Neuromyelitis optica and pulmonary tuberculosis: a case-control study1

Critique and Revised Study Design Proposal

Prepared for EPI 511 A: Introduction to Epidemiology Instructor: Walter Kukull, PhD Autumn Quarter 2012

By: Jonathan A. Childers and Veatasha H. Dorsey Date: November 27, 2012

I. Critique of Original Article 1. Design and Data Collection The subject study was conducted in Cape Town, South Africa to investigate the potential association between neuromyelitis optica (NMO) and tuberculosis (TB). Cases of NMO are rare and often occur in isolation, but reports have suggested an association with active pulmonary tuberculosis (PTB). Of particular note, a study in Western Cape, South Africa, described six patients with NMO that were found to have active PTB with no sign of TB infection in the central nervous system (CNS).2 To clarify the relationship between NMO and PTB, the study authors conducted a retrospective matched case-control study to investigate the association between PTB exposure and NMO. Given the rarity of NMO, a retrospective matched casecontrol design, despite its limitations, seems like a defensible approach to achieving the authors aims. The authors identified study cases by reviewing medical records of Cape Towns Tygerberg Hospital. Eligible cases included patients 18 years of age or older, admitted to the hospital with a diagnosis of NMO between January 1995 until February 2011, and meeting criteria for NMO as outlined by Wingerchuk et al. in 1999.3 In 2006, revised criteria for diagnosing NMO were published,4 but the study used only the earlier criteria as the revised criteria rely on technical tests that were not readily available at the subject institution. Overall, 18 cases were identified who had been admitted for NMO during the study window, but two were excluded because they did not fully meet diagnostic criteria for NMO. Two more patients were excluded from the study due to insufficient hospital records. The authors also state that they excluded patients with evidence of active TB of the CNS, or positive syphilis serology on the Cerebral Spinal Fluid (CSF), but no further mention or enumeration of such exclusion is provided. In total, 14 cases were carried forward for analysis in the study. The control group included patients admitted to Tygerberg Hospital with a diagnosis of GuillainBarre Syndrome (GBS), based on criteria established in 1985,5 as identified according to hospital records. To ensure similar background opportunity for exposure to TB, controls were chosen from the same geographic area as cases and were matched with cases for sex, age at admission (within 10 years), and date of admission (within 5 years). If multiple potential matching controls were identified for a case, the patient with age most closely matching the case at the time of admission was chosen as the control. The authors note that patients with GBS were selected as controls because onset of GBS is acute, hospital admission is involuntary, and there is no known causal association between GBS and PTB. However, as detailed below, published studies do suggest that such associations may exist between GBS and PTB, and additional studies point to potential confounding relationships between human immuno-deficiency virus (HIV) and GBS and PTB. The selection of the NMO cases probably did not appreciably bias the results of the study. The approach to selecting cases seems reasonable, given the rarity of NMO and the diagnostic criteria operable in the subject institution. The authors report that, for each case, data was collected on age, sex, date of onset of GBS symptoms, clinical features, visual evoked potentials, NMO analysis, presence or absence of a PTB Childers and Dorsey Page 1

diagnosis and the temporal relation to diagnosis of NMO, neuroimaging findings (myelography, brain computed tomography, or MRI of the brain and spinal cord), and HIV serology (although HIV status was not documented for two cases). The specific NMP screening process is not elucidated, but presumably the clinical features, visual evoked potentials, CSF analysis, and neuroimaging findings were used to confirm diagnosis of NMO. We assume that the process correctly identified eligible and ineligible NMO cases per the 1999 criteria. The exclusion of two potential cases due to inadequate records seems defensible, although some cases with missing information (HIV serology) were still included in the study. There does seem to have been the opportunity for bias in the selection of the controls to significantly affect the perceived association between PTB and NMO. By limiting the group of controls to patients with GBS, the study predisposes interpretation of the relationship between PTB and NMO to error according to any underlying relationship between GBS and PTB. While the authors state that no such relationship is known, past literature has suggested a potential association between GBS and PTB 6 , with some studies suggesting potential causality between PTB and GBS pathogenesis7 including potential invasion of tubercle baccili (tuberculous polyradiculitis) into nerve roots. 8 If such a causal relationship existed, it would be expected to increase the likelihood of finding PTB in the controls, thus yielding an overly conservative estimate of the association between HMO and PTB. Beyond the potential direct association between GBS and PTB, studies have also suggested there may be relationships between GBS and HIV,9 and PTB and HIV10 (including active PTB undiagnosed due to HIV11,12), that could substantially affect findings regarding the association of interest in the study. The authors even mention the well-described association between GBS and HIV, implying that the relationship could increase the odds of finding TB in the control group leading to a more conservative estimate of the association between PTB and NMO. Yet, the authors do not acknowledge past study findings that symptoms may not be a useful screen for PTB in patients with HIV, and that diagnosis of active PTB may be delayed in patients with HIV (Wood, et al.). Based on those findings: since HIV prevalence was higher in the GBS controls than in the NMO cases, and PTB status was not tested in asymptomatic patients, misclassification of PTB due to HIV may have led to overestimation of the association between NMO and PTB. Closely related to the selection issues discussed above, the collection of information in the subject study could have contributed to bias in the results. To allow accurate estimation of PTB exposure and adjustment for potential confounding effects of HIV, diagnostic information regarding both HIV and PTB status would have been collected for each study subject. However, HIV test results were only tabulated for 12 of the 14 cases (one was positive) while being tabulated for all 14 controls (6 were positive). For PTB, past results of positive PTB diagnosis (based on clinical features plus compatible chest radiography and/or sputum microscopy for acid-fast bacilli) were tabulated for 11 of 14 cases, and results were not tabulated for the three remaining cases with no evident indications of PTB (1 was HIV-positive). Among the controls, only 2 of 14 patients had recorded diagnoses of PTB (based on the same tests as in the cases), and investigations Childers and Dorsey Page 2

were not conducted to exclude active PTB among the other 12 patients (5 of which were HIV-positive). Irrespective of past findings that HIV could have led to delayed PTB diagnosis in some of these patients, the authors assert that tests (e.g. sputum microscopy or Mycobacterium tuberculosis culture) were not performed to exclude active PTB because it is not standard practice in their institution to investigate asymptomatic patients for TB. In short, the overall study results may have been biased by the lack of information on HIV status and/or PTB status for some cases and controls. By not testing two cases for HIV, the documented prevalence of HIV among the cases may have been artificially inflated, allowing the authors to dismiss as insignificant the differential prevalence in HIV between the cases (1 of 12, as tested) and controls (6 of 14). Furthermore, by not testing previously undiagnosed and asymptomatic patients for PTB, it is possible that PTB status was misclassified for some study subjects. Especially among HIV-positive patients, subjects previously undiagnosed and asymptomatic for PTB may have been active PTB cases. Overall, there was one case that tested positive for HIV and was assumed to be PTB-negative due to lack of past diagnosis or symptoms, and there were 5 controls that tested positive for HIV and were similarly assumed to be PTBnegative. Because this situation was more common among the controls, it is possible that false negative classification of PTB in the controls may have led to overestimation of the association between NMO and PTB. To limit bias and confounding based on background opportunity for PTB exposure, the study authors used a matched case-control design, matching GBS controls to NMO cases on a few factors including residence geography, sex, age, and admission dates. The criteria for matching, described above, could have been more rigorous, however the number of cases and controls is already so low that further narrowing the pool of study subjects would have been counter-productive. The authors comment on the very small sample size, but not the limited power, of this study. From the confidence interval provided for the calculated odds ratio (OR), the desired alpha for the study was 5%. Given that alpha, the study design with 14 matched pairs, and the measured 14.3% prevalence of PTB among controls, calculations indicate that the study only had a 32.8% power to detect the reported OR of 4.6.13 Overall, it seems the sample size for the study was too small. 2. Data Analysis and Results The authors do not report adjusting for confounding in the study analysis. As mentioned above, the study matched cases and controls on a variety of factors, so presumably the authors believed that several important confounding elements were addressed by the study design. The authors also collected a variety of data (specified above) regarding the medical condition of cases, which may have been useful for investigating differential association between PTB and particular NMO symptoms. However, it is not specified whether factors other than the main predictor and outcome variable were included in the studys statistical models. Childers and Dorsey Page 3

Due to missing data regarding HIV status and/or active PTB status in study subjects, it was not possible to adequately control for HIV as a confounder in the study. Controlling for the confounding effect of HIV status would have required coding the HIV and PTB status for all cases and controls. Since such data was not recorded, we assume no statistical adjustment was conducted regarding HIV status, despite the documented associations between HIV and the outcomes and exposure of interest. Hinting at the importance of HIV as a potential confounder, the authors do make note of the respective prevalence of HIV in the tested cases (1/12 = 0.083) and the tested controls (6/14 = .429). The authors assert that HIV prevalence in the two groups was not significantly different, citing a p-value of .089. However, we were not able to reproduce this finding. Using a Two Proportion Test, we find that the actual p-value was only .052.14 Furthermore, if the two cases with unreported HIV status in fact did not have HIV, then the prevalence of HIV in the cases (1/14 = .071) and the controls (.089) would have been significantly different, with p = .032. Finally, the authors describe the possible presence of tuberculosis meningitis (TBM) as a potential confounder regarding the association between PTB and NMO. TBM is described as a common cause of spinal cord or cranial nerve involvement in neural disease, and TBM often occurs in association with PTB. While none of the study subjects presented clinical or radiological findings suggesting TBM, the lack of CSF bacteriological studies (Ziehl-Neelsen microscopy, culture or nucleic acid assays) in the medical records made it impossible for the authors to exclude confounding presence of TBM with a higher degree of certainty. Overall, while the authors cite an OR of 4.6 (95% confidence interval (C.I.) 1.71 to 15.49) for the matched case control study, we were not able to reproduce that finding. Rather, calculating according to the standard approach for matched case-control studies, the crude OR for the study (as measured) would be have been 10 (95% C.I. 1.42 to 434). Furthermore, illustrating the importance of the HIV and PTB status of each subject: if PTB had been tested for the all of the controls, and if 5 of the HIV-positive controls with no symptoms or diagnosis of PTB had been discovered to actually have active PTB, then the crude OR for the study would been rendered insignificant at a calculated level of 2.33 (95% C.I. 0.53 to 13.98). Pathogenesis in PTB-associated NMO has not been documented, but the authors theorize that the likely causal mechanism would be immune-mediated inflammatory demyelination of the optic nerves and spinal cord triggered by pulmonary infection with M. tuberculosis. They contend that this theory is supported by observations that NMO appears to develop in patients with active (PTB) and by a previous study in rural Africa that found a mean delay of 10 weeks between PTB diagnoses and development of NMO. 15 Accordingly, the authors claim evidence of causal association in the close temporal relationship (ranging from simultaneous up to 12 weeks) between PTB and NMO diagnoses in the study cases. However, complicating the interpretation of causality, the authors also note that TB treatments (rather than active TB) have also been considered as a potential causal factors in NMO. For example, ethambutol (a bacteriostatic antimycobacterial drug used to treat TB) has been documented as causing optic neuritis. 16 To discern whether active PTB or TB treatments were causally associated with NMO would Childers and Dorsey Page 4

seemingly require assessment of the relative risk of NMO among patients treated for PTB and those with undiagnosed/untreated PTB. This is another point indicating that testing and documentation of PTB status in all of the study subjects would be ideal; allowing differential interpretation of the outcomes associated with diagnosed/treated PTB and undiagnosed/untreated PTB. 3. Interpretation and Discussion We agree that the study indicates a higher general prevalence of diagnosed PTB among the NMO cases than in the GBS controls. We are not convinced that the reported OR is correct, or that it necessarily represents the true relationship between PTB and NMO and GBS. The authors make no mention of the potential effects of confounding or bias, only noting that the small sample size may be of concern. However, as discussed above, selection bias in choosing GBS patients as the controls, and bias due to lack of information regarding HIV, PTB, and TBM status in subjects, could play a significant role in affecting the results. The above factors could have led to misclassification; of particular concern would be assigning HIVpositive controls as PTB-negative, when in fact they were PTB-positive and HIV led to delayed diagnosis. Overall, the generalizability of the subject study seems limited. The authors note that there is no known causal relationship between PTB and NMO, and the subject study cannot convincingly address the prospects of TB treatments or TNS as alternative causes of NMO in the cases. The information collected in the study was not inadequate for conclusively determining PTB status in all subjects, or for controlling potential confounding effects of HIV status. In addition, the calculations used to arrive at the study conclusions are unclear. The cited statistics do not seem reproducible. It may be that this irreproducibility is due to inclusion of additional factors as statistical controls/adjustments in the study model, but the authors do not specify such factors or adjustments. Finally, the very small sample size of the study greatly diminished the power of the study to detect a reasonable OR. On the whole, the authors seem to overstate the importance of the findings of the study, in light of the range of limitations discussed above. 4. How will your study be a methodological improvement over the assigned study? To clarify questions regarding the association between NMO and PTB, our proposed retrospective matched case-control study will be designed to pool patients from multiple institutions in order to achieve a substantially larger sample size and significantly increased power. Toward addressing questions of causality, the study will include only cases and controls with records that conclusively address PTB and TNS status. In addition, only subjects with documented HIV status will be included. Finally, the criteria used for matching cases and controls, and factors included for adjustments in statistical analysis, will be clearly documented to increase the reproducibility of our results.

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I. New Research Design A) Specific Aims 1. To conceptualize certain risk factors for Neuromyelitis optica (NMO) a. Evaluate whether the presence of certain bacteria are linked with an increased risk of developing NMO. b. Analyze the occurrence of Mycobacterium tuberculosis bacteria as a causal mechanism in both the cases and controls. 2. To evaluate what association, if any, between pulmonary tuberculosis (PTB) and Neuromyelitis optica (NMO). a. Null hypothesis: There is no association between pulmonary tuberculosis exposure and the incidence of Neuromyelitis optica (NMO). B) Research Design and Approach 1. Disease, Design and Population Base A. Research Design A common form of medical research, the design we will implore in this study is the retrospective case control study. As a rare disease, NMO commonly occurs in isolation with few associations demonstrated throughout the literature. The retrospective case control study is best in looking at incidences of rare disease as it allows us to essentially look back and determine exposure status (possible risk factors) in both the cases and controls. Another study design, retrospective cohort study, could also be used with our data collection methodology (hospital review). However, given that we have a rare disease, our best model is to compare our cases of NMO with controls similar to our cases with the exception of having NMO and determine if a potential risk factor PTB was associated with the disease. Essentially, unlike a retrospective cohort study, we are interested in associating a disease to a risk factor rather than associating a risk factor to the disease. Although retrospective case control studies are more susceptible to bias in comparison to a cohort study, given our disease of interest, we deem the retrospective case control study design is most efficacious in determining an association between PTB and NMO. B. Population Base As with any case control design, the selection of appropriate study groups within our chosen population base remains a fundamental parameter in deciphering a potential association between Childers and Dorsey Page 6

PTB and NMO. Our study location will be Cape Town, South Africa with a population of over 3.5 million people. Cape Town has a tempered climate and relatively large urban centers where the spread of communicable diseases like tuberculosis remains a considerable public health concern. Our 50 cases and 150 controls will be chosen based on hospital records of admissions, between January 2006 and December 2010, at four regional hospitals (Tygerburg, Groote Schuur, Panorama Medi-Clinic and Netcare Blaauwberg) in Cape Town. The chosen hospitals are those with the most rigorous scientific efficacy and advanced medical procedures in the city of Cape Town, with physician specialties in rare autoimmune diseases. The chosen time frame represents the period in which the hospitals began utilizing the revised criteria for diagnosing NMO (Wingerchuk, 2007), with a particular focus on using magnetic resonance imaging (MRI) of the spinal cord and NMO immunoglobulin G (IgG) antibody testing for the presence of NMO. C. Disease Description NMO is a rare inflammatory disease, usually occurring in isolation, which demyelinates the spinal cord and optic nerves. Left untreated, NMO could cause blindness, paralysis, painful spasms and loss of sensation from spinal cord damage. NMO can be reversible, but most attacks are subject to a high relapse rate and can cause permanent blindness and immobility. Given its propensity to affect the spinal cord and optic nerves, NMO is commonly misclassified with numerous CNS disorders, most notably multiple sclerosis (MS). The subtle distinctions between NMO and MS can make clear diagnosis difficult, particularly in retrospective studies in which diagnosis is dependent on historical medical technology and imaging processes. Until relatively recently, the most common approach to discerning distinctions between NMO and MS (and the method used in the original Zatjirua study) involved distinguishing the diseases via MRI imagery. Normal MRI images have a stronger link to NMO, whereas abnormal MRI images are associated with MS. However, in 2006, Wingerchuk et. al. (2007) described that patients with NMO may have additional symptoms not attributable to optic nerve or spinal cord inflammation and may have MS-like brain MRI lesions. Largely as a result of this finding, the Mayo Clinic revised their proposed absolute, major and minor criteria for NMO in 2006 omitting a provision in the original 1999 absolute criteria: no clinical disease outside of the optic nerves and spinal cord. Accordingly, toward diagnostic validation, a recent transformative study on NMO and MS diagnosis discovered a particular antibody NMO-immunoglobulin G (NMO-IgG), present in roughly 70% NMO patients, whereas MS patients do not have the antibody. This study demonstrated that use of a biomarker and biomonitoring methods 17 could provide a more efficacious form of diagnosis and treatment for NMO.

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D. Incidence and Disease Mortality Given that NMO is a rare disease, subject to misclassification with other CNS disorders like MS, the incidence and prevalence rates of NMO are not known (Wingerchuk et al., 2007). It is recognized that NMO can occur in a wide age range, in patients from 5 years old to 90 years old. Relapse of NMO is also common: 70% of NMO patients have relapses after their initial symptoms. According to the Cleveland Clinic, NMO disproportionately affects those of African ancestry, unlike MS, which is more prevalent among Caucasians. NMO also occurs more often in women, particularly the relapsing-form of NMO.18 E. Disease Duration and Treatment The incidence of NMO varies from childhood to adulthood; however, the typical age of onset is about 40 years old, which is about 10 years later than that of MS. Treatment for this disease entails aggressive therapies with plasma exchanges19 and further prevention of future attacks with immunosuppressive medications like prednisone (Chanson JB et. al.). 2. Subject Acquisition and Data Collection Methods A. Selection of Controls Our 150 controls will be selected from patients admitted to the four subject hospitals (Tygerburg, Groote Schuur, Panorama Medi-Clinic and Netcare Blaauwberg) in Cape Town, from January 2006-December 2010, matched with cases via age, sex and time of hospitalization. The original study selected controls with Guillain-Barre Syndrome, an autoimmune disorder less severe than NMO. Because the outcomes in both the cases and controls (diseases) were similar in addition to having a small sample size and roughly one match per case (n=14), the respective power and generalizability of the original study were limited. To increase the power and generalizability of the proposed study, we will select roughly 3 controls per case, admitted proportionately to the same hospital networks, individually matched for sex, age at time of admission (within 5 years) and date of admission (within a range of 3 years). We will exclude from the controls any patients that have even had NMO. The controls will also not include those admitted for any disease with known associations with the risk factor in question (PTB). Finally, selected controls will have confirmed HIV-negative status, and tested PTB status documented in their admission records. By not limiting controls to autoimmune disorders like GBS, we are sensitive to the requirement that the cases and controls must have an equal opportunity for exposure. Assessing exposure factors, it is of note that PTB transmission and exposure occurs through air droplets from Childers and Dorsey Page 8

coughing/saliva, the study population is located in a highly urbanized area of a developing country, there is high prevalence of PTB in the general population of Cape Town20 , and the controls are subject to hospitalization. Given these factors, we argue that the controls have equal opportunity for exposure to PTB, in relation to the cases, irrespective of diagnosis with an autoimmune disorder. B. Loss to Follow-up, Refusal, Non-Response Estimation Given that our methodology involves retrospective review of hospital data, loss to follow-up or participant refusal will not hinder our estimation of the association between PTB and NMO. C. Diagnostic Case Criteria/Control Criteria for Patient Subjects We will use the Mayo Clinic 2006 Revised criteria for NMO to diagnose our cases, according to data existing in hospital records. According to these revised criteria, NMO must be diagnosed by the presence of transverse myelitis and optic neuritis with at least two of the following characteristics: MRI results non-diagnostic for MS A spinal cord lesion extending over three or more vertebral segments (short segment lesions are typical of MS) A serologic test result positive for NMO-IgG. The two supportive characteristics we will use as our diagnostic criteria will be MRI and antibody testing for NMO-IgG given the high specificity in most testing completed (spec. 99%). Selected cases will also have records confirming HIV-negative status, and tested PTB status. Given the rarity of NMO we will rely on the use of prevalent cases for our analysis. The incidence of NMO in our study population is too low to generate an acceptable sample size and study power within a reasonable time period. We are also confident that our chosen methodological design, retrospective review of records from admitted prevalent cases, will limit the effect of recall or survival bias on our results. The eligibility requirements for the controls are as follows: no diagnosis of NMO; proportionately admitted to same 4-hospital network as cases; ddmitted for condition not associated with potential risk factors for NMO (never admitted for optic nerve and/or spinal cord dysfunction); and HIV-negative status and tested PTB status recorded in the hospital records. For cases and controls, we will classify exposure to PTB based on results of sputum microscopy or Mycobacterium tuberculosis culture, as documented in hospital records. 3. Confounding, Effect Modification and Analysis In order to reduce confounding to the degree possible, we will adjust for confounding initially in our study design (a priori) and then by statistical analysis once data is collected. In terms of

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our study design, we will restrict our cases to adults above the age of 18, choose controls from the same (hospital) population as cases, and individually match the cases and controls based upon similar age and gender. After data is collected, we will stratify and adjust for confounding and/or report the odds ratio for effect modification. However, as with any disease, the risk factors and causal mechanisms of interest regarding NMO do not operate in a vacuum. Particularly with rare autoimmune disorders like NMO, complex factors such as genetics may play an important role in disease etiology. Unfortunately, it is beyond the scope of the current study to address highly technical genetic and neurology-related confounders or effect modifiers. In the original methodological design, the control group comprised of only subjects with Gullian-Bare syndrome (GBS), an auto-immune disorder less severe than NMO. However, some articles suggest an association between GBS and TB (Vyravanathan et al.), and between GBS and HIV (T.H. Brannagan et. al.), which has a strong association with the occurrence of PTB. In our revised proposal, we will attempt to overcome potential confounding relationships with HIV by omitting the criteria that our controls have autoimmune disorders (which are commonly associated with HIV) and by only selecting subjects with documented HIV-negative status. Quite possibly the most critical confounding variable in our retrospective hospital review is missing or skewed data. Missing or erroneous data can make it particularly difficult to accurately determine past exposure. Although not something we can definitely control, we will attempt to overcome such problems with data by selecting a large pool of control subjects and reporting missing data in our analysis. As a general approach to statistical analysis, we will investigate the association of interest utilizing McNemars test for matched case-control studies and will further use the Mantel-Haenszel method to examine potential confounding variables. 4. Bias and Chance Effects Modes of bias, both random and systematic, are addressed in our study design. To reduce random error, we have proposed a robust sample size. Further steps have been taken to address systematic error (selection and information bias). Because the study involves retrospective review of hospital records, utilizing the consistent selection criteria in the cases and controls, some subjectbased hazards of selection (subject refusal, loss to follow-up, non-response) are not of concern to the validity of our study. However, since our study design provides that cases and controls be selected from the same study base, this feature in itself is a source of selection bias. Information bias has the potential to be implicit in our revised design, as was the case in the original study. The lack of advanced diagnostic testing of NMO in addition to the absence of test results for all subjects HIV and PTB status, were critical sources of potential diagnostic bias in the Childers and Dorsey Page 10

original study. We have attempted to limit the potential effects of such factors through our clear criteria and standardized protocols for diagnosis of NMO (as specified in the current medical literature) and by ensuring that HIV-negative status and tested PTB status has been documented for all of our study subjects. However, bias in classification of population-based PTB exposure could still readily occur if individuals with NMO or the control diseases that are exposed to PTB are more likely to be admitted to hospitals and diagnosed than individuals with the same diseases that are not exposed to PTB.21 5. Study Limitations Our proposed study is ultimately limited primarily by potential bias in the hospital records (missing data, diagnosis discrepancies) and by potential bias in the selection of our controls. To yield an accurate estimate of the association between NMO and PTB in our cases, we must ensure that the conditions for which controls were admitted did not have known associations with PTB. That screening process, designating particular diseases acceptable or unacceptable for control subjects, inherently biases the level of PTB exposure prevalent among the controls. However, with diligent screening, the level of PTB exposure in the controls will hopefully better represent the level of population-based exposure among seriously ill patients without NMO. From a public health perspective, given the rare but catastrophic character of NMO, it may be important to understand the incidence of PTB-related NMO within the general population. While we will not be able ascertain this metric directly from our proposed study, it will provide an estimate of the association that will be more defensible than those of previous studies. While a cohort study would be required to track incidence PTB-related NMO, our retrospective case-control approach is optimal for investigating a rare disease: its inexpensive, has no-subject response issues, and can readily provide a more comprehensive look into various factors in exposure (subject to record availability). Our study should provide a good estimate of the relative-risk of PTB-related NMO, and could serve as a point of reference for the future design of a more efficient and focused cohort-based study. 6. Scientific Impact In the era of increasing mobilization and expansive drug-resistant strains and mutations of disease with no current cures, retrospective case control studies, especially utilizing hospital cases and controls, have the potential to be very powerful tools in determining causal associations between diseases, especially rare, and certain exposures of interest. We applaud the original articles effort to contribute to the greater body of literature concerning neurological diseases of the spinal cord and optic nerves; conditions to which numerous people around the world are painfully Childers and Dorsey Page 11

afflicted with. We noted particular strengths in the original articles research design and analytical depthmuch of which we continued to integrate in our revised study design. This revised proposal serves to advance the methodological framework, statistical power and generalizability of the original article, incorporating metrics such as increasing sample size, using criteria for diagnosing cases which reflects the scientific advancements borne of the literature, proper selections of controls whose conditions of admittance to the hospital are independent of our exposure and overall more stringent statistical analysis and quality control measurements. C) Sample Size Estimation and Power 1. Information needed To determine the sample size needed for our study, we would need to set the desired alpha level for the study, as well as the desired power (percentage chance) for detecting an OR of particular magnitude. We would also need to know the approximate prevalence of the exposure of interest (PTB) in our controls. For the proposed study, we would use an alpha of 5% and would desire 80% power to detect an OR as low as 3.0. Since we do not know the prevalence of PTB exposure in our controls, the following table shows the sample size that would be required for the study to maintain 80% power detect an OR of 3.0, across a range of potential exposure levels. Table 1: Sample Size required for 80% Power to Detect OR of 3.0 in Proposed Study PTB Exposure in Controls 5% 10% 15% 20% 25% 30% Number of Cases and Controls 107 cases : 321 controls 62 cases : 186 controls 47 cases : 141 controls 41 cases : 123 controls 37 cases : 111 controls 36 cases : 108 controls

Based on the table above, our proposal assumes a PTB exposure level among our controls of slightly less than 15% (similar to the level of exposure in the original study). Our study is conservative in seeking to maintain strong power to detect an OR of only 3.0 (though the original study cited an OR of 4.6), thus the required sample size is about 50 cases and 150 controls. It should be possible to achieve a sample of this by pooling cases of the four study hospitals.

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D) References Zatjirua V, Butler J, Carr J, Henning F. Neuromyelitis optica and pulmonary tuberculosis: a case-control study. International Journal of Tuberculosis and Lung Disease. 2011. Vol. 15, No. 12, pp. 16751679.
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Wingerchuk MD, Hogancamp WF, OBrien PC, Weinshenker BG. The clinical course of neuromyelitis optica (Devics syndrome). Neurology. 1999. Vol. 53, pp. 11071114.
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Wingerchuk DM, Lennon VA, Lucchinetti CF, Pittock SJ, Weinshenker BG. The spectrum of neuromyelitis optica. Neurology. 2007. Vol. 6, pp. 805815.
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Gooze L, Daley C. Tuberculosis and HIV. HIV InSite Knowledge Base Chapter. University of California San Francisco Medical Center. 2003.
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Wood R, Middelkoop K, Myer L, Grant AD, Whitelaw A, Lawn SD, Kaplan G, Huebner R, McIntyre J, Bekker LG. Undiagnosed Tuberculosis in a Community with High HIV Prevalence: Implications for Tuberculosis Control. American Journal of Respiratory and Critical Care Medicine. 2007. Vol. 175, pp. 87-93.
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Boniface R, Mochabela M, Zulliger R, MacPherson P, Nyasulu P. Correlates of Delated Diagnoses among Human Immunodeficiency Virus-Infected Pulmonary Tuberculosis Suspects in a Rural HIV Clinic, South Africa. Tuberculosis Research and Treatment. 2012. Vol. 2012, pp. 1-7.
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Pronyk RM, Makhubele MB, Hargreaves JR, Tollman SM, Hausler HP. Assessing health seeking behaviour among tuberculosis patients in rural South Africa. International Journal of Tuberculosis and Lung Disease. 2001. Vol. 5, pp 619627. Chan RY, Kwok AK. Ocular toxicity of ethambutol. Hong Kong Medical Journal. 2006. Vol. 12, pp. 56 60.
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Chanson JB, de Seze J, Eliaou JF, Vincent T. Immunological follow-up of patients with neuromyelitis optica: Is there a good biomarker? Lupus. 2012 Nov 21. [Epub ahead of print] PubMed PMID: 23172901.
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