We present two cases of post-neurosurgical ventriculitis caused by carbapenem-resistant Gram-negative pathogens successfully treated with high-dose ceftazidime/avibactam. The existence of a real-time clinical pharmacological advice program, by enabling the optimization of the PK/PD targets over time at the infection site, turned out to be very helpful. Post-neurosurgical (post-NS) infections in neurocritical patients are associated with remarkable morbidity and mortality rates . In receEnterobacterales and/or Pseudomonas aeruginosa [Klebsiella pneumoniae infections treated with ceftazidime/avibactam [Ceftazidime/avibactam is a novel beta-lactam/beta-lactamase inhibitor combination with valuable activity against carbapenemase-resistant (CR) ruginosa . Nowadayruginosa . Additioruginosa , and convibactam .Here, we report two cases of post-NS infections caused by CR Gram negatives that were treated with high-dose CI ceftazidime/avibactam-based regimens. Treatment was optimized by means of a real-time clinical pharmacological advice (CPA) program focused at maximizing pharmacodynamic target attainment (PD-TA) at the infection site against CR pathogens that were isolated from the CSF.Klebsiella pneumoniae (CR-KP) strain was documented at rectal swab, with susceptibility limited to ceftazidime/avibactam (MIC 4 mg/L) and colistin (MIC 0.25 mg/L). Five days later, an episode of CR-KP bacteraemia occurred, and treatment with ceftazidime/avibactam (2.5 g over 2 h loading dose (LD) followed by 2.5 g q8h over 8 h (namely by CI)) was started. CSF chemico-physical examination was carried out, and the findings were suggestive of VPS-related infection . Consequently, VPS was replaced by an external ventricular shunt (EVD), and linezolid co-treatment was added. Soon before VPS removal and soon after EVD positioning, CSF cultures were performed. Both samples yielded CR-KP strains with the same pattern of susceptibility, and consequently, linezolid was suspended. Additionally, a real-time CPA program based on plasma and CSF therapeutic drug monitoring (TDM) was implemented for maximizing the PD-TA of ceftazidime/avibactam in both plasma and CSF over time (desired targets: steady-state concentrations (Css)/MIC ratio 4–8 for ceftazidime and 100%fT > 4 mg/L for avibactam). On day 4, both CSF pharmacodynamic targets were quasi-optimal (ceftazidime Css/MIC ratio (3.25) and avibactam CSF concentration (3.2 mg/L))  affected by post-traumatic cerebral ischemic injury that caused tetraplegia and hydrocephalus needing ventriculo-peritoneal shunt (VPS) positioning. During hospitalization, colonization with a CR 2 mg/L)) .ss/MIC ratio ranging 2.38–3.25; avibactam concentrations ranging 1.4–2.7 mg/L). Subsequent CSF culture assessments showed microbiological eradication and culture negativization persisted over time, with favorable clinical evolution and progressive reduction in both protein levels (from 1964 to 42 mg/dL) and WBC counts . Although ceftazidime/avibactam treatment was successfully completed on day 21, unfortunately, the patient passed away because of underlying complications of the cerebral damage.Ceftazidime/avibactam dosage was increased up to 2.5 g q6h over 6 h CI. The choice was supported by the finding of an augmented renal clearance at that time (measured CLCr of 192 mL/min) and by the contention that the progressive healing of the blood–brain barrier could have led to a decreased penetration rate of ceftazidime/avibactam into CSF [3, 65.9% of which being polymorphonuclear cells). Empirical treatment with cefepime plus trimethoprim-sulfamethoxazole was started. On day 3, CSF culture was positive for a CR-Pseudomonas aeruginosa strain. The strain was susceptible to ceftazidime/avibactam (MIC 4 mg/L), amikacin (MIC 8 mg/L), and ceftolozane/tazobactam (MIC 1 mg/L). Testing by agar dilution showed an MIC for fosfomycin of 64 mg/L. After EVS removal and substitution, antibiotic therapy was consequently switched to ceftazidime/avibactam (2.5 g q6h over 6 h CI after 2.5 LD) plus fosfomycin (8 g LD followed by 16 g q24h CI). A real-time CPA program was implemented for maximizing the PD-TAs of both ceftazidime/avibactam and fosfomycin into CSF  and borderline for fosfomycin  affected by a pineal neoplasm complicated by obstructive hydrocephalus. After neurosurgical exeresis, an EVS was positioned, and the post-NS course was complicated by fever coupled with headache relapse. CSF chemico-physical examination was suggestive of an EVS infection .2, and dosages of both antibiotics were increased (up to 5 g q8h over 8 h CI for ceftazidime/avibactam and 24 g q24h CI for fosfomycin). This choice was also supported by the contention that the progressive healing of the blood–brain barrier could have led to a decrease in the penetration rate of both antibiotics into CSF [ss/MIC ratio ranging from 5.675 to 8.25 and avibactam concentrations ranging from 4.7 to 7.3 mg/L). For fosfomycin, CSF-to-plasma ratios ranged between 0.42 and 0.5, and the CSF PD-TAs were always optimal as well (fAUC/MIC ratio range 104.63–126.38). Subsequent CSF assessments showed microbiological eradication and culture negativization persisted over time, with favorable clinical evolution and progressive reductions in both protein levels and WBC counts. EVS was removed on day 23 and antibiotic therapy was stopped on day 26 after achieving optimal clinical response. The patient was discharged to a rehabilitation facility, and no relapse at follow-up occurred.Estimated CLCr was 101 mL/min/1.73 mP. aeruginosa related post-NS infection was based on preclinical studies showing synergism. In a murine infection model using a high bacterial burden, the combination of ceftazidime/avibactam with fosfomycin was superior to either drug alone in significantly reducing the P. aeruginosa burden [Overall, these two cases firstly showed that the CSF penetration rates of ceftazidime and avibactam in patients with post-NS infections ranged over time around 15–30% during the first 3 weeks post-event. Additionally, our findings showed that optimizing CSF PD-TAs by means of a real-time CPA program may be valuable in tackling CR-related post-NS infections caused by borderline susceptible pathogens. Repeating TDM assessments over time may also be crucial in neurosurgical patients. This approach may allow the posology to be modulated day-by-day according to the dynamic evolution of the CNS infection . Furthera burden . NotablyIn conclusion, our findings showed that the existence of a real-time CPA program, by enabling optimization of the PK/PD targets over time at the infection site, turned out to be very helpful in granting successful treatment with high-dose CI ceftazidime/avibactam-based regimens of two cases of post-NS ventriculitis caused by CR-Gram-negative pathogens. Of course, ours is simply a proof-of-concept and we are aware that further prospective confirmatory studies are warranted before any definitive conclusion can be drawn.


Hyperuricemia (HUA) is strongly associated with abnormal glucose metabolism and insulin resistance (IR). However, the precise molecular mechanism of HUA-induced IR is still unclear. Retinol binding protein 4 (RBP4) has been shown to induce IR in type 2 diabetes mellitus. This study was designed to clarify the relationship between RBP4 and HUA-induced IR and its potential mechanisms.via intraperitoneal injection once daily for eight weeks, and gavage with adenine (100 mg/kg) once daily from the fifth week to induce the HUA model. Glucose consumption testing was performed to determine the capacity of glucose intake and consumption in 3T3-L1 adipocytes. Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect the mRNA and protein level of RBP4 and insulin receptor substrate-phosphatidylinositol 3-kinase-active protein kinase (IRS/PI3K/Akt) signaling pathway-related proteins.Patients with HUA were collected to detect the levels of plasma RBP4 and clinical biochemical indicators. Rats were fed with 10% high yeast and oteracil potassium (300 mg/kg) The levels of plasma RBP4 in both HUA patients and HUA rat models were significantly higher than that in the control groups. The level of plasma RBP4 was positively correlated with plasma uric acid, creatinine, fasting insulin, IR index, total cholesterol and triglyceride levels in patients with HUA. In HUA rats, the level of plasma RBP4 was positively correlated with plasma uric acid, IR index, and triglycerides. HUA rats also exhibited IR. After inhibition of RBP4 expression, the phosphorylation levels of the IRS/PI3K/Akt signaling pathway were increased, and IR was significantly improved.in vitro and in vivo. RBP4 may be involved in HUA-induced IR by inhibiting IRS/PI3K/Akt phosphorylation. Our findings may provide a new insight for the treatment of IR caused by HUA.HUA induced IR both  In the last few decades, the prevalence of hyperuricemia (HUA) has been rapidly increased worldwide while the age of onset has decreased , 2. AccuRetinol binding protein 4 (RBP4), belonging to the lipocalin family, is mainly synthesized in hepatocytes and the adipose tissues . PreviouIn the present study, we found that the levels of plasma RBP4 were significantly higher in patients with HUA and were positively correlated with plasma uric acid (UA), creatinine (Cr), fasting insulin (FIns), HOMA-IR index (HOMA-IR), total cholesterol (TC) and triglycerides (TG). Moreover, we verified that RBP4 promoted HUA-induced IR through inhibition of IRS/PI3K/Akt phosphorylation, which provides a new insight to understand and treat HUA-induced IR.2. Patients with the following conditions were excluded: i) an inability to walk or obvious abnormalities in understanding; ii) obvious clinical liver disease, diabetes or abnormal glucose metabolism, severe renal insufficiency (estimated glomerular filtration rate (eGFR) <30 ml/min/1.73 m2); iii) history of major cardiovascular diseases in the past three months, congestive heart failure (NYHA grade III or IV), malignant tumors diagnosed in the past five years, acute or chronic infections, history of organ transplantation or acquired immune deficiency syndrome; and iv) history of alcohol abuse or drug abuse in the past 12 months.Thirty patients with HUA and thirty healthy controls were collected from the medical center at the Second Xiangya Hospital, Central South University. The main enrollment criteria included: i) aged over 18 years; ii) diagnosed with HUA (level of UA in fasting blood >416.5 μmol/L for men and 357 μmol/L for women); and iii) body mass index (BMI) ranged from 18–40 kg/mAll subjects were investigated one by one using a questionnaire in which name, gender, age, ethnicity, hospital number (inpatient), history of illness and medication were recorded. Blood pressure was measured by benchtop mercury column sphygmomanometer and BMI was also calculated according to the Chinese Adult Overweight and Obesity Prevention and Control Guide  Standard. Plasma UA, blood urea nitrogen (BUN), Cr, TG, TC, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting plasma glucose (FPG) and FIns were detected by an automatic biochemical analyzer. The HOMA-IR index was calculated to assess IR status using the following equation: HOMA-IR = FPG × FIns/22.5. Levels of plasma RBP4 were detected by enzyme-linked immunosorbent assay (ELISA). The clinical parameters of all the participants in the study are presented in Male Sprague–Dawley rats , provided by the Department of Animal Medicine of Central South University, were used for the study. Rats were randomly divided into two groups after one week of adaptive feeding (1): CON group (n = 10) and (2) HUA group . The HUA group was fed with 10% high yeast feed (yeast dry powder: common rat feed = 1:9) and potassium oxonate was injected intraperitoneally at 300 mg/kg once a day for eight weeks. The CON group was fed with a normal diet and an equal dose of sodium carboxymethylcellulose solution was injected intraperitoneally for eight weeks . Blood sThe serum was separated and tested for RBP4 levels by ELISA . FPG was tested by the Roche blood glucose meter, and FIns, BUN and Cr were detected by ELISA . UA was tested by colorimetry. High-sensitivity C-reactive protein and Cystatin C were detected by rat high-sensitivity C-reactive protein detection kit  and rat Cystatin C Assay Kit, respectively. TC and TG were tested by enzymatic method, and LDL-C, HDL-C were tested, respectively, by homogeneous assay and elimination or PPD method, direct assay. In addition, insulin tolerance test, oral glucose tolerance test and HOMA-IR were also calculated.® CL-173™) and the mycoplasma-free state of the cells was confirmed by using a mycoplasma detection kit before the experiments were conducted. The 3T3-L1 preadipocytes were cultured with 10% fetal bovine serum (FBS), 10 mg/L insulin, 1 μmol/L dexamethasone, and 0.5 mmol/L 3-isobutyl-1-methylxanthine. After being cultured for 48 hours (h), the supernatant was discarded. Cells were then washed with PBS three times and replaced by differentiation medium B . The cells were changed 48 h later in order to be cultured with high-glucose DMEM medium, which could be used in the next experiment, when approximately 90% of the 3T3-L1 preadipocytes would exhibit an adipocyte phenotype. For transient transfection of RBP4 siRNA, RBP4 siRNA oligos (50 nM) were transfected by Lipofectamine 3000 to the 3T3-L1 adipocytes in six-well plates at a density of 2 × 105 cells per well. Differentiated mature 3T3-L1 adipocytes were grouped as needed for the experiment (1): mature 3T3-L1 adipocytes + PBS (2); mature 3T3-L1 adipocytes + 10 mg/dl UA+ scramble siRNA (3); mature 3T3-L1 adipocytes + RBP4 siRNA; and (4) mature 3T3-L1 adipocytes + 10 mg/dl UA + RBP4 siRNA . The sequences were as follows: scramble siRNA: 5’AGACCTCTCATAGCA GCTGAT 3’; RBP4 siRNA: 5’CAT CCT AGA CGT TGC TAC 3’.The 3T3-L1 preadipocytes were obtained from ATCC (ATCC−△△CT). The sequences of the primers of RBP4 and GAPDH were shown in Total RNA was extracted from the cultured 3T3-L1 cells by using trizol reagent  and RBP4 cDNA was synthesized according to the manufacturer’s instructions . RT-PCR was performed with the T100 Real-Time PCR Detection System  using the SYBR safe . The reaction conditions were 95°C for 30 s followed by 40 cycles at 95°C for 5 s, and 60°C for 30 s. RBP4 mRNA expression was normalized to the expression of GAPDH using the standard curve method . The basic value of the glucose content was based on the glucose content of the blank cells with no treatment, and finally, the glucose consumption of the cells in other groups were calculated.P <0.05 indicates statistical significance.Data analysis was performed using statistical software (version SPSS 17.0). All normal distribution digital data were expressed as mean ± standard error of the mean (SEM). Non-normal distribution data were subjected to parameter test after data conversion. The gene expression was analyzed by ANOVA variance method. Levene method was used to test the homogeneity of variance, and the comparison between groups was performed by LSD test or Tamhan’s-T2 (M) calibration test. The correlation analysis was performed using Pearson’s correlation analysis and the quantitative relationship between the indicators was analyzed by linear regression analysis. All the experiments were repeated at least three times and vs 20.70 ± 2.93, P <0.05). Besides, the levels of UA, Cr, TC, TG, FIns, and HOMA-IR in the HUA group were significantly higher than those of the CON group, while there were no significant differences of HDL-C, LDL-C and FPG between the two groups  (P >0.05).Subsequently, we generated a rat model of HUA and analyzed the correlation between the levels of plasma RBP4 and some key metabolic indicators in HUA rats. The results showed that levels of plasma RBP4 were positively associated with UA, TG, and HOMA-IR in HUA rats  Table 4.vs 26.19 ± 5.16 mg/L, P <0.001)  and Health Care Fund of Hunan Province, China (Grant No. B2015-03).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Proteus bacillus isolated from smoked horsemeat sausage. RT-qPCR was employed to evaluate the gene expression level of histidine decarboxylase (HDC) cascade-associated genes. We used HPLC to monitor histamine concentration both in pure culture as well as in the processing of smoked horsemeat sausage. Results showed that histamine accumulation was suppressed by thyme microcapsule inhibitory effect on the histamine-producing bacteria and the reduction in the transcription of hdcA and hdcP genes. Besides, compared with thyme essential oil (EO), thyme microcapsules exhibited higher antibacterial activity and had a higher score for overall acceptance. Therefore, the addition of thyme microcapsules in Xinjiang smoked horsemeat sausage inhibits histamine accumulation.Here, we explored the influences of thyme microcapsules on the growth, gene expression, and histamine accumulation by  Histamine is a low molecular weight nitrogenous organic compound that plays a pivotal role in human learning as well as memory, body temperature, and immune responses . HistamiThymus vulgaris L. has been opined to possess elevated antimicrobial, insecticidal, as well as phytotoxic features owing to the phenolic constituents, particularly carvacrol (2-methyl-5-methyl ethyl)-phenol) as well as thymol (5-methyl-2 (1methylethyl) phenol) [Botrytis spp. [The histamine content in fermented sausages depends on many factors, including the existence of microorganisms, which decarboxylate the amino acids, as well as a conducive environment for the growth of decarboxylase-producing microbes . Further phenol) ,16. Howe phenol) . The tec phenol) . Encapsutis spp. . Ginger tis spp. . Hence, However, to the best of our knowledge, there is a knowledge gap in the application of thyme microcapsules in sausages. Hence, the purpose of the research was to assess if microcapsules of thyme affect the accumulation of histamine, the growth of microorganisms, transcription of histidine decarboxylase (HDC) cascade-associated genes, and the sensory quality of smoked horsemeat sausage.Proteus bacillus, from naturally smoked horsemeat sausage in our laboratory, was used in this study . The sequences were deposited in GenBank databases under accession number MN483275. Proteus bacillus was grown on Brain-Heart Infusion Broth (BHI) media, enriched with 100 Mm histidine  at 37 °C. The strain used in this work had been previously identified by molecular methods [P. bacillus strains were used as inoculum for all fermentation assays. Each fermentation experiment was performed using the same stock culture medium and overnight culture inoculants to ensure the same CFU/mL (105–106). Thyme microcapsules and thyme essential oil were supplied by the Boao Extension Technology Co. Ltd. in Beijing. Five batches were prepared (10 mL): a batch without histidine (control); histidine and 0% thyme microcapsules (0% microcapsules); histidine and minimum inhibitory concentration (MIC) thyme microcapsules (MIC microcapsules); histidine and 1/2 MIC thyme microcapsules (1/2MIC microcapsules); and histidine and the same amount of essential oil as microcapsules . Brain-Heart Infusion Broth (BHI) media (10 mL) required the following ingredients: beef brain (20%), beef heart infusion juice (25%), peptone (1%), glucose (0.2%), NaCl (0.5%), and agar (2%). Brain-Heart Infusion Broth (BHI) media was obtained from AoBoXing Company . Samples were collected (2 mL) every four hours for 48 h. To check the microbial growth in all cultures, we measured the absorbance at 600 nm (OD600) using a multi-modal reader . A previously isolated histamine-producing microorganism,  methods . OvernigP bacillus MN483275 was cultured in BHI medium and supplemented with 100 Mm histidine to activate the transcription of the hdc gene [g for 3 min at 4 °C and resuspended in 1 mL of TRIzol reagent. TRIzol reagent  was used to extract total RNA, as previously described by del Rio et al. [g for 10 min at 4 °C. High-performance liquid chromatography (HPLC) was used to determine histamine concentration [The pH of the samples was measured using a Sartorius UB pH-meter . We calibrated the pH meter with phosphate buffer and potassium hydrogen phthalate buffer. After the calibration was completed, we washed the composite electrode with distilled water and dried the filter paper and inserted it into the sample solution to ensure that the glass bulb at the front of the electrode was in uniform contact with the standard buffer solution. After the pH meter stabilized, showing the pH value of the unknown solution, we pressed the “OK” button to measure the solution again . The meahdc gene . To harvo et al. . Sample 3 in size. Four batches of smoked horsemeat sausage were processed: a control batch without P. bacillus or thyme microcapsules (batch CK); a batch inoculated with P. bacillus (batch P); a batch inoculated with P. bacillus and thyme microcapsules (batch PMT); and a batch inoculated with P. bacillus and thyme essential oil (batch PO). Sausage preparation (10 kg/batch) required the following ingredients: basic raw materials of lean horsemeat (80%) and fat horsemeat (20%); and auxiliary raw materials of pepper (0.1%), white sugar (2%), sodium chloride (2.5%), ginger powder (0.2%), monosodium glutamate (0.1%), spiced powder (0.1%), star anise (0.1%), and a smoke solution (1%). Thyme microcapsules and thyme oil were added at a concentration of 0.156%. Each batch was kept at 4 °C for 24 h and then stuffed (300–350 g/sausage) into a natural casing  previously soaked in salt water and with a diameter of 5–6 cm [P. bacillus was suspended in sterile water (100 mL) to attain a final level of 104 CFU/g of sausage. Fermentation and ripening of the sausages was performed at a steady humidity as well as temperature in an incubator  at 90–95% relative humidity (RH) and 18–20 °C first for 2 days and then at 75–80% RH for 26 days at 10–12 °C [The horse meat was cut into cubes about 1 cm10–12 °C . We collEnterobacteriaceae were analyzed using Violet Red Bile Glucose Agar (VRBGA) for 48 h at 37 °C, whereas lactic acid bacteria (LAB) were determined on de Man Rogosa Sharpe agar (MRS) medium at 30 °C for 2 days [The bacterial numbers were enumerated during the 28 days fermentation period using a previously document protocol  with slir 2 days . AoBoXing at 4 °C and then aliquoted the supernatant into clean tubes. We added 0.5 mL isopropanol into the mixtures, gently mixed, and maintained standing for 10 min at room temperature. After that, 10-min centrifugation of the mixture was performed at 12,000× g at 4 °C, and the supernatant was discarded. The resulting precipitate was rinsed using 1 Ml 75% ethanol to precipitate it further. We air-dried the precipitate and re-suspended in DEPC-treated H2O. The RNA was inoculated with DNase  to degrade any contaminating DNA. The concentration of the RNA was estimated by measuring its absorbance at 260 nm as well as 280 nm on a NanoDrop ND-2000c UV/vis Spectrophotometer .The RNA from experimental sausages was isolated, as per protocol documented by del Rio et al.  with sli−ΔΔCt approach was employed to determine the relative gene-expression level [The 5X All-In-One RT Master Mix   was employed to convert the RNA into cDNA via reverse transcription. Thereafter, qPCR was performed using the Stratagene MX 3000 sequence detection system  in 25 μL reaction mixture, as documented previously . The hdcon level . RT-qPCRg at 4 °C and collected the supernatant. . The volume of the filtrate was adjusted to 50 mL using 0.4 M perchloric acid. Derivatization of biogenic amines was conducted using dansyl chloride according to the protocol documented by Lu et al. and Sun et al. [g for 5 min [The aggregated histamine in the experimental sausages was isolated using a previously described protocol  with slin et al. . We put or 5 min . The detThe sensory quality of the smoked horsemeat sausages was assessed at the end of the fermentation, as previously documented, with slight modifications . The senp < 0.05 signified statistical significance.The Origin 8.5 software (Origin Lab) was utilized for data analysis. Means ± standard deviations were computed from three independent replicates. The SPSS software 23 package  was employed to evaluate the differences between groups. P. bacillus growth. Moreover, compared with the thyme oil group, thyme microcapsules exhibited higher antibacterial bioactivity [p < 0.05), and microbial growth decreased with escalating thyme microcapsules.As indicated in a, the abactivity ,30. HoweThe changes in pH over 48 h in all batches are shown in P. bacillus growth as well as the HDC gene cluster expressions, thereby reducing the histamine accumulation.The thyme microcapsules group’s pH was lower compared to the other groups, which revealed that thyme microcapsules inhibit p < 0.05) in contrast with the other batches (control and 0% microcapsules). Additionally, the thyme microcapsules influence on histamine accumulation was higher compared to that of thyme EO, suggesting that thyme microcapsule significantly decreased the accumulation of histamine. Moreover, the biosynthesis of histamine decreased with increased concentration of thyme microcapsules. The accumulation of histamine may confer resistance to acid stress acquired via the consumption of intracellular protons through a decarboxylation process, as has been documented in other bacterial species [The histamine standard solution with a mass concentration of 1.00 mg/L was diluted into different low-mass concentration solutions, and samples were injected for determination after derivatization. The results showed that when the RSN was 3, the mass concentration of histamine standard solution was 0.03 mg/L; when the RSN was 10, the mass concentration of histamine standard solution was 0.12 mg/L. Therefore, the detection limit of the instrument was 0.03 mg/L, and the limit of quantification was 0.12 mg/L. The accumulation of histamine over 48 h in all batches is shown in  species ,32,33. TThe transcription of the HDC gene cluster is shown in Enterobacteriaceae initial numbers were more remarkably elevated in batches PMT and PO than in batch CK (p < 0.05), associated with the richness of P. bacillus in the initial population. In addition, the number of LAB and GCC+ in all batches rose sharply, reached the maximum on the seventh day, and then decreased slightly. Besides, there was no significant difference between the four smoked horsemeat sausage batches in the later period (p > 0.05). Thyme microcapsules have no significant bactericidal biological activity against LAB and GCC+ in all batches, which may be attributed to the fact that the sensitivity of microorganisms to thyme microcapsules varied from species to species [Enterobacteriaceae remarkably reduced in the fermentation as well as the ripening (p < 0.05). On day 28, Enterobacteriaceae was completely depleted in the PMT batch, while the count in the CK, P, and PO batches were 1.01 ± 0.03, 3.36 ± 0.01, 1.85 ± 0.029 log10 CFU g−1, respectively, revealing that thyme microcapsules repressed Enterobacteriaceae growth. Similarly, Scacchetti et al. [The alterations in bacterial numbers are indicated in  species . This isK p < 0.0, associai et al.  reportedi et al. . These ci et al. ,18,35.p < 0.05). The expression of the hdcA gene in batches CK, PO, and P was 9.58-, 18.64-, and 537.45-fold, respectively, which was higher compared to batch PMT. Similarly, the expression of the hdcp gene in batches CK, PO, and P was 10.06-, 1.89-, and 72.00-fold, respectively, which was higher than in batch PMT. Thyme microcapsules had a significantly higher effect on the expression of hdcA and hdcP compared to thyme oil. Studies have previously reported that the activity of the histamine synthesis gene cluster (HDC) affects the accumulation of histamine [P. bacillus might confer resistance to the prevailing acidic stress via the consumption of intracellular protons through a decarboxylation reaction, as documented in other bacterial species [The hdcA and hdcP gene expression is involved in the production of histamine, which is found in our previous pure-culture system. The transcription of hdcA and hdcP genes in smoked horsemeat sausage is shown in istamine . The fac species ,32. Herep < 0.05) and suggesting that the presence of thyme oil prevented the accumulation of histamine. Compared with batch (PO), there was a 33.35% reduction in the accumulation of histamine in batch PMT, indicating that thyme microcapsule had a stronger inhibitory effect than thyme oil (p < 0.05). Additionally, thyme microcapsules decreased histamine aggregation by suppressing the expression of histidine decarboxylase (hdcA) as well as histidine/histamine antiporter (hdcP) genes (p < 0.05). Our data demonstrated that the encapsulation with thyme microcapsules reduced the accumulation of histamine, thus suggesting that the thyme microcapsule application can promote the safety of smoked horsemeat sausage as well as other meat products.The changes in histamine concentration in smoked horsemeat sausage are indicated in p < 0.05), which may be attributed to the presence of nitrite. Thyme microcapsules can lower the pH value, and under acidic conditions, more nitrosomyoglobin will be produced, which plays an important role in color development [p > 0.05) in the sourness and dry scores among all sausage batches. Compared with the thyme EO group, the thyme microcapsules group had a better odor. Besides, thyme microcapsule as an encapsulated plant extract covered up the bad smell of thyme EO and also slowed down its release. This may also be attributed to the fact that thyme microcapsules reduce the accumulation of histamine. Ardö et al. [The sensory qualities of the smoked horsemeat sausages were assessed at day 28 and are shown in elopment . The gloelopment . There wö et al.  reportedö et al. ,39. TherEnterobacteria in smoked horsemeat sausage. Accumulation of histamine is not only linked to histamine-generating microbes but also associated with the histidine decarboxylase (HDC) gene cluster expression. Thyme microcapsules and thyme EO had insignificant influence on the expression of hdcRS and hdcB; however, they remarkably suppressed the transcriptional activation of hdcA as well as hdcP genes (p < 0.05). The suppressive influence of thyme microcapsules was remarkably higher (p < 0.05) relative to that in thyme EO. Additionally, the addition of thyme microcapsules enhanced the sensory quality of the smoked horsemeat sausage. Hence, the use of thyme microcapsules not only improves the smoked horsemeat sausage sensory quality but also enhances its safety.Herein, we demonstrated that both thyme microcapsules and thyme essential oil (EO) could diminish histamine levels, down-regulate the expression of HDC gene cluster, and repress the microbial growth, e.g.,


Previous studies had highlighted that α7nAChR was related to the emergency of vulnerable atherosclerotic plaques with excess inflammation cells. Thus, 18F-ASEM could be a complementary diagnostic approach to atherosclerotic plaques.Atherosclerosis is a chronic vascular inflammatory procedure alongside with lipid efflux disorder and foam cell formation. α7-Nicotinic acetylcholine receptor (α7nAChR) is a gated-calcium transmembrane channel widely expressed in neuron and non-neuron cells, such as monocytes and macrophages, activated T cells, dendritic cells, and mast cells. 18F-labeling had been performed successfully. We had established the ApoE–/– mice atherosclerotic plaques model (fed with western diet) and New Zealand rabbits atherosclerotic models . After damage of endothelial cells and primary plaque formation, 18F-ASEM imaging of atherosclerotic plaques linked to α7nAChR had been conducted. In vivo micro-PET/CT imaging of ApoE–/– mice and the control group was performed 1 h after injection of 18F-ASEM (100–150 μCi); PET/CT imaging for rabbits with atherosclerotic plaques and control ones was also performed. Meanwhile, we also conducted CT scan on the abdominal aorta of these rabbits. After that, the animals were sacrificed, and the carotid and abdominal aorta were separately taken out for circular sections. The paraffin-embedded specimens were sectioned with 5 μm thickness and stained with hematoxylin–eosin (H&E) and oil red.The synthesis of ASEM precursor and In vivo vessel binding of 18F-ASEM and α7nAChR expression in the model group with atherosclerosis plaques was significantly higher than that in the control group. PET/CT imaging successfully identified the atherosclerotic plaques in ApoE–/– mice and model rabbits, whereas no obvious signals were detected in normal mice or rabbits. Compared with 18F-FDG, 18F-ASEM had more significant effect on the early monitoring of inflammation in carotid atherosclerotic plaques of ApoE–/– mice and model rabbits. 18F-ASEM had relatively more palpable effect on the imaging of abdominal aorta with atherosclerosis in rabbits. H&E and oil red staining identified the formation of atherosclerotic plaques in model animals, which provided pathological basis for the evaluation of imaging effects.18F-ASEM as radiotracer with good imaging properties for precise identification of atherosclerotic diseases.We first confirmed  Cardiovascular disease (CVD) is a serious threat to human health; its mortality is still the main cause of death . The intin vivo imaging is of great importance to investigate the exact role of α7nAChR in AS before any theranostic approach in human setting can be justified. The exploration of α7nAChR by PET is a highly promising way not only to better understand the changes associated with the progression of AS but also to improve the development of new therapeutic strategies. Therefore, a highly specific radiotracer could be used to image differences in receptor density in different disease stages and expand our knowledge of α7nAChR’s contribution to the pathophysiology of AS.The homomeric α7 subtype of nicotinic acetylcholine receptors (α7nAChR) is a class of ligand-gated ion channels distributed throughout the brain, and the crucial role of α7nAChR in a number of specific systems and diseases has been extensively reviewed . Its abn18F-labeled probe in α7nAChR is dibenzo thiophendioxide derivative, named ASEM , has been carried out in human studies, and are proved to be the only α7nAChR PET radioligand available to humans with promising imaging characteristic , the present study reports the first PET/CT imaging with 18F-ASEM, making it feasible to image atherosclerotic plaques and to evaluate the vulnerability of plaques toward rupture. We also made pathological examination of AS to verify the reliability of 18F-ASEM in the early identification of atherosclerotic plaques.For these reasons mentioned above, α7nAChR, as a significant therapeutic target, has received increasing interest in the fields of science and clinical medicine. Therefore, great progress in quantity and quality of α7nAChR radioligands has been made over the past decades . Imagingmed ASEM . 18F-ASEteristic . Howeverculature . Therefo–/– mice were purchased from Qingzilan Technology Co., Ltd. , and New Zealand rabbits were acquired from Fuwai Hospital . C-18 light Sep-Pak cartridges and QMA light ion-exchange cartridges were obtained from Waters . The 18F isotope was acquired through Jiangsu Huayi Technology Co., Ltd. .ApoE18F-labeled ASEM were synthesized and followed the descriptions in several previous studies  and normal mice  were randomly selected; the chosen ApoE–/– mice were fed with 21% fat and 0.15% cholesterol supplement for 11–12 weeks.ApoEn = 4; weight, 2.5–3.0 kg) were fed with high-fat diet for 14 weeks. The balloon-sprained and injured experiment was carried out at the third week after 2 weeks of high-fat diet. First, rabbits were anesthetized by 10% chloral hydrate, a 3-cm incision was conducted in the right inguinal area to expose the right femoral artery; then, the right femoral artery was punctured by Seldinger technique. Second, an angiographic catheter with double lumen balloon was sent to the aortic arch under fluoroscopy. Third, the balloon was bulged and pulled down to the bifurcation of the distal iliac artery of the abdominal aorta for three times. Lastly, the catheter and sheath were removed, and the operational incisions were sutured ] was injected into tail vein of ApoE–/– mice and normal mice. ApoE–/– mice, positioned prone position, were anesthetized (1–2% isoflurane) through inhalation. After anesthesia, mice were injected with 5.56 MBq (150 μCi) 18F-ASEM through the tail vein and underwent a dynamic imaging for 1 h. Dynamic PET scanning were carried out at 5, 10, 15, 30, and 60 min after injection, and biological distributions were analyzed as previously described by our group (The high-performance liquid chromatography (HPLC)-purified ur group . Brieflyn = 4) were anesthetized by 10% chloral hydrate (2 ml/kg). Then, 18F-ASEM solution  was injected into the right ear vein of rabbits. After that, the imaging procedure was conducted 30 min after injection of 18F-ASEM and lasted for 10 min performed by PET/CT (Discovery IQ 2.0 GE). The injection of 18F-FDG  and PET/CT imaging were performed in the same strategy on the next day. The reconstruction mode was super iterative. The % ID/g value was worked out in the same protocol as mentioned above through IRW 4.0 software.First, rabbits . The scan area ranged from the aortic arch to the level of bifurcation of the iliac artery. X-ray tube current was adjusted individually. For contrast medium enhancement, automated bolus tracking was used in a region of interest within the ascending aorta, with a signal attenuation trigger threshold of 100 HU, 2-s postponed. The main scanning parameters were as follows: 0.6 mm individual detector width, 280 ms gantry rotation time, 0.20–0.50 pitch, and 200–250 mm field of view for raw image reconstruction. We used a triple-phase contrast medium injection protocol, which consisted of 5–6 ml of undiluted contrast agent  followed by a 3-ml 30%:70% mixture of contrast medium and saline and a 3-ml saline chaser bolus, all injected with flowrates of 1–2 ml/s.All original imaging date were retrospectively analyzed on the workstation  by two experienced radiologists using axial images, multiplanar reconstructions, and maximum intensity projections, as appropriate. The presence of atherosclerosis in the aorta within the scan area was analyzed. Any disagreements between the two readers were solved by consensus.en face analysis by red “O” staining.After anesthetized, the mice and rabbits were sacrificed for histological measurements and experiments. Carotid arteries of model mice and abdominal aortas of model rabbits were perfused with heparin saline, harvested under aseptic conditions, and fixed in 4% polyformaldehyde. The full length of the carotid arteries and abdominal aortas was used for H&E staining and First, paraffin-embedded slices were dewaxed in water. The slices were successively put into xylene (10 min), anhydrous ethanol (10 min), anhydrous ethanol (5 min), 95% (5 min) alcohol, 90% alcohol (5 min), 80% alcohol (5 min), 70% alcohol (5 min), and distilled water (5 min) to wash step by step. Then, slices were embedded with hematoxylin dye for 8 min, then washed with flowing water. The slices were differentiated with 1% hydrochloric acid alcohol for 10 s, washed with flowing water, retreated with 0.6% ammonium hydroxide until turning blue, then washed with running water. After hematoxylin staining the nucleus, the slices were stained for 1–3 min in eosin dye solution for eosin staining the cytoplasm. The slices were dehydrated and were transparent in 95% alcohol I, 5 min–95% alcohol, 5 min–anhydrous ethanol, 5 min–anhydrous ethanol, and 5 min–xylene. The slices were slightly dried from xylene and sealed with neutral gum for image acquisition and analysis.First, carotid arteries and abdominal aortas were prepared for frozen sectioning. Then, several frozen sections of arteries were rewarmed at room temperature, treated with fixative  for 15 min, then washed with running water and dried. Then, the slices were immersed into the oil red dye solution , kept for 10 min in a dark place, washed with distilled water, slightly differentiated with 75% alcohol, and washed with distilled water again. Afterward, the slices were dyed with hematoxylin solution  for 5 min, washed with flowing water, differentiated with differentiation solution then washed with flowing water again, retreated with returned blue solution , and washed with running water. Finally, they were sealed with glycerin gelatin  and examined with a microscope.P < 0.05 were considered statistically significant.SPSS Statistics v24  software was used. All data were expressed as mean ± SD. 18F-ASEM micro-PET/CT images of ApoE–/– mouse and normal mouse are shown in 18F-FDG, the imaging agent was more remarkable in the carotid artery of ApoE–/– mouse . This indicates that the abdominal atherosclerotic plaques can be obviously recognized with 18F-ASEM, which is better than 18F-FDG in the early stage of atherosclerotic lesions.The  18F-FDG , 18F-ASEThe coronal, sagittal, and cross-sectional CT scan imaging showed that the wall of abdominal aorta was thickened and irregular (red arrow). However, there was no significant stenosis or notable sign related with atherosclerotic plaques . MoreoveKi-value of ASEM was measured (Ki = 0.4 nM), and blocking experiment also proved that ASEM is specifically bound to α7nAChR , 15 min (4.20 ± 0.46 ID%/g vs. 2.46 ± 0.34 ID%/g), 30 min (4.05 ± 0.64 ID%/g vs. 2.40 ± 0.41 ID%/g), and 60 min (3.80 ± 0.43 ID%/g vs. 2.70 ± 0.28 ID%/g) after injection  and good specificity (BPND = 3.9–6.6)  . The bioeristics . However–/– mice and abdominal aorta in New Zealand rabbits and reported the first PET/CT imaging conducted with 18F-ASEM, which makes it feasible to image atherosclerotic plaques and to evaluate the vulnerability of plaques toward rupture. Although CT imaging modality can provide evidence of lumen stenosis and display features of remarkable plaques, its resolution is still insufficient, therefore could not identify atherosclerotic plaques in the earlier stage. 18F-ASEM had more flexibility in inflammatory disease monitoring, which not only benefit from the specificity to α7nAChR in blood vessels but also originate from better pharmacokinetics compared to nonspecificity 18F-FDG (18F-ASEM is more sensitive to detect inflammation and might present stronger signal intensity of inflamed atherosclerotic plaques compared to the classic 18F-FDG-based nuclear medicine imaging modality. In addition, we also made pathological examination of atherosclerosis to verify the reliability of 18F-ASEM in the identification of atherosclerotic plaques.In the present study, we established atherosclerotic plaques models of carotid arteries in ApoE 18F-FDG . Our res18F-ASEM might be also used to evaluate the effect of α7nAChR targeting strategies in the early diagnosis of atherosclerosis. Further clinical studies will answer the question as to whether 18F-ASEM, as the best molecular probe for α7nAChR reported in the literature, can be reliably applied clinically in noninvasive, early diagnosis and monitoring of the vulnerable atherosclerotic plaques of patients who would suffer from acute coronary syndrome. Achieving the early warning of CVEs, thus, greatly reduce the associated morbidity and mortality of patients with CADs all over the world.From the present study, we know that 18F-ASEM as the α7nAChR targeting tracer to detect the inflammation in the formation of atherosclerosis, which holds the potential to assess the vulnerability of plaques. Therefore, 18F-ASEM, as a molecular probe, has the potential to be an appropriate α7nAChR-targeted imaging radiotracer for the early identification of atherosclerotic plaques and other chronic inflammation diseases. Further studies on this radioligand series are still ongoing.The present study supported the feasibility of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Care Committee of Fu Wai Hospital.XW, ZHe, and WL designed the experiments. TY, DW, YL, FG, CW, LJ, and ZHo performed the experiments. TY, DW, and XC analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


Cerebrospinal fluid (CSF) leakage is a common complication after neurosurgical intervention. It is associated with substantial morbidity and increased healthcare costs. The current systematic review and meta-analysis aim to quantify the incidence of cerebrospinal fluid leakage in the pediatric population and identify its risk factors.The authors followed the PRISMA guidelines. The Embase, PubMed, and Cochrane database were searched for studies reporting CSF leakage after intradural cranial surgery in patients up to 18 years old. Meta-analysis of incidences was performed using a generalized linear mixed model.Twenty-six articles were included in this systematic review. Data were retrieved of 2929 patients who underwent a total of 3034 intradural cranial surgeries. Surprisingly, only four of the included articles reported their definition of CSF leakage. The overall CSF leakage rate was 4.4% (95% CI 2.6 to 7.3%). The odds of CSF leakage were significantly greater for craniectomy as opposed to craniotomy  and infratentorial as opposed to supratentorial surgery . The odds of CSF leakage were significantly lower for duraplasty use versus no duraplasty (OR 0.41 95% CI 0.2 to 0.9).The overall CSF leakage rate after intradural cranial surgery in the pediatric population is 4.4%. Risk factors are craniectomy and infratentorial surgery. Duraplasty use is negatively associated with CSF leak. We suggest defining a CSF leak as “leakage of CSF through the skin,” as an unambiguous definition is fundamental for future research.The online version contains supplementary material available at 10.1007/s00381-021-05036-8. Cerebrospinal fluid (CSF) leakage is one of the most common complications after neurosurgical intervention. CSF leakage is associated with substantial morbidity and increased healthcare costs . One stuThe exact magnitude of the problem in children, however, is still unknown and may be larger than in adults for several reasons. First, almost half of all pediatric brain tumors resides in the posterior fossa, and posterior fossa surgeries are thought to be more prone to CSF leakage , 16, 31.The authors followed the PRISMA guidelines for thisN < 10), and literature reviews were excluded. Furthermore, studies on transsphenoidal surgery, skull base reconstructions, burr hole surgery , and primary CSF diversion surgeries were excluded. Two authors (EMHS and KMvB) independently screened all records from the database search on title and abstract to identify relevant articles. All remaining full text articles were screened on their eligibility for inclusion. A consensus meeting was held to reach agreement on the included articles.Embase, PubMed, and Cochrane databases were searched until August 31, 2020 for studies reporting CSF leakage and related complications after intradural cranial surgery in patients up to 18 years old. The following search terms were used: ““children” OR “child” OR “pediatric” OR “paediatric” OR newborn OR “adolescent” OR “infant”” AND “neurosurgery” OR “craniotomy” OR “craniectomy” OR “cranial surgery” OR “tumor resection” AND ““cerebrospinal fluid leakage” OR “CSF leakage”” OR ““pseudomeningocele” OR “incisional leakage” OR “wound leakage” OR “surgical site infection” OR “surgical wound infection” OR “meningitis”” and relevant Mesh/Emtree terms. A modified version of the filter used to search pediatric studies in PubMed is used  /external ventricle drain (EVD)/ventriculoperitoneal (VP) shunt). The following surgical items were collected as proportion per study: site of durotomy , craniotomy versus craniectomy, indication for surgery , ventricular opening (yes/no), use of sealant (yes/no), use of duraplasty (yes/no), and whether a “watertight” closure of the dura was attempted or not. The following outcome parameter was collected: proportion of patients with CSF leakage .Study quality was assessed according to the National Heart, Lung and Blood Institute of National Institutes of Health (NIH) quality assessment tool for case series studies . StudiesI2 [A meta-analysis of the incidence of CSF leakage was performed using a generalized linear mixed model. Heterogeneity of the data across studies was determined using Higgins I2 .The primary outcome measure in this study is the incidence of CSF leakage with 95% confidence interval (CI). Subgroup analyses were performed for the separate surgical indications Chiari decompression  and posterior fossa tumor surgery. Secondary outcome measures are the odds ratio (OR) for CSF leakage for craniotomy versus craniectomy, supratentorial versus infratentorial surgery, cases in which a duraplasty was used or not, and studies in which watertight closure was attempted in all cases or not. Finally, three sensitivity analyses were performed (1) for studies of high quality only, (2) for studies of > 50 patients only, and (3) including the study of Jiang et al see  section All analyses were performed using SAS version 9.4 (SAS Institute Inc).The database search yielded 2123 articles of which 26 were included in this systematic review   and posterior fossa tumor surgery, as only these indications were investigated in sufficient studies. CSF leakage rates in these subgroups were 3.4% (95% CI 1.3 to 8.7%) after Chiari decompression, and 8.0% 95% CI 5.2–12.0%) after posterior fossa tumor surgery. All analyses showed substantial heterogeneity. An overview of outcomes for the primary outcome measure and subgroup analyses can be found in Table –12.0% afThe highest percentage of CSF leakage was found in patients undergoing craniectomy , with an OR of 4.7 (95% CI 1.7 to 13.4) compared to craniotomy . A CSF leakage rate of 6.4% (95% CI 4.1 to 10.0%) was found for infratentorial surgery in contrast to 1.2% (95% CI 0.4 to 3.7%) for supratentorial surgery .In patients with a duraplasty for dural closure, the incidence of CSF leakage was 5.3% whereas patients without a duraplasty had a significantly higher incidence of 11.8% ..3% as compared to 6.4% patients in studies in which watertight closure was not attempted in all cases (OR 0.3 95% CI 0.1 to 2.3). An overview of the secondary outcome measures is presented in Table In studies in which watertight closure was attempted in all cases, the CSF leakage rate was 2Separate analyses were performed: (1) for studies of high quality only, 2) for studies of > 50 patients only, and (3) including the study of Jiang et al. The overall CSF leakage rate in studies of good quality , 26, 35  for studThis meta-analysis shows that the overall incidence of CSF leakage after intradural cranial surgery in the pediatric population is 4.4%. Infratentorial as opposed to supratentorial surgery, and craniectomy as opposed to craniotomy are significant risk factors for CSF leakage . These results underline the relevance of CSF leakage in clinical practice. In the pediatric population, specifically, the burden of additional treatment that may be required for CSF leakage or related complications is substantial. In studies reporting data on treatment of CSF leakage, a total of 37 out of 114 patients with a CSF leak were treated with a ventriculoperitoneal shunt , 34, 35.There is a wide range of reported CSF leakage rates (between 0.0 and 38.0%) , 17, 28.No separate analyses could be performed per type of surgery for all these categories, nor for the risk factors like age, immune status, previous chemotherapy or previous radiotherapy, CSF diversion surgery, preoperative hydrocephalus, ventricular opening, and sealant use as there was insufficient data or only data on study level available from the included literature.Our meta-analysis shows that the proportion of CSF leakage is the highest in the subgroup of patients undergoing craniectomy 10.3%). This difference may be explained by the lack of extra counter pressure that is otherwise provided by the replaced bone flap . Replace%. This dThis meta-analysis finds a CSF leakage rate of 3.4% after Chiari decompression surgery. The relatively low leakage rate in this population is surprising considering the abovementioned surgical risk factors  as this population essentially represents a combination of these two items.On the contrary, a high leakage rate in posterior fossa tumor surgery (8.0%) is found. This type of surgery may be prone for leakage because pediatric brain tumors frequently reside in the fourth ventricle, requiring opening of the telovelar membrane and leaving a wide-open ventricle. Furthermore, postoperative hydrocephalus may contribute to the increased incidence of CSF leakage in this population .The effect of watertight closure was not significant in this study. However the effect in this analysis may be limited because it was only possible to compare studies in which all cases were closed in watertight fashion to those in which not all cases were closed with this aim . This may reflect that when careful attention is paid to optimal closure of the dura with or without augmentation such as duraplasty or sealants, the risk of CSF leakage is reduced. No distinction has been made in this study between autologous or synthetic material. A study by Hale et al. (2020) indicates that graft dural closure may furthermore be protective against hydrocephalus and wound infection in patients undergoing posterior fossa tumor surgery .Compared to adults, the incidence of CSF leakage found in children is considerably lower, which is contrary to our expectations considering the high number of craniectomies and infratentorial surgeries included. A recent meta-analysis has found that the rate of CSF leakage in adults is 8% . As is tI2-values found in the meta-analyses. It was not possible to adopt a specific definition of CSF leakage for this meta-analysis, as too few publications mention this. One study has been excluded because it included clear fluid in a low-vacuum suctioning wound drainage system as CSF leakage, resulting in an outstandingly high CSF leakage rate of 38.0% [This meta-analysis is subject to several limitations. Most importantly, the studies included are heterogenous in their definitions of the outcome measure, population, and follow-up duration. The majority of studies included in this meta-analysis do not clearly define the outcome measure CSF leakage. Those that do, use a variety of definitions, for example, being “CSF leak through the skin”  and “allof 38.0% . In a seSecondly, the risk factor analyses for duraplasty use and watertight closure were based on a limited number of studies. Therefore, caution should be applied in generalizing these results.Thirdly, we did not exclude patients with subdural-to-extracranial implants, such as subdural grid electrodes, which may influence CSF leakage, but the total influence of this population on the overall results is expected to be minimal.Fourth, the results of the risk factor analysis are potentially influenced by confounding. This is inherent to the design of the included publications and the fact that obtained data do not allow correction for potential bias. Future research should further investigate potential risk factors in a multivariate analysis.Lastly, quality assessment identified only 3 “good quality” studies out of the 26 included in the meta-analysis, compromising quality for the reported outcome measure. The sensitivity analysis shows a higher incidence of CSF leakage in studies of good quality, 7.4% vs. 4.4% found in all studies which may indicate that the CSF leakage rate in this study may be an underestimation of the true CSF leakage rate.Despite these limitations, this meta-analysis provides a representative overview of the CSF leakage rate and associated risk factors reported in the current body of literature. Moreover, it emphasizes the need for a uniform definition and future studies evaluating CSF leakage and preventative strategies in the pediatric population. CSF leakage may include both incisional leakage and pseudomeningocele (PMC). Incisional CSF leakage is defined as leakage of CSF through the skin, whereas a PMC is an extradural collection of CSF under the skin . AlthougThe overall CSF leakage rate after intradural cranial surgery in the pediatric population is 4.4%. The highest leakage rate is found in patients undergoing a craniectomy. Infratentorial surgery is also associated with higher incidence of CSF leakage, whereas the use of a duraplasty is negatively associated with CSF leak. We emphasize the need for a uniform and clinically meaningful definition of CSF leakage, suggesting “leakage of CSF through the skin.”ESM 1(DOCX 19 kb)ESM 2(DOCX 14 kb)


Objective: Guillain-Barré syndrome (GBS) is an autoimmune disease characterized by weakness in limbs or cranial nerve innervated muscles. Acute inflammatory demyelinating polyradiculoneuropathy (AIDP) is the most common variant. Electrophysiologic abnormalities and elevated cerebrospinal fluid (CSF) protein are frequently present in AIDP, but the relationship between these two parameters is not well known. We aimed to fill this gap by studying this relationship.Methods: This was a prospective cross-sectional study conducted for two years in the Department of Neurology, Dr. Ruth K. M. Pfau Civil Hospital, Karachi, Pakistan. All 90 adult patients with the AIDP variant of GBS were selected. Nerve conduction studies were performed to determine the degree of demyelination through the four electrophysiologic demyelination criteria. The CSF sample was sent to lab immediately after lumbar puncture. SPSS version 20.0 was used. The CSF protein level was measured with mean ±SD. Demyelination criteria were measured in frequency and percentages. Chi-square test was applied to a number of demyelination criteria and T-test/ANOVA was applied on mean CSF protein level.Results: We found a mean CSF protein of 37.41 mg/dl (±3.69) with one demyelination criterion, 81.87 mg/dl (±17.39) with two demyelination criteria, 119.75 mg/dl (±31.42) with three demyelination criteria, and 134.00 mg/dl (±42.87) with four demyelination criteria . Conclusion: This study demonstrates a significant relationship between CSF protein levels and degree of demyelination in the AIDP variant of GBS. This is an under-researched area in GBS and this study adds favorably to limited data in this regard. Guillain-Barré syndrome (GBS) is an autoimmune disease which is characterized by weakness in limbs or cranial nerve innervated muscles. It has an acute or subacute onset and usually presents with decreased or absent deep tendon reflexes. Cerebrospinal fluid and electrophysiologic studies demonstrate characteristic abnormalities in this disorder . The incDamage to the peripheral nervous system and the barriers, including the blood-nerve barrier (BNB) and the blood-cerebrospinal fluid barrier (B-CSF-B), is implicated in GBS. BNB and B-CSF-B are barriers between blood and nerve/CSF that result in a stable environment in their surrounding tissues. CSF proteins are elevated in GBS, presumably due to damage to these barriers . Prolongst July 2018 to 30th June 2020.Kanda et al. demonstrated the dysfunction of BNB and B-CSF-B in patients of GBS . Gonzalest July 2018 to 30th June 2020).This was a prospective cross-sectional study conducted in the Department of Neurology, Dr. Ruth K. M. Pfau Civil Hospital, Karachi, Pakistan, after approval from the institutional review board. All the patients presenting with hyporeflexic or areflexic limbs weakness with or without cranial nerves involvement who were subsequently confirmed to have an AIDP variant of GBS were selected with non-probability consecutive sampling over a period of two years , and 3 sensory nerves  were studied. It was ensured that CSF analysis and nerve conduction studies were performed within 24 hours of each other so that a more accurate relationship could be demonstrated. CSF protein level and demyelination criteria were charted on pro forma along with age, gender, and duration from the onset of the disease.The inclusion criteria of this study were i) patients with hyporeflexic or areflexic limbs weakness with or without cranial nerves involvement who met the diagnostic criteria for AIDP, ii) age more than 18 years, and iii) nerve conduction study and lumbar puncture performed within 24 hours of each other. On the other hand, i) patients who were already diagnosed with peripheral neuropathy due to any other cause, and ii) those with CSF white blood cell count of greater than 50/µl were excluded from this study.The demyelination criteria proposed by Albers and Kelly were used in this study :Prolonged distal latency in two or more motor nerves: Distal latency >130% of upper limit of normal.Conduction velocity slowing in two or more motor nerves: Conduction velocity <75% of lower limit of normal.Prolonged late responses: F response and H reflex, in one or more motor nerves: >130% of upper limit of normal.Conduction block/temporal dispersion in one or more motor nerves: a. Conduction block: proximal/distal CMAP area ratio <0.50 b. Temporal dispersion: proximal/distal CMAP duration ratio >1.15.The collected data were analyzed through SPSS version 20.0. The quantitative variables like age, CSF protein level and duration of symptoms were measured with mean ±SD. The qualitative variables like gender and number of demyelination criteria were measured in frequency and percentages. Effect modifiers like age, gender, and duration of symptoms were stratified to see the effect of these on number of demyelination criteria and mean CSF protein level. Post-stratification chi-square test was applied to the number of demyelination criteria and T-test/ANOVA was applied on mean CSF protein level. P-value <0.05 was taken as significant.A total of 90 patients were enrolled in the study. There were 71.1% male and 28.9% female patients Figure 1 . The mea26.6% of patients had one demyelination criterion, 33.3% of patients had two demyelination criteria, 26.6% of patients had three demyelination criteria and 13.3% had four demyelination criteria in nerve conduction studies Figure 3 . Sural nWe found a mean CSF protein of 37.41 mg/dl (±3.69) with one demyelination criterion, a mean CSF protein of 81.87 mg/dl (±17.39) with two demyelination criteria, a mean CSF protein of 119.75 mg/dl (±31.42) with three demyelination criteria, and a mean CSF protein of 134.00 mg/dl (±42.87) with four demyelination criteria . The stratified data of relationship between mean CSF protein and demyelination according to age, gender and duration of disease are shown in Table 1 We found a statistically significant relationship between CSF protein and electrophysiologic abnormalities in the AIDP variant of GBS. In 2015, DiCupua et al. published a retrospective study on the positive association between CSF protein and electrophysiologic findings of demyelination among GBS patients . BourqueThe male and female ratio in our study was 2.4:1. Hughes et al. studied 35 different case series of GBS patients and found a male and female ratio of 1.25:1 . This maIn our study, 26.6% of patients had only one demyelination criterion of prolonged late responses in one or more motor nerves. There were two different pairs of two demyelination criteria: 24.4% of patients had prolonged distal latency in two or more motor nerves and conduction block/temporal dispersion in one or more motor nerves; 8.9% of patients had prolonged distal latency and reduced conduction velocity in two or more motor nerves. 26.6% of patients were found to have three demyelination criteria: prolonged distal latency in two or more motor nerves, reduced conduction velocity in two or more motor nerves, and prolonged late responses in one or more motor nerves. The remaining 13.3% had all four demyelination criteria present in nerve conduction studies.Either alone or in combination, the most common demyelination criterion in our study was prolonged distal latency in two or more motor nerves which was found in 66 patients. This was followed by prolonged late responses in one or more motor nerves in 60 patients, reduced conduction velocity in two or more motor nerves in 44 patients, and conduction block/temporal dispersion in one or more motor nerves in 34 patients. Das et al. found that all their AIDP patients had prolonged distal latency, and 40% of patients had prolonged F wave latency, which is one of the late responses . Wali etSural nerve sparing is considered a fairly specific measure to differentiate GBS from closely related diseases with a specificity of up to 0.96 in GBS patients . Our stuThe findings of this study suggest that the CSF protein level correlates with the degree of demyelination in patients with the AIDP variant of GBS. The patients with three demyelination criteria, for example, were found to have more CSF protein on average compared to those with two demyelination criteria. This relationship remained intact with stratifications for age, gender and duration of symptoms.The limitation of this study is that it included patients from a single center and therefore generalization over the whole population may not be appropriate. The major strength of this study is a significant sample size spread over two years that minimizes the chances of false representation of the actual state of affairs.Earlier studies have suggested that CSF protein concentration can be used as a surrogate measure of the integrity of the BNB and B-CSF-B. The disruption to these barriers is understood to cause inflammation of the nerve roots, resulting in elevation of the CSF protein level . ConsequThis study demonstrates a significant relationship between CSF protein levels and degree of demyelination in the AIDP variant of GBS. This is an under-researched area in GBS and this study adds favorably to limited data in this regard. An increase in CSF protein is considered a surrogate measure of progressive dysfunction of barriers between blood and nerve/CSF. This study points towards these barriers as being key factors in the pathophysiology of this disease by finding that the CSF protein level rises with an increasing degree of demyelination. This may pave the way for targeting these barriers therapeutically in the future if further studies indicate this phenomenon, and may effect a major shift from the immunomodulatory therapy currently offered in this disease with mixed results , 23], , .The authors declare that they have no competing interests.


Preventing vaccine freezing is one of the biggest challenges in vaccine management. Until 2018, vaccine carriers used in the immunization program lacked features to prevent vaccine freezing. Freeze-preventive vaccine carriers (FPVCs) have an engineered liner that buffers vaccines from direct exposure to frozen ice packs. A field evaluation of three FPVCs was conducted in 24 health posts in eastern Nepal. The objective was to evaluate the FPVCs’ performance, acceptability, systems fit, and cost, to inform prequalification and introduction planning. The study was carried out in two phases: in the first phase, FPVCs containing dummy vaccines (labeled “Not for Human Use”) were transported to outreach sessions along with a standard vaccine carrier (SVC); in the second phase, the FPVCs were used for transporting vaccines taken to outreach sessions and used for vaccinating eligible children. The study gathered quantitative and qualitative data from health workers, logbooks, and electronic temperature monitors placed inside and outside the FPVCs. Results indicate the FPVCs successfully prevented temperatures below 0 °C more than 99% of the time—except at one site, where ambient temperatures were below the minimum rated testing temperature specified by the World Health Organization. Internal cool-down times for the FPVCs were highly variable, as were mean kinetic temperatures, possibly driven by the wide range of ambient temperatures and higher-than-expected variations in freezer performance, which, along with the need to transport ice packs to some locations, affected ice-pack temperatures. Almost all health workers requested smaller, lighter-weight FPVCs but appreciated the FPVCs’ ability to prevent vaccines from freezing while avoiding undue heat exposure. FPVCs had benefit-cost ratios greater than 1 and hence good value for money. Results point to the importance of understanding the intended environment of use and the need for smaller, short-range as well as long-range carriers. Haemophilus influenzae type b, reducing vaccine potency and compromising protective immunogenicity in recipients. A number of studies conducted in countries of all income levels have reported the exposure of vaccines to freezing temperatures during storage and transport in the vaccine cold chain Preventing vaccine freezing remains one of the biggest barriers in vaccine management To protect vaccines, FPVCs and SVCs must maintain internal temperatures of 0 °C to +10 °C, the accepted temperature range for passively cooled devices, which is slightly different from actively cooled refrigerators (+2 °C to +8 °C). The primary advantage of FPVCs is in preserving the potency of freeze-sensitive vaccines, even when frozen-solid (unconditioned) ice packs are used. FPVCs are also expected to simplify logistics by reducing the amount of time required by cold chain handlers (CCHs) to prepare the carriers for outreach sessions. An additional advantage could include reduction in training burden required to effectively use cool water packs or conditioned ice packs in SVCs.This study evaluated three laboratory-tested and conditionally prequalified long-range FPVCs—produced by AOV International, Leff Trade, and Blowkings—in two districts of eastern Nepal. B.P. Koirala Institute of Health Sciences (BPKIHS) in Dharan, Nepal, in collaboration with PATH, conducted the field evaluation in 24 health posts (HPs). The primary study objectives were to evaluate the performance, acceptability, systems fit, and cost of the FPVCs within an existing immunization system.Nepal was selected as the location of the field evaluation due to the strong relationship between the Nepal Ministry of Health and Population (MOHP) and PATH, and due to the country’s variety of climatic and environmental conditions. The study was conducted in two types of terrain (hilly and plains) to evaluate the FPVCs in a variety of climatic and environmental conditions. There had to be an alternate delivery mechanism to transport vaccines to HPs. In total, 24 HPs were purposively selected in conjunction with the MOHP: 12 in two areas  of Dhankuta District (hilly region) and 12 in three blocks of Sunsari District (plains region). All phases of the study were conducted in the same 24 HPs.The evaluation was conducted in two phases: phase 1 (mid-May through August 2018), phase 1a (early May through early July 2019), and phase 2 (mid-November through December 2019). The Nepal MOHP gave approval to BPKIHS for each phase of the study. The BPKIHS Institutional Review Committee and National Health Research Council provided ethical review and approval. In phase 1, the study team evaluated the AOV FPVC ; in phase 1a, the Leff Trade and Blowkings FPVCs (and paired SVCs); and in phase 2, all three FPVC types (no SVCs). In phases 1 and 1a, the FPVCs contained dummy vaccines labeled with an “X” and “Not for Human Use.” The FPVCs were transported to routine immunization outreach sessions along with the SVCs, and were opened for set periods of time to simulate actual use . The MOHP then reviewed the data and gave approval to proceed with phase 2. In phase 2, the FPVCs were used to transport real vaccines for actual use; no SVCs were used.Twelve of the HPs had a refrigerator for storing vaccines and were able to freeze ice packs on-site. The HPs without freezers obtained ice packs from a nearby cold chain point (facility that stores vaccines) or a district vaccine store. Half of the HPs were instructed to condition ice packs prior to loading them into the vaccine carriers; the other half, not to condition ice packs. To condition the ice packs, CCHs left the ice packs outside the freezers for 30 to 60 min (based on room temperature) or until the ice was slushy when shaken; unconditioned ice packs were considered frozen solid.In phase 1, the study evaluated 24 AOV FPVCs (one for each HP); in phase 1a, 12 Leff Trade and 12 Blowkings FPVCs; and in phase 2, seven AOV, nine Leff Trade, and eight Blowkings FPVCs. Of the seven HPs that used AOV, three were directed to use frozen-solid ice packs, while four were directed to use conditioned ice packs; of the nine HPs that used Leff Trade, five used frozen solid, four used conditioned; and of the eight HPs that used Blowkings, four used frozen solid and four used conditioned.Data collection in each phase occurred in two rounds corresponding to the monthly immunization schedules of the study regions. CCHs prepared the FPVCs, which were transported to session sites by vaccinators such as auxiliary health workers (AHWs) and auxiliary nurse midwives (ANMs). The AHWs and ANMs ensured placement of two temperature monitoring devices (detailed in section 2.4) inside and outside the FPVCs. The devices automatically recorded the temperature every 10 min, while the AHWs and ANMs manually recorded session start and end times in logbooks. The study team used the resulting data to calculate mean internal and mean kinetic temperature (MKT) inside the vaccine compartment, frequency and extent of temperature excursions, effect of ambient temperature on internal temperature, cool-down times and rates, and percentage vaccine vial monitor (VVM) life loss. The quantities of vaccines taken to each session were also recorded in the logbooks and used to calculate the benefit-cost ratio of the FPVCs. In addition, the temperatures of some of the freezers were collected to understand the environment in which the ice packs were frozen.Using a structured in-depth interview guide, an experienced qualitative researcher collected qualitative data on the acceptability of the FPVCs among ANMs, AHWs, and CCHs at the end of each phase . Interviews were conducted with 12 health workers at the end of phase 1 and 24 health workers at the end of phases 1a and 2. Discussions focused on how well the FPVCs performed during outreach, including storage capacity, freeze prevention, size and weight, and staffing- and training-related aspects. Similar feedback was collected from medical officers and district immunization officers.All the FPVCs used in this study have an engineered liner that buffers vaccines from direct exposure to frozen ice packs. To prevent reaching freezing temperatures from the rapid cooling that occurs when ice packs are placed inside an SVC, FPVCs cool more slowly. To address the slower cool-down rate, WHO requires verification testing confirming that it takes no longer than 8 h for the FPVC storage compartment to cool from an ambient temperature of +43 °C to +10 °C or lower after placing the ice packs inside The study used LogTag® TRIX-8 electronic temperature monitors to gauge internal and ambient temperatures. These devices are WHO PQS prequalified and are a well-accepted global standard for reliable data collection in refrigerators. The LogTag recorders were placed in freezers in 17 of the 24 HPs (7 sites were excluded as they did not have freezers) and both inside and outside the SVCs and FPVCs at all 24 sites. Block monitors downloaded the temperature data monthly for analysis.A new electronic temperature monitoring device, the Parsyl Trek 1.1, was also included, to ascertain its ability to record temperature and humidity in field conditions and to rapidly upload data. The Trek 1.1 is commercially available but not PQS prequalified. The humidity and light data were used for study analysis; the temperature data were not used to evaluate carrier performance.Before each phase, all staff involved in routine immunization at the 24 HPs were oriented on the objectives of the study and trained according to the protocol. This training included health workers and government immunization staff—including district immunization officers, block and district medical officers, and CCHs. The trainings were provided in Nepali or Hindi and conducted at the block level with 108 participants attending across both districts.Outreach sessions lasted one to three days. Vaccines experience a range of temperatures inside carriers over time. Calculating MKT provides a single temperature value based on this range that can be used to determine the potency of vaccines as if they were left at a steady temperature. Although taking a simple average of temperatures often provides a similar result, MKT is a more rigorous metric for gauging the effect of higher temperatures on the thermal degradation of vaccines. MKT is influenced by time: the longer the ice packs remain in a carrier, the lower the MKT.The study also compared the MKT of blocks assigned frozen-solid ice packs and those assigned conditioned ice packs and found no significant difference . In phase 2, for blocks with frozen-solid ice packs, the highest mean temperature was 13.8 °C. The highest mean internal temperature with conditioned ice packs was 12.3 °C. Eighteen of 24 facilities (75%) maintained an MKT between 0 °C and +10 °C .Fig. 3MeIn all phases, temperature readings were taken every 10 min while the vaccines were inside the carriers. In total, 3,618 h of temperature data were recorded.  Because passive vaccine carriers should maintain temperatures between 0 °C and +10 °C, internal temperature readings below or above this range were deemed excursions. In phase 1, low-temperature excursions  occurred in 2.7% of SVC sessions . These fPer WHO PQS specifications, an FPVC must be able to maintain an internal temperature range of 0 °C to +10 °C at an ambient temperature range of +15 °C to +43 °C. During phase 1, higher ambient temperatures appeared to affect the MKTs of the AOVs to a greater extent than they affected the SVCs. In phase 1a, higher ambient temperatures appeared to raise the MKTs in the Leff Trade and Blowkings, but not in the SVCs. In phase 2, plotting the average ambient temperature against the mean internal temperature for each HP across all carrier brands studied shows some correlation between the temperature outside and inside the FPVCs .Fig. 4MeAs per WHO PQS specifications, an FPVC must be capable of cooling from +43 °C to less than +10 °C in 8 h or less. To do so, a carrier must cool at an approximate average rate of 4 °C/hour or faster. Slower cool-down rates can still result in complete cool-downs if the starting temperature is below 43 °C. Although the primary purpose of this study was to assess the ability of the FPVCs to prevent freezing temperatures from occurring, it is important to consider their ability to protect vaccines from heat exposure. VVMs are stickers attached to vaccine vials that change color when exposed to heat over time In phases 1 and 1a, eight LogTag TRIX-8 recorders had errors or did not record internal temperatures. The devices that malfunctioned had all been placed in SVCs where high levels of condensation resulted in a pool of water at the bottom of the carrier. Though not clearly established, it is likely the water negatively impacted both the TRIX-8 and Trek 1.1 monitoring devices, so health workers began placing them in resealable plastic (Ziploc®) bags. In phase 2, only one TRIX-8 had errors and did not record ambient temperature. Yet the Trek 1.1 devices were sometimes replaced two or three times for the same HP due to functional issues. There were challenges both in updating the devices and the Parsyl mobile app with software upgrades. In addition, data could not be retrieved from four Trek 1.1 devices during the final data download and transfer process.The correct temperature range for freezers in the vaccine cold chain is –15 °C to –25 °C The study used a baseline survey and in-depth interviews of health workers in all phases to collect data on the acceptability of the FPVCs. In all phases, health workers appreciated the freeze protection offered by the FPVCs and noted, based on VVM status, that the FPVCs did not inadvertently create a new problem of heat exposure. They also appreciated the potential for reduced wastage because no vaccine vial labels peeled off due to water damage, as they did in the SVCs. Health workers instructed to use frozen-solid ice packs felt that time was saved by not having to condition the ice packs. None of the facilities reported any major FPVC malfunctions. Size and weight of the FPVCs were the most pressing issues echoed by almost all health workers and health officials across the three carriers, though the SVCs traditionally used in the health system are shorter range than the FPVCs used in the study .In phases 1 and 1a, health workers were asked to carry two vaccine carriers . In phase 1, there was some confusion because many health workers thought the AOV FPVC would always be transported with the SVC, requiring more space and staff. In addition to its size and weight, other AOV design issues (noted much less frequently) were difficulty in opening and closing the lid, difficultly removing ice packs when fully frozen, accidental tearing of the foam discs, and uncomfortable backpack and shoulder strap design.Among the health workers using the Leff Trade in phase 1a, almost half preferred the SVC, and the other half preferred the Leff Trade over the SVC in its current condition. None of the health workers reported any breakage, and there was no reported condensation in the vaccine compartment. Most health workers said the Leff Trade took almost the same time or less than the SVC to clean. The backpacks were well received. ANMs shared that carrying the FPVC would be difficult without it. All the health workers said it was easy to insert and remove the ice packs. Almost all mentioned the Leff Trade had sufficient storage capacity, although two said their vaccines did not fit; these were health workers who had to carry a week’s supply of vaccines. Health workers gave similar feedback on the Blowkings in phase 1a. Most thought the foam discs were of good quality, though a few thought they were already showing signs of wear. Almost as many health workers preferred using the Blowkings to the SVC in its current condition. Suggestions for improvement included providing a thermometer and backpack.In phase 2, health workers uniformly agreed that all three types of FPVCs enhanced safety, reduced vaccine wastage, and prevented vaccines from freezing, yet were larger and heavier than the SVCs. However, many did not find this to be a significant issue. Most health workers who mentioned size said their chief concern was the FPVCs would not fit on their scooters or bikes the way the SVCs do. All the health workers who mentioned weight as a significant issue were in the hilly areas. District-level health officials and block monitors stated that the size and weight of the FPVCs might be something to which health workers could adapt. Some ANMs mentioned that with time they might adjust if these devices were implemented and scaled up by the national MOHP. One health worker suggested that two sizes of FPVC should be developed: standard for regular sessions, and smaller for catchment areas with fewer beneficiaries. A few workers stated that the backpacks need more pockets, and one said padding should be added to the straps. ANMs, particularly in facilities using frozen-solid ice packs, pointed out that ice packs often expanded on freezing and were hard to insert into and remove from some FPVCs. Further, health workers noted that compared to the 0.4-liter ice packs used in the SVCs and Blowkings FPVCs, the 0.6-liter packs of the AOV and Leff Trade carriers were more difficult to fit into freezers. They also took longer to freeze, which was especially challenging for facilities experiencing frequent power cuts.Costs are presented in US dollars (US$). Using data from the logbooks, we obtained information from 54 outreach sessions. On average, HP staff vaccinated 15 children (range 1 to 72) and four pregnant women (range 0 to 19) during each outreach session. We then calculated the benefit-cost ratio per health facility per year . These cThere were no freezing temperatures in the FPVCs in phases 1 and 1a. As with many studies, knowing data were being collected may have positively influenced health worker practices. In phase 2, internal FPVC temperatures below 0 °C were registered in only one outreach session, in Pakhribas. At the time, Pakhribas was experiencing ambient temperatures below the PQS minimum for freeze prevention. (The mean ambient temperature for the 24 h prior to the start of the session was 11.5 °C.) There were no SVCs used in phase 2, so there is no directly relatable data indicating how an SVC would have performed at the same time. PQS testing for carriers only requires them to show freeze prevention at a minimum rated ambient temperature of 15 °C. Yet FPVCs are well suited to decrease risk in scenarios like winter vaccination sessions, where low ambient temperatures pose a greater risk of freezing the vaccines.Data on freezer temperatures point to wide variations in freezer performance that affected both ice-pack freezing and the ability of ice packs to maintain cold temperatures. Eleven of the study freezers were not PQS prequalified. In remote settings, freezer temperatures rarely reached below –10 °C and sometimes were considerably warmer. Yet freezers that consistently maintain cooler temperatures may also result in more fully frozen ice packs and increased incidence of vaccine freezing. Freezers were more consistent at supplying sub-zero temperatures in phase 2. This could be due to the lower ambient temperatures, which would keep the freezer cooler when the door was opened or during a power loss. It may also be that access to grid power is more consistent in winter.The MKTs inside the FPVCs were substantially higher than in the SVCs. Throughout the study, the hilly blocks were able to maintain MKTs within the range of 0 °C to +10 °C more often than the plains blocks, which might be due to the lower ambient temperatures in the hilly blocks. Several factors are believed to have contributed. First, in the hilly blocks, more of the HPs than in the plains blocks placed their ice packs in the carriers the day before some or all of the outreach sessions due to logistical necessity. By the time these sessions began, the carriers were already cool. Conversely, not having a freezer on-site likely prevented some sites in both districts from actually using frozen-solid ice packs, as assigned, and warmer-than-expected freezer temperatures may have preconditioned some of the ice packs. Also, because frozen-solid ice packs can bulge, health workers may have had to let them condition for longer than recommended in order to fit them into the FPVC ice-pack compartments. Study results point to the same factors for why FPVC cool-down times were more variable than anticipated. Yet during actual use in phase 2, average cool-down times were <4 h across all vaccine carrier brands and ice-pack states, and more than 90% of carriers cooled to below 10 °C by the time the vaccination session ended.Calculating VVM life loss showed that exposure to temperatures above 10 °C in the FPVCs had a negligible effect on vaccine potency. The session with the greatest VVM life loss, at 0.95%, occurred when the vaccines had remained in the carrier for multiple days. Over the course of the study, the average VVM life loss per session—including all HPs, conditioning states, and manufacturers—was just 0.25%. With the exception of the one freezing temperature event at Pakhribas, the FPVCs were successful in both preventing exposure to sub-zero temperatures and guarding against heat degradation.FPVC selection criteria should consider storage capacity, need for short- versus long-range carriers, as well as distance and means of transport for the FPVC. This study included districts in hilly and plains settings, with the intention to evaluate the acceptability and systems fit in varying environments of use, including remote areas. However, outreach visits lasted consistently <8 h even in the hilly areas. This length of outreach is better suited for short-range carriers, regardless of terrain. Also, the smaller-capacity carriers were generally sufficient for the loads  required in these areas of Nepal. The exception is remote areas lacking refrigeration, where carriers must be kept in the field for several days before changing ice packs.Data gaps occurred for several reasons, including language barriers, inconsistent terminology, inexact time recordings, and LogTag TRIX-8 malfunctions from excessive exposure to water in SVCs (phases 1 and 1a). For the Parsyl Trek 1.1, challenges in data transfer were likely due to mobile app issues or phone connectivity rather than the hardware itself. Also, data were analyzed for each vaccine carrier type assuming the category of ice pack (frozen solid or conditioned) assigned to each site was indicative of the true state of the ice packs. The frozen-solid ice packs assigned to the HPs with no freezer may have been inadvertently conditioned during transport. Regarding freezer performance, the data were trimmed to remove ambient temperature readings at the beginning and end of each recording period, as well as temperature spikes that were unlikely to occur if the LogTags had remained in the freezer. This was done to ensure greater accuracy; however, there is a risk that some legitimate readings may have been excluded, or that some incorrect readings may have remained. Also, because freezer data were measured 24 h prior to the session, we cannot be certain how this influenced ice-pack freezing, as we do not know when the ice packs were placed in the freezers. Thus, discussions of the relationship between freezer performance and carrier performance are limited to noting generalized trends.By keeping vaccines within an optimal temperature range, the vaccine carriers used in this study averted a potential loss in potency of freeze-sensitive vaccines and, important for immunization budgets, loss in dollar value of vaccines. Study results validate that long-range FPVCs are a viable means of preventing freezing temperatures in outreach vaccination settings and are a good value for money. The study also reinforced how critical it is to verify the context of use prior to introduction of any new technology. Cool-down times for the FPVCs were more variable than laboratory testing predicted. This appears to have been driven primarily by higher-than-expected variations in freezer performance. More research is needed to understand to what extent this is due to the quality of the freezers, availability of consistent power, or other factors. As new, highly efficient water pack freezers are introduced worldwide, there may be an inadvertent, increased risk of vaccine freezing in SVCs. This study’s authors urge global immunization supply chain entities to include recommendations for countries to procure FPVCs, retrain health workers on conditioning ice packs, or consider using cool water packs in SVCs instead of ice packs. Immunization programs using FPVCs will similarly have to weigh the risk of vaccines potentially being above the recommended 0 °C to +10 °C range for several hours as the device cools. The FPVCs included in this study met their performance objectives, and users appreciated their benefits; however, most health workers did not like the increased size and weight of the long-range FPVCs in contrast to the smaller, lighter SVCs. Further efforts to design FPVCs should place a high priority on minimizing carrier size and weight.Mendeley Data under the title, “Long-range freeze-preventive vaccine carriers in Nepal: thermal data” with digital object identifier https://doi.org/10.17632/j277dvs69g.1Data presented in this article are available from the online repository The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.