Objectives of this study, as part of a nation-wide HIV pre-exposure prophylaxis (PrEP) evaluation project, were to determine the incidence of infections with HIV, chlamydia, gonorrhea, syphilis, hepatitis A/B/C in persons using PrEP, and to describe the health care funded PrEP use in Germany. Additionally, factors associated with chlamydia/gonorrhea and syphilis infections were assessed.Anonymous data of PrEP users were collected at 47 HIV-specialty centers from 09/2019–12/2020. Incidence rates were calculated per 100 person years (py). Using longitudinal mixed models, we analyzed risk factors associated with sexually transmitted infections (STIs).4620 PrEP users were included: 99.2% male, median age 38 years (IQR 32–45), 98.6% men who have sex with men (MSM). The median duration of PrEP exposure was 451 days (IQR 357–488), totaling 5132 py. Four HIV infections were diagnosed, incidence rate 0,078/100py (95% CI 0.029–0.208). For two, suboptimal adherence was reported and in the third case, suboptimal adherence and resistance to emtricitabine were observed. One infection was likely acquired before PrEP start. Incidence rates were 21.6/100py for chlamydia, 23.7/100py for gonorrhea, 10.1/100py for syphilis and 55.4/100py for any STI and decreased significantly during the observation period. 65.5% of syphilis, 55.6% of chlamydia and 50.1% of gonorrhea cases were detected by screening of asymptomatic individuals. In a multivariable analysis among MSM younger age, PrEP start before health insurance coverage and daily PrEP were associated with greater risk for chlamydia/gonorrhea. Symptom triggered testing and a history of STI were associated with a higher risk for chlamydia/gonorrhea and syphilis. A significantly lower risk for chlamydia/gonorrhea and syphilis was found for observations during the COVID-19 pandemic period.We found that HIV-PrEP is almost exclusively used by MSM in Germany. A very low incidence of HIV infection and decreasing incidence rates of STIs were found in this cohort of PrEP users. The results were influenced by the SARS-CoV-2 pandemic. Rollout of PrEP covered by health insurance should be continued to prevent HIV infections. Increased PrEP availability to people at risk of HIV infection through the elimination of barriers requires further attention. Investigation and monitoring with a longer follow-up would be of value.The online version contains supplementary material available at 10.1007/s15010-022-01919-3. Infection with the human immunodeficiency virus (HIV) remains globally endemic with 1.5 million new infections annually despite continued advancements in its management, treatment, and prophylaxis , 3. WherAlthough the use of PrEP significantly reduces the risk of HIV transmission, it may change sexual behavior resulting in an elevated risk for the transmission of other sexually transmitted infections (STIs) and even HIV –9. On thPrEP was approved in Germany in 2016 and available initially on a self-paying basis . As a reThe uptake and outcome of PrEP use after introduction of coverage by the German statutory health insurance as well as any effects on the incidence of STIs were evaluated through a nation-wide study in Germany (“EvE-PrEP”) –18. The The NEPOS study was conducted by the Robert Koch Institute (RKI) in collaboration with the German association of physicians in HIV care (dagnä e.V.). All HIV-specialty care centers were invited to participate. The study was a retrospective longitudinal analysis of randomly selected PrEP users. The maximal number of PrEP users that could be documented in the study was set at 5000 for budgetary reasons. To ensure that the study sample is representative for the German PrEP using population, the centers were allocated a defined quota on the basis of their PrEP using population in reference to that of all participating centers. The randomization process at each center was performed at the discretion of the participating center.Data were collected anonymously using an electronic reporting form in the first quarter of 2021 retrospectively for the time-period of September 2019 through December 2020. Date of individual PrEP initiation before September 2019 was documented if persons started PrEP before the observation period. STI with chlamydia, gonorrhea or syphilis 6 months prior to PrEP initiation were also documented (patient file or self-reported).The study centers entered the data into the electronic reporting form from routine patient files. Data included patient characteristics , PrEP use , testing for and potential diagnosis of HIV, hepatitis and STI, as well as hepatitis A and B antibody status. Duration of PrEP use was calculated as the number of days between PrEP start and end of follow-up, the latter defined by either PrEP discontinuation or the end of the study, 31/12/2020. Aside from HIV infection, STI included chlamydia, gonorrhea and syphilis. Newly diagnosed viral hepatitis included A, B and C. The observation period was divided into five time intervals: First September through December 2019, then four quarters in 2020 (2020q1–2020q4). Gonorrhea, chlamydia and syphilis were counted for each time interval, HIV and hepatitis with a date of diagnosis.Testing for HIV and STIs was performed in all centers as recommended by the German/Austrian PrEP guidelines . In accoUnivariable and multivariable longitudinal multilevel mixed-effects logistic regression corrected for the study centers as random intercept were used to analyze risk factors associated with infections with chlamydia/gonorrhea and syphilis. The analysis was performed among all MSM with/without other risk factors being tested in the respective time interval adjusted for age, PrEP use (intermittent/on-demand vs. daily), PrEP interruptions, PrEP discontinuations, PrEP duration, PrEP start before statutory health insurance coverage, infections with Hepatitis or HIV, other STI than the one analyzed, STI history (any STI yes/no) in the past 6 months before PrEP start, the number of STI tests, observation before  or within  the COVID-19 pandemic period.In a sensitivity analysis, we analyzed persons without any event related to PrEP use  for at least two quarters before their last event to calculate a ”loss to follow-up” rate for those without documentation of PrEP discontinuation or interruption.Data were collected anonymously using an electronic reporting form programmed in C+ + . Data were stored and the dataset prepared using a MS-SQL server. Statistical analysis was performed using STATA Version 17. Figures were produced using Gfk RegioGraph and Microsoft Excel (Office 2019).Forty-seven German HIV-specialty care centers participated in the study and provided representation of PrEP use as a whole from both a geographical and a diversity perspective Fig. .Fig. 1Din = 1638, 35.5%) were documented in Berlin, which also reflects the pre-dominant location of current PrEP use.4620 PrEP users were randomly selected to be documented in this study. Nearly all (99.2%) were male, median age was 38 years (IQR 32–45), 98.6% were MSM of whom 10.6% had additional risk factors. Of the 39 PrEP users who were not male, 17 were female, 16 transgender and four non-binary gender. Characteristics are shown in Table n = 2466, 53.4%). Taking this into consideration, the median duration of PrEP exposure was 500 days (IQR 388–766), totaling 7353 py. Intermittent/on-demand PrEP was documented for 18.9%. In all, the average number of days of PrEP as prescribed divided by the number of days on PrEP (PrEP pill coverage) was 0.85 (SD = 0.23). Almost one-third  had a calculated PrEP pill coverage of less than 85%, which reflected both intermittent/on-demand PrEP use as well as a documented daily PrEP with lower adherence. The average PrEP pill coverage by mode of PrEP intake was 0.91 (SD = 0.16) for persons with PrEP prescribed for daily intake and 0.58 (SD = 0.28) for persons using PrEP intermittent/on-demand.The median duration of PrEP exposure within the period of observation (September 2019–December 2020) was 451 days (IQR 357–488), totaling 5132 py. More than one-half of PrEP users had started PrEP prior to September 2019 (Ten percent of PrEP users had at least one PrEP interruption with a median duration of 93 days (IQR 58–148) with the first interruption after a median of 238 days (IQR 119–458) after PrEP start. PrEP discontinuations were documented for 13.2% after a median time of 275 days (IQR 142–478). Reasons for PrEP discontinuation or interruption were mainly SARS-CoV-2 related ; also, PrEP interruptions were highest in March and April 2020 , at the time of the first COVID-19 lock-down  did not have any event related to PrEP use  for at least two quarters before their last event, 84.5% (n = 469) of whom did not indicate PrEP discontinuation or interruption.Twelve percent , accounting for 0.087% and an incidence rate of 0.078/100py (95% CI 0.029–0.208). HIV infections were diagnosed on day 32, 167, 270 and 295 after start of study observation, which corresponds to day 32, 167, 595 and 598 after PrEP start. For two individuals, PrEP on demand was documented, for the other two daily PrEP. The reason for infection in two cases was reported to be suboptimal adherence. In the third case, calculated PrEP pill coverage was 0.6 and resistance to FTC was observed. The fourth person, diagnosed on day 32 after PrEP initiation, reported condomless rectal intercourse a few days prior to PrEP start.Three of these patients were also diagnosed with other STIs. Two of the individuals had presented with a syphilis infection in the previous 6 months before PrEP start and three had other STIs during the observation period Table .Table 2DTwenty percent of the study population were reported to have had an STI in the 6 months prior to PrEP start. The baseline STI prevalences were 7.01% (95% CI 6.05–8.09) for chlamydia, 6.62% (95% CI 5.68–7.66) for gonorrhea and 3.63% (95% CI 2.93–4.44) for syphilis.Incidence rates during PrEP use in the observation period were 21.55/100py (95% CI 20.24–22.93) for chlamydia, 23.73/100py (95% CI 22.35–25.17) for gonorrhea and 10.08/100py (95% CI 9.18–11.03) for syphilis. The total incidence rate of any STI in our cohort was 55.36/100py (95% CI 53.24–57.54). The incidence per time period of any STI significantly decreased over time. Incidence rates over time are shown in Fig. n = 1728, 37.4%) were reported to have had at least one STI in the observation period. During the study, 1203 tests for chlamydia were positive in 21.0% (971/4620 persons). For gonorrhea and syphilis, these were 1324 positive tests in 21.4% (987/4620 persons) and 602 positive tests in 8.9% (409/4620 persons), respectively. More than one infection with chlamydia, gonorrhea or syphilis were reported for 4.2% (194/4620 persons), 5.2% (238/4620 persons) and 1.6% (75/4620 persons), respectively. Sixty-seven individuals (1.5%) had infections with all three pathogens over the course of the observation.Over one-third of the study participants , while 43.6% (n = 2016) had at least three tests for chlamydia/gonorrhea. No testing for chlamydia/gonorrhea was documented for 22.6% (n = 1046), while no testing for syphilis was reported for 1.6% (n = 76). During the observation period, the lowest number of tests for chlamydia and gonorrhea were found in the second and fourth quarter of 2020  and for syphilis in the fourth quarter of 2020 (n = 3183).Syphilis was tested for more frequently than chlamydia/gonorrhea (18 598 vs. 12 789 tests). , with PrEP start before statutory health insurance coverage , with an STI history in the 6 months before PrEP start  or an unknown STI history , and with a higher number of symptom triggered tests for chlamydia/gonorrhea . A significantly lower risk for infections with chlamydia/gonorrhea was found for persons 40–49 years of age  or 50–59 years of age , with intermittent/on-demand PrEP use , and with a higher number of tests for chlamydia/gonorrhea when persons were asymptomatic . A significantly lower risk for chlamydia/gonorrhea was also found for observations during the COVID-19 pandemic period . (Supplement Table 1).The analysis of risk factors associated with infections among MSM with syphilis showed a significantly higher risk for persons 50–59 years of age , with an STI history in the 6 months before PrEP start  or an unknown STI history , and with a higher number of symptom triggered tests for syphilis . A significantly lower risk for syphilis was found for observations during the COVID-19 pandemic period . (Supplement Table 2).In 3358 (72.7%) of individuals, hepatitis A immunity was reported, and 3664 (79.3%) had protection through antibodies against hepatitis B.During the period of observation, two infections with hepatitis B were diagnosed on days 197 and 229 after start of observation, accounting for 0.043% of persons and an incidence rate of 0.04/100py (95% CI 0.01–0.16). Thirteen cases of hepatitis C were found at a median of 267 days (IQR 137–353) after start of observation in the study, and accounting for 0.28% of persons, a positivity rate of 0.30% (4273 tests) and an incidence rate of 0.25/100py (95% CI 0.15–0.44).Additionally, one case of each, hepatitis A and B infection, and hepatitis C were diagnosed prior to or at the start of observation in the study .To our knowledge, this is the first comprehensive description and analysis of real-life PrEP use in the largest available population of PrEP users in Germany. The cohort of PrEP users studied was predominantly male, MSM and in median 38 years of age. To estimate representativeness, we compared the demographic characteristics of our study population with health insurance data representing more than 50% of all persons with statutory health insurance in Germany and found them to be similar in gender, median age, PrEP use behavior, and regional distribution , 22. To PrEP was found to be extremely effective in the prevention of HIV infection in this routine clinical setting. The observed incidence rate of 0.078/100py is comparable with and even lower than in other studies. The early iPrEX OLE study reported an HIV incidence of 1.83/100py and a strong protective effect with adequate drug levels. No HIV infection occurred in persons whose drug levels indicated an intake of four or more tablets per week . More reWe found PrEP failure to be primarily linked to suboptimal adherence, which has been reported to be strongly correlated with efficacy , 29. In The cohort of PrEP users studied was largely male, 99% MSM and 83% under the age of 50, which is similar to that in other implementation studies –28. In GPrEP use was most commonly prescribed for daily intake, with intermittent/on-demand PrEP use reported by less than one quarter. However, the calculated PrEP pill coverage suggests slightly higher intermittent/on-demand use, in addition to suboptimal adherence to documented daily PrEP. PrEP interruptions and discontinuations were reported for 10% and 13%. Since 12% of PrEP users did not have any event related to PrEP use in the last two time periods of observation, possible interruption or discontinuation may not have been reported. The actual rate may, therefore, be higher up to around 20%. Approximately one-half of the PrEP interruptions occurred in March and April 2020 at the time of the first COVID-19 lock-down, and the reasons for PrEP discontinuations or interruptions were mainly SARS-CoV-2 related. Adverse reactions played only a minimal role, as seen in other studies , 23. ThiOne major concern of PrEP availability is the potential for increasing sexual risk taking and hence for acquisition of sexually transmitted infections. Previous publications on trends in STI for Europe showed decreasing HIV incidences but increasing incidences for early syphilis, gonorrhea and chlamydia since 2014 which was partly explained by higher PrEP use . At baseThe SARS-CoV-2 pandemic had a drastic influence on social behavior and sexuality. Yet sexual contacts in private settings took place with equal risk of HIV transmission. Sentis et al. found a 56% reduction in STIs during the March/April lockdown in Spain . It was Interestingly, in our study, rates over time also differed between STIs. Infections with chlamydia decreased with the lockdowns in the second and fourth quarters of 2020 and increased with social opening in the third quarter. After the initial decrease, infections with gonorrhea, on the other hand, showed a steady increase in both the third and the fourth quarters. Infections with Syphilis differed again, with their nadir in the third quarter, likely reflecting its longer incubation time , 41, folWhen evaluating infection rates, it is important to consider testing rates. Testing for chlamydia/gonorrhea decreased in the second and fourth quarters, which may reflect the pandemic lockdowns. Syphilis testing was higher throughout and decreased over the course of 2020. Interestingly although numbers of tests differed, the positivity rates remained relatively constant. Other investigators have reported decreased testing for other infections, reduced use of health care as well as a marked reduction of spread of communicable diseases, including STI and HIV, in times of social lockdown , 43. In While almost one-half of chlamydia and gonorrhea infections were a result of screening of asymptomatic persons, this was the case for two-thirds of syphilis infections. The high rate of syphilis found solely by screening asymptomatic MSM highlights its importance in the prevention of transmission. The lower testing numbers for chlamydia/gonorrhea appear to reflect the differing approaches taken by physicians and the controversies surrounding testing and treating of asymptomatic individuals , 45.The significantly higher risk of infection with chlamydia/gonorrhea for MSM aged 16–29 years and the significantly lower risk for MSM aged 40–59 years when compared to 30–39 years may reflect higher sexual activity and more partners in younger age groups. Unexpectedly, age 50–59 years was associated with an increased risk for syphilis. This finding is not supported by the national mandatory syphilis reports, which show the highest incidence for men between 30–39 years of age . FurtherA higher risk for chlamydia/gonorrhea was found in MSM with PrEP start before its coverage by statutory health care in September 2019. It likely reflects these individuals’ involvement in the sexually active MSM community. In addition, this indicates persistent HIV risk behavior, which goes along with infection risks of other STIs. The lower risk for chlamydia/gonorrhea seen with intermittent/on-demand PrEP use, on the other hand, may in turn reflect less frequent high-risk sexual contacts.The lower risk for chlamydia/gonorrhea and syphilis during the COVID-19 pandemic period was likely caused by a number of factors as discussed above.As expected, symptom triggered testing and a history of STI was associated with a higher risk for infections with chlamydia/gonorrhea and syphilis. The latter supports the importance of comprehensive history taking before PrEP initiation.We found a high baseline protection against hepatitis A and B in approximately three-quarters of the cohort. Of note, immunity against hepatitis A may be in part self-reported and the actual percentage therefore lower supporting the call for serologic testing of immunity against hepatitis A prior to PrEP initiation. However, this is not covered by the German statutory health insurance nor recommended by the German/Austrian PrEP guidelines. Hepatitis B virus infection status on the other hand should routinely be determined prior to commencing PrEP according to the German/Austrian PrEP guideline due to the interaction between tenofovir and hepatitis B . AccordiOur study has several limitations: the evaluation period of sixteen months limits the ability to determine HIV and STI trends over time. Furthermore, detecting a hepatitis B/C infection can take several months after transmission. However, most of the participants were followed for a longer period with a median of 451 days within the observation period. The predefined four weeks for PrEP interruptions may have led to an underestimation of shorter PrEP interruptions. Also, the SARS-CoV-2 pandemic drastically affected social interaction and influenced the outcomes found in this study. Further investigation in a regular non-pandemic setting with a longer follow-up period would be valuable. The number of partners or sexual behaviors were not documented due to the retrospective chart review design of our study. Our population does not include all PrEP-users in Germany, nor all PrEP prescribing centers. For budgetary reasons participating centers could not document all PrEP users and were asked to include a random sample of their PrEP users into the study, however, no formal randomization process was implemented. This may have led to a selection bias. However, the fact that PrEP care is provided primarily by HIV-specialty care centers, many of which participated in this study with a German wide distribution and experience in clinical studies, lead us to assume that a representative sample was obtained. More than one-third of HIV-positive people receiving treatment in Germany and one-fourth of all PrEP users in Germany were cared for in the study centers , 14. In In Germany, HIV-PrEP with TDF/FTC is almost exclusively taken by MSM. We found a very low incidence of HIV infection in this cohort of PrEP users comparable to implementation projects in other countries. The main reason for PrEP failure was suboptimal adherence. We did not see an increase in infections with chlamydia, gonorrhea and syphilis; in fact, we saw a partial decrease in their incidence rates. The COVID-19 pandemic certainly influenced the findings of our study. Our results support the coverage of PrEP medication and care by statutory health insurance. Increased PrEP availability to people at risk for HIV infection through the elimination of barriers and improved access requires further attention. Further investigation and monitoring with a longer follow-up would be of value.Supplementary file1 (DOCX 26 KB)Below is the link to the electronic supplementary material.


The emergence of carbapenemase-producing bacteria (CPB) has become a major public health concern. Long-term care facilities (LTCF) are potential reservoirs for multidrug-resistant micro-organisms (MDRO). However, data on CPB is limited. The study aims to determine the prevalence of MDRO and risk factors for CPB colonization among residents of LTCFs.Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), carbapenemase-producing Enterobacterales, MDR Acinetobacter baumannii (MDR-Ab) and MDR Pseudomonas aeruginosa. Antimicrobial susceptibility testing and molecular detection of resistance genes were performed. Risk factors for colonization by carbapenemase-producing bacteria (CPB) were determined by univariate and multivariate analysis.A point-prevalence study was conducted at 14 LTCFs in Tenerife (Spain) between October 2020 and May 2021. Nasal and rectal swabs were cultured for methicillin-resistant K. pneumoniae, 26 (36.6%) MDR-Ab, 17 (23.9%) E. coli, and 1 (1.4%) C. koseri. In addition, the prevalence of colonization by MRSA was 28.1% (n = 215) and only one case of VRE was isolated. Multivariate analysis identified male sex , having a high health requirement  and previous hospitalization  as independent risk factors for CPB rectal carriage.A total of 760 LTCF residents were recruited. The prevalence of colonization by CPB was 9.3% (n = 71) with the following distribution: 35 (49.3%) LTCFs are an important reservoir for MDRO, including CPB. We have identified some predictors of colonization by CPB, which enable a more targeted management of high-risk residents. Antimicrobial stewardship programmes and infection control preventive measures are needed to stop acquisition and transmission of MDRO. Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase Enterobacterales (ESBL) [Enterobacterales (CPE), vancomycin-resistant Enterococcus spp (VRE), MDR Acinetobacter baumannii (MDR-Ab) and MDR Pseudomonas aeruginosa (MDR-Pa) [The emergence of multidrug-resistant organisms (MDROs) is a global public health problem . Initials (ESBL) . Recentl(MDR-Pa) –15. Spec(MDR-Pa) , 17.LTCFs provide residential healthcare for people with significant disabilities, chronic illness and elderly individuals who cannot care for themselves. These institutions are also the last medical resource for patients who have survived acute illnesses in hospitals. The increase in life expectancy and, therefore, ageing of the population has meant that LTCFs have become essential in the healthcare system. However, there is evidence that a stay in a LTCF is a risk factor for the carriage of MDROs , 19 and Antimicrobial stewardship programmes (ASP) have been widely implemented in hospitals , in addiThe aim of this study is to determine the prevalence of MDROs and risk factors for colonization by carbapenemase-producing bacteria (CPB) among LTCF residents in North Tenerife (Spain). In addition, the MDROs resistance mechanism was characterized in molecular terms.A multicentre point-prevalence study (October 2020–May 2021) was conducted at 14 LTCFs distributed throughout North Tenerife (Spain). In each LTCF, rectal and nasal swab were collected from all residents. The residents’ sociodemographic and clinical data were evaluated by means of a questionnaire. None of the LTCFs taking part had a specific action protocol to monitor and prevent MDRO transmission. A resident colonized by CPB was defined as a case, and a control was defined as those who were not colonized by CPB.All samples were analyzed at the Microbiology Service in Hospital Universitario de Canarias, which is the reference hospital in the northern area of Tenerife. Rectal swabs were cultured directly on selective chromogenic media ChromID® CARBA SMART and ChromID® VRE  and McConkey (bioMèrieux). Nasal swabs were cultured on ChromID® MRSA SMART (bioMèrieux) and inoculated into Brain–Heart Infusion Broth (bioMèrieux). They were reseeded in MRSA after 24 h of incubation in broth.Identification and antimicrobial susceptibility testing of the suspicious colonies were performed with the Vitek-II® system (bioMèrieux) and reduced susceptibility/resistance to imipenem or vancomycin was confirmed by Etest (bioMèrieux). Carbapenemase production was phenotypically tested by the agar tablet/disc diffusion method . Colistin resistance was tested by disk diffusion test and confirmed by broth microdilution . All results were analyzed and interpreted according to the European Committee on Antimicrobial Susceptibility Testing guidelines . ColonieEnterobacterales and P. aeruginosa; and to vancomycin for enterococcus  were genotypically characterized by multiplex polymerase chain reaction (PCR) AllplexTM Entero-DR Assay . Carbapenemase resistance in A. baumannii  and the detection of methicillin resistance genes for S. aureus  was characterized by isothermal amplification Eazyplex® with Superbug Acineto and MRSA reagents, respectively .Genes for resistance to carbapenems  for P ≤ 0.05.The sample collected from 71 cases and 689 controls offers the study a power of 90% in detecting a difference between cases and controls of relative frequencies for nominal variables of at least 20%; or 3 years for the age or days for the stay in ranges of 0–5 in bilateral tests of hypothesis at a level of statistical significance 5–P95) given its distance from a normal probability distribution verified with the Kolmogorov–Smirnov test.The characteristics of the sample as a whole are reported by summarizing its nominal variables with the frequency (relative frequency) of its component categories, and those of numerical scale with mean .All hypothesis contrast tests are two-sided at a level of statistical significance K. pneumoniae, 26 (36.6%) MDR-Ab, 17 (23.9%) E.coli and 1 (1.4%) C. koseri. In addition, 26 (36.6%) were also colonized by MRSA and two (2.8%) by MDR-Pa. Of the controls, 188 (27.3%), 12 (1.7%) and 1 (0.1%) were colonized by MRSA, MDR-Pa and VRE, respectively. The results obtained in terms of characterization of resistance mechanism are shown in Table Among 14 LTCFs (10 publics and 4 private), 764 residents were selected to participate in the study. However, we failed to obtain rectal samples from four residents, resulting in a total 760 residents included in the study (Table P = 0.025), have active infection (P = 0.025), urinary incontinence (P = 0.044), faecal incontinence (P = 0.014), previous antibiotic use (P = 0.040), high/medium health requirement (P = 0.005/0.05), prior hospital admission within the last 3 months (P = 0.002) and previous MDRO (P = 0.013).The clinical and epidemiological characteristics of the two groups are shown in Table P = 0.01), high/medium health requirement  and previous hospitalization .On multivariate analysis Table , the onlThis study was conducted due to the increasing prevalence of infections by CPBs in acute care hospitals in our geographical area. We sought to know whether these LTCFs also constitute a reservoir of CPB, in addition to identifying risk factors for colonization by CPB.In this multicentre point-prevalence study, a remarkably high rate of colonization by CPB was observed among LTCF residents in North Tenerife. In the literature, there are few recent studies about CPB in Europe showing a high geographical variation , 12. MosOur multivariate analysis identified male sex, a high or medium health requirement and previous hospitalization as important risk factors for CPB rectal colonization. Several studies have identified male sex as a risk factor for MDRO colonization , 28. HowEstablishing a level of dependence for the probability of MDRO colonization is difficult, mainly due to the variability of scores used across the studies. Some commonly used methods are Katz, Barthel, Karnofsky or the French index. In our study, all LTCFs have a bed distribution according to health requirements  and, therefore, residents were classified according to the criteria of the centres themselves. Despite the lack of a dependence threshold related to MDRO colonization, there appears to be compelling evidence to indicate that an increased level of dependence is associated with an increased risk of being colonized by MDROs .Several previous studies have identified prior hospital admission as a risk factor for MDRO colonization , 29. DepOur univariate analysis identified active infection, urinary and faecal incontinence, previous antibiotic use and previous MDRO colonization as factors associated with colonization by CPB. However, multivariate analysis did not reveal these to be independent risk factors. These are traditional factors associated with MDRO colonization in the literature and previous use of antibiotics is the main associated factor , 31–33.Since the aim of the study was to assess colonization factors exclusively by CPB and control residents by MDR-Pa and VRE were scarce, we decided to include them in the statistical analysis. Nevertheless, we performed this analysis without including theses control residents and obtained the same significant risk factors.This study has several limitations. First, as the study was performed during the COVID-19 pandemic, we had enormous difficulty accessing the centres for sample collection and filling out the questionnaires, delaying the study deadlines. Second, the cross-sectional survey design did not enable us to investigate the dynamic of MDRO colonization . Third, disk diffusion by colistin did not allow the detection of resistance to this antibiotic since there are not breakpoints for EUCAST and CLSI, requiring confirmation by another technique.Our study documents a high prevalence of colonization by MDROs, including CPB, among LTCF residents in North Tenerife. This emphasizes the role of these centres as reservoirs for MDROs. Male sex, a high health requirement and prior hospital admission were all identified as independent risk factors for CPB rectal colonization. These results strengthen the importance of establishing a standardized protocol to manage colonized patients between acute hospital centres and LTCFs. In addition, antimicrobial stewardship programmes and infection control preventive measures accounting for related risk factors in LTCFs are required to stop the acquisition and transmission of MDROs in healthcare facilities.


The aim of this study was to evaluate initial antimicrobial therapy in catsdiagnosed with upper or lower bacterial urinary tract infections atveterinary practices in the USA and Canada.Electronic medical records from a veterinary practice corporation withclinics in the USA and Canada were queried between 2 January 2016 and 3December 2018. Feline patient visits with a diagnosis field entry of urinarytract infection, cystitis and pyelonephritis, as well as variation of thosenames and more colloquial diagnoses such as kidney and bladder infection,and where an antimicrobial was prescribed, were retrieved.Prescription data for 5724 visits were identified. Sporadic cystitis was themost common diagnosis (n = 5051 [88%]), with 491 (8.6%) cats diagnosed withpyelonephritis and 182 (3.2%) with chronic or recurrent cystitis. Cefovecinwas the most commonly prescribed antimicrobial for all conditions, followedby amoxicillin–clavulanic acid. Significant differences in antimicrobialdrug class prescribing were noted between practice types and countries, andover the 3-year study period. For sporadic cystitis, prescription ofamoxicillin–clavulanic acid increased significantly and cefovecin decreasedbetween 2016 and 2018, and 2017 and 2018, while fluoroquinolone useincreased between 2017 and 2018.The results indicate targets for intervention and some encouraging trends.Understanding how antimicrobials are used is a key component ofantimicrobial stewardship and is required to establish benchmarks, identifyareas for improvement, aid in the development of interventions and evaluatethe impact of interventions or other changes. Antimicrobial stewardship aims to optimize antimicrobial therapy in patients,maximizing clinical outcomes, while minimizing adverse effects on the patient  or population . A core aspect of antimicrobial stewardship isunderstanding how antimicrobials are used, as this is required to assessanti-microbial use practices, establish benchmarks and targets, identify areas forimprovement, develop interventions, and evaluate the impact of interventions orother changes.Infectious urinary tract disease is commonly diagnosed in cats, with resultant commonuse of antimicrobials.The objective of this multicenter study was to evaluate initial antimicrobial therapyin cats diagnosed with upper or lower bacterial urinary tract infections at a subsetof veterinary practices in the USA and Canada.Electronic medical records from a veterinary practice corporation with clinics in theUSA and Canada were queried between 2 January 2016 and 3 December 2018. Felinepatient visits with a diagnosis field entry of urinary tract infection, cystitis andpyelonephritis, as well as variation of those names and more colloquial diagnosessuch as kidney and bladder infection, and where one or more antimicrobial wasprescribed, were retrieved. Based on free-text entries in the clinical diagnosisfield code, cases were classified by one author (JSW) into sporadic bacterialcystitis, chronic or recurrent cystitis (referred to herein as recurrent cystitis),pyelonephritis and other, with no further study of the ‘other’ group. Because therewere numerous descriptions in the open-entry field, a subjective determination forclassification was required in some cases. This was performed while blinded to otherdata fields . ‘Suspect’ or ‘possible’ entries were includedbecause they were accompanied by an antimicrobial prescription for that condition.When entries indicated the potential presence of a comorbidity that might impactantimicrobial decision-making  the record wasremoved. A repeat visit of the same animal within 30 days was removed unless therewas a different field code , on the assumption that a new treatment decisionwould have been made for the second disease occurrence.Signalment, body weight, practice location , visit date,diagnosis and antimicrobial drug regimens were recorded. Duration of treatment wasdetermined based on specific prescription recommendations in the record , or, when that was not specified, calculated based on therecommended dose  and the amount of drug thatwas dispensed. If inadequate detail was present to accurately determine duration,the duration field was left blank, but records were retained for analysis of drugselection. Each dose of cefovecin was considered to be a 14-day treatment duration. assigning antimicrobials to highest priority, critically importantantimicrobial (HP-CIA); critically important antimicrobial (CIA); and highlyimportant antimicrobial (HIA) groups. Prescription practices were compared with 2011International Society for Companion Animal Infectious Diseases (ISCAID) guidelines. For sporadic bacterial cystitis, two analyses were performed. One consideredamoxicillin and potentiated sulfonamides as first-line recommendations, while thesecond added amoxicillin–clavulanic acid, based on the guideline statement that thedrug is ‘acceptable’.The antimicrobial(s) prescribed and duration of treatment were the outcomes ofinterest. Antimicrobial use analysis was performed at the drug-class level . Antimicrobials were also categorized asper World Health Organization criteria,2tests , whilecontinuous data were analyzed using Wilcoxon, Steel or Steel–Dwass tests. Oddsratios (OR) and accompanying 95% confidence intervals (95% CI) were calculated. Datawere analyzed using JMP15 (SAS Institute). For all analyses, P<0.05 was considered significant.Continuous data were evaluated for normality using Shapiro–Wilk (for sample sizes⩽2000) or Kolmogorov–Smirnov test with Lilliefors correction tests (for sample sizes>2000). Non-normally distributed data were reported as median and interquartilerange (IQR). Categorical data were assessed using Fisher’s exact or χPrescription data for 5724 visits were identified. Data were from 610 clinics: 561(92%) from the USA and 49 (8.0%) from Canada. Most cat visits (n = 5028 [88%]) wereto general practices, while 301 (5.3%) were to mixed general and specialty practices(referred to henceforth as ‘combined clinics’), 218 (3.8%) were to specialtypractices, 103 (1.8%) were to feline-only practices and 74 (1.3%) were to emergencyclinics. Feline-only and specialty clinics were only represented in 2017 and 2018.Most (n = 5515 [96%]) visit records were from the USA, while 209 (3.6%) were fromCanada.Sporadic cystitis was the most common diagnosis (n = 5051 [88%]), with 491 (8.6%)cats diagnosed with pyelonephritis and 182 (3.2%) with chronic or recurrentcystitis. In 2016, 1246 (22%) cat visits resulted in the prescription ofantimicrobials, which increased to 2226 (39%) in 2017 and 2252 (39%) in 2018. Atotal of 6112 individual antimicrobial prescriptions were identified . Overall, 5370 (93.8%) of cats were prescribed a singleantimicrobial, while combination therapy was prescribed to 354 (6.2%). One or moredrugs in the HP-CIA class were prescribed to 4319 (75%) of cats. The most commonlyprescribed antimicrobials and combinations are presented in There were 4873 (96%) cats from the USA and 178 (3.5%) from Canada with sporadicbacterial cystitis, from general practices (n = 4525 [90%]), combined clinics(n = 243 [4.8%]), specialty clinics (n = 157 [3.1%]), emergency clinics (n = 72[1.4%]) and feline-only clinics (n = 54 [1.1%]). The median age of the cats was8.0 years (IQR 9.2). Most (n = 2811 [56%]) were spayed females, 1968 (39%) werecastrated males, 82 (1.6%) were intact females and 68 (1.3%) were intact males.Sex was not reported for 122 cats (2.4%). One thousand and ninety-three (22%)cats were from 2016, 1953 (39%) were from 2017 and 2005 (40%) were from2018.A total of 15 antimicrobials were prescribed; however, only cefovecin,amoxicillin–clavulanic acid, orbifloxacin, marbofloxacin and enrofloxacinaccounted for at least 1% of prescriptions each. The most commonly prescribedantimicrobials and combinations are presented in There were significant inter-year differences in the relative use of amoxicillin,amoxicillin–clavulanic acid, cefovecin and fluoroquinolones, as well as overalluse of HP-CIAs , Table2P <0.0001), amoxicillin(P <0.0001), fluoroquinolones(P = 0.0002) and cefovecin (P <0.0001).HP-CIAs were prescribed during 36% (n = 57/157) of cat visits at specialtypractices, 47% (n = 114/243) at combined practices, 77% (n = 3503/4525) atgeneral practices, 89% (n = 48/54) at feline-only clinics and 93% (n = 67/72) atemergency clinics (P <0.0001)Prescription of the most common antimicrobials by clinic type is presented inP <0.0001) and less of amoxicillin–clavulanic acid  compared with Canadianpractices, with no difference in amoxicillin (P = 0.40) orfluoroquinolones (P = 0.21). Prescription of HP-CIAs was morecommon in the USA than in Canadian practices .National comparison (USA vs Canada) was only performed using general practices,owing to small sample sizes of the other clinic types. In the USA, there wassignificantly more prescriptions of cefovecin  overall. Prescriptions for cefovecin were of asignificantly longer duration compared with all other prescriptions . When cefovecin prescriptions areexcluded, there was a non-significant trend to decreased duration in 2018 vs2017 The median duration of prescribed therapy was 14 days (IQR 0) overall, and foreach year. There was no impact of visit year on duration(P = 0.014), feline , emergency  and general  clinics. There was also a significantly shortertreatment duration for combined clinics compared with emergency(P = 0.003), feline (P = 0.003) andgeneral practices (P <0.001).There was a significant difference in duration between specialty clinics (10days) and combined .There was a significantly shorter prescribed duration in Canada  than the USA  . There was increased consistency with the 2011guidelines over the study period (P = 0.005), increasing from0.64% (n = 7/1093) in 2016 to 1.2% in both 2017 (n = 24/1953) and 2018(n = 25/2005).Only 56 (1.1%) of prescriptions were consistent with the 2011 ISCAID guidelines(7 days of treatment with amoxicillin or trimethoprim–sulfonamide).Network analysis, indicating antimicrobial combination relationships, ispresented in There were 468 (95%) cases of pyelonephritis from the USA and 23 (4.7%) fromCanada, from general practices (n = 333 [68%]), specialty clinics (n = 57[12%]), combined clinics (n = 53 [11%]), feline-only clinics (n = 46 [9.4%]) andemergency clinics (n = 2 [0.4%]). Owing to the small sample size, emergencyclinics were excluded from analysis of the impact of clinic type.The median age of the cats was 13.7 years (IQR 6). Most (n = 319 [65%]) werespayed females, 157 (32%) were castrated males, five (1.0%) were intact malesand five (1.0%) were intact females. Sex was not reported for five (1.0%) cats.One hundred (20%) cases were from 2016, 204 (42%) were from 2017 and 187 (38%)were from 2018.Forty-nine percent (n = 243/491) of cats were prescribed a fluoroquinolone, aloneor in combination with another antimicrobial, while 34% (n = 169/491) of catswere prescribed cefovecin. The most commonly prescribed antimicrobials andcombinations are presented in P <0.0001) and cefovecin(P <0.0001) (P <0.001). There was a significant decrease inprescription of cefovecin between 2016 and 2018  and a significant increase inamoxicillin–clavulanic acid use between 2017 and 2018 . There were no other significant differencesbetween the use of the main drug classes or HP-CIAs over time.There was an impact of clinic type on the use of amoxicillin–clavulanic acid(<0.0001) . CefovecP = 0.62). There was no identifiable associationbetween duration and clinic type  or country(P = 0.14).The median duration of treatment was 14 days (IQR 14). This did not change from2016 to 2018 (P = 0.16) or clinic type (P = 0.19)Ten percent (n = 49/491) of prescriptions were consistent with the 2011 ISCAIDrecommendations of 4–6 weeks for a fluoroquinolone. This did not vary with year(Recurrent cystitis was seen in 174 (95.6%) cats from the USA and eight (4.4%)from Canada. One hundred and seventy (93.4%) were from general practices, five(2.8%) from combined clinics, four (2.2%) from specialty clinics and three(1.7%) from feline-only clinics. The median age of the cats was 10.5 years (IQR9.8). Most (n = 119 [65%]) were spayed females, 53 (29%) were castrated males,two (1.1%) were intact females and two (1.1%) were intact males. Sex was notreported for six (3.3%) cats. Fifty-three (29%) cases were from 2016, 69 (38%)were from 2017 and 60 (33%) were from 2018. Analyses involving country or clinictype were not performed because of the small sample sizes.The most common drugs and combinations are presented in P = 0.22), clinic type  or country (P = 0.087) onduration.The median prescribed duration was 14 days . There was noimpact of visit year , this study identifies both areas forintervention and some positive indicators, particularly for the treatment ofsporadic cystitis. These positive indicators include increasing consistency withISCAID guidelines over the 3-year study period, with less use of third-generationcephalosporins and fluoroquinolones, and a suggestion of decreasing duration oftreatment. These changes were of relatively low magnitude, so, while encouraging,there is still a need for interventions pertaining to drug selection and treatmentduration. Comparison with ISCAID guidelines is interesting but must done cautiouslyas patient-level considerations could not be assessed . ISCAID guidelines are also largely expertopinion-based and, while useful, they do not necessarily reflect optimal treatmentsand some recommendations have changed  between the 2011guidelines that were relevant at the time of this study and the subsequent 2019guidelines. Regardless, there were clear differences in the prescription patternsnoted here compared with the recommended first-line antimicrobials.9 Similarly, cefovecin accountedfor 10–26% of prescription for urinary tract disease.10 It is reasonable to suspectthat a long-acting injectable drug such as cefovecin was often not necessary, butdifferent approaches are needed to study that. Additionally, there are efficacyconcerns associated with use of cefovecin for pyelonephritis. While cefovecinachieves high levels in urine, pyelonephritis is an infection of renal tissue, noturine. As a highly protein-bound drug, there is limited active free drug in tissue. There are no Clinical and Laboratory StandardsInstitute (CLSI) breakpoints for Enterobacterales in tissue, because of thequestionable ability of cefovecin to reach effective concentrations forEnterobacterales in tissue. Since Enterobacterales, particularly Escherichiacoli, are the most commonly diagnosed causes of pyelonephritis,cefovecin is not an appropriate choice for pyelonephritis. While the decrease in useof cefovecin for pyelonephritis during the study period was encouraging, there was aconcurrent increase in use of amoxicillin–clavulanic acid monotherapy, somethingthat is also of concern. While potentiated penicillins are excellent for bacterialcystitis because of the high drug levels in urine, CLSI guidelines indicate that Enterobacterales should be reported as resistant toamoxicillin–clavulanic acid for infections outside of the lower urinary tractbecause inadequate drug levels are achieved in tissue.Frequent use of cefovecin was not surprising, as the ease of use of thissingle-injection treatment is particularly appealing for cat owners. Yet, as athird-generation cephalosporin that is expected to provide 14 days of therapeuticeffect in urine, it is broader spectrum and longer in duration than is needed fortreatment of acute cystitis. The need for its use could not be investigated becausereasons for drug choice  were not available in the medicalrecord. Frequent use of this drug has been reported in other studies. In studies ofprimary care veterinary clinics in Canada, the UK and Australia, cefovecin accountedfor 15–54% of antimicrobial prescriptions.10 While excellentantimicrobials for bacterial urinary tract disease, fluoroquinolones are notrecommended first-choice treatments for cystitis.5 Use of the third-generationcephalosporin and fluoroquinolone drug classes is under scrutiny in veterinarymedicine, and further study of reasons for use of cefovecin, fluoroquinolones andHP-CIAs, in general, is required. Some use is indicated based on an inability totreat the cat orally  or because of a need for a once-daily treatmentregimen , but differentiating need from otherreasons is required for assessment of the indications for use and development ofeffective interventions. Reasons for use of different treatment regimens could notbe assessed in this study, but it is likely that convenience rather than need drivesfrequent use of cefovecin. This assumption is supported by the concurrentprescription of cefovecin and oral antimicrobials in a small percentage of cats,indicating that an inability to administer oral medications was not the reason forcefovecin use in those cases.Consistent with the results of this study, fluoroquinolone use has also been widelyreported in cats, both overall and for lower urinary tract disease. similar to the results of this study. A median of 10 days was prescribed whenoral medications were used, but the frequent prescription of cefovecin resulted in amedian duration of 14 days. The 10-day duration is consistent with 2011 ISCAID guidelines but higher than the 3–5-day duration recommended most recently. Shorter durations can be beneficial from many standpoints, including adverseevent risks, stress of handling and pilling (to both cat and owner), and reducedantimicrobial resistance selection pressure. Short durations facilitate the use oforal treatments by reducing the amount of effort that is required for oraltreatment. Owner preference (vs need) should not be the deciding factor whenchoosing a short course of a lower-tier drug vs an injectable third-generationcephalosporin.There has been limited study of duration of treatment of urinary tract disease incats. A Swiss study reported a median duration of 10 days for the broad category offeline lower urinary tract disease,Numerous differences were identified in prescriptions between different clinic types,with greater use of oral treatments  andshorter prescribed durations at specialty clinics. The different nature of caseloadsbetween clinic types must be considered; however, it would be reasonable to expectthat specialty clinics would deal with more complex or compromised cases, biasingtoward longer durations or use of broader spectrum antimicrobials, not the opposite.Clinics that have both primary and specialty care had results that were subjectiveintermediary between primary care and specialty clinics. This is unsurprising butperhaps supports the apparent differences between approaches in primary andspecialty care. Reasons for this are unclear and could not be investigated withthese data, but could include greater awareness of guidelines, greater confidencechanging prescribing behaviors and differences in client interactions .The differences in treatment duration and drug selection (particularly the use ofcefovecin) between prescribing in Canada and the USA were interesting. Reasons fordifferences between prescribing practices in Canadian and US clinics deserve furtherinvestigation and could relate to differences in veterinary education, continuingeducation, awareness of antimicrobial stewardship, awareness of guidelines andperceptions about clients’ expectations. However, there is still room for improvement, both in terms of the frequencyof use of fluoroquinolones overall and the use of enrofloxacin.From an adverse event risk standpoint, it was encouraging to observe limited use ofenrofloxacin vs other fluoroquinolones, based on safety issues that can be associated with use of enrofloxacin, but not other fluoroquinolones, in cats.The decrease in HP-CIA use from 2016 to 2018 for sporadic bacterial cystitis notedhere was driven by reductions of cefovecin, and is encouraging, though there was anincrease in fluoroquinolone use that raises concern. This likely reflects somechanges from cefovecin to fluoroquinolones, and highlights the potential for furtherimprovement by changing from oral fluoroquinolones to lower-tier options .Medical records-based studies such as this have many inherent limitations based ongaps in the available data. Factors that might have influenced antimicrobial choiceswere not typically available, apart from text in the diagnostic field box. Whileguidelines provide good guidance, they cannot indicate what should be done in allcases, and individual patient factors can result in logical deviations in drug orduration choices from existing guidelines. Culture data were also not available.Anecdotally, bacterial culture is only performed in a minority of cases,particularly sporadic cystitis. Lack of culture data has some impact on theassessment of drug choice. However, antimicrobials were prescribed at the time ofexamination, when culture results were not available. an earlier comparative study of lower urinary tract infection in cats where14 days of cephalexin was used as the comparison with cefovecin and the recommendation of a redosing interval of 7–14 days in the 2019 ISCAID guidelines.Assigning a duration for cefovecin use in surveillance studies can be challenging.Here, 14 days was chosen based on previously published recommendations,15 and probably accounted formany  cases in this data set. However, theobjectives of this study were to assess antimicrobial use practices when aveterinarian had decided to treat. Therefore, the inclusion of cats that did notactually have an infection or that were misclassified  does not impact assessment of clinician treatment choices. Similarly, somecases were reported as ‘possible’ or ‘suspected’ infections but were includedbecause an antimicrobial was prescribed. Changes in diagnoses made after the resultsof diagnostic testing were also not identifiable. This could have influencedduration, if antimicrobial courses were extended based on testing results. However,it still reflects how antimicrobials were initially prescribed, even if the reasonswere not clear. There was a need to interpret disease categorization in somesituations. While there were likely some errors in categorization, it is assumedthat they represented a small percentage of cases. If these resulted in outliers,the non-parametric analyses would limit their impact.Overuse of antimicrobials in cats with non-infectious urinary tract disease  is likely commonUnderstanding how antimicrobials are used is a key component of antimicrobialstewardship and is required to establish benchmarks, identify areas for improvement,aid in the development of interventions and to evaluate the impact of interventionsor other changes.


In univariate analyses, gross total resection (p = .014) and high-grade histology in MM were associated with a prolonged time to progression (p < .001). Most clinical characteristics and rates of high-grade histology were similar in both groups . Multivariate analyses showed synchronous/metachronous meningioma growth  as an independent predictor for progression. Compared to SM, risk of progression was similar in cases with two , but exponentially raised in patients with 3–4  and ≥ 5 tumors . Clinical and histological characteristics and risk factors for progression do not relevantly differ between SM and MM. Although largely constant, histology and WHO grade occasionally intra-individually vary in MM. A distinctly higher risk of disease progression in MM as compared to SM might reflect different underlying molecular alterations.Synchronous or metachronous growth of multiple tumors (≥ 2) is found in up to 20% of meningioma patients. However, biological as well as histological features and prognosis are largely unexplored. Clinical and histological characteristics were retrospectively investigated in 95 patients harboring 226 multiple meningiomas (MMs) and compared with 135 cases of singular meningiomas (SM) using uni- and multivariate analyses. In MM, tumors occurred synchronously and metachronously in 62% and 38%, respectively. WHO grade was intra-individually constant in all but two MMs, and histological subtype varied in 13% of grade 1 tumors. MM occurred more commonly in convexity/parasagittal locations, while SM were more frequent at the skull base ( NF2 or SMARCB1 as well as their monoclonal origin [Meningiomas are the most common primary intracranial neoplasms and occur in multiple intracranial or spinal locations (≥ 2) in one individual in up to 20% , 20, 31.l origin , 30, 33.l origin , 15, 23.Beyond pathophysiology and pathogenesis, treatment of MM remains a key challenge during neuro-oncological care. Few previous studies showed increased recurrence rates in MM as compared to singular lesions, suggesting different biological behavior and worse overall prognosis , 20, 35.In this series, we therefore analyzed histological and clinical characteristics of patients suffering from sporadic neuropathologically confirmed synchronous or metachronous meningiomas and additionally provide comparative analyses with individuals harboring singular intracranial lesions.Imaging, medical, and operative reports from all patients who underwent surgery for neuropathologically confirmed meningioma in our department , between 1991 and 2018, were reviewed. MM was classified in case of ≥ 2 histopathologically confirmed meningiomas or meningioma-suspicious intracranial lesions at the date of index surgery (synchronous) or during follow-up (metachronous MM). Cases with multiple tumors arising from the resection cavity/dura attachment after surgery for a single lesion were not included. Radiological diagnosis of meningioma not subjected to surgery was established in patients with the history of at least one neuropathologically confirmed meningioma and distant, synchronously or metachronously growing contrast-enhancing, extra-axial lesions. In surgically treated cases, meningioma diagnosis and grading were performed according to the 2016 classification of brain tumors in all cases : patientInitial routine postoperative MRI was scheduled 3 months after surgery and, in case of an event-free course, repeated in 12- and 6-month intervals in grade 1 and 2/3 lesions, respectively. After a progression-free interval of 5 years, follow-up imaging intervals were extended to 24 months in grade 1 and 12 months in grade 2/3 lesions [U test. Time to progression (TTP) was defined as the duration between index surgery of surgically treated meningiomas or initial diagnosis of non-surgically treated lesions and radiologically confirmed tumor recurrence/progression or, in case of an event-free follow-up, to the date of last follow-up. TTP was analyzed by the Kaplan–Meier method and compared by Log-rank tests.Statistical calculations were performed using statistic software . Data are characterized by standard statistics: Categorical variables are described by absolute and relative frequencies and compared by Fisher’s exact test, while continuous variables are described by median and range and compared by Mann–Whitney p values. All reported p values are two-sided and considered statistically significant when < 0.05. Data collection and scientific use were approved by the local ethics committee (Münster 2018–061-f-S) and in accordance with the principles of the Declaration of Helsinki (human rights).Multivariate Cox regression analyses for TTP included patients’ age, sex ), tumor location (convexity = ref), histology ), and the extent of resection (GTR = ref). The results are characterized by hazard ratios (HR), 95% confidence intervals (CIs), and Wald-test Using the above-described approach, of 1404 patients in the Muenster Meningioma Database, 95 individuals (7%) with sporadic multiple meningiomas, harboring a total of 226 lesions (mean: 2.4 tumors per patient), were identified, while 5 patients suffering from NF1/2 and 6 individuals who received cranial irradiation during childhood were excluded. The number of patients decreased as the number of tumors per patient increased . Similarly, high-grade histology was not related with tumor location (p = 0.099) or KPS at the time of initial or last presentation (p = 0.295). However, mean KPS at the date of the last follow-up was lower in patients with high-grade meningiomas than in individuals with benign lesions . Noteworthy, neuropathological analyses revealed a constant WHO grade among all analyzed samples except two patients. Those included two male patients, one suffering from six tumors, subsuming one benign, three high-grade, and two non-operated meningiomas  and was found in nine of 85 synchronous and nine of 47 metachronous lesions . Mean KPS at the date of last follow-up was 70 (SD ± 20) in patients with and 90 (SD ± 20) without tumor progression (p = 0.002). Progression was observed in 21 of 180 tumors in females but in 13 of 46 lesions in male patients . No correlations were found between progression and age at diagnosis (p = 0.662). Progression rates  and TTP  were similar comparing synchronously and metachronously diagnosed meningiomas. Among the 132 operated cases, the risk of progression did not significantly differ comparing GTR and STR , while the median TTP was shorter after STR than after GTR . Moreover, 72% of the high-grade but 15% of the grade 1 meningiomas developed disease progression , and TTP was shorter in grade 2/3 than in grade 1 lesions , tumor progression occurred in 34 of all operated and non-operated 226 MMs (15%). Progression was observed in 30 of 132 surgically treated lesions but in four of 90 lesions initially subjected to follow-up imaging alone . Frequency of high-grade histology was 7% in skull base meningiomas (N = 5 of 76) but 18%, 22%, 13%, and 100% in convexity (N = 7 of 38), parasagittal/falcine (N = 5 of 23), spinal (N = 1 of 8), and intraventricular (N = 1 of 1) tumors, respectively (p = 0.024). High-grade histology was also more commonly found in males , and not related with KPS prior index surgery (p = 0.174) or at the time of last follow-up (p = 0.139).Table p = 0.011), while sex distribution (p = 0.165), the extent of resection (p = 0.576), and the mean KPS at the dates of presentation (p = 0.161) and last follow-up (p = 0.439) did not significantly differ between patients with SM and MM. Tumor locations differed significantly comparing MM and SM . In contrast to SM, MM were lacking angiomatous, microcystic, psammomatous, chordoid, or anaplastic histology . Risk factors for progression in SM are summarized in Table Within a median follow-up of 78 months (range: 37–252 months), progression was observed in 21% of the patients with SM , synchronous or metachronous meningioma growth  and, with borderline significance, STR  as independent predictors for progression. Correspondingly, median TTP was distinctly lower in patients with multiple as compared to cases with singular lesions  meningiomas, but an exponentially increasing risk of progression in patients with 3–4 lesions  ≥ 5 tumors .Cumulative multivariate analyses Maiuri et al. [Frequency of MM (≥ 2) in our cohort was 7%, which matches the broad range of multiple meningioma incidence reported in previous series , 29, 31.i et al. .High-grade histology was found in 13% of all operated MM in our study, which was in line with the control cohort of SM and with rates reported in previous studies on MM . In consRamos-Fresnedo et al. [exponential increase is unexpected and contradicts this explanation. Analyses further elucidating underlying molecular alterations in MM, however, remain sparse. Small series and case reports showed mutations or chromosomal losses of NF2 or SMARCB1 in MM, as well as distinct somatic mutations in samples of different meningiomas of one individual [Progression of MM was observed in 16% and was associated with a reduced KPS at the date of last follow-up. Similar to observations in meningiomas in general, rates of progression were higher in males than in females, eventually reflecting a higher incidence of high-grade histology among the first . Correspo et al. . As thiso et al. , 26, 32.dividual , 30, 33.The authors are aware of some limitations of the study. Although providing analyses in a large series, our study suffers typical shortcomings from retrospective studies, e.g., selection bias. In addition, complexity of treatment of individual patients with MM causes data heterogeneity and could not be sufficiently considered in statistical analyses. For the same reason, further imaging data could not be subjected to statistical analyses. The number of patients suffering from extensive tumor burden  was low, potentially limiting interpretation of our results. Eventually, treated tumors display different biological behavior and characteristics  as compared to lesions simply subjected to observation, and cumulative analyses might have led to additional bias. Further details about irradiation were not sufficiently available and therefore not subjected to statistical analyses. Finally, samples were neuropathologically diagnosed according to the 2016 WHO classification of brain tumors , and molIn conclusion, patients with MM were found to lack distinct clinical characteristics as compared to SM. A potential predominant location of MM in non-skull base positions remains to be further investigated. Noteworthy, rates of high-grade tumors did not differ from SM, suggesting alternative underlying, e.g., molecular alterations promoting multiple tumor growth. Risk of progression in MM was generally increased as compared to SM, and exponentially raised with the number of lesions per patient.


In additional studies recombinant alkaline phosphatase was given after IRI. In an AKI-on-chronic kidney disease (CKD) ischemic rat model, ilofotase alfa was given after the three instances of IRI and rats were followed for 56 days. Ilofotase alfa in a dose dependent manner decreased IRI in WT mice, an effect prevented by ZM241,385 and partially prevented in –/–Adora2a mice. Enzymatically inactive ilofotase alfa was not protective. Ilofotase alfa rescued –/–CD73 mice, which lack a 5′-ectonucleotidase that dephosphorylates AMP to adenosine; ZM241,385 inhibited that protection. In both rats and mice ilofotase alfa ameliorated IRI when administered after injury, thus providing relevance for therapeutic dosing of ilofotase alfa following established AKI. In an AKI-on-CKD ischemic rat model, ilofotase alfa given after the third instance of IRI reduced injury. These results suggest that ilofotase alfa promotes production of adenosine from liberated ATP in injured kidney tissue, thereby amplifying endogenous mechanisms that can reverse tissue injury, in part through A2AR-and non-A2AR-dependent signaling pathways.Adenosine triphosphate (ATP) released from injured or dying cells is a potent pro-inflammatory “danger” signal. Alkaline phosphatase (AP), an endogenous enzyme that de-phosphorylates extracellular ATP, likely plays an anti-inflammatory role in immune responses. We hypothesized that ilofotase alfa, a human recombinant AP, protects kidneys from ischemia-reperfusion injury (IRI), a model of acute kidney injury (AKI), by metabolizing extracellular ATP to adenosine, which is known to activate adenosine receptors. Ilofotase alfa (iv) with or without ZM241,385 (sc), a selective adenosine A Acute kidney injury (AKI) can result from a variety of causes and is associated with high morbidity and mortality . The incEscherichia coli-induced sepsis and cecal ligation puncture-induced sepsis), administration of AP from human placenta  is one candidate molecule with the potential to inhibit multiple inflammatory signals relevant to sepsis, sepsis-associated AKI, and other forms of AKI. AP isoforms exert detoxifying effects through dephosphorylation of a variety of soluble inflammatory mediators and other deleterious compounds. In animal models of sepsis  for treatment of sepsis and sepsis-associated AKI was investigated in two small Phase 2 clinical trials and revealed improved kidney function and better survival , 15. To In addition to preclinical studies evaluating AP for efficacy in treating sepsis and sepsis-associated AKI , pr–/–CD73 mice subjected to sub-threshold ischemic conditions  underlying the therapeutic efficacy of AP have mostly focused on its ability to inactivate damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), thereby reducing inflammation. However, direct kidney protective effects during injury and disease may involve other mechanisms and other endogenous AP substrates. Adenosine triphosphate (ATP), released from damaged and dying cells into the extracellular space during tissue injury, can bind to P2 receptors and produce detrimental effects or can be a substrate for extracellular conversion to adenosine . APs, annditions . This ph2A receptor (A2AR) activation inhibits kidney injury and reduces inflammation in kidney IRI. For the present manuscript, we hypothesized that recAP’s ability to metabolize released extracellular ATP, ADP, or AMP to adenosine  mice and –/–CD73 mice, we show that AR signaling is a component of recAP’s mechanism of action and that recAP restores the deficit in adenosine production and anti-inflammatory AR signaling in –/–CD73 mice. Taken together, these results implicate the augmentation of adenosine signaling as a key component of the mechanism of action of ilofotase alfa in rodent kidney IRI and provide additional support for the potential of ilofotase alfa as a treatment option for AKI.We –32 and odenosine , 19, 24 –/–Adora2a mice  mice  and n et al.  and now n et al.  and kidnTM1LftNt5e/J (CD73–/–) mice  were originally provided by Linda Thompson  and were also purchased from The Jackson Laboratory, stock no. 018986; Jackson C57BL/6J WT mice and CD73 wild-type littermates (CD73+/+) were used as controls. An ischemic time of 20 min was used for these experiments based on prior studies  and surgery (sham vs. IRI), and the experimenter was blinded for treatment (solutions had coded labels), creatinine assay and histological scoring. Sets of control and treatment groups were always performed on the same day.2/50 μm ZnCl2, pH 7.0, were stored at −80°C and diluted (1:35) immediately before use with sterile saline to yield a working solution for the highest dose of recAP ; serial dilutions were made with a comparable dilution of the stock vehicle  to yield working solutions for injections (i.v.) with the identical composition of vehicle components. Final vehicle composition of all working recAP solutions and the solution that was used as the administered vehicle control was 0.57 mM sodium citrate/7.14 mM D-sorbitol/57 μM MgCl2/1.43 μm ZnCl2, pH 7.0.Stock solutions of human recAP , prepared in a stock vehicle of 20 mM sodium citrate/250 mM D-sorbitol/2 mM MgClA recombinant catalytically inactive mutant form of human recAP , which has been used previously , was gen–/–CD73 mice and for pharmacokinetic studies, ZM241,385  was prepared immediately before use by dissolving in PEG-400 followed by slow sequential addition of Cremophor EL then methylcellulose to yield final proportions of 15% (v/v) Cremophor EL /30% (v/v) PEG-400 (Sigma-Aldrich)/55% (v/v) 0.5% methylcellulose in DI water. Working solutions of ZM241,385 and vehicle were prepared immediately before use by dilution of stock solutions with sterile saline.For experiments in WT mice, the AR antagonist, ZM241,385 was prepared as a stock solution in DMSO (stored at −20°C). For experiments in ZM241,385 was administered subcutaneously in mice immediately before recAP; recAP and mut-recAP were administered intravenously 1 h before IRI surgery, except as noted  in some experiments.Blood was collected under isoflurane anesthesia by retro-orbital bleeding after 24 h of kidney reperfusion. Mice were euthanized , and kidneys were harvested.n = 3 per treatment group), were used. ZM241,385 was administered  in a volume of 10 ml/kg. Mice were euthanized 1, 4, 8, and 24 h later, and blood was collected by cardiac puncture. Plasma was separated by centrifugation and stored at −80°C until analysis.C57BL/6 female mice, approximately 6–8 weeks of age . Analytical standards for each analyte were prepared by solubilizing powder stocks into 1:1 DMSO:ACN and standard curves were prepared in naïve animal plasma. Samples were extracted using protein precipitation with ∼5 volumes of an internal standard cocktail (tolbutamide at 5 ng/ml) in acetonitrile. Sample blocks were vortexed for 30 s and centrifuged at 3,000 rpm for 5′ and ∼50 μl of the supernatant was diluted into three volumes of water containing 0.1% formic acid for injection onto the LC-MS.Chromatography was performed on a Waters Acquity iClass UPLC System . The autosampler and column were kept at 10 and 40°C, respectively. Separation was achieved with an Acquity UPLC HSS T3 column  and a gradient of 0.1% formic acid in water (Mobile Phase A) and 0.1% formic acid in acetonitrile (Mobile Phase B) at a flow rate of 0.600 ml/min. An initial mobile phase composition of 5% B was ramped to 95% over 2 min, held at 95% for 0.3 min, and then returned to initial 5% B for re-equilibration.Data were collected on an AB Sciex API5500 mass spectrometer  using negative Turbo IonSpray™ electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. MRM transitions for the analyte, including DP and CE, were as follows: PF-01192544 336→174 DP: −62 CE: −33. Tolbutamide was used as the internal standard for PF-01192544 (MRM transitions = 269→170 DP: −60 CE: −30). Data acquisition and processing was carried out with Analyst software version 1.6.2 .Plasma creatinine was measured by enzymatic assay according to the manufacturer’s instructions  but using twice the recommended sample volume; the accuracy of this method has been verified by LC-MS , 39. Por6 μm for each kidney section.Tubular necrosis in the kidney outer medulla was scored in H&E-stained kidney sections by an MBF Bioscience and Zeiss AxioImager Z1/Apotome microscope system for stereology and tissue morphology in a blinded fashion  by a method described previously  and usinIsoflurane was used as anesthetic for all studies in rats. Male Sprague-Dawley rats, 220–250 g (Harlan), were used in a recAP dose escalation study  in a rat unilateral kidney clamp model of ischemic AKI . Sterile saline or the dose of recAP was administered i.v. over a period of 1 min just after the 30 min kidney pedicle clamp was removed, and plasma creatinine was measured at the end of the 24 h period of reperfusion, as previously reported . Munich P < 0.05. Means were compared by post hoc multiple-comparison test (Tukey’s), and all values are presented as mean ± SEM and as individual values in dot plots. Analyses were performed with GraphPad Prism version 6 . Rats: a one-sided Student’s t-test was used for 2 group comparisons.Mice: data were analyzed using one-way or two-way analysis of variance (ANOVA) with a significant difference defined as Choosing doses based on prior studies of recAP in models of sepsis , 37, we To evaluate whether the enzymatic activity of ilofotase alfa is needed for the protection from IRI, we administered a catalytically inactive mutant form of the enzyme (mut-recAP) carrying a single S92A mutation. With equimolar concentrations of the enzymatically active and mutant proteins, only the active form of the enzyme can dephosphorylate ATP . In the 2AR) with an A2AR agonist protects kidneys from IRI  during the 24 h reperfusion period were above the Ki for Asubtypes  41, 42)2AR but wotective . Thus, w–/–CD73 mice  in the ilofotase alpha-treated groups. Ilofotase alfa also increased 24-h urinary creatinine clearance in both studies . These results showing that ilofotase alfa protected rats in this AKI model are consistent with our findings in mice and provide additional relevance for the therapeutic dosing of ilofotase alfa clinically. This dose of ilofotase alfa  was then used in the AKI-on-CKD model in which ilofotase alfa treatment was delayed for a longer period of time after ischemic insults.To demonstrate efficacy of ilofotase alfa in an additional species and model, the next series of experiments was performed in rats. Initial studies were undertaken to determine the efficacy and dose requirement of ilofotase alfa in a unilateral kidney clamp model of ischemic AKI in male Sprague Dawley rats. A dose of 1,000 U/Kg produced a decrease in plasma creatinine at 24 h after injury, and this dose was used in all subsequent studies in rats. In two subsequent studies in 10–12-week old male Sprague Dawley rats, comparing vehicle to ilofotase alfa  given immediately after a 30-min period of ischemia, plasma creatinine was significantly reduced 24 h after injury  showed that this difference persisted. Twenty four-hour urinary protein was not statistically different at 7 days following injury between the two groups (data not shown).At randomization two weeks after the second clamping procedure, the IRI rats had a plasma creatinine and 24-h urinary creatinine clearance of 0.90 ± 0.19 mg/dl and 0.22 ± 0.06 ml/min/100 g body weight compared to nephrectomized only sham rats with a plasma creatinine and 24-h creatinine clearance of 0.52 ± 0.04 and 0.65 ± 0.08, respectively. They also had increased proteinuria of 80.9 ± 37.8 vs. 28.1 ± 3.0 mg/24 h/GFR, respectively. via A2A receptors to produce protective effects that are known to reduce AKI  in preclinical and clinical studies for the treatment of sepsis-associated AKI and for reduction of mortality in sepsis has yielded promising results. Alkaline phosphatase (AP) is an endogenous enzyme that is expressed ubiquitously and that is responsible for dephosphorylating a variety of molecules. Several mechanisms have been proposed to account for the efficacy of ilofotase alfa in preventing sepsis-associated AKI when administered to animals. We now show that ilofotase alfa, a human recombinant AP, which is being tested clinically, is also efficacious in preventing kidney injury in IRI models of AKI in both mice and rats, and furthermore we show that it is efficacious when used therapeutically, i.e., after IRI, to reduce injury in rodent AKI models and in an AKI-on-CKD model in rats. Our data strongly suggest that the ability of ilofotase alfa to sequentially dephosphorylate ATP to produce adenosine , 19, 24 duce AKI ; removalDuring ischemic injury and other tissue injury processes, ATP is released from damaged and dying cells . Extrace2ARs (including activation of A2ARs on immune cells)  resulting in activation of adenylate cyclase, elevation of cAMP, and subsequent stimulation of protein kinase A. The involvement of each receptor subtype will also be dependent on the local concentration of ATP during injury and that of adenosine generated by ilofotase alfa. At lower levels of ATP and adenosine, the effect on A2ARs would predominate, whereas higher levels of adenosine would engage A2BRs. Although the protective effects of ilofotase alfa were blocked completely by ZM241,385 in WT mice, they were only partially blocked by the absence of A2ARs in –/–Adora2a mice, suggesting the possibility that other AR subtypes and/or other mechanisms are also involved. Both A2ARs and A2BRs have excess capacity to provide protection, and stimulation of either receptor (or both) could give full protection. In –/–CD73 mice, complete inhibition of the protective effects of recAP was found only at higher doses of ZM241,385 (100 mg/kg), which could be blocking A2ARs and A2BRs, and more transiently A1Rs, as supported by our pharmacokinetic studies , it is possible that ZM241,385 blocked A2ARs, A2BRs and perhaps A1Rs at some point during the course of the experiment.Adenosine protects tissues from ischemic injury and exerts anti-inflammatory effects, and kidneys are protected from IRI by activation of Ae cells) –34 or A2s)    but thisle cells . Still, In addition to reducing injury through AR-mediated effects, ilofotase alfa may also protect kidneys by metabolizing ATP, reducing levels of extracellular ATP, and thereby reducing the deleterious effects resulting from ATP’s action as a DAMP. Similarly, recAP provides protection from sepsis by dephosphorylating and detoxifying LPS , 37, and–/–CD73 mice by prior administration of ilofotase alfa, and the protection was inhibited by ZM241,385 at the highest dose. These results suggest that ilofotase alfa is sufficient to rescue the deficit in adenosine-mediated protective effects, due to CD73 deficiency, that contribute to increased kidney injury in –/–CD73 mice  and AMP to adenosine (CD73). These ectonucleotidases and endogenous AP in the kidney vasculature can metabolize AMP to adenosine . In the –/– mice  and thatThe lack of translation of successful therapies in preclinical AKI models has been a challenge in advancement of therapy for clinical AKI. However, IRI studies to date have been accomplished in model systems that utilize young and otherwise normal rodents, and the clinical studies are conducted in patients with a high risk of AKI, most of whom have CKD and are proteinuric. Therefore, we used our model of aged rats with CKD and proteinuria based on recurrent episodes of IR-induced AKI to test whether the effect of ilofotase alfa would still be present in rats with significant comorbidities including a reduced GFR. We used a right nephrectomy and left unilateral kidney pedicle clamp model in aged, proteinuric MWF rats to induce CKD. This rat strain is known to have a reduced number of glomeruli and develop progressive spontaneous proteinuria at 12 weeks in males and 16 weeks in females . A monthThe doses found to be effective in our rodent models are highly relevant from a therapeutic perspective in patients. The dose of 1,000 U/Kg was the highest dose used in the STOP-AKI trial that resulted in statistically significant improvement of kidney function at 21 and 28 days, as measured by improvement of endogenous creatinine clearance. In the same study, the dose of 1,000 U/Kg also improved survival of the patients compared to placebo. The dose of 1,000 U/Kg is currently being tested in the Phase 3 clinical trial REVIVAL (NCT04411472) for sepsis-associated AKI.2ARs. Other AR subtypes may contribute, including A2BRs and A1Rs. These mechanistic studies are supportive of clinical trials of ilofotase alfa for sepsis-associated AKI (In summary, we have demonstrated that ilofotase alfa is effective in rodents in preventing and treating both AKI and AKI-on-CKD, and our results suggest a mechanism that is dependent on adenosine-mediated tissue protective effects, which are dependent at least in part on Aated AKI , 17, 49 The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the University of Virginia Animal Care and Use Committee and the Indiana University IACUC.DR, JH, NK, JG, BM, AVE, and MDO designed the experiments. LH, SZ, DR, AB, KB, EM, JL, GH, IE, RS, and SC-B conducted the experiments and acquired and analyzed the data. DR, BM, JH, AVE, and MDO wrote and edited the manuscript. All authors approved the final version of the manuscript.The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. With the exception of the Conflicts of Interest Statement, all authors declare no other competing interests.JH, AB, KB, EM, JL, GH, NK and JG were employed by Pfizer Inc. AVE was employed by AM-Pharma B.V. MDO had a research grant from Pfizer Inc. AVE was consultant to AM-Pharma B.V. and a named inventor on patent filings related to ilofotase alfa. BM had research grants with AM-Pharma B.V. and Pfizer Inc. and was on a MAB for AM-Pharma B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Taxillus chinensis is an important medicinal and parasitic plant that attacks other plants for living. The development of haustorium is a critical process, imperative for successful parasitic invasion. To reveal the mechanisms underlying haustorium development, we performed an iTRAQ-based proteomics analysis which led to the identification of several differentially abundant proteins (DAPs) in fresh seeds (CK), baby (FB), and adult haustoria (FD). A total of 563 and 785 DAPs were identified and quantified in the early and later developmental stages, respectively. Pathway enrichment analysis revealed that the DAPs are mainly associated with metabolic pathways, ribosome, phenylpropanoid biosynthesis, and photosynthesis. In addition, DAPs associated with the phytohormone signaling pathway changed markedly. Furthermore, we evaluated the content of various phytohormones during different stages of haustoria development. These results indicated that phytohormones are very important for haustorium development. qRT-PCR results validated that the mRNA expression levels were consistent with the expression of proteins, suggesting that our results are reliable. This is the first report on haustoria proteomes in the parasitic plant, Taxillus chinensis, to the best of our knowledge. Our findings will enhance our understanding of the molecular mechanism of haustoria development. Taxillus chinensis (DC.) Danser is a member of the family Loranthaceae which consists of approximately 73 genera and 900 species and is majorly comprised of aerial hemiparasitic plants . T. chinThe initiation of haustorium development in most parasitic plants is triggered by host-derived chemical signals released after seed germination. These components include phenolic acids, flavonoids, quinones, and 2,6-dimethoxy-p-benzoquinone (DMBQ) . HoweverRapid advancement in the microarray, next-generation sequencing, and mass spectrometry- (MS-) based technologies has enabled high throughput analyses of transcriptomes, proteomes, and metabolomes, serving as powerful tools for obtaining large-scale information on transcripts, proteins, and metabolites. Earlier, the transcriptome of different stages during haustoria formation in T. chinense has been investigated which revealed the possible mechanism of haustorial development . Study oThe fresh seeds of Taxillus chinensis (DC.) Danser were collected from the parasitized mulberry trees that were planted in the experimental field of Guangxi Botanical Garden of Medicinal Plants, Guangxi, China. Seeds were accessed by the senior botanists and deposited in the herbarium of Guangxi Botanical Garden of Medical Plants (accession number: s0001794). The seeds were peeled, washed with sterile water, placed on a germination dish, and incubated in a controlled environment , as previously described [v/v) trichloroacetic acid (TCA) and incubated at -20°C overnight. After centrifugation at 6,000 g for 40 min, the supernatant was discarded. The precipitant was washed thrice with precooled acetone and air-dried. Pellet was dissolved in the Lysis buffer . Finally, the suspension was filtered through 0.22 mm filters after centrifugation at 13,000 g for 10 min at 4°C. The concentration of protein was quantified with the BCA Protein Assay Kit , and the quality of the protein sample was measured by SDS-PAGE.Total protein was extracted as described in a previous study . Brieflyμg of total protein was used for trypsin-based digestion followed by iTRAQ labeling. Protein was reduced with 10 mM DTT at 37°C for 60 min and alkylated with 55 mM iodoacetamide (IAM) at room temperature for 30 min in the dark. The urea concentration of the protein samples was diluted to less than 2 M by adding 100 mM TEAB. Next, the trypsin was added to the protein pool of each sample with the ratio of protein : trypsin = 50 : 1 (mass ratio) at 37°C overnight and 100 : 1 for a second digestion for 4 h. After trypsin digestion, peptides were desalted with the Strata X SPE column and vacuum-dried. The peptides were reconstituted in 20 μL of 500 mM TEAB and labeled according to the manufacturer's protocol. Briefly, the peptide solution was mixed with 50 μL isopropanol, and one unit of iTRAQ reagent was added to each sample. The mixture was incubated for 2 h at room temperature, pooled, and dried by vacuum centrifugation.For each sample, 100 μm, and 4.6∗250 mm). Briefly, peptides were first separated into 72 fractions using a gradient of 2% to 98% acetonitrile (pH 10). The wavelength 250 nm is used for the detection of peptides. Then, the peptides were pooled into 18 fractions and dried by vacuum centrifugation. The peptide fractions were desalted using Ziptip C18  according to the manufacturer's instructions.The peptides were reconstituted in the HPLC solution A  and fractionated using high pH reverse-phase HPLC with Waters Bridge Peptide column BEHC18  at a rate of 5 μL/min in 100% solvent A (0.1 M acetic acid in water). Next, peptides eluted from the trap column were loaded onto a reversed-phase analytical column . The gradient was comprised of solvent B (0.1% FA in 98% ACN) with an increasing concentration from 15% to 35% over 45 min, 35% to 98% for 5 min, and kept in 98% solvent B for 5 min at a constant flow rate of 300 nl/min on an EASY-nLC1000 system. The elute was sprayed via NSI source at the 2.0 kv electrospray voltage and analyzed by tandem mass spectrometry (MS/MS) in Q Exactive. A data-dependent acquisition mode in the scan range of 350-2000 m/z was carried out for the mass spectrometry analysis, and the survey scans were captured by the Orbitrap analyzer at a mass resolution of 17500. In the linear ion trap, 15 of the most intense precursor ions were selected for subsequent decision tree-based ion trap HCD fragmentation at the normalized collision energy of 32% in the MS survey scan with 10.0 s dynamic exclusion.Mass spectrometry-based analysis was performed by NanoLC 1000 LC-MS/MS using a Proxeon EASY-nLC 1000 coupled with Thermo Fisher Q Exactive. Digested fractions were resuspended using 0.1% formic acid and loaded onto a reversed-phase-column . The search parameters were used as follows: carbamidomethylation (C) was set as fixed modifications, whereas oxidation (M), and acetylation at the N-Term were set as variable modifications. Trypsin was chosen as the enzyme, and two missed cleavages were allowed. Mass tolerance of precursor ions was set as 20 ppm, and a fragment ion tolerance was 0.05 Da, resulting in a 1% false discovery rate (FDR). Differentially abundant proteins (DAPs) were identified based on the following criteria: http://www.ebi.ac.uk/GOA) [http://www.genome.jp/kegg/) [To determine the functional characterization of differentially abundant proteins (DAPs), proteins were mapped with Gene Ontology (GO) annotation base on UniProt-GOA database (.uk/GOA) . All thep/kegg/) .μm, Thermo Fisher Scientific) at a flow rate of 0.3 mL/min with a 17 min gradient elution. For multiple reaction monitoring (MRM), the phytohormones were analyzed with the negative mode by the ESI ion source.Phytohormone indole-3-acetic acid (IAA), gibberellin (GA), and abscisic acid (ABA) were quantified using liquid chromatography coupled with a mass spectrometry system . Phytohormone extraction was performed from the Taxillus chinensis seeds based on the previous method . The phyΔΔCt− method was used to evaluate the expression levels of transcripts in each sample [Total RNA was extracted from each sample using TRIzol reagent (Invitrogen), following the manufacturer's protocol. FastQuant RT Kit  was used for DNA removal and cDNA synthesis. Specific primer pairs from randomly selected 8 genes were designed for quantitative real-time PCR (qRT-PCR) using the Primer Premier 5.0 software. Sequences of the primers used in this study are given in Table h sample . Each exP < 0.05.Statistical comparisons were made using the SPSS statistics software package with a significance level of To investigate the potential mechanism involved in the haustorial development in T. chinense, an integrated approach involving LC-MS/MS and iTRAQ labeling was applied to analyze the proteomic changes. After processing MS/MS spectra in the Mascot software, a total of 325354 spectra were generated, which were segregated into 26463 matched spectra, 24367 unique spectra, 10325 matched peptides, 9462 unique peptides, and 2615 matched proteins Figure . A totalThe DAPs were classified into three main GO categories . In the early developmental stage (FB), 1546 DAPs (some proteins have more than one GO annotation) were annotated with biological process, 653 DAPs with molecular functions, and 1885 DAPs with cellular components compared to control. In the later developmental stage (FD), 2126 DAPs were annotated with biological process, 945 DAPs with molecular functions, and 2665 DAPs with cellular components compared to control. In the biological process, the most enriched categories were the cellular process and metabolic process. In the molecular function, the most abundant category was binding and catalytic activity. In the cellular component, term cell and cell part were found to be highly enriched .To further understand the molecular mechanism potentially associated with the haustorial development in T. chinense, DAPs were subjected to the KEGG pathway database. During the early developmental stage (FB), proteins with increased abundance were mainly involved in the pathways related to metabolic pathways (108 DAPs), photosynthesis (20 DAPs), and carbon fixation in photosynthetic organisms (12 DAPs). Proteins with decreased abundance were involved in only three pathways, namely ribosome (52 DAPs), systemic lupus erythematosus (3 DAPs), and flavonoid biosynthesis (3 DAPs). During the later developmental stage (FD), proteins with increased abundance were mainly involved in pathways related to metabolic pathways (158 DAPs), photosynthesis (23 DAPs), glyoxylate metabolism, dicarboxylate metabolism (15 DAPs), and phenylpropanoid biosynthesis (14 DAPs). In addition, the proteins whose abundance decreased were also associated with only three following pathways, ribosome (67 DAPs), systemic lupus erythematosus (4 DAPs), and alcoholism (4 DAPs) .Based on the proteomic analysis, we observed that the expression levels of 17 proteins associated with the phytohormone signaling pathway were altered, including auxin-binding protein, mitogen-activated protein kinase, ABC transporter protein, and gibberellin-regulated protein . We founTo elucidate the correspondence between the level of hormones and the abundance of proteins, the IAA, ABA, and GA contents were analyzed using LC-MS-based analysis. The accumulation of IAA was slightly decreased after 10 days of haustorial development. However, the content of IAA was significantly increased after 20 days of haustorial development compared to the CK and FB. In contrast, the levels of ABA gradually decreased with the haustorial development. Interestingly, GA3 contents were initially upregulated, but then downregulated, reaching their highest level at 10 days .To validate the correlation between the transcript level of mRNA and the abundance of proteins, a transcriptional analysis of 8 DAPs was analyzed by qRT-PCR. The results showed that the expressions of these genes were similar to those of the abundance of their corresponding protein. The results indicate the authenticity of the method used to confirm DAPs in our study .Branches and leaves of T. chinensis are widely used in traditional Chinese medicine for the treatment of rheumatism, hypertension, and obesity as well as for preventing miscarriages . The hauPreviously, few studies have uncovered metabolic pathways that have an important role in haustorial development. Ichihashi et al. (2018) proposed that the accumulation of very long chain fatty acid (VLCFAs) involved in the developmental reprogramming of Thesium chinense haustoria formation in the natural environment . BesidesPhytohormones, such as IAA, GAs, and ABA, play crucial roles in regulating plant growth, development, and response to various stresses as previously reported , 30. Auxn conclusion, we studied the protein and phytohormones profiles during the Taxillus chinensis haustoria development. Some crucial proteins involved in haustorial development were successfully identified in this study. More importantly, the findings of this study will improve our understanding of parasitism and contribute to the breeding program of Taxillus chinensis. We believe that further functional analysis focusing on the single crucial proteins involved in the developmental process is a critical need for the future. That will provide more precise information about the mechanisms underlying haustorial development in Taxillus chinensis.