Eleven deaths from causes unrelated to NHL or its treatment occurred in the G-CSF arm compared to five in controls. More deaths occurred from second malignancies (4 vs 2) and cardiovascular causes (5 vs 0) in the G-CSF arm. Although this pharmacovigilance study has insufficient statistical power to draw conclusions and is limited by the lack of data on smoking history and other cardiovascular risk factors, these unique long-term outcome data generate hypotheses that warrant further investigation.The effect of utilising granulocyte colony-stimulating factor (G-CSF) to maintain chemotherapy dose intensity in non-Hodgkin's lymphoma (NHL) on long-term mortality patterns has not been formally evaluated. We analysed prolonged follow-up data from the first randomised controlled trial investigating this approach. Data on 10-year overall survival (OS), progression-free survival (PFS), freedom from progression (FFP) and incidence of second malignancies were collected for 80 patients with aggressive subtypes of NHL, who had been randomised to receive either VAPEC-B chemotherapy or VAPEC-B+G-CSF. Median follow-up was 15.7 years for surviving patients. No significant differences were found in PFS or OS. However, 10-year FFP was better in the G-CSF arm (68  One of the major dose-limiting toxicities of cytotoxic chemotherapy for non-Hodgkin's lymphoma (NHL) is myelosuppression and subsequent susceptibility to infection. Haemopoietic colony-stimulating factors (G-CSF and GM-CSF) are now commonly administered as an adjunct to cytotoxic chemotherapy in patients with NHL, to reduce the incidence of these toxicities based on data accrued from several randomised controlled studies  and all cause mortality was analysed. We therefore decided to analyse long-term follow-up data from the first published randomised study of G-CSF administration in NHL patients (−2 by daily subcutaneous injection for 13 weeks apart from days preceding and during myelosuppressive chemotherapy administration) in addition to chemotherapy and 39 received chemotherapy alone. Pretreatment characteristics of the two groups were well matched for prognostic factors (see Eighty patients aged 16–71 years with high-grade NHL (Kiel) of any disease stage/performance status commencing VAPEC-B chemotherapy were entered between August 1989 and March 1991. Forty-one were randomised to receive G-CSF -230 mcg mtors see . Thirty-vs 83%) – see Patients randomised to receive G-CSF achieved 12% greater median dose intensity than control patients .The objectives of the study were to evaluate the long-term effects of the use of G-CSF and increased dose-intensity chemotherapy on overall survival and mortality patterns. Definitions of reported end points are as follows  – time from randomisation to death from any cause.Freedom from progression (FFP) – time from randomisation to first documentation of disease progression.Progression-free survival (PFS) – time from randomisation to the first of disease progression or death from any cause.All analyses were performed on an intention-to-treat basis. To assess the effects of specific causes of death, cumulative incidence curves were constructed in a competing risks framework. The cumulative incidence of death from cause x at time t is the probability of dying from x by time t in an environment where other causes of death are acting. The treatment groups were then compared using Gray's test .A further analysis was also performed by fitting a multi-state Cox illness/death model .Relative mortality models .Forty-eight of the eighty initial patients had died at the time of this analysis. The median follow up of the 36 surviving patients was 15.7 years . Of the 48 dead, 24 died in the G-CSF arm and 24 in the control arm. Ten-year overall survival figures were 51 and 46% in the G-CSF and control arms respectively and 10-year PFS 49 and 44%.The Kaplan–Meier survival curves for OS and FFP are illustrated in P=0.02). The cumulative incidence curves for lymphoma-specific death are shown in P=0.02)  .To assess whether known lymphoma-related prognostic factors may have influenced these findings, we controlled these using a multi-state formulation of Cox regression model analysis . Age, seOf the 14 non-NHL deaths in the G-CSF arm, three patients died from treatment-related infections, two from neutropenic infections during the 11-week treatment schedule and one from autopsy-confirmed invasive aspergillosis 3 months after commencement of chemotherapy. No patient in the control arm died from treatment-related infection.P=0.12)  and one from an intracerebral haemorrhage. In the control arm, one patient died from Pneumocystis carinii pneumonia 14 months after chemotherapy with no evidence of recurrent NHL and one from cirrhosis secondary to chronic hepatitis C infection. The causes of non-lymphoma deaths are summarised in One possible explanation for these findings is that because patients who were exposed to G-CSF appeared less likely to experience progression of lymphoma, they were exposed to other competing causes of death for longer than patients who did not receive G-CSF but with the proportional rate of non-lymphoma-related mortality being the same in both groups. To assess this possibility, we constructed relative mortality models for each trial arm separately using England and Wales death rates broken down by sex, age (integer years) and period  obtained from the Human Mortality Database . This anThere have been numerous randomised studies on the use of G-CSF during induction chemotherapy for high-grade NHLs (vs 47% control arm). This prompted us to further investigate the mortality patterns of the two study groups, which were well-balanced at original randomisation for age, sex and performance status (P=0.02) while five died from other causes in the control group compared with 14 in the G-CSF group (P=0.02). Despite the small size and retrospective nature of our analysis, this observation could support the hypothesis that the use of G-CSF to facilitate chemotherapy administration allows greater lymphoma-specific survival at the expense of death from other causes. However, our analysis is limited by the lack of baseline information on major risk factors for both second malignancies and cardiovascular disease including smoking and family histories, hypertension, diabetes and cholesterol levels. The random allocation of patients to treatment arm will have limited uneven distribution of these variables between the two treatment arms but we cannot exclude biased distribution as an explanation of our findings.In this report, we detail long-term pharmacovigilance follow up of the first randomised study of G-CSF during induction chemotherapy for high-grade NHL , a diffee status . NineteeAn excess of deaths from acute treatment-related infective complications were noted in the intervention arm of our study. The reasons for this are unclear. Identical cytotoxic dose modification criteria were applied in both groups and G-CSF administration reduced the risk of febrile neutropenia. No increased on-chemotherapy mortality was seen associated with G-CSF administration in the Bohlius meta-analysis (relative risk– 0.93 95% CI 0.60–1.43) (P=0.12). The occurrence of deaths from second malignancy and cardiovascular disease which account for the majority of these  are of particular interest as it is now apparent from large epidemiologic analyses of NHL databases that long-term NHL survivors are at increased risk from these treatment-related complications.Eleven patients died from other causes in the G-CSF arm compared with five in the control arm  in the first-line therapy of aggressive NHL  has been reported in one retrospective cohort study of 592 patients (−2) was still well below the conventional ceiling dose for clinically apparent cardiotoxicity of 400–450 mg m−2. It is also intriguing to note that G-CSF appears to protect from anthracycline cardiomyopathy in animal models (How G-CSF could affect the long-term incidence of cardiovascular disease is less clear. Although patients receiving G-CSF achieved a 12% increased adriamycin dose intensity, total dose (200 mg ml models .post hoc nature of this analysis limits the conclusions that can be drawn. However, this analysis emphasises the need for long-term and detailed follow-up of patients enrolled into such studies and the sharing of these data for the purposes of meta-analysis.To conclude, this unique long-term follow-up data of the first randomised study investigating the use of G-CSF in the first-line chemotherapeutic treatment of high-grade NHL suggests that although overall survival is not altered by this intervention, the pattern of mortality may be changed. Although potential long-term increased risk of second malignancy and cardiovascular disease in patients receiving G-CSF is intriguing and may have significant implications, the small size of the original study cohorts, lack of information on other known risk factors and the


Maintenance care (MC) is relatively commonly used among chiropractors. However, factual information is needed on its indications for use.This study had two objectives: 1) to describe which role patients' past history and treatment outcome play in chiropractors' decision to use MC in patients with low back pain, 2) to investigate if the chiropractors' clinical/educational background has an effect on the frequency of using MC and their indications for use of MC.An anonymous questionnaire was sent to all 413 chiropractors practising in Denmark. Its main part consisted of 3 sets of 4 questions relating to one basic case of low back pain. For each case, the chiropractors were asked if they would use MC as they self-defined the term (no/perhaps/yes). There were questions also on gender, age, educational and clinical background, and on the number of MC patients seen by these chiropractors. Their decision to recommend MC was reported. Associations between the demographic variables and 1) the frequency of MC-use and 2) their indications for use of MC were tested through multivariate analysis.The response rate was 72%. Non-indications for MC were: 1) a good outcome combined with no previous events, or 2) a past history of LBP and gradual worsening with treatment. Indications for MC were a good outcome combined with a previous history of low back pain between once a month and once a year. The mean proportion of MC patients per week were 22% (SD 19), ranging from 0% to 100%. The use of MC was highest among experienced chiropractors, those who were educated in North America, and clinic owners. However, in Denmark most chiropractors graduated before 1999, are educated abroad, whereas most chiropractors thereafter are educated in Denmark. Therefore, we cannot conclude whether this difference relates to education or years of experience. There were no associations detected between demographic variables and the indications for MC.There is relatively high consensus on when MC should and should not be used. A history of prior low back pain combined with a positive response to treatment encourages the use of MC, whereas no previous history of back pain or a worsening of symptoms discourages the use of MC. There seems to be a difference in the proportional use of MC between chiropractors with more experience educated in North America and those with less experience educated in Denmark. Presently, it is not known if or how low back pain (LBP) can be prevented from developing. Because LBP frequently is a long-lasting or recurring problem, prevention of recurrences is as relevant as primary prevention. However, also this aspect is clouded in mystery.Many patients with LBP will seek care from chiropractors ,2. Some The majority of patients with LBP who are treated by chiropractors will receive spinal manipulation. Spinal manipulative therapy has been tested in a number of trials and found to be effective for LBP, at least in the short term ,4. WhethNevertheless, this concept is fairly well accepted among chiropractors. Chiropractors in various parts of the world state that they use MC in about one third of their patients -9. HowevPresently, a research program is being conducted in the Nordic countries trying to illuminate the use of, the indications for, and the efficiency of MC. So far, it has been determined, that there is a large degree of consensus among Nordic chiropractors that the primary goal of MC is prevention of new episodes of LBP, although for some patients it can be prevention of deterioration ,11. TherSince prior history of LBP episodes, and positive response to treatment have been identified as commonly accepted indications for offering MC to patients, we designed this current study to explore this finding in more specific detail. We asked chiropractors in this pragmatic cross-sectional survey about their use of MC, as they self-defined the term, by presenting them with a series of clinical case scenarios that varied as to prior history (frequency and duration of the previous episodes) and response to treatment.In addition we were curious to see, if there were other factors that would influence chiropractors' use of MC. In a previous study from Australia, it had been shown that MC was more common among practitioners who had only few new patients as compared to those who had more new patients . Other fThis study had two objectives: 1. to describe which role patients' past history and treatment outcome play in chiropractors' attitudes to the use of MC in patients with LBP, and 2. to investigate if the chiropractors' demographic, clinical and educational background has an influence on how many patients receive MC and what type of patients are offered MC.A list of actively practising chiropractors was obtained from the Danish Chiropractors' Association. These were mailed a questionnaire plus a pre-stamped envelope in February 2007. In order to encourage participation, information on the study was given to participants at local chiropractic meetings across the country by two chiropractors and in the Danish chiropractors' professional journal. The questionnaires were returned anonymously.low back pain of 2 days duration with no additional spinal or musculoskeletal problems, and with no other health problems. His x-rays are normal for his age. There are no red flags and he seems to be in good shape both physically and psychologically. There are no aggravating factors at work or at home." The questions about this patient were constructed such that he was assumed to have received chiropractic treatment but that his past history could vary as could the outcome of the treatment.A questionnaire was designed specifically for the purpose of this study  the first scenario on the second page and fourth page, and 2) the first scenario on the third page and the third scenario on the fourth page.The self-reported use of MC was estimated by asking for its proportional use both on the day of the study and for the last working week. Both questions were asked because the data for the present day would be easier collected (just counted) than data for the past week. However, the week-data were there in case some respondents on the day of the survey had an unusual number of MC-patients.In the analyses, the variables age and clinical experience were collapsed into fewer categories, based on the distribution of data.The distributions of MC use per day and per week were analyzed with the help of frequency tables and graphs and the mean value with standard deviations of the weekly proportion of MC-patients were reported. The number of"expected" answers for each participant was calculated based on the 8 predetermined"expected" answers. On the basis of the distribution of data this variable was dichotomized into two groups, consisting of"mainstream" (giving the"expected" answer for at least 7 of these 8 scenarios) vs."non-mainstream" answers .Stata Statistical Software Release 8.2, College Station, Tex., USA) and a p-level of 0.05 or less was considered statistically significant.Associations between the demographic variables and the proportional use of MC and the proportion of"mainstream" answers were tested with bivariate analyses, and thereafter with multivariate linear regression analyses for the variables found to be significant in the bivariate analyses. Because of the possibility of strong correlations between several of the demographic variables, we tested these correlations using Pearson's r. The relationship between the proportional use and the proportion of"mainstream" answers was tested using kappa statistics. Data were analysed with STATA 8.2  returned their questionnaires, with an even distribution of men and women. Two-thirds of the participants were aged 30-49 years and almost half had graduated in the USA or Canada. The details are presented in Table The mean and median values of the self-reported proportion of MC patients were almost identical for the last full working week and the day of the survey. The reported proportion of MC-patients the past week ranged from 0% to 100%, with 15 missing replies and the mean value of MC patients per week was 22% (SD19). Based on the distribution, three subgroups were determined. The first group consisted of chiropractors who defined 0% -25% of the patients seen that week as MC patients (n = 195). The second group reported that 26% -42% of their patients were MC patients (n = 46), and the third group reported their proportion of MC patients to be 43%-100% (n = 40). For ease of reporting, these groups were called"low","medium", and"high", respectively.The study sample closely resembled the target population, i.e. all the members of the Danish Chiropractors' Association as seen in Table The internal consistency was good for one pair of identical questions  with agreement in 94% of the cases. However, the second pair of identical questions  was less convincing with 54% total agreement.In relation to the indications for the use of MC, 40% (n = 118) of the participants were classified as"mainstream" practitioners, giving the expected answer in at least 7 of the 8 cases. For each of the 8 individual cases, 57% to 89% of the participants responded as expected.Overall, the results of the first set of cases were in accordance with the expectations of the research group, as the majority of respondents found it unsuitable to provide MC to patients with no previous history of LBP at all. There was only one possible exception. In scenario 3, depicting a patient who does not improve over two months of treatment , 54% of the chiropractors responded either"perhaps" or"yes" to the consideration of MC. For this scenario, the research team expected the majority to reply"no" Table .Information collected in this part of the questionnaire did not always agree with the expectations of the research group Table . In the As expected, 87% would not offer MC to a patient with immediate and lasting positive response and no previous history of back pain. The opinions were mixed if there had been 1-2 episodes of LBP a year, with 46% answering"perhaps", whereas the research team's expected answer was"yes". The majority, however, agreed that MC would be suitable for more frequent past episodes. For monthly episodes, 70% were in favour, and 69% for weekly episodes. This was in line with the"expected" answers  graduates from USA/Canada had the highest use of MC and graduates from Denmark the lowest, 2) more years in practice was associated with a higher use of MC, and 3) clinic owners used MC more often than employees, see Tables Neither the demographic data nor the use of MC was associated with the"mainstream" answers in the analyses.The results of this study indicate that our study sample was representative of the membership of the Danish Chiropractors' Association. Among Danish chiropractors there is a wide variation in the use of MC; some not using it at all and others always. A strong determinant for this was, not surprisingly, their educational background, with the American-educated chiropractors being more likely to have more MC patients than those educated in the UK or Denmark. It is possible that the use of MC is a major differential factor between European-style and American-style chiropractic. One should therefore be careful when interpreting data on this topic from different parts of the world. However, since there is a strong correlation between country of graduation and years of clinical experience among Danish chiropractors, this difference could also be related to experience.Whether MC is a useful approach or not, is not known, but assuming that it does have an effect, it should be used in such a way as to be cost-effective for the patient. Therefore it seemed logical that good outcome should be a major prerequisite, as should a considerable risk of future LBP. This was also the result in this study as well as in previous studies.Interestingly, respondents who provided the unexpected answers could do this in both directions, i.e. not offering MC to a case with a profile assumed to be suitable or offering MC to those assumed to be unsuitable. This shows there is some diversity in the chiropractic profession regarding MC. If such a thing as"correct" answers exists to these survey questions, some chiropractors will offer MC more frequently and others less frequently. On that note, it must be remembered that the clinical scenarios presented in the survey have very limited information and many other factors  might influence the chiropractor's choice . DespiteThere seems to be a general agreement that MC should be used in patients at risk for future problems and that the past history is a predictor of that. Further, the patient should improve with treatment. As for the cut-off point for past LBP events, it lies somewhere between one episode per year and one per month, but probably closer to the latter.For clinicians, also the non-indications for MC treatment are important. Almost 90% agreed that patients who recovered very quickly, remained stable over two months, and who had no past history of LBP should not have MC. And about as many of our participants would not consider MC for patients who got gradually worse.Curiously, there was no majority"no"- response for the patients who"oscillated" (some days good some days bad) with no real improvement. Also according to previous Swedish and Danish studies, subgroups of respondents who would recommend MC also in patients who do not report a clinically relevant amount of improvement were noted ,10,11. FWe were able to reach four distinct conclusions:• About 2/3 of the Danish chiropractors reported to use MC on between 0 and 25% of their patients.• The most frequent use of MC was reported by chiropractors who were graduates from North America, experienced chiropractors and clinic owners.• For Danish chiropractors in general, the indications for MC in patients with LBP were: good short-term outcome and at least 1-2 previous episodes per year.• There were no associations between the demographic background and the indications for the use of MC.The authors declare that they have no competing interests.SHF and ALSH designed the study, collected and analyzed the preliminary data and wrote a report on part of the results as a part requirement for their masters degree in Health Sciences (Biomechanics), supervised by CLY and LH. TSJ supervised and assisted with the data analysis. CLY and LH were responsible for the final manuscript. All authors read and approved the final manuscript.Questionnaire. A copy of the questionnaire used in the survey about the use of maintenance care in Danish chiropractic practice.Click here for file


The legislation called the Transplantation of Human Organ Act (THO) was passed in India in 1994 to streamline organ donation and transplantation activities. Broadly, the act accepted brain death as a form of death and made the sale of organs a punishable offence. With the acceptance of brain death, it became possible to not only undertake kidney transplantations but also start other solid organ transplants like liver, heart, lungs, and pancreas. Despite the THO legislation, organ commerce and kidney scandals are regularly reported in the Indian media. In most instances, the implementation of the law has been flawed and more often than once its provisions have been abused. Parallel to the living related and unrelated donation program, the deceased donation program has slowly evolved in a few states. In approximately one-third of all liver transplants, the organs have come from the deceased donor program as have all the hearts and pancreas transplants. In these states, a few hospitals along with committed NGOs have kept the momentum of the deceased donor program. The MOHAN Foundation (NGO based in Tamil Nadu and Andhra Pradesh) has facilitated 400 of the 1,300 deceased organ transplants performed in the country over the last 14 years. To overcome organ shortage, developed countries are re-looking at the ethics of unrelated programs and there seems to be a move towards making this an acceptable legal alternative. The supply of deceased donors in these countries has peaked and there has been no further increase over the last few years. India is currently having a deceased donation rate of 0.05 to 0.08 per million population. We need to find a solution on how we can utilize the potentially large pool of trauma-related brain deaths for organ donation. This year in the state of Tamil Nadu, the Government has passed seven special orders. These orders are expected to streamline the activity of deceased donors and help increase their numbers. Recently, on July 30, 2008, the Government brought in a few new amendments as a Gazette with the purpose of putting a stop to organ commerce. The ethics of commerce in organ donation and transplant tourism has been widely criticized by international bodies. The legal and ethical principles that we follow universally with organ donation and transplantation are also important for the future as these may be used to resolve our conflicts related to emerging sciences such as cloning, tissue engineering, and stem cells. Kidney transplants in India first started in the 1970s and since that time, India has been a leading country in this field on the Asian sub-continent. The evolutionary history of transplants in the last four decades has witnessed a different facet of transplant emerging in each decade. The first 10 years were spent mastering the surgical techniques and immune-suppression. Its success resulted in a phenomenal rise in the numbers of transplants in the next 10 years and unrelated kidney donation from economically weaker sections started taking place with commerce in organ donation becoming an acceptable integral part of the program. After this was accepted, the ethics of transplants in India has always been on a slippery slope and all kinds of nefarious activities were accepted as normal practice. The general dictum was “when you can buy one why donate?” The next 10 years saw an outcry from the physicians of the western world at the growing numbers of these exploitative transplants being done in India. There were also protests from many sections in India. The pressure on the Government saw the passing of the Transplantation of Human Organ Act (THO) legislation that made unrelated transplants illegal and deceased donation a legal option with the acceptance of brain death.In India, despite the THO act, neither has the commerce stopped nor have the number of deceased donors increased to take care of organ shortage. The concThe main provisions of the THO act and the newly passed Gazette by the Government of India include the following:For living donation - it defines who can donate without any legal formalities. The relatives who are allowed to donate include mother, father, brothers, sisters, son, daughter, and spouse. Recently, in the new Gazette grandparents have been included in the list of first relatives. The first relatives are required to provide proof of their relationship by genetic testing and/or by legal documents. In the event of there being no first relatives, the recipient and donor are required to seek special permission from the government appointed authorization committee and appear for an interview in front of the committee to prove that the motive of donation is purely out of altruism or affection for the recipient.Brain-death and its declaration - brain death is defined by the following criteria: two certifications are required 6 hours apart from doctors and two of these have to be doctors nominated by the appropriate authority of the government with one of the two being an expert in the field of neurology.Regulation of transplant activities by forming an Authorization Committee (AC) and Appropriate Authority (AA.) in each State or Union Territory. Each has a defined role as follows:Role of Authorization Committee (AC) - The purpose of this body is to regulate the process of authorization to approve or reject transplants between the recipient and donors other than a first relative. The primary duty of the committee is to ensure that the donor is not being exploited for monetary consideration to donate their organ. The joint application made by the recipient and donor is scrutinized and a personal interview is essential to satisfy to the AC the genuine motive of donation and to ensure that the donor understands the potential risks of the surgery. Information about approval or rejection is sent by mail to the concerned hospitals. The decision to accept or reject a donor is governed by Sub Clause (3), Clause 9 of Chapter II of the THO act.Role of Appropriate Authority (AA): The purpose of this body is to regulate the removal, storage, and transplantation of human organs. A hospital is permitted to perform such activities only after being licensed by the authority. The removal of eyes from a dead body of a donor is not governed by such an authority and can be done at other premises and does not require any licensing procedure. The powers of the AA include inspecting and granting registration to the hospitals for transplant surgery, enforcing the required standards for hospitals, conducting regular inspections of the hospitals to examine the quality of transplantation and follow-up medical care of donors and recipients, suspending or canceling the registrations or erring hospitals, and conducting investigations into complaints for breach of any provisions of the Act. The AA issues a license to a hospital for a period of 5 years at a time and can renew the license after that period. Each organ requires a separate license.The Transplantation of Human Organ Act clearly lays out various procedures; for this purpose, it has thirteen different forms [Table 1]. The Central Government has amended the Transplantation of Human Organs Act, 1994 (42 of 1994) to include certain changes called the Transplantation of Human Organs Rules, 1995 (GSR NO. 51(E), dr. 4-2-1995) . Given below are important excerpts from the rules.Any donor may authorize the removal, before his death, of any human organ of his body for therapeutic purposes as specified in Forms 1(A), 1(B), and 1(C). The new forms have been made more comprehensive and are to be submitted with proof of identity and address, marriage registration certificate, family photographs, etc. with attestation by a Notary Public.The gazette states that before removing a human organ from the body of a donor before his death, a medical practitioner should satisfy himself that the donor has given authorization in Form 1(A) if the relative is a close relative i.e., a mother, father, brother, sister, son, or daughter. Form 1(B) is used for a spouse and Form 1(C) is used for other relatives. He should also confirm the following:The donor is in a proper state of health and is fit to donate the organ. The registered medical practitioner should then sign a certificate as specified in Form 2.The donor is a close relative of the recipient as certified in Form 3 and has signed Form 1(A).The donor has submitted an application in Form 10 jointly with the recipient and the proposed donation has been approved by the concerned competent authority. The relationship between the donor and recipient also needs to be examined to the satisfaction of the Registered Medical Practitioner in charge of the transplant center.In the case of the recipient being a spouse of the donor, the donor has given a statement to the effect that they are so related by signing a certificate in Form 1(B) and has submitted an application in Form 10 jointly with the recipient and the proposed donation has been approved by the concerned competent authority.In the case of a donor who is other than a close relative, the donor has signed Form 1(C), submitted an application in Form 10 jointly with the recipient, and permission from the Authorization Committee for the donation has been obtained.A registered medical practitioner shall, before removing a human organ from the body of a person after his death, confirm the following:The donor had, in the presence of two or more witnesses (at least one of whom is a close relative of the recipient), unequivocally authorized as specified in Form 5 before his death, the removal of the human organ of his body after his death for therapeutic purposes and there is no reason to believe that the donor had subsequently revoked the authority.The person lawfully in possession of the dead body has signed a certificate as specified in Form 6.A registered medical practitioner shall, before removing a human organ from the body of a person in the event of brain-stem death, confirm the following:A certificate as specified in Form 8 has been signed by all the members of the Board of Medical Experts.In the case of brain-stem death of a person of less than 18 years of age, a certificate specified in Form 8 has been signed by all the members of the Board of Medical Experts and an authority as specified in Form 9 has been signed by either of the parents the person.The new gazette clearly lays down the following guidelines:1. Where the proposed transplant is between persons related genetically , the following shall be evaluated:Results of tissue typing and other basic testsDocumentary evidence of relationship e.g., relevant birth certificates and marriage certificateDocumentary evidence of identity and residence of the proposed donor e.g., Ration Card or Voters Identity Card, Passport, Driving License, PAN Card, or Bank Account and family photograph depicting the proposed donor and the proposed recipient along with another near relativeIf the relationship is not conclusively established after evaluating the above evidence, direct further medical tests may be given as described follows.– Test for Human Leukocyte Antigen (HLA), human leukocyte antigen-B alleles to be performed by the serological and /or polymerase chain reaction (PCR) based Deoxyribonucleic Acid (DNA) methods– Test for human leukocyte antigen-Dr beta genes to be performed using PCR-based DNA methods.Tests shall be done from a laboratory accredited with National Accreditation Board for Laboratories (NABL). When the tests referred to above do not establish a genetic relationship between the donor and the recipient, the same tests should be performed on both or at least one parent, preferably both parents. If parents are not available, the same tests should be performed on relatives of donor and recipient that are available and are willing to be tested failing which, the genetic relationship between the donor and the recipient will be deemed to have not been established.When the proposed transplantation is between a married couple, the Registered Medical Practitioner i.e., the person in charge of the transplant center must evaluate the fact and duration of marriage . When the proposed donor or recipient or both are not Indian Nationals/citizens whether close relatives or otherwise, the AC shall consider all such requests. A senior Embassy official of the country of origin has to certify the relationship between the donor and the recipient. When the proposed donor and the recipient are not close relatives, the Authorization Committee shall evaluate that there is no commercial transaction between the recipient and the donor and the following shall specifically be assessed:An explanation of the link between them and the circumstances that led to the offer being madeReasons why the donor wishes to donateDocumentary evidence of the link, e.g., proof that they have lived togetherOld photographs showing the donor and recipient togetherThere is no middleman or tout involvedThe financial status of the donor and the recipient is probed by asking them to give appropriate evidence of their vocation and income for the previous three financial years. Any gross disparity between the status of the two must be evaluated with the objective of preventing commercial dealing.The donor is not a drug addict or known person with criminal recordThe next of kin of the proposed unrelated donor is interviewed regarding awareness about his or her intention to donate an organ, the authenticity of the link between the donor and the recipient and the reasons for donation.The AC should state in writing its reason for rejecting or approving the application of the proposed donor and all approvals should be subject to the following conditions:The approved proposed donor would be subjected to all medical tests as required at relevant stages to determine his biological capacity and compatibility to donate the organ in question.Psychiatrist's clearance in such cases is deemed mandatory to certify the donor's mental condition, awareness, absence of any overt or latent psychiatric disease, and ability to give free consent.All prescribed forms have been completed by all relevant persons involved in the process of the transplantation.All interviews should be video recorded.The AC is required to take a final decision within 24 hours of the meeting for grant of permission or rejection for transplant. Every authorized transplantation center must have its own website. The decision of the AC should be displayed on the notice board of the hospital immediately and on the website of the hospital or institution within 24 hours of making the decision.There shall be one State Level AC. It will provide approval or a no objection certificate to the donor and recipient to establish legal and residential status in a particular state. Additional ACs may be set up at various levels as per the requirements as follows:No member from the transplant team of the institution should be a member of the respective AC.The AC should be hospital-based in metros and big cities if the number of transplants exceeds 25 in a year at the respective transplantation centers. In small towns, there shall be state or district level committees if transplants are less than 25 in a year in the respective districts.Medical Director or Medical Superintendent of the HospitalTwo senior medical practitioners from the same hospital who are not part of the transplant teamTwo members of high integrity, social standing, and credibilitySecretary  or nominee and Director Health Services or nomineeMedical Practitioner officiating as Chief Medical Officer or any other equivalent post in a main/major government hospital of the districtTwo senior medical practitioners who are residing in the concerned district and who are not part of any transplant teamTwo senior citizens of high reputation and integrity residing in the same districtSecretary  or nominee and Director of Health Services or nomineeTo a large extent, the interpretation of the THO act by the AC and the registered medical practitioners has been flawed. To a large extent this has been addressed in the current Gazette. However, this Gazette has to be passed by the state governments before it becomes compulsory for the hospitals to follow the ruling.The provisions available in Sub Clause (3), Clause 9 of Chapter II of the THO act states “If any donor authorizes the removal of any of his human organs before his death under sub-section (1) of Section 3 for transplantation into the body of such recipient, not being a near relative as is specified by the donor, by reason of affection or attachment towards the recipient or for any other special reasons, such human organ shall not be removed and transplanted without the prior approval of the Authorization Committee” has been misused or misinterpreted by one and all over the years, since the act was passed.The clinicians wonder if the law itself provides a clause to help people whose own family members refuse to donate or those who do not have a fit or matching donor, why should they refuse any arrangement between the donor and recipient? To them the plight of the recipient overrules all objections. They also argue that it is difficult for them to understand the so-called true affection. They feel the onus of responsibility to find true affection and relationships rests under the purview of the government AC.A patient whose kidney has failed uses this clause to find an instant affection in a stranger who is willing to donate his/her organ for money but they deny any such information to the AC. Later, the same donor makes a claim to the police or the media that they were duped into the donation process and not paid the promised sum for the organ. The affection in these instances, which they expressed for the recipient in front of the AC has no meaning or relevance. The police having no knowledge that the act of donation for money is illegal instantly pulls up the middleman or doctor or the hospital. Occasionally, when there is a media expose, the authorization committee in a knee-jerk reaction, tightens its regulations and stops clearing even genuine cases. For the past 3 years, the AC in Tamil Nadu videotaped all the interviews so that these videos can be used as an evidence later if necessary.When presented provisions of the law, the AC concludes that if the recipient and donor pledge affection in front of them they should not object unless there is a complaint or some gross oversight. They also believe that since the doctor himself has sent such a case to the committee, they need verify such claims. The majority of applications to the AC are usually accepted. Most unrelated donations occur when the donor expresses their true affection for the recipient in front of the AC. Between 1995 and 2002, there were about 5,000 cases interviewed by the AC in Tamil Nadu with a rejection rate of less than 5%. In another memo issued by the Department of Health of Tamil Nadu, it indicated that during January 2000 to May 2002 they had approved 1,559 unrelated transplants out of the 1,868 applications received. The scenario in other states in the country where transplants are done is similar to Tamil Nadu.vs. Kantha and others, the prospective recipient filed a writ petition before the High Court of Karnataka against the order of the AC that rejected the application for organ donation by the sister-in-law of the recipient on the grounds that close relatives were not considered donors. The High Court while allowing the writ petition held:As per the law, any person who is aggrieved with the order of the AC is allowed to make an appeal within 30 days of the issue of the order to the State government. In, B.L. Nagaraj and others “There is no provision in the Act which prohibits the person who is not a 'near relative' by definition, from donating his kidney merely because the 'near relative' has not been considered as donors by the family for kidney transplantation. The Committee has misdirected itself in this regard while refusing permission to the petitioners.”“The Committee would ascertain from the second petitioner whether she would be donating the kidney out of ‘affection and attachment’. The donors relationship with the recipient, period of acquaintance and the degree of association, reciprocity of feelings, gratitude and other human bonds are perhaps some of the factors which would sustain ‘affection and attachment’ between two individuals. The committee has to ensure that the human organ does not become an article of commerce. The main thrust of the act is against commercial dealings in human organs.”The problem has been on how to use Sub-Clause (3), Clause 9 of Chapter II of the THO act and how to protect the exploitative element in the word affection. In 1997, Dr. M.K. Mani, a leading Nephrologist in Chennai, summarized the above very well when he wrote: “The stalwarts of the unrelated live donor program continue to do as many transplants as they did before the Legislative Assembly of Tamil Nadu adopted the Act. What is more, they do them with the seal of approval from the Authorization Committee and are therefore a very satisfied lot. The law, which was meant to prohibit commercial dealings in human organs, now provides protection for those very commercial dealings.” Dr. Mani's article is titled ‘The Law is an Ass’.After a major kidney racket in Tamil Nadu, the Department of Health issued a notification in form of a ‘Government Order’ trying to absolve all responsibilities to prove relationship or any possibility of commerce with AC. It categorically stated that the responsibility to prove such a relationship was solely on the doctors of the hospital who signed the document to request for an interview. However, this was against what the THO act itself states and the role it defines for authorization committee. When the legal standing of the order was questioned, the order was withdrawn. The new Gazette now requires videotaping of the whole proceedings of the interview. In addition, it also gives guidelines to the AC and clearly states that there should be no tout or middleman with the donor having to provide an explanation of why he wishes to donate with documentary proof of having lived together (old Photographs) and information about his vocation with financial statements from the previous 3 years. Taking away the ambiguity of the term affection and giving it the seriousness it deserves may go some way in preventing the sale of kidneys.The presence of a growing middle class, the lack of a national health insurance scheme, the growing disparity between the rich and poor, and to some extent the presence of technology in the country makes the process of commodification of organs a simple, quick, and attractive business proposition for some and a solution for others. In many affordable middle class or upper class families, even when there are relatives in good health who can donate, the general argument that is often presented is “why donate and take any risks when you can buy a kidney?” Organ trade in India like other problems such as child labor and prostitution has a societal issue to it. It relates to the exploitation of the poverty-stricken people by alluring them with financial gains that at times can be large and can meet their immediate short-term financial needs. Unlike other similar exploitative social situations, organ donation requires an invasive surgical procedure that has both physical and psychological implications.The more recent live liver donation program has also been influenced by kidney donation and unrelated living donations have been reported in the media including two deaths. AlthoughLawrence Cohen, an anthropologist from Berkeley, interviewed patients in India and like Goyal had found that most of the donors were women who were deeply in debt and most of the money was squandered by their husbands in gambling and debts and the promise of a better future was never realized. In his research, Cohen found one-way trade in some of the “kidney belt region” of southern India where he investigated the trade route from organ sellers - usually poor rural women - to hospitals and recipients - often wealthy people from Sri Lanka and Bangladesh or from the Gulf States. Cohen found that poor people sold their kidneys to get out of debt or to support their families; yet most of these families were back in debt very shortly minus their kidneys. Some of the donors when asked if they would do it again said: ‘I'd do it again. I have a family to support. What choice did I have?’ Cohen states: “In some neighborhoods, the structure of debt appeared to rest on kidney selling since lenders would advance money knowing the organs were collateral. Moreover, there was no follow-up care after the operation nor were there efforts to prevent infection in the donor”. When kidGiving in to market forces and making organs a commodity is fraught with dangers and erodes social, moral, and ethical values and is not an alternative that can be acceptable to overcome the problem of organ shortage in a civilized society. In her article on ‘The End of the Body: The Global Traffic in Organs for Transplant Surgery’, Nancy Scheper-Hughes, an anthropologist from Berkeley, states that by their very nature markets are indiscriminate, promiscuous, and inclined to reduce everything, including human beings, their labor and even their reproductive capacity to the status of commodities, to things that can be bought, sold, traded, and sometimes even stolen. Mr. Soros, the self made billionaire and a great believer of market forces, is deeply concerned with the erosion of social values and the dominance of anti-social market forces in the field of health sciences. He is of the opinion that a market economy is generally a good thing but opines that we cannot live by markets alone. Open and democratic societies require strong social institutions to serve such goals as social justice, political freedom, bodily integrity, and other human rights. The real dilemma, as Mr. Soros sees it, is one of uneven development. The evolution of the global market has outstripped the development of a mediating global society.The Bellagio Task Force from the Department of Anthropology, University of California, Berkeley with support from the Open Society Institute (from the Soros Foundation) conducted ethnographic research in sites in Brazil, India, and South Africa between 1997 and 1998. Their findings were as follows:Strong and persistent race, class, and gender inequalities and injustices exist in the acquisition, harvesting, and distribution of organsViolation of national laws that prohibit the sale of organsThe collapse of cultural and religious sanctions against body dismemberment and commercial use exist in the face of the enormous market pressures in the transplant industryThe appearance of new forms of traditional debt peonage in which the commodified kidney occupies a critical spacePersistent and flagrant human rights violations of cadavers in public morgues exist, with organs and tissues removed without any consent for international saleThe spread and persistence of narratives of terror concerning the theft and disappearance of bodies and body parts exists globallyAnother report on The Global Traffic In Human Organs: a report presented to the House Subcommittee on International Operations and Human Rights, United States Congress on June 27, 2001 states that: “The growth of medical tourism for transplant surgery and other advanced procedures has exacerbated older divisions between the North and South and between the haves and have-nots. In general, the flow of organs, tissues, and body parts follows the modern routes of capital: from South to North, from third to first world, from poor to rich, from black and brown to white, and from female to male bodies. In the very worst instance, this market has resulted in theft and coercion, as in the case of China, to a self-serving belief in rights of the rich to the “spare parts” of the poor, as in the case of the many transplant junkets arranged to carry affluent patients from Saudi Arabia, Israel, and North America to Turkey, India, Romania, and the Philippines where kidney sellers are recruited from prisons, unemployment offices, and urban shantytowns.”In an editorial in the New England Journal of Medicine, Francis Delmonico states that “The fundamental truths of our society, of life and liberty, are values that should not have a monetary price. These values are degraded when a poor person feels compelled to risk death for the sole purpose of obtaining monetary payment for a body part. Physicians, whose primary responsibility is to provide care, should not support this practice. Furthermore, our society places limits on individual autonomy when it comes to protection from harm. We do not endorse as public policy the sale of the human body through prostitution of any sort, despite the purported benefits of such a sale for both the buyer and the seller.”Cantarovitch suggests that organ transplantation depends on a social contract and social trust and it requires national and international law protecting the rights of both organ donors and organ recipients. In the lThe high demand and poor supply of kidneys in the United States has widen over the years. This has resulted in many patients traveling abroad for transplant surgery. Some of the countries that have weak regulatory mechanisms have given in to the market forces and include India, Iran, China, Pakistan, Philippines, Brazil, Turkey, Moldova, Ukraine, Russia, Bulgaria, and Romania.The World Health Organization (WHO) in its statement on the sale of organs clearly states that it violates the Universal Declaration of Human Rights as well as its own constitution: “The human body and its parts cannot be the subject of commercial transactions. Accordingly, giving or receiving payment… for organs should be prohibited.” The WHO advices physicians not to transplant organs “if they have reason to believe that the organs concerned have been the subject of commercial transactions.”More recently, the representatives of the world transplant community met in Istanbul to discuss the growing transplant donation commerce and transplant tourism. It defined ‘Transplant commercialism’ as a policy or practice in which an organ is treated as a commodity, including by being bought or sold or used for material gain. In an edIn India, where the deceased donation rate is abysmally small, there is a need to seriously explore this option and seems to be the way forward to our problem of organ shortage and to curb commerce in organs. Besides this swap or donor exchange in living transplants should be explored as a feasible alternative.There are currently over 120 transplant centers in India performing approximately 3,500 to 4,000 kidney transplants annually. Out of these transplant centers, four centers undertake approximately 150 to 200 liver transplants annually while some of these centers also do an occasional heart transplant. Presently, approximately 50 liver transplants are done from deceased donors and the rest are from living donors. So far, 100 heart transplants have also been done.In 1998, India had 1% of the world's road vehicles and 6% of the world's road accidents. These acThere have been pockets of success with the deceased donation program and organ sharing among various hospitals. Five hospitals in Tamil Nadu and approximately eight hospitals in Hyderabad from the year 2000 to 2008 have successfully shared over 450 organs (170 organs in Hyderabad and 280 in Tamil Nadu) under the Organ Sharing Network that was initiated by a Non-Governmental Organization (NGO) called MOHAN Foundation.17 In the pUnderstanding the ethics of organ donation is important if we are to tackle the moral and ethical challenges that are emerging with cutting edge regenerative medicine such as stem cells transplants, cloning, and tissue re-engineering. The ethical principles of organ donation is an acid test that will help us in evolving and resolving many of the future moral issues that we are likely to encounter.The THO act despite having been passed 15 years ago has neither curbed commerce in organs nor helped promotion of the deceased donation program to take care of the organ shortage. The gap between the numbers of organs available and the number of patients joining the waiting list for a kidney transplant is widening globally. The high demand of organs has led to its commodification, more so in countries where there is a large proportion of the population below the poverty line with weak regulatory authorities. The resulting transplant tourism has caused an outcry from many international bodies. In India, the potential for deceased donation is huge due to the high number of fatal road traffic accidents and this pool is yet to be tapped. Few hospitals and committed NGOs in the country have shown that deceased donation as a feasible option. The ethics of kidney donation has important bearings on the society as this would form the basis to resolve many conflicts in emerging regenerative sciences.


Macrophage migration inhibitory factor (MIF) plays an essential pathophysiological role in septic shock, but its role in central nervous system infection (CNS) remains to be defined.We investigated cerebrospinal fluid (CSF) levels of MIF in 171 patients who were clinically suspected of having meningitis on admission. Of these, 31 were found to have purulent meningitis of known aetiology, 20 purulent meningitis of unknown aetiology, 59 lymphocytic meningitis and 11 encephalitis, whereas 50 were suspected of having but had no evidence of CNS infection.P = 0.003), patients with lymphocytic meningitis  and patients suspected of having but without evidence of CNS infection . Also, patients with encephalitis  had higher CSF MIF than did patients without CNS infection (P < 0.01). Among patients with purulent meningitis, CSF MIF levels were significantly higher in patients infected with pneumococci than in those with meningococcal infection , in patients who required versus those not requiring assisted ventilation , and in patients with versus those without impaired consciousness . CSF MIF levels correlated significantly with meningeal inflammation (P < 0.05) but not with systemic inflammatory response (P > 0.05) in patients with purulent meningitis of known aetiology, those with lymphocytic meningitis and those with encephalitis.CSF MIF levels were significantly higher in patients with purulent meningitis of known aetiology  than in patients with purulent meningitis of unknown aetiology (2,209  ng/l; Mann-Whitney test, MIF was significantly increased in the CSF of patients with purulent meningitis and encephalitis, and was to some degree associated with severity of the infection. Our findings indicate that MIF may play an important role in CNS infection. Macrophage migration inhibitory factor (MIF) is a cytokine that participates in both innate and adaptive immune responses. It is released from a wide range of cells, including macrophages and T lymphocytes, and when released it counter-regulates the inhibitory effect of corticosteroids on the release of pro-inflammatory cytokines from lipopolysaccharide-stimulated monocytes. This suggests a role of MIF in both the initiation of and sustaining the inflammatory cascade . The Bacterial meningitis is characterized by an overwhelming inflammatory cascade, primarily localized to the subarachnoidal space, which continues to evolve after eradication of the bacterial pathogen by antibiotic therapy . Anti-inThe aim of the present study was to investigate CSF MIF levels in 171 patients clinically suspected of having meningitis on admission and to study the use of CSF MIF in the differential diagnosis of meningitis and as a prognostic factor.Since 1988 CSF samples have been collected prospectively from patients undergoing lumbar puncture at the Department of Infectious Diseases at Hvidovre University Hospital. All lumbar punctures were performed as routine diagnostic procedures and were done in accordance with the hospital's ethical standards. Immediately after lumbar puncture, all CSF samples were transported directly to the Department of Clinical Biochemistry, where they were centrifuged, and the supernatants were collected and stored at -20°C within 15 to 30 minutes. The following day, CSF samples were registered and transferred for storage at -80°C. Because of the non-interventional nature of the study, blood samples were not routinely collected together with the CSF sampling. However, in six patients a blood sample was available that was obtained on admission and at the same time as the CSF sample. All protocols were approved by the local scientific ethics committee and the Danish Data Protection Agency (1995–1200/95–517); because the study required no intervention in addition to routine care, informed consent from patients was not required.Mycobacterium tuberculosis infection.Patients' demographic and clinical characteristics, as well as biochemical and microbiological data, were obtained retrospectively from medicals records. All patients who were clinically suspected of having meningitis, in whom CSF samples was available that had been obtained at admission from a diagnostic lumbar puncture conducted during the period from 1988 to 2002, were included, except patients infected with HIV or A total of 205 patients were identified. However, 34 patients  were excluded, because MIF could not be measured for lack of CSF. Thus, the study comprised 171 patients. Based on clinical, microbiological and biochemical characterization, patients could be divided into five diagnostic groups.Neisseria meningitidis. Fifteen cases were due to N. meningitidis, 11 to Streptococcus pneumoniae, two to Haemophilus influenzae, one to Klebsiella pneumoniae, one to Staphylococcus aureus and one to Listeria monocytogenes.The first group included those with purulent meningitis of known bacterial aetiology (n = 31). Twenty-five patients had positive CSF Gram stain or CSF culture  and six had neutrophil pleocytosis  and positive blood culture or a significant increase in antibody titres against Patients in the second group, purulent meningitis of unknown bacterial aetiology (n = 20), had negative CSF and blood cultures, neutrophil pleocytosis , who were treated for bacterial meningitis in accordance with local recommendations.9 cells/l. Antibiotic therapy was initiated before the lumbar puncture in 23% (7/31) of patients with purulent meningitis of known bacterial aetiology and in 15% (3/20) of those with an unknown bacterial aetiology. The initial or empirical antibiotic therapy for purulent meningitis was intravenous ceftriaxone and ampicillin. If bacteria were demonstrated in the CSF and/or blood, then antibiotic therapy was changed in based on the susceptibility profile of the pathogen. The duration of antibiotic therapy was usually 7 to 10 days.Five of the 20 patients (20%) with negative cultures and one out of the 31 patients (3%) with known bacterial aetiology had CSF WBC count under 1,000 cells/μl, CSF/blood glucose ratio above 0.3, CSF protein levels under 1 g/l and blood WBC count under 12 × 10The third group included patients with lymphocytic meningitis . All of these cases were due to acute aseptic meningitis. A known viral aetiology was established in 15 cases (enterovirus in 14 and herpes simplex virus-2 in one). The majority of patients recovered fully without antibiotic treatment, except for 14 patients who received one dose of antibiotics immediately after the lumbar puncture was performed and before the results of the CSF analysis were known . Two patients also received one dose of aciclovir.The fourth group included patients with encephalitis . A viral aetiology was established in two cases (herpes simplex virus and varicella zoster virus [VZV]). All patients were treated with aciclovir .N. meningitidis and in one it was due to Escherichia coli), seven had pneumonia, three had acute tonsillitis, one had acute otitis media, two had urinary tract infection, one had hepatitis due to cytomegalovirus, 25 patients had fever of unknown origin and seven had cephalgia.The fifth diagnostic group included patients suspected of having meningitis but without evidence of CNS infection . Four patients had septic shock , and optical density at 450 nm was read on an ELISA reader. Lower limit of detection was 20 pg/ml.MIF levels wee measured using a commercial available ELISA , in accordance with the manufacturer's instructions. In brief, MaxiSorp plates  were coated and incubated overnight at 4°C with a murine monoclonal anti-MIF antibody (R&D Systems), diluted in phosphate-buffered saline  to a final concentration of 2 μg/ml. After the plates were washed three times with wash buffer (PBS with Triton X-100), the unbound sites were blocked by adding 300 μl blocking buffer  for 90 minutes at room temperature. After another three washes, standards (recombinant human MIF [R&D Systems] in triplicates) or CSF and blood samples (at least in duplicate) diluted in dilution buffer  were added and incubated for 2 hours at room temperature. After another three washes, MIF was detected by adding a biotinylated goat anti-human MIF antibody (R&D Systems) diluted in dilution buffer to a final concentration of 0.2 μg/ml and incubating for 2 hours at room temperature. After washing five times, streptavidin horseradish peroxidase (R&D Systems) diluted 1:1,000 in dilution buffer was added and incubated for 20 minutes at room temperature with subsequent washing five times and adding substrate solution, tetramethylbenzidine , for 30 minutes. The reaction was stopped with 1.2 mol/l HP < 0.05 were considered statistically significant.All data are expressed as medians and interquartile ranges. For continuous data, comparisons between two groups were performed using the Mann-Whitney test, whereas the Kruskal-Wallis test was used for comparisons between more than two groups. Fisher's exact test was used for comparisons between categorical data. When appropriate, correction with a Bonferroni's coefficient of 10 was used to compensate for multiple comparisons between the five diagnostic groups. For correlation analysis, the nonparametric Spearman's test was used. S. pneumoniae meningitis, one with N. meningitidis meningitis, one with S. aureus meningitis, one with K. pneumoniae meningitis, one with purulent meningitis of unknown aetiology, two with encephalitis of unknown aetiology, and two without meningitis [meningococcaemia and pneumonia]).Clinical and demographic data for the 171 patients with available CSF samples on admission are shown in Table P < 0.0001; Figure P = 0.003), those with lymphocytic meningitis  and those suspected of having meningitis but without evidence of CNS infection . Also, patients with encephalitis  had significantly higher CSF MIF levels than did those with lymphocytic meningitis (P = 0.004) and patients without meningitis (P < 0.001). Moreover, patients with pneumococcal meningitis had significantly higher CSF MIF levels than did those with meningococcal meningitis . Before lumbar puncture, two out of 171 patients – one with pneumococcal meningitis  and one with VZV encephalitis  received therapy with prednisolone 5 mg/day for treatment of rheumatoid arthritis and chronic myeloid leukaemia, respectively.A total of 163 patients had measurable CSF MIF levels, whereas eight patients had MIF levels under 20 ng/l. There was a significant difference between the five diagnostic patient groups , and one patient with pneumococcal meningitis with accompanying bacteraemia had lower MIF levels in CSF than in blood . Three patients without CNS infection had lower MIF levels in CSF than in blood , whereas one patients with hepatitis due to cytomegalovirus had higher MIF levels in CSF than in blood .P = 0.003), and in patients with impaired consciousness than in those without . However, CSF MIF levels were not significantly higher in patients who died from the infection than in surviving patients  or in patients who developed septic shock than in those who did not . In purulent meningitis of known bacterial aetiology, the following were all associated with high CSF MIF levels: positive Gram stain , positive CSF culture  and positive blood culture .Among patients with purulent meningitis, CSF MIF levels were to some degree associated with severity of the infection. CSF MIF levels were significantly higher in patients who required assisted ventilation than in those who did not .The two patients who died from encephalitis had high CSF MIF levels , but the difference in CSF MIF between these patients and those who survived  was not statistically significant (P = 0.03).CSF MIF levels correlated with number of days hospitalized  but not with the systemic inflammatory response (P > 0.05). In contrast, no such association was observed in patients with purulent meningitis of unknown aetiology (P > 0.05). In patients without CNS infection, CSF MIF correlated significantly with CSF protein levels (P < 0.05).Associations between CSF MIF levels at admission and biochemical parameters in CSF and blood are summarized in Table In the present study we found that CSF MIF levels were significantly higher in patients with purulent meningitis and encephalitis than in patients with lymphocytic meningitis and patients suspected of having meningitis but without evidence of CNS infection. In accordance with our findings, a previous study  showed tCSF MIF levels were significantly higher in patients with pneumococcal meningitis than in those with meningococcal meningitis. Pneumococcal meningitis carries a higher mortality than meningococcal meningitis, and differences in bacterial virulence factors may account for higher CSF MIF levels in pneumococcal meningitis than in meningococcal meningitis. Moreover, CSF MIF levels were to some degree related to disease severity, being significantly higher levels in patients with purulent meningitis who were unconscious or who required assisted ventilation, but CSF MIF levels were not correlated with mortality or presence of septic shock. Previous studies found that serum levels of MIF were related to mortality in sepsis -5. UnforIn the present study, we found a significant correlation between CSF MIF levels and CSF WBC count in meningitis and encephalitis patients, whereas no association was found between CSF MIF levels and the systemic inflammatory response (blood WBC count). Moreover, Arjona and coworkers  found thMIF is produced by a wide range of cell types including macrophages  and actiApart from its critical role in sepsis through potentiating septic shock, MIF is a pituitary-derived antagonist of glucocorticoids . AdjunctThe role played by MIF has been studied extensively in experimental disease models using pharmacological intervention (stimulation with MIF or inhibition with antibodies to MIF) or by use of MIF gene-deficient animals. Inhibition or lack of MIF attenuated development of septic shock ,6, encepMIF levels were significantly increased in CSF of patients with purulent meningitis of known aetiology or with encephalitis, and they were to some degree associated with severity of the infection. Our findings indicate that MIF may play a pathophysiological role in CNS infection.• CNS infections cause increased CSF levels of MIF.• Patients with purulent meningitis of known aetiology or with encephalitis had significantly higher CSF MIF levels than did patients with lymphocytic meningitis or patients with no CNS infection.• CSF MIF levels were associated with disease severity in patients with purulent meningitis.CNS: central nervous system; CSF: cerebrospinal fluid; ELISA: enzyme-linked immunosorbent assay; MIF: macrophage migration inhibitory factor; PBS: phosphate-buffered saline; VZV: varicella zoster virus; WBC: white blood cell.The authors declare that they have no competing interests.CØ designed the study, collected and analyzed data, and drafted the manuscript. TB participated in collection of data and in revising the manuscript.


Transplantation is the only treatment offering long-term benefit to patients with chronic kidney failure. Live donor nephrectomy is performed on healthy individuals who do not receive direct therapeutic benefit of the procedure themselves. In order to guarantee the donor's safety, it is important to optimise the surgical approach. Recently we demonstrated the benefit of laparoscopic nephrectomy experienced by the donor. However, this method is characterised by higher in hospital costs, longer operating times and it requires a well-trained surgeon. The hand-assisted retroperitoneoscopic technique may be an alternative to a complete laparoscopic, transperitoneal approach. The peritoneum remains intact and the risk of visceral injuries is reduced. Hand-assistance results in a faster procedure and a significantly reduced operating time. The feasibility of this method has been demonstrated recently, but as to date there are no data available advocating the use of one technique above the other.The HARP-trial is a multi-centre randomised controlled, single-blind trial. The study compares the hand-assisted retroperitoneoscopic approach with standard laparoscopic donor nephrectomy. The objective is to determine the best approach for live donor nephrectomy to optimise donor's safety and comfort while reducing donation related costs.This study will contribute to the evidence on any benefits of hand-assisted retroperitoneoscopic versus standard laparoscopic donor nephrectomy.Dutch Trial Register NTR1433 Transplantation is the only treatment offering long-term benefit to patients with chronic kidney failure. As the number of patients suffering end stage renal disease (ESRD) increases, the recruitment of more kidney donors is important . Live kiLive donor nephrectomy is performed on healthy individuals who do not receive direct therapeutic benefit of the procedure themselves. In order to guarantee safety for the donors, it is important to optimise the surgical approach. Recently we demonstrated the benefit of laparoscopic nephrectomy (LDN) to the donor. However, this method is characterised by higher in-hospital costs, longer operating times and requires a well-trained surgeon . The hanWe recently proved that laparoscopic kidney donation is beneficial for the donor. In comparison to minimally invasive open techniques, laparoscopic kidney donation is associated with a better quality of life, less pain, shorter in hospital stay and earlier return to work. This method is expensive for the hospital, has a long operating time and requires an experienced, well-trained, surgeon ,8,9. OthThe hand-assisted retroperitoneoscopic approach may be a viable alternative. With this method the surgeon inserts his hand to create a retroperitoneal space, which is thereafter insufflated with gas. The peritoneum stays intact and tactile sensation remains. The chance of a complication to the intestines is very small. Furthermore, this technique is easier and quicker to learn for the surgeon than the laparoscopic approach. There is no randomized controlled trial comparing both techniques for the effectiveness -6.To determine the best approach for live donor nephrectomy i.e. to optimise donor's safety and comfort while reducing donation related costs.The HARP-trial is a multi-centre, randomised controlled, single-blind trial. We have stratified per centre. The study started on July 24th 2008 and the duration of the inclusion will be approximately 3 years. The study compares hand-assisted retroperitoneoscopic donor nephrectomy and standard laparoscopic donor nephrectomy. In total 190 live kidney donors will be included in the study. Approval of the medical ethical committee of both centres was obtained.Randomisation will take place after endotracheal intubation, by means of telephonic consultation of the study coordinator. A computer generated randomisation list with hidden block size is made for each centre by the statistician. The donor and the physicians involved in the postoperative period are blinded to the surgical technique until one year after donor nephrectomy. The operating theatre is not accessible for people who do not join the operating team. An independent surgeon evaluates the donor on the outpatient clinic before operation. As the extraction incision is similar in both techniques, we did not attempt to cover the wounds with a standard pattern of bandages . All donAll, properly Dutch speaking, live kidney donors who are medically capable of donating their left kidney can be included in the HARP-trial. Informed consent is mandatory. All types of live kidney donors can participate in the study, i.e. related, unrelated, altruistic and cross-over live kidney donors.Exclusion criteria are a history of kidney surgery or adrenal gland surgery on the left side.All potential donors are informed about the study at the outpatient clinic. For further information the patient can refer to the research fellow, transplant surgeon, or the independent physician. If a patient does not sign the informed consent form, the patient is not included in the study and therefore will be operated via the standard protocol. A live kidney donor can always withdraw his or her consent at anytime during the study.The left-sided hand-assisted retroperitoneal approach will lead, with a similar or better quality of life, to fewer complications, and reduced operating times and costs.Primary Question: Does left-sided hand-assisted retroperitoneoscopic donor nephrectomy lead to a similar or better quality of life as compared to left-sided transperitoneal laparoscopic donor nephrectomy?Secondary Question: Is the retroperitoneoscopic technique safer (conversions and complications) than the transperitoneal technique?Other secondary endpoints: pain perception, return to work, operation time, cost-effectiveness from both a societal and health care perspectives .The Erasmus Medical Centre Rotterdam harbours one of the largest European live kidney donor transplantation programs. On a yearly basis 75 to a 100 laparoscopic donor nephrectomies are performed. Our strategy is to improve the results of live kidney donation by optimizing the surgical technique with decreased complication rates and costs. The infrastructure, raised in earlier studies, led to the formation of a multidisciplinary team, with a high standard of care and surgery.In the previous study on live kidney donation running at our centre we compared the laparoscopic technique to the mini-incision muscle-splitting technique . LaparosA pilot study with 60 live kidney donors showed the feasibility of the retroperitoneal hand-assisted approach . Even in2O pressure. A 30° video endoscope and 3 to 4 additional trocars were introduced. The colon was mobilized and displaced medially. Opening of the renal capsule and division of the perirenal fat was facilitated using an ultrasonic device . After identification and careful dissection of the ureter, the renal artery and the renal vein, a pfannenstiel incision was made. An endobag  was introduced into the abdomen. The ureter was clipped distally and divided. The renal artery and vein were divided using an endoscopic stapler and the kidney was placed in the endobag and extracted through the pfannenstiel incision.Both procedures were performed with the donor placed in right-decubitus position. LDN was performed as described earlier . Shortly2 was insufflated retroperitoneally to 12 cm H2O pressure. Two other 10-12 mm trocars, respectively just outside the midline inferior to the costal margin and in the flank, were inserted to create a triangular shape. For dissection the aforementioned ultrasonic device was used. Dissection of the kidney and renal vessels was similar to transperitoneal donor nephrectomy but with hand-assistance and from a slightly different angle. The kidney was removed manually.In HARP we started with a 7-10 cm pfannenstiel incision . After bPrimary outcome: Physical functioning as a measure for quality of life one month after donor nephrectomy. This is one of the dimensions of the standardized SF-36 questionnaire. Physical functioning is measured by means of the complaints with daily physical activities, like walking stairs, carrying grocery bags, walking 500 meter, etc. The SF-36 is in our opinion a suitable parameter to measure post-operative recovery .Secondary outcome: Direct costs . Other secondary endpoints: conversion to open surgery, complications, pain perception, work resumption, other dimensions of quality of life (SF-36), cost-effectiveness from hospital and health care perspective.In this effectiveness analysis the effect of the surgical procedure on quality of life of the donor and the costs for the hospital are the most important outcome measures. The donor is the central point of interest; therefore we chose quality of life as primary endpoint for the power calculation. Saving money, but with a worse quality of life for the donors is indeed not relevant.A difference of 7.5 point in physical functioning (SF-36) is a relevant difference. In international literature, a five point difference is the minimal clinically relevant difference . In our The donor is prehydrated with intravenous crystalloids before operation. On the morning of surgery antithrombotic stockings are given. The anaesthetist uses a standard protocol for live kidney donation anaesthesia (remifentanyl and propofol), intravenous policy, and respiration. One hour after the beginning of the operation, 20 mg mannitol is infused. During the operation the research-fellow notes warm-ischemia time, blood loss, and complications. Postoperative pain medication is measured through a Patient Controlled Analgesic  device. If the patient does not use the PCA for 6 hours, the PCA is stopped. The dosage regimen is registered. Patients can be discharged when they meet with the following criteria:- The donor tolerates a normal diet- The donor is mobile - The donor is adequate and oriented- The donor does not use intravenous medicationAll live kidney donors will be seen at the outpatient clinic after four weeks, three months and yearly thereafter. All donors are asked to fill out different questionnaires; we measure pain and nausea scores (VAS-score), quality of life , and a questionnaire on work and homecare Table .The live kidney donor is informed at the outpatient clinic on the background of the study, possible complications and the chance of conversion to an open approach. After both operations the donor may wake up having an extra scar caudal to the costal margin if the operation is converted to an open approach.If a donor has post-operative pain or discomfort of the bandages, they can be removed. All documentation will be marked with the HARP-trial logo. Information about the operation technique will be sealed in an envelope in the medical file. In case of unexpected events this envelope may be opened. Unexpected events are reported to the responsible physician and the study coordinator.All personal data are coded into numbers (1 to 190). The coordinating investigator and the principle investigator are the only ones who have access to the coding system. All data are imported in a database, which is managed by the coordinating investigator. At the end of the trial all data are analysed together with the trial statistician.The hand-assisted retroperitoneoscopic technique may be an alternative to a complete laparoscopic, transperitoneal approach. The feasibility of this method has been demonstrated recently, but as to date there are limited data available advocating the use of one technique above the other -7. This In comparison to minimally invasive open techniques, laparoscopic kidney donation is associated with a better quality of life, less pain, shorter in-hospital stay and earlier return to work. This method is time consuming, leading to high hospital costs, and requires an experienced surgeon. Other studies showed a possibly increased rate of major, life threatening, complications, such as injuries to the intestines and bleeding .et al compared the LDN (n = 11), hand-assisted laparoscopic technique (HALS) (n = 14), and the HARP (n = 18). The operation time with the HARP was significantly shorter than the LDN  [et al performed a prospective study, comparing HARP (n= 11), LDN (n= 14) and open donor nephrectomy (n= 11). Hand-assisted retroperitoneoscopic donor nephrectomy had a significantly shorter operation time compared to LDN  [et al performed a retrospective study, comparing HARP (n= 11), LDN (n= 15) and open donor nephrectomy (n= 25). Reduced operation time was observed for the HARP group compared with the LDN (166 min vs 244 min) [et al performed a prospective study, comparing 20 left-sided HARP procedures and 40 left-sided LDNs. Median operation time and WIT were shorter in HARP  [The hand-assisted retroperitoneoscopic approach may be a viable alternative. The chance of a complication to the intestines is very small, and hand-assistance could be beneficial for the control of bleedings. There is no randomized controlled trial comparing both techniques. Only three small studies compare left-sided hand-assisted retroperitoneoscopic with laparoscopic donor nephrectomy, but only with respect to clinical parameters -5,7. WadLS) n = 1, and theAll other studies only described surgical outcome, and did not address quality of life of live kidney donors. Our main point of interest is to know whether the donors operated on with the hand-assisted retroperitoneal technique have a good quality of life afterwards. Therefore we will perform this randomised controlled trial comparing laparoscopic to hand-assisted retroperitoneoscopic technique, with physical function as a primary outcome.The authors declare that they have no competing interests.LFCD and NFMK drafted the manuscript. JNMIJ co-authored the writing of the manuscript. All other authors participated in the design of the study during several meetings and are local investigators at participating centres. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2482/10/11/prepub


Bacterial meningitis is an acute disease with high mortality that is reduced by early treatment. Identification of the causative microorganism by culture is sensitive but slow. Large volumes of cerebrospinal fluid (CSF) are required to maximise sensitivity and establish a provisional diagnosis.We have utilised nuclear magnetic resonance (NMR) spectroscopy to rapidly characterise the biochemical profile of CSF from normal rats and animals with pneumococcal or cryptococcal meningitis. Use of a miniaturised capillary NMR system overcame limitations caused by small CSF volumes and low metabolite concentrations. The analysis of the complex NMR spectroscopic data by a supervised statistical classification strategy included major, minor and unidentified metabolites.Reproducible spectral profiles were generated within less than three minutes, and revealed differences in the relative amounts of glucose, lactate, citrate, amino acid residues, acetate and polyols in the three groups. Contributions from microbial metabolism and inflammatory cells were evident. The computerised statistical classification strategy is based on both major metabolites and minor, partially unidentified metabolites. This data analysis proved highly specific for diagnosis , provided those with visible blood contamination were excluded from analysis; 6–8% of samples were classified as indeterminate.This proof of principle study suggests that a rapid etiologic diagnosis of meningitis is possible without prior culture. The method can be fully automated and avoids delays due to processing and selective identification of specific pathogens that are inherent in DNA-based techniques. Bacterial meningitis is an acute disease with a high mortality Alternative approaches based on chemicals in CSF due to production by the infection causing microorganism or due to the immune response of the host are potentially useful for the diagnosis of a variety of neurological diseases, including meningitis The metabolite composition of normal CSF has been studied intensively by NMR spectroscopy Computerised methods utilize the whole NMR spectrum and include unnoticed (or unknown) compounds. In a pilot study of acute meningitis, CSF spectra from small numbers of controls, patients with viral meningitis and bacterial/fungal meningitis were distinguished using an unsupervised cluster analysis method Cryptococcus neoformans and Streptococcus pneumoniae.In clinical practice, rapid identification of a specific bacterial or fungal pathogen is important as it enables immediate initiation of appropriate antimicrobial therapy based on predictable patterns of antibiotic susceptibility and hence improves clinical outcomes. We have utilised a metabolomic approach based on NMR spectra from CSF of animal models to evaluate if metabolites produced by an infection-causing microorganism or a specific immune response can be utilised for diagnosis. This could provide the basis for further development of the method on human CSF specimens. The two disease models used were meningitis caused by Cryptococcus neoformans (N = 31) or Streptococcus pneumoniae (N = 30) into the cisterna magna. CSF samples were collected from these animals after first signs of meningitis and at several time points thereafter. Control CSF samples were repeatedly collected from sham injected animals , N = 5) or prior infection.Meningitis was induced in Fisher 344 rats by injection of 4 cfu of C. neoformans and S. pneumoniae, respectively. Resonances in the 1D NMR spectra from each pathology were assigned to respective metabolites using 2D NMR correlation spectra from at least five independent samples per category  and polyol signals (mannitol and glycerol resonances (3.90–3.95 ppm) for 20% of C. neoformans infections).Although the major metabolites in CSF samples from all three categories were qualitatively similar for control and post-infection CSF samples from individual animals, quantitative differences were noted  are summarised in C. neoformans and S. pneumoniae meningitis but not to that between C. neoformans meningitis and controls, presumably because polyols overlap with glucose resonances, and glucose levels are higher in normal CSF than in that from animals with cryptococcal meningitis.A statistical classification strategy (SCS) C. neoformans  or S. pneumoniae . Both new isolates were classified mostly correctly (one isolate was misclassified and one was fuzzily assigned).After identification of the most discriminatory regions in the NMR spectra of CSF, these regions were utilised to distinguish between the two pathologies and the uninfected rats. The final classification accuracy was achieved after repeated re-development and validation of the pair-wise classifiers. The first set of classifiers were based on all CSF samples (N = 101) and was still relatively inaccurate . AlthougRepeated CSF collections from the same infected animal (N = 27) were mainly used for classifier validation. The time of collection of CSF after onset of meningitis did not influence class assignment. One possible explanation is that quantitative changes in metabolite composition between day 4 and 11 after infection in an individual animal were similar to differences in CSF composition between different animals collected at the same time point.Micro-NMR spectroscopy of CSF when analysed by a supervised classification method distinguished rapidly and reproducibly between controls, rats with pneumococcal meningitis and rats with cryptococcal meningitis. This proof of principle study in experimental rats indicates that the etiology of meningitis can potentially be established rapidly without prior culture of the infection-causing microorganism using a very small amount of CSF. If able to be translated into clinical practice and extended to other pathogens, this is potentially a major diagnostic advance that would assist in the rapid selection of appropriate antimicrobial therapy and hence improve patient outcomes.Human CSF is rich in NMR detectable metabolites. More than hundred have been identified based on NMR spectroscopy in CSF from patients with various neurological, metabolic and other non-neurological diseases S. pneumoniae and levels in pneumococcal meningitis exceed those produced in individual human cases of staphylococcal and cryptococcal meningitis C. neoformansSemi-quantitative analysis revealed that elevation of lactate and reduction in glucose levels were the dominant CSF metabolite changes in rats with meningitis. These changes are also documented in human cases of bacterial and fungal meningitis, but are not specific Although some metabolic differences were detected by analysing subjectively chosen spectral regions, discrimination between the two pathologies and controls would not have been possible if based solely on these major metabolites. In the present study, the selection of the most discriminatory regions of the NMR spectra by GA-ORS suggests that glutamine (2.25–2.27/2.3 ppm) may have contributed most to the distinction between the three pathologies , which was in principle confirmed by the comparison of individual integral regions . Those metabolites can often only be identified by time consuming 2D correlation spectra  to some samples (N = 30) for evaluation of spectral quality (resolution) after shimming. No significant differences were found between these samples and sample acquisition performed without shimming. Thus the micro-volume NMR system represents a powerful advance. The stability of CSF samples at room temperature for some hours makes the combination of the NMR system with automatic sample changers feasible, allowing rapid, high-throughput data acquisition and analysis.A desirable precondition for the incorporation of NMR spectroscopy into clinical diagnostics is automation of sample handling and data analysis, and reduction of the sample volume to a minimum. Even more important is the utilization of small volumes for repeated monitoring in animal models. We therefore chose a 1 mm micro-NMR probe for acquisition of NMR spectra. Low volumes of CSF samples (5 to 35 µl) were directly collected into 1 mm micro NMR tubes. Subsequent snap-freezing and later, automated acquisition of NMR spectra resulted in a satisfactory signal-to-noise ratio and spectral quality. Comparison between data acquisition using the 1 mm micro NMR probe with those in conventional 5 mm tubes confirmed previous findings of dramatically improved signal-to-noise ratios and equal or better spectral resolution and water suppression by using 1 mm micro tubes C. neoformans and S. pneumoniae infections that diagnosis of meningitis according to etiology of the infection causing microorganisms is possible without prior culture of the microorganism using a metabolomics based approach. The method can be fully automated and is suitable for incorporation in clinical diagnosis. This would potentially accelerate and hence greatly improve turn-around-times compared with current diagnostic procedures once it has been extended to other meningitis-causing pathogens and validated on clinical samples.We have demonstrated for Animal experimentation was carried out according to the Australian National Health and Medical Research Council Guidelines and with ethical approval from the University of Sydney Animal Ethics Committee .Clinical samples were studied in compliance with ethical approval granted by the Human Ethics Review Committee of Sydney Western Area Health Service.4 cfu of clinical isolates of Cryptococcus neoformans  or Streptococcus pneumoniae , followed by rapid withdraw of the needle. Sham injection was performed for five animals by injection of sterile PBS. For validation of the classification five animals per pathogen were injected using an additional clinical isolates of C. neoformans (isolate WM1128) and S. pneumoniae (isolate 99-235-2193).A total number of 175 CSF samples were collected from 76 animals  in this study. Rats were anaesthetized by inhalation of 2% halothane  in oxygen delivered from a precision out-of-circuit vaporiser using a tight-fitting facemask. Following induction of deep anaesthesia, a 25-gauge needle was inserted into the cisterna magna and 15–35 µl of CSF was removed by suctioning the hub of the needle using an insulin syringe. These CSF samples were used as control (sterile) specimens. Meningitis was induced by the slow injection of 15–35 µl PBS containing 10C. neoformans and seven S. pneumoniae) by culture of five different dilutions on horse blood agar plates for 24 hours (S. pneumoniae) and on Sabouraud dextrose agar plates for 48 hours (C. neoformans) at 35°C. Three inoculated animals were excluded from the study due to an absence of microorganisms in CSF samples.CSF samples (15–35 µl) from infected rats were harvested when animals first showed signs of meningitis  or after day eight if the animals remained asymptomatic. Additional collection of CSF was performed three days thereafter for all animals that had not been euthanized (N = 27). CSF was collected as before from the cisterna magna. Part of the CSF sample from all animals was used for confirmation of the presence of microorganisms using standard microbiological tests (10–15 µl CSF). Only CSF samples from which the respective microorganisms had been cultured were included in the NMR study. The presence of cryptococcal organisms in CSF was confirmed by thin smears of CSF (10 µl), which were stained with a rapid Romanowsky stain . Typically, large numbers of organisms were evident in stained smears. Plate counts were performed on CSF samples from thirteen animals . The samples were transferred within 15 minutes into liquid nitrogen and stored at −70°C for up to 60 days for NMR experiments. Samples were carefully thawed before NMR experiments and centrifuged for 30 seconds using a manual centrifuge to avoid air bubbles in the micro tubes. Deuterated water (2–4 µl) was added to 30 samples to adjust for low filling heights.1H NMR spectra for SCS analysis were acquired using a Bruker Biospin Avance 400 spectrometer. Two-dimensional (2D) correlation NMR spectra for resonance assignment were acquired using a Bruker Biospin Avance 600 spectrometer. An appropriate TXI 1 mm MicroProbe with z-gradients was used on either NMR spectrometer. In addition, NMR spectra were also acquired from CSF samples collected at the terminal time point (volume 50–150 µl) using a Bruker Biospin Avance 600 spectrometer equipped with a 5 mm {1H, 13C} inverse-detection dual-frequency probe. All measurements were carried out without spinning at 37°C. 1D 1H NMR spectra were acquired with 16 k data points, a spectral width of 10 ppm, a repetition time of three seconds and accumulation of 64 averages. Residual water was suppressed using an 1D NOESY presaturation sequence with a mixing time of 100 ms NMR experiments were carried out as described 1H NMR resonances to respective metabolites  were used to develop three pair-wise classifiers  as described previously Figure S1(0.34 MB DOC)Click here for additional data file.Table S1(0.03 MB DOC)Click here for additional data file.Table S2(0.03 MB DOC)Click here for additional data file.