Highly porous carbon electrodes for supercapacitors with high energy storage performance were prepared by using a new precursor blend of aromatic polyimide (PI) and polyvinylidene fluoride (PVDF). Supercapacitor electrodes were prepared through the electrospinning and thermal treatment of the precursor blends of aromatic PI and PVDF. Microstructures of the carbonized PI/PVDF nanofibers were studied using Raman spectroscopy. Nitrogen adsorption/desorption measurements confirmed their high surface area and porosity, which is critical for supercapacitor performance. Energy storage performance was investigated and carbonized PI/PVDF showed a high specific capacitance of 283 F/g at 10 mV/s (37% higher than that of PI) and an energy density of 11.3 Wh/kg at 0.5 A/g (27% higher than that of PI) with high cycling stability. For energy storage devices such as lithium-ion batteries (LiBs) or supercapacitors, electrode materials are one of the most critical components in determining energy storage performance. For LiBs, high porosity of the electrode materials provides a large surface area for Faradaic reaction and short distances for charge diffusion . It is aCarbon nanofibers (CNFs) have gained considerable attention as promising electrode materials due to their superior electrochemical properties, chemical and thermal stability, and ready synthesis ,4,5. TheTo produce CNFs, polyacrylonitrile (PAN), polybenzimidazole (PBI), aromatic polyimides (PIs), and polyvinylidene fluoride (PVDF) have typically been reported as precursor polymers ,12,13,143–) groups, which can facilitate polymer chain rearrangement during stabilization [3– groups can generate a large number of pores. It was reported that CNFs derived from 6FDA-based PIs exhibit a large surface area and high porosity compared to those derived from PAN or PBI with sacrificial polymers [Due to the energy storage mechanism, the surface area and porosity of electrode materials are some of the most critical factors in improving the number of active sites. Among precursor polymers, hexafluoroisopropylidene diphthalic anhydride (6FDA)-based PIs have a high free volume due to the presence of bulky trifluoromethyl  diphthalic anhydride , 2,3,5,6-tetramethyl-1,3-phenyldiamine , anhydrous dimethyl acetamide (99.8%), triethylamine (99%), and ethylene diamine were purchased from Tokyo Chemical Industry Co., Tokyo, Japan, and acetic anhydride was purchased from Sigma-Aldrich, St. Louis, MO, USA. 1-Methyl-2-pyrrolidinone  obtained from Tokyo Chemical Industry Co. was used as a co-solvent for 6FDA–durene and PVDF . To evaluate the electrochemical properties, coin cells were assembled using CR2032 coin blanks  with glass microfiber filters  as a separator and potassium hydroxide  as an electrolyte.®, NanoNC, Seoul, Korea). There was a significant difference between the molecular weight of the synthesized PI  and the purchased PVDF . Thus, to have appropriate electrospinnability and comparable fiber diameters, the concentration of PI/PVDF and PI solutions were set as 16% and 20%, respectively. The applied voltage was controlled in the range of 9 to 15 kV depending on environmental conditions such as relative humidity in the electrospinning chamber. Electrospun nanofibers were stabilized at 350 °C in a box furnace  for 2 h under air, and carbonized at 800 °C in a tube furnace  for 30 min under nitrogen.6FDA–durene was synthesized according to the literature procedures . The mol−1. The surface morphology of the electrospun and carbonized PI/PVDF nanofibers was observed using scanning electron microscopy (SEM) with an SU8220  after sputter coating with gold. Raman spectroscopy was recorded to study the carbon microstructures of carbonized PI/PVDF using an inVia reflex  with a 780 nm laser. Surface area and porosity of CNFs were obtained using nitrogen adsorption/desorption measurement on an Autosorb-iQ and a Quadrasorb SI . Specific surface area was calculated from nitrogen adsorption isotherms using the Brunauer–Emmett–Teller (BET) equation, and pore size distribution was calculated using density functional theory (DFT) methods.Chemical structures of the electrospun nanofibers were investigated by attenuated total reflectance Fourier transform infrared . Thermal stability was measured using thermogravimetric analysis  by heating up to 800 °C at a heating rate of 10 °C minCsp, F/g) was measured from CV measurements using Equation (1) as follows:m is the mass of both electrodes (g), I the discharge current (A), and ν the scan rate (V/s). Capacitance retention after 5000 cycles under the voltage range of −0.5 to 0.5 V and a scan rate of 100 mV/s was measured to confirm the cycling stability.Supercapacitor performance was investigated through cyclic voltammetry (CV), cycling performance, and galvanostatic charge–discharge using a WBCS3000S . They were measured by assembling symmetric coin cells using two identical CNF electrodes without the use of binders or additives. Glass fiber membrane and 6.0 M potassium hydroxide (KOH) were introduced as a separator and an electrolyte, respectively. The specific capacitance  and power density  were calculated using Equations (2) and (3), respectively, as follows:dI is the discharge current (A), dt the discharge time (s), and V the voltage window (V). Electrochemical impedance spectra (EIS) were measured over the frequency range from 100 kHz to 0.1 Hz using a 0.01 V amplitude on a PEC-L01 .From a galvanostatic charge–discharge test under a voltage range of 1.0 to 0 V and a discharge current density range of 0.5 to 5 A/g, energy density  were blended and then electrospun to produce precursor blend nanofibers. The nanofibrous morphology of electrospun and carbonized PI and PI/PVDF was observed via SEM, as shown in −1 (D-band) and 1590 cm−1 (G-band). It is known that D-band and G-band represent the presence of disordered carbon and oriented graphitic carbon, respectively [ID/IG) was also measured, and PI/PVDF-derived CNFs show a lower ID/IG ratio of 1.7 than that of PI-derived CNFs (1.9), which means that more ordered carbon is formed in PI/PVDF-derived CNFs.Electrospun PI and PI/PVDF were stabilized and carbonized to produce CNFs, and their carbon microstructures were observed by Raman spectroscopy, as shown in ectively . The rel3–) groups [ID/IG ratio. Abeykoon et al. reported the fabrication of CNFs using PBI/PI blends, and CNFs-derived PBI/PI blends contained more ordered carbon than those from PBI only [6FDA-based PIs are considered as an outstanding candidate for CNF precursors due to their high free volume caused by the presence of bulky trifluoromethyl (CF) groups . During ) groups . The addPBI only .3/g of nitrogen at 0.99 of the relative pressure, which is a 55% increase over carbonized PI. It can also be seen that CNFs derived from PI/PVDF present a high SSA of 1559 cm2/g, which is significantly higher than those from PI only (1210 cm2/g), as shown in CNFs were characterized by nitrogen adsorption/desorption analyses as shown in Among energy storage devices, supercapacitors store energy at relatively high rates compared to batteries due to their simple energy storage mechanism of ion adsorption/desorption at the electrode/electrolyte interface . Thus, m6FDA-based PIs are considered to be excellent precursor polymers for highly porous carbon ,11,40. DCoin cells were assembled with free standing carbonized PI and PI/PVDF nanofibers without using additives or binders, and the supercapacitor performances of the CNF electrodes were studied using CV and galvanostatic charge–discharge testing. To optimize the blend ratio, CNFs derived from PI/PVDF with different ratios of 95:5, 90:10 and 85:15 were prepared, and their electrochemical properties were measured.Recently, several 6FDA-based PIs have been reported as precursor polymers of CNF electrodes, showing excellent energy storage performance. Porous CNF electrodes prepared by using 6FDA–durene only without the use of any sacrificial compounds showed a specific capacitance of 139 F/g at 10 mV/s with an ionic liquid electrolyte, which is higher than those from PAN or PBI with the pore-generating materials . CNFs deOne of the great advantages of supercapacitors is their long cycle life, since they store charges physically in the electrochemical double layers formed at the electrode surface without irreversible redox reactions . In FiguGalvanostatic charge–discharge was conducted to study energy storage performance of CNF electrodes. Optimization of a blend ratio of PI and PVDF was also done with a charge–discharge test under a current density of 0.5 A/g and a potential range of 1.0–0 V, as shown in ® blends were good precursor candidates for CNF electrodes, showing superior surface and electrochemical properties [® was partially damaged, while its blends with PBI were successfully converted to carbon. The addition of PBI improved the thermal stability of the blend matrix through hydrogen bonding between PBI and PI. Matrimid®, which is thermally unstable compared to PBI, and was partially decomposed during thermal treatment, which resulted in the creation of pores in the CNF surface and thus improving the surface area and porosity. As a result, the addition of 25% Matrimid® led to 48% and 54% increases in specific capacitance and energy density, respectively. 6FDA-based PI was also used to prepare PBI/PI blends for CNF precursors [Polymer blends have been studied to produce highly porous CNF electrodes with good energy storage performance. Previously, PBI/PI blends were chosen as precursors for supercapacitor electrodes since both PBI and PI were well-known carbon precursor polymers. It was reported that PBI/Matrimidoperties . During ecursors . It was Recently, biopolymers such as cellulose acetate or lignin have gained attention as carbon precursors since they are widely available and are low-cost renewable sources. Blends of PAN and these biopolymers were studied as CNF precursors for high thermal stability and processability ,47. The The electrochemical impedance of the CNF electrodes was measured as shown in The large surface area of the electrode materials leads to short distances for charge diffusion, low resistance, and good cyclic stability in supercapacitors . It can Another approach to improving energy storage performance is to introduce pseudocapacitance, which involves reversible faradaic reactions. For pseudocapacitors, metal oxides or conductive polymers have been investigated as electrode materials. Moreover, metal oxide/carbon composite electrodes are widely studied ,53,54,55Highly porous CNFs were fabricated through the electrospinning and thermal treatment of precursor blends of 6FDA-based PI and PVDF. Thermally stable PI enabled successful conversion to carbon, while PVDF played a role in the creation of pores on CNF surfaces due to its relatively lower thermal stability. Nitrogen adsorption/desorptiinon data confirmed that the addition of PVDF to PI successfully developed a larger pore volume and a wider pore size distribution on the CNF surface. According to CV and galvanostatic charge–discharge testing, the best composition of PI/PVDF (90:10) was selected as a CNF precursor. CNFs derived from PI/PVDF showed a high specific capacitance of 283 F/g at 10 mV/s (37% higher than those from PI) and an energy density of 11.3 Wh/kg at 0.5 A/g (27% higher than those from PI) with high cycling stability, confirming the successful fabrication of highly porous CNF electrodes using a new precursor blend of PI and PVDF. Further improvement of supercapacitor performance was expected by fabricating composite electrodes with these porous CNF electrodes as a carbon matrix and using conductive polymers or metal oxides for pseudocapacitance.


The World Health Organization recommends intravenous amikacin for the treatment of MDR-TB at a dose of 15 mg/kg. However, higher doses are associated with significant toxicity.Patients with MDR-TB treated at our institution receive amikacin at 8–10 mg/kg, with dose adjustment based on therapeutic drug monitoring. We conducted a retrospective cohort study of patients with MDR-TB who received amikacin between 2010 and 2016.Forty-nine patients were included in the study. The median starting dose of amikacin was 8.9 mg/kg , and target therapeutic drug levels were achieved at a median of 12 days . The median duration of amikacin treatment was 7.2 months , and median time to sputum culture conversion was 1 month . Six patients (12.2%) experienced hearing loss based on formal audiometry testing (95% CI 4.6–24.8%); 22.2% had subjective hearing loss (95% CI 11.2–37.1%) and 31.9% subjective tinnitus (95% CI 19.1–47.1%). Ten patients (23%) had a significant rise in serum creatinine (95% CI 11.8–38.6%), but only 5 patients had a GFR < 60 at treatment completion. 84% of patients had a successful treatment outcome (95% CI 84–99%).Low dose amikacin is associated with relatively low rates of aminoglycoside-related adverse events. We hypothesize that low-dose amikacin can be used as a safe and effective treatment for MDR-TB in situations where an adequate regimen cannot be constructed with Group A and B drugs, and where careful monitoring for adverse events is feasible. Despite decades of efforts at eliminating tuberculosis (TB), an estimated 10 million people were infected with TB and over 1.2 million people died of TB in 2018 [Updated WHO guidelines for the treatment of drug-resistant TB have classified amikacin as a Group C drug, in part due to the significant toxicity associated with its use . Previoumax/MIC (maximum concentration to minimum inhibitory concentration) ratio is thought to be the most relevant PK/PD parameter to monitor their efficacy [Van Altena and colleagues reported using low-dose amikacin and kanamycin with drug dosing based on therapeutic drug monitoring, using a target Cmax/MIC ratio of > 20. Their patients received a median dose of 6.5 mg/kg, achieved a mean weighted Cmax/MIC ratio of 31.2 with amikacin, and had lower reported rates of ototoxicity, while still achieving good treatment outcomes [Although data regarding the pharmacokinetic (PK) and pharmacodynamic (PD) parameters in TB are relatively scarce, there is emerging evidence to support the use of lower-dose aminoglycoside therapy in the treatment of MDR-TB. Aminoglycosides are known to show concentration-dependent activity and thus the Cefficacy . Van Altmax/MIC ratio of 25–35. These targets were selected based upon recommended peak serum levels for nontuberculous mycobacterial infections [Prior to the wide-spread use of bedaquiline and availability of all-oral regimens for treatment of MDR-TB, amikacin was a standard component of the MDR-TB regimen. In our TB referral hospital in Toronto, Canada, patients received amikacin at a dose of 8–10 mg/kg, with dose adjustment based on therapeutic drug monitoring using 30-min post-dose levels, targeting a Cfections  . The first culture positive specimen from each patient routinely underwent phenotypic drug susceptibility testing to first line drugs. Specimens demonstrating multi-drug resistance also underwent phenotypic drug susceptibility testing to amikacin and other standard second-line medications at the Public Health Ontario Laboratory. Individual MICs for amikacin were not reported, however the laboratory reported susceptibility or resistance to amikacin using a critical concentration of 1.0 mg/L. Repeat drug susceptibility testing was routinely performed if specimens were still culture positive after 3 months of treatment, or at the request of the treating physician.max/MIC ratios of 25–30 and 30–35, respectively. Follow-up therapeutic drug levels were drawn monthly, and doses adjusted accordingly.Amikacin was administered intravenously via peripheral inserted central catheter (PICC) line daily (7 days/week) for a minimum of 3 months. Thereafter, once sputum culture conversion had been achieved, administration was thrice weekly. Each amikacin dose was delivered over a 30-min period. The distribution phase of amikacin was predicted to be complete at 0.5 h following the end of infusion, as amikacin would be in first order elimination after this time point in accordance with aminoglycoside PK. Therefore, peak concentration levels were taken 30-min post drug infusion and steady state expected after the initial dose, as the half-life of amikacin is expected to be 2.5 h or less , 10. IniWe monitored for ototoxicity using formal audiometry testing. Baseline audiometry was performed following amikacin initiation, and monthly audiometry testing was recommended thereafter for the duration of amikacin therapy. Testing was performed at 250, 500, 1000, 2000, 4000, 8000, 10,000 and 12,000 Hz. Hearing loss was defined as ≥20 dB decline in hearing threshold from baseline in either ear at any frequency . Renal tEnd of treatment outcomes were defined according to the WHO , and incDescriptive statistics were calculated for baseline variables and treatment characteristics, reporting proportions and medians with interquartile range (IQR), as appropriate. For binary treatment outcomes, exact binomial confidence intervals were reported. We performed a priori exploratory analyses to assess whether age, diabetes, or cumulative dose of amikacin were associated with hearing loss using Fisher’s exact tests, and logistic models for categorical and continuous variables, respectively. We assessed whether nephrotoxic drugs were associated with nephrotoxicity using Fisher’s exact test; lastly, we assessed whether diabetes, low body mass index (< 18.5), nephrotoxicity, or hearing loss were associated with treatment success using Fisher’s exact test. Given the low event rate, no multivariable analysis was possible. All analyses were performed using Stata V15 .Patient characteristics are described in Table Treatment details are outlined in Tables Details of adverse events are described in Table 2 at the time of treatment completion. Patients receiving other nephrotoxic medications  were more likely to experience a significant rise in creatinine during amikacin treatment, and less likely to have recovery of renal function by the end of MDR-TB treatment . However, this observation was based on a small number of patients.The median baseline serum creatinine was 64 , and 10 (23%) patients had nephrotoxicity (95% CI 11.8–38.6%) to a median peak creatinine of 90 . 20% of these patients had recovery of serum creatinine by the end of treatment. Much of the described nephrotoxicity was subclinical; only 5 patients had a GFR < 60 ml/min/mTreatment outcomes are detailed in Table Mycobacterium tuberculosis [Prior to 2018, amikacin was considered a Group B drug and therefore widely used in MDR-TB treatment. The introduction of bedaquiline and all-oral regimens for MDR-TB treatment has relegated amikacin to a Group C drug. However, aminoglycosides remain important drugs in MDR-TB treatment, as they exhibit concentration-dependent bactericidal activity against rculosis , 16 and rculosis . Of all max) to minimum inhibitory concentration (MIC) ratio [max/MIC ratio is determined to be the most relevant PK/PD parameter to monitor amikacin efficacy based on the principle of aminoglycoside concentration-dependent killing [max/MIC ratio of at least 10 at the site of infection is necessary to optimize bactericidal activity for amikacin, with MIC ranging from 0.5 to 2 mg/L. [max/MIC ratio. By this method, we targeted and achieved a serum Cmax/MIC of well over 10 (25–30 with daily dosing and 30–35 with thrice weekly dosing), while still delivering lower than conventional doses of amikacin and avoiding unnecessary toxicity. Van Altena et al targeted a serum Cmax/MIC of > 20 with amikacin, and achieved a mean weighted Cmax/MIC of 31.2 [max/MIC ratio, or the ratio needed to achieve a Cmax/MIC of > 10 at the site of infection , remains unknown. Srivastava et al hypothesized that a Cmax/MIC ratio of 67–89 would be necessary to achieve the target Cmax/MIC ratio of 10.3 in the lung based on a hollow-fiber model, but this was based upon a reported serum to bronchial secretion ratio of 0.135 [max/MIC ratio of approximately 34, much closer to the targets we utilized [Extended interval aminoglycoside dosing regimens are used to optimize bactericidal activity by achieving desired peak concentration (CC) ratio . Cmax/MI killing , 5. Expe 2 mg/L. , 18, 19  2 mg/L. . Since t of 31.2 , very si of 31.2 . Therefoutilized . More reOtotoxicity remains one of the most debilitating side effects related to aminoglycoside use. In a similar setting to ours but using standard dose aminoglycosides, ototoxicity based on regular audiogram testing occurred in 18% of patients in the Netherlands  28% in tHair cell injury in the inner ear by aminoglycosides also results in vestibular toxicity, which can manifest as disequilibrium and dizziness , 32, andAminoglycosides are renally excreted and known to cause nephrotoxicity. This is triggered by uptake of drug by renal tubular cells after glomerular filtration, leading to intra-cellular accumulation and subsequent tubular necrosis . Nephrotmax/MIC ratios. Although monthly audiometry was routinely arranged for patients, a median of only 3 audiometry tests were administered despite a median treatment duration of 7 months of amikacin. Although we may have missed some patients with mild hearing loss, we believe that many patients did not attend audiology appointments because they did not have overt concerns with their hearing.Our study has several limitations that require discussion. As this was a consecutive cohort study with no control group, we could not evaluate the treatment effectiveness of low-dose amikacin in comparison to regimens with standard dosing or regimens excluding amikacin entirely. In addition, our sample size was modest to estimate the proportion of patients with sensorineural hearing loss. Due to the fact that our Public Health laboratory does not report individual MICs for amikacin, we used critical concentration to estimate an MIC of 1 for amikacin susceptible isolates, and therefore likely underestimated our achieved CThe treatment outcomes in our patient cohort were similar to those in other high-income settings  and in sAlthough recent guidelines have relegated amikacin to a group C drug, we believe that it remains an important treatment option for MDR-TB in patients with complex resistance patterns or those with toxicity to Group A and B drugs. We demonstrate that low-dose amikacin with dose adjustment based on therapeutic drug monitoring has fairly limited toxicity with good treatment outcomes. Future prospective trials evaluating lower dose aminoglycosides and therapeutic drug level monitoring-guided dosing in MDR-TB would be beneficial.


Cannabis, probably aiming at relieving their symptomatology. However, it is still largely unknown whether and how Cannabis or its psychotomimetic compound Δ9-tetrahydrocannabinol (THC) attenuates the aversive/traumatic memory outcomes. Here, we seek to review and discuss the effects of THC on aversive memory extinction and anxiety in healthy humans and PTSD patients.Posttraumatic stress disorder (PTSD) may stem from the formation of aberrant and enduring aversive memories. Some PTSD patients have recreationally used Medline, PubMed, Cochrane Library, and Central Register for Controlled Trials databases were searched to identify peer-reviewed published studies and randomized controlled trials in humans published in English between 1974 and July 2020, including those using only THC and THC combined with cannabidiol (CBD). The effect size of the experimental intervention under investigation was calculated.At low doses, THC can enhance the extinction rate and reduce anxiety responses. Both effects involve the activation of cannabinoid type-1 receptors in discrete components of the corticolimbic circuitry, which could couterbalance the low “endocannabinoid tonus” reported in PTSD patients. The advantage of associating CBD with THC to attenuate anxiety while minimizing the potential psychotic or anxiogenic effect produced by high doses of THC has been reported. The effects of THC either alone or combined with CBD on aversive memory reconsolidation, however, are still unknown.Current evidence from healthy humans and PTSD patients supports the THC value to suppress anxiety and aversive memory expression without producing significant adverse effects if used in low doses or when associated with CBD. Future studies are guaranteed to address open questions related to their dose ratios, administration routes, pharmacokinetic interactions, sex-dependent differences, and prolonged efficacy. The formation of intense and long-lasting aversive memories after threatening or stressful events affects the individual’s quality of life when it triggers the development of posttraumatic stress disorder PTSD , 2;), wh;, wh2];)Fear extinction is a form of inhibitory learning that suppresses the expression of the original aversive/traumatic memory. As a consequence, individuals express less fear responses. In both laboratory animals and healthy humans, prolonged and repeated exposures to conditioned cues without presenting the aversive stimulus can induce it , 8. PrecUpon recall, a consolidated memory can become labile again and, thus, its content is destabilized and gradually reconsolidated after that, being susceptible to intervention-induced changes during this period. In both laboratory animals and healthy humans, short exposure to conditioned cues can induce memory destabilization and reconsolidation . The neuIn preclinical studies, fear conditioning is a standard procedure for investigating the process of fear memory extinction and reconsolidation. It has a translational value from laboratory animals to healthy humans, and onwards to anxious/PTSD patients. However, the stimuli, the primary outcome measures, and the populations typically used differ between animal and human studies . BesidesA low “endocannabinoid tone” has been reported in PTSD patients. Relative to healthy controls, they present reduced circulating concentrations of anandamide and 2-arachidonoylglycerol (2-AG), and lower hair concentration of palmitoylethanolamide (PEA), oleoylethanolamide (OEA) and stearoylethanolamide (SEA) –42. TheyCannabis use in PTSD patients to relaxation, sleep improvement, attenuation of hyperarousal and anxiety  and [Fear extinction/memory extinction/fear memory/memory reconsolidation or anxiety] for human studies and controlled trials published between 1974 and July 2020 in the English language. The reference lists of articles included and previous systematic reviews were checked for relevant publications. Primary studies presenting data from oral or smoked THC or THC plus CBD effects on fear-related memory or anxiety were identified.Medline, PubMed, Cochrane Library and Central Register for Controlled Trials databases were searched using the keywords and MeSH terms [ΔAbstracts were identified and independently analyzed by two reviewers. Three reviewers independently conducted data extraction and coding disagreements arising were discussed, and consensus coding applied. The following information was extracted for each study: age, gender, the health status of subjects, drug(s) used, doses and its management, protocols adopted, and the main results found. Figure Means, standard errors, and the number of subjects per group (“n”) were collected to calculate the effect size of treatment in the selected studies  between two groups. A d ≥ 0.5 and ≤ 0.8 was considered a medium effect size while a large one happened when d > 0.8 [d values were all presented as positive values, and when the study presented repeated time points of the evaluated parameter, the d expressed in the text was the highest achieved.The effect size of behavioral results was calculated using the formula for Cohen’s  d > 0.8 . When thTable d = 0.81) the extinction process in subjects submitted to a cued fear conditioning, as inferred by a reduced skin conductance response (SCR) during extinction recall 24 h later [d not calculated [Oral administration of 7.5 mg of dronabinol before fear extinction potentiated .d = 0.55) and the retention test on day 1 (d = 0.55), but not on day 7. In the last case, however, the drug-treated group presented a significant increase in the functional coupling among the vmPFC, hippocampus, and dACC, which was associated with lower spontaneous recovery of fear. Moreover, healthy volunteers submitted to a neutral face presentation associated with an aversive sound and treated with 10 mg of dronabinol before extinguishing that association presented a transient reduction in SCR (d = 0.58), but unaltered fear-potentiated startle response [Dronabinol effects on extinction have already been evaluated at a more remote time point. In the study by Hammoud et al., 2019 , subjectresponse .In summary, in four of five studies, dronabinol induced significant changes in extinction-related autonomic or behavioral responses, with medium to large effect sizes . Therefore, this acute pharmacological intervention is associated with clinically relevant effects. There were no sex-dependent differences in dronabinol-induced facilitating effects on aversive memory extinction. However, such action will possibly require periodic associations of drug administration with extinction to be preserved. Noteworthy, the effects of repeated THC/dronabinol administration on aversive/traumatic memory extinction are still unknown. Despite the current knowledge about aversive memory reconsolidation and the well-documented role of the endocannabinoid system in this process in laboratory animals , 76, no d = from 0.84 to 2.52 [No human studies or clinical trials investigating the effects of associating THC with CBD on aversive/traumatic memory extinction or reconsolidation were identified. However, they are foreseen since the combination of THC and CBD presents advantages in comparison with THC/dronabinol alone, such as fewer and less intense adverse effects and greater safety , 78. Acc to 2.52 –83;), 50 to 2.52 , 86. How to 2.52 , 88).Table Cannabis consumption produced no changes in the anxiety state measured by the Hopkins Symptom Checklist Scores [d = 0.82), and amygdala activation when viewing fearful faces [d = from 0.93 to 2.52 [d = 0.63 [d = 1.32 [d = from 0.92 to 1.42 [Oral administration of 2.0 mg of nabilone to healthy subjects with no history of t Scores . In contul faces , 91. THCul faces , 91. Whe to 2.52 –83, 92;)d = 0.63 ;), POMS  to 1.42 , 83;). T to 1.42 , 81. It Cannabis consumption, smoking a cigarette containing 1.8% (~ 15 mg) of THC produced no changes in the anxiety state [d not calculated [Cannabis users and non-users , but the induction of psychotic symptoms was less intense in the former group [Cannabis differed significantly [In healthy subjects with a history of ty state , 95. In lculated , 95, 96;er group . Of noteficantly , 83. Thed not calculated [d not calculated; 100) divided into two phases: the first being open-label with five patients receiving nabilone on an escalating dose regimen starting with 1.0 mg and not exceeding 10 mg, for 28 days; and the second being double-blind, placebo-controlled with 20 patients receiving nabilone 1.0 mg twice a day for 28 days. The adverse effects reported were drowsiness and dry mouth and eyes [In anxious patients, oral administration of 1.0 or 2.0 mg of nabilone attenuated  the anxiety response measured using POMS, an effect accompanied by increased heart rate. At doses of 4.0 and 5.0 mg, nabilone produced orthostatic hypotension without further anxiety reduction . Reducand eyes .d = 1.16) and global state. A recent double-blind study [In an open-label study using PTSD patients , orally nd study  investignd study .In summary, in most of the studies above described, THC/dronabinol or nabilone induced statistically significant effects on anxiety-related responses, with medium to large effect sizes . However, the outcome (anxiolytic or anxiogenic effect) depends on the dose, regimen of treatment, and psychiatric status. When considered, there were no sex-related differences in drug-induced effects on anxiety.Cannabis [d = from 4.33 to 4.59), but not 1:0.5 or 1:0.33 [d = 3.41; THC:CBD dose ratio of 1:2 and d = 1.90 THC:CBD dose ratio of 1:1 [d not calculated [d = 0.58 [d not calculated) than the one containing only THC 3.6%. In contrast, smoking a cigarette with THC 1.8% (~ 15 mg) alone produced no changes in anxiety, but it was increased when the same THC dose was associated with CBD 1.0% [As detailed in Table Cannabis , 95, 96.r 1:0.33 , 93, 95.o of 1:1 ;). FurthIn summary, CBD attenuates or even prevents the anxiogenic action produced by higher THC doses when given in a dose similar to or higher than that of THC. This outcome, however, depends on their absolute quantity and route of administration. Importantly, no study has already evaluated the effect of chronic use of CBD and THC on anxiety. When considered, there were no sex-related differences in THC-induced effects on anxiety, as well as in the potential counteracting CBD action.The facilitating effects of dronabinol (7.5–10 mg) in humans undergoing aversive memory extinction agree with laboratory animal data showing that the activation of CB1 receptors plays a crucial role in fear memory extinction –104. ForClinicalTrials.gov Identifier: NCT03008005; ClinicalTrials.gov Identifier: NCT04080427). The preliminary results indicate that PTSD patients treated with THC (7.5 mg) exhibited better recall of the extinction of a cued aversive memory, an effect accompanied by increased hippocampal activation [Cannabis by individuals with PTSD is associated with worse clinical outcomes [The studies focusing on aversive memory extinction in healthy humans and laboratory animals are compelling. However, future studies are guaranteed to examine the effects of acute and chronic THC/dronabinol administration on the extinction of traumatic memories. Currently, there are two double-blind and placebo-controlled study investigating THC effects on extinction learning in PTSD patients  while lower doses produced either no changes or anxiety reduction. Although the anxiogenic and psychotic effects of THC could have clinic impact , the dose range in which the beneficial and detrimental THC effects predominates is not necessarily the same. Therefore, its therapeutic value depends on the dose. Another aspect to keep in mind is the treatment regimen. Repeated administration of 1.0 or 5.0 mg of THC reduced anxious symptoms in anxious or PTSD patients, but acute treatment produced minor or no effects. In Cannabis users, the effects on anxiety are more variable because of the influence of the previous emotional state of individuals, how long have they used Cannabis, and when in life they started its use. Of note, inhalation is an alternative route to deliver THC. THC bioavailability after oral administration is around 10% [Cannabis users, possibly having some degree of tolerance to the anxiogenic effect of THC. Besides, chronic users of Cannabis present altered anxiety levels relative to non-users [Cannabis users.Regarding the effects of THC, dronabinol or nabilone on anxiety in subjects with no history of ound 10% , and aftound 10% . Based oon-users . Thus, tThe biphasic effects of THC on anxiety have also been shown in laboratory animals. Low doses of THC  did not alter the anxiety response in rodents tested in the elevated plus-maze, but doses between 1.0 and 10 mg/kg produced an anxiogenic-like effect . In anotCannabis use history of participants. In humans, oral pretreatment with CBD 600 mg attenuated the psychotic symptoms induced by THC 10 mg [Associating CBD with THC could be advantageous as CBD can minimize or even counteract some adverse effects of THC, such as the anxiogenic and psychotic effects. Oral administration of THC plus CBD in a dose ratio 1:2 attenuated the THC-associated anxiogenic effect. When using a similar THC:CBD ratio, the THC-associated anxiogenic effect was no longer observed with the use of low, but not high, doses of these drugs, a result that also depended on the previous HC 10 mg . SimilarHC 10 mg . PreclinHC 10 mg , 151. BaThere is evidence suggesting that CBD interferes with the pharmacokinetics of THC. Indeed, CBD can inhibit the hepatic metabolism of THC , 153. AsTHC, dronabinol or nabilone could help with hyperarousal symptoms, insomnia, anxiety, and extinction deficits related to PTSD . Indeed,There is a lack of studies with PTSD patients using THC alone and associated with CBD focusing on aversive memory extinction and reconsolidation. Further, most studies evaluated the acute effects of THC or THC plus CBD. Therefore, it is unknown whether chronic treatment is still advantageous. Besides, some studies do not address potential sex-dependent differences in THC-induced effects, which would provide further information on whether or not it is a potential issue in humans. Animal data have shown the detrimental effects of THC following high doses. Based on that, human studies have selected an appropriate dose range of THC and, thus, neither worsening of PTSD symptoms nor strengthening of aversive memories after the use of THC has been reported. Few studies have investigated the effects of associating THC with CBD in varying dose ratios yet. Altogether, the findings encourage future controlled studies evaluating the effects of low doses of THC to attenuate aversive/traumatic memory expression in PTSD patients.Additional file 1 Supplementary Table 1. Raw data used for calculate the effect sizes of behavioral parameters from studies detailed in Table Additional file 2 Supplementary Table 2. Raw data used for calculate the effect sizes of behavioral and autonomic parameters from studies detailed in Table


Secondary prevention of ischaemic heart disease (IHD) is an important aspect of healthcare. To improve the prognosis of and control risk factors for IHD patients, we created a unique referral system called the Shizuoka IHD patient registry. n = 32), coronary event group (n = 195), and event-free group (n = 1013) without overlapping.  From 2009 to 2013, we enrolled 1240 patients; they participated in follow-up until 2018. The risk factor target values were as follows: low-density-lipoprotein cholesterol, <100 mg/dl; glycated haemoglobin of diabetes patients, <7%; systolic blood pressure, <130 mmHg; and diastolic blood pressure, <80 mmHg . The cumulative incidence rates were 10.8% for all-cause death , 15.7% for coronary events, and 2.6% for major bleeding. Patients were separated into the major bleeding group (P=0.002) and between the coronary event and major bleeding groups (P=0.026); there was no significant difference between the event-free and coronary event groups.  We observed significant differences in age, rate antithrombotic drug use, and mortality. A Kaplan–Meier analysis of all-cause death showed significant differences between the event-free and major bleeding groups ( Major bleeding events were stronger predictors of long-term mortality than coronary events during the long-term follow-up of stable IHD. There are established evidence and guidelines for patients with ischaemic heart disease (IHD) to achieve secondary prevention. Nevertheless, the long-term prognosis of these patients is unknown, and the current evidence is based on trials mainly performed at specialised centres with experienced cardiologists –3. HowevSecondary prevention of IHD is an unobtrusive yet important aspect of healthcare , 5. BecaThis study aimed to introduce our unique audit referral system and present the long-term prognosis for IHD patients undergoing optimal secondary prevention therapy for a decade.The Shizuoka Ischaemic Heart Disease registry was established in 2009 to connect cardiologists in Shizuoka City Hospital with more than 200 general practitioners in Shizuoka City. The registry is for patients who undergo interventions and those who need only optimal medical therapy.To audit the treatment provided by general practitioners, we adopted a circulation-type cooperative form. General practitioners were required to present registered patients at least once per year even if no cardiac events occurred. They were also asked to examine electrocardiographs every 3 months and order blood sampling tests for risk factors every 6 months. We created a simple form for referral to determine whether the patients' risk factors were well controlled . The firData from yearly follow-up visits, including vital signs and physical examination findings, electrocardiograms, chest X-rays, and laboratory tests, were collected by the investigators upon hospital visits. Additional follow-up information, including vital signs, mortality, additional hospitalisations, and the status of antiplatelet therapy, were collected by contacting the patients, their relatives, or referring physicians via a questionnaire or telephone calls (patients only). Data of patients who were lost to follow-up were removed from the follow-up dataset on the last day of the study.The study was conducted in accordance with the Declaration of Helsinki and was approved by an institutional review board. Oral informed consent was obtained from the enrolled patients.In 2015, the registry was expanded to a multicentre study involving three core hospitals in Shizuoka city. However, for this study, we used the data extracted from only our institute.We followed up a total of 1240 patients  who were divided into three groups based on major bleeding events, coronary events, and absence of any events. The median follow-up interval was 2106 days .Death was defined as cardiac in origin unless obvious noncardiac causes could be identified. Any death during the index hospitalisation was regarded as cardiac death. Angina pectoris (AP) and myocardial infarction (MI) were evaluated according to the latest guidelines of the Japanese Circulation Society based on the universal definition . CoronarBleeding events were recorded when the patient required discontinuation of antiplatelet drugs, hospitalisation, or blood transfusions. Major and minor bleeding were defined by the thrombolysis in MI bleeding criteria .The recommended antiplatelet regimen after PCI was aspirin (100 mg daily) indefinitely and thienopyridine (200 mg ticlopidine or 75 mg clopidogrel daily or 3.75 mg prasugrel daily) for at least 1 year. The duration of dual antiplatelet therapy (DAPT) was based on the discretion of attending cardiologist at the hospital. We set a target value that adheres to the latest Japanese guidelines for the secondary prevention of IHD . Statinst-tests for continuous variables and chi-squared tests for categorical variables. A multivariate analysis was performed using binary logistic regression when the univariate analysis showed statistically significant differences. To compare all three groups, a nonparametric analysis was performed because equal variance could not be assumed. Cumulative incidence rates were calculated using the Kaplan–Meier method.Categorical variables were expressed as numbers and percentages. Continuous variables were expressed as means ± standard deviation unless otherwise indicated. The characteristics of patients across two groups were compared using P-values were two-sided, and P < 0.05 was considered statistically significant. Significant predictors of clinical events were presented with odds ratios (OR) and 95% confidence intervals (CI).All statistical analyses were performed using SPSS Statistics 21 . All reported Among the 1286 patients who were registered from 2009 to 2013, 925 attended consultations at the hospital based on their predetermined medical examinations. Data regarding clinical events were available for 315 nonconsulted patients from the primary care physician or through telephone interviews. Forty-six patients were lost to follow-up. Therefore, we followed up 1240 patients, all of whom survived with a confirmed prognosis until 2018. The mean age was 77.3 ± 10.6 years and 76.3% were men. Patients with MI accounted for 39.6%, and 33% had diabetes at registration. The DAPT rate was 52.3%. The statin prescription rate was 75.7%. Patients were prescribed the following: mineralocorticoid receptor antagonists, 60.9%; beta-blockers, 24.1%; and calcium channel blockers (CCB), 55%. Risk factor control at registration showed the following results: SBP, 126.6 ± 17.5 mmHg; DBP, 69.5 ± 12.6 mmHg; LDL-C, 93 ± 28.5 mg/dL; and HbA1c in diabetes patients, 6.7 ± 1.0%. Baseline characteristics of the patients are presented in The proportion of patients diagnosed with diabetes at the latest follow-up increased to 36.1%, whereas that of patients who required antihypertensive drugs and statins increased to the 90% range. The DAPT rate decreased to 52.3%. Oral anticoagulation (OAC) therapy was administered to 11.7% of patients. The results of changes in morbidity and medications are shown in P < 0.001). HbA1c control in diabetes patients significantly worsened . Moreover, there was a significant increase in both SBP  and DBP .The average LDL-C level significantly decreased from baseline to follow-up . Cardiac death occurred in 1.5% of patients (n = 18) and MACCE in 19.8% (n = 246). The proportions of patients according to the classification of MACCE, except cardiac death, were as follows: MI, 1.6% (n = 20); AP with hospitalisation, 4.6% (n = 57); any lesion revascularisation, 4.5% (n = 56); target lesion failure (TLF), 12.9% (n = 160); CHF, 3.0% (n = 37); ventricular arrhythmia, 1.2% (n = 15); and cerebral infarction, 1.1% (n = 13) (n = 195).Over the course of the 5- to 10-year follow-up period, the cumulative incidence of all-cause death was 10.8% ((n = 13) . We defin = 32), intracranial haemorrhage occurred in 0.9% (n = 11), gastrointestinal bleeding occurred in 2.0% (n = 19), and other complications occurred in 0.2% (n = 2) during follow-up (Major bleeding events occurred in 3.1% of patients (ollow-up . There wn = 34), followed by infection, including pneumonia and sepsis , cardiac death , and major bleeding . Despite every effort, the cause of death could not be identified for 28.9% of patients. Classifications of all causes of death are shown in The most common cause of death was malignancy . The incidence rates of MI , ventricular arrhythmia , and intracranial haemorrhage  were higher in the all-cause death group than in the event-free group.Age at baseline was significantly older in the all-cause death group than in the event-free group , MI , ventricular arrhythmia , and intracranial haemorrhage  .P < 0.001; ventricular arrhythmia, P=0.011; and intracranial haemorrhage, P < 0.001) , antithrombotic drug use (P < 0.001), and mortality (P=0.017). The Kaplan–Meier analysis of all-cause death showed a significant difference between the event-free group and the major bleeding group (log-rank P=0.002) and between the coronary event group and the major bleeding group (P=0.026); however, no significant difference existed between the event-free group and the coronary event group  was used to evaluate the group differences. The analysis showed significant differences among these groups in age . The major bleeding group had a significantly lower rate of single antiplatelet therapy (SAPT) than the event-free group  despite having a significantly higher rate of combined SAPT and OAC use than the event-free group   .To the best of our knowledge, this is the first study to reveal the long-term prognosis of IHD patients in the real world using a unique audit referral system. Using this method, general practitioners could follow up these patients in the same manner as cardiologists. Our results suggest that we could audit at least four times as many patients using this approach than with the conventional outpatient system at our hospital. The number of physicians per 1000 people in the Shizuoka Prefecture is 1.9, which is the fourth smallest ratio in Japan. Therefore, especially in these circumstances, the referral system may contribute significantly to patient healthcare.The development of interventional cardiology has been remarkable. Despite the advent of new devices and methods, objective end point rates have not notably improved for several years. Nevertheless, in the meta-analysis of more versus less intensive statin therapy, the all-cause mortality was reduced by 10% per 1.0 mmol/L decrease in LDL, largely reflecting significant reduction in deaths due to coronary heart diseases and other cardiac causes . AlthougRecent outcomes of secondary prevention after an IHD event with conventional optimal medical therapy were reported by several international, multicentre, prospective, randomised studies. In the FOURIER study, the cumulative incidence of cardiac death in the control group was 1.7% and that of MI was 4.6% within a 2.2-year follow-up period . After 6P=0.015), whereas those with unplanned revascularisation did not have a significantly higher risk [We compared the coronary event, major bleeding, and event-free groups. Unexpectedly, neither the nonparametric analysis nor the Kaplan–Meier analysis showed significant differences between the coronary event group and the event-free group. A subanalysis of mortality with or without MI before and after registration also showed no significant difference. According to the KiCS PCI registry (2009–2011), the incidence of acute coronary syndrome (ACS) was 6.4%, the unplanned revascularisation rate was 5.9%, and the all-cause mortality rate was 3.9%. In the propensity matched cohorts, patients with a subsequent admission for ACS had an increased risk of mortality . In addition, no significant difference was found in the incidence of stroke between the treatment arms (P=0.23). Similarly, no significant difference was observed in the incidence of stent thrombosis between the treatment arms (P=0.08) [In the current study, major bleeding events were more important factors than coronary events. Intracranial haemorrhage directly caused death in many cases. Other major bleeding events always led to the cessation of antiplatelet therapy, either temporarily or permanently. Discontinuation of antiplatelet therapy possibly caused new cardiovascular events, including stent thrombosis. Secondary events worsened the survival rate of the major bleeding group. Coincidentally, the major bleeding group and coronary event group had no overlap of patients in our study. Mortality was estimated to occur more frequently in the major bleeding group than in the coronary event group. The DAPT score is a novel decision tool that was recently developed to identify those who are more likely to benefit from long-term therapy among patients eligible for long-term DAPT . In the (P=0.08) . Indeed,(P=0.08) . Hence, There were some limitations to this study. One major limitation involved patient selection. Hospital cardiologists selected and registered patients who were asked to comply with the referral system. The cardiologists tended to follow up patients who were at “high risk” with low ejection fraction (EF); patients with recurrent cardiovascular disease, including those who were postcardiovascular surgery and/or device implantation, and patients with poor adherence and/or poor risk factor control, especially those requiring follow-up for endocrine and metabolic conditions. This study was a prospective, nonrandomised trial. Generally, echocardiographic findings, especially EF, are significant predictors of the prognosis for IHD patients. We did not have adequate resources to perform yearly echocardiography examinations for every referred patient. Our referral system only recommends better medication to the practitioners; there is no enforcement mechanism. The preference of treatment varies based on the practitioners and cardiologists, and treatment strategies are thought to be inhomogeneous. Nevertheless, this diversity is the essence of this research.We continue to enrol and follow up patients in the referral system. Propensity score matching may be considered for registered patients and patients controlled by hospital visits as a method of evaluating and confirming the usefulness of this system.This unique referral system for secondary prevention of cardiovascular disease followed by general practitioners in Shizuoka city showed a good risk factor control and a moderate event rate compared to the previous major studies conducted by cardiologists only. In this study, MI, ventricular arrhythmia, and intracranial haemorrhage after registration were significant predictors of all-cause death. Furthermore, major bleeding events were stronger predictors than coronary events.


Our results demonstrate that the presence and number of CLs are robustly associated with cognitive dysfunction in Asian MS patients.Cortical lesions (CLs) have a low prevalence and are associated with physical disabilities in Japanese patients with multiple sclerosis (MS). However, the contribution of CLs to cognitive impairment remains unclear in Asian MS. Sixty-one prospectively enrolled MS patients underwent three-dimensional double inversion recovery MR imaging, the Brief Repeatable Battery of Neuropsychological Tests (BRB-N), the Apathy Scale (AS), the Fatigue Questionnaire (FQ), and the Hospital Anxiety and Depression Scale (HADS) within a 1-week period. The cognitive impairment index (CII) score was calculated to measure patients’ overall cognitive impairment. MS patients with CLs had poorer scores than those without CLs in most BRB-N tests, but scored comparably in the FQ, AS, and HADS. The number of CLs correlated negatively with all BRB-N test scores and positively with total CII scores. Leukocortical lesions were more extensively associated with cognitive dysfunction in various domains than intracortical lesions. Stepwise multiple regression analysis revealed that potential confounding factors for the highest quartile of CII score were the number of CLs (odds ratio 2.38,  In MS, information processing speed, visual memory, visuospatial perception, and working memory are particularly impaired from the early phase of the disease1. No large-scale epidemiological studies have investigated the prevalence of cognitive impairment among Japanese patients with MS. However, in a small case series, attention deficits characterized by slow automatic information processing, a very early sign of cognitive impairment in MS, were found in 14 of 22 (66.7%) patients5. Several studies have also reported significantly lower scores in the cognition test batteries, including the Brief Repeatable Battery of Neuropsychological Tests (BRB-N) used in the present study, among Japanese MS patients compared with healthy controls9.Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system that affects not only motor, but also cognitive and psychiatric functions. Cognitive impairment has been reported in 43–70% of MS patients10. In Caucasians, there are several lines of evidence demonstrating that CLs contribute to cognitive dysfunction in patients with MS15. Cognitively impaired relapsing-remitting MS (RRMS) patients have higher numbers and greater volumes of intracortical lesions (ICLs) compared with cognitively normal MS patients11. Prospective studies have shown that CLs accumulate over time in association with cognitive decline13. One study reported that leukocortical lesions (LCLs) affecting both grey matter (GM) and white matter (WM) make a greater contribution to cognitive impairment than ICLs in MS13. Recent 7-tesla MRI studies have reported that LCLs are more extensively associated with various domains of cognitive impairment than ICLs15.The advent of new magnetic resonance imaging (MR) sequences, such as double inversion recovery (DIR) and phase-sensitive inversion recovery, have facilitated the identification of cortical lesions (CLs) in patients with MS, even in the early phase of the disease17, who present features distinct from those of European patients with MS19. We recently reported that CLs were found at a relatively low frequency (29.7%) on DIR imaging of 3-tesla MRI among Japanese patients with RRMS16 compared with that among Caucasian patients with RRMS (≥ 60%)23. Moreover, the number of CLs was significantly associated with motor disability in Japanese patients16. Furthermore, because two human leukocyte antigen (HLA) risk alleles, HLA–DRB1*15:01 and HLA–DRB1*04:05, are prevalent in Japanese MS patients24, we also studied the influence of these HLA alleles on CLs and found that HLA–DRB1*15:01 increases CLs, whereas HLA–DRB1*04:05 decreases CLs16. This finding is consistent with the result from a previous pathological study, which found that HLA–DRB1*15:01 was associated with greater cortical inflammation in autopsied MS materials25, suggesting an involvement of HLA class II molecules in CL development27. Another small-scale study of Japanese MS patients that assessed cognitive function reported that the attention of 13 CL-positive patients was significantly worse than that of six CL-negative patients; however, the study did not classify CLs into ICLs and LCLs and did not evaluate the relationship between CL quantity and cognitive function17.Despite the serious impact of cognitive decline on the quality of life of patients with MS, only a few studies have investigated cognitive impairment and CLs in Asian patients with MSHLA–DRB1 genotype and cognitive impairment in Japanese patients with MS.Therefore, it is possible that despite the lower quantity of CLs in Japanese MS patients compared with Caucasian MS patients, CLs may have similar effects on cognitive function in Japanese and Caucasian MS patients. The effects of different CL subtypes, particularly ICLs and LCLs, on cognitive dysfunction remains to be elucidated in Asian patients, including Japanese patients. Therefore, we aimed to elucidate the effects of CLs on cognitive functions by using a comprehensive, prospective assessment of these functions, combined with DIR neuroimaging. In particular, our focus was the differential effects of ICLs and LCLs on cognition and the association between the The participants consisted of 61 MS patients in the remission phase (42 RRMS and 19 secondary progressive MS [SPMS]) , SPART (p < 0.0001), SPART-D (p = 0.0169), SDMT (p < 0.0001), PASAT-3 (p < 0.0001), PASAT-2 (p < 0.0001), and the WLG test (p < 0.0001).We first compared our MS patients with age- and sex-matched healthy controls previously subjected to BRB-N who were included in our previous studyp = 0.0201), SPART (p = 0.0335), SPART-D (p = 0.0082), SDMT (p = 0.0080), PASAT-3 (p = 0.0033), and PASAT-2 (p = 0.0042). Similarly, CII scores were significantly poorer in MS patients with CLs than in those without CLs (p = 0.0019). However, there were no significant differences in AS, FQ, HADS-A, or HADS-D scores between MS patients with and without CLs.Japanese MS patients with CLs had significantly lower scores than those without CLs in the following tests Table : SRT-LTSp = 0.0043), SPART-D (p = 0.0325), PASAT-3 (p = 0.0117), PASAT-2 (p = 0.0075), and WLG test (p = 0.0126) and higher CII scores (p = 0.0065) than those without ICLs (Table p = 0.0262), SPART (p = 0.0179), SPART-D (p = 0.0072), SDMT (p = 0.0077), PASAT-3 (p = 0.0020), and PASAT-2 (p = 0.0084) and higher CII scores (p = 0.0033) than those without LCLs (Table p = 0.0315) and WLG scores  than those with LCLs but lacking ICLs , whereas FQ scores were correlated with age at examination and with the EDSS score . HADS-A and HADS-D scores were weakly associated with age at examination . The associations of CII scores with EDSS scores and with the number of CLs were the most robust among all the comparisons   Fig. .Figure 1HLA–DRB1*15:01 or HLA–DRB1*04:05 alleles .Univariate and multivariate regression analyses were performed to elucidate the factors contributing to poor cognitive function in Japanese MS patients Table . Univari1. The lower SRT-LTS and WLG scores suggest impairment of verbal memory and verbal fluency in our MS patients, which is observed in around half of Caucasian MS patients1. In the present study, WLG score did not correlate with age. We believe this lack of correlation in part resulted from the age distribution of this cohort, which included patients from 16 to 72 years of age and lacked very elderly participants. Similarly, Niino et al. reported that WLG scores did not correlate with age in either 184 MS patients or in 163 healthy controls with a similar age distribution6. However, WLG negatively correlated with disease duration, EDSS score, and the number of CLs, ICLs, and LCLs, suggesting that verbal fluency was affected more profoundly by the disease course and disability progression of MS as well as the presence of CLs than by aging. These observations suggest the patterns of cognitive impairment in Japanese patients with MS are largely similar to those in Caucasian patients.In the present study, SDMT, PASAT-2, and PASAT-3 scores were significantly lower in our MS patients compared with controls, indicating impairments of attention, information processing, and working memory. Additionally, lower SPART and SPART-D scores indicated visual memory impairment in Japanese MS patients, an aspect of cognition that is also commonly affected even during the early phase of the disease in Caucasian MS patients13 and with an earlier study of Japanese MS patients, which reported an association between the presence of CLs and cognitive dysfunction17. The presence and number of CLs were strongly associated with the overall CII score in our study, which is also consistent with a previous study in Caucasian MS patients21. The results of multivariate regression analysis also indicate that both the number of CLs and the EDSS score are independent predictors of the most severe cognitive decline in Japanese MS patients. The presence and the number of CLs could therefore be relevant risk factors for cognitive impairment in Japanese patients with MS, even though Japanese patients have CLs less frequently than Caucasian patients.Although the prevalence and the number of CLs, ICLs, and LCLs were relatively low in our subjects compared with Caucasian MS patients, we found that the number of CLs in Japanese MS patients was significantly associated with all of the BRB-N tests. This result is in accord with previous studies of Caucasian MS patients21. Therefore, CLs are more strongly related to the cognitive subdomains that are preferentially affected in the early phase of the disease1, which further suggests that CLs are an important risk factor for early cognitive impairment in MS.As for the cognitive subdomains assessed, our study revealed that in Japanese MS patients, the presence and number of CLs were more significantly associated with scores in the SDMT, PASAT-3, and SPART-D than with SRT-LTS scores. This finding suggests that CLs contribute more to impairments of attention, information processing, and working and visual memory than verbal memory. A study of Caucasian MS patients also reported a more robust association of CL numbers with PASAT and SDMT scores than with SRT-LTS scores28. However, type III subpial lesions are only detectable by 7-tesla high-resolution MRI, not conventional 3-tesla MRI14. LCLs were reported to affect total cognitive function more severely than ICLs, based on 3-tesla MRI of Caucasian MS patients13. A recent study using 7-tesla MRI demonstrated that type I CLs (LCLs) were most frequently associated with impairments in various domains of cognitive function, such as visuospatial, learning/memory, processing speed, and semantic language domains, while type III and IV CLs (ICLs) were associated with poor performance in learning/memory and semantic language domains14. In the present study, we found that LCLs in patients were more extensively associated with various domains of neuropsychological assessments than ICLs, although the effects on overall CII scores were comparable. However, ICLs, including in patients who had both ICLs and LCLs, were more significantly associated with lower scores in the SPART and WLG test than LCLs. This finding suggests that the presence of ICLs may relate to impairments of verbal and visual memory. Given the association of type III and IV CLs, which are included in the ICL category, with impairment of learning/memory and semantic language tests in Caucasian MS patients14, it is possible that ICLs may relate to impairments in somewhat distinct domains of cognitive functions from LCLs. This point should be clarified in future large-scale studies. Overall, although we could not assess type III subpial lesions by 3-tesla MRI, these results are roughly consistent with results from studies of Caucasian MS patients14.Concerning the subtype of CLs, pathologically classified type I lesions correspond to LCLs, while type II–IV lesions correspond to ICLsHLA–DRB1*1501 and HLA–DRB1*04:05 alleles. Although we previously reported that HLA–DRB1*15:01 increased CLs, whereas HLA–DRB1*04:05 decreased CLs16, the MS-susceptible HLA–DRB1 genotypes did not influence the neuropsychological outcome in the present study. CLs contribute to cognitive impairment, but such an impairment could be influenced by much broader damage to brain structures. For example, cognitive dysfunction is also associated with losses of volume in subcortical structures, such as the caudate29 and the thalamus30. Therefore, although the HLA alleles for MS susceptibility can facilitate CL development, they are not so influential as to modulate cognitive decline.In this study, we did not find any significant differences in the neuropsychological status of carriers and non-carriers of the 31 and the difficulties in enrolling patients prospectively to undergo both MRI and neuropsychological studies within a 1-week period in a single institution. Second, because 3-tesla MRI was used, subpial lesions could not be assessed and evaluating which CLs extended into the subcortical WM was occasionally difficult, even though two examiners independently assessed the CLs. Third, the relationship between the location of CLs and cognitive functions was not assessed because of the small number of enrolled patients; this should be investigated in future studies. Fourth, volumetric measurement of WM, GM, and T2 lesions were lacking, because the present study was initially designed to determine whether CLs imaged with 3-tesla 3D-DIR MRI might be an indicator of cognitive impairment in real-world settings in the Japanese population. Thus, such volumetric analysis should be performed in future studies to further evaluate factors contributing to cognitive dysfunction.There are several limitations to the present study. First, the sample size was relatively small, mainly because of the low prevalence of MS in JapanNonetheless, in this study we demonstrated the contribution of CLs to overall cognitive impairment in Japanese MS patients. LCLs were more significantly associated with the impairment of cognitive domains preferentially affected by MS, including attention, information processing, and working and visual memory, while ICLs may contribute more to verbal and visual memory impairments. The presence and number of CLs could be relevant risk factors for cognitive decline in Japanese MS patients.32. All patients were carefully followed-up at a single MS centre in the district, the Department of Neurology at Kyushu University Hospital. Patients were clinically stable with no relapse at least for 1 month prior to MRI (including 3D-DIR imaging) and neuropsychological assessment, which were both performed within a 1-week period. This study was approved by the Ethics Committee of Kyushu University and was conducted according to the World Medical Association Declaration of Helsinki.Sixty-one patients with MS who gave written informed consent prior to their inclusion in this study were prospectively and consecutively enrolled in the period from February 1, 2016, to May 31, 2017 (Table 16: repetition time (TR)/echo time (TE) = 5500/255–284 ms, inversion time (TI) = 450/2550 ms, echo train length (ETL) = 173, matrix size = 208 × 208, and field of view (FOV) = 250 mm × 250 mm. Lesions were identified based on consensus between two examiners, an experienced neuroradiologist (A.H.) and a neurologist (K.S.), with special attention to the recommendations made by Geurts et al.33. CLs were classified into ICLs localized in the cortical GM and LCLs encompassing both GM and WM  with the following sequence parameters, as previously described6, which comprises the following tests: the Selective Reminding Test (SRT) of verbal learning memory; the 10/36-Spatial Recall Test (SPART) of visuospatial memory and learning; the Paced Auditory Serial Addition Test (PASAT) and Symbol Digit Modalities Test (SDMT) of attention, information processing, and working memory; and the word list generation (WLG) test of verbal fluency. The battery of tests was performed in the following order: SRT, SPART, SDMT, PASAT, delayed recall of SRT (SRT-D), delayed recall of SPART (SPART-D), and WLG. These tests included the following variants: for the SRT, the long-term storage (SRT-LTS), consistent long-term retrieval (SRT-CLTR), and delayed recall (SRT-D) tests; for the SPART, the immediate recall (SPART) and delayed recall (SPART-D) tests; and for the PASAT, both the 2-s and 3-s versions (PASAT-2 and PASAT-3). Cognitive impairment index (CII) scores were computed by calculating the mean and the SD of the control data6, as previously described34. The results obtained from the controls in our previous study6 were used for comparison with the total MS patient group in this study. Apathy was assessed with the Apathy Scale (AS)35, using a translated version of the scale that has been previously validated in Japanese36. Fatigue was evaluated with the Japanese version of the Fatigue Questionnaire (FQ)37. Depression and anxiety were assessed with the Japanese version of the Hospital Anxiety and Depression Scale (HADS-A and HADS-D)38.Participants underwent the Japanese version of BRB-NHLA–DRB1 alleles were analysed in all patients by using hybridization between the polymerase chain reaction amplification products of the genes and the sequence-specific oligonucleotide probes, as previously described39.The genotypes of the HLA–DRB1*15:01 allele, and presence of the HLA–DRB1*04:05 allele. Given the small sample size, forward stepwise selection was performed to reduce the number of predictors incorporated into the multivariate analysis40. A p-value < 0.05 was considered statistically significant.Statistical analysis was performed with JMP Pro 12.2.0  or GraphPad Prism 7.0d . Fisher’s exact test was used to compare categorical variables, and the Wilcoxon rank-sum test was used to analyse continuous scales. Correlations among continuous scales were calculated by using Spearman’s rank correlation coefficient. To perform the regression analysis, we divided the CII scores of patients into quartiles defined by the following ranges: 0–1, 2–4, 5–9, and ≥10. Univariate and multivariate logistic regression analysis was performed to assess possible factors associated with the lowest quartile of the CII score, including sex, age at examination, disease duration, Kurtzke Expanded Disability Severity Scale (EDSS) score, number of CLs, presence of the Supplementary Information.


An epidemic of chronic kidney disease of unknown cause (CKDu) is occurring in rural communities in tropical regions of low-and middle-income countries in South America and India. Little information is available from Southern African countries which have similar climatic and occupational characteristics to CKDu-endemic countries. We investigated whether CKDu is prevalent in Malawi and identified its potential risk factors in this setting.n = 821) without diabetes, hypertension, and proteinuria. Estimates of glomerular filtration rate (eGFR) were calculated using the CKD-EPI equation. Linear and logistic regression models were applied with potential risk factors, to estimate risk of reduced eGFR.We conducted a cross-sectional study from January–August 2018 collecting bio samples and anthropometric data in two Malawian populations. The sample comprised adults > 18 years  0.1, 0.9); the prevalence of eGFR< 90 was 5% . We observed a higher prevalence in the rural population ), versus urban ). Age and BMI were associated with reduced eGFR< 90 [Odds ratio (OR) (95%CI) =3.59  per ten-year increment]; [OR (95%CI) =2.01  per 5 kg/m2 increment] respectively. No increased risk of eGFR < 90 was observed for rural participants [OR (95%CI) =1.75 ].The mean eGFR was 117.1 ± 16.0 ml/min per 1.73mReduced kidney function consistent with the definition of CKDu is not common in the areas of Malawi sampled, compared to that observed in other tropical or sub-tropical countries in Central America and South Asia. Reduced eGFR< 90 was related to age, BMI, and was more common in rural areas. These findings are important as they contradict some current hypothesis that CKDu is endemic across tropical and sub-tropical countries. This study has enabled standardized comparisons of impaired kidney function between and within tropical/subtropical regions of the world and will help form the basis for further etiological research, surveillance strategies, and the implementation and evaluation of interventions. An epidemic of chronic kidney disease of unknown cause (CKDu) is occurring in rural communities in an increasing number of low-and middle-income countries (LMICs) , 2. The 2 in the absence of traditional risk factors such as diabetes, hypertension, heavy proteinuria, and structural renal disease, based on two measures of serum creatinine 3 months apart , males, n = 286 [90%]), approximately 57% of which were classified as agricultural workers . 56% of the population completed ≥10 years of formal education.There was an 82% employment rate in the population , for higher income, non-regular meat-eaters (those eating meat 0 days per week) with decreasing fat-free mass, and HIV seropositivity. We observed small site and sex-specific differences in mean eGFR; the mean was 117.6(±14.9) for urban participants, and 116.9(±16.5) for rural participants and 116.8 (±16.7), 117.3 (±15.6) for males and females, respectively. Figure The mean eGFR was 117.1 ± 16.0 ml/min per 1.73mThe prevalence of eGFR< 60 in the study population was 0.2% 95%CI 0.1, 0.9), and the prevalence of eGFR< 90 was 5%  = 3.2, 6.3). We observed small differences in reduced eGFR (< 90) prevalence by site, with 3%  in (urban) Area 25, versus 5%  in  Bonje , we also repeated the models minimally adjusted for key risk factors and confounders to test for inflation of relative risk due to sparse data. There was little difference between the findings from the fully adjusted and minimally adjusted models, therefore we report results from the fully adjusted models only. Table We first conducted linear and logistic regression models which included all potential risk factors and confounders; however, due to the small number of participants with an eGFR < 90 , the odds of reduced eGFR< 90 increased with age [OR = 3.59 ] per ten-year increase, and with increasing BMI [OR = 2.01 ] per 5 kg/mAs CKDu had been found to be a predominantly rural disease in areas where it is endemic, we conducted regression analyses to assess site-specific risk factors. We report linear and logistic regression results from fully adjusted models for Bonje . In Bonje, education < 5 years was associated with higher eGFR [4.19 ]. After further stratification by age however, this association was lost, suggesting age was confounding this relationship due to a higher proportion of younger participants in this category having an eGFR> 90 in comparison to the other education-level categories. In logistic regression, increasing BMI was associated with risk of eGFR< 90 (models 1–2). No associations were observed between reduced eGFR and sex or occupational status within either study site.We report the distribution of eGFR in participants without hypertension, diabetes, or heavy proteinuria, and estimate the prevalence and potential risk factors of reduced eGFR in an urban and rural study population.2, suggesting that low eGFR is not generally a health burden in this population. As we observed only two cases of eGFR< 60 we conducted analyses using the cut-off eGFR< 90. Observing so few cases of eGFR< 60 in Malawi was unexpected, as the country has a similar climate (within a 2-4 °C range of endemic regions) and agricultural-led economy to other countries  where the rural population is at high risk  from CKD. Therefore - as is commonly experienced in epidemiological studies - we may have misclassified some AKI as reduced eGFR and overestimated reduced eGFR (< 90) prevalence. There is debate surrounding the diagnostic criteria for CKDu which largely surrounds the exclusion of hypertension and proteinuria, both of which are both causes and consequences of advanced CKD (and CKDu). At early stages of the disease, proteinuria is uncommon in CKDu, and hypertension is not present, by definition; however, both are common in other forms of CKD (including diabetic glomerular disease), and at the population level, most disease is likely to be detected at earlier stages. Currently, the exclusion criteria for hypertension when defining CKDu varies by geographical location, and there is not yet an internationally accepted definition of CKDu . For exaPerhaps most crucially, the CKD-EPI equation has not yet been validated for the African population, which is a major limiting factor in these analyses.The main strengths of the study are the use of a random selection of population-based participants. Furthermore, DEGREE CKDu definitions were used , which aIn conclusion, our findings indicate that in Malawi reduced eGFR consistent with the definition of CKDu is non-existent. eGFR< 90 in the absence of known CKD risk factors was present, but these cases were associated with traditional CKD factors, age, and BMI. Contrary to the current literature that CKDu is a rural disease disproportionately affecting males, we observed a low prevalence of lower eGFR amongst rural males, and no rural-urban or sex-specific associations with lower eGFR or risk of eGFR< 90 were found. These findings suggest that due to the absence of site, occupation, and sex associations, CKD(u) is not present at least to the extent that it has been observed in other tropical or sub-tropical countries in Central America and South Asia.This research is informative to the CKDu research field, as we did not observe the disease in a region which has a similar agricultural-led economy and climate to CKDu-endemic regions. These analyses are also important in informing the geographical distribution of CKDu suggesting that the disease is localized to specific countries, as opposed to the entire tropical or subtropical region.Additional file 1 : Table S1. Sociodemographic and anthropometric characteristics of overall study participants .Additional file 2 : Table S2. Linear and logistic regression models, showing both minimally and fully adjusted models Bonje (n = 578).Additional file 3 : Table S3. Linear regression models, showing both minimally and fully adjusted models, Area 25 (n = 243).Additional file 4 : Table S4. A comparison of socioeconomic and environmental factors between non-endemic Malawi and endemic regions of Central America and South Asia.


RPGR exon ORF15 variants are one of the most frequent causes for inherited retinal disorders (IRDs), in particular retinitis pigmentosa. The low sequence complexity of this mutation hotspot makes it prone to indels and challenging for sequence data analysis. Whole-exome sequencing generally fails to provide adequate coverage in this region. Therefore, complementary methods are needed to avoid false positives as well as negative results. In this study, next-generation sequencing (NGS) was used to sequence long-range PCR amplicons for an IRD cohort of African ancestry. By developing a novel secondary analysis pipeline based on de novo assembly, we were able to avoid the miscalling of variants generated by standard NGS analysis tools. We identified pathogenic variants in 11 patients (13% of the cohort), two of which have not been reported previously. We provide a novel and alternative end-to-end secondary analysis pipeline for targeted NGS of ORF15 that is less prone to false positive and negative variant calls. The inisive ar, 5–50% or sive ar, 5–50% or %) trait .TM, https://sph.uth.edu/retnet/). In addition to the exceptional genetic heterogeneity, phenotypic variability also occurs, complicating molecular analysis even further. However, it can be generalised that xlRP cases display some of the most severe RP phenotypes in males, with early age of onset (childhood to late teen years) and rapid disease progression [RP2 and RPGR, and they account for 10–20% and 50–90% of cases, respectively [OFD1, has been identified in one family with severe non-syndromic xlRP [A genetic diagnosis for IRD patients can confirm a clinical diagnosis, provide a clearer prognosis, form the basis of genetic counselling for families, and allow participation in gene-based therapy trials . Howevergression ,6. To daectively ,6,7,8,9.ad trait. The incorrect assumption of disease inheritance  may cause false negative results.The systematic screening of all RP genes in a cohort of patients using a candidate approach is challenging and costly due to the marked phenotypic and genetic heterogeneity described. Thus, next-generation sequencing (NGS) approaches such as whole exome sequencing (WES) or targeted resequencing are revolutionising molecular diagnostics in heterogeneous Mendelian diseases such as IRD, and specifically RP ,12. StilRPGR gene was identified as causing xlRP in 1996. Four years later, an RPGR transcript with a novel 3′ terminal exon, known as exon open reading frame 15 (ORF15), was identified [RPGR exon ORF15 is difficult to sequence, using conventional Sanger sequencing as well as NGS approaches, due to the highly repetitive sequence exceeding 1 Kb in length [RPGR ORF15, which evade capture-based technologies [One limitation of capture-based NGS is that a critical xlRP mutational hotspot is inadequately captured. The entified . ORF15 wentified ,23. ORF1entified . Addition length . Huang enologies .n = 285) [RPGR exon ORF15 in those four probands identified two mutations in three families: c.2790_2791delGG; p.Glu931GlyfsTer147 and c.2964_2965delGG; and p.Glu989GlyfsTer89 (In an effort to elucidate the genetic landscape of IRD among the indigenous African populations of South Africa (SA), WES was previously performed in a cohort of 16 families, and a molecular diagnosis obtained for approximately 40% through an analysis of all reported IRD genes at that time n = 285) . Leverag5 . Levern = 285) ,30,31 yin = 285)  and assin = 285) . SubsequyfsTer89 . Both muNGS data analysis workflow can be divided into three stages of analysis: primary, secondary, and tertiary . Brieflyn = 65), probands with a family history suggestive of adRP, arRP, or xlRP (n = 17) and probands with recessive or sporadic macular degeneration (n = 15).The University of Cape Town (UCT) IRD registry currently contains biological material, clinical information, and demographic data of 3506 individuals from 1617 SA Families with IRD. Informed consent was obtained according to the 2013 Declaration of Helsinki for all participants from whom samples have been archived. Ethics approval was granted by the Human Research Ethics Committee of the UCT Faculty of Health Sciences (REC REF: 226/2010 and 768/2013). Samples from the registry have been investigated over the years in various research projects, and only genetically unresolved indigenous African IRD cases were included in the present study. All individuals with pedigrees displaying male-to-male transmission of disease were excluded. The cohort comprised isolated males with RP  and processed according to the manufacturers’ instructions.The UCT IRD registry and biobank has archived biological material from patients for three decades; therefore, various DNA extraction methods have been used over the years. Blood samples were collected in EDTA tubes and processed immediately, mainly using a salting out method , althougw/v) agarose gels containing 0.5–1 g agarose , 50 mL of 1X Tris Borate EDTA (TBE) buffer and 5 µL SYBR® Safe DNA Gel Stain (Thermo Fisher Scientific).DNA samples were quantified using the Nanodrop ND-1000 Spectrophotometer  and subsequently diluted to a final concentration of 20 ng/µL in Sabax water . After dilution, the DNA integrity was confirmed using 1–2% weight/volume , and variant calling was performed by GATK . Additionally to this standard secondary analysis pipeline, FASTQ files also underwent de novo assembly with SPAdes (St. Petersburg genome assembler) . The resRPGR ORF15 sequence with MAFFT v7 (Multiple Alignment using Fast Fourier Transform) and screened for variants using SNP-sites for single-nucleotide variants (SNVs) and an in-house Python 3 (https://www.python.org) script for indels [https://jupyterlab.readthedocs.io/en/stable/). Patient NGS data are not available, due to privacy or ethical restrictions. The vast majority of reported pathogenic mutations in ORF15  are frameshift or nonsense variants [Then, the final contigs were aligned to the reference r indels ,40. Finar indels . The entvariants ,43,44. W® kit , using the following cycling conditions: 95 °C—15 min, 45 cycles of {94 °C—45 s, 60 °C—1 min, 72 °C—3.5 min}, 72 °C—10 min, 10 °C hold.Likely pathogenic variants were confirmed by Sanger sequencing and cosegregation analysis was performed in available familial samples. Moreover, assumed false positives and negatives generated by the standard pipeline were verified by Sanger sequencing when possible. A 1953 bp fragment spanning the coding region of ORF15 was amplified from 200 ng genomic DNA, using previously published primers ORF15-F3 and ORF15-R6 . PCR was® Terminator v3.1 Ready Reaction Mix (Applied Biosystems by Thermo Fisher Scientific) and Q-Solution (QIAGEN).Following purification with FastAP™ Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific) and Exonuclease I (Thermo Fisher Scientific), sequencing was performed with published primers ORF15-R7b, -R8b, -R8c, or -R9  . Capillary electrophoresis was performed on an ABI Prism 3130xl Genetic Analyser (Applied Biosystems by Thermo Fisher Scientific). Output.ab1 files were aligned to the reference ORF15 sequence (NM_001034853.2) using Bioedit  and manuAmplification of the ORF15 region of RPGR was possible for 82 of the 97 samples included in this study (85%). It was not possible to associate sample characteristics  to unsuccessful amplification. It is possible that variants in the primer-binding regions prevented amplification, at least in a subset of these samples.The average DNA concentration after PCR for the successful samples was 35.1 ng/μL (range 5.4–132.0 ng/μL). The average maximum read depth in the ORF15 region after BWA-MEM alignment equalled 5346 reads.RPGR’s ORF15 repeatedly proved itself not only challenging in terms of sequencing data generation, but also during subsequent data analysis. The standard NGS pipeline, consisting of reads alignment to the reference genome with BWA-MEM followed by variant calling with GATK, is not sufficient for this low-complexity region. Specifically, it often resulted in erroneous “heterozygous” calls in males, and it was particularly poor at detecting larger indels (≥20 bp). In fact, there were 606 total and 43 unique variants called across all sequenced samples that passed all quality filters; of the 606 variants, 546 were called as heterozygous, and of these, 432 (79%) were found in male samples. Furthermore, the standard analysis pipeline produced both false positives and false negative results.To overcome this obstacle, we created an alternative Python-based pipeline  = 0.0016). The underlying sequence for either variant combination is the same; these are merely two different ways to call the variants compared to the reference sequence.In contrast, Assuming all low-quality calls are false positive, the standard pipeline resulted in 516 false positive calls, whilst the novel pipeline produced 65 false positives. Unfortunately, it was not possible to test all discrepancies by Sanger sequencing. Instead, at least one carrier sample per discrepant variant was selected for Sanger sequencing to confirm the presence or absence of the respective variant.Variants c.3074T>G, c.3062T>G, c.2863T>G, c.2758A>G, and c.2499T>G were called in most samples by the standard pipeline , but they were found only once by the novel pipeline . Moreover, the standard pipeline resulted in heterozygous calls exclusively, which we considered a low-quality variant marker. Sanger sequencing revealed these variants to be false positives in the tested subset (8 samples).Variants c.2829T>G and c.2829T>A show a similar pattern; they were called in 50 and 30 samples, respectively, from the standard pipeline and only once by the novel pipeline. Both variants were confirmed to be false positive in 4 and 3 samples, respectively. Interestingly, these variants overlap with the c.2820_2840dup variant that has been identified by the novel pipeline in a subset of the corresponding samples. Even more striking is the overlap of samples carrying the c.2639A>G, c.2634G>A, c.2633G>A, and c.2589A>G variants in the standard pipeline’s outputs with those carrying the c.2606_2632dup variant in the novel pipeline’s output. All of the former variants were confirmed as false positives in a subset of the carriers, and the latter was confirmed as being present. These findings highlight that some of the standard pipeline’s false positive cluster around the region, where the novel pipeline identified indels.All remaining low-quality call variants were not found in the novel pipeline’s output , and all those that could be verified by Sanger sequencing were confirmed to be false positives.In contrast, all high-quality call variants tested could be confirmed, except for 2 variants from sample Afr-31, which is one out of five samples with an average coverage lower than 100×. It is noteworthy that most variants identified in these low-coverage samples are confirmed false positives , suggesting that very deep coverage might be needed to result in high-quality variant calls with the novel pipeline.Variants c.3407G>A, c.3396C>T, c.3264G>A, c.3219C>T, and c.2667_2669del were identified in one additional sample by standard analysis compared to the novel pipeline outputs; all missing samples were females called as heterozygous by the standard pipeline. These results seem to suggest that the novel pipeline might produce false negatives in female carriers. However, other heterozygous variants in female carriers were detected by the novel pipeline .Eight pathogenic variants were confirmed in 11 families by Sanger sequencing . The lacRPGR ORF15 variants in 13% (n = 11/82 probands) of an African IRD cohort comprising isolated affected males and families lacking male-to-male disease transmission. A total of 32 unique variants were identified , including novel pathogenic frameshift mutations.In this study, we identified pathogenic RPGR exon ORF15 (57 in 82 samples sequenced in this cohort) indicate that the replication of this region in vivo is demanding and error-prone. This might explain, at least partly, the challenge of amplification in vitro. In fact, PCR amplification failed several times, for both NGS  and Sanger protocols . On the other hand, it is possible that variants in the primer-binding regions prevent successful amplification. Most publications only report “positive results” and do not comment on amplification failure. ORF15 is a known troublesome region, and it would be valuable to know what proportion of ORF15 amplifications fail, in particular for older DNA samples, in other laboratories.The prevalence of small indels in Heterozygous variant calls resulting from the standard secondary analysis pipeline (BWA-MEM and GATK) in male samples highlighted problems that occurred during alignment and/or variant calling across this low-complexity region. Furthermore, we identified 516 potential false positives produced by the standard pipeline, which passed all quality filters . We were able to circumvent the problem by developing an alternative secondary analysis pipeline based on de novo assembly. This analysis allowed for the identification of otherwise missed variants, in particular indels larger than 15 bp. By comparing the results of both data analysis pipelines, we showed that the novel method is more sensitive (in particular for indels) and less prone to false positive calls, as illustrated in RPGR’s ORF15 by NGS and to perform the analysis on affected males where possible.However, the continued identification of false positives in the novel pipeline’s output warrants caution in results interpretation. All of these (except for c.2820A>G) were present in the standard pipeline’s output, too. Furthermore, the unavailability of verified true heterozygous variants in female probands (16 included in this study) made it impossible to verify how the pipeline will perform in the presence of two alleles. Sanger sequencing ORF15 in females can be very complicated due to the high frequency of small indels , which shift the frame of the Sanger traces and can hinder its analysis. Moreover, this problem affects all other current methodologies in the case of ORF15. Based on this, we recommend the parallel use of both pipelines for the analysis of n = 15/32 as listed on Our analysis of the African cohort identified a large number  and/or other treatments and interventions in order to preserve vision.Finally, the genetic diagnosis achieved by our new sequence data analysis pipeline will impact the care and counselling for these 11 patients and their extended families. Identification of the underlying mutations allows for diagnostic closure and enables calculation of the risk of developing the disease, thereby facilitating decision making and life choices. Importantly, a molecular diagnosis may also determine eligibility for a potentially available gene-based and personalised therapy (