Due to the complexity of health system reform in the post-conflict, post-disaster, and development settings, attempts to restructure health services are fraught with pitfalls that are often unanticipated because of inadequate preliminary assessments. Our proposed Integrated Multimodal Assessment – combining quantitative and qualitative methodologies – may provide a more robust mechanism for identifying programmatic priorities and critical barriers for appropriate and sustainable health system interventions. The purpose of this study is to describe this novel multimodal assessment using emergency medicine in post-conflict Serbia as a model.Integrated quantitative and qualitative methodologies – system characterization and observation, focus group discussions, free-response questionnaires, and by-person factor analysis – were used to identify needs, problems, and potential barriers to the development of emergency medicine in Serbia. Participants included emergency and pre-hospital personnel from all emergency medical institutions in Belgrade.Demographic data indicate a loosely ordered network of part-time emergency departments supported by 24-hour pre-hospital services and an academic emergency center. Focus groups and questionnaires reveal significant impediments to delivery of care and suggest development priorities. By-person factor analysis subsequently divides respondents into distinctive attitudinal types, compares participant opinions, and identifies programmatic priorities.By combining quantitative and qualitative methodologies, our Integrated Multimodal Assessment identified critical needs and barriers to emergency medicine development in Serbia and may serve as a model for future health system assessments in post-conflict, post-disaster, and development settings. In June 1991, a series of civil wars began as the Socialist Federal Republic of Yugoslavia dissolved violently into the four independent republics of Slovenia, Croatia, Bosnia-Herzegovina, the Former Yugoslav Republic of Macedonia, and the semi-autonomous nations of Serbia and Montenegro. Kosovo remains a province of Serbia but under the administration of the international community. The events of the last decade in the Balkans have significantly impacted the health care system of Serbia. The nation and its health care system were devastated by international sanctions, soaring unemployment, political instability, near economic collapse, and the North Atlantic Treaty Organization (NATO) air campaign .A country's emergency medical services are a particularly important component of the health care system for they provide an essential front-line resource for trauma, medical emergencies, and those without other access to health care. Unfortunately, the system of emergency services in Serbia did not escape the devastation of the 1990s and continues to suffer from problems common throughout the health care system. Limited health care funding has resulted in a lack of necessary equipment, supplies, medications, personnel wages, and an economic infrastructure unable to support necessary health care reforms in the public and private sectors. Furthermore, the country has received little external support for health system reform, underscoring the need for focused assessments to best determine priorities for health care development.There is a desperate need for reorganizing and restructuring emergency medical services throughout Serbia. To better understand the problems, needs, and obstacles to development, a team of investigators from Johns Hopkins University Schools of Medicine and Public Health collaborated with the Belgrade University Emergency Center to perform a multimodal assessment of emergency medicine in Serbia. Our goal in this study was to identify the most pressing needs and achievable goals, as perceived by emergency health care personnel, in order to ensure that development programs and available funds are appropriately directed .Evaluating health care systems through the use of demographic data collection, surveys, or focus groups alone – as is often done in health system assessments – cannot adequately elucidate the intricacies of a country's health care system. In order to fully appreciate the complex dynamics of the health care system, it is necessary to perform a complete initial assessment; include strong local participation; identify and address significant barriers to change; and identify unique local needs and other cultural dimensions. To do this effectively, it is important to avoid simply introducing a "carbon copy" of a Western health care system that would further burden the system with inappropriate and unsustainable programs. We propose for the rapid assessment of health care services an Integrated Multimodal Assessment that uniquely combines diverse methodologies, whose individual effectiveness has been well described in the literature.This study used a multimodal approach to assess the strengths, needs, problems, and obstacles related to the development of emergency medical services in Belgrade. The four study modalities were 1) demographic information and observational data; 2) focus group discussions with 68 emergency medicine personnel; 3) individual, free-response questionnaires with 39 emergency medicine personnel; and 4) by-person factor analysis (or Q-methodology) of the attitudes and opinions of 36 emergency medicine personnel regarding the development of emergency medical services. Participants in the study were a non-probabilistic sampling of emergency medicine physicians, nurses, and department directors from all pre-hospital and hospital-based emergency medicine institutions in Belgrade. Many participants took part in more than one study modality. Data collection occurred during July-August 2002. The authors received institutional review board approval and the written informed consent of all participants.The capacity of emergency medical services in Serbia to provide for the needs of the country's citizens was characterized through health system data collection. Health system information was obtained through meetings with key officials in established health surveillance institutions such as the Emergency Center of the Clinical Center of Serbia; World Health Organization Belgrade office; Public Health Institute of Belgrade; Republic of Serbia Ministry of Health; European Agency for Reconstruction; Belgrade University, Institute of Social Medicine, Statistics and Health Research; as well as several non-governmental organizations with offices in Belgrade. Key officials were asked a series of quantitative, closed-response questions about the Serbian health care system and emergency medical services. Developed from a pilot health care assessment in Serbia the previous year, the questions focused on health system organization; patient population size and demographics; health care personnel; medical education and training; materials, supplies, and medications; and provider aptitude and morale .The two-fold purpose of the focus group discussions was 1) to better understand the needs, problems, and obstacles to system development as perceived by those involved directly in providing emergency medical services and 2) to collect a wide variety of opinions and attitudes for subsequent by-person factor analysis. The focus group discussions were held with 68 emergency medicine physicians, nurses, and directors through a non-probabilistic sample taken from the institute for pre-hospital emergency services, the Emergency Center of the Clinical Center of Serbia, and four of Belgrade's six community emergency departments. The discussions were conducted by the primary author in the Serbian language. The audio recordings of the 30- to 60-minute discussions were transcribed and translated into English by a native-English speaker fluent in Serbian (BDN) and a native-Serbian speaker fluent in English (MŠ). The translation was later verified by additional native-Serbian speaking co-authors fluent in English (MV, LM).The investigative field team (BDN, KD, MŠ, MV, LM) summarized the final transcripts and extracted common themes and divisive statements for by-person factor analysis.Free-response questionnaires enabled assessment of emergency health care needs and priorities for change as perceived by those most directly involved in patient care: the emergency health care providers. A non-probabilistic sample of 39 participants was obtained by open invitation, without compensation, through the director's office of each institution. As with the focus group discussions, the institute for pre-hospital emergency services, the Emergency Center of the Clinical Center of Serbia, and four of the city's six community emergency departments were represented. All subjects were administered a written Serbian-language questionnaire comprised of 10 free-response items  were designated respondent type "definers" and closely reviewed to assist in characterizing each respondent type.By-person factor analysis, or Q-methodology, avoids many of the limitations of other modalities by allowing the grouping of participants based on their subjective responses to an issue while preventing investigator preconceptions from influencing the grouping structure. In a letter to ectivity ,7. In ouectivity  followedzdravstvene stanice) and primary health centers (domovi zdravlja). In the capital city of Belgrade, there exists a network of 6 part-time, hospital-based (kliničko-bolnički centri) emergency departments that are supported by around-the-clock pre-hospital emergency services (hitna pomoć) and the academic Emergency Center (Urgentni centar) at the Clinical Center of Serbia. Like many European countries, Serbia has adopted a largely Franco-German model of emergency medicine in which pre-hospital emergency services are provided in the field by physician-staffed ambulances [Emergency medicine is not a recognized health care specialty in Serbia. In smaller communities, emergency services are provided by primary health stations , internal medicine (29%), anesthesia (12%), general medicine (7%), emergency medicine (3%), other (15%)). Discussions were held at the institute for pre-hospital emergency services, the Emergency Center, and four hospital emergency departments.The participants' comments from the focus group discussions are collectively summarized in Table In addition, physicians and medical directors report that their emergency medical system is in significant need of improved education and training programs. Suggested improvements include further training in emergency medicine during medical school, bedside training to develop practical skills, development of standardized treatment protocols, and access to continuing professional education.Questionnaires were completed by 39 physicians at the institute for pre-hospital emergency services, the Emergency Center, and four hospital emergency departments , internal medicine (31%), anesthesia (13%), general medicine (10%), emergency medicine (5%), other (10%)).Respondents show several areas of high agreement – particularly remarkable considering the questionnaire's free-response format. The most frequently reported strength of Serbia's current health care system are the health care workers (69%). Worker enthusiasm (33%) and the collaboration of various medical specialties providing emergency services (21%) are most frequently cited as the greatest positive attributes of emergency service providers. In addition, 46% of respondents report that the various emergency medical service providers  are other strengths of emergency medical services in Serbia.Thirty percent of the participants surveyed believe that lack of incentives to enter the specialty, difficulty of the work, and poor financial compensation are important barriers to becoming a physician in emergency medicine in Serbia. The poor reputation of physicians in emergency medicine relative to other physicians (reported by 46% of participants) may contribute to lower interest in specializing in emergency medicine. Poor organization (26%) and insufficient opportunities for professional development (15%) were also reported as major impediments to being an emergency service provider.Participants were asked what they believed were major problems of emergency medical services in Serbia. Inadequate finances, medications, medical supplies, and modern medical equipment were cited by 54% of the emergency medicine personnel as being a major problem , the absence of treatment protocols (27%), insufficient numbers of emergency medical physicians (17%), and the lack of government regulation (13%) were the most frequently cited priorities for improving the organization of emergency medical services. Other concerns include lack of quality control measures, burden of caring for non-emergent cases, inadequate admission and triage areas, and insufficient hours of operation of hospital-based emergency departments.Many emergency medicine personnel are also critical of the education and training of physicians in emergency medicine. Although 58% of those surveyed feel that physicians receive good theoretical training, 77% report that their educational system is in significant need of improved medical school and postgraduate training programs. Of the individuals advocating for improved education and training, 27% feel that further training in emergency medicine should be included in the medical school curriculum, and 53% believe that additional bedside training to develop practical skills is imperative. Furthermore, 56% of participants in this study express a need for better access to continuing professional education, including shared practical experiences and training between health care institutions. A large number of emergency medicine personnel (36%) stress the need for greater access to international journals, conferences, seminars, and the internet. Many (15%) also communicate a need for international collaboration and training programs between Serbia and foreign medical institutions.Survey participants cite numerous obstacles to the development of emergency medical services in Serbia, including insufficient funding and resources (69%), poor organization of emergency services (36%), lack of governmental support (26%), and inadequate education and training (18%). Also vital to development efforts are the perceptions and attitudes of the people impacted by and involved in these efforts – the public, health care community, and government officials. A slight majority (62%) of individuals surveyed believe that the health care community will support further development of emergency medical services. However, only 46% and 41% of participants feel that the public and government, respectively, will respond positively to development.Of the 36 emergency medicine personnel invited to participate in factor analysis, 33 individuals (91.7%) completed the exercise correctly , internal medicine (27%), anesthesia (27%), general medicine (6%), emergency medicine (6%), other (12%)). The institute for pre-hospital emergency services, the Emergency Center, and two hospital emergency departments participated in this component of the study.Factor analysis followed by manual factor rotation determined levels of agreement among the 33 participants and identified three unique types of respondents. Table In addition to assessing levels of agreement among participants, by-person factor analysis allowed us to identify three different respondent types, each involving multiple respondents with diverse demographics but who share common and unique perspectives relative to the remaining participants. To aid discussion of the three respondent types, each was assigned a brief label: "Utilize", "Develop", and "Invest".Respondent type "Utilize" was heavily loaded by a total of 5 respondents (15.2% of participants) and defined by 4 (12.1%). The principal concerns of the "Utilize" respondent type include the poor utilization of emergency medical services. These individuals believe that emergency personnel are well trained, educated, and prepared to address the emergent needs of the community. However, they consider their role ill-defined and misunderstood, leading to the ineffective use of their services by the public.Respondent type "Develop" was characterized by 6 loaders (18.2%) and 3 definers (9.1%). These individuals support the further development of emergency medicine as an independent specialty in Serbia. They do not consider inadequate finances, supplies, or equipment to be the chief concern of the system of emergency medicine. Instead, the "Develop" respondent type encourages the development of standardized treatment guidelines, improved coordination between the specialties providing emergency care, and additional education and training in the specialty of emergency medicine.The third respondent type, "Invest", included 4 loaders (12.1%) and 2 definers (6.1%). These individuals consider the system of emergency medicine appropriately organized but lacking adequate investment. They believe greater resources should be applied towards equipment and professional training and development. They are also concerned with an apparent lack of coordination between institutions providing primary health care and emergency services.Additional respondent types were defined by single respondents, which prevented adequate characterization. However, the demographic data on each of these isolated respondent types were closely examined to assure that the individual was not in a unique position of authority that could disproportionately influence future health system reform. The specific responses of respondent types "Utilize", "Develop", and "Invest" to the 23 Q-statements are listed in Table The purpose of a rapid assessment is to provide organizations with timely and reliable information to aid targeted interventions. Many rapid assessment methodologies have been utilized, each with its own strengths and weaknesses. One of the most commonly used methodologies is the KAP (Knowledge, Attitudes, and Practices) survey, which is traditionally a standard questionnaire with pre-developed, closed-response questions . BecauseThe use of more detailed assessment tools – employing a combination of qualitative and quantitative methodologies – can more accurately characterize the country's health care needs and the significant political and cultural barriers to sustainable health care reform. Our proposed Integrated Multimodal Assessment utilizes a diverse approach for strong internal validity and by-person factor analysis to uniquely identify critical subpopulations. By understanding the concerns of subpopulations, more targeted interventions can be developed to directly address these concerns. A trial study evaluating primary health care in Serbia has demonstrated the advantages of using a combination of qualitative and quantitative tools for health system assessment . Each ofOut of the near collapse of the Serbian health care system comes the opportunity to establish a health system more effective than ever in meeting the needs of its citizens. Our focus group discussions, questionnaires, individual surveys, and by-person factor analysis reveal that most emergency medical physicians believe the greatest problems in their system are poor organization of emergency medical services; lack of essential funding, medical supplies, medication, and technical equipment; and inadequate education and training. Several respondents also share the idea that poor incentives for specializing in emergency medicine, difficulties of emergency service work, and inadequate compensation are significant barriers to advancing the field of emergency medicine in Serbia. Although many of these are problems ubiquitous to developing health systems, this study illuminates specific opportunities for emergency medicine providers, the Serbian government, and international institutions to work together to address these issues.According to the majority of emergency medicine providers and administrators surveyed, of foremost priority is reorganization of emergency medical services. In order to begin addressing this need, several participants propose the development of triage and treatment protocols. To facilitate improved routing of patients within the hospitals and to the appropriate emergency health care facility, all health care providers, including those within the primary health centers, should receive formal training in triage. Several participants suggest that leadership development and management training should be provided to medical directors and leading health care providers to improve the organization within and between emergency health care centers. Public education programs should also be developed to increase awareness of the level of care that each health care institution provides so that the public can more properly utilize available health care services.Study participants further reported that the attainment of necessary medications, supplies, and equipment should be a top priority in the development of emergency services. The professionals working in the health care system of Serbia are severely limited by a shortage of these resources and, therefore, are unable to apply their knowledge and skills for proper diagnosis and treatment. Obsolete medical equipment further constrain their diagnostic capabilities, and lack of basic medications and supplies impedes their treatment strategies.This multimodal assessment also shows that the education and training of physicians in emergency medicine need further development. Elements of the educational system needing improvement include medical school and postgraduate bedside training programs; training between health care institutions; access to continuing professional education materials including international journals, conferences, seminars, and internet access; and international collaboration between Serbia and foreign medical institutions, including opportunities for Serbian physicians to gain international clinical experience. Several participants also suggest that all health care workers, including physicians working in the primary health centers, receive periodic training in Basic Life Support (BLS), Advanced Life Support (ALS), and emergency triage.Further development of emergency medicine in Serbia will be complex and will face numerous challenges. Reform will require funding, political commitment, supportive legislation, a revised medical curriculum, multi-phasic implementation, and post-interventional evaluation. Respondents believe that the funding needed for development of emergency services represents a significant barrier to change, but that, however, it is not the universal solution.Although a majority of the emergency medicine personnel surveyed feel that the health care community will support further development of emergency services, just under half of the participants expect similar support from the public and government. Additional studies specifically assessing the needs and concerns of the public and government are recommended.The use of multiple assessment modalities and the resulting complexity of this study involve several limitations that require consideration. All participants in this study were invited to participate by the director's office at each hospital or clinic. The study participants were aware that their participation was entirely voluntary and that they would not receive compensation. Although the study did not provide an economic incentive for participation, it did provide the opportunity for those providers with strong opinions to openly voice their concerns and suggestions. As a result, the study population may not represent all emergency health care providers. While every emergency medicine institution in Belgrade participated, this study was limited to the pre-hospital and hospital-based institutions within the capital city (covering 14.9% of the population of Serbia (excluding Kosovo)). Nevertheless, the participants represent the breadth of medical specialties in Serbia, and they constitute a large number of the stakeholders involved in further development of emergency medicine.Despite the challenges, development of emergency services in Serbia can be accomplished through the dedication and commitment of providers. As a result of studies on emergency services in Kosovo, developmental programs similar to those suggested here have been successfully implemented to improve the training in and provision of Kosovo's emergency medical services . VisitinIt is the hope of the authors that similar reforms will also be implemented to address the specific situation in the Republic of Serbia and that this multimodal assessment will assist by identifying the critical needs, barriers, and priorities for sustainable development. By utilizing both quantitative and qualitative methodologies, the authors submit that this Integrated Multimodal Assessment tool offers a more robust alternative to standard surveys, KAP studies, or the use of anecdotal information for quickly identifying priorities in health system reconstruction in the post-conflict, post-disaster, or development setting.KAP: knowledge, attitudes, and practicesRAP: rapid assessment proceduresThe authors declare that they have no competing interests.Each author has participated sufficiently in the work being reported to take public responsibility for the content. BDN, KD, and MVR conceived and designed the study and obtained research funding. MVR and MM supervised the completion of the study. BDN, KD, MS, MV, and LM undertook recruitment of participating centers and physicians. BDN, KD, and MS completed the data collection, statistical analysis, and first draft of the manuscript. All authors reviewed and contributed to the revision of the manuscript.The pre-publication history for this paper can be accessed here:


Increasing antimicrobial resistance among the key pathogens responsible for community-acquired respiratory tract infections has the potential to limit the effectiveness of antibiotics available to treat these infections. Since there are regional differences in the susceptibility patterns observed and treatment is frequently empirical, the selection of antibiotic therapy may be challenging. PROTEKT, a global, longitudinal multicentre surveillance study, tracks the activity of telithromycin and comparator antibacterial agents against key respiratory tract pathogens.In this analysis, we examine the prevalence of antibacterial resistance in 1,336 bacterial pathogens, isolated from adult and paediatric patients clinically diagnosed with acute bacterial sinusitis (ABS).S. pneumoniae isolates were susceptible to penicillin, cefuroxime, and clarithromycin respectively. Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of S. pneumoniae isolates (128 isolates were resistant to penicillin [MIC >= 2 mg/L], 72 intermediate [MIC 0.12–1 mg/L]) while 99.5% and 95.5% of isolates were susceptible to telithromycin and amoxicillin-clavulanate, respectively. In total, 88.2%, 87.5%, 99.4%, 100%, and 100% of H. influenzae isolates were susceptible to ampicillin, clarithromycin, cefuroxime, telithromycin, and amoxicillin-clavulanate, respectively. In vitro, telithromycin demonstrated the highest activity against M. catarrhalis (MIC50 = 0.06 mg/L, MIC90 = 0.12 mg/L).In total, 58.0%, 66.1%, and 55.8% of in vitro activity of against pathogens commonly isolated in ABS, together with a once daily dosing regimen and clinical efficacy with 5-day course of therapy, suggest that telithromycin may play a role in the empiric treatment of ABS.The high  The incidences of both the acute and chronic forms of sinusitis have been increasing, and between 10 and 15% of the population of central Europe are affected annually -4. AcuteS. pneumoniae is the most common pathogenic bacterium responsible for ABS, isolated in 30–50% of cases, followed by H. influenzae, isolated in 20–40% of cases. Moraxella catarrhalis is isolated in 5–10% of cases, beta haemolytic streptococci in less than 5%, and Staphylococcus aureus in less than 10% although it is often found co-infecting with other bacteria [Treatment options for ABS are controversial as up to 40% of patients recover spontaneously, however, studies have shown that treatment with an antibacterial reduces the time to recovery from sinusitis, improves symptoms, and helps to prevent complications ,12. GuidSecond line, or alternative antibacterial agents of choice, are clarithromycin or second-generation cephalosporins such as cefuroxime and cefpodoxime, third-generation cephalosporins such as cefdinir, and trimethoprim-sulphamethoxazole ,5,16,17.in vitro activity and demonstrated clinical efficacy against bacterial pathogens causing community-acquired respiratory tract infections (RTI's) [in vitro activity against the common community-acquired RTI pathogens [However, due to increasing levels of resistance in bacterial respiratory tract pathogens to these commonly used antibacterials (particularly the rapid emergence of penicillin-and macrolide-resistant strains of pneumococci), new agents are required that have high  (RTI's) -22. Teliathogens . Clinicaathogens -26.The PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study is a longitudinal, global multicentre surveillance study designed in part to determine the activity of telithromycin against community-acquired RTI isolates, in relation to the frequency of prescribing, in the regions where the study is conducted . The aimDetailed study design, including patient selection and methodology for isolate identification and storage in the PROTEKT study has been described previously . The isoS. pneumoniae and S. aureus, ≤ 1 μg/ml is susceptible, 2 μg/ml is intermediate, and ≥ 4 μg/ml is resistant; for Haemophilus influenzae, ≤ 4 μg/ml is susceptible, 8 μg/ml is intermediate, and ≥ 16 μg/ml is resistant [MIC susceptibility status was determined, using the National Committee of Clinical and Laboratory Standards (NCCLS) breakpoints, at a central laboratory (GR Micro Ltd, London, UK) from a panel of existing and new antibacterials, using the NCCLS broth microdilution method and lyophilised microtitre plates (Sensititre, Trek Diagnostics) . NCCLS besistant .2 test.Statistical analysis was performed using a χn = 652), North America (n = 14), Latin America (n = 207), Asia (n = 464), Eastern Europe (n = 68), Australia (n = 2), and South Africa (n = 126) in the PROTEKT study from years 2000–2001 and 2001–2002 for analysis of the susceptibility of bacterial pathogens isolated from patients with acute sinusitis. Gender distribution was 52.2% male (695 patients), 46.7% female (624 patients); gender was not provided for 1.3% of patients. Almost two thirds (66.3%) of patients were in the 0–12 year age group, one third (29.7%) in the 13–65 year age group, 2.7% in the over 65 year age group and age was not specified in 1.3% of patients. S. pneumoniae was the pathogen most frequently isolated (47.9% of isolates) followed by H. influenzae (24.6% of isolates) . Of note, 3 isolates of S. pneumoniae were non-susceptible to telithromycin (2 isolates intermediate with an MIC of 2 mg/L, 1 isolate resistant with an MIC of 8 mg/L). This represented 0.5% of isolates, a value that is significantly (p < 0.001) lower than those obtained by erythromycin (44.1%), clarithromycin (44.2%) and cefuroxime (33.9%).Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of H. influenzae isolates, 34 (10.3 %) were positive for β-lactamase production. All isolates of H. influenzae were susceptible to amoxycillin-clavulanate and telithromycin with an MIC90 of 1 and 2 mg/L, respectively. Amoxycillin-clavulanate and telithromycin were more potent and had greater activity than clarithromycin (MIC90 = 16 mg/L, 87.5% susceptible). This activity was comparable to azithromycin (MIC90 = 2 mg/l, 100% susceptible).Of the 329 S. aureus isolated from the total number of specimens was small (116/1366 isolates), telithromycin was as efficacious as comparators. Of the 116 isolates, 11 were resistant to methicillin (MRSA) and 105 were methicillin susceptible (MSSA). Ninety-nine (94.3%) of the MSSA isolates and 5 of the 11 MRSA isolates were susceptible to telithromycin. Of note, all of the S. pyogenes isolates were inhibited by ≤ 1 mg/L telithromycin, despite 17.7% resistance to erythromycin and clarithromycin.Although the number of M. catarrhalis with an MIC50 of 0.06 mg/L and MIC90 of 0.12 mg/L. β-lactamase production was detected in 97.6% of these isolates.Telithromycin was the most potent antimicrobial against in vitro activity against bacterial pathogens isolated from a large, globally distributed population of patients diagnosed with ABS. Telithromycin was the most active and potent agent against all isolates of the pathogens isolated from patients with ABS with 99.4% of isolates susceptible. Not surprisingly, high levels of penicillin resistance, macrolide resistance, and combined penicillin and macrolides resistance were prevalent in S. pneumoniae although prevalence varied widely between geographical regions.The data in this analysis demonstrates that telithromycin has high H. influenzae and M. catarrhalis found in the present study demonstrate compromised in vitro efficacy of amoxicillin against these isolates. Although the cephalosporins (cefuroxime, cefpodoxime and cefdinir) showed high activity against H. influenzae (including beta-lactamase positive strains), resistance to these agents was high in S. pneumoniae: >30% for cefuroxime and cefpodoxime – cefdinir was not tested against pneumococci in PROTEKT, however susceptibility is usually similar to the other cephalosporins reported here [Amoxicillin has been the treatment of choice in ABS because of its general effectiveness, safety, tolerability, low cost and narrow spectrum . The higS. pneumoniae of 44.1% to erythromycin, azithromycin, and clarithromycin was found. Trimethoprim-sulfamethoxazole activity was low for S. pneumoniae (56.3% susceptible) and decreased for H. influenzae (84.5% susceptible).Similarly, macrolides are prescribed in various countries for ABS and an overall resistance rate for Respiratory fluoroquinolones are recommended second-line treatment options in some countries (references needed to support this statement). However, recent evidence suggests that resistance to fluoroquinolones is rapidly developing in pneumococci and other pathogens  High rates of clinical cure and bacteriological eradication have been demonstrated using telithromycin against sinus isolates of S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus [The high . aureus -26. 2) T. aureus -39. 3) T. aureus ,41. This. aureus . 5) Teli. aureus ,45.S. pneumoniae isolates from patients with community-acquired pneumonia, they may then only send H. influenzae from patients with ABS to fulfill their quota for this organism. Thus, the potential exists to over or under estimate the prevalence of a species in a particular disease. A further limitation of this study is it is restricted to the major bacterial pathogens causing sinusitis and does not therefore assess anaerobic bacteria, which are also known to be involved in this disease. However, a recent study of sinus puncture specimens demonstrated that telithromycin had good in vitro activity against anaerobes involved in sinusitis [Although this study provides valuable information on the overall antimicrobial profile of bacteria causing ABS care should be taken when interpreting data related to specific demographics. A major limitation of this study, inherent to most surveillance studies, is the requirement for collecting centers to fulfill a specified quota of isolates over a defined time period (1 year). If, for instance, 1 center manages to fulfill their quota for inusitis .Streptococcus pneumoniae and Haemophilus influenzae. Additionally, there are regional differences in the susceptibility patterns observed and, as therapy is usually empirical, choosing an effective therapy can be challenging [The inclusion of nasopharyngeal specimens is a potential limitation of this study and the higher rate of resistance compared to sinus aspirates may indicate some isolates were nasopharyngeal flora rather than pathogens. However, the difference in resistance prevalence between nasopharyngeal specimens and sinus aspirates was not great for any species/antibiotic combination, and assuming the majority of isolates were the responsible pathogen, significant bias of resistance patterns is unlikely. The treatment of ABS is complicated by a difficulty in establishing the causative pathogen(s). Sampling of infected fluid using sinus puncture is a painful and rare procedure . Nasophallenging ,20,49.in vitro activity against S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus respiratory pathogens commonly isolated in ABS. It is as active as or more active than antibacterial agents that are currently used in this clinical setting. The development of resistance will always be a threat to the usefulness of antibacterial compounds, however surveillance studies such as PROTEKT allow the rapid detection and characterization of resistance mechanisms and highlight the need for and examine the in vitro efficacy of newer antibacterial agents. Providing careful surveillance for the development of resistance is maintained telithromycin currently offers a useful therapeutic option in the treatment of AS.In summary, the data presented here demonstrates that telithromycin has good JD, RCa and RCo reviewed the data and provided clinical and microbiological interpretation and discussion. DF and DJF participated in the design of the study, supervised the scientific testing, and provided data analysis, microbiological interpretation and discussion. All authors drafted the manuscript. All authors read and approved the final manuscript.


Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.The M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19 kDa) in Escherichia coli. IFN-γ production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19 kDa. Overall, the mean response to MBP-19 kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-γ after stimulation with either WCL or MBP-19 kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne's disease were used in immunoblot analysis. Reactivity to MBP-19 kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows.MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-γ production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay.Collectively, these results demonstrate that while the 19-kDa protein from  Mycobacterium avium subsp. paratuberculosis (referred to hereafter as M. avium subsp. paratuberculosis) and induces a chronic enteritis in ruminants. The disease signs include weight loss, diarrhea, and decreased milk production. In the United States alone the economic burden of Johne's disease is estimated at over $200 million in lost annual revenue to the dairy industry [M. avium subsp. paratuberculosis [Paratuberculosis (Johne's disease) is caused by industry . Prevalerculosis . Neonatarculosis ,5. Durinrculosis . Vaccinerculosis -9.M. tuberculosis genome that possess a characteristic amino-terminal acylation motif [Mycobacterium tuberculosis is immunodominant in both mice [+ T cell proliferation as well as the release of IL-2, IFN-γ, and IL-12 [M. tuberculosis 19-kDa protein inhibits innate immune responses, such as the release of TNF-α, IL-6, and IL-10 from macrophages, but does not affect antibody binding [M. tuberculosis-infected monocytes [M. tuberculosis residing within the phagolysosomal compartment [M. bovis, M. avium, and M. intracellulare but are absent from M. phlei, M. smegmatis, M. fortuitum, M. gordonae, and M. leprae [Lipoproteins have long been considered immunomodulators and mycobacteria are especially rich in these post-translationally modified proteins. There are approximately 100 open reading frames identified in the oth mice -13 and hoth mice ,15 and hnd IL-12 ,17. Acyl binding -20. The onocytes  as well onocytes . Finallyonocytes ,23. Expepartment . Homolog. leprae .M. avium subsp. paratuberculosis proteins involved in metabolism, cell wall synthesis, macrophage entry and survival, disease pathogenesis, or host immune evasion. However, several antigens have recently been identified and their immunogenicity examined by serodiagnostic and/or lymphocyte stimulation assays [M. avium subsp. paratuberculosis recently defined [M. avium subsp. paratuberculosis enable us to select and characterize genes of interest. A major goal of this laboratory is to define a complete catalog of immunodominant antigens in M. avium subsp. paratuberculosis. With the immunostimulatory capabilities of the M. tuberculosis 19-kDa antigen in mind, the objective of this study was to determine if the 19-kDa protein of M. avium subsp. paratuberculosis possessed a similar capacity. In this study, the 19-kDa lipoprotein from M. avium subspecies paratuberculosis was cloned, expressed, and characterized. In addition, the purified recombinant protein was used to assess cellular immune responses in subclinically infected cattle as well as humoral immune responses in cattle with clinical Johne's disease.Despite decades of research, little is known about the n assays -30. With defined , all proM. avium subsp. paratuberculosis genome project as MAP0261c. Comparison of amino acid sequences from other species of mycobacteria show that this gene product is conserved. Sequence alignment shows the N-terminal half is more variable and the region between amino acids 99 and 123 is highly conserved  detected a signal peptide generated from a putative cleavage site between amino acids 34 and 35, to expose an N-terminal serine -19 kDa fusion protein that was affinity-purified from E. coli lysates. MBP-19 kDa was analyzed by SDS-PAGE  that detects the MBP affinity tag . By comparison, sera from 9 of 14 infected cattle reacted specifically with the 19-kDa protein, but not MBP alone. Antibody reactivity to the 19-kDa protein from 3 clinical cows is shown in Figure The M. avium subsp. paratuberculosis sonicated whole cell lysate (WCL), MBP, or MBP-19 kDa and IFN-γ production was assayed by ELISA. These cattle were selected from a larger group because they showed no IFN-γ stimulation in response to MBP alone. IFN-γ production in response to WCL stimulation allowed for the segregation of infected cattle into three groups: suspect, positive, and high positive. After subtracting the IFN-γ responses of non-stimulated cells, suspect animals had less than 0.1 absorbance units of IFN-γ production, positive animals had 0.1 – 0.3 absorbance units of IFN-γ production, and high positive animals had more than 0.3 absorbance units of IFN-γ production. IFN-γ responses by blood mononuclear cells from infected cattle exceeded responses from control cattle for both the WCL and MBP-19 kDa  or WCL, which may not be specific for M. avium subsp. paratuberculosis. Recent completion of the M. avium subsp. paratuberculosis genome has already advanced efforts to identify novel antigens [M. avium subsp. paratuberculosis 19-kDa protein, as well as assess its immunostimulatory capabilities in cattle.A majority of the research on individual mycobacterial proteins has been performed in antigens ,34. FurtM. avium subsp. paratuberculosis can be readily overexpressed as a fusion protein in E. coli. This is not true for many other proteins encoded by M. avium subsp. paratuberculosis [E. coli. Previous studies have suggested the M. tuberculosis 19-kDa protein undergoes posttranslational modification by the addition of fatty acids to form a lipoprotein [E. coli host. It is unclear whether posttranslational modification of this protein would affect its immunological activity. The recombinant antigen was detected by sera from cattle with Johne's disease; however, it was not as strong a stimulator of proliferative T-cell responses as has been reported for its counterpart in M. tuberculosis [M. tuberculosis was shown to induce both cellular and humoral immune responses from mice and humans [In this study, we show that the 19-kDa protein of rculosis  and suggoprotein . Althougrculosis . Furtherd humans ,14. ThesM. avium subsp. paratuberculosis-infected cattle are biphasic, with IFN-γ responses detected early and antibody responses detected late in infection. However, evidence suggests that an unknown M. avium subsp. paratuberculosis protein can be detected by antibodies from cattle just 3 weeks after infection [M. avium subsp. paratuberculosis (WCL), and IFN-γ production was measured. Results from this study suggest that while the 19-kDa protein is a stimulator of IFN-γ production, it is not as potent when compared to WCL. Additionally, we found that a majority (9 of 14) of infected cattle produced antibodies to the 19-kDa protein, as determined by immunoblot analysis. By comparison, sera from the majority of control, non-infected cattle (8 of 9) did not react to the 19-kDa protein. The single non-infected cow that did show reactivity to the MAP0261c gene product may be attributed to exposure of environmental mycobacteria such as M. avium subsp. avium, which has a similar protein .It is generally accepted that cellular and humoral immune responses of nfection . As a meM. tuberculosis 19-kDa protein was reported to contain a 21 amino acid N-terminal signal peptide [M. avium subsp. paratuberculosis identified a putative 34 amino acid N-terminal signal sequence. Furthermore, the computer algorithm PSORTb predicts a putative signal sequence, but it cannot determine if the protein is actually secreted. Antibodies will be produced against the recombinant MBP-19 kDa protein to determine if the protein is secreted by M. avium subsp. paratuberculosis.The  peptide , howeverM. avium subsp. paratuberculosis to identify infected cattle but should not be used as a "stand alone antigen" in new diagnostic assays.The results from this study show that the recombinant 19-kDa protein stimulates a weak host immune response in infected cattle. The 19-kDa protein may be used in conjunction with other antigens from M. avium subsp. paratuberculosis strain 19698-1974  was grown in Middlebrook 7H9 liquid media (pH 6.0) supplemented with 10% oleic acid albumin dextrose complex (Becton Dickinson Microbiology, Sparks, MD), 0.05% Tween 80 (Becton Dickinson Microbiology), and 2 mg/ml mycobactin J (Allied Monitor Inc., Fayette, MO). M. avium subsp. paratuberculosis cultures were grown to log phase at an optical density 540 nm (OD540) of 0.4, at 37°C without shaking. Escherichia coli DH5α cells were routinely grown in Luria-Bertani (LB) broth or LB agar plates at 37°C supplemented with ampicillin (100 μg/ml) for selection.M. avium subsp. paratuberculosis. The animals used in IFN-γ experiments were placed in four groups consisting of 9 non-infected healthy cows, 9 suspect subclinical cows (IFN-γ responses < 0.1), 6 positive subclinical cows (IFN-γ responses 0.1 – 0.3), and 6 high-positive cows (IFN-γ responses > 0.3). The non-infected control cows were characterized by repeated negative fecal cultures performed quarterly over a 3- to 5-year period. In addition, these animals were negative on all immunological assays (i.e. ELISA and IFN-γ production) performed during that period. Subclinical cattle (suspect, positive, and high-positive) were characterized by shedding less than 10 CFU/g of feces and were intermittently positive by IFN-γ assays (response > 0.1) performed quarterly over a 3- to 5-year period. The institutional Animal Care and Use Committee approved all animal procedures described in this study.The Johne's Disease Research Project at the National Animal Disease Center has a repository of sera from cattle that were euthanized with clinical signs of Johne's disease, which included shedding, weight loss and diarrhea. Fecal samples from each of these clinical cattle were found to contain more than 100 CFU per gram of feces as determined by colony counts on Herrold's egg yolk media (HEYM) agar slants by standard culture methods . Sera frM. tuberculosis, M. bovis, M. avium and M. intracellulare were obtained from the NCBI nucleotide sequence database. The sequences were assembled and compared using MegAlign software (DNASTAR, Inc., Madison, WI).The nucleotide sequences for the 19 kDa coding regions from M. avium subsp. paratuberculosis 19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector (New England Biolabs, Beverly, MA). To amplify the 19-kDa coding region from M. avium subsp. paratuberculosis, primers were designed directly from the MAP0261c sequence. MAP0261c was amplified with Expand High Fidelity PCR system using the primers 19-kDa-pMal5' (5'-GCGCCAGCTGACGATCGCGGTCGCGGGCGCGGC-3') and 19-kDa-pMal3' (5'-GCGCAAGCTTCAGGTCACATCGATCTCGAAC-3'). The 19-kDa-pMal5' and 19-kDa-pMal3' primers contained Pvu II and Hind III restriction sites (underlined) for cloning, respectively. This primer set amplified the 19-kDa coding sequence minus the N-terminal 4 amino acids and the C-terminal 3 amino acids. The pMal-c2 vector was digested with Xmn I and Hind III and the PCR amplicon was digested with Pvu II and Hind III. The two products were ligated overnight at 12°C with T4 DNA ligase (Life Technologies Inc., Rockville, MD), which resulted in an in-frame fusion between the vector-encoded malE gene and a majority of the 19-kDa gene. The resulting recombinant plasmid, designated pMal-19-kDa, was transformed into E. coli DH5α competent cells. Recombinant clones were selected by plating on LB-ampicillin plates overnight at 37°C. Individual clones were picked and inserts were confirmed by DNA sequencing. The resulting fusion protein was overexpressed and purified by maltose affinity chromatography using amylose resin (New England Biolabs). A detailed expression and purification protocol has been published previously [A maltose binding protein (MBP) fusion of the eviously . ExpressE. coli lysates expressing MBP-19 kDa were prepared as previously described [escribed . SDS-PAGM. avium subsp. paratuberculosis sonicate (WCL), 10 μg/ml of MBP, or 10 μg/ml of MBP-19 kDa. The WCL was prepared by sonication of bacilli and centrifugation exactly as described previously [2 humidified atmosphere. Plates containing blood-antigen samples were centrifuged at 500 × g for 15 minutes and the plasma was harvested from each well. Plasma samples were frozen at -20°C until being analyzed for IFN-γ concentration by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Bovigam, BioCor, Omaha, NE) as recommended by the manufacturer. Samples were analyzed in duplicate and were determined to be positive for IFN-γ production if the absorbance of the stimulated sample (WCL, MBP, MBP-19 kDa) was 0.1 units greater than the absorbance of the nonstimulated well for that animal. This classification of IFN-γ positive samples has been previously reported by our laboratory as well as others [Blood was collected from the jugular vein of subclinically-infected cattle into sodium heparin vacutainer blood collection tubes. One ml aliquots of whole blood from each animal were plated into 4 wells of 24-well culture plates and cultured alone (non-stimulated) or with 10 μg/ml of eviously . Blood-as others ,42.ANOVA and unpaired t tests were performed to analyze the IFN-γ stimulation data. Analyses were performed to compare average stimulation of control, non-infected cattle to the infected cattle groups (suspect, positive, high positive). Differences were considered significant when P < 0.05.JFH carried out all the experiments and drafted the manuscript as part of his PhD dissertation. JRS provided advice, participated in its design and coordination and helped edit the manuscript. JPB conceived of the study, participated in its design and helped to draft the manuscript.


Multiple instruments exist to measure dementia behaviors, but the nursing staff perspective on those behaviors and their level of burden has not been well measured. The goal of this study was to examine the psychometric performance of the Modified Nursing Care Assessment Scale (M-NCAS), a 28-item nurse rating of burden associated with care for institutionalized individuals with dementia. Nurses rate items in terms of extent to which the behavior or characteristic is present ("attitude" domain), and extent to which it is a burden ("strain" domain).Data from 282 patients enrolled in a 12-week, double-blind, randomized clinical trial comparing risperidone treatment to placebo was used to evaluate M-NCAS item performance, internal consistency reliability, and construct validity. Empirical subscales were identified via exploratory factor analysis (EFA).Four poorly-performing items were deleted from further analyses. EFA identified 3 "attitude" subscales and 5 "strain" subscales. Cronbach's alphas were 0.65 and above. Correlation with the Cohen-Mansfield Agitation Inventory and the BEHAVE-AD, clinical ratings of dementia behaviors, were low to moderate.The M-NCAS provides a valid and reliable means of obtaining care burden ratings from formal caregivers in long-term care, and provides a method for evaluating dementia interventions from the perspective of nursing staff. Caring for the institutionalized dementia patient can be challenging for nursing staff ,2. PersoThere are measures relevant to the burden of paid/formal caregivers (i.e., nursing staff caring for dementia patients), but most focus on such overt behaviors as "forgetting what day it is" or "pull away" only  ,10 was dThe M-NCAS was used in a clinical trial of an atypical antipsychotic, risperidone, for treatment of nursing home patients with behavioral and psychological signs and symptoms in dementia. This preliminary investigation describes the psychometric evaluation of the M-NCAS using data from the clinical trial; thus, it is a post-hoc assessment of reliability and validity. Assessment of psychometric properties included exploration of item performance, subscale assignment, and assessment of reliability and validity based on the available data from the clinical trial.The psychometric evaluation used data from the baseline assessment of a multi-site, double-blind, placebo-controlled trial of risperidone in the treatment of behavioral and psychological symptoms among institutionalized patients with dementia. Fourteen sites throughout Australia and New Zealand recruited patients from the various nursing homes associated with each site. Patients were included in the clinical trial if they were 55 years of age or older, residing in a nursing home environment for one month, with a DSM-IV diagnosis of either dementia of the Alzheimer's type with behavioral disturbance, vascular dementia with behavioral disturbance, or mixed dementia, had scores less than or equal to 23 on the Mini-Mental Status Exam (MMSE) , and metThe Behavioral Pathology in Alzheimer's Disease Rating Scale (BEHAVE-AD), the CMAI, and the M-NCAS were administered to the nurse carers of the individual patients by the investigator or site coordinator at baseline, week 4, week 8, and week 12 . Nurse carers could have more than one patient participating in the trial, and they completed questionnaires for each individual patient.The M-NCAS is was adapted from the original NCAS instrument developed in Sweden, and contains 32 items based in part on the 21 original items of the NCAS. Additional items were selected based on comments made by long-term care nurses regarding their experience caring for dementia patients with dementia in long-term care facilities, and added to make the instrument more comprehensive.The M-NCAS contains 32 items, with two domains per item: one addresses the occurrence and intensity of the behavior, in which the staff members indicate the extent to which they agree that the patient exhibits the behavior (the "attitude" domain); and one which addresses staff rating of the difficulty of coping with the behavior (the "strain" domain). Responses are measured on a four-point Likert-type scale (ranging from Agree to Don't Agree for the Attitude domain, and Very Easy to Very Difficult for the Strain domain). Lower scores are better for both domains. Domain total and subscale scores are calculated separately; total and subscale scores are calculated as an average of the individual item scores. The M-NCAS was translated from the original Swedish to English using standard forward and backward translation techniques.For subscale analyses, if < 50% of the scale items were missing, the scale was scored with the mean score of the non-missing items for that individual used to impute a score for the missing items. If > 50% of the items were missing, no scale score was calculated; the subscale score was considered missing. Total scores were calculated only if < 50% of the items were missing. Missing item criteria was based on that recommended by Ware et al. .The CMAI is a 29-item scale specifically developed to assess agitated and disruptive behaviors, as well as care-related problems, occurring in demented subjects residing in nursing homes. The scale measures the frequency of behaviors, and systematically assesses agitation. The scale's 29 activities are rated on a 7-point scale indicating the frequency of a particular activity . The activities are organized into 4 subscales: physical/aggressive, physical/non-aggressive, verbal/aggressive, and verbal/non-aggressive. The total aggression score is the sum of the physical and verbal aggression subscales. The total non-aggression score is the sum of the verbal and physical non-aggression subscales. A recall period of one week was used. The CMAI was used to assess the construct validity of the M-NCAS.The BEHAVE-AD is a 25-item instrument designed to assess the severity of behavioral disturbances in subjects with dementia, based upon the caregiver's observation and report of the subject's behavioral problems. It consists of 7 subscales: paranoid and delusion ideation, hallucinations, activity disturbances, aggressiveness, diurnal rhythm disturbances, affective disturbances, and anxieties and phobias. Symptoms are rated on a scale from 0 (absence of the symptom) to 3 (greatest severity). A global item is included, in which a single judgment is made as to how troubling or dangerous the subject's behavior has been to the caregiver. A recall period of one month was used. The BEHAVE-AD was used to assess the construct validity of the M-NCAS.We evaluated the item performance, scaling characteristics, reliability, and construct validity of the M-NCAS using baseline assessment data.Item performance was examined to identify the presence of items that may reduce the instrument's ability to detect changes over time, and to discriminate between groups. The characteristics of individual M-NCAS items examined were mean, minimum and maximum responses, percent missing, floor and ceiling effects, and item-total correlations. These data were used to determine if some items should be deleted for subsequent analyses . ExplorInternal consistency reliabilities of total and subscale scores were estimated using Cronbach's coefficient α. Data to examine test-retest reliability was not available.Correlations were determined for total and subscale scores for attitude and strain domains and the CMAI physical and verbal aggression and non-aggression subscales. Correlations were also calculated between M-NCAS scores and the total score, seven subscales, psychotic symptoms subtotal and global rating question of the BEHAVE-AD, and the CGI-S and CGI-C scores. Pearson correlations were used unless otherwise specified. Correlations were expected to be low to moderate (0.20 ≤ r < 0.40).All statistical analyses were conducted using SAS statistical software version 8.0. A significance level of 0.05 and 2-tailed tests were used unless otherwise noted. No adjustments were made for multiple comparisons.Two hundred and eighty-two patients had evaluable baseline data for the psychometric evaluation. The majority of patients were female (72.4%) and Caucasian (98.4%); 11.5% were between the ages of 65 and 74, while 87.1% were over age 74. Most subjects had a diagnosis of Alzheimer's Disease (59.1%), while 28.3% had vascular dementia and 12.5% had mixed dementia.Nurse carers were primarily Caucasian. Further demographic data and information about the number and type of carers were not available.Items and their distributional characteristics were reviewed. See Table Examination of any floor or ceiling effects on the strain domain demonstrated no outliers. Item-to-item correlations appeared acceptable, as did item-to-total correlations, all generally above 0.30.With the number of factors unspecified, no clear factor pattern was discernible. Next, a six-factor solution for both dimensions (strain and attitude) was examined, based on the suitability of six factors for the original instrument . ExaminaBased on overall item analysis results, including floor and ceiling effects, item-to-item and item-to-total correlation results, and a review of factor analysis results, four poorly-performing items were deleted from the attitude domain: item 5 tries to influence others in order to maintain control of his/her own life; item 12 submissive/gives in to everything done to him/her; item 19 has little control over his/her difficult behavior; and item 27 has patterns of behavior you can foresee. The removal of these items resulted in three distinct subscales for the attitude domain, demonstrating good approximation of simple structure. The three factors, all with variable loadings of 0.30 or above were "Attention-seeking" , "Autonomy" (e.g., "does things for a reason", "is apathetic/seems to have limited emotions"), and "Difficulty" (e.g. "is unpredictable" and "is friendly"). Items were reverse-coded as appropriate.To ensure inter-domain consistency, we removed the same four items from the strain dimension, thus resulting in an acceptable five-factor solution for this domain. The five factors, all with variable loadings of 0.30 or above, are "Affect" , "Job satisfaction" (e.g., "is grateful for what is done for him/her", "gives no job satisfaction"), "Neediness" (e.g., "is selfish", "is manipulative"), "Predictability" (e.g., "knows what he/she wants and stands up for his/herself", "is aggressive/resistive"), and "Self-direction" . See Tables Following the removal of these 4 items, item-to-subscale correlations were examined for all subscales. In all cases, items correlated moderately to highly with the subscales to which they were assigned via the factor analysis. See Tables The internal consistency reliability  for the attitude and strain total scores was good – 0.79 and 0.95, respectively. Internal consistency reliability for subscales in both domains was excellent in general. See Table Table Table Table Table The M-NCAS demonstrated good psychometric properties based on an analysis of baseline data collected during a clinical trial of risperidone versus placebo, as reported in this preliminary investigation. Item performance, particularly floor and ceiling effects , suggested that four items on the attitude domain were performing poorly from a psychometric standpoint. Two items performed poorly on item correlations as well as factor analysis, while item 12 had a high floor effect as well as poor correlations. The remaining 28 items still capture sufficiently comprehensive data on the domains of interest, based on content review and on the empirical performance of subscales derived from them. The collection of additional data would enhance the confidence of conclusions regarding M-NCAS performance.In general, the M-NCAS demonstrated excellent internal consistency reliability, with only the autonomy subscale producing a Cronbach's alpha below 0.70. Cronbach's alpha values of 0.70 or greater are considered suitable for use in the analyses of group comparisons. Supporting construct validity of the M-NCAS were the moderate and significant correlations to the CMAI and BEHAVE-AD. Correlation with these measures was not expected to be high, given the more expansive focus of the M-NCAS relative to the CMAI and BEHAVE-AD.These results support the use of the M-NCAS for the collection of valid, reliable, and comprehensive information on the burden experienced by nurse carers when caring for dementia patients in long-term care settings. The instrument can assist in staff management by identifying nurse carers with the greatest levels of burden – ideally so that remedial action could be taken, either through extra support for staff members or through the shifting of caseloads from particularly difficult patients for nurses at risk for attrition.There are several limitations to this study. Of major concern is the lack of information on the nurse carers themselves. No data was available as to the type of carer, the duration they had cared for the patient, and their sociodemographic characteristics. The inability to characterize the respondents to the M-NCAS is a limitation in interpreting generalizability of results. No control was attempted for nurse raters, so the possibility exists that measurement properties of the M-NCAS differed across nurse raters – therefore skewing the results. The extent to which rater effects limit the validity of results is likely to be minimal, however, given that multiple subjects were being rated by each nurse rater. Future examination of between-subject properties would be helpful. The sample size for the study is small, relative to the standard recommendations regarding the number of subjects per item for factor analysis. However, we set criteria for relatively moderate to high factor loadings. Of note is that the final recommendations for the measure length are closer to that guideline. Because this was a post-hoc analysis of clinical trial data, the instruments selected for use in assessing construct validity may not have been optimal, in that they did not assess nursing strain per se. However, they did assess the propensity of exhibiting certain behaviors – as does the M-NCAS. Additional data on stability across time (test-retest reliability) is desirable for a full psychometric description of the M-NCAS, and was not available from this study. Finally, this sample of nurse carers was almost exclusively Caucasian. Therefore, the generalizability of these results to nurse carers of different ethnicities, and in different locales is limited.The M-NCAS enables the detection of the presence or absence of specific behaviors similar to checklists , but extends that information by providing a rating of the degree of burden of each aspect rated. It possesses good psychometric properties for use with nurse carers working with Alzheimer's patients in long-term care facilities. The M-NCAS provides a unique approach to identifying both positive and negative behaviors, and to quantifying the amount of stress felt by carers as a result of these behaviors. The nursing staff perspective on residents with dementia is unique , and theBehavioral Pathology in Alzheimer's Disease Rating Scale (BEHAVE-AD)Cohen-Mansfield Agitation Inventory (CMAI)Exploratory Factor Analysis (EFA)Mini-Mental Status Exam (MMSE)Modified Strain in Nursing Care Assessment Scale (M-NCAS)Strain in Nursing Care Assessment Scale (NCAS)LK participated in the data analysis design, data interpretation, and drafting of the manuscript. LF participated in the design, data interpretation, and drafting of the manuscript. GC performed the statistical analyses. MR conceived of the study and participated in data interpretation. HB conceived of the study and participated in both data collection and interpretation. All authors read and approved the final manuscript.


Geohelminth infection is a major health problem of children from rural areas of developing countries. In an attempt to reduce this burden, the Department of Health of the province of KwaZulu-Natal (KZN) established in 1998 a programme for helminth control that aimed at regularly treating primary school children for schistosomiasis and intestinal helminths. This article describes the baseline situation and the effect of treatment on geohelminth infection in a rural part of the province.Ascaris lumbricoides, and Trichuris trichiura, treated twice with 400 mg albendazole and re-examined several times over one year after the first treatment in order to assess the impact of treatment and patterns of infection and re-infection.Grade 3 schoolchildren from Maputaland in northern KZN were examined for infections with hookworm, T. trichiura and especially A. lumbricoides prevalences (57.2 and 19.4%, respectively) were much lower than elsewhere on the KZN coastal plain. Single dose treatment with albendazole was very effective against hookworm and A. lumbricoides with cure rates (CR) of 78.8 and 96.4% and egg reduction rates (ERR) of 93.2 and 97.7%, respectively. It was exceptionally ineffective against T. trichiura (CR = 12.7%, ERR = 24.8%). Re-infection with hookworm and A. lumbricoides over 29 weeks after treatment was considerable but still well below pre-treatment levels.The hookworm prevalence in the study population (83.2%) was considerably higher than in other parts of the province whereas T. trichiura infection however demands consideration of alternative treatment approaches.High geohelminth prevalences and re-infection rates in the study population confirm the need for regular treatment of primary school children in the area. The low effectiveness of single course albendazole treatment against  Geohelminth infections are among the most prevalent diseases in developing countries. Children are the group with the highest prevalences and infection intensities and are also very vulnerable to the effects of worm infection. These include nutritional deficiencies ,2 and imConsequently in 1998 the KwaZulu-Natal (KZN) Department of Health initiated a pilot helminth control programme which aimed to regularly apply treatment for intestinal helminths and schistosomiasis to primary school children . This grOur objectives were to describe the initial pattern of geohelminth infection, assess the impact of treatment with 400 mg albendazole and to monitor re-infection after treatment in order to develop recommendations for further local and regional control efforts.The study was conducted in central Ingwavuma district in northern KwaZulu-Natal Figure . This arThe study population was limited to one grade in order to keep disturbance of the school routine low. Grade 3 was selected because it should represent the infection situation in a primary school relatively well . All pup®, SmithKline Beecham). Treatment in the studied schools was administered in April and in October 1998. The study team assisted the nurses with treatment and also recorded those of the study population who were treated and those who were not. During the first round of treatment the pupils were also treated for schistosomiasis with 40 mg/kg praziquantel (Biltricide®, Bayer). However, results regarding schistosomiasis will be reported elsewhere. After the end of the study the participants were included in the normal treatment routine of the control programme.Treatment in all primary schools in the entire district was carried out by school nursing teams from the two local hospitals as part of the helminth control programme. Consenting children from all grades were treated for intestinal helminths with 400 mg albendazole  were nevertheless included in the analysis in order not to increase bias.A. lumbricoides and T. trichiura eggs within one day after preparation [Specimens were kept cool until preparation of the slides. Duplicate 50 mg Kato-Katz cellophane thick smears were prepared from each faecal sample. They were examined by trained microscopists twice: first for hookworm eggs within one hour of preparation and later for paration . AccuratDiarrhoeal specimens and slides that were too dark were not examined. Instead the respective pupils were asked to provide another specimen. Infection intensities are expressed as eggs per gram of faeces (EPG) calculated as the arithmetic mean number of eggs per thick smear multiplied by 20.Data were double entered into Microsoft Excel 97 and corrected for data entry errors. Data analysis was carried out in SPSS 10.0.5 for Windows.Prevalence ratios (PR) and their 95% confidence intervals were calculated using the SPSS "Tables" procedure . Cure raOnly data from the first of the two samples we received per pupil in each post-treatment survey were used for the above calculations in order to make them comparable to the baseline survey. Otherwise differences in sensitivity between surveys would have caused misleading results. Re-infection was however calculated for both obtained samples because the main purpose was not to compare with the baseline situation but to estimate trends in re-infection reliably.A. lumbricoides infection. With a PR of 0.71, males were about 30% less likely to be Ascaris infected than females and they also had lower infection intensities. The prevalence and intensity of hookworm infection were slightly  higher in male pupils and the prevalence of T. trichiura was nearly identical in the two groups.The cumulative prevalence of all three geohelminths combined was as follows: At baseline 90.0% of the pupils were infected with one or more species, 55.9% were infected with two or more species and 30.9% of the children were infected with all three geohelminths. Figure A. lumbricoides infection  was about 27% which is still about 2% higher then the ERR that we found in this study.In contrast, the CR and ERR for T. trichiura: 6 months after treatment the treated pupils had the same prevalence (61%) as a placebo treated control group [A study that was conducted in Durban only about 350 km south of the study area found that albendazole reduced the median EPG of children infected before treatment by 44.1% . The samol group .T. trichiura infection warrant attention. It is unlikely that the low CR and ERR in our study are due to low compliance: Tablet intake was monitored by the school nurses and the study team and only pupils recorded as treated were included in the analysis. Moreover, if a considerable number of children had only pretended to take the tablets, this would also have affected the impact of treatment on hookworm and A. lumbricoides infection. It is also unlikely that the low efficacy is due to drug resistance because of prior treatment. There were no previous control programmes in the area, to our knowledge hospitals and clinics had not been using albendazole or any other benzimidazole until recently and benzimidazoles were also not available in local shops.Although the CR and ERR as measured in most field studies including ours are notoriously unreliable and can only approximately indicate the true treatment success , the verT. trichiura was only 10.5% [High infection intensities, as they are frequently found in schoolchildren, are often mentioned in the literature as a reason for low cure rates ,26 and tly 10.5% . But evely 10.5%  but abouT. trichiura infection as single dose treatments, but other studies show that two or three repeated doses of albendazole on consecutive days are more effective than a single dose [Currently there are no drugs available that are highly effective against gle dose ,26,28,29gle dose .The proportion of children treated in our study can not be regarded as representative for the control programme in general, because study participants were more informed about it than their schoolmates. Therefore it was not analysed statistically. However, it should be noted that although only seven pupils openly refused to be treated, absenteeism was unusually high during the first round of treatment. This improved greatly in the second treatment, when – according to the opinion of school staff – pupils and their parents had realised that treatment was beneficial.A. lumbricoides is also in accordance with the pre-treatment situation.In accordance with the pre-treatment infection levels, hookworm re-infection over 29 weeks after the second treatment was relatively high and thus confirms the need for regular treatment. The moderate re-infection with A. lumbricoides is not too surprising. As opposed to hookworm that is restricted to the humid tropics and subtropics, this helminth also occurs in temperate climates [The markedly higher re-infection with hookworm during the 18 weeks after the second treatment when compared to the 16 weeks after the first treatment is most likely only partly due to the slightly longer re-infection period. The difference in season could also be important ,31: The climates . Its eggT. trichiura complicates conclusions, the data seem to indicate that re-infection was low. Therefore, if a more effective treatment approach could be utilised, the resulting decrease in prevalence and intensity of T. trichiura infections might be relatively long-lasting.Although the low efficacy of treatment for This study has shown that the high prevalence and intensity of geohelminth infection necessitate regular treatment of primary schoolchildren in the study area according to WHO criteria . This isBecause high prevalences in primary schoolchildren may also indicate relatively high community prevalences  it shoulA. lumbricoides infection, but that it is much less effective for the treatment of T. trichiura infection in the study area. It would be important to find out whether albendazole treatment is similarly ineffective against T. trichiura in other parts of the province and to test the effectiveness of treatment alternatives.Our study has also demonstrated that a single course of treatment with 400 mg of albendazole is a powerful tool to control hookworm and To answer the question whether hookworm transmission is really seasonal as this study seems to suggest would necessitate a cohort study over at least one year. Nevertheless it might be worthwhile to investigate when in the year transmission is highest in order to schedule treatment accordingly.None declared.ES conceived of the study and designed it with input from all authors. He conducted the field work with contributions from the other authors, did the statistical analysis and drafted the manuscript. All authors contributed to the final version of the manuscript and read and approved it.The pre-publication history for this paper can be accessed here:


Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of “acquired” resistance.EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec).We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.In patients with tumors bearing gefitinib- or erlotinib-sensitive  A specific secondary mutation in the kinase domain of the epidermal growth factor receptor can render cells insensitive to the two kinase inhibitors. This mutation was found in resistant tumors from three of six patients studied KRAS mutations have been associated with some cases of primary resistance to gefitinib or erlotinib [Somatic gain-of-function mutations in exons encoding the epidermal growth factor receptor (EGFR) tyrosine kinase domain are found in about 10% of non-small cell lung cancers (NSCLCs) from the United States ,3, with rlotinib , mechaniAcquired resistance to kinase-targeted anticancer therapy has been most extensively studied with imatinib, an inhibitor of the aberrant BCR-ABL kinase, in chronic myelogenous leukemia (CML). Mutations in the ABL kinase domain are found in 50%–90% of patients with secondary resistance to the drug (reviewed in ). Such mAlthough imatinib inhibits different kinases in various diseases  (reviewed in ), some tEGFR exons 18 to 24 in tumors from five patients who initially responded but subsequently progressed while on these drugs. These exons were also assessed in tumor cells from a sixth patient whose disease rapidly recurred while on gefitinib therapy after complete gross tumor resection. Because of the association of KRAS mutations with primary resistance to gefitinib and erlotinib [KRAS in tumor cells from these six patients. In an effort to explain the selective advantage of cells with a newly identified “resistance” mutation in EGFR—a T790M amino acid substitution—we further characterized the drug sensitivity of putatively resistant EGFR mutants versus wild-type or drug-sensitive EGFR mutants, using both a NSCLC cell line fortuitously found to contain the T790M mutation and lysates from cells transiently transfected with wild-type and mutant EGFR cDNAs.To determine whether lung cancers that acquire clinical resistance to either gefitinib or erlotinib display additional mutations in the EGFR kinase domain, we have examined the status of rlotinib , we alsoTumor specimens, including paraffin blocks, fine needle biopsies, and pleural effusions, were obtained through protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (protocol 92–055  and protEGFR (exons 18–24) and KRAS2 (exon 2) analyses were as published [Genomic DNA was extracted from tumor specimens, and primers for ublished ,7. All sEGFR wild-type) for calibration, this assay detects the presence of the T790M mutation when H1975 DNA comprises 3% or more of the total DNA tested, compared to a sensitivity of 6% for direct sequencing (data not shown).A specific exon 20 mutation (T790M) was also detected by length analysis of fluorescently labeled (FAM) PCR products on a capillary electrophoresis device , based on a new NlaIII restriction site created by the T790M mutation (2369 C→T), using the following primers: EGFR Ex20F, 5′-FAM-CTCCCTCCAGGAAGCCTACGTGAT-3′ and EGFR Ex20R 5′-TTTGCGATCTGCACACACCA-3′. Using serially mixed dilutions of DNA from NSCLC cell lines , as per manufacturer's instructions. Minipreps of DNA from individual clones were sequenced using the T7 priming site of the cloning vector.The following primers were used to generate EGFR cDNAs using a QuikChange Site-Directed Mutagenesis Kit  and cloned into expression vectors as described [Two numbering systems are used for EGFR. The first denotes the initiating methionine in the signal sequence as amino acid −24. The second, used here, denotes the methionine as amino acid +1. Commercial suppliers of antibodies, such as the Y1068-specific anti-phospho-EGFR, use the first nomenclature. To be consistent, we consider Y1068 as Y1092. Likewise, the T790M mutation reported here has also been called T766M. Mutations were introduced into full-length wild-type and mutant escribed . The folescribed . All mutescribed . Cells wSee 2. For viability studies, cells were seeded in complete growth medium in black 96-well clear bottom ViewPlates (PerkinElmer, Wellesley, Massachusetts, United States) at a density of 5,000 (H1975 and H2030) or 7,500 cells per well (H3255). Following overnight incubation, cells were grown for 24 h in the supplemented RPMI-1640 medium with 0.1% serum. Cells (in supplemented RPMI-1640 medium containing 0.1% serum) were then incubated for 48 h in the continued presence of gefitinib or erlotinib.The NSCLC cell lines H1650, H1975, H2030, H2347, H2444, H358, and H1734 were purchased from American Type Culture Collection (Manassas, Virginia, United States). H3255 was a gift of B. Johnson and P. Janne. Cells were grown in complete growth medium  supplemented with 10% fetal calf serum, 10 units/ml penicillin, and 10 μg/ml streptomycin) at 37 °C and 5% COCell viability was assayed using Calcein AM . Following incubation with gefitinib or erlotinib, monolayers were washed twice with PBS  and incubated with 7.5 μmol Calcein AM in supplemented RPMI-1640 (no serum) for 30 min. Labeling medium was removed, and cells were washed three times with PBS. Calcein fluorescence  was detected immediately using a Victor V multi-label plate reader (PerkinElmer). Three independent experiments were performed for each cell line; each experiment included four to eight replicates per condition.EGFR mutations in three of six individuals whose disease progressed on either gefitinib or erlotinib  initially presented with bilateral diffuse chest opacities and a right-sided pleural effusion. Transbronchial biopsy revealed adenocarcinoma. Disease progressed on two cycles of systemic chemotherapy, after which gefitinib, 250 mg daily, was started. Comparison of chest radiographs obtained prior to starting gefitinib .This 55-y-old woman with a nine pack-year history of smoking underwent two surgical resections within 2 y (right lower and left upper lobectomies) for bronchioloalveolar carcinoma with focal invasion. Two years later, her disease recurred with bilateral pulmonary nodules and further progressed on systemic chemotherapy. Thereafter, the patient began erlotinib, 150 mg daily. A baseline CT scan of the chest demonstrated innumerable bilateral nodules .This 55-y-old female “never smoker” was treated for nearly 4.5 y with weekly paclitaxel and trastuzumab  for adenEGFR mutations, by direct DNA sequencing of exons 19 and 21 [EGFR mutation by direct sequencing .All three specimens from patient 2, including the original lung tumor and the two metastatic samples from bone and lung, showed an exon 19 deletion involving elimination of 11 nucleotides (2238–2248) and insertion of two nucleotides, G and C . These nBoth of the available tumor samples from patient 3 contained a deletion of 15 nucleotides (2236–2250) in exon 19  encoding the EGFR catalytic region in the available tumor specimens.To determine whether additional mutations in the EGFR sequence . However, we did not find any additional mutations in exons 18 to 24 of EGFR, including the C→T change at position 2369 (data not shown). These results imply that alternative mechanisms of acquired drug resistance exist.In three additional patients (case histories not described here) with lung adenocarcinomas who improved but subsequently progressed on therapy with either gefitinib or erlotinib, we examined DNA from tumor specimens obtained during disease progression. In all three patients, we found KRAS2 occur in about one-fourth of NSCLCs. Such mutations rarely, if ever, accompany EGFR mutations and are associated with primary resistance to gefitinib or erlotinib [KRAS mutations confer acquired resistance to these drugs, we performed mutational profiling of KRAS2 exon 2 from tumor specimens from patients 1 to 3, as well as the three additional patients lacking evidence of the T790M mutation. None of the specimens contained any changes in KRAS  and KRAS exon 2 in eight established NSCLC lines .In our own analysis of H1975 (exons 18 to 24), the mutant 2369 T peak resulting in the T790M amino acid substitution was dominant, suggesting an increase in copy number of the mutant allele in comparison to the wild-type allele. The ratio of mutant to wild-type peaks was similar to that of the mutant 2573 G (corresponding to the L858R amino acid substitution) to wild-type T peaks  generated by the specific missense mutation. After PCR amplification with exon-20-specific primers spanning nucleotide 2369, wild-type sequence contains specific NlaIII sites, which upon digestion yield a 106-bp product A. PresenEGFR;We first used DNA from the H1975 cell line (which contains both T790M and L858R mutations) to confirm the specificity of the PCR-RFLP assay. As expected, analysis of these cells produced both the 97- and 106-bp fragments. By contrast, analysis of DNA from H2030 (which contains wild-type EGFR exons 18 to 24 by direct sequencing (data not shown).We next used this PCR-RFLP assay to assess various patient samples for the presence of the specific 2369 C→T mutation corresponding to the T790M amino acid substitution. DNA from the progressing bone and lung lesions in patient 1 produced both the 97- and 106-bp fragments, but DNA from the original lung tumor did not B. The raEGFR cDNAs that encoded the exon 21 and 19 mutations found in patients 1 and 2, respectively. Corresponding proteins  were then produced by transient transfection with expression vectors in 293T cells, which have very low levels of endogenous EGFR [To determine how the T790M mutation would affect EGFR proteins already containing mutations associated with sensitivity to EGFR tyrosine kinase inhibitors, we introduced the specific mutation into Gefitinib inhibited the activity of wild-type and L858R EGFRs progressively with increasing concentrations of drug, as demonstrated by a reduction of tyrosine-phosphorylated proteins A and a dEGFR TK domain mutations (50 of about 0.01 μmol (50 of about 1 μmol (EGFR (exons 18 to 24) and mutant KRAS  appears to be extremely rare. We have not identified this mutation in 155 tumors (see above), and among nearly 1,300 lung cancers in which analysis of erformed ,5,6, onl. However, based upon analogous studies in CML, it is also possible that NSCLC subclones bearing this secondary mutation pre-exist within the primary tumor clone in individual patients, albeit at low frequency [How tumor cells bearing the T790M mutation emerge within gefitinib- or erlotinib-treated patients is a matter of investigation. Subclones bearing this mutation could arise de novo during treatmentrequency . In eithFrom analysis of the crystal structure of the EGFR kinase domain bound to erlotinib, it is has been shown that the wild-type threonine residue at position 790 is located in the hydrophobic ATP-binding pocket of the catalytic region, where it forms a critical hydrogen bond with the drug . The relThe T790M mutation could also affect the kinase activity or alter the substrate specificity of mutant EGFRs, such that a proliferative advantage would be conferred upon cells bearing the mutation. Consistent with this, the H1975 NSCLC cell line reported here to contain both T790M and L858R did not to our knowledge undergo any prior treatment with gefitinib or erlotinib; the doubly mutated cells must have become dominant over time through multiple passages in vitro. This scenario could explain the seemingly contradictory report by others who found the H1975 cell line to be highly sensitive to gefitinib ; our H19Recently, new small-molecule inhibitors have been identified that retain activity against the majority of imatinib-resistant BCR-ABL mutants. The new drugs bind to ABL in an “open” conformation, as opposed to imatinib, which binds ABL in a “closed” conformation ,13. AnalIn some of the patient specimens analyzed, the actual sequencing peaks demonstrating the T790M mutation were smaller than originally anticipated. These results differ from those of acquired resistance mutation in CML , GIST 1,27, and EGFR, either within or outside the tyrosine kinase domain, are likely to exist. It is also possible that EGFR amplification itself plays a role in acquired resistance, since imatinib-resistant clones have been shown to lack resistance mutations but contain amplified copies of BCR-ABL [Since tumor specimens from three additional patients with acquired resistance to EGFR tyrosine kinase inhibitors did not demonstrate the T790M mutation, this specific lesion does not account for all mechanisms of acquired resistance to gefitinib or erlotinib. Given the paradigm established with imatinib, other drug-resistance mutations in  BCR-ABL ,28. NoneFigure S1(A) Patient 1. Serial chest radiographs from before (day 0) and during gefitinib treatment (14 d and 9 mo), demonstrating initial response and subsequent progression.(B) Patient 2. Serial CT studies of the chest before (day 0) and during erlotinib treatment (4 mo and 25 mo), demonstrating initial response and subsequent progression.(C) Patient 3. Serial chest radiographs before (day 0) and during adjuvant gefitinib treatment (3 mo), following complete resection of grossly visible disease. The left-sided pleural effusion seen at 3 mo recurred 4 mo later, at which time fluid was collected for molecular analysis.(951 KB PPT).Click here for additional data file.Figure S2EGFR exon 21 in tumor specimens from patient 1. DNA from the growing lung lesion and the pleural effusion demonstrated a heterozygous T→G mutation at position 2573, leading to the common L858R amino acid substitution.(A) Status of (B) All three specimens from patient 2 showed the same heterozygous exon 19 deletion, removing residues 747–749 and changing the alanine at position 750 to proline.(104 KB PPT).Click here for additional data file.Figure S3See legend for (153 KB PPT).Click here for additional data file.Protocol S1(566 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink/) accession number for the KRAS2 sequence discussed in this paper is 3845; the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the KRAS2 sequence discussed in this paper is NT_009714.16. Reference EGFR sequence was obtained from LocusLink accession number 1956 and GenBank accession number NT_033968.The LocusLink  and erlotinib , have been developed to inhibit activated EGFR, and studies have shown that they can shrink tumors in some patients. Most patients who respond to these drugs have tumors that carry an alteration (or mutation) in the EGFR gene, which somehow makes their tumors responsive to the drugs.In those patients in whom the drugs work, the tumors shrink initially, but after a while they stop responding and the cancer comes back. The cancer has, as researchers describe it, become resistant to the drugs. Understanding how tumors become resistant is important to develop new and better drugs.They asked patients who initially responded to erlotinib or gefitinib but then became resistant to consent to studies allowing further analysis of tumor tissue during and after drug treatment. They then re-examined the EGFR gene in these tumor samples.They found that tumors from all patients carried mutations in the EGFR gene that are known to make them responsive to the drugs. In addition, three of the post-treatment tumors had an identical second mutation in their EGFR gene. Biochemical studies showed that these secondary alterations made the original drug-sensitive EGFR less sensitive to drug treatment. The numbers are small but suggest that this secondary resistance mutation could be quite common. Tumor cells from the three other patients didn't have this mutation, which suggests that there are other ways for lung cancers to become resistant to gefitinib and erlotinib.Larger studies are needed to confirm that this particular mutation is a major cause of resistance against the two drugs. It is also important to find out what causes resistance in the other cases. And knowing about this resistance mutation will help researchers to develop drugs that will work even against tumors with the mutation.The following pages contain some information on the EGFR kinase inhibitors.http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htmU. S. Food and Drug Administration information page on Iressa (gefitinib): http://www.cancerhelp.org.uk/help/default.asp?page=10296Cancer Research UK information page about erlotinib (Tarceva):