerase chain reaction (PCR). Five drops of blood were drawn from the infant by heel stick using a sterile lancet and a Guthrie card with five wells and stored as dried blood spots (DBS). The DBS was collected by trained CHC nursing staff and couriered to an independent laboratory at the University of Cape Town, Division of Pharmacology for confirmation of HIV serostatus. Infant survival status, defined as miscarriage or death, by 12 months postpartum was obtained from clinic records. To analyse the number of HIV-free surviving infants, the number of HIV-infected infants were not included in analyses. To analyse the number of infants who were either HIV-infected or died at 12-months, a variable was created to combine HIV infection or mortality.Sociodemographic information. Sociodemographic characteristics included age, level of education, employment status, relationship status, income, and number of children.Health information. Health-related characteristics included whether participants had been diagnosed with HIV during the current pregnancy, time since HIV diagnosis, time since initiation of ART, whether the current pregnancy was planned, and children’s HIV status.Partner Information. Partner-related characteristics included partner’s HIV status and disclosure of HIV status to partner.Depressive symptoms. Depression was assessed using the Edinburgh Postnatal Depression Scale 10 (EPDS-10)[(EPDS-10), a 10-it(EPDS-10). A totalHIV stigma. HIV stigma was assessed using the AIDS-Related Stigma Scale (ARSS) [e (ARSS) , a 9-iteFamily planning knowledge. Family planning knowledge was assessed using 8 multiple choice questions adapted from the Safer Conception Knowledge, Attitudes & Practices (SCKAP) and Family Planning Survey [g Survey . Each itIntimate partner violence. Intimate partner violence (IPV) was assessed using an adapted version of the Conflict Tactics Scales 18 (CTS-18) [(CTS-18) , which ia priori power analysis was conducted to ensure that an adequate sample size was used to assess intervention, phase, and phase x intervention differences, taking into account expected levels of attrition and mortality, as previously described[t-tests for continuous variables meeting distributional assumptions. As needed, Fischer’s Exact Tests or Mann-Whitney U tests were used as alternatives. Variables found to be significantly associated with attrition at p < 0.01 were included in all subsequent analyses.An described. UnivariTo examine the effect of baseline demographic and psychosocial characteristics on rates of HIV infection and survival, unadjusted (bivariate) and adjusted (multivariable) logistic regression models were used. To adjust for the intraclass-correlation for the dependency within community health centers, random effects for the intercept and random slope were estimated. To estimate effect sizes, odds ratios were used . To handOriginally about 1424 women were identified to be eligible for recruitment into the study. Only about 18 eligible women declined to participate and 7 were lost as a result of technical issues, resulting in a total of 1399 participants at baseline. The Consolidated Standards of Reporting Trials (CONSORT) diagrams are presented in Figs Women were a mean age of 28 years (SD = 5.82). Nearly half (48%) of women had completed 10 to 11 years of education, and as part of study eligibility, all women had a partner; 54% were unmarried living separately from their partner, 26% were cohabiting, and 20% were married. Two-thirds (64%) of participants had a monthly income of at least 1000 ZAR (~USD$70). Just over half (55%) of the women had been diagnosed with HIV during the current pregnancy, 50% reported that their pregnancy was unplanned, 79% had at least one child, 27% reported that their partner was HIV-infected, and out of women who reported having children, 6% indicated that they had an HIV-infected child. On average, it had been 24 months (SD = 35.9) since women had been diagnosed with HIV, and 15 months (SD = 25.4) since they had initiated ART. Male involvement and family planning knowledge were moderate, and HIV-related stigma was low. The average EPDS-10 score  was 11.7 (SD = 6.01), which translated to 45% of women being above the clinically significant cutoff for depression. Approximately 15% of women reported having more than 2 alcoholic drinks in the past month, and 61% reported having disclosed their HIV status to their partner. Overall, by12 months postpartum, 5% of infants tested positive for HIV and 9% had died. At 12-months postpartum, 91% of infants were alive and HIV-free; 13% were dead or HIV-infected at 12-months. Further detail on the baseline demographic and psychosocial characteristics of women is presented in n = 1159 infants at 12-months postpartum out of N = 1399 participants at baseline, and not known for n = 240 infants; these were imputed. Alive status was not known for n = 91 infants and known for n = 1308; therefore, alive status was imputed for n = 91 infants. Either HIV or alive status was known for n = 1344 participants out of N = 1399; as such, only n = 55 were not known.Attrition analyses using multivariable logistic regression indicated that women who were retained were more likely to be older, have greater income, have an HIV-infected partner, have a greater time since ART initiation and HIV diagnosis, greater family planning knowledge, and lower depressive symptomatology. Comparisons of women who were lost as opposed to retained are presented in Phase 1 had significantly more HIV-infected infants than Phase 2 at 12-months postpartum . In addition, increased depressive symptoms were associated with infant HIV infection at 12-months postpartum . No other significant associations with infant HIV infection were found see .Decreased depressive symptoms were associated with infant survival at 12-months postpartum . No other significant associations with infant survival were found. Neither intervention nor phase were associated with infant survival at 12-months see .Decreased depressive symptoms were associated with infant HIV-free survival at 12-months postpartum . No other significant associations with infant HIV-free survival were found. Neither intervention nor phase were associated with infant HIV-free survival at 12-months see .Phase 1 had a significantly greater proportion of dead and HIV-infected infants than Phase 2 at 12-months postpartum . Depressive symptoms were also associated with infant HIV infection and mortality at 12-months postpartum . No other significant associations with infant HIV infection and mortality emerged. Furthermore, the intervention was not associated with infant HIV infection and mortality at 12-months see .This study examines male partner involvement in the antenatal period as well as other factors that may impact first year MTCT and survival among HIV-exposed infants. To our knowledge, this is one of the first experimental studies conducted in South Africa to assess the impact of MPI on MTCT and survival amongst HIV-exposed infants. More infants seroconverted in Phase 1 of the study compared to Phase 2. Since Phase 2 enrolled women together with their male partners, this suggests that male engagement in PMTCT improves infant outcomes. It must be highlighted that in the first phase of the study MPI was measured through self-report by women enrolled in the study using a male involvement index. However in the second phase the male partners had to also be enrolled in the study and thus this phase went beyond measuring self-reported MPI and also emphasised actual male partner participation in antenatal care.Furthermore, women who were recruited in the second phase of the study together with their partners being participants in the study showed much lower rates of attrition and loss to follow-up in the study. Although there was no association between intervention and HIV infection or survival, male participation in the intervention may have promoted greater MPI overall, including in PMTCT and child nurturing, leading to decreased risk of infant HIV infection and mortality. Male involvement therefore should be emphasized in areas with high rates of HIV transmission during or after pregnancy to enhance infant outcomes among HIV-exposed infants.The results from this study corroborate findings from other studies which found that MPI greatly improves the health outcomes of infants and decreases chance of mortality among HIV-exposed infants. For example, in an experimental study conducted in Kenya, it was found that infants born to mothers without MPI in antenatal care were almost four times as likely to die or seroconvert by 6 weeks postpartum . SimilarEven though most studies exploring MPI report generally positive perceptions from both male and female participants, the actual rates of men following through are generally low ,30. WithThis study also found that antenatal depression in HIV-infected women was associated with perinatal HIV transmission and infant mortality. Although screening for depression is recommended during pregnancy, prevalence of depression screening is suboptimal, including in South Africa . Our resThe present study is not without limitations. The main limitation of this study is that we are reporting significant associations that may not necessarily imply causality. Other limitations included high attrition of study participants, which limited the number of infants who could be tested at 12-months, and those who had a survival status in the clinic records by 12-months postpartum. However this study went a long way in giving a deeper understanding of the socio-economic context of South African communities, on issues around infants and maternal health.Male partner involvement in antenatal care is critical in ensuring infant survival and prevention of HIV infection among children born to HIV-positive mothers. In addition, this study highlights the high risk for new-borns associated with antenatal depression and underscores the need of screening for depressive symptoms during pregnancy in order to improve treatment adherence and decrease infant HIV infections and infant mortality.S1 Consort(PDF)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.S1 Data(SAV)Click here for additional data file.


The aim of this systematic review was to determine if the human colon, through the lower gut-liver axis, drives PSC activity by assessing the progression of the disease in patients with and without colectomy for colonic disease.The gut-liver axis is involved in the pathogenesis of liver disease. Abnormal immune-mediated responses to intestinal microbiome are implicated in primary sclerosing cholangitis (PSC) however the mechanisms remain poorly understood. Currently, no single animal model recapitulates all attributes of PSC in humans and this limits further studies of gut-liver interactions. A systematic search of PubMed, Medline, and Scopus was performed for articles that contained the terms “colectomy” or “bowel resection” AND “primary sclerosing cholangitis” up to 15th April 2018. Articles were reviewed by 2 reviewers and raw data collated. A Forest plot was used to illustrate the effect of colectomy on subsequent liver transplantation for PSC. Linear regression was used to estimate mortality risk. Colectomy appeared to have no effect on PSC progression, although high-quality studies were lacking. Rates of liver transplantation or transjugular intrahepatic portosystemic shunt for PSC were not affected by colectomy . Mortality risk following colectomy in patients with PSC is 2.11% per year . Current evidence is limited but suggests colectomy does not affect the progression of PSC in patients with colonic disease. Pathogenic micro-organisms or antigens that drive PSC may not be limited to the lower gut. The gut-liver axis, a concept thought to be first introduced in 1987 by Volta et al , describ4β7 positive (CCR9+ α4β7+) T-lymphocytes to abnormally upregulated mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), vascular adhesion protein-1 (VAP-1) and gut homing chemokine (C-C motif) ligand (CCL25) on hepatic endothelial cells, and the up-regulation of toll-like receptors on biliary epithelial cells and T-Helper type 17 (TH17) cells as responses of the gut-liver axis specific to PSC , were included for further evaluation when available. All patients with a confirmed diagnosis of PSC were included. Patients who had PSC but not undergone colectomy were used as a control group, and total colectomy or procto-colectomy were considered as an identical intervention, where applicable. The exclusion criteria for meta-analyses were: (i) studies that did not report sufficient primary data, (ii) studies with irrelevant content and (iii) studies that were not accessible by the UK Access Management Federation.Data extraction Two authors (JO and MFB) independently reviewed all titles and abstracts then assessed articles against the inclusion criteria for analysis. A third reviewer (YAN) resolved any differences. The primary outcome measure was the progression of PSC as determined by serological or histological evidence, defined pragmatically on the specific criteria used within each study. Secondary outcome measures were the rate of liver transplantation (including re-transplantation) or transjugular intrahepatic portosystemic shunts (TIPSS) when liver transplantation was unsuitable, and mortality rates. Data were extracted independently by authors JO and MFB using a standardized form. Data extracted included serological or histological progression of PSC, transplantation rates, and mortality rates. Quality of the studies included was assessed using a modified Newcastle–Ottawa Scale.Data synthesis and analysisForest plots were used to illustrate effect size between studies, where possible. Each study included had an odds ratio (OR) with respective 95 percent confidence interval (CI) calculated. A Mantel-Haenszel (M-H) statistical method was performed, calculating an overall OR for respective outcomes: an OR less than 1.00 inferred a worse survival for the colectomy group versus the control group, whilst an OR greater than 1.00 inferred a better survival. The significance level was set to 5% for all tests and alternative hypotheses were two-sided. High heterogeneity between studies was presumed and a random-effects model was used. A funnel plot was employed to assess for potential publication bias . All-cause mortality following colectomy was plotted for each study against respective mean study follow up. A mean-weighted linear regression was performed to estimate the change in all-cause mortality per year.Statistical analysis was performed using Review Manager (RevMan) Version 5.3  and GraphPad Prism 5.0 .675 studies were identified from the initial literature search . FollowiRate of PSC ProgressionOverall, colectomy did not appear to have an effect on PSC progression. Two studies directly comparing patients with PSC following colectomy versus no colectomy were identified  but therOne study  followedFive further observational studies were identified with a follow-up period ranging between 4.3 to 11 years , 17-20. Rates of Liver Transplantation or TIPSS Six studies -25 reporAll-Cause Mortality Rates 2 = 0.722) for patients with PSC who have undergone colectomy.Seven studies were identified that reported all-cause mortality rates following colectomy  . Mean-weTwo studies , 23 direOur results suggest that cumulatively there is limited evidence in the current literature to demonstrate any beneficial effect of colectomy on the disease activity of PSC. Nordenvall et al  reportedWe were able to calculate an estimated mortality rate, based on the included studies, for patients with PSC following colectomy at 2.11% per year. Putting this into context, mortality rates for all PSC patients reported in the literature ranges between 3.3-5.8% -39, and The main limitation of this systematic review was the lack of high-quality studies for meta-analyses in current literature. No randomized control trials were identified and only a small proportion of studies had a control group so that an effect size could be estimated. Another limitation was the high heterogeneity between studies, which was in part due to varying criteria of how disease progression was measured and defined within these studies. In this regard, it precluded meaningful quantitative analyses in this systematic review. Lastly, though the statistical analyses performed herein were robust, effects from small sample sizes in the meta-analyses and publication bias cannot be excluded completely. Nonetheless, the main aim of this systematic review was to study PSC activity after the interference of the lower gut-liver axis is achieved through a "surgical knock-out" of the colon in humans. As animal models remain sub-optimal, observational studies such as case-control and association studies, although do not demonstrate causality, still provide important information and have contributed too much of our understanding of PSC . HoweverMoving away from animal models and clinical observational studies, in vitro longitudinal and high throughput screens of the microbiome in the lower GI tract offers the possibility of identifying the pathogenic microorganisms in PSC as the cost of interrogating the microbiome becomes more affordable. Unfortunately, even if a pathogen is identified, the transition from mechanistic studies to identify a target for drug action and then to drug safety studies preclude the use of any pharmacological treatments in the near future. In considering novel and potential treatments for PSC that are on the horizon, faecal transplantation is perhaps the closest at being introduced into clinical practice. Fecal transplantation could potentially reverse gut dysbiosis in PSC and provide a means of controlling PSC progression where current drug treatments have failed. Interestingly, a clinical trial assessing the effects of faecal transplantation in patients with PSC is currently in progress  and the results of this study are eagerly awaited. Alternatively, advances in regenerative medicine ,41 have In summary, the colon as part of the lower gut-liver axis is likely to be involved in the pathogenesis of PSC but is unlikely to be the key factor driving the disease and higher-quality larger clinical trials are required. The pathogenic microbiome or antigens could potentially be identified in the midgut, since total colectomy has shown little effect on PSC activity. Though our understanding of the immune-mediated disease remains poor, rapid advances in regenerative medicine, high throughput screening of the gut microbiome, and research targeting the gut-liver axis are likely to make headway in developing new treatments for the disease.