Comparison of Droplet Digital PCR and Metagenomic Next-Generation Sequencing Methods for the Detection of Human Herpesvirus 6B Infection Using Cell-Free DNA from Patients Receiving CAR-T and Hematopoietic Stem Cell Transplantation

Figures & data

Table 1 The Sequences of the Primers and Probes Used in ddPCR

Genes	Primers/Probes	Sequences
HHV-6B	Forward primer	5’-TCCATTGTTTGATTGATTTCCGTAT-3’
	Reverse primer	5’-AACGCGGGATGTTCTATTGG-3’
	Probe	5’-FAM-CTTGAGCTTGTAGATAAT-3’
POP4	Forward primer	5’-GGCGGTGGTCCTGGAGTACT-3’
	Reverse primer	5’-AGAGGCCTTTGGCTTTCTTCTT-3’
	Probe	5’-VIC-ACCCGCCACAAGC-3’

Figure 1 Evaluation of the HHV-6B probe primer set using plasmid standards. (A) Dilution curve of plasmid standard quantification by ddPCR. The correlation between the expected and observed copy numbers is shown. Each black dot represents a single replicate well of the dilution experiment, while the regression line is based on the average concentration at each dilution. (B) The lower detection limit of droplet digital PCR. The results are presented as the means ±1 SD.

Table 2 Characteristics of Infection in Patients with HHV-6B

Study Population		N (%)
Age (Years)
	Median (range)	43 (20~67)
Sex
	Female	12 (44.44)
	Male	15 (55.56)
Diagnosis
	NHL	19 (70.37)
	HL	1 (3.70)
	B-ALL	2 (7.41)
	ANKL	1 (3.70)
	AA	4 (14.82)
Neurological symptoms
	Yes	13 (48.15)
	No	14 (51.85)
Treatment
	CAR T+HSCT	9 (33.33)
	CAR T	10 (37.04)
	HSCT	8 (29.63)

Abbreviations: NHL, non-Hodgkin’s lymphoma; HL, Hodgkin’s lymphoma; B-ALL, B cell-acute lymphoblastic leukemia; ANKL, aggressive NK cell leukemia; AA, aplastic anemia.

Figure 2 Comparison between ddPCR and mNGS for HHV-6B detection. (A) Scatter plot and correlation of ddPCR and mNGS results in HHV-6B detection. (B) Comparison of ddPCR and mNGS results for HHV-6B detection. The blue bar shows that both ddPCR and mNGS detected HHV-6B positivity; the burgundy bar shows that ddPCR detected HHV-6B positivity, while mNGS results were negative; the gray bar indicates that neither ddPCR nor mNGS detected HHV-6B virus.

Table 3 Clinical Characteristics of Patients Treated with CAR T for Continuous HHV-6B Monitoring

Patient ID	Sex	Age	Disease	ddPCR Sample Type	Neurological Symptoms	CRS Grade	ICANS Grade	Time to First mNGS Test After CAR-T Treatment
Pt.1	Female	24	DLBCL	PB	Yes	4	3	14
Pt.2	Male	43	PCNSL	PB\U	Yes	2	0	8
Pt.3	Male	33	DLBCL	PB	No	1	0	18
Pt.4	Female	25	PTCL	PB	Yes	2	0	19
Pt.5	Female	44	DLBCL	PB\U	No	1	0	12
Pt.6	Female	40	DLBCL	PB	No	2	0	15
Pt.7	Male	39	PMBCL	PB	No	2	0	11
Pt.8	Female	48	DLBCL	PB	Yes	3	0	22
Pt.9	Female	52	HL	PB	No	1	0	16
Pt.10	Female	50	DLBCL	PB\U	Yes	3	0	17

Abbreviations: DLBCL, diffuse large B cell lymphoma; PCNSL, primary central nervous system lymphoma; PTCL-NOS, peripheral T cell lymphoma, not otherwise specified; PMBCL, primary mediastinal large B-cell lymphoma; HL, Hodgkin’s lymphoma; PB, peripheral blood; U, urine.

Figure 3 Time of onset and levels of HHV-6B after CAR T-cell therapy. (A) Time of onset of HHV-6B and CRS in 10 patients after CAR T-cell infusion. Red dots represent the onset of HHV-6B infection; blue dots indicate the end of HHV-6B infection; black dots represent HHV-6B levels that were greatly reduced but that did not drop to 0; green solid lines indicate ranges of CRS (days); black dotted lines indicate ranges of HHV-6B infection (days). (B) HHV-6B copy number levels after CAR T-cell therapy. Arrows represent the first mNGS monitoring time point after CAR T-cell infusion; red dots/red solid lines indicate HHV-6B detection in a peripheral blood sample; dark green dots/dark green solid lines represent HHV-6B detection in a urine specimen; black dots represent antiviral therapy. A-J each represent a lymphoma patient treated with CAR T-cell therapy.

Figure 4 Differentiation of viral encephalitis caused by HHV-6B activation and ICANS after CAR T-cell therapy. (A) One case of HHV-6B encephalitis after CAR T-cell therapy. (B) Another case of ICANS after CAR T-cell therapy. Dynamic changes in IL-6 levels and HHV-6B copy number in two patients after CAR T-cell infusion. The arrows represent the first mNGS monitoring time point after CAR T infusion.