Antibiotic resistance in our pathogens is medicine’s climate change: caused by human activity, and resulting in more extreme outcomes. Resistance emerges in microbial populations when antibiotics act on phenotypic variance within the population. This can arise from either genotypic diversity , or from differences in gene expression due to environmental variation, referred to as adaptive resistance. Adaptive changes can increase fitness allowing bacteria to survive at higher concentrations of antibiotics. They can also decrease fitness, potentially leading to selection for antibiotic resistance at lower concentrations. There are opportunities for other environmental stressors to promote antibiotic resistance in ways that are hard to predict using conventional assays. Exploiting our previous observation that commonly used herbicides can increase or decrease the minimum inhibitory concentration (MIC) of different antibiotics, we provide the first comprehensive test of the hypothesis that the rate of antibiotic resistance evolution under specified conditions can increase, regardless of whether a herbicide increases or decreases the antibiotic MIC. Short term evolution experiments were used for various herbicide and antibiotic combinations. We found conditions where acquired resistance arises more frequently regardless of whether the exogenous non-antibiotic agent increased or decreased antibiotic effectiveness. This is attributed to the effect of the herbicide on either MIC or the minimum selective concentration (MSC) of a paired antibiotic. The MSC is the lowest concentration of antibiotic at which the fitness of individuals varies because of the antibiotic, and is lower than MIC. Our results suggest that additional environmental factors influencing competition between bacteria could enhance the ability of antibiotics to select antibiotic resistance. Our work demonstrates that bacteria may acquire antibiotic resistance in the environment at rates substantially faster than predicted from laboratory conditions. Neither reducing the use of antibiotics nor discovery of new ones may be sufficient strategies to avoid the post-antibiotic era. This is because bacteria may be exposed to other non-antibiotic chemicals that predispose them to evolve resistance to antibiotics more quickly. Herbicides are examples of some of the most common non-antibiotic chemicals in frequent global use. We previously showed that in some combinations the herbicides we tested made bacteria phenotypically resistant to higher concentrations of antibiotics, while in other combinations bacteria became susceptible at lower antibiotic concentrations. Here we demonstrate that in both cases the herbicides worked with antibiotics to accelerate genotypic resistance evolution. Unfortunately, antibiotic resistance may increase even if total antibiotic use is reduced, and new ones are invented, unless other environmental exposures are also controlled.As fundamental tools for infection control, antibiotics underpin diverse human systems ranging from hospital care to concentrated animal feeding operations through to crop and pollinator disease management. The loss of this tool due to antibiotic resistance will result in higher mortality and morbidity, but also deny access to many routine medical procedures for risk of subsequently untreatable infections . AntibioOne stewardship strategy is reduction of use which might help increase longevity . If bactacquired genes or alleles through mutation or horizontal gene transfer . Also, organisms can have innate differences between them, e.g., due to differences in permeability.Variation in antibiotic responses can be caused by either genetic or physiological differences between individual bacteria. The toxic effect of an antibiotic may occur at different concentrations for different individuals because some have adaptive (change in phenotype) response  containing 500 g/L dimethyl salt of dicamba, and Roundup Weedkiller  containing 360 g/L isopropylamine salt of glyphosate. Herbicide concentrations are reported in parts per million acid equivalent (ppm ae) of the active ingredient to allow for comparison with other formulations. Antibiotic and herbicide concentrations used are specified in the main text or in figure legends.Strains and plasmids are listed in R by removing the HindIII and BamHI fragment of pBR322 and inserting cat from pACYC184 at the PstI site within bla. The resulting plasmid conferred resistance to chloramphenicol, but not to ampicillin or tetracycline . RSF1010 .pBR322  was used RSF1010  was the E. coli or S. typhimurium were grown for 24 h in 10 mL liquid LB medium containing Cip, herbicide, both, or neither. Initial densities were ca. 105 cfu/mL for the former treatment and ca. 106 cfu/mL (E. coli) and 105 cfu/mL (S. typhimurium) for the latter three. The experiment was abandoned at this point if cultures containing Cip but not herbicide were visibly turbid. This was because in this experiment the chosen Cip concentration was above MIC in cultures not exposed to herbicide, and growth was therefore interpreted as due to a resistant mutant in the starting culture. Herbicide and antibiotic concentrations used are detailed in the Results section. Cultures were then diluted as above in the same conditions and incubated again for 24 h. A 10-fold dilution in LB without herbicides or antibiotics followed to ensure observed effects were not due to herbicide induced adaptive responses. Titers (cfu/mL) were determined at the end of each incubation step on both LB and on Cip at an initially non-permissive concentration (ca. 2×MIC) (For the experiments described in the section “Evolution of acquired antibiotic resistance in cultures with herbicide-induced increases in MIC” . 2×MIC) . The freExperiments described in the section “Herbicide-induced changes in MSC can occur without a change in MIC” were conducted similarly. However, in this case the culture supplemented with only Cip was able to grow because the chosen Cip concentration was below MIC.Individual colonies of strains grown on solid LB medium were used to inoculate 100 µL of liquid LB in a 96 well plate. The plate was incubated with aeration to saturation before ca. 4 µL samples were stenciled onto LB plates containing the appropriate antibiotics. Plates were incubated at 37 °C for 18 h. Strains were scored positively for growth if growth was tangible.E. coli differing only in the MIC phenotype  and an additional selection marker encoded on a low copy number plasmid were co-incubated in liquid LB medium containing herbicide, antibiotic, both, or neither and grown to saturation before dilution by a factor of 103 in the same conditions. The antibiotic concentration chosen was below NOEL for both strains. NOEL is defined here as the highest antibiotic concentration for which no effect on survival or selection were observed. The titer of each culture was determined by plating on non-selective medium after each incubation step. After 5 rounds of incubation, the ratio of strains was determined by selecting for the second—competition irrelevant—marker.Isogenic strains of p = est∕(c + est), where t = time, p = proportion of resistant individuals, s = strength of selection, c = constant describing the initial proportion of resistant individuals, c = (1∕p0) − 1. The constant p0 is the proportion of resistant individuals at t0 (the start of the experiment). t is defined as the number of generations, using the generation time for the more resistant strain. By rearranging this formula, the strength of selection is s = ln(pc∕(1 − p))∕t.Natural selection was defined as the difference in the exponential growth rate of the two strains . Under t600 was determined every 6 min for 16 h and averaged over five biological replicates. Cultures were started at densities of ca. 106 cfu/mL. Antibiotic and herbicide concentrations used are detailed in the legend of Growth curves were established at 37 °C in liquid LB medium supplemented with herbicide, antibiotic, both, or neither using a FLUOstar Omega microplate reader . The ODR was used for all statistical analyses . For expFor mixed culture experiments (changes to MSC), two sets of analyses were performed. First, we determined whether adding antibiotics increases the strength of selection at different herbicide concentrations. Second, we determined whether adding herbicides increases the strength of selection at different antibiotic concentrations. Each set of questions was tested using contrasts performed with the glht function in the multcomp package  with a tt = 48 min and t = 150 min was measured. A visual inspection of the plots revealed that before t = 48 min graphs were not linear. After t = 150 min, growth slowed as cells were entering stationary phase.To estimate carrying capacity (k), a logistic growth model on the raw data using non-linear least squares (nls) was fit in R. Differences in population growth between strains in different treatments were estimated by estimating r, the intrinsic growth rate, of each strain. Data sets were log transformed and the slope of the growth curve between We tested for differences between the two strains in both r and k by calculating contrast in an ANOVA (aov). Residual plots were used to test for violations of assumptions for ANOVA. Assumptions of normality and equal variances were met in all data sets. In two data sets there were a small number of outliers. These had small influences on parameter estimates and were hence not removed. Contrasts between treatments were calculated using the glht package in R using the sequential Bonferroni correction.E. coli and S. typhimurium compared to exposure to only the antibiotics. This was due to adaptive changes in exposed bacteria. From these observations we hypothesized that particular combinations of antibiotics and these formulations alter the MSC or MIC of the antibiotic in a way that favours genetically resistant variants. To test this hypothesis, we conducted short-term evolution in antibiotics with and without additional exposures to commercial herbicide formulations.As reported previously, simultaneous exposure to a variety of antibiotics and either various commercial herbicide formulations  or to eiBecause the effect of the herbicide exposure was specific to the combination of herbicide and antibiotic, primarily two kinds of experiments were done. Firstly, in the case where the herbicide raised the MIC of the antibiotic, we tested whether antibiotic survivors could give rise to antibiotic resistant variants. In this case, only bacteria simultaneously exposed to both the herbicide and antibiotic would survive when the antibiotic concentration exceeded the MIC of the bacteria measured without the addition of the herbicide. Secondly, in the case where the herbicide decreased the MIC of the antibiotic, we tested whether the herbicide decreased the MSC of the antibiotic, leading to a greater rate of resistance evolution at lower concentrations of antibiotic because of exposure to the herbicide formulation.E. coli or S. typhimurium to the herbicide formulation Roundup increased the MIC of the fluoroquinolone antibiotic ciprofloxacin, as did exposing S. typhimurium to the herbicide formulation Kamba .Expectedly, cultures supplemented only with ciprofloxacin did not grow, and genotypic resistant variants were not detected. Cultures that grew for 25 generations without ciprofloxacin supplementation produced resistant variants at similar low rates regardless of exposure to the herbicide formulations. This indicated that the herbicides were not mutagens at these concentrations. In a separate standard test of mutagenicity , bacteri2 times higher for the combination of Cip+Kamba+S. typhimurium to 105 times higher for Cip+Roundup+E. coli , 27 isolates from Kamba+Cip and 29 isolates from Roundup+Cip treatments. The parental strain and 2 isolates from each LB, Kamba, and Roundup treatments, also isolated on 0.05 µg/mL Cip, were included as controls.Ciprofloxacin resistant colonies were isolated at the end of each experiment from all treatments. We determined MICs for 56 The parental strain and six evolved isolates did not grow at 0.07 µg/mL, a concentration just above the selection concentration. We observed MICs of 0.1 µg/mL ciprofloxacin for 21 isolates and 0.2 µg/mL ciprofloxacin for 28 isolates. Only seven isolates displayed higher MICs , the highest being 1.25 µg/mL ciprofloxacin reached by one isolate recovered from a Roundup+Cip culture. We observed no correlation between level of resistance and original treatment, which indicates that there are no qualitative differences between the ciprofloxacin resistant variants arising in the different treatments.Antibiotic resistant bacteria are becoming a fixed part of many environments despite the concentration of antibiotics often being very low . We hypoE. coli were created to represent pre-existing antibiotic resistance heterogeneity within natural environments. The phenotypic differences were created using bacteria carrying isogenic plasmids with different alleles of tetA , indicating that the ciprofloxacin concentration met or exceeded the MSC. The addition of Kamba neutralized the selective effect of ciprofloxacin by shifting the MSC to a higher level without changing the MIC.Acquired resistance rates were the same for cultures grown in LB, LB+Kamba or LB+Cip+Kamba  or tetracycline  MICs were competed for about 40 generations. The frequency of each strain after competition was measured. The concentration of the appropriate antibiotic did not vary between cultures, but the concentration of herbicide did. Combinations were chosen that lead to a decrease in MIC with the chosen herbicide, namely Str+Kamba, and Tet+Roundup.Pairs of isogenic strains of E. coli  with eitThere was no differential in the fitness of paired strains when they were co-cultured in standard rich medium . In cultIn each test, the combination of antibiotic and herbicide reduced MSC. The more resistant strain invariably increased relative to the isogenic competitor in a herbicide dose-dependent manner when both antibiotic and herbicide were in the environment. Selection coefficients were statistically significantly different from LB medium at 183 parts per million acid equivalent (ppm ae) Kamba and 311 ppm ae Roundup.high and Tethigh strains in environments with sub-lethal concentrations of antibiotic for both strains in each pair. This was shown by measuring the growth rate of the strains in monoculture rather than in competition  and p = 4.7 ×10−10 (Roundup+Tet)).The observed fitness differential was due to the faster reproductive rate of the Strpetition . No diffp < 2 ×10−16; low strain population grew to higher final optical densities in LB, LB+Str, and LB+Kamba treatments. In cultures with Roundup+Tet, Tethigh and Tetlow had similar densities  values between 48 and 150 min, the period between the grey dashed vertical lines. In this period log(OD) increased approximately linearly with time indicating exponential growth.  determination of the carrying capacity k of Str high∕low (AH204/AH211) and Tet high∕low (AH201/AH214), respectively. The gray line represents OD 600 values; the dotted black line represents the estimated logistic curve calculated by fitting a logistic growth model using non-linear least squares. Antibiotics were used 0.25 μg/mL for Str and 0.05 μg/mL for Tet; herbicide concentrations were 1,830 ppm ae Kamba and 311 ppm ae Roundup. determination of the growth rate r of Str high∕low (AH204/AH211) and Tet high∕low (AH201/AH214), respectively. The gray line represents log transformed ODClick here for additional data file.10.7717/peerj.5801/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.5801/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.5801/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.5801/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.5801/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.5801/supp-7Supplemental Information 7Click here for additional data file.


There is no doubt that the use of waste rubber in concrete applications is a genius alternative because Styrene is the main component of rubber, which has a strong toxicity and is harmful to humans. Therefore, it will significantly reduce impacts on the environment when waste rubber can be recycled for genuine uses. In this paper, the dynamic properties of high-strength rubberised concrete have been investigated by carrying out various experiments to retain the compressive strength, tensile strength, flexural strength, electrical resistivity, and damping characteristics by replacing fine aggregates with micro-scale crumb rubber. Over 20 variations of concrete mixes have been performed. The experimental results confirm that a decrease in the compressive strength can be expected when the rubber content is increased. The new findings demonstrate that the high-strength concrete can be enhanced by optimal rubber particles in order to improve splitting tensile and flexural strengths, damping properties, and electrical resistivity. It is therefore recommended to consider the use of rubberised concrete (up to 10 wt. % crumb rubber) in designing railway sleepers as this will improve the service life of railway track systems and reduce wastes to the environment. At the beginning of the last century, with the development of science and technology, the reduction of forest resources led to the production of steel and concrete railway sleepers rather than wood ,2,3. SinRecently, many researches have proposed and encouraged the use of waste rubber in concrete to improve its performance ,6,7. MorThe purpose of this paper is to highlight the sensitivity of dynamic properties of the high-strength concrete to crumb rubber inclusion as well as its capability in meeting the standards of railway concrete sleepers’ manufacturing. Different proportions of crumb rubber with different particle sizes  have been used in order to analyse their effects on the compressive strength, splitting tensile and flexural strengths, electrical resistivity and damping properties of the rubberised concrete.Conventional rail track structure can be divided into superstructure and substructure . The supThe sleepers are usually manufactured by timber or concrete composite nowadays. Timber sleepers are often used in North America which have superior elasticity, better mitigation and absorption of vibration, but their life cycle is no more able to meet today’s market requirements. Consequently, concrete sleepers have recently become the most widely used due to their low maintenance cost and longer life cycle in comparison with timber sleepers .The railway concrete sleepers are expected to service for 50 years ,14. HoweThe large amount of abandoned waste tyres  while haAs early as 1976, there was a patent for old tyres usage in the United States ,22. NowaMost studies have shown that the replacement of the fine aggregates by crumb rubber in concrete will reduce its strength to a certain extent, and the degree of decrease in strength depends on both the particle size and the amount of addition . Still, The emphasis of this study is placed on the application of recycled crumb rubber to enhance dynamic damping of structural concrete for railway applications. To enable the industry application, specific requirements must be complied , & is the maximum load (N), L is the length of the line of contact of the specimen (mm), and D is the designated cross-sectional dimension (mm).According to BS EN 12390-6 , the splSimilarly to the compressive strength results, the increase in silica fume content clearly improved the splitting tensile strength by 30% comparing to conventional concrete, reaching a maximum value of 3.85 MPa at 28 days as shown in cff is the flexural strength (MPa), F is the maximum load (N), I is the length between roller supports (mm), and d1 and d2 are the cross-sectional dimensions of specimen (mm).Likewise, the flexural strength has been calculated following the British standard BS EN 12390-5 :(2)fcf=FAs shown in The tensile and flexural ratio can be calculated by:The tensile ratio and flexural ratio have been calculated by Formulas (3) and (4) shown in Generally, there are two main methods to measure the electrical resistance of cementitious materials: two-electrode method and four-electrode method . In thisR is the resistance of concrete, k is a geometrical factor which depends on the size and shape of the specimen, A is the cross-sectional area perpendicular to the current, L is the height of the specimen.After 28 days, the electrical resistance was measured then calculated using the resistance equation :(5)ρ=k∗RAs it can be seen from The vibration tests have been conducted in the civil engineering laboratory to avoid vibration from surrounding environment. Prism specimens were used to carry out these experiments using the following equipment: computer with PROSIG hammer system, DATS modal analysis software, and an accelerometer. The specimens’ surfaces have been cleaned to eliminate bias in tests results ,53, and Generally, there are two methods to analyse the damping ratio and both have been considered in this study. The first method is called exponential curve fitting method (ECFM); this method is based on calculating the damping ratio directly using the natural frequency and the vibration response of the concrete samples from the FRF graph, as can be seen from The acceleration-time graph is then plotted and the exponential line is fitted with the peak amplitude according to the decreasing trend of the vibration response, while the equation of the exponential line is generated from the Microsoft Excel program as illustrated in tA is the amplitude, A0 is the peak amplitude, ζ is the damping ratio, nw is the natural frequency (rad/s) equal to 2 nπf where nf is the natural frequency in Hz, and t is the time in seconds. The calculation of the damping ratio of the SF5%–CR10% sample presented by the graph of Finally, the equation obtained from the vibration graph  is compaδ is the damping index, A0 is the peak amplitude, nA is the amplitude after n number of cycles, and ζ is the damping ratio.The second method is used to verify the value obtained from the ECFM and in order to make sure that the tests’ results are more reliable. It is called the logarithmic decrement method (LDM), and the damping ratio in this case is calculated using the logarithmic decrement formulas (Equations (5) and (6)):The damping ratio estimated from the LDM for the same sample tested before using the ECFM is 0.0323 (3.23%), which presents a small error of 0.31%. The results regarding all the samples are plotted in the graph of According to Kaewunruen et al., the dynamic flexural modulus of elasticity demonstrates the elastic behaviour of material under flexural loading. The dynamic flexural modulus can be estimated from Equation (9) ,59,60,61Railway traffic environment is aggressive and the railway infrastructure has to experience heavy-haul or high-speed rail operation. Therefore, material for railway infrastructure needs to be strong, durable, resilient and tough to withstand various unexpected damage. Waste tyre is a global environmental problem which is non-biodegradability, flammability and chemical composition. Waste tyre cannot be solved as a normal method like disposal. This research study aimed to develop an environmentally friendly concrete using crumb rubber, recycled form waste rubber tyres (75 micron and 180 micro mixed with 400 micron), as micro-filler to make concrete sleepers that meet with the railway requirements. Although the emphasis is placed on concrete sleepers in ballasted railway tracks, the application of this recycled concrete can also be applied to concrete slabs in ballastless tracks. Based on diverse proportions of crumb rubber and silica fume, eleven different types of concrete were casted according to British standard. The first experiments carried out on the concrete specimens tested their compressive strength at 7 and 28 days, as it presents the most important property of this material. Similar to previous studies’ findings, it has been deducted that the increase in the rubber content results in a drop in compressive strength. However, the rubber concrete containing a proportion of 10% silica fume and 5% crumb rubber has shown considerable results by reaching 57.30 MPa and 59.20 MPa at 28 days in both rubber particle cases 75 micro and the mix of 180 and 400 micro respectively, thus meeting the standard for railway sleepers manufacturing (55 MPa). Likewise, it has been observed a significant deterioration in both splitting tensile and flexural strengths when increasing the crumb rubber proportion in concrete. Still, the proportions that seemed to show better splitting tensile performance than plain concrete are the ones containing 10% silica fume: mixed with 5% crumb rubber in the case of 180 and 400 micro rubber, and 10% in the case of 75 micro rubber (3.75 MPa and 3.54 MPa respectively).On the other hand, the specimens containing a combination of 10% silica fume and whether 5% of the 180&400 micro rubber or 10% 75 micro rubbers have shown acceptable flexural strengths superior to plain concrete by 8 and 13%: 6.19 MPa and 6.49 MPa, respectively. In terms of electrical resistivity, the concrete samples containing crumb rubber have shown the best results: 374.25 kΩ and 371 kΩ for SF10%/CR10% (180&400) and SF10%/CR5% (75) respectively. Therefore, the addition of crumb rubber is capable of enhancing the electrical resistivity of conventional concrete up to 47%. Finally, by conducting vibration tests on rubberised concrete and using both the exponential curve fitting method (ECFM) and the logarithmic decrement method (LDM), it has been concluded that the crumb rubber has clearly improved the concrete’s damping ratio, especially when using the 180&400 micro which resulted in an improvement of 100% comparing to normal concrete. It is therefore recommended from the results of this study to use crumb rubber in concrete as micro-filler, and more precisely the SF/10%–CR/5% (400&180) as it showed the best performance in all tests and meets the requirements of railway concrete sleepers. From the environmental perspective, the use of crumb rubber in concrete presents one of the best alternatives while dealing with rubber waste, as it will help both the environment protection and also the reduction in railway sleepers cost.


To evaluate the trends in AIDS detection and the AIDS-attributed death rate in Brazil between 2007 and 2015 and to determine the population characteristics associated with AIDS detection.Surveillance data including demographics, mode of HIV transmission, AIDS cases, and AIDS-attributed deaths were analysed. A logistic regression model was fitted to assess the trends in AIDS cases by mode of HIV transmission throughout the study period. A segmented Poisson regression model was used to assess changes in the trends of AIDS detection and mortality rates before (2007–2014) and after (2015) the introduction of the Test and Treat Initiative.p-value < 0.001). The observed AIDS detection rate in 2015 was 7.7 cases per 100,000, which was 60% lower than the regression estimate of 19.8 for the hypothetical absence of the Test and Treat Initiative. The reduction in AIDS-attributed deaths from 2007 to 2014 was 8.0% annually . The observed AIDS mortality rate in 2015 was 0.49 cases per 100,000, which was 73% lower than the regression estimate of 2.1 for the hypothetical absence of the Test and Treat Initiative in 2015.In the segmented Poisson regression, the decline in the AIDS detection rate from 2007 to 2014 was 2.0% annually  for annual decline was 0.98 , and a median CD4 count at ART enrolment. Further evaluation of the effects of the Test and Treat Initiative on AIDS diagnosis and mortality is needed and must be strengthened with additional data from subsequent years. Approximately 42% of the people living with HIV/AIDS (PLWHA) in Latin America can be found in Brazil , with 84The reported number of AIDS deaths in Brazil has remained stable from 11,020 cases in 2004 to 12,298 cases in 2015. The AIDS mortality rate has oscillated from 5.9 AIDS-related deaths per 100,000 persons in 2006 to 5.6 per 100,000 persons in 2015, although the trends present regional variations : AIDS cases, epidemiological characteristics ; 2) the Mortality Information System [Sistema de Informaçoes sobre Mortalidade (SIM)]: death information such as cause of death, the date, and the municipality of death were recorded from the declaration of death document; 3) the Logistic Treatment Control System [Sistema de Controle Logistico de Medicamentos (Siclom)]; and 4) the Laboratory Testing Information System [Sistema de Informação de Exames Laboratoriais (Siscel)].We analysed AIDS cases and related variables reported to the Brazilian surveillance system from 2007 to 2015. Data was obtained from several electronic surveillance subsystems: 1) the Notifiable Disorders Information System [3. Until 2003, HIV testing was performed by enzyme-linked immunosorbent assay (ELISA). All positive results were confirmed with a second ELISA and Western blot. After 2003, two rapid tests were performed sequentially on different finger prick specimens. If the results between the two tests were discordant, the testing was repeated. We included cases that were at least 15 years of age.The national AIDS definition was used to report AIDS cases to the national surveillance system . This deAge, sex, mode of HIV exposure, education level, vital status, race/colour, and date of AIDS diagnosis were collected. The potential mode of HIV transmission was determined based on several variables: 1) sexual activity of the following categories: sex with men, sex with women, sex with men and women, no sexual transmission; 2) intravenous drug users (IDUs) (Yes/No/Unknown); 3) haemophilia treatment or transfusion (Yes/No/Unknown); 4) vertical transmission (Yes/No/Unknown); and 5) biological material accident with seroconversion within 6 months (Yes/No/Unknown). Death was determined based on the recorded date of death. Key indicators related to the cascade of HIV care, such as the number of rapid tests, the number of new HIV diagnoses, the number of PLWHA on ART, and the median CD4 count at ART enrolment, were obtained from national programme data .Stata V11.0  was used for the analysis. Data was described using frequencies or percentages or means with standard deviations (SDs).Population estimates were obtained for the years 2007–2015 to calculate mortality and AIDS rates . These estimates were obtained from the Institute of Statistics in Brazil (IBGE – Instituto Brasileiro de Geografia e Estatística). Trends in the AIDS detection rate were evaluated by sex and age group, and trends in the AIDS detection rate and AIDS-attributed death rates were evaluated by sex. Trends in sociodemographic characteristics, mode of HIV exposure, and death were evaluated according to the proportion of AIDS diagnoses over time. The analysis of the mode of HIV exposure was conducted separately for heterosexuals, homosexual/bisexual men, IDUs, vertical transmission, and others. Homosexual/bisexual men were defined as men who reported having sex with men or with men and women as the potential exposure route. IDUs were participants who reported this as the potential transmission route. Participants who reported sexual intercourse with the other sex as the potential exposure route were classified as heterosexuals; homosexual/bisexual women were included in this group. Homosexual/bisexual men and heterosexuals with a history of being IDUs were part of the homosexual/bisexual men, heterosexual, and IDU analyses. The “others” mode of transmission included transfusion, biological accidents, and haemophilia.p-value < 0.05 was considered significant.The Test and Treat Initiative was adopted in December 2013 and implemented during 2014. Changes in the trends of the AIDS detection rate and the AIDS mortality rate after the introduction of the Test and Treat Initiative in relation to the period before the intervention (2007–2014) were assessed through a segmented regression Poisson model of an interrupted time series. The year 2015 was used as the interruption year for the interrupted trend regression analysis . A contiThis analysis is part of the Ministry of Health’s strategic plan to enhance HIV testing and was not assessed by an ethical committee board. In Brazil, according to law CFM n° 1.865/96, authorization or consent from parents or a responsible person is not required to perform an HIV test on individuals over 12 years of age if the test is voluntary and consent is provided by the adolescent, provided that the adolescent has the capacity to evaluate their problem and take actions accordingly. Administrative permission to access the raw data of the datasources used were obtained from the Department of STI, AIDS and Viral Hepatitis of the Brazilian Ministry of Health.From January 2007 to December 2015, a total of 329,154 AIDS cases were registered in the national surveillance system. The mean patient age was 38.0 years (SD: 11.5), and 63.2% were men. Table Heterosexual intercourse was reported as the mode of transmission in 60.7 and 33.8% of AIDS cases in women and men, respectively. Overall, 25.3% of AIDS cases were reported in homosexual/bisexual men. AIDS cases due to IDU represented 2.5% of cases in women and 6.0% of cases in men. In both sexes, a small proportion of cases (< 1%) occurred via vertical transmission and other modes of HIV exposure. Regarding the level of education, the highest proportions of cases were found in women with incomplete post-secondary education (43.4%) and in patients with at least a post-secondary education (40.0%). The highest proportion of AIDS cases was found in white people , followed by brown people . Death occurred in 15.7% of female cases and 18.5% of male cases.p-value < 0.001), and the predicted AIDS detection rate in 2015 without implementation of the Test and Treat Initiative in 2015 would have been 19.8. Thus, the estimated drop in 2015 (adjusted for the pre-2015 trend) was (1–7.7/19.8) = 60%.The AIDS detection rate per 100,000) before the Test and Treat Initiative (2007–2014) was 20.45; right after its implementation (2015), this rate was 7.70 , and the predicted AIDS mortality rate in 2015 without implementation of the Test and Treat Initiative in 2015 would have been 2.1. Thus, the estimated drop in 2015 (adjusted for pre-2015 trend) was (1–0.49/2.2) = 73%.The mortality rate was 2.32  before the Test and Treat Initiative (2007–2014); right after its implementation (2015) . This decline was observed in both sexes. This change might be explained by the adoption of the Test and Treat Initiative in December 2013 and its subsequent implementation in 2014, offering early treatment to all HIV-positive adults, regardless of their CD4 cell counts or viral load; in this context, treatment is also used as a form of prevention (TasP).TasP is likely the major driver of the reductions in AIDS morbidity and mortality. Amongst treated HIV-infected patients, ART leads to immune reconstitution, preventing the emergence of AIDS events and death –15.3 (from 17% in 2013 to 37% in 2016) [Brazil adopted the Test and Treat Initiative two years ahead of the standard WHO “test and wait”, which was modified in 2015. The early adaption in Brazil correlated with increases in HIV diagnoses and patients receiving treatment; therefore, the initiative might be a major driver behind the observed reduction in AIDS cases. The number of HIV rapid tests increased over time, with acceleration from 2012. Likewise, the number of new HIV diagnoses and the number of PLWHA on ART  showed increasing trends with acceleration from 2013 . As emphin 2016) . The medin 2016) . This inThe significant decrease in AIDS-attributed deaths in the final year might also be attributed to the TasP strategy –15. ScalLive Better Knowing), which was implemented in 2013 [Brazil has adopted combination prevention, which is a strategy that involves post-exposure prophylaxis (PEP); PrEP; mass campaigns to encourage condom use and testing; distribution of condoms; and specific HIV prevention actions with key populations . Indeed, in 2013 . The pro in 2013 . The Dep in 2013 . BetweenA major challenge for the country will be expanding periodic HIV testing, specifically by targeting the most at-risk populations. To expand ART for HIV-infected individuals and increase survival, estimates of HIV incidence must be incorporated into HIV/AIDS surveillance activities .The period of 2007 to 2013 displayed an increasing trend in the AIDS detection rate in men. When assessed by education, there was found to be an increase only in well-educated people. Changing patterns of risk behaviours could have influenced our results. Notably, there was a significant increase in diagnosed AIDS cases amongst homosexual/bisexual men in Brazil; this population is driving the HIV epidemic in the south of the country. It is likely that the intense macho culture that prevails discourages homosexual/bisexual men from identifying as such and therefore from adopting preventive measures. Brazil began distributing PrEP in January 2018 as part of their combined prevention initiatives [By contrast, AIDS cases in IDUs have significantly decreased over time. In Brazil, IDU has been progressively supplanted by crack cocaine. However, 5% of crack users in Brazil have HIV-AIDS, a rate 12 times higher than that of the general population .AIDS cases amongst indigenous people have remained low and stable. In all age groups, AIDS was more prevalent amongst men than women. The age group most affected was 25- to 44-year-olds.We have analysed data on AIDS cases reported to the national surveillance system, which is subject to some limitations. We cannot rule out the underreporting of AIDS cases and AIDS-attributed deaths rates, particularly in a context where a substantial number of infected subjects are unaware of their HIV status. Mortality may have multiple causes, and determining the attribution of HIV or other aetiologies is difficult.In conclusion, our results show fairly stable levels of AIDS detection rate and of AIDS-attributed deaths over 2007–2014, with sudden significant drops in 2015. The drop in the AIDS detection rate might be related to the scale-up of HIV rapid testing , the increasing trend in the number of PLWA on ART, which reflects improved survival (with a more rapid increase beginning in 2013) and the median CD4 count at ART enrolment . Given that the reduction in the number of AIDS diagnoses is apparent for only one year thus far, this observation and its interpretation require further confirmation and must be strengthened with similar analyses using additional years of data (2016 and 2017). Future studies could explore whether this decrease leads to a reduction in HIV transmission at the population level [Brazil must continue expanding ART and achieving universal access to therapy. Although gains in ART coverage have been reported, ART-eligible individuals in northern Brazil are notably less likely to obtain ART than those in southern Brazil. Other improvements, such as improved availability of the combined triple-dose, 3-in-1 medication, and use of the preferred first-line regimen in 2015, might enhance the decreasing trends in the AIDS detection and AIDS-attributed death rates.


In persons with cystic fibrosis (CF), decreased airway microbial diversity is associated with lower lung function. Conflicting data exist on the impact of short-term antibiotics for treatment of acute pulmonary exacerbations. However, whether differences in antibiotic exposure impacts airway microbiome changes has not been studied. We hypothesized that subtherapeutic beta-lactam antibiotic exposure, determined by the pharmacokinetics and pharmacodynamics (PK/PD) after intravenous (IV) antibiotic administration, would be associated with different patterns of changes in CF airway microbial diversity. Eligible children were enrolled when well; study assessments were performed around the time of pulmonary exacerbation. Plasma drug concentrations and bacterial minimum inhibitory concentrations (MICs) were used to determine therapeutic versus subtherapeutic beta-lactam antibiotic exposure. Respiratory samples were collected from children, and extracted bacterial DNA was amplified for the V4 region of the 16S rRNA gene. Twenty children experienced 31 APEs during the study; 45% (n = 14) of antibiotic courses were deemed therapeutic. Those in the therapeutic group had more significant decreases in alpha diversity at end of treatment and post-recovery compared to baseline than those in the subtherapeutic group. Therapeutic and subtherapeutic beta-lactam use is associated with different patterns of changes in CF airway microbial diversity following antibiotic administration. Children and adults frequently require hospitalization for severe recurrent lung infections, known as acute pulmonary exacerbations (APE)4. Antibiotic use is typically directed at specific pathogens, such as Pseudomonas aeruginosa5. However, prior studies have shown that in vitro susceptibilities do not correlate with clinical outcomes6.More than 30,000 people in the United States are living with cystic fibrosis (CF), a severe autosomal recessive disease that leads to recurrent lung infections and chronic suppurative lung disease associated with significant morbidity and mortalityPrevotella spp., Veillonella spp., and Gemella spp. that have commonly been identified in CF airways by culture-independent sequencing13. These obligate and facultative anaerobic bacteria may be positively associated with the development of APE7. The impact of antibiotics on microbial diversity and dominant core bacteria within the airway is unclear. Some studies suggest minimal changes within the bacterial community overall and resilience of core bacteria15, while others show decreases in Pseudomonas spp. when present17.This inconsistency may be due in part to the numerous other bacteria such as 19. Bacterial killing by beta-lactam antibiotics is time-dependent, meaning that the serum concentration of the antibiotic must be above the minimum inhibitory concentration (MIC) of the bacteria for a certain period of time (T > MIC)24. Due to higher beta-lactam antibiotic clearance in CF persons, the CF Foundation (CFF) and the European consensus guidelines recommend higher dosing regimens to achieve the needed serum concentrations26. However, surveys of CF centers in the US found that IV anti-pseudomonal beta-lactams were dosed below these guidelines 38–53% of the time27. No studies have evaluated IV beta-lactam antibiotic pharmacokinetics (PK) and pharmacodynamics (PD), specifically T > MIC, on relative bacterial abundance and alpha diversity.The variations in treatment response and airway microbiome changes to intravenous (IV) antibiotic therapy also may be due in part to subtherapeutic antibiotic exposure. The most common IV antibiotics used for treatment of APE in CF is a combination of an anti-pseudomonal beta-lactam antibiotic and an aminoglycoside30. However, the impact of antibiotics for acute treatment of APE on microbial diversity is less clear, with prior studies showing conflicting results during antibiotic treatment17. Furthermore, the impact of temporary decreases in microbial diversity with short courses of antibiotics with rebound to pre-treatment levels is not known. We hypothesized that subtherapeutic IV beta-lactam antibiotic exposure determined by PK/PD, and defined as insufficient time above the minimum inhibitory concentration (MIC) of the bacteria grown in culture, would be associated with different patterns of changes in CF airway microbial diversity. Specifically, we expected that subtherapeutic beta-lactam antibiotic exposure would have less of an impact on bacterial communities and diversity due to a lack of change in relative abundance of the targeted bacteria compared to therapeutic exposure.Identifying risk factors for decreasing microbial diversity in the CF airway is important because decreased alpha diversity is clearly associated with disease progressionTwenty study participants age 1 to 21 years of age were treated with 31 courses of IV antibiotics during the study period. The majority of participants (n = 13) had only one APE, but 5 participants had 2 APEs, 1 participant had 3 APEs, and 1 participant had 5 APEs.Of the 31 antibiotic regimens administered, 25 included a single beta-lactam antibiotic. Five courses included coverage with two beta-lactams, while one course included 3 beta-lactams for more than 72 hrs. All beta-lactams were intermittently dosed and none were given via extended or continuous infusion. Time above MIC (T > MIC) was determined for all beta-lactams; those patients with at least one beta-lactam with appropriate PD indices were considered therapeutic. Of the 5 patients who grew MRSA, three were on appropriate MRSA-directed therapy. The other two patients were already categorized as subtherapeutic based on their beta-lactam T > MIC indices and did not have to be re-categorized. Of note, 81% of all study participants also received an aminoglycoside as part of their antibiotic regimen that had been adjusted per our institutional guideline to meet goal peak concentrations. As aminoglycoside use was not different between the therapeutic and subtherapeutic groups , it was not considered in our categorization of therapeutic versus subtherapeutic.Ultimately, 45% (n = 14) of the antibiotic treatment regimens achieved optimal PD indices and were considered therapeutic, while 55% (n = 17) did not.Respiratory samples  were collected for 16S rRNA sequencing. Across all samples collected, 35% (n = 18) in the therapeutic group were sputum and 65% (n = 33) were OP swabs. In the subtherapeutic group 79% (n = 41) of the samples were sputum, 17% (n = 9) were OP swabs, and 4% (n = 2) were from BAL fluid. A sensitivity analysis of sample type on diversity measures was performed on samples from 4 participants with discordant sample types collected . Based on these findings, we excluded one OP swab and one sputum sample from further analysis. Lastly, for eleven time points, respiratory samples were not collected (n = 10) or failed sequencing (n = 1) and were also excluded from subsequent analysis.1) and forced vital capacity (FVC) compared to those in the subtherapeutic group .There was no significant difference in age, gender, race/ethnicity, or CFTR genotype between therapeutic and subtherapeutic participants Table . Those iEnterobacteriaceae_unclassified compared to the therapeutic group . There was also a trend that those with therapeutic group had higher alpha diversity measures at baseline compared to those in the subtherapeutic group . Alpha diversity was also significantly higher in the exacerbation samples of those in the therapeutic group compared to the subtherapeutic group  was used more often in the therapeutic group , while meropenem  was used less often  , Clostridiales_unclassified , and Lachnospiraceae_unclassified . Interestingly, a lower relative abundance of Fusobacterium  and Pseudomonas  were found in the baseline samples compared to treatment samples. No significant differences were found between baseline and treatment samples for those in the subtherapeutic group. There were also no significant differences identified between baseline and post-recovery samples for either group.We also evaluated for significant changes  in the abundance of bacterial genera between baseline and treatment and baseline and post-recovery for the therapeutic and subtherapeutic groups separately. Those in the therapeutic group had a higher relative abundance of the following bacterial genera in baseline versus treatment samples: Analysis of beta diversity using Morisita-Horn showed no significant differences between those in the therapeutic and subtherapeutic groups . This corroborates our findings previously published on this same cohort of patients where significant differences were found in non-normalized pulmonary function between the two groups, with higher recovery in those achieving therapeutic beta-lactam drug exposure31.Lastly, we analyzed the impact of therapeutic versus subtherapeutic beta-lactam exposure on changes in normalized pulmonary function , followed by an increase in diversity by day 8–10 of antibiotic therapy in another study17.Prior studies of the impact of antibiotic use for treatment of APE on airway microbiome have varied in their findings. One study found that the community structure within the CF microbiome did not significantly change in response antibiotic use for APE30. One study of longitudinal samples obtained in children and adults found that older adults with decreased lung function had more uneven bacterial abundance, measured by Pielou’s evenness, and that alpha diversity, measured as Inverse Simpson’s diversity index, decreased over time29. Another study found a strong correlation between poor lung function and low species richness8, while two others showed that lower alpha diversity, measured by Shannon diversity index, was associated with worsening pulmonary function30. Our previously published analysis of this patient cohort comparing antibiotic PK and lung function found subtherapeutic beta-lactam antibiotic exposure was associated with less improvement of lung function31. In this study, we also saw similar trends using normalized lung function data. Thus, the conclusion should not be drawn that because subtherapeutic antibiotics have less impact on changes in microbial diversity, it could be used as a strategy to prevent declining lung function. Further work needs to be done to determine if a strategy of using more targeted antibiotic therapy to assist in maintaining community diversity might impact CF disease progression32.Several studies have shown that decreased microbial diversity is associated with more severe lung diseasePseudomonas spp. was strongly associated with decreasing diversity. In our cohort there was no significant difference noted for Pseudomonas between the therapeutic or subtherapeutic groups when looking at culture data obtained at the onset of exacerbation. Interestingly, in our patient population, the microbiome of those patients in the subtherapeutic group had a higher relative abundance of Enterobacteriaceae at the onset of exacerbation compared to those in the therapeutic group. Other studies have shown increased airway inflammation in the presence of Enterobacteriaceae in culture11, and Enterobacteriaceae have been associated with the production of beta-lactamases in CF patients33.In the above studies, the presence of Lastly, the microbial composition of samples obtained from patients achieving therapeutic antibiotic exposure was significantly different from those achieving subtherapeutic exposure. The increased dissimilarity of microbial composition among those achieving therapeutic beta-lactam antibiotic exposure may reflect the influence of alpha diversity, as those in the therapeutic group had higher diversity at baseline and exacerbation onset yet had more significant decreases in alpha diversity at end of treatment and recovery. Surprisingly, the use of broad-spectrum beta-lactam antibiotics did not seem to play significant role in this decrease in diversity, as those in the therapeutic group were more likely to be receiving ceftazidime than meropenem.Limitations of this study include the small study size that included a range of children with different disease severity. Those with therapeutic beta-lactam antibiotic exposure were less likely to be on inhaled antibiotics, had higher baseline diversity, and had higher lung function. These baseline differences in severity of disease may have contributed to higher bacterial MICs in the subtherapeutic group, thus making it more difficult for those with more severe disease to achieve therapeutic beta-lactam concentrations. We also found high levels of inter-patient variability, which is consistent with previously published studies. However, our analysis attempted to counter these baseline and inter-patient differences by evaluating changes of each individual study participant over time instead of raw values of microbial diversity and lung function. Future studies that evaluated beta-lactam PK/PD differences in a more homogenous cohort would help corroborate the findings of this study.Pseudomonas aeruginosa11. However, while in our study there were more swab samples collected in those patients achieving therapeutic antibiotic exposure, they actually had a higher relative abundance of Pseudomonas, suggesting our findings are unlikely to be limited by an inability to detect this bacterium. We also performed a sensitivity analysis to remove samples from analysis thought to be adversely affected by sample type. Future studies using similar respiratory sample type for all measurements would help determine the significance of using variable sample types on our study findings.Another limitation was the use of both OP swab and sputum samples to describe the airway community. Some studies have shown that OP swab samples may underrepresent the presence of more inflammatory pathogens, such as P. aeruginosa34, regional variability35, or antibiotic resistance within the bacterial community at large37. It is likely that one main driver behind achieving therapeutic antibiotic exposure is the bacteria’s antibiotic susceptibility as opposed to differences in antibiotic PK alone. Further studies incorporating metagenomics could provide more detailed information on bacterial drug resistant variants in the bacterial population and overcome some of the limitations of culture based methods39.Lastly, we also relied on MIC breakpoints reported by the clinical lab, and this limited our ability to determine if higher doses of beta-lactam antibiotics could be used to overcome antibiotic resistance. This study also does not address either the variability of MICs reported within different sub-colonies of In conclusion, we found that subtherapeutic beta-lactam was associated with different patterns of changes in microbial diversity in response to antibiotic treatment. While therapeutic patients had more significant drops in alpha diversity, they trended toward improved recovery of pulmonary function. This is likely a reflection of disease severity, antibiotic exposure, and antibiotic resistance. Controlling for these factors will be important for accurately interpreting the impact of antibiotic therapy on microbial diversity and pulmonary function. Moreover, as microbial diversity is associated with disease progression, identifying additional factors that influence diversity is critical. Future studies are needed to validate the impact of antibiotic use on decreasing microbial diversity and identify ways that the CF airway microbiome might be used as a tool to better optimize antibiotic treatment for acute pulmonary exacerbations.This study was a prospective, longitudinal study that was conducted over 18 months at Children’s National Health System (CNHS), a tertiary care pediatric hospital serving the greater Washington, DC area. Inclusion criteria were age <21 years, having a confirmed diagnosis of CF (either genetic or positive sweat test), and having been hospitalized for IV antibiotics in the past three years .Institutional Board Review (IRB) at CNHS was obtained prior to beginning the study . The principles outlined in the Declaration of Helsinki were followed. Written informed consent was obtained from study participants ≥18 years of age and parental permission for those <18 years of age. Assent was obtained from participants 7–17 years of age.12. Inhaled maintenance antibiotics were not considered exclusionary. The second encounter began at a clinic visit or hospital admission when IV antibiotics were started for an acute pulmonary exacerbation (E). The third encounter occurred at the end of the antibiotic treatment course (T). The fourth encounter occurred at the next follow up visit in pulmonary clinic after completing IV antibiotic therapy, post-recovery (R). During each encounter, participants provided a respiratory sample  for microbiome analysis. During the second encounter (E), the respiratory sample was collected the day IV antibiotic therapy was started. During the third encounter (T), the respiratory sample was collected before completion of antibiotic therapy. Serum samples were also collected for determination of antibiotic PK during the second and third encounters (E and T)40. Fuchs’ criteria were used to define APE during the second encounter (E)41. Study participants who experienced more than one APE requiring IV antibiotics during the study period were asked to participate for each treatment course . Lung function testing was collected clinically following ATS spirometry guidelines42 and were reported using NHANES III reference values43.Study participants were evaluated at four types of research encounters. The first encounter (enrollment) represented their baseline state of health (B), and required that they had not received antibiotic treatment for an APE in the 30 days prior44. The following data was collected: age, gender, race/ethnicity, CFTR genotype, weight, height, medications, pulmonary function tests, and clinical culture data. A multiplex liquid chromatography mass spectrometry (LC-MS) assay in the PharmacoAnalytical laboratory  was used to determine plasma drug concentrations of the beta-lactam antibiotics received. PK modeling of beta-lactam antibiotics to determine T > MIC was performed using Bayesian estimation in MW/Pharm 46.REDCap electronic data capture tools were used to collect and store study data37. A full description of the determination of T > MIC has been published elsewhere, but in brief the highest MIC identified within a patient’s exacerbation culture against the beta-lactam antibiotics used for treatment was used31. If the patient grew only normal respiratory flora, the MIC used was the median MIC of the study patients who grew P. aeruginosa. MIC estimates from the literature were used for methicillin-sensitive S. aureus (MSSA). Participants on multiple beta-lactam antibiotics were deemed therapeutic if at least one beta-lactam antibiotic met the T > MIC parameters. Subjects who also grew methicillin-resistant S. aureus (MRSA) also had to receive directed anti-MRSA therapy to be considered therapeutic.The participants were stratified into therapeutic and subtherapeutic groups, on the basis of whether their beta-lactam exposure met appropriate T > MIC parameters g × 10 minutes). Supernatants were removed and pellets were frozen at −80 °C until they underwent DNA extraction.Spontaneously expectorated sputum samples and oropharyngeal swabs were collected by clinical and research staff according to standardized procedures. BAL samples were only collected by clinical staff. Sputum and BAL samples were processed by washing first 1:1 v/v with sterile normal saline, mixing the sample 1:1 v/v with Sputasol , vortexing for 1 minute, and placing in a 37 °C heated water bath to homogenize the sample. The media of swab samples were transferred into a sterile microcentrifuge tube. All samples were then pelleted through centrifugation  using the DSP Virus/Pathogen Midi Kit and the Complex800_V6_DSP protocol. Prior to DNA extraction, pellets were thawed and a chemical lysis step was performed. Specifically, the bacterial pellet was suspended in 500 μl of combination enzyme solution, which included lysozyme (20 mg/mL)  and lysostaphin (200 μg/mL)  in nuclease free water. The pellet was then incubated at 37 °C for 30 minutes. The tube was then briefly centrifuged to remove drops from the inside of the lid before placing on the QIAsymphony.47. Default settings were used to minimize sequencing errors48. Clean sequences were aligned to the SILVA_v123 bacterial reference alignment at http://www.mothur.org. Sequences were clustered into OTUs at the 0.03 threshold. Samples were subsampled  to the smallest sample size  to remove the effect of sample size bias on community composition. Alpha diversity was measured by the number of genera , abundance coverage estimator , Chao (q = 0), Shannon Diversity Index (q = 1), and the Inverse Simpson’s Index (q = 2). No. genus, Ace, and Chao give more weight to richness. Shannon weights richness and evenness equally, while Simpson provides more weight to evenness49. Beta diversity between samples was measured using Morisita-Horn49. The OTU and taxonomy tables were also imported into Rstudio for subsequent analyses using phyloseq and DESeq2 to determine differential abundance and create principle coordinates analysis (PCoA) plots51. A sensitivity analysis of sample type  was performed to allow for elimination of samples whose diversity was potentially affected by the method of sample collection.DNA (300 ng) was amplified for the V4 region (approximately 250 bp) of the 16S rRNA gene and libraries were sequenced on the MiSeq sequencing platform at the University of Michigan (Ann Arbor MI). Raw FASTQ files were processed in mothur (version 1.39.5)Baseline participant characteristics between those with therapeutic and subtherapeutic beta-lactam antibiotic exposure were evaluated using chi-squared and t-test. We used Generalized Estimating Equations to compare measures that varied by exacerbation to account for the repeated exacerbations within patients, modeling an exchangeable correlation structure. For dichotomous outcomes, we assumed the binomial family and the log link function. For continuous outcomes, we assumed the Gaussian family and the identity link. We used robust variance estimators due to the small sample sizes.12. Since we did not expect a linear or even a quadratic change across encounters we chose to look at the change between any pair of encounters. When assessing significance, to account for the fact that all exacerbations are not independent (some patients have more than one exacerbation) we used the GLS random-effects model where exacerbation number acts as our indicator of time. We used robust variance estimates to account for the smaller sample size.We wanted to assess the impact of treatment on our primary outcomes across four measurement encounters: baseline (B), at the beginning of an exacerbation (E), at the end of treatment (T) and at the next post-recovery follow up visit (R)DESeq2 to establish differential abundance were adjusted by the Benjamini-Hochberg adjustment51. PCoA plots using Bray and Jaccard distances with log transformed counts were created to visualize dissimilarities in microbial composition between therapeutic versus subtherapeutic antibiotic exposure and time of sample collection . Permanova was used to evaluate differences in overall bacterial distributions. Significance was set at p < 0.05 and was determined using the adonis function of vegan in R52.P values used to determine significance when using Supplemental Information


The central sensitization inventory (CSI) comprises 25 items and is commonly used to measure somatic and emotional symptoms related to central sensitization symptoms. CSI was developed as an easy-to-administer screening instrument for patients at high risk of developing central sensitization in whom it was essential to quickly evaluate the condition. The purpose of the present study was to develop a short form of CSI and evaluate its psychometric properties using a contemporary approach called Rasch analysis.A total of 505 patients with musculoskeletal disorders were recruited in this study. The CSI, pain intensity, pain interference, and the health-related quality of life (QOL) were evaluated for each participant. The original CSI items were consecutively analyzed using the Rasch model. Successive Rasch analyses were performed until a final set of items satisfied the model fit requirements. We also analyzed the psychometric properties of the original and short forms of CSI.Four consecutive Rasch analyses identified the removable items. Finally, the shortest questionnaire obtained that maintained the correct psychometric properties based on the Rasch model contained only 9 items (CSI-9). Rasch analysis showed that the CSI-9 had acceptable internal consistency, exhibited unidimensionality, had no notable differential item functioning, and was functional on the category rating scale.The nine-item short form of CSI has acceptable psychometric properties and is suitable for use for patients with musculoskeletal pain. Thus, the CSI-9 can be used as a brief instrument to evaluate central sensitization. The International Association for the Study of Pain defines central sensitization (CS) as an increased responsiveness of nociceptive neurons in the central nervous system to normal or subthreshold afferent input . SeveralThe CSI total scores have been shown to distinguish between patients with chronic pain and control subjects , 10. A hCSI has been translated into many languages and has been validated by a variety of practitioners, 16–21. The Rasch model , which iIndividuals aged between 20 and 80 years were consecutively recruited and screened by orthopedists to receive physical therapy from an orthopedic clinic. Those with acute (pain that lasts less than 3 months) or chronic (pain that lasts for at least 3 month) musculoskeletal pain disorders  were included. All participants underwent X-ray examination before receiving physical therapy. At the screening stage by orthopedists, participants with fracture, sprain, cancer, multiple sclerosis, brain or spinal cord injury, history of stroke, or history of psychiatric disease  as diagnosed by a psychiatrist were excluded.All evaluations were performed before physical therapy. Of the initial sample of 510 participants, 5 participants were excluded because they did not complete all the items in the questionnaires. A total of 505 individuals were included; all these patients were Japanese. Their characteristics are summarized in Ethical approval was obtained from the Institutional Ethics Committee of Konan Woman's University. Written informed consent was obtained from all the participants before the study, and the study was conducted in accordance with the tenets of the Declaration of Helsinki.All participants were assessed for demographic data , pain duration, CSI, health-related quality of life (QOL), pain intensity, and pain interference. CSI-J consists of Parts A and B. Part A consists of a 25-item self-report questionnaire designed to assess health-related symptoms that are common to CSS. Each item is rated on a 5-point Likert-type scale, with total scores ranging from 0 to 100. Part B (which is not scored) asks the participants whether one or more specific disorders, including seven separate CSSs, have been diagnosed previously .Health-related QOL was measured using the EuroQol 5-dimension (EQ-5D) instrument . EQ-5D wPain intensity and interference were measured using the Brief Pain Inventory (BPI) , 43. BPIThe original CSI was consecutively analyzed using the Rasch model by Winsteps software . Chi-square fit statistics were used to determine how well each CSI item contributed to total CSI scores . The mosWe investigated the psychometric properties of the original and shortened questionnaires, and compared the following components:We assessed category order to ensure the Likert scale functioned as expected. The CSI has five response categories . Category probability curves, and average measure and category fit statistics (infit and outfit) were used to explore rating scale functioning. Fit statistics are recommended to be between 0.6 and 1.4, 50. MorScale-to-sample targeting was made by comparing person locations from the sample with item locations of the scale. Their means should be close to 0 logits. In addition, we investigated targeting by visualizing the person-item map and comparing the means of the items and person measures. This was analyzed by plotting the person-item location threshold distribution graph with distributions of persons on the top half of the graph and item thresholds at the bottom half of the graph. Good targeting means that the distributions of persons and distributions of item thresholds match visually.Winsteps provides a person reliability index and Cronbach’s alpha as indicators of internal consistency  and bothWe intended to summate the CSI to provide an overall measure of CS-related symptomology. Individual items should share this unidimensional construct yet be sufficiently different to warrant their inclusion. Assessment of fit evaluates unidimensionality by identifying items that function unexpectedly. The principal component analysis of residuals (PCA) identifies unexpected clusters of items suggestive of a secondary dimension that could threaten measurement of the primary dimension. The PCA residual correlation matrix was visually inspected to identify clusters of items that would be suggestive of a second dimension. An eigenvalue greater than 2.0 for the PCA of residuals suggests a second dimension . ResponsThe dimensionality of both versions was also explored using exploratory structural equation modeling (ESEM) with geomin oblique rotation in Mplus 8. We tested various models: from a one-factor model to a five-factor model in the CFI-25 and from a one-factor model to a two-factor model in the CFI-9. A minimum cutoff of 0.95 for Comparative Fit Index (CFI), a maximum cutoff of .08 for Root Mean Square Error of Approximation (RMSEA), and a maximum cutoff of 0.08 for Standardized Root Mean Square Residual (SRMR) were considered as indicative of acceptable fit –58. ModeParticipants with outfit residuals greater than 1.5 logits were examined to determine the reason for poor fit. They were compared with those who fit the model using Fisher’s exact test  of signiItems should function in a similar manner for all people of similar levels of agreeability. We assessed for DIF across 5 subgroups: gender, age , pain intensity from BPI , pain duration , and pain interference from BPI . DIF was tested using a Mantel-Haenszel chi-square test with significance level set at p = 0.01 for each item. Item bias was explored if an item yielded a significant difference of greater than 0.5 logits between subgroups [3,1 values 0–0.40 were considered to indicate fair reliability, 0.41–0.60 moderate reliability, 0.61–0.80 substantial reliability, and 0.81–1.00 almost perfect reliability [CSI reliability was assessed using scores obtained from a second round of the questionnaire administered to participants within 2 weeks of the first questionnaire completion. An intraclass correlation coefficient (ICC) 2-way mixed model with absolute agreement was used to determine measurement reliability. ICCCorrelation analysis was evaluated using the Statistical Package for Social Sciences Version 25 . Data distribution was tested for homoscedasticity using the Kolmogorov-Smirnov test. A series of correlations was performed examining the relationships between the original and the short form of CSI total score and pain intensity, interference, and QOL. These correlations were investigated with Spearman’s correlation coefficient.3,1 and the 95% confidence interval for agreement. ICC3,1 values 0–0.40 were considered to indicate fair reliability, 0.41–0.60 moderate reliability, 0.61–0.80 substantial reliability, and 0.81–1.00 almost perfect reliability [In addition, the correlation between the original and the short form of CSI total score was investigated by calculating the ICCFive severity levels of the original CSI  were recommended to evaluate symptom severity or assess clinical changes in response to treatment . To deteThe differences in age, pain duration, pain intensity, pain interference, and health-related QOL were also compared with the shortened CSI severity level group using ANOVA and Tukey post-hoc analysis. Gender was compared with the shortened CSI severity level group using the chi-squared test. We adjusted alpha to 0.008 because we undertook six separate analyses.Four consecutive Rasch analyses identified the items that could be removed from the questionnaire . FinallyThe sample was not well targeted by both versions of CSI. The average person endorsability of CSI-25 and CSI-9 (mean ± SD logits) were -1.42 ± 0.91 and -1.09 ± 1.11, respectively. The item endorsability averages of CSI-25 and CSI-9 (mean ± SD logits) were 0 ± 0.72 and 0 ± 0.79, respectively. Visually, the shifting of person agreeability of CSI-25 and CSI-9 to the left when compared with item endorsability indicated that participants with low levels of central sensitization were not well targeted by the scale .In CSI-25 and CSI-9, 4 (0.7%) and 7 participants (1.3%), respectively, scored zero for all the items. None of the participants for CSI-25 and CSI-9 scored full points on all items.The average agreeability measures of the respondents advanced as expected across the rating scale categories in both CSI-25 and CSI-9, and there was neither excessive positive nor negative fit statistics in CSI-9, suggesting the category structure is adequate, but, in CSI-25, there was no excessive outfit statistics .Category 4 in CSI-25 was underutilized, which suggests the respondents experienced difficulty differentiating “often” from “always” . Each caPCA of the residuals indicated that the unexplained variance of the first contrast of CSI-25 and CSI-9 was 2.2 and 1.6 eigenvalue units, respectively, indicating multidimensionality of CSI-25 and unidimensionality of CSI-9. The tested five-factor ESEM models in CSI-25 and two-factor model in the CFI-9 were the most fitted to the data .The person reliability of CSI-25 and CSI-9 was 0.90 and 0.77, respectively. Cronbach’s alpha of CSI-25 and CSI-9 were 0.89 and 0.80, respectively. McDonald’s Omega of CSI-25 and CSI-9 were also 0.89 and 0.80, respectively. These results suggest that both versions of CSI have good internal consistency.Analysis of person fit of CSI-25 and CSI-9 identified 73 participants (14.4%) and 70 participants (13.8%) with excessive positive outfit (>1.5 logits), respectively. In CSI-25, the CSI-25 score of participants with misfit were significantly higher than those without misfit .There was differential item functioning for age  in CSI-25, but there was no differential item functioning for age, gender, pain intensity, pain interference or pain duration in CSI-9.3,1 of 0.86  and 0.79 , respectively.Of the participants who reported no change (the difference between first and second round of pain intensity during movement is under 10 mm) in pain intensity during the past 2 weeks (n = 100), in CSI-25 and CSI-9, there was excellent agreement between test and retest total scores, with an ICC3,1 of the CSI-25 and CSI-9 was 0.69 , suggesting substantial agreement.The CSI-9 was significantly correlated with CSI-25  . The CSIOf the 505 participants, 372 (73.6%) reported 0, 96 (19.0%) reported 1, 24 (4.7%) reported 2, and 13 (2.5%) reported ≥ 3 CSS diagnoses. The CSI-9 mean score was 9.9 ± 5.5 in the no CSS group, 13.0 ± 5.0 in the 1 CSS group, 13.7 ± 6.5 in the 2 CSS group, and 20.5 ± 5.7 in the 3 or more CSS group. The CSI score in patients with no CSS was significantly lower than in those with 1, 2, and 3 or more CSS. There was no significant difference in CSI-9 total score between the 1 and 2 CSS groups.ANOVA identified that there was consistent worsening in pain intensity, pain interference, and health-related QOL with respect to the CSI-9 severity levels . The numThe current study aimed to develop a short form of CSI and evaluate its psychometric properties. This is the first study to develop and validate a shortened CSI. The shortened CSI was obtained by reduction of the long and well-validated questionnaires by removing selected items using Rasch analysis. Rasch analysis has been proposed as an objective and optimal method to construct measurement scales and has recently been used to reduce the number of items in other questionnaires. In the present study, CSI-9 had acceptable internal consistency, unidimensional attributes, no notable DIF, test–retest reliability, and was functional on the category rating scale. Overall, CSI-9 showed adequate psychometric properties for use in evaluating health symptoms related to CSS in patients with musculoskeletal pain disorders. Therefore, CSI-9 might be useful in treatment planning when a shortened version of a questionnaire is needed, such as in a time-limited situation or for a specific population.As result of Rasch analysis, 9 items were selected. The face validity of the CSI-9 items is very good. For instance, the items correspond very well with the current criteria for fibromyalgia, using the Widespread Pain Index  and Symp-1.42 logits and -1.09 logits, respectively. Both versions covered average and high levels of somatic and emotional symptoms related to central CSS and are therefore best suited for use with people who have health symptoms related to CSS.The average person endorsability of CSI-25 and CSI-9 were Rating categories complied with the set criteria for category functioning and step measures endorsed monotonically from easy to hard across category responses, supporting proper category order in CSI-9.Although PCA analysis of the residuals of CSI-25 suggests the presence of a second dimension , CSI-9 has unidimensional attributes . This result supports the notion that CSI-9 is appropriate with respect to Rasch analysis. Therefore, a meaningful comparison of CS across patients should be conducted using CSI-9.The internal consistency of CSI-9 was noteworthy  and aligned well with the original English version  [CSI-9 adequately comprises all five components of CSS  in the Japanese version . Therefo3,1 of the CSI-25 and CSI-9 was 0.69, suggesting substantial agreement, thereby making CSI-9 a promising measure.For a satisfactory short form, the reduction in the correlation scores of the short form should not drop by more than 0.10 from the original version . Our resIn the present study, patients were divided into subgroups according to the number of CSS diagnosis. The CSI-9 total score in patients with no CSS was lower than that in patients with 1, 2, and 3 or more CSS, but there was no difference in the CSI-9 total score between participants with 1 and 2 CSS. Therefore, we determined three levels of severity in CSI-9, which included subclinical (0–9), mild (10–19), and moderate/severe (20–36). The study results suggest that a strong relationship exists between the CSI severity groups and pain intensity, disability, and health-related QOL. The lowest CSI severity group reported lower pain intensity, disability, and health-related QOL, whereas the highest CSI severity group reported higher pain intensity, disability, and health-related QOL. These results suggest that the CSI severity groups in CSI-9 have high concurrent and construct validity. Therefore, clinically relevant symptom severity levels provide more useful information to healthcare providers in assessing CS/CSS-related symptoms.Our results are not intended to suggest abandoning CSI-25. CSI-25  has been psychometrically validated in many languages, and its dimensionality and reliability have been established, suggesting that CSI could detect comprehensive traits of CS. The CSI-9 would be a valid and reliable option if researchers or clinicians want to use an abbreviated measure of CSS-related symptoms, such as screening to rule out a disorder or epidemiological surveys.There were several limitations to our study. First, our study included individuals between the age of 20 and 80 years. Therefore, it is possible that age, sex, pain intensity, and pain duration influenced the results of psychometric properties. However, a notable DIF was not found for any of the items in CSI-9, suggesting that there is no effect of participant age, sex, or pain intensity and duration. This finding indicates that these factors might not affect the model. Second, this study included a convenience sample of participants with musculoskeletal disorders who were recruited from a single clinic. Future studies, in which data is compared across multiple centers, are warranted to verify the findings of this study. Third, this study utilized a questionnaire that was psychometrically validated in a Japanese sample and therefore validation of the other language version of the short form of CSI is needed. Fourth, the short form of CSI was extracted from one administration of the full CSI. Further research to investigate the specific CSI short form might be required for more definitive conclusions. Fifth, CSS diagnoses were established from the same self-reported instrument as the CSI scores. This procedure is limited as a validation, similar to internal measure based on two different procedures . Absence of a non-self-reported type and number of clinical diagnosis of CSS is needed in the further study.A nine-item short form of CSI has acceptable psychometric properties and is suitable for use for patients with musculoskeletal pain. Thus, CSI-9 can be used as a brief instrument to evaluate CS.S1 Table(DOCX)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.


MET (or c-MET) gene amplification has long been known as an important resistance mechanism to first- or second-generation EGFR-TKIs in addition to the appearance of T790 M mutation. Recent preclinical and clinical studies have suggested that MET amplification and/or protein hyperactivation is likely to be a key mechanism underlying acquired resistance to third-generation EGFR-TKIs such as osimertinib as well, particularly when used as a first-line therapy. EGFR-mutant NSCLCs that have relapsed from first-generation EGFR-TKI treatment and have MET amplification and/or protein hyperactivation should be insensitive to osimertinib monotherapy. Therefore, combinatorial therapy with osimertinib and a MET or even a MEK inhibitor should be considered for these patients with resistant NSCLC carrying MET amplification and/or protein hyperactivation.Treatment of non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) activating mutation with EGFR-TKIs has achieved great success, yet faces the development of acquired resistance as the major obstacle to long-term disease remission in the clinic.  Lung cancer is the leading cause of cancer death among both men and women and accounts for one third of all cancer deaths worldwide. Non-small cell lung cancer (NSCLC) constitutes over 80% of lung cancer cases and has a low 5-year survival rate of about 18% [The majority of EGFR-activating mutations (~ 90%) primarily present as an exon 19 deletion  or exon 21 point mutation L858R (~ 30%). The prevalence of these mutations is ~ 15% and ~ 40% in Western and Asian populations with NSCLC, respectively [MET (c-MET) gene amplification is another important mechanism and is detectable in approximately 5–22% of NSCLC patients with acquired resistance to first-generation EGFR-TKIs [MET amplification causes EGFR-TKI resistance by activating EGFR-independent phosphorylation of ErbB3 and downstream activation of the PI3K/AKT pathway, providing a bypass pathway in the presence of an EGFR inhibitor. This redundant activation of ErbB3 permits cells to transmit the same downstream signaling in the presence of EGFR-TKIs. Thus, concomitant inhibition of both EGFR and MET would be required to overcome resistance to EGFR inhibitors by MET amplification [MET amplification can occur with the EGFR T790 M mutation, about 60% of MET amplification is found without T790 M mutation. There is an inverse correlation between the presence of T790 M and MET gene copy number, suggesting a complementary or independent role of the two mechanisms in the acquisition of resistance [Acquired resistance to first-generation EGFR-TKIs is often caused by the acquisition of the T790 M mutation, which accounts for approximately 60% of resistant cases. In addition, GFR-TKIs . MechaniTM), rociletinib (CO1686), olmutinib (HM61713), nazartinib (EGF816), naquotinib (ASP8273), mavelertinib (PF-0647775), and avitinib (AC0010) are examples of third-generation EGFR-TKIs, which selectively and irreversibly inhibit the common “sensitive” EGFR mutations, Del19 and L858R, and the resistant T790 M mutation while sparing wild-type (WT) EGFR  protein that is a membrane tyrosine kinase receptor. The initially encoded preproprotein is proteolytically processed to generate α and β subunits that are linked via disulfide bonds to form the mature receptor. The binding of MET to its ligand, hepatocyte growth factor (HGF) secreted by stromal cells, induces dimerization and activation of the receptor. Therefore, the activated MET is a heterodimer linked by an extracellular α chain and a transmembrane β chain that contains a SEMA (sema homology region) domain, a PSI (plexin-semaphorin-integrin) domain, four IPT (immunoglobulin-like regions in plexins and transcription factors) domains, a transmembrane domain, a juxtamembrane domain, a tyrosine kinase domain, and a C-terminal tail region. The SEMA domain is the site where HGF binds directly to MET, and PSI can stabilize this interaction. When HGF binds MET, autophosphorylation of Y1234 and Y1235 in the intracellular tyrosine kinase domain occurs, resulting in Y1349 and Y1356 autophosphorylation in the C-terminal multifunctional docking site. This induces the recruitment of several intracellular effector adaptor proteins such as growth factor receptor-bound protein 2 (GRB2), GAB1, SRC, and PI3K and consequently the activation of downstream signaling pathways  also possessed amplified MET gene and hyperactivated MET protein, evidenced by increased p-MET levels in comparison with the parental cell line, and were resistant to not only rociletinib, but also erlotinib [MET expression restored the ability of osimertinib to effectively inhibit the growth of both HCC827/ER and HCC827/AR cells in vitro and in vivo and to inactivate ErbB3 or suppress ErbB3 phosphorylation [MET gene amplification and protein hyperactivation are likely a common resistance mechanism to both first- and third-generation EGFR-TKIs. Moreover, our results also suggest that monotherapy with osimertinib or other third-generation EGFR-TKIs will likely be ineffective for the treatment of EGFRm NSCLCs with acquired resistance to first-generation EGFR-TKIs due to MET amplification and/or protein hyperactivation.The initial preclinical link between MET and resistance to third-generation EGFR-TKIs came from our observation that an EGFR mutant (EGFRm) NSCLC cell line with acquired resistance to erlotinib (HCC827/ER), which has amplified rlotinib . In agrerylation . Our finMET amplification with increased protein expression and were resistant to osimertinib [MET gene with increased levels of both MET protein and p-MET and were cross-resistant to afatinib, osimertinib, and rociletinib [MET amplification was also detected accompanied with elevated levels of both MET and p-MET. This resistant line was cross-resistant to gefitinib but sensitive to the combination of naquotinib with a MET inhibitor (crizotinib or SGX532) [Consistently, several recent studies have generated similar observations. HCC827 cells resistant to erlotinib, which were established in a different laboratory, exhibited mertinib . Similariletinib . In a na SGX532) .MET dysregulation in human cancer tissues can be detected at the gene level  and at the protein level as discussed below. Beyond, MET alterations in ctDNA should be another way for detecting the dysregulation Table .Table 1AThe increase in copy number of the MET gene can occur in both polyploidy and amplification. Polyploidy is the duplication of chromosomes, and multiple copies of chromosome 7 are present in tumor cells. Polyploidy is not a driving gene in biology. Amplification is the duplication of local or regional genes, and the fault-fusion-bridge mechanism is the main cause of gene amplification. Compared with polyploidy, MET amplification may serve as a driving gene and is one of the main mechanisms of EGFR-TKIs resistance. The MET gene copy number is a continuous variable, and the definition of a positive threshold affects the incidence, the rate of overlap with other genotypes, and the ability to predict the efficacy of MET inhibitors .MET amplification can be detected using a FISH method that detects the MET/CEP7 value to distinguish polyploidy from amplification. In polyploidy, MET copy genes have corresponding centromeres, and MET/CEP7 values do not change despite an increase in the number of MET copies. NGS can also be used for amplification detection and requires comparison with normal diploid. There is no consensus on how many copies of the gene are MET positive. Current practice divides MET/CEP7 into low-level amplification , medium-level amplification , and high-level amplification (≥ 5) .MET overexpression can be caused by gene amplification, gene mutation, and transcriptional enhancement or by post-transcriptional mechanisms. IHC can be used to detect MET overexpression in tissue specimens. Due to differences in antibodies and thresholds, the proportion of MET overexpression in NSCLCs varied greatly in different studies, ranging from 15 to 70%. The proportion of MET overexpression was much higher than that of MET mutation and amplification.P = 0.0002) [Beyond tissue specimen, a recent study using digital sequencing of ctDNAs from 438 patients analyzed clinical associations of MET alterations in the plasma of patients with diverse malignancies including NSCLC and showed that MET ctDNA alterations were associated with a poorer prognosis, higher numbers of genomic abnormalities, and bone metastases. This study has demonstrated that detection of MET alterations by liquid biopsy is feasible. MET alterations were observed in 7.1% patients, which is higher than in the frequency in tissues  .MET amplification in EGFRm NSCLCs after relapse from osimertinib or other third-generation EGFR-TKIs was also reported. An early case report documented that a patient who developed resistance to osimertinib after a confirmed partial response for 9 months had a high level of MET amplification post-osimertinib treatment [MET amplification have varied.In line with our preclinical findings, clinical detection of reatment . This obMET amplification (4%) was detected among 25 NSCLC patients positive for EGFR T790 M that developed resistance to osimertinib [MET amplification. Le et al. [MET amplification (14%) were detected among 42 cases of progression following treatment with osimertinib. Piotrowska et al. [MET amplification, but only 6 patients (19%) with acquired EGFR C797S. Another study by Oxnard et al. [MET amplification were detected. Analysis of plasma samples from 73 patients with resistance to osimertinib second-line treatment in the large phase III clinical study AURA3 showed that MET amplification was the most common (19%) resistance mechanism, followed by EGFR C797 secondary mutation (15%), with 10 cases of C797S and 1 case of C797G [While only one case of mertinib , other se et al.  reporteda et al.  analyzedd et al.  documentMET amplification were positive; however, C797S was detected only in two (17%) of 12 assessed patients [MET amplification (31%) were detected [MET amplification was detected through core needle biopsy and next-generation sequencing (NGS) in another cohort of 9 Chinese patients after progression with osimertinib treatment although C797S or C797G was detected in 5 patients [MET amplification (5.4%). Moreover, two other cases containing rare mutations of MET P97Q and I865F were also detected although the biological functions of these mutations are unknown [In a cohort of Chinese NSCLC patients with T790 M enrolled in the AURA trial, 5 (50%) of 10 patients assessed for patients . In a didetected . Interespatients . Another unknown .MET copy number in 11 patients (26%) [MET amplification was observed only in 7.6% (5/66) of patients with acquired resistance to rociletinib [MET amplification was detected in only one case (6.25%), but EGFR C797S mutation was not detected [Similar observations have been made in studies with other third-generation EGFR-TKIs. Although the EGFR C797S mutation was initially detected in ∼ 32% of patients after relapse from osimertinib treatment , analysits (26%) . In a modetected .MET amplification in resistance to first-line osimertinib treatment for EGFR mutation-positive advanced NSCLC is limited, largely due to its recent approval for this indication. In the first reported trial, MET amplification was detected in one case (5.3%) among 19 patients with detectable circulating plasma tDNA [MET amplification (15%) followed by EGFR C797S mutation (7%) [The majority of studies reported so far have focused on the development of resistance to osimertinib or other third-generation EGFR-TKIs as second-line treatment. Information regarding sma tDNA . In a stion (7%) .MET gene amplification and protein hyperactivation [MET amplification after osimertinib resistance tended to have inferior median progression-free survival (PFS) and median overall survival (OS) than patients without the appearance of or increase in MET amplification [MET-amplified NSCLC that has relapsed from first- or second-generation EGFR-TKI treatment or patients who develop acquired resistance to osimertinib due to MET amplification and protein hyperactivation.Our preclinical studies suggest that monotherapy with osimertinib or other third generation EGFR-TKIs will likely be ineffective for the treatment of EGFRm NSCLCs with acquired resistance to first- or second-generation EGFR-TKIs due to tivation . In the tivation . Moreovefication . TherefoMET amplification causes EGFR-TKI resistance is associated with the activation of EGFR-independent phosphorylation of ErbB3 and downstream activation of the PI3K/AKT pathway, providing a bypass signaling pathway even in the presence of an EGFR-TKI  is also a very effective strategy in overcoming MET-mediated acquired resistance to osimertinib . Differe results . This isMET amplification or overexpression as highlighted below. Therefore, MET may still remain a rational target for therapy in patients with EGFR TKI-resistant and MET-amplified NSCLCs [MET inhibitors can be divided into three categories: the small molecule MET receptor inhibitors  Fig. , the METd NSCLCs .Fig. 5ChIn the MARQUEE phase III study, 1048 patients with advanced non-squamous NSCLC previously treated with one to two systemic regimens, including a platinum doublet, were randomly assigned in a 1:1 ratio to receive erlotinib plus tivantinib (E + T) or erlotinib plus placebo (E + P) until disease progression. OS was not improved with E + T versus E + P  even though PFS increased . Exploratory subgroup analyses suggested OS improvement in patients with high MET expression . It needMET amplification, the median PFS in the T + G group was more than five times longer than that in the chemotherapy group. In patients with MET amplification, T + G was 66.7% effective while chemotherapy was 42.9% effective in terms of response rate. Among patients with MET protein overexpression, the response rate of T + G was 68.4% versus 33.3% in the chemotherapy group. Subgroup analysis showed that patients with MET gene amplification receiving T + G treatment had a median PFS of 21.2 months, much longer than the 4.2 months among those receiving chemotherapy. T + G treatment was generally well tolerated [At the 2018 ESMO conference, Dr. Wu’s group presented the first trial comparing the efficacy of tepotinib and gefitinib combination (T + G) with chemotherapy for EGFR+/MET + NSCLCs. In patients with olerated .MET-amplified tumors, with a phase II ORR of 47% in patients with a MET gene copy number ≥ 6 [In a recently reported phase Ib/II study investigating the safety and efficacy of capmatinib plus gefitinib in patients with EGFR-mutated, MET-dysregulated (amplified/overexpressing) NSCLC who experienced disease progression while receiving EGFR-TKI treatment, 61 patients were treated in phase Ib, and 100 were treated in phase II. Preliminary clinical activity was observed, with an overall response rate (ORR) across phase Ib/II of 27%. Increased activity was seen in patients with high mber ≥ 6 . The majn = 66) treated with O + E showed improvement in both PFS and OS. Conversely, clinical outcomes were worse in MET-negative patients treated with O + E. Therefore, O + E was associated with improved PFS and OS in the MET-positive population but worse outcomes in MET-negative patients [MET copy number (mean ≥ 5 copies/cell by FISH); however, the benefit was maintained in “MET IHC-positive”/MET FISH-negative patients [A phase II clinical trial compared the treatment of patients with recurrent NSCLC with a combination of onartuzumab and erlotinib (O + E) versus erlotinib alone. Tumor tissue was required to assess MET status by immunohistochemistry (IHC). The study showed no improvement in PFS or OS in the overall population. However, MET-positive patients  and for the therapy of NSCLCs with activating EGFR mutations. However, about 20% of these patients do not respond well to osimertinib. Based on our preclinical findings, NSCLCs with MET amplification and MET protein expression are usually detected in the clinic. However, there is no study that detects phosphorylated MET (p-MET), which represents activated MET protein, in EGFRm NSCLC tissues or those with acquired resistance to EGFR-TKIs and its impact on patient response to EGFR-TKIs. In our preclinical studies, MET-amplified EGFRm NSCLC cell lines possess not only high levels of MET, but also elevated levels of p-MET [of p-MET . TherefoMET amplification and/or hyperactivation or patients relapsed from osimertinib due to MET amplification and/or hyperactivation, combinatorial therapy with a MET or MEK inhibitor may be explored based on preclinical and some clinical pilot studies   or programmed death-1 (PD-1) has shown dramatic success in some lung cancer patients –48. Howe