CMV viraemia and 12-week mortality among hospitalised adults with HIV-associated TB in Khayelitsha Hospital, South Africa: A prospective cohort study A. Ward1, D. Barr2, C. Schutz1, R. Burton,3 A. Boulle1, G. Maartens1, R.J. Wilkinson1, G. Meintjes1 1University of Cape Town, Cape Town, South Africa, 2University of Liverpool, The Wellcome Trust Liverpool Glasgow Centre for Global Health Research, Liverpool, United Kingdom, 3Khayelitsha Hospital, Cape Town, South Africa Good day I am Dr Amy Ward from the university of CT and today I will be presenting: Cytomegalovirus viraemia and 12 week mortality among hospitalised adults with HIV-associated tuberulosis in Khayelitsha, South Africa: A prospective cohort study

Background HIV-TB: High mortality in hospitalised patients despite treatment 15-50%(1,2) Cytomegalovirus (CMV) organ disease demonstrated post-mortem(3) CMV infection is treatable Is CMV viraemia associated with mortality? 1Kumar, IJTLD 2012 2Crump, CID 2012 3Martinson, AIDS 2007 Despite the availability of Anti Retroviral Therapy and of anti- TB therapy, mortality in hospitalised patients with HIV-associated TB remains high ranging from 15-50%. REASONS behind this high mortality rate are not well defined. One hypothesis is that Cytomegalovirus or CMV infection could be contributing to mortality. CMV organ disease is found in autopsy STUDIES of patients dying with severe HIV associated tuberculosis. CMV viraemia may have a negative impact on host immune response to HIV and/or TB bacilli. In other severe immunodeficiency conditions, such as after organ transplant, CMV viraemia is directly related to increased mortality, And When CMV viraemia is found in these transplant patients its is treated with ganciclovir to prevent progression to clinical disease and death. If CMV viraemia causes immunopathology in severe HIV-tuberculosis co-infection, this would suggest similar interventions for reducing mortality could be trialed in this patient group. THEREFORE We looked for an association of CMV viraemia with mortality in patients hospitalised with HIV-associated TB.

Khayelitsha Hospital Participants were recruited from Khayelitsha Hospital, a large district hospital in the township of Khayelitsha on the outskirts of Cape Town

Methods Inclusion criteria Baseline day 0 Age ≥ 18 HIV-infected CD4 ≤ 350 New TB diagnosis (microbiological or clinical) Baseline day 0 Clinical assessment Blood tests including plasma CMV viral load Endpoint: Mortality at 12 weeks on anti-TB therapy HIV-infected adult inpatients with a CD4 of 350 or less and new diagnosis of microbiologically proven or clinical TB were enrolled from January 2014-June 2015 into an prosprective cohort study Baseline characteristics including plasma CMV quantitative PCR were obtained at day 0. The primary endpoint was 12 week mortality on anti-TB therapy

Methods CMV viraemia Analysis Any viral load reported as detectable on the Argene CMV R-gene®platform Analysis Cox proportional hazards models Kaplan-Meier We defined CMV viraemia as any CMV viral load detected on Argene’s CMV R-gene platform, and no CMV viraemia when the viral load was undetectable. The dataset was analysed using Cox proportional hazard models and survival analysis was done using Kaplan-Meir analysis

Time line We screened HIV+ patients whose CD4 counts at last assessment was 350 or less and enrolled those who either had a new microbiologically confirmed diagnosis of TB or were clinically suspected of having TB on admission. On enrolment a full history and physical examination including fundoscopy to look for CMV retinitis was done. Blood, sputum and urine were collected and a CXR was done to determine baseline characteristics and confirm a TB diagnosis. Patients were followed up daily while in hospital and then telephonically at week 4 and in person or telephonically at week 12 to determine vital status.

Cohort results Age median= 36 (IQR 31 to 44) Sex 51% female On ART 35% ART naïve 21% Previous ART 44% CD4 median= 64 (IQR 24 to 117) 348 patients were screened for inclusion. 256 participants were included in the analysis after excluding 84 patients who did not in fact have a diagnosis of TB, those whose CD4 was >350 and 8 patients for which we did not have CMV data 5 patients were lost to follow up. The median age of the cohort was 36 years and just over half were female sex. At enrolment only 35% of patients were on ART, 21% were ART naïve and 44% had previously been on ART. The median CD4 was 64

Cohort results HIV log viral load median= 5.2 (IQR 3.6 to 5.8) BMI median= 20.5 (IQR 18.7 to 23.8) TB BC + 101/256 (40.6%) Rif resistance 19/256 (7.4%) The cohorts median HIV VL was log 5.2 And a median BMI or body mass index of 20.5 40.6% of patients had a positive TB blood culture and 7.4% of the cohort had rifampicin resistant TB on baseline TB tests.

CMV results CMV viraemia 30.9% Among this group: CMV VL level median= 1250 copies/ml (IQR 500 to 4838) CMV VL ≥1000 53.2% Almost a third of patients were classified as CMV viraemic and among this group The median CMV viral load was 1250 copies/ml and 53.2% had a CMV VL of 1000 or more

Retinal examinations No cases of CMV retinitis detected All images reviewed by an ophthalmologist Other HIV-related retinal pathologies found in a subset of patients Not one case of CMV retinitis was detected on retinal examination with a panoptic fundoscope and all the images were reviewed by an ophthalmologist But a range of other HIV-related retinal pathologies were wound in a subset of patients Examples can be seen here and include HIV retinopathy

12-week mortality results All 23.0% CMV+ 38.0% CMV- 17.8% p= 0.008 by Fisher exact test The resluts for the primary outcome of week 12 mortality are seen in this table. The overall 12 week mortality for the cohort was 23%. 38% of those with CMV viraemia had died by week 12 compared to 17.8% of those who did not have a detectable CMV Viral load

Risk factors for 12-week mortality Cox Proportional Hazards Regression models Univariate HR Lower 95% CI Upper 95% CI p value Age, per 10 years 1.75 1.41 2.19 <0.001 Male sex 0.84 0.50 0.505 Decreasing CD4, per 50 cellsx106/L 1.27 1.04 1.56 0.017 HIV viral load, Log10 copies/mL 0.90 0.80 1.03 0.117 TB blood culture positive 1.48 0.88 2.49 0.138 Decreasing Albumin, per 5 g/L 1.37 1.09 1.73 0.006 CMV viraemia 2.09 1.24 3.53 0.004 We wanted to know if this association between CMV viraemia and mortality was independent of other important clinical variables thought to relate to mortality in severe HIV associated tuberculosis. In univariate Cox regression analysis CMV viraemia had a hazards ratio 2 fold greater for 12-week mortality In addition, increasing age, lower CD4 count, and lower albumin were associated with increased mortality. We included these variables , along with HIV viral load, TB blood culture +, and sex in a multivariate model…

Risk factors for 12-week mortality Cox Proportional Hazards Regression models Multivariate: n= 232 with complete observations HR Lower 95% CI Upper 95% CI p value Age, per 10 years 1.70 1.34 2.15 <0.001 Male sex 0.69 0.41 1.19 0.185 Decreasing CD4, per 50 cellsx106/L 1.25 1.00 1.56 0.052 HIV viral load, Log10 copies/mL 0.94 0.82 1.07 0.316 TB blood culture positive 0.56 1.76 0.990 Decreasing Albumin, per 5 g/L 0.93 1.52 0.177 CMV viraemia 1.67 0.95 2.93 0.077 After adjustment for all these variables, CMV viraemia hazard ratio for mortality was decreased and was no longer statistically significant although there was still about a 70% increase in mortality showing a trend that is independent of co-variates, including CD4 count. In the multivariate model increasing age remains a strong predictor for mortality in our cohort.

CMV viral load and 12-week mortality CMV VL (copies/ml) Mortality <1000 33.3% ≥1000 34.1% p=1.0 by Fisher exact test In addition, in those patients that had detectable CMV viraemia, a higher level of viraemia did not appear to confer greater risk of mortality. As seen in this table there was no significant difference in mortality between those with a CMV VL less than1000 compared to those with a VL of 1000 or more. And as seen in this figure there was no real difference in the log CMV VL between those that survived 12 weeks and those that didn’t in other words we found no “dose-response” relationship between CMV status and mortality. *By Wilcoxon Rank Sum test

Results stratified by age Older Age (≥36 years) Younger Age (<36 years) *p Value Mortality 32.8% 14.1% p=0.001 CD4<50 48.5% 37.9% p=0.11 CMV+ 38.0% 26.3% p=0.02 *by Fisher exact test To try and tease out the effect of age on the relationship between CMV viraemia and mortality, we performed an analysis stratified by median age cut off. Those aged 36 years and more had a significantly higher mortality of 32.8% compared to 14.1% of those less than 36 years. While a greater proportion of the older group did have a CD4 count of less than 50, this was not a significant finding BUT in those aged 36 years and above we did see a significantly higher proportion of CMV viraemia compared to the younger group This could indicate that independent of CD4 count there are other factors in this group that are causing immune suppression resulting in an increase in both CMV viraemia and mortality

Survival stratified by CMV status The interaction between age, CMV and mortality was also examined on Kaplan Meier plots. This first graph represents survival for whole cohort. As per the key those with CMV viraemia are seen in the solid line at the bottom and those without in the broken line on top So as stated before CMV viraemia did associate strongly with mortality prior to adjustment for other important variables. Key: CMV - CMV+

Disaggregated by age Key: CMV - CMV+ These graphs represent survival with the cohort divided into older and younger groups by median age cut off. In graph B on the left you can see those aged less than 36 years and there is no significant difference in the mortality of those with CMV viraemia and those without The graph on the right hand side shows those 36 years and older and here we can see the difference in survival between the two CMV groups and mortality did associate with CMV viraemia. Key: CMV - CMV+

Conclusions (1) CMV viraemia association with mortality reduced after adjusting for relevant co-variates No “dose response” between CMV and mortality Age ≥ 36 years:  Mortality  CMV viraemia In conclusion for this hospitalised HIV-TB coinfected adult cohort from Khayelitsha we found that: CMV viraemia was associated with higher mortality, but the strength of association was reduced after adjusting for potential confounders and was no longer statistically significant No dose response was observed between CMV viral load and mortality risk Those patients who were 36 years and above had a higher mortality and were more likely to have CMV viraemia, both findings perhaps reflecting premature ageing of the immune system Premature ageing immune system?

Conclusions (2) CMV viraemia likely a marker immunodeficiency, rather than a direct contributor to mortality Larger studies needed to examine trend So CMV viraemia is likely a marker of more severe immunodeficiency rather than a direct contributor to mortality, but larger studies are required to address this question as we did note a trend towards increased mortality in those with CMV viraemia after controlling for relevant co-variates.

Thank you! IDM Rene Goliath Kathryn Wood MK Mpalali Jane Kawadza, Ophthalmology TBH NHLS GSH staff Funders Wellcome Trust South African Dept. of Science and Technology South African MRC KDH Administrative Staff Clinical Staff The Patients I would like to thank my colleagues and collaborators at UCT, funders and the staff and especially the patients at Khayelitsha Hospital Thank you

Questions?