Longitudinal study on oral shedding of human betaherpesviruses 6 and 7 in renal transplant recipients reveals active replication

Figures & data

Table 1. Sequences of primers and DNA probes used for standard curves for qPCR.

		HHV-6 sequence (5´-3´)	HHV-7 sequence (5´-3´)
Probes	VIC-TTAGATGGTGGTGAGCTGGGATCGGT- NFQ-MGB		NED – CTCGCAGATTGCTTGTTGGCCATG- NFQ-MGB
Primers (Sense)	AAAGACCTAAATTGCCGCTACCT		CGGAAGTCACTGGAGTAATGACAA
Primers (Anti-sense)	GCAAGCTCATGAACATCGTCA		CCAATCCTTCCGAAACCGAT
Standard Curve probe	TTCGTGCAAGCTCATGAACATCGTCACGTATACCGATCCCAGCTCACCACCATCTAAATGCGTAGGTAGCGGCAATTTAGGTCTTTCTGATA		TTCGTCCAATCCTTCCGAAACCGATCGTATCATGGCCAACAAGCAATCTGCGAGATGCGTTTGTCATTACTCCAGTGACTTCCGCTGATA

Table 2. Sequences of nPCR primers.

	HHV-6 sequence (5´- 3´)	HHV-7 sequence (5´- 3´)
Primers – (Sense) F1	5ʹ-CTCGGAGCCCGAGCTACGAAATCTG-3’	5ʹ-TTGCGGAGGATAGAGAATATGGAAC-3’
Primers – (Anti- Sense) R1	5ʹ-GCAACAAGTCTGGCCCTAAGCTG-3’	5ʹ-TCGTTCTGCATGTTCTTTTTCAAAC-3’
Primers (sense) F2	5ʹ-CGATGAACACAGAAAACGCTCG-3’	5ʹ-AGAAACTCTTCTGGGAGCAGTTCA-3’
Primers (Anti-sense) R2	5ʹ-GTAGAAGTCGGTGCTATGCCATC-3’	5ʹ-CGACACCAGAGCTGTGGCAA-3’

Figure 1. HHV-6 and HHV-7 viral loads at pre and post-transplantation periods in oral fluids of renal transplant patients (T1: Pre transplantation and without immunosuppression treatment; T2: 15–20 days after transplantation; T3: 45 to 60 days post-transplantation).

Figure 2. Detection of actively replicating HHV-6 and HHV-7 based on mRNA analysis in oral fluid before and after kidney transplantation. (T1: pre-transplant and without immunosuppressive treatment; T2: 15–20 days after transplantation; T3: 45 to 60 days after transplantation).

Table 3. Association of HHV-6 and HHV-7 replication with transplant times.

Virus	Time period	Positive n (%)	Negative n (%)	TOTAL	p^[Citation1]
HHV-6	Pre-transplantation	1 (3.1)	31 (96.9)	32 (100)
	15–20 days after transplantation	3 (9.4)	29 (90.6)	32 (100)	0.625
	45 to 60 days after transplantation	6 (18.8)	26 (81.3)	32 (100)	0.063
HHV-7	Pre-transplantation	8 [25]	24 (75)	32 (100)
	15–20 days after transplantation	5 (15.6)	27 (84.4)	32 (100)	0.508
	45 to 60 days after transplantation	23 (71.9)	9 (28.1)	32 (100)	0.001*

[1] McNemar Test, ‘p’ values correspond to the significance of pre-transplant data relative to 15–20 days and 45–60 days after transplantation.

* Statistical significance (p < 0.05).

Table 4. Relationship between HHV-6 and HHV-7 DNA levels and mRNA detection.

mRNA Herpesvirus		Quantitation			p^(b)
		Mean ± Standard deviation (DNA copies/mL)	N^a	Rank Mean
HHV-6	Negative	4.58 10^5 ± 1.22 10^6	86	46.64	0.055
	Positive	1.45 10^6 ± 2.80 10^6	10	64.50
HHV-7	Negative	7.48 10^5 ± 1.24 10^6	60	40.38	<0.001*
	Positive	3.91 106 ± 7.63 10^6	36	62.04

^arepresents the number of samples evaluated. (b) Mann-Whitney Test * Statistical significance (p < 0.05).

Figure 3. Correlations between HHV-6 and HHV-7 viral DNA quantities (Spearman correlation: N = 96, Correlation coefficient = 0.781, p < 0.001).

Table 5. Immunosuppressive regimens adopted for the study participants.

Immunosuppressive regimen	n	%
Tacrolimus + Azathioprine + Prednisone	15	46.9
Cyclosporine + Azathioprine + Prednisone	5	15.6
Tacrolimus + Everolimus + Prednisone	7	21.9
Tacrolimus + Mycophenolate sodium + Prednisone	5	15.6
TOTAL	32	100

Note: No regimen or individual use of medication was associated with viral replication of HHV-6 or HHV-7 (Fisher’s Exact Test, p > 0.05).

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