The effect of short-course antiretroviral therapy initiated in primary HIV-1 infection on Interleukin-6 and D-dimer levels

Elizabeth HAMLYN1,2, Wolfgang STÖHR3, David A COOPER4, Martin

FISHER5, Giuseppe TAMBUSSI6, Mauro SCHECHTER7, Jose M MIRO8,

Fiona VANOBBERGHEN3,9, Abdel BABIKER3, Jonathan WEBER1, Myra

MCCLURE1, Kholoud PORTER*3 and Sarah FIDLER*1 on behalf of the

SPARTAC Investigators

* contributed equally

1Imperial College London, UK2Kings College Hospital NHS Foundation Trust, London, UK3Medical Research Council Clinical Trials Unit at University College London,

London, UK4Kirby Institute, University of New South Wales, Sydney, Australia5 Brighton and Sussex University Hospitals, Brighton, UK6Ospedale San Raffaele, Milan, Italy7Projeto Praça Onze, Hospital Escola São Francisco de Assis, Universidade

Federal do Rio de Janeiro, Rio de Janeiro, Brazil8Hospital Clinic–Institut d’investigacions Biomèdiques August Pi I Sunyer,

University of Barcelona, Barcelona, Spain9London School of Hygiene & Tropical Medicine, London, UK

Corresponding author:Dr E Hamlyn, Caldecot Centre, Kings College Hospital, London SE5 9RS

Email: e.hamlyn@nhs.net

Telephone: +44 20 3299 1119

Fax: +44 20 3299 3486

Word count: abstract (249), text (1798); Figures (2) plus one appendix figure

Conflicts of interest and sources of funding: The SPARTAC trial was

funded by Wellcome Trust grants WT069598MA and 069598/Z/02/B. These

1

data were presented in part at European AIDS Clinical Society Conference

(EACS), Belgrade, Serbia, 2011

2

ABSTRACT

Objective

Interruption of ART in chronic HIV disease is associated with increased

mortality, predicted by elevations in Interleukin-6 (IL-6) and D-dimer. The

effect of ART interruption in Primary HIV-1 Infection (PHI) on these

biomarkers is unknown.

Methods

Plasma samples from 200 HIV seroconverters enrolled in the SPARTAC trial

of deferred ART (standard of care; SoC), 12 or 48 week ART (ART12 or

ART48, respectively) were analysed for IL-6 and D-dimer at weeks: 0, 12, 16,

48, 52, 60 and 108 after randomisation. Changes in log10-levels from weeks 0-

12 were analysed using linear regression, as were changes from baseline to 4

weeks after stopping ART. Areas under the biomarker-time curves (AUC) to

week 108 were adjusted for baseline values, and compared across all arms.

Results

Median (IQR) baseline IL-6 and D-dimer were 1.45 (0.88, 2.41) pg/mL and

0.34 (0.20, 0.50) μg/L, respectively. At week 12, D-dimer levels were

significantly lower among treated compared to untreated individuals (p<0.001)

whereas IL-6 levels were similar (p=0.23). Within 4 weeks from stopping ART,

IL-6 and D-dimer levels rose by 22% and 18% reaching pre-ART levels. Over

108-week follow-up, there was no difference between arms in IL-6 AUC

(p=0.53), but D-dimer AUC was significantly lower for ART12 and ART48

compared to SoC (overall p=0.008).

Conclusions

Stopping ART in PHI leads to inflammatory biomarker rebound to pre-

treatment levels. However, over 108-week follow-up we found no evidence

that biomarker levels were higher for those interrupting ART, compared to

those remaining ART-naïve, and D-dimer levels were significantly lower.

3

Key words: Primary HIV infection, seroconversion, interleukin-6, D-dimer,

antiretroviral medication, SPARTAC

4

INTRODUCTION

Untreated HIV infection is associated with systemic inflammation, which

persists, to a lesser extent, with viral control on antiretroviral therapy (ART)(1,

2). In the SMART trial, where individuals with chronic HIV infection were

randomly allocated to interruption or continuation of ART, those interrupting

treatment had higher risk of death compared to continuous therapy (3).

Baseline levels of inflammatory and pro-coagulatory biomarkers interleukin-6

(IL-6) and D-dimer were predictive of all-cause mortality and interruption of

ART was associated with significant rises in these biomarkers 4 weeks

following treatment discontinuation (4), providing a likely biological

explanation for the adverse outcomes seen.

Whilst ART interruption in chronic HIV infection is no longer recommended,

recent data from VISCONTI and others (5-7) reporting unexpected rates of

sustained post treatment viral control after discontinuing ART initiated in

Primary HIV Infection (PHI), has led to interest in the use of early transient

ART to achieve functional cure. Few data exist examining the effect of

initiating ART in PHI and subsequently interrupting it on IL-6 and D-dimer.

Using data from participants enrolled in the Short Pulse Anti-Retroviral

Therapy At HIV Seroconversion (SPARTAC) trial, we examine the effect of

transient ART commenced in PHI on these biomarkers.

METHODS

SPARTAC participants with defined PHI within a maximum of 6 months of

seroconversion were randomly allocated to deferred therapy (standard of

care, SoC), 12-week (ART12) or 48-week ART (ART48) (8).

Plasma samples were taken at baseline (randomisation; week 0) and weeks

12, 16, 48, 52, 60, and 108. We analysed stored samples from sites in

Australia, Brazil, Italy, and the UK, where HIV RNA assays had a lower limit of

detection of 50 copies/mL. Plasma IL-6 and D-dimer levels were measured

using Quantikine HS600B IL-6 immunoassay (R&D Systems, Minneapolis,

5

Minnesota, USA) and Innovance D-dimer (Siemens Healthcare Diagnostics,

Tarrytown, New York, USA).

Changes in IL-6 and D-dimer levels from week 0 to week 12 were compared

between participants who started ART and those who did not, using linear

regression models in an as-treated analysis, adjusting for baseline values. We

examined biomarker changes over this period by week 12 HIV RNA level,

categorised as <50, 50-<400, and ≥400 copies/mL and also considering HIV

RNA as a continuous variable.

We compared biomarker levels 4 weeks after stopping ART with pre-ART

levels using linear regression, restricting analysis to participants initiating and

stopping ART at scheduled time-points with <50 copies/mL at ART stop. We

also looked at changes from ART stop to 4 weeks later, and compared

baseline to ART stop. We calculated areas under the biomarker-time curves

(AUC) to week 108, adjusted for baseline values, and compared these across

the 3 trial arms using an intention-to-treat analysis.

For all analyses, values of both biomarkers were log10 transformed. For

presentation of regression results, coefficients were exponentiated to obtain

percent differences.

To enable comparison between SPARTAC and the SMART trial, samples

from 10 SPARTAC participants were shipped to the University of Vermont,

where SMART biomarker evaluations took place. A chemiluminescent assay

for IL-6 (R&D systems Q6000B, Minneapolis, Minnesota, USA) and

immunoturbidimetric assay for D-dimer (STA-R analyszer, Stago, Parsipanny,

NJ, USA) were used. Bland-Altman plots were generated to assess the level

of agreement between measurements at SPARTAC and SMART laboratories.

6

RESULTS

Samples were available for 200/299 (87%) eligible participants (72 SoC, 63

ART12, and 65 ART48). Of 128 randomised to ART, one did not start

treatment. Of the remainder, all but one initiated therapy within 7 days, and

94% within 3 days of randomisation.

Baseline characteristics

Participant characteristics and factors associated with IL-6 and D-dimer levels

at baseline have previously been described (9). Overall, 191 (96%) were

male, median (IQR) age was 34 years (29, 42) and median time from

estimated seroconversion was 11 weeks (7, 14). Median CD4 T-cell count

and HIV-RNA levels were 559 (443, 700) cells/µl and 4.66 (3.84-5.25) log10

copies/ml, respectively. Median (IQR) baseline IL-6 and D-dimer were 1.45

(0.88,2.41) pg/mL and 0.34 (0.20,0.50) μg/L, respectively.

IL-6 and D-dimer at week 12

IL-6 and D-dimer values at week 12 were available for 172 and 164

participants, respectively, of which 57 and 56 were untreated. IL-6 levels at

week 12 were similar to baseline levels for both treated and untreated

participants, with mean (95% CI) and percent change from baseline of -0.09 (-

0.15 to -0.04) log10 pg/mL and -19.6% for those treated and -0.04 (-0.11 to

+0.04) log10 pg/mL and -8.3% for those untreated (p=0.23), both adjusted for

baseline levels. In contrast, D-dimer levels significantly dropped from baseline

by week 12 for participants on ART, but increased for those not on ART, with -

0.09 (-0.14 to -0.04) log10 μg/L and -18.7% for treated, and +0.10 (+0.02 to

+0.17) log10 μg/L and +25.2% for untreated participants (p<0.001).

Biomarker changes from baseline to week 12 are presented in Figure 1 by

treatment status and HIV RNA levels for treated individuals. There was a

suggestion of a greater decrease in IL-6 levels for those achieving lower HIV

RNA (continuous variable) at 12 weeks (p=0.072) but no such association for

D-dimer levels (p=0.89).

7

IL-6 and D-dimer 4 weeks after ART stop

Of participants who stopped ART correctly and had biomarkers measured 4

weeks later, 70 (70%) achieved HIV RNA <50 copies/mL at ART stop. Four

weeks after stopping, median (IQR) HIV RNA for these participants was 3.63

(2.93, 4.47) log copies/mL.

Biomarker levels significantly increased from ART stop to 4 weeks later, with

an overall mean change for IL-6 of +0.09 log10 pg/mL (95% CI +0.003,+0.17;

p=0.044) (+22% mean percent on log10 scale), and for D-dimer +0.07 log10

μg/mL (95% CI +0.003,+0.14; p=0.041) (+18% mean percent on log10 scale).

Overall, for treated individuals with HIV RNA <50 copies/mL at ART stop,

biomarker levels 4 weeks after stopping were similar to pre-ART levels, with a

mean difference of +0.03 log10 pg/mL (95% CI -0.07,+0.13; p=0.53) for IL-6,

and -0.05 log10 μg/L (95% CI -0.12.,+0.03; p=0.20) for D-dimer (Figure 1).

IL-6 and D-dimer over time by trial arm

Median biomarker levels over time are shown according to trial arm in Figure

2. Over 108 weeks follow-up, overall IL-6 AUC was -0.03 (95% CI -0.07,0.02)

log10 pg/mL, with no difference between the three arms (p=0.53). In

comparison, D-dimer AUC was significantly lower for ART12 (-0.11, 95% CI -

0.20,-0.02) and ART48 (-0.13, 95% CI -0.23,-0.04) log10 μg/mL, respectively,

compared to SoC (overall p=0.008).

Inter-laboratory variation

We found highly significant correlations between biomarkers measured in the

SPARTAC and SMART laboratories (Pearson correlation coefficient for log10

transformed values = 0.97, p<0.0001, and 0.77, p=0.014 for IL-6 and D-dimer,

respectively). However, absolute values differed between the two assays for

both biomarkers and were consistently higher for IL-6 and lower for D-dimer at

the SMART laboratory compared to the SPARTAC laboratories (Appendix).

8

DISCUSSION

This is the first study to describe the effect of starting, and subsequently

stopping, ART in PHI on two biomarkers associated with all-cause mortality.

The reason why individuals start or defer ART in PHI may not be random, and

may be driven by factors which themselves are linked to biomarker changes.

The study design of random allocation to ART deferral, initiation and

cessation avoids the bias that is unavoidable amongst many PHI cohort

studies.

Initiation of ART within six months of seroconversion significantly reduced D-

dimer after 12 weeks, but not IL-6 levels overall, in keeping with findings from

the SMART study (10). We found evidence of a greater reduction in IL-6 with

lower HIV RNA suggesting that IL-6 levels drop only upon attainment of viral

suppression, whereas the drop in D-dimer may be more immediate upon ART

initiation. This finding is also in keeping with SMART which demonstrated a

significant decline in IL-6 and D-dimer amongst those achieving viral

suppression by 6 months (10). Of note, we were able to examine HIV RNA

categories <50 and 50-400 copies/mL separately (in SMART, suppression

was defined as <400 copies/mL) and found no evidence of an association

between achieving 50-400 copies/mL at week 12 and a reduction in either

biomarker. The reduction in IL-6 observed in SMART among those achieving

<400 copies/mL may, therefore, be driven by those achieving <50 copies/mL.

Stopping ART in SPARTAC was associated with rises in both biomarkers 4

weeks after discontinuing therapy, lower for IL-6 than those experienced by

SMART participants interrupting therapy (22% vs. 30%) but similar for D-

dimer (18% vs. 16%) (4). Median IL-6 level 4 weeks after ART cessation was

1.51 pg/mL in SPARTAC, lower than seen in SMART (4). However, we found

consistently higher IL-6 levels at the SMART laboratory, compared to those

measured on parallel samples from the same patient and time point at the

SPARTAC laboratory, demonstrating the need for caution when comparing

biomarker results between studies, as results may be affected by the assay

used, laboratory techniques, or storage conditions. Absolute levels for D-

9

dimer 4 weeks after stopping ART for SPARTAC participants were similar

(median 0.32 µg/L) to SMART controls (0.26 µg/L) and substantially lower

than in those who had died (0.49 µg/L) in SMART (4). Given our finding of

consistently lower D-dimer levels at the SMART laboratory, we may have

underestimated the differences between biomarker levels in those initiating

and then stopping ART in PHI compared to chronic infection. We have

previously described lower HIV RNA rebound following ART cessation

initiated in PHI compared to chronic infection (11), and theorise that this may

also be true for biomarker rebound. As ART in PHI was restricted to a

maximum of 48 weeks in SPARTAC, we cannot comment on whether ART of

longer duration may positively impact on biomarker levels upon cessation.

Over 108 week follow up, overall IL-6 levels were comparable across the 3

arms, while D-dimer was significantly lower for participants randomised to the

2 ART arms compared to SoC. This may, in part, be due to the lower viral set

point observed in participants receiving ART48 (8), although no change in

viral setpoint was seen amongst ART12 individuals suggesting an alternative

explanation. It is not possible to extrapolate from this analysis whether the

observed reduction in D-dimer confers any clinical benefit.

In SPARTAC, in contrast to SMART, no significant clinical events were

reported following cessation of therapy (8). The use of ART in PHI may have

several benefits in reducing viral reservoirs (12, 13), preserving immunological

function (8), limiting long-term immunological damage(13-16) and reducing

onward HIV transmission (17). Reports of long-term viral control following

discontinuation of early treatment have led to renewed hopes for sustained

clinical benefits in some individuals (5). Our data provide evidence that

treatment interruption following either 12 or 48 weeks therapy has no

significant adverse effects on IL-6 and D-dimer levels up to 2 years after PHI

compared to untreated individuals. While higher post-seroconversion IL-6, but

not D-dimer, predicts HIV disease progression (9), the clinical significance of

biomarker rebound following ART interruption in this setting remains

uncertain.

10

ACKNOWLEDGEMENTSAuthor contributions: E.H., K.P., A.B., J.M., J.W. and S.F. designed the study.

D.C., G.T., M.S., M.F, and S.F. enrolled participants. W.S. and F.V. performed

the statistical analyses. E.H. and M.M. did the laboratory analyses. E.H., W.S.

and K.P. wrote the first draft of the article. All authors contributed to

subsequent drafts and approved the final version. J.W. was Principle

Investigator for the SPARTAC study.

We thank all the participants and staff at all the sites participating in the

SPARTAC trial. SPARTAC was funded by Wellcome Trust grants

WT069598MA and 069598/Z/02/B. The Biomedical Research Centre provided

staff and infrastructure support in the UK. Abbott Laboratories provided

Kaletra/Aluvia (lopinavir and low-dose ritonavir) for the African sites of

SPARTAC. We would like to thank Elaine Cornell and Russ Tracy (University

of Vermont, USA), for helping to compare SMART study laboratory techniques

with those in SPARTAC.

SPARTAC Trial Investigators: Trial Steering Committee (TSC) Independent

Members: A Breckenridge (Chair), P Clayden, C Conlon, F Conradie, J

Kaldor*, F Maggiolo, F Ssali Country Principal Investigators: DA Cooper, P

Kaleebu, G Ramjee, M Schechter, G Tambussi, J WeberTrial Physician S

Fidler Trial Statistician A Babiker Data and Safety Monitoring Committee

(DSMC) T Peto (Chair) A McLaren (in memoriam), V Beral, G Chene, J Hakim

Co-ordinating Trial Centre MRC Clinical Trials Unit, London (A Babiker, K

Porter, M Thomason, F Vanobberghen, M Gabriel, D Johnson, K Thompson,

A Cursley*, K Donegan*, E Fossey*, P Kelleher*, K Lee*, B Murphy*, D

Nock*) Central Immunology Laboratories and RepositoriesThe Peter Medawar

Building for Pathogen Research, University of Oxford, UK (R Phillips, J Frater,

L Ohm Laursen*, N Robinson, P Goulder, H Brown) Central Virology

Laboratories and Repositories Jefferiss Trust Laboratories, Imperial College,

London, UK (M McClure, D Bonsall*, O Erlwein*, A Helander*, S Kaye, M

Robinson, L Cook*, G Adcock*, P Ahmed*) Clinical Endpoint Review

Committee N Paton, S Fidler Investigators and Staff at Participating Sites

Australia: St Vincent’s Hospital, Sydney (A Kelleher), Northside Clinic,

11

Melbourne (R Moore), East Sydney Doctors, Sydney, (R McFarlane), Prahran

Market Clinic, Melbourne (N Roth), Taylor Square Private Clinic, Sydney (R

Finlayson), The Centre Clinic, Melbourne (B Kiem Tee), Sexual Health

Centre, Melbourne (T Read), AIDS Medical Unit, Brisbane (M Kelly), Burwood

Rd Practice, Sydney (N Doong) Holdsworth House Medical Practice, Sydney

(M Bloch) Aids Research Initiative, Sydney (C Workman) Coordinating centre

in Australia: Kirby Institute University of New South Wales, Sydney (P Grey,

DA Cooper, A Kelleher, M Law) Brazil: Projeto Praça Onze, Hospital Escola

São Francisco de Assis, Universidade federal do Rio de Janeiro, Rio de

Janeiro (M Schechter, P Gama, M Mercon*, M Barbosa de Souza, C Beppu

Yoshida, JR Grangeiro da Silva, A Sampaio Amaral, D Fernandes de

Aguiar, M de Fátima Melo, R Quaresma Garrido) Italy: Ospedale San

Raffaele, Milan (G Tambussi, S Nozza, M Pogliaghi, S Chiappetta, L Della

Torre, E Gasparotto,), Ospedale Lazzaro Spallanzani, Roma (G D’Offizi, C

Vlassi, A Corpolongo) South Africa: Cape Town: Desmond Tutu HIV Centre,

Institute of Infectious Diseases, Cape Town (R Wood, J Pitt, C Orrell, F

Cilliers, R Croxford, K Middelkoop, LG Bekker, C Heiberg, J Aploon, N Killa, E

Fielder, T Buhler ) Johannesburg: The Wits Reproductive Health and HIV

Institute, University of Witswatersrand, Hillbrow Health Precinct,

Johannesburg. (H Rees, F Venter, T Palanee), Contract Laboratory Services,

Johannesburg Hospital, Johannesburg (W Stevens, C Ingram, M Majam, M

Papathanasopoulos) Kwazulu-Natal: HIV Prevention Unit, Medical Research

Council, Durban (G Ramjee, S Gappoo, J Moodley, A Premrajh, L Zako)

Uganda: MRC/Uganda Virus Research Institute, Entebbe (H Grosskurth, A

Kamali, P Kaleebu, U Bahemuka, J Mugisha*, H F Njaj*) Spain: Hospital

Clinic-IDIBAPS, University of Barcelona, Barcelona (JM Miro, M López-

Dieguez*, C Manzardo, JA Arnaiz, T Pumarola, M Plana, M Tuset, MC Ligero,

MT García, T Gallart, JM Gatell) UK and Ireland: Royal Sussex County

Hospital, Brighton (M Fisher, K Hobbs, N Perry, D Pao, D Maitland, L Heald),

St James’s Hospital, Dublin (F Mulcahy, G Courtney, S O’Dea, D Reidy),

Regional Infectious Diseases Unit, Western General Hospital and

Genitourinary Dept, Royal Infirmary of Edinburgh, Edinburgh (C Leen, G

Scott, L Ellis, S Morris, P Simmonds), Chelsea and Westminster Hospital,

London (B Gazzard, D Hawkins, C Higgs), Homerton Hospital, London (J

12

Anderson, S Mguni), Mortimer Market Centre, London (I Williams, N De

Esteban, P Pellegrino, A Arenas-Pinto, D Cornforth*, J Turner*) North

Middlesex Hospital (J Ainsworth, A Waters), Royal Free Hospital, London (M

Johnson, S Kinloch, A Carroll, P Byrne, Z Cuthbertson), Barts & the London

NHS Trust, London (C Orkin, J Hand, C De Souza), St Mary’s Hospital,

London (J Weber, S Fidler, E Hamlyn, E Thomson*, J Fox*, K Legg, S

Mullaney*, A Winston, S Wilson, P Ambrose), Birmingham Heartlands

Hospital, Birmingham (S Taylor, G Gilleran) Imperial College Trial & DSMC

Secretariat S Keeling, A Becker Imperial College DSMC Secretariat C

Boocock

(* Left the study team before the trial ended)

13

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Figure 1. Changes in absolute IL-6 and D-dimer levels from baseline to week 12, by treatment status and HIV RNA level at week 12 (a and b) and from pre-ART (baseline) levels to stopping ART initiated in PHI (c and d)a)

b)

Legend to Figure 1a and 1b: Mean change (95% confidence intervals) from

baseline to week 12, adjusted for baseline D-dimer or IL-6, respectively, by

ART status and HIV RNA levels (copies/mL) at week 12. Note: HIV RNA at

week 12 missing for 2 participants therefore excluded from these plots.

16

c)

-0.15

0.02

-0.02

0.04

-0.30

-0.25

-0.20

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

ART stop 4 weeks later ART stop 4 weeks later

ART12 ART48

IL-6

cha

nge

from

bas

elin

e (lo

g 10 p

g/m

L)

d)

-0.11

-0.04

-0.16

-0.05

-0.30

-0.25

-0.20

-0.15

-0.10

-0.05

0.00

0.05

0.10

ART stop 4 weeks later ART stop 4 weeks later

ART12 ART48

D-d

imer

cha

nge

from

bas

elin

e (lo

g 10 m

g/L)

Legend to Figure 1c and d: Mean change (95% CI) in IL-6 and D-dimer from

baseline to ART stop (12 and 48 weeks for ART12 and ART48, respectively),

and from baseline to 4 weeks after stopping (16 and 52 weeks for ART12 and

ART 48, respectively) for participants on 12 week ART (ART12) and 48 week

ART (ART48).

17

Figure 2. Total (AUC) IL-6 and D-dimer levels by treatment arm over 108 weeks

a)

-0.10

0.00

0.10

0.20

0.30

IL-6

(log

10 p

g/m

L)

Week 0

Week 12

Week 16

Week 48

Week 52

Week 60

Week 108

SoC

ART12

ART48

IL-6

Number of patients:

Week 0 12 16 48 52 60 108

SoC 72 56 60 55 57 61 50

ART12 63 58 59 48 53 59 48

ART48 65 58 56 58 57 58 51

Note: 2 participants had baseline values only

18

b)

-0.70

-0.60

-0.50

-0.40

-0.30

D-d

imer

(log

10

/L)

Week 0

Week 12

Week 16

Week 48

Week 52

Week 60

Week 108

SoC

ART12

ART48

D-dimer

Number of patients:

Note: 1 participant had baseline values only

Legend to Figure 2: Mean log10 levels plus 95% CI of a) IL-6 and b) D-dimer

during follow-up, by randomisation arm.

Note week 16 and week 52 correspond to 4 weeks after ART stop for ART12

and AR T48, respectively

19

Week 0 12 16 48 52 60 108

SoC 68 55 56 52 54 58 47

ART12 60 56 56 45 49 56 45

ART48 63 53 51 54 56 57 49

Appendix Figure: Bland-Altman plots of biomarkers measured at SPARTAC and SMART laboratories

(a) IL-6

(b) D-dimer

The mean of the 2 measurements (x axis) is plotted against the difference between the 2

measurements (y axis). The horizontal lines demonstrate the mean difference and limits of

agreement (2 Standard Deviations of mean difference). For IL-6, the limits of agreement are -

2.30 and -0.36 pg/ml, indicating that IL-6 measurement in the SPARTAC laboratory is

consistently lower than in the SMART laboratory and demonstrating lack of agreement. For D-

dimer, the limits of agreement are -0.01 and 0.35, indicating that that D-dimer measurements

may be between -0.01 mg/L lower and 0.35 mg/L higher in the SPARTAC laboratory, again

demonstrating lack of agreement. 20

21