1. 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

2. 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.

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

4. 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

5. 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

6. 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.

7. 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

8. 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 /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.

9. CMV results CMV viraemia 30.9% Among this group:
CMV VL level median= 1250 copies/ml
(IQR 500 to 4838)
CMV VL ≥ %
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

10. 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

11. 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

12. 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…

13. 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.

14. 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

15. 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

16. 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+

17. 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+

18. 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?

19. 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.

20. 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

21. Questions?