Advances in Infectious Diseases, 2013, 3, 1-9
http://dx.doi.org/10.4236/aid.2013.31001 Published Online March 2013 (http://www.scirp.org/journal/aid)
1
Evaluation of Pathogen Reduction Systems to Inactivate
Dengue and Chikungunya Viruses in Apheresis Platelets
Suspended in Plasma
Li Kiang Tan1*, Sally Lam2*, Swee Ling Low1, Fang Hui Tan2, Lee Ching Ng1#, Diana Teo2
1Environmental Health Institute, National Environment Agency, Singapore City, Singapore; 2Blood Services Group, Health Sciences
Authority, Singapore City, Singapore.
Received January 12th, 2013; revised February 13th, 2013; accepted March 13th, 2013
ABSTRACT
The risk of blood-borne transmission of infectious diseases has led to an increasing awareness of the need for a safe and
effective pathogen reduction technology. This study evaluated the efficacy of 2 pathogen reduction systems to inacti-
vate dengue virus (DENV-2) and chikungunya virus (CHIKV) spiked into apheresis platelets (APLT) concentrates.
Double-dose APLT collections (n = 3) were split evenly into two units and spiked with 107 infectious units of DENV-2
or CHIKV. APLTs samples were assayed for viral infectivity before and after Amotosalen photochemical treatment
(PCT) or Riboflavin pathogen reduction treatment (PRT). Viral infectivity was determined by plaque assays. Platelet
(PLT) count, pH and residual S-59 were measured during the storage of 5 days. Amotosalen PCT showed robust effi-
cacy and complete inactivation of both viruses in APLTs, with up to 3.01 and 3.75 log reductions of DENV-2 and
CHIKV respectively. At similar initial concentrations, Riboflavin PRT showed complete inactivation of CHIKV with
up to 3.73 log reduction, much higher efficacy than against DENV-2 where a log reduction of up to 1.58 was observed.
All post-treated APLTs maintained acceptable PLT yields and quality parameters. This parallel study of 2 pathogen
reduction systems demonstrates their efficacy in inactivating or reducing DENV and CHIKV in APLTs and reaffirms
the usefulness of pathogen inactivation systems to ensure the safety in PLTs transfusion.
Keywords: Pathogen Inactivation; Dengue; Chikungunya; Transfusion-Transmitted Disease; Platelets
1. Introduction
Arboviruses have increasingly caught world-wide atten-
tion in the last decade. Among them, dengue and chi-
kungunya viruses (DENV and CHIKV) are of major in-
ternational public health concerns with extensive geo-
graphical spread that has expanded from tropical regions
to some temperate places such as Italy, France, Nepal
and China [1-6]. While the transmission of CHIKV ex-
tends from Africa, to Asia and Europe, DENV transmis-
sion is active in every single continent on the globe. The
primary mode of dengue and chikungunya transmission
is through bites of Aedes mosquito—Aedes aegypti and
Aedes albopictus.
Dengue illness causes an estimate of 50 - 100 million
infections annually, leading to half a million hospitaliza-
tion with life threatening dengue haemorrhagic fever/
shock syndrome (DHF/DSS syndrome) [7,8]. Its causa-
tive agent, DENV, is a ssRNA positive-strand virus of
the family Flaviviridae, genus Flavivirus. DENV is clas-
sified into four antigenically related but immunologically
distinct serotypes, namely DENV-1, -2, -3 and -4 [9,10].
Aside from the bite of infectious mosquitoes, trans-
missions via dengue contaminated blood or blood prod-
ucts have been documented in 3 publications [11-13]. In
Singapore, three patients received blood products do-
nated by a donor who developed symptoms of dengue
fever a day after donation [11]. Two of the recipients
developed dengue symptoms while the third was asymp-
tomatic and developed IgM and IgG antibodies. DENV-2
was detected by PCR assay in the donated blood products,
the donor and two of the symptomatic recipients. In
Hong Kong, an elderly lady acquired dengue from a
blood product donated by a donor prior to the onset of
dengue fever symptoms [12]. The donor was one of the
six cases confirmed to be epidemiologically linked to the
small Ma Wan outbreak in 2002 and DENV-1 was found
in the donated blood product. The third incident occurred
during the 2007 dengue outbreak in Puerto Rico [13]. A
recipient of contaminated blood became febrile on the
*The two authors contributed equally to the work.
#Corresponding author.

Evaluation of Pathogen Reduction Systems to Inactivate Dengue and Chikungunya Viruses in
Apheresis Platelets Suspended in Plasma
2
third day post-transfusion, subsequently developed DHF
and tested positive for DENV-2 by PCR. Donor’s blood
was found to contain a matching virus and was one of 12
asymptomatic donors that were detected through a post-
outbreak batch screening process.
Considering the incidence of dengue in endemic coun-
tries, the high proportion of asymptomatic infection (up
to 87%) and the median 5-day viremia, transfusion-as-
sociated dengue transmission may be more widespread
than documented [14-16]. Predictions on the risks of ac-
cidental transfusion of dengue-infected blood products
from asymptomatic carrier donors in Singapore have
been reviewed [17,18]. Using a mathematical modeling
on cases reported during the 2005 outbreak, Wilder-
Smith and colleagues estimated the risk for dengue-in-
fected blood transfusion to be 1.625 - 6/10,000, assuming
a ratio of 2:1 to 10:1 for asymptomatic to symptomatic
infections [18]. Interestingly, S. L. Stramer et al. (2012)
suggested that there could be an overestimation on the
risk of viremic donations in Puerto Rico from 1995-2010
(7 per 10,000) as not all the donated products detected
with viral RNA are considered infectious and associated
with dengue transmission [13].
Chikungunya fever struck several islands off the In-
dian Ocean, Southeast Asian, and European countries
causing large-scale epidemics [2-5,19-21]. The causative
agent, CHIKV, is an enveloped (+) ssRNA belonging to
family Togaviridae, genus Alphavirus. It causes a non-
fatal, self-limiting disease characterized by abrupt onset
of high fever, severe arthralgia, often associated with
skin rash. In 2008, there were several outbreaks of chi-
kungunya fever in Singapore that resulted to 718 labora-
tory-confirmed cases [19,20]. Interestingly, during the
surveillance of the first outbreak, a positively-diagnosed
patient developed fever only on the subsequent day [20].
Unlike dengue fever, there has been no report of trans-
fusion-transmitted cases involving chikungunya nor do-
cumented case of contaminated blood product to date
[22]. Transfusion-related transmission of CHIKV is high-
ly possible since CHIKV has been transmitted to a health
care worker drawing blood from an infected patient [23].
Concern is heightened due to the high infection rates
during outbreaks and high viremia titer that can last up to
six days [24]. Furthermore, at the height of the outbreak
at Reunion Island in 2006, the estimated transfusion risks
was estimated to be as high as 150 per 10,000 donations
[21].
While dengue and chikungunya transmissions in Sin-
gapore have been controlled through a comprehensive
integrated vector control program, transmission by trans-
fusion of contaminated blood or blood products is being
recognized as a threat to the current blood supply system.
The current donor selection and deferral policy for ex-
clusion of dengue and chikungunya infection in Singa-
pore is primarily based on the clinical history of infection,
onset of fever and travel history. However, an oversight
of revealing details by donors, particular those with late
symptom onset or asymptomatic cases, could lead to the
contamination of blood supplies with these viruses. There-
fore, in the absence of effective routine screening tests
for DENV and CHIKV in Singapore, the use of a patho-
gen reduction system may help in ensuring safety of
blood and its product.
Many studies have reported the efficacy of individual
pathogen reduction system on platelets (PLTs). Here we
evaluate the efficacy of 2 pathogen reduction systems
concurrently to inactivate the DENV or CHIKV spiked
in apheresis human platelet (APLT) concentrates. The
first system, Amotosalen photochemical treatment (Amo-
tosalen PCT, Cerus Corporation), uses amotosalen HCl
(a photoactive compound, formerly designated as S-59)
and long-wavelength UVA illumination to photochemi-
cally treat blood products. A compound adsorption de-
vice (CAD) is used to remove the residual S-59 and me-
tabolites after illumination. The second system, Ribofla-
vin pathogen reduction technology (Riboflavin PRT,
Terumo BCT), uses riboflavin (vitamin B2), a naturally
occurring, non-toxic compound, combined with UV light
to substantially reduce the level of disease-causing agents
in blood components. Over the storage of 5 days, the
viral infectivity and PLT in vitro properties, including
PLT yield, pH and residual S-59, were measured to as-
sess the impact of photochemical treatments to the vi-
rus-spiked APLTs.
2. Materials and Methods
2.1. Treatment of Double-Dose APLT
A schematic diagram on the experimental design is illus-
trated in Figure 1. The PLTs used were obtained from
donors who qualify for the donation criteria of double-
dose APLT collection. APLT was used for this pathogen
reduction study instead of whole blood derived PLTs as
the Singapore Health Sciences Authority, Blood Services
Group do not prepare pooled PLTs from whole blood
donations. To facilitate comparison, double-dose APLTs
were used to reduce donor-specific variability in the
samples subject to the two pathogen reduction processes.
The double-dose APLT donations were randomly se-
lected based on the fulfillment of the following quality
indicators as starting source material for pathogen reduc-
tion processing. All donors of the APLTs used in this
study were tested negative for past and recent dengue and
chikungunya infection by using Panbio IgM capture
ELISA and indirect IgG ELISA kits (Inverness Medical

Evaluation of Pathogen Reduction Systems to Inactivate Dengue and Chikungunya Viruses in
Apheresis Platelets Suspended in Plasma
3
Double–dose APLT
from apheresis
donor (>6 × 10
11
)
Donor’s
serum
sample
Antibody
tests for
DEN and
CHIK
Spilt into 2 units (APLT1,
APLT 2) with equal PLT dose
of >3 × 10
11
(Pre-treatment Day 1)
Add 10
7
units of
virus into APLT 1
Add 10
7
units of
virus into APLT 2
2mL aliquot
for PLT QC
Riboflavin PRT
Add Riboflavin
and subject to 3 J
per cm3 UVA light
(320 - 400nm)
Amotosalen PCT
Add S-59 and
subject to 6.2 J
per cm
2
UV light
(265 - 370 nm)
Place units in PLT incubator with agitation
at 22˚C for 5 days
2mL
aliquot
taken
within 16
hrs after
CAD
incubation
for
residual
S-59 test
2 × 2 mL aliquots taken for virus
infectivity tests and PLT QC assessment
(Post-treatment Day 1)
2 × 2 mL aliquots taken for virus infectivity
tests and PLT QC assessment
(Post-treatment Day 3 and Day 5)
Figure 1. Schematic flow diagram showing the experimental
design.
Innovations, Australia) and EUROIMMUN AG Chikun-
gunya IgM and IgG IIFT (Lübeck, Germany), in respec-
tive.
2.2. Cell Cultures and Virus Preparation
DENV-2 (strain ST10) were propagated in Aedes al-
bopictus cells (C6/36, ATCC CRL 1660) at 33˚C for 4 -
5 days and titrated by plaque assay. C6/36 cultures were
grown in Leibowitch L-15 medium (Invitrogen Corp.,
Carlsbad, CA), supplemented with 10% heat-inactivated
Fetal Calf Serum (FCS), 1% L-glutamine solution, 100
mM Penicillin/Streptomycin, at 28˚C. Maintenance me-
dium was similarly prepared as above but supplemented
with 3% FCS.
CHIKV (strain EHI0067Y08) was propagated in Baby
Hamster Kidney cells (BHK-21, Clone 15, courtesy of
NITD) at 37˚C for 3 days and titrated by plaque assay.
The cells were grown and maintained in RPMI media
(Sigma-Aldrich Corp., St. Louis, MO, USA), supple-
mented with 10% FCS, 1% L-glutamine solution, 100
mM Penicillin/Streptomycin, 7.5% sodium bicarbonate,
10 mM HEPES, at 37˚C with 5% CO2.
2.3. Pathogen Reduction Treatments, Sampling
and Storage of APLT Concentrates
To test the efficacy of each pathogen reduction treatment,
each APLT unit (about 260 mLs) was inoculated with
approximately 107 infectious units (or plaque forming
unit, PFU) of DENV-2 or CHIKV in 1 - 2 mL of culture
supernatant. The pre-treatment concentration was ex-
pected to be about 104 PFU/mL.
In units treated with the Amotosalen PCT system,
APLT spiked with virus was subjected to amotosalen
HCL (S-59) at 150 μmol per L of APLT and exposed to 3
J·per·cm3 UVA light (320 - 400 nm) for approximately 5
min. The PCT treated APLTs were subjected to a mini-
mum of 16 hours agitation in a storage container with
Compound Adsorption Device (CAD) to remove residual
S-59. In units treated with the Riboflavin PRT system,
spiked APLT was treated with 500 μM of riboflavin and
exposed to 6.2 J·per·cm2 UV light (265 - 370 nm) for
approximately 10 min. Both pathogen reduction treat-
ments were performed according to the manufacturers’
instructions. Three units of spiked APLTs were tested in
each system. For each system, a unit of APLT spiked
with virus and subjected to chemical treatment without
UV light exposure was used as a positive control (Pos-C,
n = 1). APLTs that received no virus, and no chemical
and UV treatments were referred as negative controls
(Neg-C, n = 5). All treated APLT units were maintained
at 22˚C on a PLT incubator for 5 days. For each APLT
unit, two aliquots of 2 mL were collected aseptically on
pre-treatment Day 1, followed by post-treatment Day 1, 3
and 5, for determination of in vitro PLT quality indica-
tors and/or for virus assays. The viral infectivity was
determined by plaque assays.
2.4. In Vitro PLT Quality Parameters
PLT yield and pH were determined in all pre- and
post-treated APLTs to assess the impact of inactivation
processes on APLTs. PLT samples were uncapped and
measured one at a time. PLT count was determined by
ADVIA 120 Haematology System and TIMEPAC re-
agents (Siemens Healthcare Diagnostics Inc, Germany)

Evaluation of Pathogen Reduction Systems to Inactivate Dengue and Chikungunya Viruses in
Apheresis Platelets Suspended in Plasma
4
on neat samples and pH was measured at 22˚C ± 1˚C
with Accumet Basic AB15 pH meter (Fisher Scientific,
USA) in an open system. The microprobe used was cali-
brated with 3 pH buffer solution at pH 4.0, 7.0 and 10.0
before use.
2.5. Virus Quantitation by Plaque Assay
A series of ten-fold dilutions (10−1 to 10−7) were made
with each treated and control APLT aliquot collected and
assayed using BHK monolayer cells on 24-well plates.
One hundred microliters of the neat or diluted aliquots
were added into each well, and incubated for 1 hr at 37˚C
with gentle rocking at 15 min intervals to facilitate ab-
sorption of virus and to prevent the drying out of cells.
After infection, supernatants were aspirated and 1 mL of
1% carboxymethylcellulose (Sigma-Aldrich) was over-
laid onto each well. The plate was incubated at 37˚C for
5 days till plaque development. After fixing the cells
with 20% formalin, overlaid medium was removed and
cells were stained with 1% napthol blue black stain
(Sigma-Aldrich). Plaques were counted and viral titer
was expressed as log PFU/mL.
2.6. Residual S-59 Analysis
S-59 level was assessed by Cerus Corporation using liq-
uid-liquid phase extraction method followed by HPLC
Tandem Mass Spectrometry (HPLC3408) with a detec-
tion limit of 0.125 μmol/L.
2.7. Statistical Analysis
The level of virus inactivation was calculated as log re-
duction: Log reduction = Log (pre-treatment titer/post-
treatment titer). If no virus was detected after treatment,
the viral titer was expressed as <1/V infectious units,
where V is the total PLT volume assayed [25].
In this study, the absence of plaques for each experi-
ment was confirmed on 4 replicate wells (total volume
assayed was 0.2 mL). The residual viral titer was <1/0.2
PFU/mL, which is equal to <5 PFU/mL or <0.70 log, for
each inactivation experiment. This detection limit is
equivalent to <1300 PFU in the total 260 mLs of APLT.
3. Results
3.1. Amotosalen PCT Reduced DENV-2 and
CHIKV Inoculated in APLTs to below
Detection Limits of All Assays
A distinct reduction of DENV-2 and CHIKV was ob-
served in all APLTs treated with Amotosalen PCT (Ta-
ble 1, samples A-C, Table 1 samples E-G). Results
Table 1. Inactivation of DENV-2 and CHIKV inoculated in
APLTs using Amotosalen PCT.
Pre-treatment
Day 1
Post-treatment
Days 1/3/5
Virus/APLTs
PA(Log titer, PFU/mL)
Extent of
log-reduction
(Pre- vs
post-treatment Day 1)
1. DENV-2
A 3.71 <0.7/<0.7/<0.7 3.01
B 1.7 <0.7/<0.7/<0.7 1.00
C 2.44 <0.7/<0.7/<0.7 1.74
D (Pos-C)2.3 2.30/<0.7/<0.7 0
2. CHIKV
E 4.4 <0.7/<0.7/<0.7 3.70
F 4.36 <0.7/<0.7/<0.7 3.66
G 4.45 <0.7/<0.7/<0.7 3.75
H (Pos-C)4.63 NC/4.15/3.36 0.48 (vs Day 3)
Even in the absence of detectable IgG, variable DENV-2 concentrations
were detected immediately after the virus inoculation (pre-treatment Day 1).
PA = plaque assay; PFU/mL = plaque-forming units per millimetre; Pos-C =
positive control; NC = not collected.
showed that the extent of log reduction of the system was
as high as 3.01 and 3.75 for DENV-2 and CHIKV re-
spectively. In this Amotosalen PCT reduction experiment,
the highest levels of viruses in APLTs were 103.71 PFU/
mL and 104.45 PFU/mL for DENV-2 and CHIKV respect-
tively. This treatment reduced the viral load to below
detection limits of plaque assay, <5 PFU/mL or <0.70
log.
We noticed that pre-treatment DENV-2 titers dropped
after spiked into APLTs. This might be attributed to the
surface receptor Fragment of Crystallization Receptor
(FcR) presented on PLTs that uptake DENV-2 [26].
3.2. Riboflavin PRT Showed Variable Degrees
in Reduction of DENV-2 and CHIKV
Inoculated in APLTs
Variable degrees of DENV-2 reduction, from 0.76 to
1.58 log, were noticed in APLTs that had undergone Ri-
boflavin PRT (Table 2, samples A-C). Only one of the
APLTs (sample B), which had the lowest initial DENV-2
inoculum titer of 101.88, showed complete viral inactiva-
tion of 1.18 log in plaque detection assay.
Likewise to Amotosalen PCT, Riboflavin PRT showed
complete inactivation of CHIKV with up to 3.73 log re-
duction (Table 2, samples E-G). The positive control
(sample H) showed presence of infective CHIKV through-
out the storage period, indicating the validity of this inac-
tivation experiment.

Evaluation of Pathogen Reduction Systems to Inactivate Dengue and Chikungunya Viruses in
Apheresis Platelets Suspended in Plasma
5
3.3. APLTs Treated with Pathogen Reduction
Systems Maintained Acceptable Yields and
Quality Parameters
The PLT yields of all test and control units were main-
tained between 2.63 to 3.74 × 1011 during storage (Table
3), in approximate range of the AABB acceptable criteria
of 3.0 × 1011 at end of storage lifespan [27]. Our results
show no significant loss of PLTs during the 5 days of
storage following pathogen reduction treatment, except
for one DENV-2 inoculated APLT that lost PLT concen-
trates of up to 22.47% following Amotosalen PCT (pre-
treatment Day 1 vs post-treatment Day 5, 3.69 × 1011 vs
2.86 × 1011, p = 0.046, Table 3). Other treated samples
of both systems showed variable percentages of PLT loss
as compared to the Neg-C (1.68% to 3.89%) during stor-
age.
The pH values of all test and control units were above
7.0 in the entire experiment (Table 4), well above the
acceptable pH value of ≥6.2 of the AABB standards for
PLT recovery [28,29]. The initial pHs of most test units
(prior virus inoculation) were higher as compared to
Neg-C (p < 0.026). Most test units had exhibited statisti-
cally significant pH reductions following treatments or
during storage as compared to pre-treatment Day 1 (p ≤
0.42) but overall the pH range is still within AABB
standard.
The residual S-59 of amotosalen-treated APLTs met
the 2 µM absolute value (0.5 µM per test unit) as rec-
ommended by CERUS Corporation (personal communi-
cation with Melody Holtan) during the entire storage
(Table 5). Following CAD treatment, the detected resid-
ual S-59 was maintained at the level much lower than the
recommendation during storage.
Table 2. Inactivation of DENV-2 and CHIKV inoculated in
APLTs using Riboflavin PRT.
Pre-treatment
Day 1
Post-treatment
Days 1/3/5
Virus/APLTs
PA (Log titer, PFU/mL)
Extent of
log-reduction
(Pre- vs
post-treatment Day 1)
1. DENV-2
A 3.46 1.88/<0.7/<0.7 1.58
B 1.88 <0.7/<0.7/<0.7 1.18
C 2.76 2.00/<0.7/<0.7 0.76
D (Pos-C) 2.95 2.54/<0.7/<0.7 0.41
2. CHIKV
E 4.34 <0.7/<0.7/<0.7 3.64
F 4.43 <0.7/<0.7/<0.7 3.73
G 4.4 <0.7/<0.7/<0.7 3.70
H (Pos-C) 4.52 4.04/3.46/3.36 0.48
PA = plaque assay; PFU/mL = plaque-forming units per millimetre; Pos-C =
positive control.
Table 3. Modifications in PLT yie l d during storage period.
Platelet count (×1011)
Post-treatment
Tr eat m en t/
Virus Pre-treatment
Day 1 Day 1 Day 3 Day 5
1. Amotosalen PCT
DENV-2 3.69 ± 0.223.24 ± 0.11
(12.27)
3.08 ± 0.11
(16.62)
2.86 ± 0.12
(22.47)*
CHIKV 3.27± 0.123.37 ± 0.12
(−3.06)
3.13 ± 0.03
(4.08)
3.03 ± 0.15
(7.14)
2. Riboflavin PRT
DENV-2 3.66 ± 0.023.33 ± 0.17
(9.75)
3.28 ± 0.16
(10.21)
3.38 ± 0.11
(7.47)
CHIKV 3.13 ± 0.122.83 ± 0.12
(9.57)
3.43 ± 0.12
(−9.57)
3.37 ± 0.12
(−7.45)
3. Neg-C3.60 ± 0.22- 3.54 ± 0.25
(1.68)
3.46 ± 0.24
(3.89)
Results are expressed as mean ± SEM, n = 3 per treatment group, and n = 5
for negative control (Neg-C); ( ) refers to percentage reduction as compared
to first collected sample. *Compared with Amotosalen PCT DENV-2 pre-
treatment Day 1, p = 0.046, Student’s t-Test.
Table 4. Modifications in pH during storage period.
Post-treatment
Treatments/
Virus
Pre-treatment
Day 1 Day 1 Day 3 Day 5
1. Amotosalen PCT  
DENV-27.64 ± 0.09 7.60 ± 0.09 7.45 ± 0.04 7.21 ± 0.00†
CHIKV7.74 ± 0.05* 7.64 ± 0.03 7.13 ± 0.06† 7.04 ± 0.10†
2. Riboflavin PRT  
DENV-27.60 ± 0.06* 7.55 ± 0.07 7.42 ± 0.03 7.12 ± 0.12†
CHIKV7.49 ± 0.02* 7.40 ± 0.01† 7.12 ± 0.06 7.12 ± 0.05
3. Neg-C7.33 ± 0.05 - 7.37 ± 0.03 7.26 ± 0.03
Results are expressed as mean ± SEM, n = 3 per treatment group, n = 5 for
untreated group. *Compared with Neg-C Day 1, p < 0.022, †Compared with
respective group of pre-treatment Day 1, p ≤ 0.042, Student’s t-Test.
Table 5. Quantity of residual S-59 detected in the APLTs
treated with Amotosalen PCT. SEM indicates standard
error mean.
Post-CAD level : S-59 (µmol/L)
Treated APLTs (n = 6)Day 1 Day 3 Day 5
Mean ± SEM 0.248 ± 0.015 0.305 ± 0.013 0.370 ± 0.019
Median 0.23 0.29 0.35
Range 0.22 - 0.26 0.28 - 0.35 0.32 - 0.42

Evaluation of Pathogen Reduction Systems to Inactivate Dengue and Chikungunya Viruses in
Apheresis Platelets Suspended in Plasma
6
4. Discussion
The two pathogen inactivation systems have independ-
ently shown to be effective in inactivating enveloped
viruses, including some flaviviruses and alphaviruses [25,
30-32]. In this comparative study, we used similar assays
to demonstrate the effectiveness of these systems in inac-
tivating two globally important viruses, DENV-2 and
CHIKV, that have posed increasing threats to the blood
supply [17,18,22].
Donor selection and deferral based on clinical and
travel history (to countries experiencing disease out-
breaks) are in practice to reduce the risk of transfusion-
transmitted dengue or chikungunya infection via blood
supply in the absence of effective screening tests [17,
21,32]. These strategies, however, may exacerbate the
ongoing demand of blood products, particularly during
disease outbreak situations. Furthermore, the significant
proportion of asymptomatic DENV and CHIKV infec-
tions [11,16,20] may result in the collection of contami-
nated blood or blood product from viraemic non-febrile
donors. Accompanying the global resurgence and emer-
gence of dengue is the increase in modal age of the cases
[22], suggesting that the blood donors age group is now a
susceptible population globally, and could contribute to
an increased risk of DENV contaminated blood product.
Altogether, these advocate the employment of pathogen
reduction treatments on blood products to sustain con-
tinual supply of blood products and reduce risk of disease
transmission via contaminated blood.
Interestingly, despite millions of reported cases each
year, globally there have only been a few reports of
blood and organ associated dengue transmission, and no
report of chikungunya transmission associated with
blood and organ donations. This is in contrast to the
situation of West Nile virus (WNV), also a vector-borne
flavivirus transmitted by mosquitoes, in the United States,
where 23 patients were reported to have acquired the
disease through transfusion in 2002 and where transfu-
sion-transmitted infectious disease (TTID) continues
despite implementation of nucleic acid-amplification
testing (NAT) [33,34]. A likely explanation is that the
dengue and chikungunya cases have gone unrecognized
as transfusion associated, due to the challenge of epide-
miological confirmation in endemic countries where the
risk of acquiring dengue from the community is high.
Another plausible reason for dengue is that dengue trans-
mission in endemic areas had largely occurred among
children, with most blood donors, who are adults, having
acquired immunity in their childhood. The high level of
seroprevalence among adult donors had in fact limited
and delayed this study. A high proportion of APLTs
(about 50%) was tested positive for anti-dengue IgG and
IgM, which interfered with the study by neutralizing the
spiked viruses even before treatment (our data, unpub-
lished).
In this comparison study, both systems showed com-
plete inactivation to CHIKV with reductions of up to
3.73 - 3.75 log. While a recent report on Riboflavin PRT
showed almost similar reductions of 3.5 log activity to
CHIKV [31], a higher log reduction of up to 6 log had
previously been documented on Amotosalen PCT [30].
Amotosalen PCT showed slightly better reductions effi-
cacy of up to 3 log for DENV-2 as compared to Ribofla-
vin PRT. Riboflavin PRT showed up to 1.58 log reduc-
tions to DENV-2, almost comparable to the 1.45 log re-
ductions reported by Faddy H et al. [35].
In this study, due to the limited availability of APLTs
that are free from dengue IgG, the other serotypes of
dengue virus were not tested. Limited tests, comprising
of only duplicates in separate experiments (instead of
triplicates in a single experiment) were done on DENV-1
(refer supplementary table, Table S1). Two separate ex-
periments on DENV-1 revealed a similar pathogen re-
duction trend as of the study on DENV-2. Amotosalen
PCT showed completely inactivation of DENV-1 with
log reduction of up to 4.18 while Riboflavin PRT re-
duced DENV-1 of up to 1.87 log. A recent study was
presented by Faddy H et al. on the inactivation of the
dengue serotypes 1, 2, 3 and 4 using Riboflavin PCT
which showed log reductions of 1.28, 1.45, 1.71 and 1.81
respectively [35]. Those results, consistent with our ob-
servations, demonstrate that log reduction titers obtained
by the Riboflavin system are relatively independent of
serotype. Taken together, both systems are likely to in-
duce similar inactivation effect on all four serotypes of
DENV.
It has been a challenge to define the required threshold
for transfusion transmitted infection to occur or to deter-
mine the amount of residual pathogen in the inactivated
blood product that is deemed safe for transfusion. Re-
ports have revealed asymptomatic blood donors of Hon-
duran could have dengue viral loads range of fewer than
3 × 104 to 4.2 × 104 copies per mL as quantitated by RT-
PCR [15] and Puerto Rico blood donors who were as-
ymptomatic were found to contain up to 8.12 × 107 cop-
ies per·mL using similar assay [14]. A recent report by
Stramer SL et al. has highlighted the viral load of as-
ymptomatic blood donors responsible for transfusion-
transmitted infections [13]. During the 2007 dengue out-
break in Puerto Rico, the blood of 12 donors were found
to contain DENV titers of 105 - 109 copies per·mL and
viral infectivity was demonstrated in mosquito cultures.
The blood implicated in the documented dengue transfu-
sion incident had contained 1.35 × 108 copies per·mL of
DENV-2. While a known viral titer was reported in this
transfusion-transmitted case, genomic copy numbers are

Evaluation of Pathogen Reduction Systems to Inactivate Dengue and Chikungunya Viruses in
Apheresis Platelets Suspended in Plasma
7
generally not equivalent to infective virus count. In a
recent study of Vanlandingham DL et al., they revealed
that the CHIKV viral RNA copies of 108 per·mL is
equivalent to 105.5 PFU/mL (considering that only 1 of
200 genome copies are infectious) and Riboflavin PRT
could not completely eliminate the risk of CHIKV infec-
tion if this high titer is found in the viremia blood [31].
Even so, the viral titer threshold to cause transfusion in-
fection is still unclear. For other arboviruses, asympto-
matic donors could have lower viral loads of 800
PFU/mL of CHIKV [20] or 0.06 - 0.5 PFU/mL of WNV
that has been documented to cause infection [36]. The
incidence of a PLT transfusion-associated transmission
of WNV was attributed to an asymptomatic donor who
has an estimated viremia of 0.11 PFU/mL of virus in the
implicated component. Taken together, the pathogen
viral loads could vary drastically among asymptomatic
individuals and the high virulence of certain pathogens
could cause transfusion-transmitted infection even at
minute level.
The efficiency of a pathogen inactivation technology
has been constantly contested and complicated by the
safety aspect and clinical efficacy of the treated PLTs
[30]. Assessment of post-treatment damage to the PLTs
includes the functionality and survival of transfused cells.
Our study had shown that Amotosalen PCT had caused
more than 20% of PLT lost in DENV-2 study. However,
out of the 6 APLTs treated by Amotosalen PCT, only 3
of them had PLT count marginally below acceptable
concentrate guideline of 3.0 × 1011 after 5 days of storage,
namely, 2.63, 2.80 and 2.96 × 1011. PLT decline has been
a common observation in pathogen reduction technolo-
gies. Significant PLT loss of 11% to below demanded
guideline dose due to photochemical treatments had been
previously reported in study of pathogen inactivation
technology [37]. Besides treatment effects, platelet ag-
gregation and the addition of culture media (and virus)
might also affect PLT yield further. This is in particular
that PLT aggregation by CHIKV [38] and binding of
DENV-2 to PLTs surface receptor FcR [26] had been
reported. Overall, our study had shown that the qualities
of all post-treated APLTs were maintained at around
acceptable ranges and the residual active pathogen re-
duction ingredient was lower than recommended.
In summary, data of this comparative study indicate
that both Amotosalen PRT and Riboflavin PRT can ef-
fectively inactivate the CHIKV spiked in APLTs. Amo-
tosalen PRT can completely eliminate DENV but Ribo-
flavin PCT has a lower efficacy to reduce DENV in con-
taminated APLTs. Together with previous individual treat-
ment studies on similar or other pathogens, this study
reaffirms the usefulness of pathogen inactivation systems
to ensure the safety in PLTs transfusion.
5. Acknowledgements
The authors are grateful to CERUS Corporation and Te-
rumo BCT for the provision of pathogen reduction dis-
posal sets and illuminator units to facilitate this pathogen
reduction study. This work is funded by the National
Environment Agency, Singapore. The funders had no
role in study design, data collection and analysis, deci-
sion to publish, or preparation of the manuscript. The
authors declare that they have no conflicts of interest
relevant to the manuscript submitted to Advances Infec-
tious Disease.
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Supplementary Data
Table S1. Inactivation of DENV-1 inoculated in APLTs using Amotosalen PCT and Riboflavin PRT.
Pre-treatment Day 1 Post-treatment Days 1/3/5
Treatment/APLTs PA (Log titer, PFU/mL)
Extent of log-reduction
(Pre- vs post-treatment Day 1)
1. Amotosalen PCT
Expt 1
M 4.44 <0.7/<0.7/<0.7 3.74
N (Pos-C) 4.7 4.44/3.74/3.30
Expt 2
O 4.88 <0.7/<0.7/<0.7 4.18
P (Pos-C) 4.74 4.72/3.57/2.30
2. Riboflavin PRT
Expt 1
M 4.51 3.78/<0.7/<0.7 0.73
N (Pos-C) 4.74 4.63/3.35/2.88
Expt 2
O 5.31 3.44/2.70/1.70 1.87
P (Pos-C) 4.78 1.83/2.81/2.70
APLTs were inoculated with 107 infectious units of DENV-1. The maximum number of IgG free APLTs obtained was two. Each inactivation process was
evaluated in two separate experiments. Amotosalen PCT showed complete inactivation of DENV-1 with log reduction of up to 4.18. Riboflavin PRT reduced
DENV-1 of up to 1.87 log. PA = plaque assay; PFU/mL = plaque-forming units per millimetre; Pos-C = positive control.