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1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Churchyard GJ, Stevens WS, Mametja LD, et al. Xpert MTB/RIF versus sputum microscopy as the initial diagnostic test for tuberculosis: a cluster-randomised trial embedded in South African roll-out of Xpert MTB/RIF. Lancet Glob Health 2015; 3: e

2 Web appendix Supplement to: Churchyard GJ, Stevens W, Lerole D, et al. Xpert MTB/RIF replacing sputum microscopy as the initial diagnostic test for tuberculosis: a cluster randomised trial embedded in South African roll out Selection of clinics We selected two primary care clinics served by each laboratory. Clinics were assessed for eligibility using data on the number of sputum specimens sent to the National Health Laboratory Service in Clinics were eligible for selection if the estimated number of diagnostic specimens in a three-month period (the planned recruitment period for the study) was greater than 200. For two laboratories, clinics with an estimated threemonth diagnostic specimen load of at least 150 were chosen as there was a paucity of clinics with a load of greater than 200. Xpert MTB/Rif implementation After initiation of testing with Xpert MTB/RIF at each laboratory, the National Priorities Programme of the National Health Laboratory Service monitored stock inventory, machine utilization rate, results (including error and invalid rates) and turn-around-time at each site, using remote connectivity. Periodic refresher training was held for laboratory staff, and directed interventions were conducted in response to high error or invalid rates from specific sites. A national stock-out of Xpert MTB/RIF cartridges occurred in May 2013 (due to a global stock-out), but through careful re-distribution of existing stock, and delaying commencement of enrolment into XTEND in province A, this did not affect the progress of the XTEND study. Data management At enrolment, a unique participant number was generated. Completed case report forms were submitted in an encrypted format via the cell phone network into a central repository database maintained by an independently contracted software company. These data were available on a password protected website for real-time review by investigators, statisticians and data managers. Weekly downloads via secure FTP server to the Aurum Institute database facilitated review of data, and data cleaning. Statistical analysis Primary outcome: mortality Mortality was measured as a risk over the six-month follow-up period (182 days) from enrolment. Participants whose vital status was not known at six months were assumed to be alive. A sensitivity analysis was conducted, excluding participants whose vital status was not known at six months. Secondary outcomes 1. Proportion with a positive (microscopy or Xpert MTB/RIF) index specimen: This analysis was restricted to participants with a positive or negative (microscopy or Xpert MTB/RIF) index specimen result. A scanty microscopy result was assumed to be positive, in accordance with National guidelines. 2. Proportion not started on tuberculosis treatment within 28 days of enrolment among those with positive index specimen This analysis was restricted to participants with a positive (microscopy or Xpert MTB/RIF) index specimen. A scanty microscopy result was assumed to be positive. The outcome was defined as not starting tuberculosis treatment within 28 days of enrolment. If a participant had died during the first 28 days from enrolment and not started on tuberculosis treatment prior to death they were included in the denominator and numerator. 1

3 3. Proportion starting tuberculosis treatment over a six-month period from enrolment. This analysis was based on all participants recruited into the cohort. Starting tuberculosis treatment was measured as a risk over the six-month follow-up period (182 days) from enrolment. Participants who started treatment on the same day as enrolment contributed to the numerator and denominator. Analysis adjusting for randomisation strata All analyses gave each cluster equal weight. A log transformation was applied to the risk/rate for each cluster, where necessary. For clusters with no events, one event was added to all clusters in order that the log transformation could be conducted. The mean and standard deviation of these log risks/rates were used to obtain the geometric mean and associated 95% confidence interval (CI) for each arm of the study. The risk/rate ratio was estimated using linear regression of the log risks/rates on stratum and arm. Prior to randomisation, clusters were grouped into four strata, based on province (A, B, C and D). For the analysis three strata (A, C, and B and D combined) were included in the statistical analysis, as planned at the time of randomisation. B and D were similar with respect to urban/rural status. An approximate standard error for the log (risk ratio) based on geometric means of cluster risks between arms was obtained based on the residual mean square from a two-way analysis of variance of the cluster log prevalence/rate on stratum, study arm and the interaction between stratum and study arm. The 95% CI was calculated from this standard error, using a t-statistic with 14 degrees of freedom. Fully adjusted analysis controlling for randomisation strata and individual-level baseline factors Depending on the outcome to be analysed, Poisson/ logistic regression was used to adjust for confounders at the individual level adopting a two-stage approach(1). In stage 1 the regression model included terms for the adjustment factors and strata, but not study arm. For each cluster the fitted model was used to obtain the ratio of observed to expected (O/E) events, and a log transformation applied to this ratio, where appropriate. In stage 2 linear regression of the log(o/e) on stratum and arm was used to estimate the risk/rate ratio. The variance for the ratio of mean O/E was calculated from the residual mean square from an ANOVA of log(o/e) on stratum, arm and the interaction between stratum and arm. The 95% CI was calculated from this variance, using a t- statistic with 14 degrees of freedom. Analysis of risk factors for death within six months from enrolment A risk factor analysis using logistic regression was conducted to identify individual-level factors, including index specimen result, associated with six-month mortality risk. As the number of outcomes was limited (193 deaths among 4411 participants), controlling for cluster using a fixed-effect in the regression model was not possible. Instead all analyses were adjusted for province. A priori, sex and age group were included in the fullyadjusted model. 1. Hayes RJ, Moulton LH. Cluster randomised trials: Chapman & Hall;

4 Supplementary Table 1: six-month mortality risk by cluster (n=4656) Province Cluster code Deaths n % Microscopy A % A % A % A % B % D % C % C % C % C % Overall % Mean of cluster risks 5.0% Xpert A % A % A % A % B % D % C % C % C % C % Overall % Mean of cluster risks 3.9% 3

5 Supplementary Figure 1: South African algorithm for the investigation of tuberculosis using Xpert MTB/RIF 4

6 Supplementary Figure 2a: South African algorithm for tuberculosis diagnosis in a new case using smear microscopy and culture No previous TB or less than 4 weeks of TB treatment Send 2 sputum specimens for smear microscopy: 1 st spot specimen taken at the health facility under supervision 2 nd early morning specimen HIV- negative or HIV status unknown HIV positive Provide antibiotics. Send 3 rd specimen for smear and culture. If no improvement: Send 3 rd specimen for sputum smear and culture Do chest x-ray. Do chest x-ray. Provide antibiotics. TB on chest x-ray and No TB on chest x-ray TB on chest x-ray and No TB on chest x-ray HCW decision to treat on clinical grounds HCW decision to treat on clinical grounds Aim to provide full course of treatment. Review clinical picture and progress when culture result available for smear negative cases. Aim to provide full course of treatment. Review clinical picture and progress when culture and drug sensitivity available for smear negative cases. 5

7 Supplementary Figure 2b: South African algorithm for investigation of tuberculosis among persons at high risk of or previous treated for tuberculosis using smear microscopy, culture and drug sensitivity testing Previous TB (4 or more weeks of TB treatment) and high risk TB suspect (MDR contact, health care personnel, prisoner) Send 2 sputum specimens: 1 st spot specimen taken at the health facility for sputum smear microscopy 2 nd early morning specimen for sputum smear microscopy, culture and drug susceptibility testing (DST) HIV-negative or HIV status unknown HIV positive Provide antibiotics. Send 3 rd specimen for smear. If no improvement, send 3 rd specimen for sputum smear and do chest x-ray. Review drug sensitivity Do chest x-ray. Provide antibiotics TB on Chest x-ray and No TB on Chest x-ray TB on Chest x-ray and No TB on Chest x-ray HCW decision to treat on clinical grounds HCW decision to treat on clinical grounds Aim to provide full course of treatment. Review clinical picture and progress when culture available for smear negative cases. Review DST. Aim to provide full course of treatment. Review clinical picture and progress when culture available for smear negative. 6

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