If the United States' Human Cloning Prohibition Act of 2003 (H.R. 534) becomes law, American researchers practicing any form of cloning could face up to ten years in prison and a minimum $1 million fine. The bill criminalizes a research procedure, called somatic cell nuclear transfer, that involves removing the DNA from a fertilized egg and replacing it with the DNA of a body (soma) cell. While the procedure could theoretically be used to clone a human being, used therapeutically its great promise lies in yielding a renewable source of stem cells to repair and regenerate tissue damaged by disease or injury. Embryonic stem cells appear most suited to this task, but some researchers are finding that adult stem cells could perform similar duties in certain tissues. And adult stem cells, it appears, are responsive to genetic manipulation. H.R. 534 does not threaten researchers working with adult stem cells.The precise origin of adult stem cells is unclear, though some propose that they are “set aside” during embryonic development and sequestered in mature tissue. These cells, which can make identical copies of themselves or give rise to specialized cells, serve primarily to replace damaged or injured cells. Skeletal muscle has a remarkable capacity to regenerate following exercise or injury and harbors two different types of adult stem cells to accomplish the job: satellite cells and adult stem cells that can be isolated as side population (SP) cells. Like embryonic stem cells, the adult cells commit to a certain fate once particular genes are activated.It was thought that only satellite cells could mediate skeletal muscle regeneration until recently, when scientists found that adult stem cells not only participate in muscle tissue regeneration but also spawn satellite cells. A certain population of these stem cells, which are recognized by the cell surface proteins CD45 and Sca1 (stem cell antigen-1), is involved in normal muscle tissue repair, but is only triggered into the muscle cell development pathway by injury. The question then arises: what molecular factors turn these adult stem cells into muscle cells? Now Michael Rudnicki and colleagues have shown that one gene,  Pax7,  plays a crucial role in directing the differentiation of these adult stem cells into skeletal muscle cells.Pax7,In previous studies, Rudnicki's group demonstrated that  Pax7  is required to turn adult stem cells into myogenic cells during regeneration. Here, the researchers worked with mouse models and in vitro experiments to investigate which cell populations  Pax7  targets and how the gene initiates muscle cell formation in injured tissue. They show that CD45:Sca1 cells taken from regenerating muscle in mice lacking the  Pax7  gene could not become muscle cells. And they show that by putting  Pax7  back into CD45:Sca1 cells taken from uninjured muscle, they can generate a population of proliferating myoblasts that readily differentiate into muscle cells. When CD45:Sca1 cells engineered to express  Pax7  proteins were injected into the muscles of mice lacking dystrophin (the protein defective in muscular dystrophy), the cells differentiated, forming dystrophin-expressing muscle cells in the defective muscle. This shows that engineered “donor cells” can differentiate in living tissue and help repair dystrophic muscle. When the researchers injected  Pax7  (using a gene therapy virus) into the damaged muscle of mice lacking  Pax7 , they observed the production of muscle-forming cells that not only gave rise to differentiated muscle cells, but also aided in tissue repair.Pax7Pax7Pax7Pax7Pax7Pax7Pax7The researchers argue that these results “unequivocally establish”  Pax7  as a key regulator of muscle cell differentiation in specific populations of adult stem cells during muscle tissue regeneration. If therapeutic strategies that activate  Pax7  in adult stem cells can turn them into muscle cells, effectively replenishing injured or diseased muscle tissue, there's hope of reversing the debilitating effects of progressive muscle-wasting diseases. Though the clinical efficacy of such an approach will require intensive investigation, the results on these adult stem cells are encouraging—especially in this political climate.Pax7Pax7


Health professionals, says Herxheimer, must share their understanding of the benefits and harms of any treatment with patients and their families

Everything that doctors and other health workers do involves communication about the benefits and harms to be expected from interventions—whether they are therapeutic, diagnostic, or prophylactic. As health-care professionals, we need to share our understanding and perceptions of benefits and harms with patients and their families as fully as we can. We also have to share them with other professionals. When we do so we have to remember that how we personally value particular benefits and harms may well differ from how another person values them.A clinician who recommends an intervention does so in the belief that its benefits outweigh the harms that it can cause. In most consultations there is little time in which to explain in detail what these benefits and harms are, or to find out what the patient thinks about them. Moreover, most clinicians are not trained or practised at describing and explaining benefits and harms clearly to patients, and much of the time they also lack important information about these aspects.“Risk” Versus “Harm” The problems begin with the word “risk”. Very often people use it when they mean “harm”, and this causes ambiguities and confusion. The widely used expression “benefit/risk ratio” is meaningless—no such ratio exists. Before a decision is made to use an intervention, its benefits and harms must be weighed, ideally by the clinician and the patient together. Other advantages and disadvantages, such as convenience and cost, may also be relevant. This analysis requires use of the same dimensions for considering both benefits and harms. These dimensions have not been generally recognised or taught, though they seem obvious enough.
 Health professionals need to share their understanding of harms and benefits with patients and their families (Illustration: Margaret Shear, Public Library of Science) In this context any benefit or harm has four dimensions (see sidebar). The clinician is expected to know or find out about the nature and probability of each benefit and harm, and how to maximise benefits and minimise harms. A great many clinicians do not meet this expectation, and often that is not their fault. But only patients can say how they regard the hoped for benefits and the possible harms, though they may need help to think clearly about them. Clinicians should identify how much the benefits matter to their patient—for example, are the benefits of taking a medicine or having an operation “worth the trouble”?—and whether a specific harm is particularly threatening or would be intolerable to that particular patient. People's fears, wishes, and priorities differ greatly and unpredictably. The deepening of the voice that occurs with long-term use of tamoxifen for breast cancer, and that is usually irreversible, is an example of a side effect that prescribers, manufacturers, and drug regulators have considered trivial and have largely ignored. While this side effect does not bother most women, for professional or keen amateur singers it is a disaster—it can rob them of what they enjoy most. A patient who is offered a treatment with serious implications needs time and encouragement to think, and to talk to other people, before making a decision. Three major issues are important in helping patients with decision-making: obtaining reliable information about benefits and harms, effectively communicating probabilities to the patient, and determining what to do to reverse or mitigate harmful effects when they occur.
“Risk” Versus “Harm”
The problems begin with the word “risk”. Very often people use it when they mean “harm”, and this causes ambiguities and confusion. The widely used expression “benefit/risk ratio” is meaningless—no such ratio exists. Before a decision is made to use an intervention, its benefits and harms must be weighed, ideally by the clinician and the patient together. Other advantages and disadvantages, such as convenience and cost, may also be relevant. This analysis requires use of the same dimensions for considering both benefits and harms. These dimensions have not been generally recognised or taught, though they seem obvious enough.In this context any benefit or harm has four dimensions (see sidebar). The clinician is expected to know or find out about the nature and probability of each benefit and harm, and how to maximise benefits and minimise harms. A great many clinicians do not meet this expectation, and often that is not their fault. But only patients can say how they regard the hoped for benefits and the possible harms, though they may need help to think clearly about them. Clinicians should identify how much the benefits matter to their patient—for example, are the benefits of taking a medicine or having an operation “worth the trouble”?—and whether a specific harm is particularly threatening or would be intolerable to that particular patient. People's fears, wishes, and priorities differ greatly and unpredictably.The deepening of the voice that occurs with long-term use of tamoxifen for breast cancer, and that is usually irreversible, is an example of a side effect that prescribers, manufacturers, and drug regulators have considered trivial and have largely ignored. While this side effect does not bother most women, for professional or keen amateur singers it is a disaster—it can rob them of what they enjoy most.A patient who is offered a treatment with serious implications needs time and encouragement to think, and to talk to other people, before making a decision. Three major issues are important in helping patients with decision-making: obtaining reliable information about benefits and harms, effectively communicating probabilities to the patient, and determining what to do to reverse or mitigate harmful effects when they occur.Explaining Uncertainties and Probabilities Innumeracy is very widespread. Many people cannot handle percentages, and most are unclear about the meaning of “relative risk”, “absolute risk”, “odds ratio”, and “number needed to treat”, including many clinicians who want to tell their patients about the likelihood of benefits or harms [ 1 ]. Gigerenzer has shown that information on outcomes presented as natural frequencies (for example, “a one in five chance”) is much easier to understand than information expressed in probabilities (for example, “a 20% chance”) [ 2 ]. The reason, he suggests, is that “natural frequencies result from natural sampling, the process by which humans and animals have encountered information about risks during most of their evolution” ([ 1 ], p. 48–49). So, to put it in its simplest form, it is most effective to say to a patient “this treatment is effective in eight patients out of ten”, or “this drug causes nausea or vomiting in three people out of every ten who use it”. The Four Dimensions of Any Benefit or Harm
 Its  nature , described by its quality, its intensity, and its time course (onset, duration, and reversibility). The  probability  that it will occur. Its  importance to the person experiencing it.
 How the benefit can be  maximised , or the harm  prevented or minimised.
Explaining Uncertainties and Probabilities
Innumeracy is very widespread. Many people cannot handle percentages, and most are unclear about the meaning of “relative risk”, “absolute risk”, “odds ratio”, and “number needed to treat”, including many clinicians who want to tell their patients about the likelihood of benefits or harms [ 1 ]. Gigerenzer has shown that information on outcomes presented as natural frequencies (for example, “a one in five chance”) is much easier to understand than information expressed in probabilities (for example, “a 20% chance”) [ 2 ]. The reason, he suggests, is that “natural frequencies result from natural sampling, the process by which humans and animals have encountered information about risks during most of their evolution” ([ 1 ], p. 48–49). So, to put it in its simplest form, it is most effective to say to a patient “this treatment is effective in eight patients out of ten”, or “this drug causes nausea or vomiting in three people out of every ten who use it”.The Four Dimensions of Any Benefit or Harm
 Its  nature , described by its quality, its intensity, and its time course (onset, duration, and reversibility). The  probability  that it will occur. Its  importance to the person experiencing it.
 How the benefit can be  maximised , or the harm  prevented or minimised.
The Four Dimensions of Any Benefit or Harm
Its  nature , described by its quality, its intensity, and its time course (onset, duration, and reversibility). The  probability  that it will occur. Its  importance to the person experiencing it.
 How the benefit can be  maximised , or the harm  prevented or minimised.Its  nature , described by its quality, its intensity, and its time course (onset, duration, and reversibility).natureThe  probability  that it will occur.probabilityIts  importance to the person experiencing it.importance to the person experiencing it.How the benefit can be  maximised , or the harm  prevented or minimised.maximisedprevented or minimised.Obtaining Reliable Information The effectiveness of an intervention (the extent to which a treatment produces a beneficial effect when implemented under the usual conditions of clinical care for a particular group of patients) is most readily estimated from controlled clinical trials. With the rise of evidence-based medicine, there are now many more critical analyses, systematic reviews, and meta-analyses of the best evidence, as in the rapidly growing Cochrane Database of Systematic Reviews ( www.cochrane.org ). Nevertheless, there are huge gaps—we still lack reliable evidence about many important interventions. Even in the case of common conditions for which many high-quality trials have been published, trial reports have not addressed some elementary questions, for example, on optimal drug dosage and duration of treatment. Almost all drug effects are related to dose [ 3 ], but we rarely learn what the lowest effective dosage is in different circumstances, and how far it is worth increasing the dosage if the effect is insufficient. Details of dose–response relationships are hardly ever published. They are usually studied early in the development of a drug in a relatively small number of volunteers, and are used to decide on the dosages to be used in the major clinical trials that will support the licensing application. They are regarded as internal working data of the company, which is not interested in publishing them. Regulatory agencies do not appear to ask for them or examine them critically. Everybody now habitually uses means and group differences to judge effectiveness, although individuals commonly differ greatly in their sensitivity to both beneficial and harmful effects of drugs. This thoughtless reliance on means and group differences, which ignores an important dimension of evidence, is now embedded in “evidence-based practice”. Marketing departments prefer a “one size fits all” approach: it is hard to sell a drug whose dose may need to be titrated. Another important unanswered question is the variation in response between individuals. Because controlled trials compare treatments they usually report only group means and test their significance. This gives clinicians no help in treating people who are more or less sensitive to the drug than average. Reliable information on harms is for several reasons even harder to get [ 4 ]. Far less research is done to investigate them. Companies do not want to do more work than regulators require, and once they have marketed a drug they hesitate to pay for more research, especially if the results might be inconvenient. Independent public funding hardly exists. Many kinds of harm—often unforeseen and uncommon—need to be first detected and then diligently investigated and analysed. And the available research designs yield less robust evidence than can be obtained for predefined therapeutic effects. Here, too, dose–response data are almost completely lacking.
 We still lack reliable evidence about many important interventions.
 Thus, much of the time prescribers and patients are poorly informed, and have to rely on cautious exploration, common sense, and personal experience. Nevertheless, as Yoon Kong Loke has pointed out, there are certainly some situations at the bedside when it is particularly important to base treatment decisions on as precise an estimate as possible of the balance of benefits and harms [ 5 ]. An example is when there is a narrow margin between benefit and harm, such as giving aspirin to a patient with a stroke who has a past history of gastrointestinal haemorrhage. Another example is when there are several efficacious treatments with differing safety profiles, such as warfarin versus aspirin in a healthy, middle-aged patient with lone atrial fibrillation.
Obtaining Reliable Information
The effectiveness of an intervention (the extent to which a treatment produces a beneficial effect when implemented under the usual conditions of clinical care for a particular group of patients) is most readily estimated from controlled clinical trials. With the rise of evidence-based medicine, there are now many more critical analyses, systematic reviews, and meta-analyses of the best evidence, as in the rapidly growing Cochrane Database of Systematic Reviews ( www.cochrane.org ). Nevertheless, there are huge gaps—we still lack reliable evidence about many important interventions.Even in the case of common conditions for which many high-quality trials have been published, trial reports have not addressed some elementary questions, for example, on optimal drug dosage and duration of treatment. Almost all drug effects are related to dose [ 3 ], but we rarely learn what the lowest effective dosage is in different circumstances, and how far it is worth increasing the dosage if the effect is insufficient.Details of dose–response relationships are hardly ever published. They are usually studied early in the development of a drug in a relatively small number of volunteers, and are used to decide on the dosages to be used in the major clinical trials that will support the licensing application. They are regarded as internal working data of the company, which is not interested in publishing them. Regulatory agencies do not appear to ask for them or examine them critically. Everybody now habitually uses means and group differences to judge effectiveness, although individuals commonly differ greatly in their sensitivity to both beneficial and harmful effects of drugs. This thoughtless reliance on means and group differences, which ignores an important dimension of evidence, is now embedded in “evidence-based practice”. Marketing departments prefer a “one size fits all” approach: it is hard to sell a drug whose dose may need to be titrated.Another important unanswered question is the variation in response between individuals. Because controlled trials compare treatments they usually report only group means and test their significance. This gives clinicians no help in treating people who are more or less sensitive to the drug than average.Reliable information on harms is for several reasons even harder to get [ 4 ]. Far less research is done to investigate them. Companies do not want to do more work than regulators require, and once they have marketed a drug they hesitate to pay for more research, especially if the results might be inconvenient. Independent public funding hardly exists. Many kinds of harm—often unforeseen and uncommon—need to be first detected and then diligently investigated and analysed. And the available research designs yield less robust evidence than can be obtained for predefined therapeutic effects. Here, too, dose–response data are almost completely lacking.We still lack reliable evidence about many important interventions.We still lack reliable evidence about many important interventions.Thus, much of the time prescribers and patients are poorly informed, and have to rely on cautious exploration, common sense, and personal experience. Nevertheless, as Yoon Kong Loke has pointed out, there are certainly some situations at the bedside when it is particularly important to base treatment decisions on as precise an estimate as possible of the balance of benefits and harms [ 5 ]. An example is when there is a narrow margin between benefit and harm, such as giving aspirin to a patient with a stroke who has a past history of gastrointestinal haemorrhage. Another example is when there are several efficacious treatments with differing safety profiles, such as warfarin versus aspirin in a healthy, middle-aged patient with lone atrial fibrillation.Checking Effectiveness and Detecting and Dealing with Harms Doctor and patient need to work together to check that the treatment is as effective as intended, and to detect possible harm promptly. Monitoring can be left to patients if they (or the family) can understand what to watch for and what to do if a problem arises. If not, or if examination or lab tests are necessary, then monitoring and follow-up by a nurse or doctor will need to be arranged. Here is a checklist of points for clinicians—and of course also drug regulators—to consider. (1) When an adverse effect occurs, should the dose be reduced, or the drug changed? (2) If reduced, by how much? (3) Is reducing the dose possible and practicable with the available preparations? (4) How and over what time should the effect of the change be observed and assessed? (5) Should the patient, as well as the clinician, keep the records of adverse effect(s) and their intensity and timing? Such notes can help both the patient and current and future doctors. Medication experiences can remain relevant for life. (6) Should an adverse effect be reported to a local or national adverse drug reaction register? (If in doubt, the answer is yes).
Checking Effectiveness and Detecting and Dealing with Harms
Doctor and patient need to work together to check that the treatment is as effective as intended, and to detect possible harm promptly. Monitoring can be left to patients if they (or the family) can understand what to watch for and what to do if a problem arises. If not, or if examination or lab tests are necessary, then monitoring and follow-up by a nurse or doctor will need to be arranged.Here is a checklist of points for clinicians—and of course also drug regulators—to consider. (1) When an adverse effect occurs, should the dose be reduced, or the drug changed? (2) If reduced, by how much? (3) Is reducing the dose possible and practicable with the available preparations? (4) How and over what time should the effect of the change be observed and assessed? (5) Should the patient, as well as the clinician, keep the records of adverse effect(s) and their intensity and timing? Such notes can help both the patient and current and future doctors. Medication experiences can remain relevant for life. (6) Should an adverse effect be reported to a local or national adverse drug reaction register? (If in doubt, the answer is yes).Conclusion Effective communication about harms and risks is an essential component of care, and it requires learning, preparation, and rehearsal. The onus lies with professionals to persuade and to teach patients to play their part in coming to an informed decision about treatments.
Conclusion
Effective communication about harms and risks is an essential component of care, and it requires learning, preparation, and rehearsal. The onus lies with professionals to persuade and to teach patients to play their part in coming to an informed decision about treatments.


Background Tuberculous meningitis (TBM) is a major global health problem, and it is sometimes difficult to perform a differential diagnosis of this disease from other diseases, particularly partially-treated pyogenic meningitis (PTPM). In an earlier study, we demonstrated the presence of a 30-kD protein antigen in cerebrospinal fluid (CSF) of TBM patients. We have also shown that lymphocytes from CSF of TBM patients respond differently to this antigen than do those from PTPM patients. The purpose of this study was to develop an assay that can discriminate between TBM and PTPM.
Background
Tuberculous meningitis (TBM) is a major global health problem, and it is sometimes difficult to perform a differential diagnosis of this disease from other diseases, particularly partially-treated pyogenic meningitis (PTPM). In an earlier study, we demonstrated the presence of a 30-kD protein antigen in cerebrospinal fluid (CSF) of TBM patients. We have also shown that lymphocytes from CSF of TBM patients respond differently to this antigen than do those from PTPM patients. The purpose of this study was to develop an assay that can discriminate between TBM and PTPM.Methods We developed a cell enzyme-linked immunosorbant assay (Cell ELISA) to quantitatively measure production of antibodies against the 30-kD protein in B cells from CSF of TBM and PTPM patients.
Methods
We developed a cell enzyme-linked immunosorbant assay (Cell ELISA) to quantitatively measure production of antibodies against the 30-kD protein in B cells from CSF of TBM and PTPM patients.Results The cell ELISA yielded 92% (11/12) sensitivity and 92% (11/12) specificity for the differential diagnosis of TBM from PTPM.
Results
The cell ELISA yielded 92% (11/12) sensitivity and 92% (11/12) specificity for the differential diagnosis of TBM from PTPM.Conclusion When induced with the 30-kD protein antigen, B cells derived from CSF of TBM patients respond to IgG production within 24 h while those derived from PTPM patients do not respond.
Conclusion
When induced with the 30-kD protein antigen, B cells derived from CSF of TBM patients respond to IgG production within 24 h while those derived from PTPM patients do not respond.

Background Tuberculous meningitis (TBM) is an infection of the central nervous system (CNS) that is prevalent in both under-developed and developing countries. An increased incidence of TBM has occurred in recent years due to the growing number of people infected with human immunodeficiency virus (HIV). Diagnosis of TBM remains problematic despite many new, advanced diagnostic methods [ 1 , 2 ]. Previous clinical studies have clearly demonstrated that the timing of TBM treatment is the most critical factor in determining the ultimate outcome, which underscores the importance of early diagnosis [ 3 ]. The laboratory confirmation for the diagnosis of TBM is based on the detection of acid-fast bacilli (AFB) in the cerebrospinal fluid (CSF) and by culturing CSF for Mycobacterium tuberculosis bacilli (MTB) [ 4 ]. However, the sensitivity of direct AFB smears from CSF ranges from 5–10% and culturing techniques take 4–6 weeks. It has been recently reported that the staining efficiency of the AFB smear test can be increased to detect up to 50% of TBM cases, but this technique requires a very large amount of CSF [ 5 ]. Clinical as well as CSF features are helpful for diagnosing TBM, but they cannot be used to differentiate TBM from other infectious and non-infectious disorders [ 6 , 7 ]. In particular, clinicians often encounter difficulty when performing a differential diagnosis of TBM from partially-treated pyogenic meningitis (PTPM) cases. Both the results from biochemical and pathological analysis of CSF and the clinical presentation of TBM are often similar to those of PTPM, which results in frequent misdiagnosis. In an earlier study, we reported the presence of a diagnostic 30-kD protein antigen in CSF of confirmed and suspected TBM patients [ 8 ]. Immunological methods such as antibody-capture enzyme-linked immunosorbant assay (ELISA) have been previously used for diagnosing TBM [ 9 ]. The cell ELISA method allows further confirmation of the results obtained by antibody-capture ELISA. Cellular immune function is characterized by the existence of various types of lymphoid cells. As lymphocytes participate in the production of humoral immunity, they may respond to the 30-kD protein antigen in TBM and PTPM patients. We have developed a cell ELISA to study the response of B cells derived from CSF of TBM and PTPM cases following challenge with the 30-kD protein antigen. The purpose of the present study was to evaluate the antibody response to the 30-kD protein antigen in CSF of TBM and PTPM patients by cell ELISA and to determine whether this method may be used in differential diagnosis of TBM from PTPM.
Background
Tuberculous meningitis (TBM) is an infection of the central nervous system (CNS) that is prevalent in both under-developed and developing countries. An increased incidence of TBM has occurred in recent years due to the growing number of people infected with human immunodeficiency virus (HIV). Diagnosis of TBM remains problematic despite many new, advanced diagnostic methods [ 1 , 2 ]. Previous clinical studies have clearly demonstrated that the timing of TBM treatment is the most critical factor in determining the ultimate outcome, which underscores the importance of early diagnosis [ 3 ]. The laboratory confirmation for the diagnosis of TBM is based on the detection of acid-fast bacilli (AFB) in the cerebrospinal fluid (CSF) and by culturing CSF for Mycobacterium tuberculosis bacilli (MTB) [ 4 ]. However, the sensitivity of direct AFB smears from CSF ranges from 5–10% and culturing techniques take 4–6 weeks. It has been recently reported that the staining efficiency of the AFB smear test can be increased to detect up to 50% of TBM cases, but this technique requires a very large amount of CSF [ 5 ].Clinical as well as CSF features are helpful for diagnosing TBM, but they cannot be used to differentiate TBM from other infectious and non-infectious disorders [ 6 , 7 ]. In particular, clinicians often encounter difficulty when performing a differential diagnosis of TBM from partially-treated pyogenic meningitis (PTPM) cases. Both the results from biochemical and pathological analysis of CSF and the clinical presentation of TBM are often similar to those of PTPM, which results in frequent misdiagnosis.In an earlier study, we reported the presence of a diagnostic 30-kD protein antigen in CSF of confirmed and suspected TBM patients [ 8 ]. Immunological methods such as antibody-capture enzyme-linked immunosorbant assay (ELISA) have been previously used for diagnosing TBM [ 9 ]. The cell ELISA method allows further confirmation of the results obtained by antibody-capture ELISA.Cellular immune function is characterized by the existence of various types of lymphoid cells. As lymphocytes participate in the production of humoral immunity, they may respond to the 30-kD protein antigen in TBM and PTPM patients. We have developed a cell ELISA to study the response of B cells derived from CSF of TBM and PTPM cases following challenge with the 30-kD protein antigen. The purpose of the present study was to evaluate the antibody response to the 30-kD protein antigen in CSF of TBM and PTPM patients by cell ELISA and to determine whether this method may be used in differential diagnosis of TBM from PTPM.Methods Patients and sample collection The Central India Institute of Medical Sciences (CIIMS), Nagpur, is a tertiary referral center. CSF was collected from patients who were suspected of having TBM or other infections before they received any treatment. For patients undergoing cranial surgery, analysis of CSF was performed if they were suspected of having meningitis. These patients were already on broad-spectrum antibiotics, such as third-generation cephalosporins and aminoglycosides. To establish a diagnosis of meningitis, 2–5 ml CSF was withdrawn from patients using a lumbar puncture. CSF was then subjected to routine biochemical analysis and pathological analysis including Gram staining, India ink staining, and AFB staining and culturing. One milliliter of CSF was used for the cell ELISA study, and 1 ml was used for detection of the 30-kD protein band by SDS-PAGE analysis in 12 randomly selected TBM and PTPM patients. Diagnosis of TBM and PTPM was based on the criteria described below. Diagnostic criteria 1. Tuberculous Meningitis (TBM) Presence of Mycobacterium tuberculosis in CSF by staining and/or culture, OR Clinical meningitis with the following observations: A. Sub-acute or chronic fever with features of meningeal irritation such as headache, neck stiffness, and vomiting with or without other features of CNS involvement B. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.5), and/or pleocytosis with lymphocytic predominance C. Presence of the 30-kD protein band in CSF on SDS-PAGE analysis D. Good clinical response to antituberculous drugs None of the 12 TBM patients had positive AFB staining. 2. Partially-treated pyogenic meningitis (PTPM) Presence of pathogenic bacteria in CSF by staining and/or culture, OR Clinical meningitis with the following observations: A. Fever and/or signs of meningeal irritation (patients who have undergone cranial surgery to treat tumor(s), stroke, or head injury and who have received antibiotics), OR High fever and/or signs of meningeal irritation with or without CNS manifestations (patients who received broad-spectrum antibiotics) B. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.2), and/or pleocytosis with a predominance of polymorphonuclear cells; CSF may resemble that of chronic meningitis patients C. Absence of the 30-kD protein band in CSF on SDS-PAGE analysis D. Good clinical response to broad-spectrum antibiotics 3. Control group Peripheral blood samples from six healthy volunteers were also analyzed and included as negative controls. Laboratory studies Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE CSF samples obtained from confirmed and suspected TBM cases were subjected to SDS-PAGE. SDS-PAGE was performed with a vertical slab gel electrophoresis system (Broviga, India) using the standard Laemmali method (10). A 4% stacking gel and 10% running gel were used. Electrophoresis was carried out at 250 volts/50 mAmps. Gels were developed by staining with Coomassie brilliant blue GR-250 and the protein profiles were then studied. Band size (i.e., molecular weight) was estimated using molecular weight markers (Genei, Bangalore, India) in a parallel lane. Antigen (30-kD) preparation Following separation of proteins from CSF of confirmed TBM patients (AFB-positive) by SDS-PAGE, the 30-kD protein band was sliced out of the gel and pre-equilibrated in elution buffer (0.15 M phosphate-buffered saline [PBS], pH 7.4) and then electro-eluted in a whole gel eluter system (Biotech, India) for 90 min at 30 volts (11). The sample was then harvested from the unit and dialyzed against PBS and the protein content was measured using a Bio Lab KIT. Protein purity was checked using native PAGE and was then used to evaluate the antibody response of B cells derived from CSF of TBM and PTPM patients. Preparation of CSF Cells One milliliter of CSF collected from TBM and PTPM patients was centrifuged at 400 rpm for approximately 20 min. The supernatant was then discarded and the cell pellet was washed two times with PBS and then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum. Preparation of Blood Cells Heparinized blood samples were obtained from six healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by standard Ficoll-Hypaque gradient centrifugation. PBMCs were dissolved in PBS and centrifuged at 400 rpm for approximately 15–20 min, and the PBMCs were then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum. Cell ELISA Flat-bottomed, 96-well ELISA plates were coated with 10 μg 30-kD antigen/ml diluted in PBS (pH 7.2). Following overnight incubation, the plates were washed with PBS and then coated with 5% BSA-PBS for 4 h. The plates were then washed five times with PBS. Two-hundred μl of the cell preparation derived from CSF of patients with TBM or PTPM were then added to the wells and coated. Each sample was prepared in duplicate. Plates were maintained overnight at 37°C in 5% CO 2  in a carbon dioxide incubator. The following day, the plates were washed with PBS and horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (1:10,000) was then added to the plates. After a 2-hr incubation at 37°C, the plates were washed again with PBS and 100 μl tetramethylbenzidine (TMB)/H 2 O 2  were added. The TMB/H 2 O 2  served as a substrate for HRP. After a 15-min incubation, 100 μl stop solution (2.5 N sulphuric acid) were added and the plates were then read with an ELISA reader at 450 nm (12).
Methods
Patients and sample collection The Central India Institute of Medical Sciences (CIIMS), Nagpur, is a tertiary referral center. CSF was collected from patients who were suspected of having TBM or other infections before they received any treatment. For patients undergoing cranial surgery, analysis of CSF was performed if they were suspected of having meningitis. These patients were already on broad-spectrum antibiotics, such as third-generation cephalosporins and aminoglycosides. To establish a diagnosis of meningitis, 2–5 ml CSF was withdrawn from patients using a lumbar puncture. CSF was then subjected to routine biochemical analysis and pathological analysis including Gram staining, India ink staining, and AFB staining and culturing. One milliliter of CSF was used for the cell ELISA study, and 1 ml was used for detection of the 30-kD protein band by SDS-PAGE analysis in 12 randomly selected TBM and PTPM patients. Diagnosis of TBM and PTPM was based on the criteria described below.
Patients and sample collection
The Central India Institute of Medical Sciences (CIIMS), Nagpur, is a tertiary referral center. CSF was collected from patients who were suspected of having TBM or other infections before they received any treatment. For patients undergoing cranial surgery, analysis of CSF was performed if they were suspected of having meningitis. These patients were already on broad-spectrum antibiotics, such as third-generation cephalosporins and aminoglycosides.To establish a diagnosis of meningitis, 2–5 ml CSF was withdrawn from patients using a lumbar puncture. CSF was then subjected to routine biochemical analysis and pathological analysis including Gram staining, India ink staining, and AFB staining and culturing. One milliliter of CSF was used for the cell ELISA study, and 1 ml was used for detection of the 30-kD protein band by SDS-PAGE analysis in 12 randomly selected TBM and PTPM patients. Diagnosis of TBM and PTPM was based on the criteria described below.Diagnostic criteria 1. Tuberculous Meningitis (TBM) Presence of Mycobacterium tuberculosis in CSF by staining and/or culture, OR Clinical meningitis with the following observations: A. Sub-acute or chronic fever with features of meningeal irritation such as headache, neck stiffness, and vomiting with or without other features of CNS involvement B. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.5), and/or pleocytosis with lymphocytic predominance C. Presence of the 30-kD protein band in CSF on SDS-PAGE analysis D. Good clinical response to antituberculous drugs None of the 12 TBM patients had positive AFB staining. 2. Partially-treated pyogenic meningitis (PTPM) Presence of pathogenic bacteria in CSF by staining and/or culture, OR Clinical meningitis with the following observations: A. Fever and/or signs of meningeal irritation (patients who have undergone cranial surgery to treat tumor(s), stroke, or head injury and who have received antibiotics), OR High fever and/or signs of meningeal irritation with or without CNS manifestations (patients who received broad-spectrum antibiotics) B. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.2), and/or pleocytosis with a predominance of polymorphonuclear cells; CSF may resemble that of chronic meningitis patients C. Absence of the 30-kD protein band in CSF on SDS-PAGE analysis D. Good clinical response to broad-spectrum antibiotics 3. Control group Peripheral blood samples from six healthy volunteers were also analyzed and included as negative controls.
Diagnostic criteria
1. Tuberculous Meningitis (TBM) Presence of Mycobacterium tuberculosis in CSF by staining and/or culture, OR Clinical meningitis with the following observations: A. Sub-acute or chronic fever with features of meningeal irritation such as headache, neck stiffness, and vomiting with or without other features of CNS involvement B. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.5), and/or pleocytosis with lymphocytic predominance C. Presence of the 30-kD protein band in CSF on SDS-PAGE analysis D. Good clinical response to antituberculous drugs None of the 12 TBM patients had positive AFB staining.
1. Tuberculous Meningitis (TBM)
Presence of Mycobacterium tuberculosis in CSF by staining and/or culture, ORClinical meningitis with the following observations:A. Sub-acute or chronic fever with features of meningeal irritation such as headache, neck stiffness, and vomiting with or without other features of CNS involvementB. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.5), and/or pleocytosis with lymphocytic predominanceC. Presence of the 30-kD protein band in CSF on SDS-PAGE analysisD. Good clinical response to antituberculous drugsNone of the 12 TBM patients had positive AFB staining.2. Partially-treated pyogenic meningitis (PTPM) Presence of pathogenic bacteria in CSF by staining and/or culture, OR Clinical meningitis with the following observations: A. Fever and/or signs of meningeal irritation (patients who have undergone cranial surgery to treat tumor(s), stroke, or head injury and who have received antibiotics), OR High fever and/or signs of meningeal irritation with or without CNS manifestations (patients who received broad-spectrum antibiotics) B. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.2), and/or pleocytosis with a predominance of polymorphonuclear cells; CSF may resemble that of chronic meningitis patients C. Absence of the 30-kD protein band in CSF on SDS-PAGE analysis D. Good clinical response to broad-spectrum antibiotics
2. Partially-treated pyogenic meningitis (PTPM)
Presence of pathogenic bacteria in CSF by staining and/or culture, ORClinical meningitis with the following observations:A. Fever and/or signs of meningeal irritation (patients who have undergone cranial surgery to treat tumor(s), stroke, or head injury and who have received antibiotics), OR High fever and/or signs of meningeal irritation with or without CNS manifestations (patients who received broad-spectrum antibiotics)B. CSF findings showing increased proteins, decreased glucose (CSF:blood glucose ratio <0.2), and/or pleocytosis with a predominance of polymorphonuclear cells; CSF may resemble that of chronic meningitis patientsC. Absence of the 30-kD protein band in CSF on SDS-PAGE analysisD. Good clinical response to broad-spectrum antibiotics3. Control group Peripheral blood samples from six healthy volunteers were also analyzed and included as negative controls.
3. Control group
Peripheral blood samples from six healthy volunteers were also analyzed and included as negative controls.Laboratory studies Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE CSF samples obtained from confirmed and suspected TBM cases were subjected to SDS-PAGE. SDS-PAGE was performed with a vertical slab gel electrophoresis system (Broviga, India) using the standard Laemmali method (10). A 4% stacking gel and 10% running gel were used. Electrophoresis was carried out at 250 volts/50 mAmps. Gels were developed by staining with Coomassie brilliant blue GR-250 and the protein profiles were then studied. Band size (i.e., molecular weight) was estimated using molecular weight markers (Genei, Bangalore, India) in a parallel lane. Antigen (30-kD) preparation Following separation of proteins from CSF of confirmed TBM patients (AFB-positive) by SDS-PAGE, the 30-kD protein band was sliced out of the gel and pre-equilibrated in elution buffer (0.15 M phosphate-buffered saline [PBS], pH 7.4) and then electro-eluted in a whole gel eluter system (Biotech, India) for 90 min at 30 volts (11). The sample was then harvested from the unit and dialyzed against PBS and the protein content was measured using a Bio Lab KIT. Protein purity was checked using native PAGE and was then used to evaluate the antibody response of B cells derived from CSF of TBM and PTPM patients. Preparation of CSF Cells One milliliter of CSF collected from TBM and PTPM patients was centrifuged at 400 rpm for approximately 20 min. The supernatant was then discarded and the cell pellet was washed two times with PBS and then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum. Preparation of Blood Cells Heparinized blood samples were obtained from six healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by standard Ficoll-Hypaque gradient centrifugation. PBMCs were dissolved in PBS and centrifuged at 400 rpm for approximately 15–20 min, and the PBMCs were then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum. Cell ELISA Flat-bottomed, 96-well ELISA plates were coated with 10 μg 30-kD antigen/ml diluted in PBS (pH 7.2). Following overnight incubation, the plates were washed with PBS and then coated with 5% BSA-PBS for 4 h. The plates were then washed five times with PBS. Two-hundred μl of the cell preparation derived from CSF of patients with TBM or PTPM were then added to the wells and coated. Each sample was prepared in duplicate. Plates were maintained overnight at 37°C in 5% CO 2  in a carbon dioxide incubator. The following day, the plates were washed with PBS and horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (1:10,000) was then added to the plates. After a 2-hr incubation at 37°C, the plates were washed again with PBS and 100 μl tetramethylbenzidine (TMB)/H 2 O 2  were added. The TMB/H 2 O 2  served as a substrate for HRP. After a 15-min incubation, 100 μl stop solution (2.5 N sulphuric acid) were added and the plates were then read with an ELISA reader at 450 nm (12).
Laboratory studies
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE CSF samples obtained from confirmed and suspected TBM cases were subjected to SDS-PAGE. SDS-PAGE was performed with a vertical slab gel electrophoresis system (Broviga, India) using the standard Laemmali method (10). A 4% stacking gel and 10% running gel were used. Electrophoresis was carried out at 250 volts/50 mAmps. Gels were developed by staining with Coomassie brilliant blue GR-250 and the protein profiles were then studied. Band size (i.e., molecular weight) was estimated using molecular weight markers (Genei, Bangalore, India) in a parallel lane.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE
CSF samples obtained from confirmed and suspected TBM cases were subjected to SDS-PAGE. SDS-PAGE was performed with a vertical slab gel electrophoresis system (Broviga, India) using the standard Laemmali method (10). A 4% stacking gel and 10% running gel were used. Electrophoresis was carried out at 250 volts/50 mAmps. Gels were developed by staining with Coomassie brilliant blue GR-250 and the protein profiles were then studied. Band size (i.e., molecular weight) was estimated using molecular weight markers (Genei, Bangalore, India) in a parallel lane.Antigen (30-kD) preparation Following separation of proteins from CSF of confirmed TBM patients (AFB-positive) by SDS-PAGE, the 30-kD protein band was sliced out of the gel and pre-equilibrated in elution buffer (0.15 M phosphate-buffered saline [PBS], pH 7.4) and then electro-eluted in a whole gel eluter system (Biotech, India) for 90 min at 30 volts (11). The sample was then harvested from the unit and dialyzed against PBS and the protein content was measured using a Bio Lab KIT. Protein purity was checked using native PAGE and was then used to evaluate the antibody response of B cells derived from CSF of TBM and PTPM patients.
Antigen (30-kD) preparation
Following separation of proteins from CSF of confirmed TBM patients (AFB-positive) by SDS-PAGE, the 30-kD protein band was sliced out of the gel and pre-equilibrated in elution buffer (0.15 M phosphate-buffered saline [PBS], pH 7.4) and then electro-eluted in a whole gel eluter system (Biotech, India) for 90 min at 30 volts (11). The sample was then harvested from the unit and dialyzed against PBS and the protein content was measured using a Bio Lab KIT. Protein purity was checked using native PAGE and was then used to evaluate the antibody response of B cells derived from CSF of TBM and PTPM patients.Preparation of CSF Cells One milliliter of CSF collected from TBM and PTPM patients was centrifuged at 400 rpm for approximately 20 min. The supernatant was then discarded and the cell pellet was washed two times with PBS and then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum.
Preparation of CSF Cells
One milliliter of CSF collected from TBM and PTPM patients was centrifuged at 400 rpm for approximately 20 min. The supernatant was then discarded and the cell pellet was washed two times with PBS and then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum.Preparation of Blood Cells Heparinized blood samples were obtained from six healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by standard Ficoll-Hypaque gradient centrifugation. PBMCs were dissolved in PBS and centrifuged at 400 rpm for approximately 15–20 min, and the PBMCs were then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum.
Preparation of Blood Cells
Heparinized blood samples were obtained from six healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by standard Ficoll-Hypaque gradient centrifugation. PBMCs were dissolved in PBS and centrifuged at 400 rpm for approximately 15–20 min, and the PBMCs were then diluted in RPMI 11640 tissue culture medium containing 10% fetal calf serum.Cell ELISA Flat-bottomed, 96-well ELISA plates were coated with 10 μg 30-kD antigen/ml diluted in PBS (pH 7.2). Following overnight incubation, the plates were washed with PBS and then coated with 5% BSA-PBS for 4 h. The plates were then washed five times with PBS. Two-hundred μl of the cell preparation derived from CSF of patients with TBM or PTPM were then added to the wells and coated. Each sample was prepared in duplicate. Plates were maintained overnight at 37°C in 5% CO 2  in a carbon dioxide incubator. The following day, the plates were washed with PBS and horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (1:10,000) was then added to the plates. After a 2-hr incubation at 37°C, the plates were washed again with PBS and 100 μl tetramethylbenzidine (TMB)/H 2 O 2  were added. The TMB/H 2 O 2  served as a substrate for HRP. After a 15-min incubation, 100 μl stop solution (2.5 N sulphuric acid) were added and the plates were then read with an ELISA reader at 450 nm (12).
Cell ELISA
Flat-bottomed, 96-well ELISA plates were coated with 10 μg 30-kD antigen/ml diluted in PBS (pH 7.2). Following overnight incubation, the plates were washed with PBS and then coated with 5% BSA-PBS for 4 h. The plates were then washed five times with PBS. Two-hundred μl of the cell preparation derived from CSF of patients with TBM or PTPM were then added to the wells and coated. Each sample was prepared in duplicate. Plates were maintained overnight at 37°C in 5% CO 2  in a carbon dioxide incubator. The following day, the plates were washed with PBS and horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (1:10,000) was then added to the plates. After a 2-hr incubation at 37°C, the plates were washed again with PBS and 100 μl tetramethylbenzidine (TMB)/H 2 O 2  were added. The TMB/H 2 O 2  served as a substrate for HRP. After a 15-min incubation, 100 μl stop solution (2.5 N sulphuric acid) were added and the plates were then read with an ELISA reader at 450 nm (12).Results Detailed clinical data for TBM and PTPM patients are presented in Table  1 . Out of the 12 PTPM patients, two cases harbored microorganisms, which were cultured (gram-positive cocci in one case and gram-negative bacilli in the other case). Among the 12 patients who fulfilled the criteria for TBM (shown in Table  1 ), CSF of all these patients was positive for the 30-kD protein antigen and was negative for AFB. None of the patients had a previous history of extra-CNS tuberculosis. In addition to the patients described in Table  1 , we also tested an additional 700 CSF samples, including 150 from TBM patients. The 30-kD protein antigen was observed in >90% of these TBM patients (data not shown). Figure  1  shows the presence of the 30-kD protein band in the CSF of suspected TBM cases. This band was markedly absent from the CSF of PTPM patients. Table 1 Clinical and CSF Findings for TBM and PTPM Patients CSF Analysis Case No. Age (years)/Sex TLC P % L % Protein (mg/dl) Sugar (mg/dl) CSF:blood sugar ratio Neck Stiffness Duration of fever (weeks) Headache TBM 1* 16/f 30 - 100 83 27 0.32 Present 16 Present 2 8/m 90 5 95 105 30 0.31 Absent 8 Present 3 54/f 25 2 98 47 67 0.54 Absent 3 Present 4 16/f 450 18 82 535 21 0.24 Absent 12 Present 5* 26/m 120 60 38 143 43 0.66 Present 12 Present 6 58/m 14 - 100 68 37 0.44 Present - Absent 7 31/f 180 12 88 203 86 0.53 Absent 1 Present 8 55/f 112 10 90 231 22 0.38 Present 4 Present 9 65/m 150 1 99 131 23 0.33 Present 4 Present 10 56/m 121 12 88 217 41 0.47 Present 2 Present 11 7/f 32 35 65 68 31 0.32 Present 4 Present 12 43/f 60 - 100 97 39 0.48 Present 2 Present PTPM 1 # 38/m 220 90 10 96 26 0.20 Present 1 Present 2 # 63/f 1600 61 38 401 15 0.14 Present 1 Present 3**# 23/m 61 95 5 270 33 0.20 Absent 6 Present 4 +π 25/m 180 88 12 203 86 0.53 Present 1 Present 5 # 48/m 450 77 32 868 32 0.21 Present 8 Present 6 π 14/f 150 84 11 61 38 0.31 Absent 1 Present 7 ψ 52/m 140 92 8 471 12 0.14 Absent - Absent 8 # 56/m 430 80 20 518 17 0.22 Absent - Present 9 ++ψ 6/m 36 82 9 61 107 0.33 Absent - Absent 10 # 62/f 40 73 27 142 25 0.25 Absent - Present 11 # 4/m 50 90 5 71 24 0.19 Absent - Absent 12 π 27/m 200 78 22 131 21 0.18 Absent 2 Present %P- Polymorphs, % L- Lymphocytes. *Pulmonary tuberculosis (on chest x-ray) **Consolidation (on chest x-ray) # Cranial surgery post operative.  ψ  Post Head injury,  π  Presented to CIIMS as PTPM. +  gram-negative bacilli observed,  ++  gram-positive& non-capsulated cocci pairs observed. Figure 1 SDS-PAGE electrophoretogram of CSF from control (lanes B, C, E) and suspected (lane D) TBM subjects. Molecular weight marker is shown in lane A. The arrow indicates the 30-kD band, which represents the 30-kD protein antigen The ELISA absorbance values of IgG to the 30-kD protein antigen in CSF from TBM and PTPM patients are presented in Figure  2 . The cut-off value (OD at 450 nm) for positivity to the 30-kD protein antigen IgG in the control CSF is 0.6. High-titer values for IgG antibody production against the 30-kD protein antigen were observed in 11 out of 12 TBM patients. However, the titer in PTPM patients was much lower than that observed in TBM patients. IgG antibody production (expressed as ELISA absorbance value) ranged from 0.7 to 2.0 for cells derived from CSF of TBM patients, with the exception of case no. 5 (ELISA absorbance value, 0.59), and from 0.05 to 0.38 for cells derived from CSF of pyogenic meningitis cases, with the exception of case no. 4 (ELISA absorbance value, 0.79). The sensitivity of the cell ELISA was 92% and the specificity was 92% for differential diagnosis of TBM from PTPM. No IgG antibodies to the 30-kD protein antigen were produced by PBMCs from six healthy individuals within 48 h of exposure to the 30-kD protein antigen. Figure 2 B Cell response (IgG reactivity) to the 30-kD protein antigen in CSF cells derived from tuberculous meningitis (TBM) and partially-treated pyogenic meningitis (PTPM) patients and peripheral blood cells from control subjects
Results
Detailed clinical data for TBM and PTPM patients are presented in Table  1 . Out of the 12 PTPM patients, two cases harbored microorganisms, which were cultured (gram-positive cocci in one case and gram-negative bacilli in the other case). Among the 12 patients who fulfilled the criteria for TBM (shown in Table  1 ), CSF of all these patients was positive for the 30-kD protein antigen and was negative for AFB. None of the patients had a previous history of extra-CNS tuberculosis. In addition to the patients described in Table  1 , we also tested an additional 700 CSF samples, including 150 from TBM patients. The 30-kD protein antigen was observed in >90% of these TBM patients (data not shown). Figure  1  shows the presence of the 30-kD protein band in the CSF of suspected TBM cases. This band was markedly absent from the CSF of PTPM patients.Clinical and CSF Findings for TBM and PTPM PatientsCSF AnalysisCase No.Age (years)/SexTLCP %L %Protein (mg/dl)Sugar (mg/dl)CSF:blood sugar ratioNeck StiffnessDuration of fever (weeks)HeadacheTBMPTPM%P- Polymorphs, % L- Lymphocytes.*Pulmonary tuberculosis (on chest x-ray) **Consolidation (on chest x-ray)# Cranial surgery post operative.  ψ  Post Head injury,  π  Presented to CIIMS as PTPM.+  gram-negative bacilli observed,  ++  gram-positive& non-capsulated cocci pairs observed.The ELISA absorbance values of IgG to the 30-kD protein antigen in CSF from TBM and PTPM patients are presented in Figure  2 . The cut-off value (OD at 450 nm) for positivity to the 30-kD protein antigen IgG in the control CSF is 0.6. High-titer values for IgG antibody production against the 30-kD protein antigen were observed in 11 out of 12 TBM patients. However, the titer in PTPM patients was much lower than that observed in TBM patients. IgG antibody production (expressed as ELISA absorbance value) ranged from 0.7 to 2.0 for cells derived from CSF of TBM patients, with the exception of case no. 5 (ELISA absorbance value, 0.59), and from 0.05 to 0.38 for cells derived from CSF of pyogenic meningitis cases, with the exception of case no. 4 (ELISA absorbance value, 0.79). The sensitivity of the cell ELISA was 92% and the specificity was 92% for differential diagnosis of TBM from PTPM. No IgG antibodies to the 30-kD protein antigen were produced by PBMCs from six healthy individuals within 48 h of exposure to the 30-kD protein antigen.Discussion During the past decade, several conventional immunoassays including ELISA, dot immunobinding assays, immunoblot assays, and various molecular methods such as the polymerase chain reaction (PCR) have been reported as adjuncts in the diagnosis of TBM [ 4 , 13 - 15 ]. However, difficulties have been encountered when using many of the aforementioned techniques to differentiate TBM from PTPM. CSF TLC, DLC (total and differential leukocyte count), protein, and glucose estimation are helpful parameters for establishing a TBM diagnosis and for differentiating other infectious and non-infectious neurological disorders, but these tests are non-specific and often cannot differentiate TBM from PTPM in patients in whom organisms are not observed. Delays in diagnosis and treatment are regarded as major contributing factors to the high mortality and morbidity of TBM, and any delay in starting appropriate medication for TBM and PTPM worsens the outcome. We previously used SDS-PAGE to demonstrate the presence of a 30-kD protein antigen in the CSF of TBM patients that is specific to  M. tuberculosis  and may be considered to be a diagnostic marker for TBM. In this study, we used this 30-kD protein antigen to evaluate the IgG antibody response of B cells derived from CSF of TBM and PTPM patients and from peripheral blood samples from six healthy volunteers. A cell ELISA was developed for the quantitative measurement of antibody production against the 30-kD protein antigen by these cells. Higher titers of IgG antibody production were observed in TBM patients compared to PTPM patients. The cells obtained from CSF of TBM patients gave an early response, presumably because they were already sensitized against the TBM antigen. However, when challenged with the 30-kD protein antigen, the cells obtained from PTPM patients and healthy volunteers gave a delayed response since they are not sensitized against this antigen. Therefore, an early response on this time scale is indicative of TBM. We have thus shown that cell ELISA is a sensitive technique for the differential diagnosis of TBM from PTPM. This method involves the demonstration of active antibody production by cells, particularly those derived from the affected site [ 16 ]. Previously, we standardized cell ELISA methodology in our laboratory using standard culture filtrate protein of  M. tuberculosis  (H37Rv strain) received from Colorado State University, Fort Collins USA (data not shown). The only limitation of this method is the time period (24–30 h) involved. However, the sensitivity of the test overcomes this drawback since it the only reported method that can discriminate TBM from PTPM. The sensitivity and specificity of IgG antibody in differential diagnosis of TBM from PTPM using the 30-kD protein antigen by cell ELISA was found to be 92% (11/12). We have also demonstrated that antibody production against the 30-kD protein antigen is higher in cells derived from CSF of TBM patients compared to PTPM patients. Various methods have been developed in our laboratory that yield a high specificity and sensitivity for diagnosis of TBM, but a small number of false positive results have been observed in pyogenic meningitis cases, particularly PTPM cases [ 17 , 18 ]. The cell ELISA method developed in our laboratory using the 30-kD protein antigen marker can potentially provide additional information to the treating physician that may enable a differential diagnosis of TBM from PTPM. The cell ELISA method for diagnosing TBM is based on the assumption that local synthesis of humoral antibodies against MTB antigen occurs. Various researchers have shown that CSF-derived cells have a significantly higher proliferation response to purified protein derivative (PPD) in patients with TBM, which is suggestive of an intrathecal immune response [ 11 , 19 ]. Our data can be summarized by the following observations: first, cell ELISA is a useful method for differentiating TBM from PTPM using the 30-kD protein antigen; second, the method of challenging B cells from CSF of suspected TBM patients with the 30-kD protein antigen can be helpful in confirming a TBM diagnosis; and third, the cell ELISA allows several samples to be analyzed simultaneously. Hence, the cell ELISA should be a very useful tool for the differential diagnosis of TBM from PTPM.
Discussion
During the past decade, several conventional immunoassays including ELISA, dot immunobinding assays, immunoblot assays, and various molecular methods such as the polymerase chain reaction (PCR) have been reported as adjuncts in the diagnosis of TBM [ 4 , 13 - 15 ]. However, difficulties have been encountered when using many of the aforementioned techniques to differentiate TBM from PTPM. CSF TLC, DLC (total and differential leukocyte count), protein, and glucose estimation are helpful parameters for establishing a TBM diagnosis and for differentiating other infectious and non-infectious neurological disorders, but these tests are non-specific and often cannot differentiate TBM from PTPM in patients in whom organisms are not observed. Delays in diagnosis and treatment are regarded as major contributing factors to the high mortality and morbidity of TBM, and any delay in starting appropriate medication for TBM and PTPM worsens the outcome.We previously used SDS-PAGE to demonstrate the presence of a 30-kD protein antigen in the CSF of TBM patients that is specific to  M. tuberculosis  and may be considered to be a diagnostic marker for TBM. In this study, we used this 30-kD protein antigen to evaluate the IgG antibody response of B cells derived from CSF of TBM and PTPM patients and from peripheral blood samples from six healthy volunteers. A cell ELISA was developed for the quantitative measurement of antibody production against the 30-kD protein antigen by these cells. Higher titers of IgG antibody production were observed in TBM patients compared to PTPM patients. The cells obtained from CSF of TBM patients gave an early response, presumably because they were already sensitized against the TBM antigen. However, when challenged with the 30-kD protein antigen, the cells obtained from PTPM patients and healthy volunteers gave a delayed response since they are not sensitized against this antigen. Therefore, an early response on this time scale is indicative of TBM.M. tuberculosisWe have thus shown that cell ELISA is a sensitive technique for the differential diagnosis of TBM from PTPM. This method involves the demonstration of active antibody production by cells, particularly those derived from the affected site [ 16 ]. Previously, we standardized cell ELISA methodology in our laboratory using standard culture filtrate protein of  M. tuberculosis  (H37Rv strain) received from Colorado State University, Fort Collins USA (data not shown). The only limitation of this method is the time period (24–30 h) involved. However, the sensitivity of the test overcomes this drawback since it the only reported method that can discriminate TBM from PTPM.M. tuberculosisThe sensitivity and specificity of IgG antibody in differential diagnosis of TBM from PTPM using the 30-kD protein antigen by cell ELISA was found to be 92% (11/12). We have also demonstrated that antibody production against the 30-kD protein antigen is higher in cells derived from CSF of TBM patients compared to PTPM patients.Various methods have been developed in our laboratory that yield a high specificity and sensitivity for diagnosis of TBM, but a small number of false positive results have been observed in pyogenic meningitis cases, particularly PTPM cases [ 17 , 18 ]. The cell ELISA method developed in our laboratory using the 30-kD protein antigen marker can potentially provide additional information to the treating physician that may enable a differential diagnosis of TBM from PTPM.The cell ELISA method for diagnosing TBM is based on the assumption that local synthesis of humoral antibodies against MTB antigen occurs. Various researchers have shown that CSF-derived cells have a significantly higher proliferation response to purified protein derivative (PPD) in patients with TBM, which is suggestive of an intrathecal immune response [ 11 , 19 ].Our data can be summarized by the following observations: first, cell ELISA is a useful method for differentiating TBM from PTPM using the 30-kD protein antigen; second, the method of challenging B cells from CSF of suspected TBM patients with the 30-kD protein antigen can be helpful in confirming a TBM diagnosis; and third, the cell ELISA allows several samples to be analyzed simultaneously. Hence, the cell ELISA should be a very useful tool for the differential diagnosis of TBM from PTPM.Conclusion The presence of a 30-kD protein antigen in CSF of TBM patients indicates that this protein carries the candidate marker antigen which is specific to  M. tuberculosis . We have demonstrated that by using cell ELISA, we can differentiate TBM patients from PTPM patients, which should be helpful for diagnosing TBM. Additionally our results suggest that lymphocytes from CSF of TBM patients when challenged with 30 kD protein give a quick response by producing IgG antibodies when compared with that of PTPM and healthy volunteers. This may be because lymphocytes from TBM patients have already been exposed to 30 kD MTB antigens.
Conclusion
The presence of a 30-kD protein antigen in CSF of TBM patients indicates that this protein carries the candidate marker antigen which is specific to  M. tuberculosis . We have demonstrated that by using cell ELISA, we can differentiate TBM patients from PTPM patients, which should be helpful for diagnosing TBM. Additionally our results suggest that lymphocytes from CSF of TBM patients when challenged with 30 kD protein give a quick response by producing IgG antibodies when compared with that of PTPM and healthy volunteers. This may be because lymphocytes from TBM patients have already been exposed to 30 kD MTB antigens.M. tuberculosisCompeting interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions RSK carried out the study design, data collection, statistical analysis, data interpretation, literature search, and manuscript preparation; NPA, RPK, and RMS assisted in data analysis collection; NHC assisted in data collection, statistical analysis, and data interpretation; HJP participated in the preparation of the manuscript, data interpretation, and study design; GMT provided assistance in preparation of the manuscript, data interpretation, study design, and funds collection; and HFD supervised the study design, statistical analysis, data interpretation, manuscript preparation, and literature search.
Authors' contributions
RSK carried out the study design, data collection, statistical analysis, data interpretation, literature search, and manuscript preparation; NPA, RPK, and RMS assisted in data analysis collection; NHC assisted in data collection, statistical analysis, and data interpretation; HJP participated in the preparation of the manuscript, data interpretation, and study design; GMT provided assistance in preparation of the manuscript, data interpretation, study design, and funds collection; and HFD supervised the study design, statistical analysis, data interpretation, manuscript preparation, and literature search.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: