The aim of this study was to determine and to verify the correlation between the amount of prolactin (PRL) levels in the blood and in the cerebrospinal fluid (CSF) by various causes of death as an indicator for acute hypoxia in autopsy cases. It is to confirm the cause of the change in prolactin level in CSF by in vitro system.In autopsy materials, the PRL levels in blood from the right heart ventricle and in the CSF were measured by chemiluminescent enzyme immunoassay, and changes in the percentage of PRL-positive cells in the pituitary gland were examined using an immunohistochemical method. Furthermore, an inverted culture method was used as an in vitro model of the blood-CSF barrier using epithelial cells of the human choroid plexus (HIBCPP cell line) and SDR-P-1D5 or MSH-P3 (PRL-secreting cell line derived from miniature swine hypophysis) under normoxic or hypoxic (5% oxygen) conditions, and as an index of cell activity, we used Vascular Endothelial Growth Factor (VEGF).Serum PRL levels were not significantly different between hypoxia/ischemia cases and other causes of death. However, PRL levels in CSF were three times higher in cases of hypoxia/ischemia than in those of the other causes of death. In the cultured cell under the hypoxia condition, PRL and VEGF showed a high concentration at 10 min. We established a brain-CSF barrier model to clarify the mechanism of PRL transport to CSF from blood, the PRL concentrations from blood to CSF increased under hypoxic conditions from 5 min. These results suggested that PRL moves in CSF through choroidal epithelium from blood within a short time. PRL is hypothesized to protect the hypoxic/ischemic brain, and this may be because of the increased transportation of the choroid plexus epithelial cells. Prolactin (PRL) is a hormone primarily secreted by lactotrophs in the anterior pituitary gland, with the exception of placental PRL . PRL secTherefore, we examined PRL using several autopsy samples and a cell culture of the blood–CSF barrier model to clarify the significance of existence PRL in the blood and CSF under hypoxic conditions. We established hypoxic culture using pituitary cells, and a blood–CSF barrier model was prepared to investigate changes in blood and CSF PRL levels under hypoxic conditions.We evaluated the cell activity under hypoxic conditions according to the experimental model. In our examination, VEGF was measured together with PRL, though several markers of cell activity are known . VEGF isThis study was evaluated by the Independent Ethics Committee of the Osaka City University Graduate School of Medicine. According to the Independent Ethics Committee of the Osaka City University Graduate School of Medicine, informed consent from opt-out for the autopsy data analysis was approved.Serial autopsy cases were examined within 48 h postmortem at our institution. There were 118 cases , and the median age was 70 years . The specimens were collected aseptically using syringes. Blood was collected from the right heart chamber and the cerebrospinal fluid (CSF). The blood samples were centrifuged immediately to separate the serum and then stored at −20°C until use , 17. The®  according to the manufacturer’s protocol. For these measurements, the clinical serum reference range was 4.98−26.4 ng/mL for males and 1.74−26.8 ng/mL for females. Amount of PRL in the conditioned medium of SDR-P-1D5 cells was measured by prolactin EIA Kit , and in the conditioned medium of MSH-P3 cells was measured by pig prolactin ELISA KIT . Amounts of vascular endothelial growth factor (VEGF) in the conditioned medium of SDR-P-1D5 cells was measured by rat VEGF ELISA KIT , and in the conditioned medium of MSH-P3 cells was measured by pig VEGF 164 ELISA KIT .Amount of PRL in serum and CSF were measured by chemiluminescent enzyme immunoassay using PATHFASTTM, Ambion, Austin, TX, USA) and stored at −80°C until use. Total RNA was isolated using Isogen  according to the manufacturer’s instructions. cDNA copies of total RNA were synthesized using the High-Capacity RNA-to-cDNA kit . The reaction mixture included 9 μL samples of total RNA, 10.0 μL 2× RT buffer, and 1.0 μL 20× RT enzyme mix. Conditions for reverse transcription were as follows: 37°C for 60 min and 95°C for 5 min . Because HIBCPP cells have been reported to change doubling time with increasing passages , only ceter well . Becauseter well . Subsequhe cells .p-value <0.05 was considered significant.Spearman’s rank correlation coefficient was used to compare two values, including PRL levels, age of the subjects, and postmortem time. For comparisons between groups, we used the nonparametric Mann−Whitney U test. Games−Howell test was used for analyses involving multiple comparisons. In this test, the line in each box represents the median, and the lines outside each box represent the 90% confidence interval. The maximum PRL levels in serum and CSF were log-transformed for graphical presentation only. Diagnostic relevance was estimated according to the values obtained for sensitivity, specificity, and accuracy (proportion of subjects correctly predicted). Youden’s index (sensitivity + specificity -1) was used to determine the best cut-off value. We evaluated the usefulness of PRL levels in the serum and CSF for differentiating between death due to hypoxia/ischemia by asphyxia and other causes of death, using receiver operating characteristic curves and the respective areas under the curve. The results are presented as medians. All analyses were performed using the SPSS 9.0 statistical package . A r = 0.221, p < 0.05). The PRL level was significantly higher in the serum  than in the CSF  (p < 0.0001) (A slight correlation was observed between serum and CSF PRL levels ( 0.0001) .p < 0.01, respectively). A slightly correlation was observed between the postmortem period and PRL levels of the serum  and CSF . No relationship was found between PRL levels and age or survival period in either the serum or CSF, respectively, except for serum PRL levels and survival period   and CSF  ( < 0.05) .p < 0.05 to p < 0.001) (No significant differences in serum PRL levels and the causes of death were observed . Serum P< 0.001) . A cut-o< 0.001) . However< 0.001) .PRL expression was not associated with CSF PRL levels, although PRL expression was associated with serum PRL levels. There was no significant relationship between PRL expression and the causes of death  or MSH-P3 (swine pituitary) cells under hypoxic conditions (5% oxygen) for 90 min. Both SDR-P-1D5 and MSH-P3 cells showed high levels of PRL and VEGF following incubation for 10 min under hypoxic conditions . HoweverChoroid plexus from cases of asphyxia-induced hypoxia/ischemia was found to be immunocyte chemical staining positive for PRL , but immPRL mRNA levels among the different causes of death. However, PRL levels in serum decreased according to the survival period. Furthermore, there was no significant difference in PRL-positive cell ratio as determined by immunocytochemical staining among the causes of death. On the other hand, PRL levels in CSF were higher in cases of hypoxia/ischemia caused by asphyxia than in cases involving other causes of death.No significant differences were observed in serum PRL levels and PRL levels among the various causes of death were observed.PRL is secreted in the hypophyseal portal system by the anterior pituitary gland, and the half-life of the PRL is approximately 50 min. CSF PRL levels have been reported to correlate with blood PRL levels . HoweverPRL mRNA level in the pituitary gland were not increased, however amount of PRL increased in CSF under hypoxia. These results suggested that PRL cells do not have the ability to synthesize PRL under hypoxic conditions. Instead, PRL stored in pituitary gland cells were released, PRL level in the CSF is thought to be increased by it passing through the BCB by hypoxic stimulation. In our previous study, we thought that the transportation of hormones  from blood to the CSF under hypoxic conditions is not increased. Only a prolactin is increased in CSF under hypoxia condition [In a hypoxic environment, PRL level in the blood and ondition –33. The ondition , 34, 35.ondition . On the ondition . Althougondition . Variousondition . The maiondition . The posIn the present pituitary cell culture studies, culture cells were always exposed to new culture fluid under the circumfusion culture system, and hormone secretion from cultured cells does not influence the half-life or autocrine action of PRL . With thIn conclusion, our observations form the postmortem PRL levels and cultured cell studies suggest that PRL is transported selectively from the blood to the CSF during the early stages of hypoxia.Therefore, it is suggested that post-mortem CSF PRL levels are markers of hypoxia/ischemia in medico-legal autopsy cases. In addition, the possibility of assessing the severity of hypoxic encephalopathy in the clinical field was also suggested.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


Globally, trachoma is the leading cause of infectious blindness. In Ethiopia, the overall Trachomatous Trichiasis (TT) surgical coverage is 41%. Identifying determinants for not utilizing TT surgery among TT patients is important to design and monitor effective intervention programs. Therefore, this study aimed to identify determinants for not utilizing TT surgery among TT patients in Mehalsayint District, North East Ethiopia.p<0.2 for the final model. Finally, multivariable logistic regression analysis was employed to identify significant factors (p<0.05) for not utilizing TT surgery. Respondents’ age of 16–30 years  and widowed respondents , time to reach the service , unavailability of TT surgeon , symptoms of trichiasis , duration of the problem , the affected eye , epilation practice , and place of TT surgery given  were significant determinants for not utilizing TT surgical services.A community based unmatched case control study was employed from March 30, 2017 to April 13, 2017. A total of 482 study participants (241 cases and 241 controls) with age of ≥15 years were included in the study. The data were entered with Epi info version 7.2 software and exported to SPSS version 20 for analysis. Bivariate analysis was fitted to screen candidate variables with In this study, TT surgery against trachoma is very low and TT remains public health problem in the district. Being younger age and widowed, time taken to reach the service, absence of TT surgeon, symptoms of trichiasis, duration of problem, the affected eye, epilation practice, and service place were determinants for the inability of TT surgical services. The findings of this study would help in designing effective interventions to reduce trachoma in that district. Chlamydia trachomatis that might effect in chronic inflammation of the eyelids, which produces scarring of the conjunctiva that can consequently cause entropion trichiasis, resulting in interned eyelashes. The interned eyelashes as well as other changes of the eye, harm the cornea causing severe pain, corneal opacity and resulting vision loss. Over a million people in Ethiopia are estimated to have Trachomatous trichiasis (TT). Trachomatous trichiasis surgery is the backbone treatment option. Though the provision of free surgical services in the country exists, utilization rates are very low. Identifying the determinants for not utilizing the service is mandatory to take measures towards surgical uptake. A total of 482 study participants (241 cases and 241 controls) with age of ≥15 years were included in the study. The determinants for not use of surgical services were respondents in the younger age group (16–30 years) and widowed participants, lengthy distance from the service, unavailability of TT surgeon, no trichiasis symptoms, long time knowing the problem, right/left eye affected, no experience of epilation practice, and participants who knew place of service was given at health center.Trachoma is the common ophthalmic infection and cause of blindness worldwide. It is caused by ocular infections with causative agent of  Chlamydia trachomatis that might effect in chronic inflammation of the eyelids. Based on WHO grading system for trachoma, the disease classifies into five grades. These are Trachomatous Inflammation-Follicular (TF), which mostly requires topical treatment; Trachomatous Inflammation-Intense (TI), which topical and systemic treatments are considered; Trachomatous Scarring (TS)-when scars are visible as in the tarsal conjunctiva and which may obscure tarsal blood vessels; Trachomatous Trichiasis (TT)-when an individual is referred for eyelid surgery; and Corneal Opacity-a stage during which a person is irreversibly blind [Globally, trachoma is the leading cause of infectious blindness of the eye . It is cly blind . At the ly blind . Epidemily blind . In Ethily blind .Low uptake of TT surgery has always been a concern for the success of the surgical services in trachoma-control strategy. Awkwardly, surgical coverage in affected communities of Africa is lower than 50% . In EthiVarious studies have examined why people with TT do not access the surgical services. Some of the reported causes of poor uptake of surgical services were unaware of how to access services, fear of surgery, burden of household tasks, indirect cost of surgery, longer walking distance, sex, family size, doubt surrounding the outcome, and absence of a companion and mildness of the condition –12.To control and prevent this problem, international strategy had been created to eliminate trachoma as a blinding disease. The WHO-developed strategy is a combination of interventions known by ‘SAFE”. SAFE—stands for surgery for trichiasis (interned eyelashes), antibiotics, facial cleanliness and environmental improvement . In EthiIn Amhara region, in support of primary eye care services including elimination of blinding trachoma, integrated eye care team was established and members of the team were assigned to play different roles towards TT surgical services. The health cadres assigned as Integrated Eye Care Workers (IECWs) are registered nurses or health officers with additional training to enable them perform trichiasis surgery. They are selected from public health facilities using set criteria as described in the trainees’ manual. They are the ones on whom everyone relies to clear the backlog of TT in the region by delivering surgical services. Program managers are zonal health departments’ and district health offices’ prevention of blindness focal persons, zonal program coordinators, and health centers’ heads. Health care providers are Integrated Eye Care Workers and TT surgery assistants. Health extension workers, kebele leaders and health development army volunteers are support staff. Quality control and monitoring and evaluation roles are given to zonal focal persons, The Carter Center staff  and mid-level eye care workers. The surgical services are given at static and outreach. This initiative has been working in collaboration to The Carter Center Ethiopia .To reduce trachoma, several countries, including Ethiopia, have made considerable efforts to improve surgical services in the recent years. Unluckily, despite this increased service delivery, the number of operated cases was less than expected. This is due to a range of service and patient-specific barriers . DespiteThis study used a case control study design with patients of untreated trichiasis being cases and those operated being controls. It was conducted in Mehalsayint district, 190 kilometers from Dessie, 675 kilometers East from Bahir Dar and 591 kilometers from Addis Abeba, the capital of Ethiopia. The district had a total population of 83,024 of which 40,848 (49.2%) were men . In the Worento”) [All backlogs of the district were the target populations which were 2,665 obtained from Carter Center’s survey report. The study populations were all previously operated and non-operated TT patients registered in the service beneficiary registration book and in TT screened registration book in 2016 respectively living in the district. The previously operated TT patients were 1,018 and considered as controls and the non-operated TT patients were 902 and considered as cases. The study included both controls and cases whose ages were greater than 15 years and registered in 2015/16. Structured and pretested questionnaire was employed for data collection. Data were collected from participants using systematic random sampling technique during March 30, 2017 to April 13, 2017. Trachomatous trichiasis is defined as if at least one eye lash rubbing the eye ball or history of epilation secondary to trachoma . EpilatiThe sample size was calculated based on two population proportion formula using Epi Info version 7.2. From previous case control studies done in North Ethiopia on determinants of uptake of surgical treatment for trachomatous trichiasis, the major reasons for not using trichiasis surgery by respondents were walking distance and symptoms of interned eye lash. Walking distance from the nearest health facility for < 30 minutes was 33.3% and for >30 minutes was 66.7% with OR of 0.44 . Symptoms of the interned eye lash with persistent pain which was 29.7% and with no persistent pain was 70.3% with OR of 0.57  . The varp-value < 0.2 for the final model. To end, multivariable logistic regression analysis model, using backward stepwise method, was employed to identify significant factors for not utilizing TT surgical services. Hosmer and Lemshow goodness of fit test, with p-value>0.05, was done to test model fitness. Adjusted odds ratio (AOR) with 95% CI and p-value of <0.05 was used to identify independent determinants for not utilizing TT Surgical services.Data were edited, coded and entered into Epi info version 7.2 software packages. It was then exported to Statistical Package for Social Sciences (SPSS) version 20 for analysis. Descriptive analysis was presented using frequency tables, figures, and percentages. In logistic regression, bivariate analysis model was fitted to screen candidate variables with This study was approved by Ethical Review Committee of College of Medicine and Health Sciences, Wollo University. Written field permits were obtained from South Wollo Zone Health Department and Mehalsayint District Health Office. All study subjects were adults. Verbal/oral consents were taken from the study participants. Since TT cases included in this study will not be stigmatized and even the issue is not sensitive, we preferred oral consents to save time. The consents were taken when interviewing the respondents. In general, it was done with respect to the principles of Declaration of Helsinki.A total of 241 cases and 241 controls of trichiasis were interviewed. Respondents’ mean age was 53.87  years. Women constitute 153(63.5%) of cases and 149(61.8%) of controls in the study. The majority, 202(83.8%) of cases and 210(87.1%) of controls were unable to read and write. One hundred seventy six (72.6%) of cases and 155(64.3%) of controls were married .Among the total respondents, 137(56.8%) of cases and 212(88%) of controls had spent less than or equal to two hours to reach to the surgical services area from their homes. Conversely, 71(29.5%) of cases and 21(8.7%) of controls of the respondents have been reach in greater than two hours walk. One hundred ten (45.6%) of cases faced difficulty of transportation. Twenty three (9.5%) of cases and 3(1.2%) of controls went to surgery sites and returned home by absence of TT surgeon .Of the total respondents, 86(35.7%) of cases and 124(51.5%) of controls had trichiasis on both eyes. One hundred sixty five (68.5%) of cases and 190(78.8%) of controls were having eye illness for less than five years. Thirty one (12.9%) of cases and five (two percent) of controls had no symptoms of trichiasis. Among the study subjects, 145(60.2%) of cases and 206(85.5%) of controls were practiced epilation. Of which, 93(38.6%) of cases and 142(58.9%) controls for more than one year. Among the study participants, 195(80.9%) of cases and 174(72.2%) of controls have minor trichiasis (≤ 5 lashes). Concerning the outcome of surgery, only 73(30.3%) of cases and 8(3.3%) of controls knew recurrence, whereas 157(65%) of cases and 232(96%) of controls knew good outcome. Almost all of cases and controls are volunteered to get the service if transport costs are covered, even if they get food and shelter service in free of charge for overnight until the dressing is removed .As shown in Respondents who had not symptoms of trichiasis were 7.49 times more likely to not get TT surgery as compared to those who had symptoms of trichiasis . Respondents who know the problem for > 5 years were 2.56 times more likely to not go to TT surgical services than those that know the problem for < 5 years . Respondents who didn’t practice epilation were 3.22 times more likely to not get TT surgical services compared to those who practiced epilation . Participants whose right eyes with the problem were 2.14 times more likely to not get the TT surgical services than participants whose both eyes affected . Moreover, participants whose left eyes with the problem were 2.03 times more likely to not get the TT surgical services than participants whose both eyes affected . Respondents who respond as “the service was given at health center” were 4.21 times more likely to not get TT surgery than those who know the service was given at health post .This study identified significant factors for no uptake of TT surgery. The determinants for not use of surgery were respondent’s younger age group and married respondents, long distance from the service, unavailability of TT surgeon, no symptoms of trichiasis, long time knowing the problem, single eye affected, no experience of epilation practice, and they know as “place of service is given at health center".This finding revealed that the younger respondents were tenfold affected to not get the TT surgical services compared to the elders group (> 60 years). This may be due to doubt by younger age group on the outcomes of surgery for their cosmetics reasons. In addition to the mildness of the condition, they had no severe pain and not to affect their vision. But, as age increases, probably severity of symptoms and disease progresses increase and they go to surgery to alleviate their pain as well as to prevent from suffering from blindness. This finding is in line with a study done in seventeen-outreach campaigns in Amhara Region . In addiThis finding revealed that married participants were more likely to not get the surgical services than their counterparts. This may be due to most married participants mistakenly believed that the surgical wound needs up to 2 months to heal. During this time they should avoid exposure to fire or smoke. Married participants have greater childcare responsibilities, agricultural and domestic work and no body supports the above activity and to prepare food for their family. If they exposed to fire or smoke, they believe the disease recurs. Furthermore, most married women have not the right to take surgery without the willingness of their husband. Attendants who had no symptoms of trichiasis were more likely to not get TT surgical services as compared to their counterparts. This may be due to most respondents in this category had severe form of trichiasis with effect on vision and severe discomfort. Patients with severe discomfort have problem on their vision secondary to the presence of lash on the cornea and the tearing. Such patients consider warranted surgery and want to take the trouble for treatment at present. If most patients reported ‘‘having symptoms”, this may reflect prioritization of eye care over work or farming , 21, 22.This study shows that respondents who knew the problem for short period were better to go to TT surgical services than those that knew the problem for longer periods. This result contradicts with a study done in North Ethiopia, in which the uptake of trichiasis surgery increased with duration of illness . This maRespondents who didn’t practice epilation were more likely to not get TT surgical services when compared to those who practiced epilation. This may be due to clients who practice epilation perceived that the disease is severe and utilize surgery as a choice of treatment for long time. They tested as epilation was not permanent treatment and go to TT surgery site to get the service to relief from severe pain and to prevent from blindness. Whereas those respondents who did not practice epilation believe that the disease had no severe pain and did not affect their vision to initiate them for early surgery. Participants whose single eye had the problem were more likely to not get the TT surgical services than those whose both eyes affected. The uptake of surgery in those having bilateral TT was good. This may be due to severity of symptoms increases as the disease attacks both eyes. When both eyes were affected it was indicative of the severity of trichiasis grading and early corneal opacity. This finding is in line with a study done in Tanzania, individuals with bilateral trichiasis were more likely to have surgery than those with unilateral trichiasis . DespiteThis study indicated that respondents who went to surgery site and return by absence of TT surgeon were more likely to not get TT surgery in other times than those who did not miss the Surgeon. This is similar with the study done in Tanzania and Ethiopia , 21. If The study shows that patients who live in the nearby health facility were better to have had surgery as compared to those that are distant. This is similar with a study done in North Ethiopia . It may The findings of this study have valuable policy implications for health programs scheme and interventions. Training and deploying of HEWs and Community-based screeners on house to house screening and awareness creation activities; expanding the service to health post level with campaign and outreach services; and posting the service days on different places may be important to reduce the problem. In other words, this findings supreme important to Mehalsayint district health office and the respective zonal health department, and partners to develop interventions programs against this neglected tropical disease.


The objective of this study was to evaluate the performance of lateral flow immunoassay (LFA) against latex agglutination (LA), India ink and culture in point-of-care diagnosis of cryptococcus meningitis (CM). We conducted cross-sectional study among HIV-positive patients with suspected CM at Mbagathi Hospital, Nairobi, April–July 2017.Of 124 capillary blood and serum and 99 cerebrospinal fluid (CSF) samples, LFA and LA had a concurrence on serum of 94.4%, kappa (0.88), sensitivity (100%) and specificity (91%). LFA and LA on CSF, was 97.9%, kappa (0.96), sensitivity (100%) and specificity (96%). LFA and India ink was 96.9%, kappa (0.94), sensitivity (100%) and specificity (94.1%). On CSF culture, concurrence was 72.7%, kappa (0.43), sensitivity (100%) and specificity (64%) and of LFA on capillary blood, serum and CSF was 100% with kappa (1.00), sensitivity and specificity of 100%. Cryptococcus meningitis (CM) is a life-threatening opportunistic infection among HIV-infected persons . In AfriCulture is the gold standard diagnostic method for CM, but it has poor sensitivity, requires approximately 100 µl of cerebral spinal fluid (CSF), technical expertise, and laboratory infrastructure , 8. MicrCryptococcus species complex (Cryptococcus neoformans and Cryptococcus gattii) [Lateral flow immunoassay (LFA) is a point-of-care (POC) qualitative test to detect capsular polysaccharide antigens of  gattii) –11. LFA  gattii) .Most studies evaluating LFA are focused on use of serum and CSF , 12–14. We conducted a hospital-based cross-sectional study from April to July 2017.The study was conducted at Mbagathi Hospital, a referral facility located in Nairobi County. The evaluation targeted patients ≥ 18 years scheduled for lumbar puncture (LP) and routine blood sample collection for CM diagnosis.LP requested by health care provider, availability of remnant serum and CSF (≥ 500 µl) after routine Cryptococcus LA or culture was performed on HIV-positive patients ≥ 18 years with the ability and willingness to provide informed consent.Patients involuntarily incarcerated in the hospital for psychiatric or physical illness, any patient/patient with a guardian who was deemed mentally unstable or unable to provide informed consent. Patients on any antifungal treatment and patients who were having repeat LPs.The sample size of 125 participants was calculated using Fisher’s formulae, assuming the expected proportion of agreement between LFA and other methodologies (p) was 97.7% and the precision (P) of 3% .We reviewed records for 6 months at Mbagathi Hospital and obtained an average of 95 cases of suspected CM in a month. To achieve a sample size of 125 patients using the estimated sampling frame of 285 within a period of 3 months, every second HIV-positive patient ≥ 18 years suspected of CM and scheduled for routine LP and blood collection for CM diagnosis was enrolled after giving written consent to obtain an additional capillary blood and use of the remnant CSF and serum for evaluation of LFA.The attending laboratory technologist collected blood samples from patients as part of routine testing requested by the clinician. Serum was centrifuged and separated for LA and LFA assays. CSF samples were collected by LP and centrifuged. The supernatant was used for LFA and LA assays and the pellet for culture. Sera were tested by LA, and CSF (where available) was tested by India ink microscopy and culture for clinical management of the patient. Leftover sera and CSF samples were used for the laboratory evaluation. A minimum of 500 µl of the remaining sample was aliquoted into 1.8 ml cryogenic vials. The samples were stored at 4 °C for a maximum of 72 h or at − 20 °C awaiting transportation to the Central Microbiology Reference Laboratory (CMRL). Additionally, a non-routine finger prick capillary blood sample was requested from all enrolled patients. Using standardized lancets and micro capillary tubes, ~ 50 µl of blood was transferred into micro centrifuge tubes containing LFA specimen diluent. The test was performed at the sample collection site per manufacturer instructions. Sera and CSF sample processing were done at CMRL per laboratory standard operating procedures and manufacturer instructions  coefficients of India ink, LA, LFA and culture in sera, CSF and capillary blood. Interpretation of k was per standard guidelines [Data on the test type and test results in different sample types were entered and cleaned in MS-Excel version 2013. Statistics were calculated using GraphPad QuickCalcs 2017  with categorical data analysis to assess sensitivity, specificity, predictive values, confidence intervals (CIs) of proportion, overall percent agreement, and kappa  using CSF, the sensitivity and specificity were 100% (95% CI 92.6–100) and 94.1% (95% CI 84.1–97.9), PPV and NPV at 94.1% and 100% with a total agreement of 97% and a kappa-value of 0.94 (95% CI 0.87–1.00). On comparison of LFA to culture on CSF, the sensitivity and specificity were 100% (95% CI 86.2–100) and 64% (95% CI 52.7–73.9), PPV and NPV at 47.6% and 100% with a total agreement of 72.7% and a kappa-value of 0.46 (95% CI 0.32–0.61).Comparison of culture to LA using CSF, the sensitivity and specificity were 95.8 (95% CI 79.8–99.3) and 65.3 (95% CI 54.1–75.1) with a total agreement of 72% and a kappa value of 0.45 (95% CI 0.30–0.60). On comparison to India ink, the sensitivity and specificity were 95.9% (95% CI 79.8–99.3) and 66.7% (95% CI 55.4–76.3) with a total agreement of 73% and a kappa value of 0.47 (95% CI 0.31–0.62).Comparison of LFA on capillary blood to LFA on sera, the sensitivity and specificity were 100%, PPV and NPV with a total agreement of 100%, and a kappa-value of 1.00 (95% CI 1.00–1.00). LFA on capillary blood was compared to LFA on CSF, the sensitivity, specificity and predictive values were all 100% with a total agreement of 100% and a kappa-value of 1.00 (95% CI 1.00–1.00).Our results show high agreement between LFA, LA and India ink in different samples and a perfect agreement between LFA in different samples. The high agreement shows that LFA is a reliable POC diagnostic test. The results on individual tests show that there was almost perfect agreement between LFA and LA on CSF and serum. The test demonstrated high level of sensitivity and specificity of LFA compared to LA on sera and CSF. These findings are consistent with similar studies conducted in South Africa and USA that show high sensitivity using CSF and serum , 18. ComThe findings from comparison of LFA to India ink microscopy using CSF demonstrated high sensitivity, specificity, and predictive values. This is in contrast to the findings from the expert opinion and other studies that documented lower sensitivity and NPV for CSF microscopy against LFA , 9. The On comparison of LFA to culture using CSF, there was high sensitivity, low specificity, and moderate agreement with a weak kappa value. The findings were consistent with other studies that documented high sensitivity and low specificity , 19. TheLFA on capillary blood was compared with LFA on serum and CSF. The LFA results on capillary blood had an ideal concordance with LFA serum and CSF results. LFA had a very high positive and negative predictive values both on serum and CSF, a characteristic that makes it good for an accurate diagnosis of cryptococcal meningitis. The high sensitivity and specificity of the test and its ability to be easily performed at the bedside and giving accurate results rapidly allows for prompt and timely initiation of treatment . The finThe evidence of the perfect agreement between LFA on capillary blood, serum and CSF, high sensitivity and specificity, ease of performance, along with rapid results may indicate LFA using capillary blood POC test as the method of choice for CM diagnosis. Our results show that LFA meets World Health Organization assured criteria for POC diagnostic tests in resource-limited settings. Therefore, we recommend use of LFA test as a POC test in resource limited settings for the diagnosis of CM.The limitations of this study include participants not yielding CSF sample due to dry taps, thus reducing the CSF samples that were analysed. The difference on CSF samples analysed had no major implications to the overall evaluation since there were more than one sample type used in the evaluation. Capillary blood could only be used on LFA test thus, there was no uniform use of the sample type across other testing procedures.


Loss of the sensory Pkd2l1 channel nearly abolishes CSF-cN calcium activity and single channel opening. Recording from isolated CSF-cNs in vitro, we show that CSF-cNs are mechanosensory and require Pkd2l1 to respond to pressure. Additionally, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. These results indicate that CSF-cNs are mechanosensory cells whose Pkd2l1-driven spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to maintenance of natural curvature of the spine.Defects in cerebrospinal fluid (CSF) flow may contribute to idiopathic scoliosis. However, the mechanisms underlying detection of CSF flow in the central canal of the spinal cord are unknown. Here we demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the  Alteration of cerebrospinal fluid (CSF) flow and cilia defects are clinically associated with idiopathic scoliosis. This study shows that transient receptor potential channel Pkd2l1 is required for mechanosensory function of neurons detecting CSF flow and normal spine curvature development in zebrafish. The CSF contains nutrients, extracellular vesicles, peptides, and proteins that can modulate development and function of the nervous system6. Flow of the CSF distributes these molecules throughout the brain ventricles and central canal of the spinal cord. Motile cilia that line the epithelium surrounding the CSF could contribute to this distribution by shaping the flow8.Cerebrospinal fluid (CSF) is secreted by the choroid plexus and circulates within the ventricular system of the brain and in the central canal of the spinal cord10 as well as spine morphogenesis in juveniles and adults11. In mutants with impaired cilia motility, CSF flow in the brain ventricles is dramatically reduced, and adult mutants exhibit pronounced torsion of the spine similar to clinical presentation of idiopathic scoliosis1. Observations linking CSF flow and spine morphogenesis in zebrafish are corroborated by observations from patients with Chiari malformations, in which structural defects in the brain lead to CSF flow defects and scoliosis13. Active CSF circulation or signaling cues distributed by motile cilia may instruct proper body axis formation, though these mechanisms are currently unknown14. Elucidating how physical and biochemical properties of CSF can impact morphogenesis requires an understanding of how CSF flows in the spinal cord and how its physical and chemical properties are detected by local receptor cells lining the central canal.Multiple lines of evidence from zebrafish indicate that cilia or CSF flow defects influence body axis formation at embryonic stages16. In zebrafish, mouse, and macaque, CSF-cNs also specifically express the transient receptor potential channel Pkd2l1 (polycystic kidney disease 2-like 1), also known as TRPP318. Previous studies showed these cells utilize Pkd2l1 to sense pH changes and respond to bending of the spinal cord during locomotion21. However, whether the cells can directly direct mechanical perturbation or sense CSF flow has not been proven.Primary candidates for sensory cells to detect changes in CSF flow or content are the CSF-contacting neurons (CSF-cNs) that line the brain ventricles and central canal of the spinal cord. Originally characterized in over 100 vertebrate species by Kolmer and Agduhr, these cells share a similar morphology and possess an apical extension that contacts the central canalcfap298tm304, previously known as kurly, reduction of cilia motility22 slows transport of particles down the length of central canal. Using calcium imaging, we show that ventral CSF-cNs in this mutant lose spontaneous activity in vivo. In a pkd2l1 null mutant, in which CSF-cNs lack the sensory channel Pkd2l1, ventral CSF-cNs also lose spontaneous calcium activity. Whole-cell patch clamp recordings further demonstrate that opening of a single channel leads to depolarization that is sufficient to trigger action potentials in wild-type CSF-cNs. Due to the link between disrupted CSF flow and CSF-cN activity, we recorded from isolated zebrafish CSF-cNs to determine whether CSF-cNs respond directly to mechanical pressure. Mechanical stimulation that increases single channel opening in wild-type CSF-cNs abolishes channel activity in pkd2l1 mutants. Finally, we demonstrate that Pkd2l1 is required for maintenance of a straight spine in adulthood.Here we investigate flow of the CSF within the spinal cord, detection of flow by CSF-cNs, and how altered flow can lead to spinal defects. Via full-field OCT and imaging of fluorescent beads, we find that CSF flows bidirectionally in the central canal of the spinal cord. In the zebrafish mutant Tg(β-actin:Arl13b-GFP)23 at regularly spaced planes along the dorsoventral axis of the central canal . In order to confirm that the circulation of exogenous beads reflected endogenous flow, we implemented FF-OCT, which allows detection of objects with a refractive index contrast based on interference patterns from backscattered photons .We took advantage of the embryonic zebrafish, which is transparent and has easily accessible ventricular structures to study the interactions between the CSF flow, cilia motility, and receptor cells. To examine the dynamics of motile cilia in vivo, we imaged cilia in 24 h post-fertilization (hpf) transgenic zebrafish  that line the brain ventricles and central canal of the spinal cord. Using population calcium imaging in 24–30 hpf paralyzed wild-type embryos, we found that CSF-cNs located near ventral cilia within the central canal were highly active  that enabled extensive single channel opening to be measured in whole-cell mode in vivo  zebrafish larvae  and the National Ethics Committee based on E.U. legislation. All experiments were performed on Tg(β-actin:Arl13B-GFP) embryos were mounted dorsal side up in 1.5% agarose and paralyzed with ~2 nL of 500 µM α-bungarotoxin injected into caudal lateral muscle. A spinning disk confocal microscope  was used to focus on a single optical section at the ventral-most extent of the central canal and a 15 s time-lapse was acquired at 33 Hz imaging frequency. Subsequent time lapses were acquired at 2 µm steps moving from the ventral to dorsal limits of the central canal  to yield the values displayed in Fig. Because fluorescence intensity changes depending on transgene copy number and fluctuations in imaging conditions, it was necessary to normalize the standard deviation plot by dividing it by the average intensity plot to yield an intensity-corrected motion index. Finally, a background subtraction was carried out on the normalized plots  and the integral was approximated using the 2, 10 HEPES, 10 glucose, 2 CaCl2; 290 ± 3 mOsm/L, pH adjusted to 7.8 with NaOH) then briefly sonicated. 1–3 nL of the solution were pressure injected in the hindbrain ventricle. Images were acquired using spinning disk confocal  for 20 s at 10 Hz using Slidebook software . Motion of beads or endogenous particles were analyzed in Fiji45. Videos were rotated and cropped to reduce the field of view to the central canal. Images were resliced in Fiji, then a 1.5–1.7-µm-wide region at 30 or 85% of the dorsoventral axis was projected as a kymograph to obtain time trajectories of individual beads. Custom MATLAB code was used to obtain start and end points for individual beads and calculate velocities.Embryos were manually dechorionated then embedded in 1.5% low-melting point agarose at 24–26 hpf and paralyzed by injection of ~2 nL of 500 µM α-bungarotoxin in the caudal musculature of the tail. Yellow-green 0.02 µm fluorescent FluoSpheres  were diluted to a 2% concentration by volume in artificial CSF  or Tg(pkd2l1:GCaMP5G) transgenic lines.The FF-OCT path is based on a Linnik interference microscope configuration illuminated by a temporally and spatially incoherent light source. A high power 660 nm LED  provided illumination in a pseudo Köhler configuration. A 90:10 beam splitter separates the light into sample and reference arms. Each arm contains a ×40 water immersion objective . In the reference arm, the light is focused onto a flat silicon wafer with a reflection coefficient of about 23.5% at the interface with water. FF-OCT detects any structure that reflects or backscatters light within the sample arm. The backscattered and reflected coefficients depend on the refractive index, size, and shape of the imaged structures. Light returning from both arms is recombined by the entrance beam splitter. The two beams interfere only if the optical path length difference between both arms remains within the coherence length of the system, ensuring efficient optical sectioning. A 25-cm focal length achromatic doublet focuses the light to a high speed and high full well capacity CMOS camera . The overall magnification of the FF-OCT path is ×50. The measured transverse and axial resolutions were 0.525 μm and 4 μm, respectively. Camera exposure was 9.8 ms, and images were acquired at 100 Hz. Direct images are used to reduce the mechanical vibrations caused by movement of the piezo actuator. Due to the low coherence of the setup, it is only sensitive to intensity changes that happen at a given depth inside the coherence gate of the microscope. We acquired sequences of consecutive direct images and computed the standard deviation on groups of images to cancel the incoherent light that does not produce interference. The image obtained therefore corresponds to intensity fluctuations occurring at a given depth, similar to what is obtained by modulating the piezo position. This method allows detection of phase fluctuations caused by small-particle displacement occurring in the 10–100 Hz range. The full-field optical coherence tomography (FF-OCT) setup used is described in additional detail in a separate publicationBeads were injected into the hindbrain ventricle as described above. Only embryos in which the beads reached the central canal from the ventricle were selected for analysis for both genotypes. To develop an unbiased method to determine the position of the end of the dye front, regions of interest (ROI) were first defined for each somite in the field of view using a transmitted light image. A plot profile was then generated in the green channel for each somite ROI. If the central canal had been infiltrated by the fluorescent beads at this rostrocaudal somite position, a distinct peak would be apparent in this plot profile. To determine the maximum extent of the dye front in an unbiased fashion, a ratio of the peak central canal fluorescence intensity was divided by the green channel fluorescence adjacent to the central canal. A cutoff value of 1.2 was used to define a somite as infiltrated vs. not infiltrated.Thirty hpf embryos were killed using 0.2% tricaine, and fixed in 4% paraformaldehyde for 4 h at 4 °C. After three washes in 1× PBS, embryos were blocked in PBS 0.7% Triton, 1% DMSO, 10% normal goat serum, and 1 mg/mL bovine serum albumin overnight at 4 °C. The following primary antibodies were then incubated overnight at 4 °C using the following concentrations in blocking buffer: anti-Pkd2l1 , anti-RFP , and anti-GFP . Washes were performed in 1% DMSO and 0.3% Triton-PBS, and secondary antibodies were incubated for 2.5 h at room temperature. All secondary antibodies  were used at 1:500 dilution in blocking buffer.45. ROIs were manually selected based on a standard deviation Z-projection and ventromedial position in the spinal cord. ΔF/F and normalized integral of activity were calculated with custom scripts written in MATLAB.Embryos were staged to 24–26 hpf, dechorionated manually, then embedded in 1.5% low-melting point agarose. All imaging was performed following injection in caudal axial muscle with ~2 nL of 500 µM α-bungarotoxin (Tocris Bioscience). Calcium imaging was performed at 4 or 5 Hz with a spinning disk confocal  for 4 or 8 min. Images were acquired using Slidebook software  and reconstructed online using FijiTg or Tg(olig2:DsRed2)47 embryos in aCSF. Embryos were pinned through the notochord with 0.025 mm tungsten pins. Skin and muscle from two to four segments between segments five and twelve were dissected using a glass suction pipette. A MultiClamp 700B amplifier, a Digidata series 1440 A Digitizer, and pClamp 10.3 software (Axon Instruments) were used for acquisition. Raw signals were acquired at 50 kHz and low-pass filtered at 10 kHz. Patch pipettes  with a tip resistance of 7–9 MΩ were filled with internal solution . Holding potential was −85 mV, away from the calculated chloride reversal potential (−51 mV). Analysis of electrophysiological data was performed offline using Clampex 10 software (Molecular Devices). Single channel events were identified using a threshold search in Clampfit (Molecular Devices), with a threshold triggered at −8 pA from the baseline and a rejection at −30 pA. Only events lasting longer than 1.5 ms were included for analysis. For CSF-cNs in pkd2l1+/+ embryos, a 20-s window was used to identify channel events from a gap-free voltage-clamp recording from the first 2–5 min of recording.Whole-cell recordings were performed in 25–30 hpf Tg(pkd2l1:TagRFP)-positive zebrafish embryos were then placed in Hank’s balanced salt solution  and anesthetized with 0.02% tricaine. The chorion, yolk, head, and caudal tail were removed. Dissected embryos were then placed into 1 mL HBSS, which was replaced by collagenase type IA solution (2 mg/mL) once the spinal cords were dissected and incubated for 45 min at 37 °C with trituration occurring half time using a P1000 tip. Dissociated tissue was centrifuged for 3 min at 1000 rpm. The supernatant was removed and the pellet resuspended in 1 mL HBSS. The solution was then passed through a 40 µm filter.Tg(pkd2l1:TagRFP)-positive zebrafish were plated on top of spinal cord cells cultured from 2 dpf wild-type zebrafish (to improve adherence) on 12 mm Corning Bioboat Coverslips precoated with Laminine and Poly-Lysine. Cells were then cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum, 50 units/mL penicillin–streptomycin, 25 mM glucose, 2 mM L-glutamine, 25 ng/mL NGF, and 4 ng/mL GDNF and incubated overnight at 37 °C in 5% CO2.Dissociated spinal cord cells from 4 dpf Tg(pkd2l1:TagRFP)-positive CSF-cNs were patch clamp recorded within 1 day of plating. Recordings were performed using borosilicate electrodes (Harvard Apparatus) having a resistance of 5–8 MΩ when filled with a solution containing (mM): 130 KCl, 10 HEPES, 4 CaCl2, 1 MgCl2, and 10 EGTA, 4 Mg-ATP and 0.4 Na-GTP . The extracellular solution consisted of (mM): 140 NaCl, 10 HEPES, 10 glucose, 1 KCl, 1 MgCl2, and 2.5 CaCl2 . Whole-cell recordings were made at 24 °C using an Axopatch 200B amplifier (Axon Instruments), filtered at 1–2 kHz, and digitally sampled at 20 kHz. Voltage errors were minimized using 75–80% series resistance compensation. Data acquisition was performed with pClamp 10.2.48. Mechanical stimulation was achieved using a fire-polished glass micropipette positioned at 65° from the horizontal plane and cemented to a piezo-electric actuator . Voltage-clamped CSF-cNs were mechanically stimulated by the displacement of the probe toward the selected neuron using increments of 0.2–0.5 µm. The probe had a velocity of 800 μm/s during the ramp segment of the command for forward motion. Raw data were analyzed using paired t test or Mann–Whitney test depending on the experimental design. Analysis used a combination of Clampfit 10.2 (Molecular Devices), Origin 7.0 (OriginLab), and PRISM 4.0 (GraphPad) softwares. All values are shown as mean ± standard error of the mean (s.e.m.) and n represents the number of cells examined.Piezoelectrically driven mechanical stimulation was used to apply focal force onto the neuronspkd2l1+/+ siblings were used as controls. For analysis of 2 dpf pkd2l1 mutant embryos, the angle of the tail was calculated based on a point placed midway through the eyes, the swim bladder, and on the tip of the tail.Male and female adult zebrafish aged 5, 12, or 19 months were briefly anesthetized in 0.02% tricaine and lateral images were obtained. Curvature was calculated in MATLAB using the LineCurvature2D function. Curvature analysis was performed blinded to fish genotype. pkd2l1+/+ and pkd2l1−/− fish were killed at 20 months of age in 0.1% tricaine and subsequently fixed in 4% paraformaldehyde for 1 h at room temperature. Fish were then eviscerated, cleared, and stained using 1 mg/mL alizarin red as described in ref. 49. After staining, fish were photographed using a Nikon digital single-lens reflex camera fitted with an AF-S Micro Nikkor 60 mmF/2.8G ED lens.Male and female adult Tg(pkd2l1:GCaMP5G) zebrafish embryos were pinned through the notochord with 0.025 mm tungsten pins and bathed in aCSF. Images were acquired with a 488 nm laser on a spinning disk confocal  at 4 Hz, as performed for calcium activity experiments in paralyzed embryos. Offline analysis to determine contraction amplitude was performed in MATLAB. In order to estimate the timepoint of motion artifacts, the average fluorescence signal for all ROIs was calculated to obtain a single time series. The threshold for motion artifacts was set to be when the absolute value of the time derivative was greater than three times the standard deviation of the single time series. In order to prevent increases in calcium activity being detected as a motion artifact, a filter was applied to impose the motion artifact values to be below the median value of the signal.Unparalyzed 24–30 hpf Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6


Background: While primary progressive aphasia (PPA) is associated with frontotemporal lobar degeneration (FTLD) pathology due to tau or TDP, clinical-pathological studies also demonstrate many cases have Alzheimer's disease (AD) pathology. The logopenic variant of PPA (lvPPA) is most often associated with AD pathology, but this has proven to be the least reliable PPA to diagnose using published clinical criteria. In this study, we used cerebrospinal fluid (CSF) analytes to identify patients with likely AD pathology, and relate phenotypic features of lvPPA to CSF.Methods: We studied 46 PPA patients who had available CSF analytes, including 26 with a clinical diagnosis of lvPPA, 9 with non-fluent/agrammatic variant (naPPA), and 11 with semantic variant (svPPA). We identified patients with likely AD pathology using amyloid-beta 1–42 (Aβ1−42) < 192 pg/ml and assessed MRI gray matter atrophy in these patients.Results: We found that 23 (49%) of 46 PPA patients have a low CSF Aβ1−42 level consistent with AD pathology. Twenty-one (91%) of 23 patients had a lvPPA phenotype, and 18 (79%) of 23 cases with an elevated CSF Aβ1−42 level did not have a lvPPA phenotype. Patients with a lvPPA phenotype demonstrated GM atrophy in the left lateral temporal lobe, and this was also seen in those with a CSF Aβ1−42 level < 192 pg/ml.Conclusion: The lvPPA clinical phenotype may be a useful screen for CSF analytes that are a surrogate for likely AD pathology, and may help establish eligibility of these patients for disease-modifying treatment trials. Primary progressive aphasia (PPA) refers to a syndrome of declining language ability that results from a neurodegenerative disease. Three variants of PPA have been identified: non-fluent/agrammatic (naPPA), semantic (svPPA), and logopenic (lvPPA) . It is v1−42) to identify PPA patients with likely AD pathology, and assessed whether this corresponds to PPA patients with a lvPPA phenotype. Several previous studies have examined Aß1−42 in PPA. In one study, patients with a clinical diagnosis of lvPPA had lower Aß1−42 levels than controls and naPPA patients  analytes have been shown to serve as a sensitive biomarker for AD pathology , this stpatients . In a lapatients . Here wen = 26, svPPA: n = 11, naPPA: n = 9) (1−42 level that was < 192 pg/mL and AD pathology. One autopsy case with FTLD-Tau pathology had a CSF of Aß1−42 level that was >192 pg/mL and did not have a lvPPA phenotype. Some of these cases have participated in other CSF biomarker studies.Patients included for this study were seen in the Department of Neurology out-patient clinic of the Penn FTD Center and data were retrieved from The Integrated Neurodegenerative Disease Database (INDD)  at the U: n = 9) . All patWe assessed the neuropsychological profile for each subtype of PPA . We evalAll procedures, including CSF collection and MRI, involved participation in an informed consent procedure, and were performed in accordance with the Helsinki Agreement and the rules of the Institutional Review Board at the University of Pennsylvania.1−42, p-tau, and t-tau levels, as previously described (CSF samples were obtained by routine lumbar puncture according to standard operating procedures of the Alzheimer's Disease Neuroimaging Initiative (ADNI) . In brieescribed , and a sescribed .1−42 level, and examined this cut-point in our cohort. The variables utilized in this analysis were the categorical variable “clinical phenotype” and the continuous variable “CSF Aß1−42 level.” A receiver operating characteristic (ROC) curve analysis used sensitivity and specificity to define the cut-point between these groups, and provided the area under the curve (AUC). In both the analysis using the predetermined cut-point and the empirically determined cut-point in our cohort, we tabulated the frequency of patients with lvPPA compared to PPA patients with another phenotype in the two CSF-determined groups, and used chi-squared analyses to assess whether there was a statistically significant difference between phenotypes within and between CSF-defined groups.In the first analysis, we grouped patients according to the predefined cut-point of 192 pg/ml. Since the cut-point based on an AD phenotype may not generalize to non-amnestic cases with AD pathology, we also implemented a second analytic approach. Here we used a k-means cluster analysis with a two-group solution to identify the groups among all PPA patients in our cohort according to their CSF Aßp > 0.1). MRI exclusion criteria included poor-quality MRI at visual inspection . Briefly, participants underwent a structural T1-weighted MPRAGE MRI acquired from a SIEMENS 3.0T Trio scanner with an eight-channel coil using the following parameters: repetition time (TR) = 1,620 ms; echo time (TE) = 3 ms; 160 1.0 mm slices; flip angle = 15°; matrix = 192 × 256; and in-plane resolution = 0.9766 × 0.9766 mm. T1 MRI images were preprocessed using antsCorticalThickness  with 10,000 permutations that is equivalent to an analysis protecting for multiple comparisons. We report clusters that survived p < 0.01 with a minimum of 150 adjacent voxels. We report two sets of t-tests: First, we examined each patient group  relative to controls; and second, we examined each CSF-subgroup relative to controls .High resolution T1-weighted MRI scans were available for 20 lvPPA, 10 naPPA, and 11 svPPA, and we compared these PPA patients with 69 demographically matched control participants. The PPA patients with MRI matched the clinical and demographic characteristics of those without MRI  of these cases had an lvPPA phenotype, revealing significantly more cases of clinically diagnosed lvPPA than non-lvPPA among PPA patients with a lower CSF Aß1−42 level (p < 0.001). The sensitivity for low CSF Aß1−42 level to identify lvPPA compared to non-lvPPA is 91%; the specificity is 89%; the positive predictive value is 87%; and the negative predictive value is 92%. Of the 2 non-lvPPA cases with a lower Aß1−42 level, one had an svPPA phenotype (CSF Aß1−42 level = 167 pg/ml) and the other had a naPPA phenotype (CSF Aß1−42 level = 183 pg/ml). The svPPA patient with CSF Aß1−42 level < 192 pg/mL had an age at onset of 68. Not only was this well-above the mean age of onset of lvPPA patients with CSF Aß1−42 < 192 pg/mL, but it was also well above the mean age at onset of the svPPA patients with CSF Aß1−42 level > 192 pg/mL , suggesting the possibility of AD co-pathology. Duration of disease at the time of obtaining CSF was shorter in this svPPA patient (1 year) and longer in this naPPA patient (7 years), and differed from the mean disease duration of lvPPA patients with CSF Aß1−42 < 192 pg/mL  and from that of PPA patients with CSF Aß1−42 level >192 pg/mL . The svPPA and naPPA patients with CSF Aß1−42 < 192 pg/mL levels did not differ from lvPPA patients with CSF Aß1−42 < 192 pg/mL, and did not differ from PPA patients with CSF Aß1−42 level >192 pg/mL with regards to education level.A summary of CSF analyte values is provided in 1−42 level < 192 pg/mL, significantly greater than the number of lvPPA cases with CSF >192 pg/mL (p < 0.001). The sensitivity for lvPPA to identify low CSF Aß1−42 level compared to elevated CSF Aß1−42 level is 81%; the specificity is 75%; the positive predictive value is 91%; and the negative predictive value is 78%. The mean CSF Aß1−42 level of the 5 lvPPA cases with CSF >192 pg/mL was 317 pg/mL (SD = 186). The lvPPA patients with CSF Aß1−42 < 192 pg/mL did not differ significantly from those with CSF Aß1−42 level >192 pg/mL with regards to education level and disease duration at the time that CSF was obtained. The age at onset, however, differed significantly , with the 5 lvPPA patients with an elevated CSF Aß1−42 level having an older age at onset  than lvPPA cases with CSF Aß1−42 < 192 pg/ml .Twenty-one (80.8%) of 26 cases with a clinical diagnosis of lvPPA had a CSF Aß1−42 level in non-amnestic AD with early-onset disease may differ from the CSF Aß1−42 level associated with later-onset amnestic AD, we also used a cluster analysis to partition the entire cohort of PPA patients (n = 46) according to the CSF Aß1−42 level. One cluster included PPA patients with a lower Aß1−42 level , and the second cluster included PPA patients with a higher Aß1−42 level . A ROC curve analysis in this sample defined a cutpoint at 204.2 pg/mL, yielding 91% sensitivity, 89% specificity, and an area under the curve = 0.914. Independent samples t-test showed that the Aß1−42 level of the cohort with likely AD pathology to be significantly lower than that of the cohort less likely to have AD pathology .Since CSF Aß1−42 level and a high Aß1−42 level. This demonstrated distinct areas of atrophy that stratified the two groups  is present in the CSF of many PPA patients, suggesting that these PPA patients may have AD pathology (1−42 level in the range consistent with likely AD pathology have a clinical phenotype of lvPPA.We found that a low Aβathology . It is pathology . In our athology , which i1−42 level had lvPPA, this phenotype alone cannot be used reliably to identify patients with likely AD pathology. For example, in our cohort we found two patients with a low CSF Aß1−42 level who did not have a lvPPA phenotype: One patient had svPPA and another had naPPA. Without autopsy evidence, we can only speculate about the basis for this discrepancy. One possibility is that these cases in fact have AD pathology but in an anatomic distribution more consistent with these non-lvPPA phenotypes. Several patients with atypical presentations of AD pathology have been reported with svPPA or naPPA phenotypes (While a large majority of cases with a low CSF Aßenotypes , 25. A senotypes , and we enotypes .1−42. The five lvPPA cases with elevated CSF Aß1−42 levels had an age of onset that was older than that of the lvPPA patients with lower CSF Aß1−42 levels. With the caveat that we did not have a pathologic diagnosis in our cases, our findings are consistent with the claim that a clinical evaluation for lvPPA can be an inexpensive way to screen PPA patients for those who may be eligible for participation in disease-modifying therapies targeting the misfolded proteins contributing to AD pathology. Additional biomarker data would be helpful to confirm the diagnosis of PPA associated with likely AD such as amyloid-PET, although it should be noted that amyloid-PET is also associated with false-positive and false-negative findings (Another important consideration is that 26 cases in our cohort had a lvPPA phenotype, but only 21 of these cases with a lvPPA phenotype had low CSF Aßfindings .1−42 level cohort by showing nearly identical areas of reduced GMP in the lvPPA cohort and the low Aβ1−42 level cohort (1−42 cohort, including the left middle-superior temporal gyrus, left parietal region, and left precuneus. This anatomic distribution of atrophy is a subset of regions typically affected in clinical AD. Importantly, our cohort did not have significant medial temporal lobe atrophy, emphasizing that these patients had PPA and not a language-dominant variant of AD. Despite the small cohort of PPA patients with elevated CSF Aβ1−42 consistent with a non-AD form of PPA, the pattern of atrophy in this group is distinctly different from that of the low CSF Aβ1−42 cohort with lvPPA. The areas of atrophy found in the group with elevated CSF Aβ1−42 include the left anterior temporal region, and the left inferior frontal-insula region. These are areas associated with svPPA and naPPA, respectively (The MRI analysis was consistent with the finding that the lvPPA phenotype is most prominent in the low Aβl cohort . The patl cohort , 26, 28.l cohort  and audil cohort  that areectively .1−42, t-tau, and p-tau181 in a small cohort of PPA patients, AD patients, and healthy controls (181/Aß1−42 ratio allowed separating AD and non-AD patients, although there was no available converging evidence such as imaging or autopsy to support this finding. Another study also found lower levels of CSF Aβ1−42 in clinically-diagnosed lvPPA compared to other PPA patients, discriminating between lvPPA and naPPA/svPPA with 86% sensitivity and 69% specificity (Other groups have explored the utility of using CSF analytes to differentiate PPA phenotypes. One study evaluated CSF levels of Aßcontrols . They focificity . In a smcificity . In a lacificity . An impocificity , 31. In cificity , for exacificity . Serum ncificity , and othcificity  [also secificity , 33. Con1−42 is not uncommon in patients with PPA, and that there is a statistical association between a low CSF Aβ1−42 level and the lvPPA phenotype. The link between lvPPA phenotype and a surrogate marker of AD pathology was further supported by MRI imaging. The potential use of the lvPPA clinical phenotype to screen for CSF analytes as a surrogate for likely AD pathology may help establish eligibility of these patients for disease-modifying treatment trials.Several caveats should be kept in mind when considering our results. First, a relatively small number of patients participated in our study, although PPA is a relatively rare condition, and we adhered narrowly to the published criteria for PPA to determine the usefulness of AD CSF analytes within the scope of these criteria. Other CSF analytes may be informative in PPA and have been reported in some patients from this cohort elsewhere , and addThis study was carried out in accordance with the recommendations of the Institutional Review Board of the University of Pennsylvania with written informed consent from all subjects and witnessed by a responsible caregiver. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board of the University of Pennsylvania.AH, CJ, MU, DI, and MG data collection. CN, CJ, CM, and KC data analysis. CN, CM, DI, KC, and MG manuscript writing and editing.MG is an Associate Editor of Neurology, participates in clinical trials sponsored by Biogen, Alector, and Eisai, and receives support for his role as a consultant for Biogen, UCB, Ionis and Bracco. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We used various methods for identifying and prioritizing patient-centered outcomes (PCOs) for comparative effectiveness research (CER).We considered potential PCOs (“benefits” and “harms”) related to (1) gabapentin for neuropathic pain and (2) quetiapine for bipolar depression. Part 1 (April 2014 to March 2015): we searched for PCO research and core outcome sets (COSs). We conducted electronic searches of bibliographic databases and key websites and examined FDA prescribing information and reports of clinical trials and systematic reviews. We asked patient and clinician co-investigators to identify PCOs. Part 2 : in 2015, we surveyed members of The TMJ Association, Ltd., a patient group associated with temporomandibular disorders (4130 invitations sent). Participants prioritized (1) the importance of six potential benefits and (2) 21 potential harms selected by the investigators in part 1, using stated preference methods. We calculated descriptive statistics.N = 746) and successfully prioritized both benefits and harms. Participants did not identify many benefits other than those we identified in part 1; several participants identified additional harms.In part 1, we identified a COS for pain, the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) recommendations. The COS identified several important benefits, but it lacked specific recommendations about which potential harms to include in CER. We did not identify a COS for bipolar depression. Research reports, prescribing information, and patient co-investigators helped identify but not prioritize outcomes. We abandoned our electronic search for PCO research because we found it would be resource-intensive and yield few relevant reports. In part 2, surveying patients was useful for prioritizing PCOs. Members of The TMJ Association, Ltd., completed the survey  contains supplementary material, which is available to authorized users. The role of patients in health research has been expanding since the 1980s, when activists succeeded in prioritizing patient interests for AIDS research , 3, reviidentified, it is not clear how the “most important” PCOs should be prioritized, either for primary research  or for systematic reviews ) on September 30, 2014  in May 2014, to identify COSs. The James Lind Alliance is a nonprofit organization dedicated to identifying research priorities related to intervention effects.For each condition, we asked co-investigators and colleagues to identify COSs related to neuropathic pain and bipolar disorder. We searched the Core Outcome Measures in Effectiveness Trials (COMET) database  in AprilBecause previous CER includes many outcomes that patients might consider important, we recorded the outcomes included in reports of clinical trials that were eligible for our overall study . Search Because prescribing information might include outcomes that are important to patients, even if patients are unaware of those outcomes, we examined all outcomes in the prescribing information for both case studies.https://www.accessdata.fda.gov/scripts/cder/daf/.) For a given drug, prescribing information described the specific health conditions that the drug might be used to treat, and it included information that might apply to people who use the drug for any condition.We examined the Drugs@FDA website in April 2014 using the generic drug names “gabapentin” and “quetiapine” to identify prescribing information .  on January 22, 2015. DRUGDEX includes information about on- and off-label uses of medications; the Centers for Medicare and Medicaid Services (CMS) may use DRUGDEX to make reimbursement decisions for anti-cancer treatments [Like prescribing information, drug compendia might include outcomes that are important to patients. Thus, we examined the compendium “DRUGDEX” (which was available at eatments . Like FDhttp://www.patientslikeme.com) on April 29, 2014. Information on PatientsLikeMe is entered by patients. PatientsLikeMe allows users to search for (“filter”) information about potential benefits for both intervention and indication. We searched for information about gabapentin used for treating “neuropathic pain” and quetiapine used for treating “bipolar disorder.” For each indication-intervention pair, patients assess the intervention’s perceived “effectiveness” using a 5-point scale . PatientsLikeMe allows users to search for information about potential harms  only by intervention. For each intervention, patients assess the broad label side effects using a 4-point scale . Although patients can list specific side effects , they cannot rate specific side effects using the scales above. PatientsLikeMe creates summary tables that report the number of patients who say they experienced each specific side effect . Finally, PatientsLikeMe includes patient narratives , which we did not review because of time constraints.We examined the website PatientsLikeMe  selected “outcome domains” , 22 relaThe survey first asked participants to rank the importance of the six potential benefits of interventions used for pain. In part 1 of our study, we identified a relatively small number of potential beneficial outcome domains  grouped terms describing potential harms into 21 items. For example, we grouped terms related to skin into one item “skin problems .” We then reviewed these categorizations with patient and clinician co-investigators and modified the groupings if appropriate.To minimize the number of items we asked each participant to rank on the survey, we asked each participant, depending on his or her birth month, to rank only seven of the 21 harms. To do this, we made 12 sets of seven harms; each harm appeared with equal frequency across the survey “sets” . For each set of seven harms, we asked participants to assign values from 1  to 7 (would least affect your decision to use an intervention).After participants ranked seven harms, the survey questions asked if there were any other potential harms they would want to know about before starting treatment. Participants could identify additional harms in a text box.We then asked patients to select whether (1) “the likelihood that the medication will reduce your symptoms”  or (2) “the likelihood that you will experience side effects”  was more important in making a decision about treatment.The survey concluded with questions about participant characteristics. We asked each participant for his or her year of birth, gender, types of pain experienced currently and in the past, “present pain intensity” (PPI), current and previous pain treatments, duration of pain, and harms they had experienced because of treatment.On June 17, 2015, TC sent an email invitation to 4130 people on the TMJ Association mail list. To be included, people had to be subscribed to the TMJ e-newsletter and must have opened at least one e-newsletter in 2015. TC sent a reminder on July 1, 2015, and a second reminder on July 31, 2015. We considered invitees to have declined participation if we received a declining email or no response. The number of surveyed individuals (sample size) was opportunistic; it was determined by our ability to contact members of this patient group in collaboration with our patient investigators.or ranked the importance of seven potential harms; and (3) reported their age, sex, and current PPI  indicated that their pain condition(s) included “TMJ (temporomandibular joint and muscle disorders),” (2) ranked the importance of the six benefits and one side effects item  indicateWe did not include two Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) recommendations outcomes in part 2 survey of TMJ Association members: (1) “participant ratings of global improvement and satisfaction with treatment” was considered inappropriate for the survey because it was a composite outcome including multiple outcome domains and (2) “participant disposition” was not considered a potential PCO because it was defined described as “information regarding participant recruitment and progress through the trial, including all information specified in the CONSORT guidelines” .To summarize the results, we first averaged individual patient rankings to derive an overall ranking of potential beneficial outcome domains and the single outcome item side effects from 1 to 7.To summarize the individual part 2 survey participant rankings of harms, we used best-worst scaling (BWS) to compute a BWS score for each of the seven harms ranked by each survey participant . First, Because each part 2 survey participant ranked only seven of the 21 possible harms, we compared the characteristics of the 12 groups to check our assumption that the 12 groups did not differ and that computing an overall ranking would be acceptable.We compared the results of the part 2 survey with the results of the methods used in part 1, including the opinions of our patient and stakeholder co-investigators.All analyses were performed using Stata 14 .Using systematic searches to identify published studies that aimed to identify or to prioritize PCOs for neuropathic pain and bipolar depression, we obtained a large number of titles and abstracts for each condition. For example, we retrieved 11,326 titles and abstracts in MEDLINE (Ovid) related to neuropathic pain and we retrieved 8801 titles and abstracts in the PubMed searches related to bipolar depression , many participants in IMMPACT meetings have been employed by pharmaceutical manufacturers. Other participants have worked in academia or government. Between zero and three patients have contributed to each of the 20 IMMPACT meetings, representing at most 3/39 (8%) of participants. IMMPACT recommends that six core outcome domains be assessed in studies of chronic pain: pain , physical functioning, emotional functioning, participant ratings of global improvement and satisfaction with treatment, symptoms and adverse events, and participant disposition (Table One of our co-investigators (JH) identified a COS for pain, the IMMPACT recommendations , 25. IMMon Table .Table 1PWe did not identify a COS for bipolar disorder through the COMET database, the James Lind Alliance, by contacting colleagues, or from patient and clinician co-investigators.Clinical trial sources described potential benefits (mainly symptom reduction) and hundreds of potential harms (adverse events or “side effects”) of gabapentin for neuropathic pain and quetiapine for bipolar depression. Benefits and harms were measured and reported differently. Both benefits and harms were described in public sources and non-public sources; however, non-public sources included many more potential harms than public sources. We found 19 gabapentin-neuropathic pain trials reporting outcomes in our pre-specified time-windows , and we found 7 quetiapine-bipolar depression trials; two gabapentin trials were less than 4 weeks duration and we did not extract their outcomes following our pre-specified protocol.Outcome domains examined for potential benefits in each case study are listed in Table Prescribing information for FDA-approved indications included some outcomes from clinical trials submitted to FDA for those indications, including “pain intensity” for trials of gabapentin and “depression” for trials of quetiapine. There was little information about potential benefits related to outcomes other than the labeled indications, and prescribing information did not include any potential benefits that we did not also locate in publicly available published reports of the clinical trials. For gabapentin, prescribing information did not include any information about potential benefits noted to be relevant to off-label uses included in our study  .Prescribing information included many potential harms. It was often unclear whether information about harms was applicable to any condition or only specific conditions. Prescribing information included some potential harms that we would not have identified if we had relied only on reports of clinical trials and systematic reviews. For example, prescribing information for quetiapine indicated that it might be associated with increased risk of cataract, information that came from dog studies .As noted in the “Using PatientsLikeMe, we did not identify potential benefits related to our case studies other than the patient ratings of “effectiveness” for the conditions “bipolar disorder” and “neuropathic pain.” We were able to identify potential harms and the possible frequency of harms, but because the importance of each harm was not recorded, we did use PatientsLikeMe to prioritize specific harms associated with gabapentin or quetiapine. For example, some rare events, such as serious AEs, may be more important than common events.all harms that could be associated with treatment, including those that they would not have identified themselves .Patient and clinician co-investigators confirmed that the outcome domains we identified using the sources of information described in the “The two patient co-investigators in our study agreed to rate potential outcome domains by relative importance using a survey we developed for part 1. The two patients categorized many potential beneficial and harmful outcomes as “definitely analyze” . From the analysis of potential benefits, we also excluded 101 participants who did not rank all of the six specific benefits and the item “side effects.” From the analysis of potential harms, we excluded 110 participants who did not rank all seven potential harms. Thus, 388/746 (52%) participants were included in the analysis  completed some or all of the survey. From all analyses, we excluded participants who did not provide demographic characteristics or rate their present pain intensity ; “improvement in your ability to do normal activities” and “changes in the overall quality of your life” were ranked second and third most important, respectively  named other potential benefits they would want to know about before starting treatment  named other potential harms they would want to know about before starting treatment  with potential harm . Most of the 383 respondents to this question said that symptom reduction was more important when deciding whether to use a treatment ; a minority said that side effects were more important  or that symptom reduction and side effects were equally important .Comparing the (1) COS and (2) the ratings of patient co-investigators (part 1) with (3) results of the patient survey (part 2), we found that all three groups said pain intensity, physical functioning, emotional function, and adverse events were important to people with pain. The COS for pain included little information about specific harms; by contrast, patient and clinician co-investigators, and participants in the patient survey, identified many specific harms. We found that part 2 survey participants named benefits not identified using other sources, such as “able to socialize better,” “added energy,” and “to eventually stop meds––in other words, a medication that will cure!”We found that various methods and sources were useful for identifying PCOs for CER, but the list of PCOs we identified was long. When we attempted to reduce the number of items on the list, we encountered challenges in prioritizing outcomes. In part 1, we identified PCOs associated with gabapentin for neuropathic pain and quetiapine for bipolar depression. Patient co-investigators said that we should include almost all of the outcomes we identified, even those not traditionally viewed as patient-centered . We considered conducting a systematic search of existing patient engagement research, but we found this was not feasible for our study. Thus, we undertook part 2 to prioritize the PCOs we would examine in MUDS. Predictably, by surveying many patients, we identified a range of views about the relative importance of potential benefits and potential harms, including potential benefits and harms that our patient and stakeholder co-investigators did not identify.We found that the possible benefits of treatment could be captured in a short list, so it was relatively straightforward for patients to prioritize them. However, our patient co-investigators found it challenging to prioritize potential harms because hundreds of potential harms were associated with each drug. In addition, many patients reported that they considered all potential harms important. We are not aware of any evidence-based methods for prioritizing such a large number of PCOs. Moreover, as patient co-investigators pointed out, harms differ in type  as well as degree  and duration ; considering these differences would lead to an even longer long list.Our findings highlight several challenges for researchers. For example, we thought it would be logical to review PCO research before undertaking a new study, yet we found that a systematic search would have required more resources than we could commit to this task. Unless common terms are used to index studies about patient preferences , PCO research may remain difficult to locate and use for clinical research. Furthermore, we know that clinical trials and systematic reviews should assess core outcomes where possible, but we found a COS for only one of two conditions: few patients were involved in that COS, and the COS included little information about potential harms. The challenges we faced when identifying and prioritizing harms are especially important because 40% of patients surveyed in part 2 believed potential harms were as important or more important than potential benefits. Given the differences between prioritizing potential benefits and harms, these two types of outcomes might be included in separate COSs.Using any or all the methods we used to identify PCOs could be impractical for other studies, so it is important to identify which methods are most informative and most efficient. We found our part 2 survey most useful for prioritizing outcomes, but this was possible because one of our patient co-investigators led the patient group we surveyed. Reliable COSs would limit the need for each individual trial or review to identify PCOs . In the This study has several limitations. First, the availability of sources of evidence  might differ for other health conditions and interventions. Second, sources other than those we examined might include useful information that we did not assess. For example, we examined the website PatientsLikeMe, which is a for-profit business that receives funding from industry; websites dedicated specifically to neuropathic pain or bipolar disorder might have included more detailed information. Third, because we surveyed members of the TMJ Association in part 2 and because temporomandibular joint and muscle disorders are not neuropathic conditions, patients included in the survey were from a different population than the patients in the clinical trials we included in the MUDS study. Fourth, we compared harms that differ in type; we did not consider the effects of differences in the severity and duration of these harms. Fifth, the outcomes we asked patients to prioritize in part 2 were limited to those selected by the investigators from the list we compiled in part 1; BWS, like other stated preference methods, allowed limited opportunities for patients to add outcomes. Our patient co-investigators and survey participants stated that all potential harms were important to them; if stated preference methods depend on researchers to identify potential PCOs, and if researchers do not ask about all possible outcomes, then stated preference methods could overlook PCOs that are important to patients. Further research is needed to compare BWS with other methods for prioritizing PCOs and to evaluate the generalizability of our results.Using several sources of information, we identified many potential benefits and harms of interest to patients taking gabapentin for neuropathic pain and quetiapine for bipolar depression. Including patients as co-investigators was valuable because patients identified important outcomes and provided ongoing feedback about the conduct of the study. While the methods used in part 1 were useful for identifying outcomes, they were less useful for prioritizing outcomes, and patient co-investigators were apprehensive about prioritizing outcomes for a large population of patients. By engaging a patient group through an exploratory survey, we were able to prioritize potential beneficial outcomes for gabapentin-neuropathic pain. Prioritizing potential harms was more complicated. Increasing the availability and use of COSs, preferably those that include meaningful patient engagement, could be an important strategy for improving the patient-centeredness of CER,  especialAdditional file 1:Search strategies. (PDF 2549 kb)Additional file 2:Patient and clinician co-investigator survey. (PDF 597 kb)Additional file 3:Part 2 Survey. (PDF 905 kb)Additional file 4:Patient co-investigator responses. (PDF 2693 kb)Additional file 5:TMJ survey participant characteristics. (PDF 1546 kb)Additional file 6:All “other” potential benefits and harms reported by survey participants in open-ended questions. (PDF 3420 kb)Additional file 7:Baseline characteristics of survey participants included in the final analysis by the reported month of birth. (PDF 1966 kb)


Neurofilaments are the major cytoskeletal components of neurons, and cell injury leads to their release into the surrounding area. The aim of this study was to compare the cerebrospinal fluid (CSF) and serum (S) concentrations of neurofilament light chains (NFLs) and phosphorylated neurofilament heavy chains (pNFHs).Neurofilament concentrations were measured in CSF and S samples from 172 patients using three enzyme‐linked immunosorbent assays. Excel, Stata version 13, MedCal version 17.9.7., and NCSS 2007 software were used for the statistical analysis.rs = 0.748; n = 89; P < 0.001), but Passing‐Bablok regression showed systematic deviation between the values obtained using the two assays. This indicates that the assays were not interchangeable. CSF pNFH and S pNFH concentrations showed low correlation. The kappa statistic showed moderate conformity between CSF pNFH and CSF NFL concentrations (κ = 0.556).There was a statistically significant correlation between the concentrations of CSF NFL and CSF pNFH (The CSF NFL and CSF pNFH assays gave clinically consistent results that reflected the degree of axonal damage, independent of any particular neurological diagnosis. The S pNFH assays had a lower predictive value due to the low correlation coefficient and the kappa index of the CSF pNFH method. NFs are selectively expressed in the nervous system and are found at the highest levels in long projection axons. They are composed of four subunits, namely NF light (NFL), NF medium (NFM), and NF heavy (NFH) chain subunits plus an unstable alpha‐internexin subunit. These subunits have different molecular weights and functional properties. The NFL gene is on chromosome 8p21, and the NFL protein, which has a molecular mass of 61.5 kDa, consists of 543 amino acids. The NFM gene is also on chromosome 8p21; it consists of 916 amino acids and is important for radial axonal growth. The NFH gene is on chromosome 22q12.2, and the protein, which has a molecular mass of 112.5 kDa, consists of 1020 amino acids. NFH is important for protein‐protein interactions, which are regulated locally in the axon by phosphorylation.Enzyme‐linked immunosorbent assays (ELISAs) or more sensitive techniques, such as electrochemiluminescence immunoassays and single molecule arrays (SIMOAs), can be used to determine NF levels.Natalizumab‐treated patients show a 3‐fold decrease in NFL, indicating that this treatment not only has an immunomodulatory effect but may also reduce axonal damage.The aims of the study were to compare the cerebrospinal fluid (CSF) concentrations of NFL and pNFH and the CSF and S concentrations of pNFH and to evaluate the correlation of these parameters with the following diagnoses: MS; CIS; inflammatory diseases of the peripheral nervous system (IDPNS); and other inflammatory central nervous system diseases (OIND), noninflammatory neurological diseases (NIND), and no evidence of organic nervous system disease .22.1We examined a total of 172 patients from the Moravian‐Silesian region of the Czech Republic who had CSF and S samples sent for analysis to the Institute of Laboratory Diagnostics, Department of Clinical Biochemistry, University Hospital Ostrava. The study was approved by the Ethics Committee of the University Hospital Ostrava, Czech Republic, and was conducted in accordance with the ethical standards of the Helsinki Declaration of 1975 as revised in 2000. The average age of the entire group of subjects was 47.0 ± 16.44 years. The group consisted of 113 women (65.7%) with an average age of 46.4 ± 16.30 years and 59 men (34.3%) with an average age of 48.2 ± 16.64 years. The files of patients from the University Hospital Ostrava with available clinical data (n = 101) were further subdivided into diagnosis groups: MS , CIS , OIND , IDPNS , NIND , and Control . For diagnosis of multiple sclerosis, we used the 2017 Revisions of the McDonald Criteria.2.2g for 10 minutes at room temperature, and the S samples were centrifuged at 2500 × g for 6 minutes at 4°C. Both the CSF and S samples were aliquoted into at least three vials  and stored at −70°C until the analysis.Neurofilament light, pNFH, and albumin concentrations were determined in CSF samples that were collected into a polypropylene tube (Sarstedt) and in S samples that were collected into a Serum Gel with Clotting Activator tube (Sarstedt). S and CSF samples were drawn on the same day. The CSF samples were centrifuged at 390 × 2.3® [Neurofilament light] ELISA, REF 10‐7001, IVD CE, UmanDiagnostics AB) and pNFH ‐high sensitive ELISA, REF EQ6562‐9601, For Research Use Only, Euroimmun AG) were determined by ELISA. A patient sample was used for precise and reproducible measurement of NFL, as the diagnostic kit did not include a quality control sample; for measuring pNFH, the manufacturer of the diagnostic kit supplied two quality control samples. The kit manufacturers stated that the analytical sensitivity for NFL was 32 ng·L−1, 27 ng·L−1 for pNFH and 6 ng·L−1 for pNFH sensitive (pNFHs). All samples were measured in duplicate, and the mean intra‐assay coefficients of variation for CSF NFL, CSF pNFH, and S pNFH were 1.9%, 3.3%, and 4.2%, respectively.The concentrations of NFL .3First, we partially verified diagnostic kits for NFL and pNFH determination. When we evaluated whether the measurements were precise and reproducible, both diagnostic kits showed variation coefficients that were comparable to the values supplied by the manufacturer Table .A total of 172 patient samples were included in the analysis that evaluated the correlation between NF levels and clinical diagnoses. The analytical characteristics of the studied group are presented in Table rs = 0.748; n = 89; P < 0.001). The regression relationship between these parameters was evaluated using Passing‐Bablok regression . This indicated that at high CSF NFL concentrations, the CSF pNFH concentration was approximately half of the CSF NFL value.There was a statistically significant correlation between CSF NFL and CSF pNFH concentrations  and control  diagnosis groups, and between CSF pNFH and S pNFH in the IDPNS  and NIND diagnosis groups  and between CSF pNFH and S pNFHs in the NIND diagnosis groups  , probably due to the differences in the biological material that was analyzed . The positive value for the concentration of CSF NFL was set at >900 ng·L−1 based on the study of Arrambide et al.−1 can be considered to be significantly elevated and indicative of axonal damage. For CSF pNFH, the positive value was >610 ng·L−1  6 months after lumbar puncture versus the subgroup of patients with EDSS scores of 0 to 2  score of 2.5 or higher  and one to determine the concentrations of pNFH in S and CSF (Neurofilament (pNf‐H) ELISA, Euroimmun). The other ELISA diagnostic kits for S pNFHs determination (Neurofilament (pNf‐H)‐high sensitive ELISA, Euroimmun) are for research use only. All three assays were suitable for precise measurement of NFs. There was a statistically significant correlation between CSF NFL levels and CSF pNFH levels. However, the CSF pNFH assay showed much lower values than the values for CSF NFL. The reason for this difference may be the relative molar ratio of the individual NF subunits, which is approximately 5:2:1 for NFL:NFM:NFH.We demonstrated that the CSF NFL concentrations correlated well with the CSF pNFH concentrations, especially in the Control and in patients with NIND. We found no correlation for the other diagnosis groups, possibly due to the small number of patients, especially in the IDPNS group. The correlation between the S and CSF concentrations of pNFH was lower. The SIMOA method is a suitable alternative for testing these analytes in CSF and S. Kuhle et alr = 0.652).When we evaluated the correlations of these methods with the different diagnoses, the assays showed the best correlations with each other in the IDPNS and NIND groups of patients. Similar results were obtained by De Schaepdryver et al,When we evaluated these assays according to clinical findings, there was moderate conformity between the CSF NFL and pNFH concentrations, but only fair conformity between the CSF pNFH and the S pNFH concentrations. One possible explanation is that NFs are heteropolymers that form aggregates. Thus, precise determination of NFH concentrations by immuno‐based methods can be influenced by several factors: the ability of the aggregate to mask the NFH epitope; the aggregate's decreased solubility; the difference in stability of NFH monomers in solution versus NFH in aggregates; and the ability of the antibody to bind to soluble NFH.We performed one‐way analysis of variance of the individual analytes for all diagnoses and for paired comparisons of patients with MS and CIS versus the control group. A statistically significant difference was found only for the concentration of CSF pNFH in the group that included all diagnoses. This finding was not a surprise, because a number of studies have shown that NFs are markers of axonal damage rather than markers of a specific diagnosis. Increased levels of NF have been observed, for example, in ALS,We studied the correlation of CSF NFL with clinical data as well. The CSF NFL concentration in the MS and CIS diagnosis groups was significantly higher in the subgroup of patients who had EDSS scores of 2.5 or higher 6 months after sampling versus the subgroup of patients with EDSS scores of 0 to 2. These data showed the suitability of using CSF NFL to predict disease severity. Similar results were obtained in the study by Disanto et al,5In this study, we tested three diagnostic kits for the determination of NF concentrations in biological fluids. The NFL ELISA assay had lower sensitivity and was suitable only for CSF analysis, while the pNFH ELISA assay had satisfactory sensitivity and was suitable for S and CSF analysis, the pNFHs only for S analysis. The data showed good correlation and moderate conformity between CSF NFL and CSF pNFH concentrations, indicating that the results can be considered to be consistent. However, the low correlation coefficient and the kappa index found between the S pNFH, even if using a high‐sensitivity ELISA assay and CSF pNFH meant that the S pNFH and S pNFHs assays gave a lower predictive value. When assessing the relationship of NF concentrations and diagnosis, correlations were found between the concentration of CSF NFL and CSF pNFH in the NIND diagnosis group and in the control group of patients, between the CSF and S pNFH in the IDPNS and NIND diagnosis groups, and between the CSF and S pNFHs in the NIND diagnosis groups. The results confirmed that NFs, whether NFLs or pNFHs, represent an etiologically nonspecific indicator of tissue damage and that it is better to determine their levels in CSF than in S.The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.