PAPER TITLE
ANALYSIS OF BLOOD PARAMETERS & RT-PCR
RESULTS IN DENGUE SUSPECTED PATIENTS FROM
SRI LANKA
D.B.G.M. Ekanayake 1, 2, H.K.I. Perera1 A.N.B. Ellepola3 and S.B.P. Athauda1
1
Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka
Postgraduate Institute of Science, University of Peradeniya, Sri Lanka,
3
Department of Bioclinical Sciences, Faculty of Dentistry, Kuwait University, Kuwait
2
Corresponding author:
Dr. H.K.I. Perera, Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri
Lanka
E mail: kumudup@pdn.ac.lk, Fax: 94-81-2389106, Telephone: 94-81-2396329
ABSTRACT
Background & Objectives: Early laboratory confirmation of dengue viral infection is vital to minimize its
fatal outcomes. There is no information on the relationship between reverse transcriptase polymerase chain
reaction (RT-PCR) results and different blood parameters of patients with dengue fever (DF) in Sri Lanka.
Therefore the objective was to analyze the blood parameters and RT- PCR results in clinically suspected
dengue patients.
Materials and Methods: Blood samples were obtained from 111 in-ward clinically suspected adult male
dengue patients during the febrile phase of the infection. RNA was extracted from serum samples and
single tube multiplex RT-PCR was performed. Total white cell count (WBC), differential count, platelet
count, haemoglobin concentration (Hb), pack cell volume (PCV), aspartate aminotransferase concentration
(AST) and alanine aminotransferase concentration (ALT) were quantified.
Results: Of the 111 samples, 48 were positive with RT-PCR. All positive samples were of DEN-2
serotype. WBC, platelet counts, percentage of neutrophils and lymphocytes, Hb, PCV, AST and ALT
showed a significant difference (p< 0.05) between RT-PCR positive and negative samples. Increased ALT
levels was the most significant as more than 50 IU/ L of ALT levels was observed in 90% of the RT-PCR
positive patients whereas its increase was only in 3% in RT-PCR negative patients.
Conclusions: ALT was the most significantly affected parameter in RT-PCR positive group and appears to
be the best serological parameter among the tested, in early identification of patients with DF.
Key words:
Dengue fever, RT-PCR and ALT
INTRODUCTION
Dengue Fever (DF) is now endemic in more
than 100 countries and 100 million cases of DF and
half a million cases of dengue hemorrhagic fever
(DHF) occurs annually in the world1. Tropical
countries are most seriously affected by DF as
environmental conditions of the tropics favor the
development and proliferation of Aedes Aegypti, the
principal vector2.
Dengue virus is a single-stranded RNA virus in
the genus Flavivirus, family Flaviviridae. There are
four closely related serotypes of dengue virus (DEN
1-4) classified according to biological and
immunological criteria. Infection with any of these
serotypes may result in asymptomatic infection or
may cause a range of manifestations from nonspecific fever to DHF which is a syndrome
characterized by increased vascular permeability
and thrombocytopenia1. In severe cases, the
increased vascular permeability may result in lifethreatening dengue shock syndrome (DSS).
Infection with one serotype confers life-long
immunity to that serotype but not to heterologous
serotypes. Repeated (secondary) infection with a
different strain of the four serotypes of dengue
virus can result in more severe disease.
The first major epidemic of DHF occurred in Sri
Lanka in 1989-90 and its magnitude has continued
to increase ever since, documenting hundreds to
thousands of cases of DHF every year3, 4. DF is
currently the most important mosquito-borne viral
infection in Sri Lanka and data from the
Epidemiology Unit, has reported that the number of
cases recorded for the year 2009 exceeded 32000 5,
6
. Recent dengue epidemics were reportedly
associated with DHF and DSS leading to a
significantly high case fatality rate.
Early diagnosis of dengue is critical in the
absence of any licensed antiviral therapy and
prophylaxis. Patients recover fully without
progressing into DHF and DSS, if they are
managed appropriately. Hence early diagnosis of
dengue infection is vital. Its diagnosis on the basis
of clinical symptoms is not reliable, because most
infected patients are either asymptomatic or have a
mild
undifferentiated
fever7.
Therefore,
confirmation of DF is achieved by the detection of
the specific virus, viral antigen, genomic sequence,
and/or virus specific antibodies. After the onset of
illness, dengue virus is found in serum or plasma,
blood cells, and tissues of the immune system, for
approximately 2 to 7 days, corresponding to the
period of fever8. DEN-2 RNA has been isolated in
the liver, spleen and lymph nodes from fatal cases9.
Molecular diagnosis based on reverse transcriptase
polymerase chain reaction (RT-PCR), has gradually
replaced the virus isolation method as the new
standard for detection of dengue virus in acutephase serum samples10. Single-step multiplex RTPCR offers a sensitive and specific diagnostic
procedure for detection and serotyping of dengue
virus in clinical samples to make a definitive
diagnosis11, 12.
Blood parameters such as WBC and platelet
count have been used for diagnosis of DF as well.
However, information on the relationship between
these parameters and RT-PCR results has not been
analyzed previously in Sri Lanka. Furthermore,
analysis of blood parameters is less laborious, less
time consuming and less expensive and therefore
may be useful in early identification of true DF
patients until a positive confirmation is made by
RT-PCR. Hence the objective of this study was
firstly to confirm the clinically suspected dengue
patients at the acute phase using RT-PCR and
secondly to correlate the results of blood
parameters to RT-PCR positive and negative
groups.
negative controls were used in each batch of
analysis.
Total white cell counts (WBC), differential
count, platelet count, haemoglobin concentration
(Hb) and pack cell volume (PCV) were performed
using manual methods 13, 14, 15. Aspartate
aminotransferase
(AST)
and
alanine
aminotransferase (ALT) levels were analyzed using
assay kits (Catalog No. 12011 and 12012 Human
GmbH Germany). Data are presented as mean ±
standard deviation.
Blood parameters of RT-PCR positive and
negative groups were compared and were analyzed
using student’t- test [R console R version 2.13.1
(2011)]. Mean values of total study group (n=111)
were compared with the respective reference values
obtained from a group of 111 healthy adult male
volunteers (Reference), and with published
reference values15.
MATERIALS AND METHODS
Ethical clearance and permission from the
relevant authorities and informed written consent
from the patients were obtained.
Blood samples were collected from clinically
suspected male patients between the ages 20-50 y,
admitted to three hospitals during a period of one
year. Samples were collected within the first 3-5
days of febrile phase, who had at least two of DF
related symptoms (skin rash, retro orbital pain,
myalgia, arthralgia) and whose platelet count was
less than 150x109/ L. The demographic details of
the patients were taken from bed head tickets and
through questioning. Blood was collected in to a
sterile tube for RNA extraction and to an EDTA
coated tube for blood parameter analysis.
RNA was extracted from serum samples as
previously described using guanidine thiocyanate
method and purified using silica gel and eluted with
RNase-free sterile deionized water. Single tube
multiplex RT-PCR was carried out according to the
method described by Harris et al 11. The 5’ Primer
targeted a region conserved in all four dengue virus
serotypes and the four 3’ primers are targeted
sequences unique to each serotype. Reverse
transcriptase was used in the same tube. Forty
cycles were used to amplify cDNA. Expected sizes
of the RT-PCR products were, DEN-1: 482 bp,
DEN-2: 119 bp, DEN- 3: 290 bp and DEN-4: 389
bp. RT-PCR products were separated using
electrophoresis and the serotype was identified
based on the size of the products. Positive and
RESULTS
Forty eight samples out of 111 samples tested
(43%) were RT-PCR positive whereas the balance
63 samples (57%) were RT-PCR negative. All the
positive samples were of DEN-2 serotype.
The results of all the blood parameters tested in
RT-PCR positive and negative patient groups are
given in Table 1. Reference values of these
parametres of healthy males and previously
reported reference values15 are also given in this
table.
The lowering of the mean WBC count,
neutrophil percentage, platelets and Hb in RT-PCR
positive group compared to that of RT-PCR
negative group was statistically significant
(p=0.000 to p=0.003). These values in total
clinically suspected dengue sample were also
significantly lower than that of reference values.
However, the mean percentage of eosinophils of
RT-PCR positive group was not significantly
different from that of RT-PCR negative group.
Mean percentage of lymphocytes, PCV, AST
and ALT of RT-PCR positive group was
significantly higher than that of RT-PCR negative
group (p=0.000 to p=0.001). These values in
clinically suspected dengue patients was also
significantly higher compared to that of reference
values.
Percentage of samples showing measurements
below the lower limits of the normal ranges and
above the upper limits of the normal ranges has
been analyzed (Table 2). Accordingly, the most
significantly affected blood parameter observed in
relation to RT-PCR positivity was ALT level
among the tested parameters. Approximately 1/3rd
of RT-PCR positive samples had ALT > 100 IU/L.
Highest ALT level observed was 201 IU/L.
DISSCUSION
In the present study, out of the 111 samples
analyzed, 48 (43%) samples were RT-PCR
positive. All RT-PCR positive samples were of
DEN-2 serotype. Though the predominant serotype
prevailed during the study period within the regions
was DEN-2, this serotype as well as DEN-3 have
been previously identified as major circulating
serotypes prevailing in Sri Lanka1, 4, 16.
In our study, reduction of several blood
parameters such as WBC, platelets, Hb and
percentage of neutrophils below the normal range
was more prominent in RT- PCR positive samples
compared to that of RT-PCR negative samples
(Table 1). Elevation of several blood parameters
such as ALT, AST, PCV and percentage
lymphocytes (however, total lymphocyte count was
lower than that of RT-PCR negative group, as the
total WBC count was lower) above the normal
range was more prevalent in RT-PCR positive
samples compared to that of RT-PCR negative
samples (Table 1).
According to a study conducted in Sri Lanka
using adult patients, mean WBC count and platelet
count were started to fall from the 2nd day of fever
with the lowest counts observed on the 5th to 7th
day17. A review on features that differentiate DF
from other febrile illnesses indicated that low
platelet count and decreases in WBC and
neutrophils were independently associated with the
presence of dengue, in both adults and children18.
Our results were compatible with those findings
showing significant reduction of these parameters
in RT-PCR positive group.
In our study, most significantly affected blood
parameter observed in relation to RT-PCR
positivity was the ALT level among the tested
parameters. This finding is novel to Sri Lanka as
the blood parameters were compared after
confirming the samples using RT-PCR, with acute
phase samples. In a study conducted in Sri Lanka
using children with clinical features suggestive of
DF, 49% had high ALT and 67% had high AST 19.
However, method of confirmation of DF was based
on detection of IgM and IgG after the 7th day.
Several other studies have also reported elevations
of AST and ALT in majority of DF patients2, 20, 21, 22
but mostly after the acute phase of the illness. In
these studies, confirmation of DF was based on
detection of IgM and IgG2, 20, 21. One study used
virus isolation method for acute serum and
detection of IgM and IgG for samples collected
between 10th to 14th days after the onset of illness22.
Number of patients seen with elevated AST level in
DF in these studies was greater than that of ALT
level. However, in our study, number of patients
with elevation of ALT was greater than that of
AST. Aminotransferase levels increases from 1 st to
3rd day after the onset of illness and peak during the
second week of illness23. In our study, we collected
the samples during 3rd to 5th day of febrile phase
and therefore, before the appearance of peak
aminotransferase. We have observed elevations of
<201 IU/L. Higher ALT concentrations may have
been observed during the second week of illness.
The predominant elevation of ALT (compared to
that of AST) seen in our study may also indicate
more acute nature of the liver damage releasing
mostly the cytosolic markers. Another feature
revealed was that samples which had elevated AST
in RT-PCR positive group had elevated ALT too.
This feature was not seen in RT-PCR negative
group. This finding suggests that the liver is likely
to be compromised in DF as ALT is more specific
to liver where as AST observed in RT-PCR
negative samples may have been originated from
extra hepatic sources such as skeletal muscle23.
Liver is one of the major target organs
involved in acute DF. Involvement of the liver in
the pathogenesis of dengue virus has been indicated
by mild-to moderate increases in aminotransferase
levels7, 24. The pathogenesis of hepatic involvement
during DF is poorly understood.
Potential
mechanisms include direct effects of the virus or
host immune response on hepatocytes, ischemia
due to circulatory collapse, hepatotoxic effects of
drugs and tissue tropism of viral serotypes or
genotypes23. DEN-2 virus has been shown to
replicate actively and cause severe cytopathic
effects in liver cell lines25. Antibodies against non
structural proteins such as NS1 of dengue virus
have been shown to play a role in liver damage26.
In our study, immune response may not have
contributed to the elevation of ALT as a major
cause, as the blood samples were collected at a
stage where antibodies against the virus could not
have been detected unless the DF was due to a
secondary infection. There is experimental evidence
to suggest that liver damage could be serotypedependent and the damage has been found to
correlate with DEN-2, 1 and 3 in that order7.
Hence, whether ALT changes seen in our study are
more specific to DEN-2 or common to other
serotypes, need to be addressed.
Based on our findings, only 43% of the
clinically suspected dengue patients were
confirmed to have DF. Previous study conducted in
Sri Lanka17, suggested that a combination of
clinical picture, thrombocytopenia, leukopenia and
elevated liver enzymes could be used as markers
for early diagnosis of dengue infection. Our
findings support those findings and further, identify
ALT as the most distinctly affected blood
parameter in these patients. Therefore, our findings
could be considered as an early diagnostic method
to be implemented in clinically suspected DF
patients.
CONCLUSION
All the positive DF samples were of DEN-2
serotype. Compared to the RT-PCR negative group,
RT-PCR positive group showed a decline in WBC
count, platelets, Hb and percentage of neutrophils
and an increase in ALT, AST, PCV and percentage
of lymphocytes. ALT was the most significantly
affected parameter in RT-PCR positive group
and appears to be the best parameter among the
tested, in early identification of patients with DF.
Acknowledgments
Authors acknowledge the consultants and staff of
General Hospital Kandy, Base Hospitals
Mawanella and Padaviya and all patients enrolled
in the study.
REFERENCES
1.
2.
Gurugama P, Garg P, Perera J, Wijewickrama
A, Seneviratne SL: Dengue viral infections.
Indian J Dermatol 2010; 55(1): 68-78.
Souza LJ, Nogueira RMR, Soares LC, Soares
CEC, Ribas BF, Alves FP, et al. The Impact of
Dengue on Liver Function as Evaluated by
Aminotransferase Levels. Braz J Infect Dis
2007; 11(4): 407-10.
3. Messer WB, Vitarana UT, Sivananthan K,
Elvtigala J, Preethimala LD, Ramesh R, et al.
Epidemiology of dengue in Sri Lanka before
and after the emergence of epidemic dengue
haemorrhagic fever. Am J Trop Med Hyg.
2002; 66: 765-73.
4. Kanakaratne N, Wahala WMPB, Messer WB,
Tissera HA, Shahani A, Abeysinghe N, et al.
Severe Dengue Epidemics in Sri Lanka, 2003–
2006. Emerg Infect Dis 2009; 15(2): 192–9.
5. www.epid.gov.lk -Sri Lankan Government
official website.
6. www.healthedu.gov.lk
-Sri
Lankan
Government official website.
7. Vázquez-Pichardo M, Rosales-Jiménez C,
Rojas-Espinosa O, López-Martínez I, MorenoAltamirano MMB:
Is Liver Damage
Dependent on the Serotype of Dengue Virus? –
A Study in Mexico. Dengue Bulletin 2006; 30:
114-20.
8. World
Health
Organization,
Dengue
haemorrhagic fever: diagnosis, treatment,
prevention and control, ed 2, World Health
Organization, Geneva, Switzerland, 1997.
9. Sariol CC, Pelegrino JL, Marinez A, Arteaga
E, Kouri G, Guzman MG: Detection and
genetic relationship of dengue virus sequences
in seventeen-year-old paraffin embedded
samples from Cuba. Am J Trop Med Hyg 1999;
61: 994-1000.
10. Shu PY, Huang JH: Current Advances in
Dengue Diagnosis. Clin diagnostic lab
immunol 2004; 11(4): 642–50
11. Harris E, Roberts TG, Smith L, Selle J, Kramer
LD, Valle S, et al. Typing of dengue viruses in
clinical specimens and mosquitoes by singletube multiplex reverse transcriptase PCR. J
Clin Microbiol 1998; 36(9): 2634-9.
12. Mishara B, Sharma M, Pujhari SK, Appannna
SB: Clinical applicability of single-tube
multiplex reverse-transcriptase PCR in dengue
virus diagnosis and serotyping. J Clin Lab
Analysis 2011; 25: 76-8.
13. World Health Organization. Laboratory
diagnosis of Dengue hemorrhagic fever.
Diagnosis, treatment, prevention and control.
ed 2, 1997, vol 4, pp 34-47.
14. Cheesbrough M: District Laboratory Practice
in Tropical Countries, Cambridge University
Press, United Kingdom, 1998.
15. Lewis SM, Bain BJ, Bates I: Dacie and Lewis
Practical Haematology, ed 10, Elsevier Ltd.
2006.
16. Kularatne SAM, Seneviratne SL, Malavige
GN, Fernando S, Velathanthiri VGNS,
Ranatunga PK, et al. Synopsis of Findings
from Recent Studies on Dengue in Sri Lanka.
Dengue Bulletin 2006; 30: 80-6.
17. Kularatne SA, Gawarammana IB, Kumarasiri
PR: Epidemiology, clinical features, laboratory
investigations
and
early
diagnosis
of dengue fever in adults: a descriptive study in
Sri Lanka. Southeast Asian J Trop Med Public
Health 2005; 36(3): 686-92.
18. Potts JA, Rothman AL: Clinical and laboratory
features that distinguish dengue from other
febrile illnesses in endemic populations. Trop
Med Int Health 2008;13(11):1328-40.
19. Malavige GN, Ranatunga PK, Velathanthiri
VGNS, Fernando S, Karunatilaka DH, Aaskov
J, et al. Patterns of disease in Sri Lankan
dengue patients. Arch Dis Child 2006; 91: 396400.
20. Chhina RS, Goyal O, Chhina DK, Goyal P,
Kumar R, Puri S: Liver function tests in
patients with dengue viral infection. Dengue
Bulletin 2008; 32: 110-7.
21. Jain PK, Sharma AK, Agarwal N, Siddiqui
MZ, Pawal P, Gaba R, et al. A prospective
clinical study of incidence of hepatorenal and
hematological complications in dengue fever
22.
23.
24.
25.
26.
and management of symptomatic bleed in
bundelkhand region of Northern India with
fresh whole blood. J Infect Dis and Immunity
2011; 3(7): 124-33.
Souza LJ, Alves JG, Nogueira RMR, Neto CG,
Bastos DA, Siqueira EWS, et al.
Aminotransferase Changes and Acute Hepatitis
in Patients with Dengue Fever: Analysis of
1,585 Cases. Braz J Infect Dis 2004; 8(2): 15663.
Trung DT, Thao LTT, Hien TT, Hung NT,
Vinh NN, Hien PTD, et al. Liver Involvement
Associated with Dengue Infection in Adults in
Vietnam. Am J Trop Med Hyg 2010; 83(4):
774-80.
Seneviratne SL, Malavige GN, de Silva HJ:
Pathogenesis of liver involvement during
dengue viral infections. Trans R Soc Trop Med
Hyg 2006; 100: 608-14.
Lin YL, Liu CC, Lei HY, Yeh TM, Lin YS,
Chen RM, et al. Infection of five human liver
cell lines by dengue-2 virus. J Med Virol 2000;
60(4): 425-31.
Lin CF, Wan SW, Chen MC, Lin SC, Cheng
CC, Chiu SC, et al. Liver injury caused by
antibodies against dengue virus nonstructural
protein 1 in a murine model. Lab Invest 2008;
88(10): 1079-89.
Table 1. Blood parameters from RT-PCR positive and negative groups
Blood Parameter
RT-PCR positive
Mean values (
SD)
RT-PCR negative
Mean values (
SD)
Reference from
healthy males
Mean values (
SD)
Published
reference15
WBC (×106 / L)
3161 (542)
4354 (1065)
7255 (1729)
400010000
Neutrophil %
45 (4)
48 (5)
51 (10)
40 - 80
Lymphocyte %
53 (4)
50 (5)
46 (9)
20 - 40
Eosinophil %
1.8 (1.3)
1.8 (1.6)
4 (2.7)
1–6
Platelet (x109/L)
102 (25)
138 (32)
258 (57)
280 (130)
Hb (g/dL)
13.7 (2)
14.7 (1.6)
14.6 (1.4)
15 (2)
PCV %
49 (5)
44 (5)
45 (5)
45 (5)
AST (IU/L)
44 (13)
32 (9)
25 (6)
0 - 50
ALT (IU/L)
88 (36)
34 (10)
25 (7)
0 - 50
Reference values are given for comparison.
Table 2. Percentage of RT-PCR positive and negative samples showing abnormal blood parameters
Blood parameter
Cut off value
RT-PCR Positive
RT-PCR Negative
WBC (×106 / L)
< 4000
93.8
41.3
Neutrophil %
< 41
20.8
11.1
Platelet (x109/L)
< 150
95.8
63.5
Platelet (x109/L)
< 100
48
1.6
Hb (g/dL)
< 13
37.5
11.1
Lymphocyte %
> 55
31.3
12.7
PCV %
> 50
31.3
4.8
AST (IU/L)
> 50
23
3
ALT (IU/L)
> 50
90
3
ALT (IU/L)
> 100
31.25
0