David J. Speicher1,2, Newell W. Johnson2
1School of Dentistry and Oral Health, Griffith University, Southport, Queensland, Australia
2Griffith Health Institute, Griffith University, Queensland, Australia

Introduction: Salivary Detection of HHV-8

Kenyan Field Study:

Saliva is a window into the body’s health, and combined with PCR is a powerful tool for the detection of many viruses. With the reservoir ofHHV-8 being the oropharynx viral transmission is via saliva making detection, and treatment monitoring, ideal in whole mouth fluid (WMF).
Traditionally, salivary detection of HHV-8 has involved the collection of WMF. Samples are frozen without additives or in Qiagen’s RNAprotect
solution. Throat epithelial cells can be collected with throat gargles with 10 mL of PBS, which is then preserved by freezing. All of these sample
types are then extracted via spin columns before examination by PCR. While these methods work well in developed nations that havesufficient resources they are not useful in field studies in developing nations or resource poor settings. DNAGenotek offers a commercial
salivary collection kit useful for examining human genomic DNA, but are expanding into microbial diagnostics. Our study evaluated theDNAGenotek prototype kit (P-021) designed to stabilise microbial DNA, in both in vitro spiking studies and a Kenyan field trial to compare theDNAGenotek kit against traditional methods to quantitate HHV-8 oral shedding in HIV-positive patients in Kenya and determine HHV-8
subtypes.

• All 10 Kenyan HIV-positive patients had good oral health with no evidence of tumours or other soft tissue lesions• 30% had hyposalivation (average: 0.227 mL/min; range: 0.199-0.286 mL/min)• Total DNA concentration: DNAGenotek Kits > WMF > PBS-TG (Table 1)• When extraction volumes were normalized to 100 µL WMF the total DNA concentration were comparable frozen WMF& DNAGenotek kits (Confirmed by GAPDH qPCR)• DNA purity was excellent in all samples no matter how they were stored or extracted• HHV-8 was detected in all samples , but only in low concentrations (Table 2)• WMF had a higher positive detection rate due to the slightly higher viral loads as a result of the DNAGenotek kits
being extracted via ethanol precipitation of the supernatant with the pellets being discarded• PBS-TG had the lowest positive detection rated possibly due to the huge dilutions factor when using 10 mL PBSMethods:Table 1: Total DNA concentration (ng/µL) and purity of DNA extracted from the Kenyan oral samples frozen andin the DNAGenotek P-021 kits for 14 months.

Effect of Storage Duration on WMF using the DNAGenotek Kit• WMF was spiked with HHV-8 produced from BCBL-1 cell lines that are HHV-8 positive/EBV negative• Free HHV-8 virion (4.38x108 copies/mL) was purified from the supernatant while cell-associated HHV-8 (1.33x1011 copies/mL) was obtained
from the cell pellets• Spiked WMF from 3 subjects were stored in the DNAGenotek P-021 up to 9 months and examined by qPCR for HHV-8 ORF73 & ORF26• Samples were extracted via the DNAGenotek method, which involves ethanol precipitation and purification of DNA• All HHV-8 concentrations were normalised to copes HHV-8/mL WMF
Kenyan Field Trial• WMF and Phosphate Buffered Saline Throat Gargles (PBS-TG) were collected from 10 Kenyan HIV-positive patients on HAART (males:females, 2:8) presenting at the School of Dental Science, University of Nairobi in Nairobi, Kenya• Samples were stored frozen and in DNAGenotek P-021 kits at room temperature in Kenya• Specimens were processed 14 months later in Australia.
• Frozen samples were extracted via Qiagen DNeasy Blood and Tissue Kit while DNAGenotek P-021 kits were ethanol precipitated.
• DNA concentration and purity was determined via NanoDrop, while viral loads were determined by qPCR for HHV-8 ORF73 & ORF26
Sample Processing using the DNAGenotek collection Kit• 10-fold serial dilutions of pGEM-T/ORF73 & pGEM-T/ORF26 performed in TE Buffer and WMF (stored frozen & in DNAGenotek P-021 kits)• qPCR for HHV-8 ORF73 & ORF26 was performed directly on spiked TE Buffer• Spiked WMF stored frozen was extracted via Qiagen DNeasy Blood and Tissue Kit• Spiked WMF stored in DNAGenotek P-021 kits was extracted via ethanol precipitation and Qiagen DNeasy Blood and Tissue Kit• qPCR for HHV-8 ORF73 & ORF26 was performed on all samples in triplicate to determine efficiency, linearity and dynamic range

Frozen Samples

Griff 5
Griff 6
Griff 7
Griff 8
Griff 9
Griff 10
Griff 11
Griff 12
Griff 13
Griff 14
Average
St. Dev

WMF
PBS-TGWMF
PBS-TG[DNA]/100 µL[DNA]/100 µL[DNA]/Total [DNA]/100 µL[DNA]/Total [DNA]/100 µL
PurityPurityPurityPurity(ng/µL)(ng/µL)(ng/µL)(ng/µL)(ng/µL)(ng/µL)
25.40
1.90
5.66
1.72
120.82
48.33
1.99
7.29
2.92
1.90
18.82
1.67
3.85
2.93
33.4213.37
1.81
4.50
1.80
1.77
13.31
1.85
4.74
2.72
50.94
20.38
2.11
3.17
1.27
1.06
27.20
1.70
4.09
1.98
50.64
20.26
1.93
2.63
1.05
1.79
30.44
1.74
4.78
2.45
99.72
39.89
1.95
2.90
1.16
2.63
68.55
1.90
6.08
2.15
248.80
99.52
1.94
6.39
2.56
1.29
20.08
1.89
6.73
2.03
120.73
48.29
1.99
7.11
2.84
1.51
30.13
2.06
6.14
1.66
130.27
52.11
2.05
17.05
6.82
1.92
19.22
1.95
1.59
58.15
1.42
2.86
Table 2: 4.28
Extent of disease
asdetermined23.26
by HHV-81.93
IHC and 3.56
verified by rtPCR.
13.51
1.69
3.86
55.99
22.40
1.99 positive
10.90before transplant.
4.36
2.19
Explanted
liver was1.74
examined
to see if hostwas HHV-8
26.67
1.84
5.02
2.10
96.95
38.78
1.97
6.55
2.62
1.89
15.97
0.13
1.05
0.46
63.94
25.58
0.08
4.51
1.81
0.56

Table 2: Normalized HHV-8 viral loads in Kenyan oral samples that were frozen and stored in the DNAGenotek
kit presented as HHV-8 copies/mL WMF. Values of “ND” indicate that HHV-8 was “not detected” in these samples.

Effect of Storage Duration on WMF using the DNAGenotek Kit:

Frozen Samples• Viral loads were stable in the DNAGenotek P-021 kits for 6 months for cell-associated HHV-8 but 9 months for free HHV-8 virion• 31% decrease in cell-associated HHV-8, but only 9.9% decrease in free HHV-8 virion from 6 months to 9 months• % loss of cell-associated HHV-8 (max loss: 48.27%) is much higher than free HHV-8 virion (max loss: 26.27%)• Difference possibly due to the ethanol precipitation method discarding cell-associated virus with the cell pellet

Serial #Griff 5
Griff 6
Griff 7
Griff 8
Griff 9
Griff 10
Griff 11
Griff 12
Griff 13
Griff 14

Serial #Griff 5
Griff 6
Griff 7
Griff 8
Griff 9
Griff 10
Griff 11
Griff 12
Griff 13
Griff 14A.

HHV-8 ORF73
4.92E+04
3.39E+06
ND
3.61E+06
2.14E+06
6.00E+06
2.70E+06
1.16E+06
3.95E+05
2.80E+06

HHV-8 ORF73
ND
ND
1.67E+06
ND
ND
ND
ND
ND
ND
ND

DNAGenotek Kit Processing Methods:• Standard curves produced in spiked WMF stored in the DNAGenotekkits and purified via ethanol precipitation had poor efficiency andlinearity• Extraction of the DNAGenotek kits with Qiagen spin columns
displayed excellent efficiency and linearity• Efficiency and linearity (R2) is as good as plasmid diluted in TE
Buffer and WMF• Linear range and lower limit of detection is slightly better than TE
Buffer and WMF for both assays.

B.

DNAGenotek Kit

Figure 1: HHV-8 concentration in DNAGenotek kits spiked 1:10 with (A) 1.33x1011 copies/mL

cell-associated HHV-8 or (B) 4.38x108 copies/mL of free HHV-8 virion over 9 months.

WMF
HHV-8 ORF26
Average Viral Load
GAPDH
HHV-8 ORF73
3.90E+05
2.19E+05
4.48E+08
ND
2.97E+05
1.84E+06
1.02E+09
1.61E+06
ND
ND
4.16E+08
2.13E+06
7.60E+05
2.18E+06
9.95E+08
ND
ND
2.14E+06
1.41E+09
ND
1.51E+06
3.75E+06
3.17E+09
ND
ND
2.70E+06
3.49E+08
ND
2.02E+05
6.79E+05
1.03E+09
ND
3.39E+05
3.67E+05
4.61E+08
1.18E+06
1.53E+05
1.47E+06
4.70E+08
3.67E+06
Average GAPDH (Total)
9.59E+08
DNA Genotek Kit
WMF
HHV-8 ORF26
Average Viral Load
GAPDH
HHV-8 ORF73
ND
ND
9.78E+08
ND
ND
ND
5.54E+08
ND
6.64E+05
1.17E+06
5.64E+08
ND
4.26E+05
4.26E+05
1.06E+09
ND
ND
ND
8.46E+08
ND
ND
ND
3.52E+09
2.18E+05
ND
ND
6.22E+08
ND
3.10E+05
3.10E+05
1.67E+09
2.34E+06
7.56E+04
7.56E+04
8.60E+08
ND
3.80E+05
3.80E+05
6.00E+08
ND
Average GAPDH (Total)
1.13E+09

PBS Throat Gargle
HHV-8 ORF26
Average Viral Load
ND
ND
4.38E+05
1.02E+06
2.39E+05
1.18E+06
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.51E+05
7.66E+05
ND
3.67E+06
Average GAPDH (Total)

GAPDH
1.16E+08
6.05E+07
1.49E+07
5.65E+07
9.00E+07
1.32E+08
7.70E+07
5.35E+07
1.26E+08
1.54E+07
7.41E+07

PBS Throat Gargle
HHV-8 ORF26
Average Viral Load
ND
ND
ND
ND
ND
ND
ND
ND
2.96E+05
2.96E+05
ND
ND
1.89E+05
1.89E+05
2.28E+06
2.31E+06
1.18E+05
1.18E+05
NDND
Average GAPDH (Total)

GAPDH
1.13E+08
4.18E+07
1.09E+07
2.94E+07
5.72E+07
1.06E+08
1.68E+08
1.48E+07
1.44E+08
2.56E+07
7.10E+07

Table 3: qPCR dynamics including efficiency, R2, and linear rang e for the HHV-8
ORF73 & ORF26 qPCR assays on pGEM-T/ORF73 & pGEM-T/ORF26 plasmids diluted in
TE Buffer and WMF stored and extracted with 2 different methods.

Sample Medium and Processing Methods Efficiency (%) R^2 Value
HHV-8 ORF73 qPCR assay
TE Buffer
97.544
0.99664
WMF (23/05/2011)
95.95
0.99887
DNAGenotek (EtOH precipitation)
164.220.89071
DNAGenotek (Qiagen Spin Columns)
104.520.99542
HHV-8 ORF26 qPCR assay
TE Buffer
100.4
0.99407
WMF (23/05/2011)
99.13
0.98179
DNAGenotek (EtOH precipitation)
167.310.84137DNAGenotek (Qiagen Spin Columns)
97.46
0.99625

1A.

2A.

3A.

4A.

1B.

2B.

3B.

4B.

Linearity Range
6.34E+03 to 6.34E+10
2.66E+02 to 1.45E+09
Non-linear
2.44E+01 to 1.12E+09
1.95E+02 to 1.95E+10
3.39E+01 to 1.12E+09
Non-linear
5.89E+01 to 1.56E+09

Figure 2: pGEM-T/ORF73 & pGEM-T/ORF26 plasmids diluted 10-fold serially in (1) TE Buffer and (2, 3, 4) WMF. Spiked WMF stored in the DNAGenotek P-021 kit and then extracted via (3) ethanol precipitation yields a nonlinear standard curve for both for (A) HHV-8 ORF73 and (B) HHV-8 ORF26 qPCR assays. When the same samples stored in the DNAGenotek P-021 kit are extracted via (4) via Qiagen spin columns the linear range andsensitivity is as good or better than plasmids diluted in (1) TE Buffer or (2) WMF and extracted immediately with Qiagen spin columns. GAPDH qPCR (blue line) show that cellular DNA is similar in all samples proving thatthe differences in HHV-8 copy number is due to the serial dilutions and not a difference in amount of sample extracted.

Conclusions:
1. DNAGenotek kits are excellent for preserving oral specimens collected in the field for up to 14 months.
2. DNAGenotek kits produce high quality DNA with yields as high or higher than frozen WMF.
3. Standard curves produced from spiked WMF stored in the DNAGenotek kits and extracted with theQiagen DNeasy Blood and Tissue Kit are as good or better than plasmids diluted in TE Buffer or WMF
extracted with Qiagen spin columns.
4. 24.4 copies HHV-8/µL can be detected when the DNAGenotek kits are extracted with spin columns.

The DNAGenotek system has been a great asset in the detection of HHV-8 in our field studies in Kenya where room temperature canexceed 30oC. When extracted with spin columns serial dilutions show near perfect linearity down to 24.4 copies of HHV-8/µL.

References:1.
2.
3.

Mancuso, R., R. Biffi, M. Valli, M. Bellinvia, T. Athanasia, S. Ferrucci, L. Brambilla, S. Delbue, P. Ferrante, C. Tinelli, and M. Clerici. 2008. HHV8 a subtype is associated with rapidly evolving classic Kaposi'ssarcoma. J Med Virol 80:2153-60.
Park, N. J., T. Yu, V. Nabili, B. M. Brinkman, S. Henry, J. Wang, and D. T. Wong. 2006. RNAprotect saliva: An optimal room- temperature stabilization reagent for the salivary transcriptome. Clin Chem 52:2303-4.
Pauk, J., M. L. Huang, S. J. Brodie, A. Wald, D. M. Koelle, T. Schacker, C. Celum, S. Selke, and L. Corey. 2000. Mucosal shedding of human herpesvirus 8 in men. N Engl J Med 343:1369-77.