Supplemental digital content to
Frye et al, Kinetics of Torque Teno Virus-DNA plasma load predict rejection in lung transplant recipients
Table S1: Patients’ characteristics separated by rejections type A0 through A2.
A2 rejection A1 rejection A0 rejection
Age [years] Mean 57.7 57.1 56.8
Median 60.5 58.0 61.0
IQR 12 9.3 11.3
Sex M/F 3/3 4/3 15/6
Underlying disease
ILD (n / %) 3 / 50 6 / 86 12 / 57
COPD 3 / 50 1 / 14 8 / 38
Others 0 / 0 0 / 0 1 / 5
CMV (R/D) -/- (n / %) 2 / 33.3 0 / 0 3 / 14.3 -/+ (n / %) 0 / 0 2 / 28.6 4 / 19.0 +/- (n / %) 1 / 16.7 3 / 42.8 6 / 28.6 +/+ (n / %) 3 / 50 2 / 28.6 8 / 38.1
Table S2: infections leading to medical treatment (antibiotic, antiviral or antifungal medication, change in immunosuppression, hospitalization) in lung transplant recipients
Rejection- free group
Rejection group Bacterial infection of
the lungs
(pneumonia, bronchitis)
total 9 7
Staphylococcus aureus 2 1
Streptococcus pneumonia 1
Klebsiella species 2 2
Pseudomonas aeruginosa 3 1
Others 2 2
Aspergillus pneumonia
1 1
Norcardia infection 1
Viral infections total 7 8
CMV viremia 4 4
Rhinovirus
Human metapneumovirus 1
RS virus 1 2
Parainfluenza vius 1
Corona virus 1
Noro virus 1
Figure S1
Eosinophils [%]
0 1 2 3 4 5 6 7
t=0 t=60 t=120 t=180 t=240 t=300 t=360
D
Dotted light gray line: w/o biopsy-proven rejection Irregular broken dark gray line: with biopsy-proven rejection
Lymphocytes [%]
0 5 10 15 20 25 30 35
t=0 t=60 t=120 t=180 t=240 t=300 t=360
C
0 3 6 9 12 15 18 21
Tacrolimus [ng/ml] t=60 t=120 t=180 t=240 t=300 t=360
A
Leucocytes G/l
4 5 6 7 8 9 10 11
t=0 t=60 t=120 t=180 t=240 t=300 t=360
B
p< 0.05
*
*
p< 0.05
* p= 0.05
#
* #
* p calculated in Mann-Whitney U test
Figure S1: Patients with lung transplant rejection and patients without rejection episodes do not differ in their white blood cell counts and immunosuppressive trough levels. Median values of 60-day intervals are depicted.
A. Immunosuppressive trough levels after lung transplantation are shown as medium of 60-day intervals and are comparable for the rejection free group (dotted light gray line) and the rejection group (irregular broken dark gray line).
B. Total white blood cell counts after lung transplantation are significantly lower at day 180 and day 240 after transplantation in the rejection group (irregular broken dark gray line) compared to the rejection free group (dotted light gray line).
C. Percentage of lymphocytes after lung transplantation for the rejection free group (dotted light gray line) and the rejection group (irregular broken dark gray line) do not differ.
D. Percentage of eosinophils after lung transplantation for the rejection free group (dotted light gray line) and the rejection group (irregular broken dark gray line) differed significantly only at day 300 after transplantation.
*
Figure S2
A B
Leucocytes G/l
4 5 6 7 8 9 10 11
t=0 t=60 t=120 t=180 t=240 t=300 t=360
0 3 6 9 12 15 18 21
Tacrolimus [ng/ml] t=60 t=120 t=180 t=240 t=300 t=360
p=0.09 for A2 vs A0 p=0.06 for A2 vs A0
#
*
#
Figure S2: Basal laboratory parameters for patients after lung transplantation separated according to grade of rejection (A1 rejection group vs >A1 rejection group), shown as medium of 60-day intervals.
A. Immunosuppressive trough levels after lung transplantation are shown as medium of 60-day intervals and are similar for the rejection free group (dotted light gray line) and the A1rejection group (regular broken medium gray line).
The immunosuppressive drug levels are slightly higher in the >A1rejection group (straight dark gray line).
B. Total white blood cell count after lung transplantation did not differ significantly between all 3 groups, ie, rejection free group (dotted light gray line), the A1rejection group (regular broken medium gray line) and the >A1rejection group (straight dark gray line).
Figure S3
LDH[U/l]
250 300 350 400 450 500 550 600
t<0 t=60 t=120 t=180 t=240 t=300 t=360
p< 0.05
A
CRP [mg/l]
0 10 20 30 40 50 60 70
t<0 t=60 t=120 t=180 t=240 t=300 t=360
B
IgG[mg/dl]
0 300 600 900 1200 1500 1800 2100
t<0 t=60 t=120 t=180 t=240 t=300 t=360
C
* *
* * p= 0.05
*
*
p< 0.1
*
Dotted light gray line: w/o biopsy-proven rejection Irregular broken dark gray line: with biopsy-proven rejection
* p calculated in Mann-Whitney U test
Figure S3: Blood chemistry findings in lung transplant recipients after lung transplantation shown as medium of 60-day intervals.
A. Patients with biopsy-proven rejection (irregular broken dark gray line) exhibit a slightly but significantly higher LDH level compared to patients without biopsy- proven transplant rejection (dotted light gray line).
B. CRP was significantly elevated in patients with biopsy-proven rejection (irregular broken dark gray line) compared to patients without biopsy-proven rejection (dotted light gray line) at 1 time point.
C. IgG levels were not different between rejection (irregular broken dark gray line) and no-rejection (dotted light gray line) groups after lung transplantation.
Figure S4
1 10 1E2 1E3 1E4 1E5 1E6 1E7 1E8 1E9 1E10
0 5 10 15 20 25 30
Tacrolimus [ng/ml]
TTV-DNA copies
r² = 0.002 p = 0.51
1 10 1E2 1E3 1E4 1E5 1E6 1E7 1E8 1E9 1E10
0 5 10 15 20 25 30
TTV-DNA copies
r² = 0.02 p = 0.20
Tacrolimus [ng/ml]
B A
Figure S4:
TTV-DNA levels and immunosuppressive drug trough levels do not correlate in lung transplant recipients.
A. Immunosuppressive drug trough levels and TTV-DNA levels for all individuals between month 3 and months 12 after lung transplantation did not correlate.
B. Immunosuppressive drug trough levels and TTV-DNA levels for all individuals with biopsy-proven rejection between month 3 and months 12 after lung transplantation did not correlate.
SDC, Materials and Methods Quantitative real-time PCR
Viral DNA was extracted from 200 µl of plasma / serum using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) in a QIAcube automated extractor (Qiagen) according to the manufacturer`s instructions.
Real-time polymerase chain reaction (RT-PCR) of a TTV conserved region was performed in a LightCycler Nano Instrument (Roche Diagnostics, Mannheim, Germany). The assay was carried out in a 25 µl reaction volume using Platinum Taq DNA Polymerase Kit (ThermoFisher Scientific, Darmstadt, Germany). A primer concentration of 0,4 µM for each primer and a probe concentration of 0,2 µM were used. The cycling profile was as follows: 2 min at 94°C, 45 cycles at 94°C for 15 s and 60°C for 30 s. Primers and probe were as follows: TTVf, 5`-GTT TTC TAC GCC CGT CC-3`; TTVr, 5`-CCT TGA CTC CGG TGT GTA A-3`; TTV/TTMVpb, (6-FAM) 5`- ACT CAC CTH CGG CAC CCG C-3`(TAMRA) 18. For further information in real-time PCR please refer to supplementary methods.
For simultaneous detection of KIPyV- and WUPyV-DNA RT-PCR reactions were performed using a LightCycler Nano Instrument (Roche Diagnostics, Mannheim, Ger- many). The assay was carried out in a 25 µl reaction volume using QIAGEN OneStep RT-PCR Kit (Qiagen, Hilden, Germany). A primer concentration of 0,4 µM for each primer and a probe concentration of 0,2 µM for each probe were used. The PCR cyc- ling conditions were as follows: 15 min at 95°C, 45 cycles at 94°C for 15 s and 60°C for 30 s. Primers and probe were as follows: KIPyV2263F, 5`-TTGGATGAAAAT- GGCATTGG-3`; KIPyV2404R, 5`TAACCCTTCTTTGTCTAAAATGTAGCC-3`;
KIPyVP, (HEX) 5`ACATTACTTGTGCAGATATGCTTGGAACAGC-3`(BHQ1);
WUPyVP, (FAM) 5`-CATAACTTGTGCTGACCTTTTGGGAGTTAAC-3`(BHQ1)19.
Quantitative BK-Virus-, JC-Virus- and CMV-PCRs were carried out using RealStar PCR Kits (altona Diagnostics GmbH, Hamburg, Germany) according to the manufacturer’s instructions. RT-PCRs were performed on an Abbott m2000 real-time instrument system (Abbott Molecular, Wiesbaden, Germany).
Each run contained a negative control and a positive control (plasmid DNA as a copy number standard). Comparison between calculated threshold cycle (Ct) of each sample with Ct of quantification curve points allowed the determination of viral loads in copies/ml or IU/ml.
Statistical methods
Statistical analyses were performed with StatView (Version 5.0.1 by SAS Institute Inc.).
Data are in general presented as means with error bars showing 1 standard error of the mean if not otherwise indicated. Values of p were calculated with Wilcoxon signed rank test or Mann Whitney U test as appropriate. Values of p < 0.05 were considered statistically significant.