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International Journal of Microbiology

Volume 2020, Article ID 6658445, 6 pages
https://doi.org/10.1155/2020/6658445

Research Article
Correlation of Clinical Severity and Laboratory Parameters with
Various Serotypes in Dengue Virus: A Hospital-Based Study

Pooja Rao ,1,2 Achappa Basavaprabhu ,2,3 Suchitra Shenoy ,1,2

Nikhil Victor Dsouza ,2,3 Basavaiah Sridevi Hanaganahalli ,2,4 and Vaman Kulkarni 5

1
Department of Microbiology, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal,
Karnataka-575001, India
2
Manipal Center for Infectious Diseases, Prasanna School of Public Health, Manipal Academy of Higher Education, Manipal,
Karnataka 576104, India
3
Department of Internal Medicine, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal,
Karnataka-575001, India
4
Department of Pathology, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal,
Karnataka-575001, India
5
Department of Community Medicine, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal,
Karnataka-575001, India

Correspondence should be addressed to Pooja Rao; [email protected]

Received 18 October 2020; Accepted 5 December 2020; Published 15 December 2020

Academic Editor: Faham Khamesipour

Copyright © 2020 Pooja Rao et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Objectives. Dengue fever, being hyperendemic with analogous presentations as in many other acute febrile illnesses, poses a challenge
in diagnosis during the acute stage. Additionally, the coexistence of multiple serotypes further complicates the disease prognosis. The
study was undertaken to determine the dengue virus serotypes, clinical, and laboratory markers as predictors in the severity of
infection. Methods. A prospective study was conducted among 106 patients admitted with acute febrile illness having positive NS1
antigen/IgM ELISA. Clinical data were extracted from medical records including demographics, presence of comorbid conditions,
clinical presentation, laboratory investigations, and course including length of hospital stay and outcome. Detection of dengue
serotypes was done by multiplex reverse transcriptase polymerase chain reaction (RT_PCR). Results. Out of 106 RT-PCR-confirmed
cases, DENV-3 was the most common serotype found in 56 (52.8%) patients, followed by DENV-3 and DENV-4 coinfection in 27
(25.4%) patients. Coinfection with more than one serotype was witnessed in our study. Raised liver enzymes and increased ferritin are
good biomarkers in differentiating dengue from severe dengue with cutoff levels for AST (134 U/L), ALT (88 U/L), and ferritin
(3670 ng/ml). Musculoskeletal, followed by gastrointestinal, manifestations were comparatively higher than respiratory and cu-
taneous manifestations. Conclusion. This study provides more information on the dengue serotypes. The clinical spectrum along with
laboratory parameters such as ferritin, liver enzymes, platelet can be used as potential biomarkers in prediction of dengue severity.
The data demonstrated will be useful in early detection and monitoring of the disease.

1. Introduction Four antigenically different dengue virus serotypes

(DENV-1, DENV-2, DENV-3, and DENV-4) are known to
Dengue fever (DF) is a major public health issue. It is a cause infections in humans. Chances for developing dengue
common mosquito-borne illness that is endemic in the coastal hemorrhagic fever-dengue shock syndrome (DHF-DSS)
region of South India. Global burden estimate indicates 390 increases significantly with a history of the previous infec-
million infections per year. Among these, 70% actual burden tion with one of the four serotypes. Early diagnosis, sero-
was seen in Asia [1]. It is an important cause of mortality and typing, and providing timely warning of dengue fever
morbidity among the vector-borne illness. epidemics to the concerned authorities become very
2 International Journal of Microbiology

important for better patient outcomes and to curb the rapid Bio, Japan) were utilized in this study. A multiplex assay was
spread of the virulent serotypes within the community [2]. performed for detecting the 4 serotypes DENV-1, DENV-2,
The hyperendemicity with coinfection of two or more se- DENV-3, and DENV-4. The PCR amplification was carried
rotypes during the same period has been widely suspected as out with thermal cycling conditions as per the CDC Dengue
one of the major causes of disease severity in dengue patients Kit protocol: hold at 30 min at 50°C, holding cycle 2 : 2.0 min
in India [3]. Dengue surveillance becomes important when at 95°C; florescence acquiring at 15 sec at 95°C and 1 min at
multiple serotypes are prevalent in one individual [4]. 60.0°C for 45 cycles. The amplified products were accounted
In the current study, we aimed (1) to identify the dengue for an increase in fluorescence detection in a specific
virus serotypes in clinically suspected cases with dengue, as channel. The positive control was provided in the kit.
its distribution in this region is not documented, (2) to The study was conducted after the approval from the
determine the different serotype-specific clinical manifes- institutional ethics committee and with the informed con-
tations and its association with disease severity, and (3) to sent from the patients.
study the association of markers such as ferritin, platelet
count, and differential leucocyte count as risk predictors in
assessing the severity of dengue. 2.2. Statistical Analysis. The categorical data were analyzed in
the form of frequency and proportion. The quantitative data
were analyzed in the form of mean, median, and proportion.
2. Methods The complete data were entered and analyzed in SPSS version
A prospective study was conducted in patients admitted to 17. For the quantitative data, receptor operator curve (ROC)
the tertiary care center in Mangalore, India, with clinical was plotted to establish the cutoff concentration. For ferritin,
suspected signs and symptoms of dengue during the dengue Kruskal–Wallis test was used to study the relation with severity
outbreak 2019 in Dakshina Kannada district of Karnataka. in dengue. A p value <0.05 was considered as significant.
This region is endemic for dengue, and a steep rise in cases is
seen during every rainy season. Patients above 18 years were 3. Results
included in the study. Cases that were treated on an out-
patient basis or those who were less 18 years of age were Among 106 confirmed positive cases, dengue was pre-
excluded from this study. dominantly seen in males (68) than in females (38) (Fig-
Out of 3,801 suspected patients with fever during the ure 1). According to age-group-wise distribution, more
period of July to October 2019, 991 were tested positive by positive cases were seen in the age group of 21–30 years (36)
NS1/IgM ELISA. Serotype analysis was performed for 106 followed by 31–40 years (29), 51–60 years (14), 18–20 years,
patients. Patients were categorized based on WHO classi- 41–50 years (11), and above 61 years (5). The mean age was
fication as stage 1—dengue without warning signs, stage 35.27.
2—dengue with warning signs, and stage 3—severe dengue. The incidence of different dengue serotypes in this re-
A sample size of 106 was calculated considering a power of gion is shown in Figure 2.
80%, confidence level of 95%, proportion of DENV-1 to be The various comorbidities associated with dengue were
68.8%, and absolute precision of 5%. diabetes mellitus (10.4%), diabetes with hypertension (0.9%),
The formula used is and malignancy (0.9%). According to WHO dengue clas-
sification, 70.8%, 24.5%, and 4.7% belonged to stages 1, 2,
4PQ and 3, respectively.
N� , (1)
D2 All cases have presented with fever ranging from 99°C to
104.5°C followed by symptoms such as headache, malaise,
where N � sample size, P � proportion of interest (68.8%),
chills, and rigors. Musculoskeletal manifestations such as
Q � 1 − P, and D � absolute precision (5%) [3].
myalgia and backache followed by gastrointestinal manifes-
tations including vomiting, abdominal pain, and ascites were
2.1. Sampling Technique: Convenient Sampling the next common clinical presentation, with the least number
presenting with respiratory manifestations such as cough and
2.1.1. Serological Diagnosis. Acute phase serum samples cold and cutaneous manifestations such as rash and erythema.
within 7 days of onset of symptoms which were positive for DENV-3 followed by mixed serotypes DENV-3 and DENV-4
dengue NS1 rapid immunochromatographic test/IgM pos- presented with the above symptoms. Rash and cough was not
itive by ELISA (Panbio) were analyzed from 106 patients. a manifestation seen in the serotypes commonly found in the
The samples were stored at −20°C. present study (Figure 1 and Table 1).

2.1.2. Molecular Diagnosis. The viral RNA was extracted 3.1. Laboratory Parameters
using QIAamp Viral RNA Mini Extraction Kit (Qiagen,
Germany) as per the kit protocol. The extracted RNA was 3.1.1. Ferritin. In our study, the association of ferritin
stored at −80°C until use. The multiplex one-step reverse levels in patients categorized according to WHO clas-
transcriptase PCR was carried out for dengue serotype sification of dengue was significant with a p value of
confirmation. The CDC dengue primers and probes and the 0.009 and interquartile range from 925 ng/ml–13,829 ng/
™
master mix One Step Prime Script RT-PCR Kit (Takara ml. The maximum ferritin level rise being 87,945 ng/ml
International Journal of Microbiology 3

Suspected dengue case

3801
Excluded from study
outpatient department
NS1/IgM positive -991 <18 years of age

Hospitalized

106 patients analyzed by

random sampling

Stage 1 Stage 2 Stage 3

Dengue without warning signs: 75 Dengue with warning signs: 26 Severe dengue: 5

Figure 1: Flowchart depicting the case distribution.

Figure 2: Geospatial mapping of positive dengue cases by RT-PCR in this region.

was noticed in a severe dengue case. The mean ferritin 16,250–31,500, and stage 3 was 22,000–33,000. The maximum
levels in dengue without warning signs, dengue with platelet drop during the stay in the hospital did not show any
warning signs, and severe dengue were 2,304.48, 12,431.00, significance as a predictor of dengue severity. A total of 9
and 54655.25 ng/ml, and the cutoff values were 640, 2458, patients had platelet count above 1,50,000 cells/mm3.
and 35,930 ng/ml, respectively. The cutoff for ferritin to
differentiate between dengue and severe dengue was
3670 ng/ml. The area under the curve (AUC) for ferritin 3.1.3. Liver Enzymes. Serum ALT and AST observed were in
obtained in predicting dengue versus severe dengue is the higher range with a maximum value of ALT and AST
0.849 with 95% CI (0.712, 0.985). being 502 (IQR, 28–153 U/L) and 1737 (IQR, 40–240 U/L),
respectively. On plotting the receiver operating character-
istics (ROC), the cutoff for AST was 134 U/L and ALT was
3.1.2. Platelet Levels. The lowest platelet level observed 88 U/L. The AUC for AST and ALT obtained in predicting
during the stay in the hospital in stage 1 was 3000, stage 2 was dengue versus severe dengue is 0.757, 0.731 with 95% CI
46,076, and stage 3 was 58,400 cells per mm3. The median (0.586, 0.928) and (0.551, 0.911) (Figure 3).
platelet count in stage 1 was 85,000, stage 2 was 31,500, and An association USG gall bladder with the severity of
stage 3 was 33,000. The p value was significant with <0.05. The dengue was significant (p value 0.001). The fatty liver change
AUC for maximum platelet drop obtained in predicting was a common feature observed in our study. Hemopha-
dengue versus severe dengue is 0.635 with 95% CI (0.454, gocytic lymphohistiocytosis (HLH) syndrome was seen in
0.817). IQR in stage 1 was 53,000–85,000, stage 2 was 8.5% of infected individuals (Table 2).
4 International Journal of Microbiology

Table 1: Clinical and laboratory profile in dengue infection.

Present (%) Absent (%)
Symptoms
Fever 100 NIL
Headache 58.50 41.5
Vomiting 39.6 60.4
Myalgia 55.7 44.3
Chills/rigors 51.9 48.1
Backache 23.6 76
Rash 7 99
Cough/cold 7.5 98.5
Chest pain/palpitations NIL 100
Giddiness 13.2 86.8
Abdominal pain 10.4 93.4
Postural drop 5.7 99
Loose stools 3.8 96.2
Signs
Decreased Urine output 18 98.1
Ascites 13.2 86.2
Bleeding 9 99
Lab Parameters
Cytopenia 56 44
Thrombocytosis 9 99.1
Thrombocytopenia 99.1 9
ALT 85 21
AST 84 22

ROC curve
1.0

0.8

0.6
Sensitivity

0.4

0.2

0.0
0.0 0.2 0.4 0.6 0.8 1.0
1 – specificity

Ferith Max platelet drop

AST Reference line
ALT
Figure 3: ROC curve for various biomarkers as predictors of dengue severity.

3.2. Serotype Analysis. The most common monotypic se- DENV-3 was a common serotype in these cases. The se-
rotype in this region was DENV-3 seen in 56 (52.8%) pa- rotype distribution pattern was DENV-3 serotype and was
tients, followed by multiple serotypes DENV-3 and 4 in 27 found in 93 patients, DENV-4 in 39 patients, and DENV-2
(25.4%) patients; all 4 serotypes in 8 (7.5%) patients; and in 21 patients followed by DENV-1 in 12 patients (Table 3).
DENV-2, 3, and 4 in 7 (6.6%) of patients (Table 3). Multiple No association was found between the severity of disease
serotypes, with more than one type, were a feature, and and serotype, as the percentage of severe dengue was less in
International Journal of Microbiology 5

Table 2: Ultrasonography abdomen findings in dengue.

Dengue classification
Ultrasound
Dengue without warning signs (75) Dengue with warning signs (26) Severe dengue (5)
Fatty liver 68 (79.1%) 14 (16.3%) 4 (4.7%)
Gall bladder stones 2 (25.0%) 5 (62.5%) 1 (12.5%)
Gall bladder wall thickening 3 (42.9%) 4 (57.1%) 0
Splenomegaly 6 (7.9%) 8 (32.6%) 3 (60%)
Hepatomegaly 14 (16.3%) 5 (62.5%) 0

Table 3: Distribution of dengue serotype.

Serotype Number
DENV-3 56
DENV-3 and DENV-4 27
DENV-2, 3, and 4 08
DENV-2 and DENV-3 04
All 4 serotypes 08
DENV-4 02
DENV-1 01

number. DENV-3 was found in 8 out of 9 cases of HLH High prevalence of gastrointestinal and musculoskeletal
syndrome. The least common serotype was DENV-1. No symptoms was seen in DENV-3 in a study conducted in
fatalities were observed in the patients included in this study. Malaysia and India which is similar to our study, and cu-
taneous and respiratory symptoms were observed in DENV-
4 which altered as DENV-4 was not prevalent in the current
4. Discussion study [8, 10]. In a study conducted in the Taiwan population,
Dengue fever has a dynamic pattern ranging from mild DENV-3 cases presented with rash compared to DENV-2
febrile illness to a spectrum of manifestations including [11]. Another study by Kumaria showed DENV-4 presenting
hemorrhage, multiorgan dysfunction, HLH, and death. In with hemorrhagic manifestations, but with low positivity
the present study, the pattern of the clinical and laboratory DENV-4 rate [6]. A steady rise in the ferritin levels along
parameter in patients during the monsoon season 2019 with levels of 2,304 ng/ml in stage 1 DF and the highest level
dengue outbreak in Dakshina Kannada was studied. With up to 87,945 ng/ml seen among severe dengue with HLH
991 dengue IgM confirmed cases during four months, syndrome correlated well with studies conducted by Roy
dengue continues to be an endemic disease. The male Chaudhuri et al. and Soundaravally et al. [12, 13]. Thus,
population has dengue predominately in the ratio of 2 : 1. In ferritin can be an important serological biomarker in the
other studies, a similar male preponderance was seen [4]. diagnosis and prognosis of DF. Total cell count was di-
The probability is because this region is endemic for dengue minished in DF but did not draw parallel with the severity
and exposure of males to Aedes mosquito at their work place index. The liver enzymes such as serum ALT and AST were
or while traveling. In our population, dengue was seen in age significantly increased asserting that these parameters could
group between 20 and 40 years which could be attributed to also be used as markers for prediction of dengue in acute
increased movement of adults for earning their livelihood, febrile illness cases. Hyperferritinemia along with raised
easy accessibility to health care facilities, and awareness ALT and AST levels were noted as stated in a study by Ho
about high prevalence of mosquito-borne illness in the area. et al. [14]. Increase in ALT and AST levels were seen as a
On clinical evaluation, WHO stage 1-type DF was found marker to differentiate from other AFI excluding other
in a higher number of cases than severe dengue of stage 2 causes such as liver abscess and acute hepatitis [15].
and stage 3, which was comparable with studies conducted Thrombocytopenia was a feature in most of the cases with
in Bali [5]. All four serotypes existed during the study period. DENV-3 in our study which was similar to a study by Tsai
The data show the most common serotype prevalent in this et al. [11].
geographical region is DENV-3 which was seen in a study in
Indonesia and in different parts of India such as Kerala and 5. Conclusion
Uttar Pradesh [4, 6]. In Delhi, the DENV-2 serotype was
predominant [7]. There was no association of serotypes with DENV-3 serotype and DENV-3 and DENV-4 serotype
the severity of the disease. There is considerable variation in coinfection were prevalent in the Dakshina Kannada region
virulence of infecting serotypes which is dynamic. A com- of Karnataka, India. The present study concludes that
parative analysis of molecular serotyping could not be made coinfections with more than one serotype were not asso-
as ours was the first study on dengue serotyping in this ciated with disease severity in dengue infection. The study
region. Studies have shown the coexistence of multiple se- also showed that biomarkers such as ferritin and serum AST
rotypes in a single patient as a contributing factor in in- and ALT can be better predictors to assess the disease se-
creased severity of dengue in these cases [8, 9]. verity, and serial estimation of these markers should be
6 International Journal of Microbiology

considered. These outcomes provide novel data of clinical manifestations,” PLoS Neglected Tropical Diseases, vol. 6, no. 5,
and serotype characteristics for better management of p. e1638, 2012.
dengue in the population. [11] J. J. Tsai, K. S. Chan, J. S. Chang et al., “Effect of serotypes on
clinical manifestations of dengue fever in adults,” Journal of
Microbiology, Immunology, and Infection\Wei Mian Yu gan
Data Availability Ran Za Zhi, vol. 42, no. 6, pp. 471–478, 2009.
[12] S. Roy Chaudhuri, S. Bhattacharya, M. Chakraborty, and
The data sets used to support the findings of this study are
K. Bhattacharjee, “Serum ferritin: a backstage weapon in
available from the corresponding author upon request. diagnosis of dengue fever,” Interdisciplinary Perspectives on
Infectious Diseases, vol. 2017, Article ID 7463489, 6 pages,
Conflicts of Interest 2017.
[13] R. Soundravally, B. Agieshkumar, M. Daisy, J. Sherin, and
The authors declare that they have no conflicts of interest. C. C. Cleetus, “Ferritin levels predict severe dengue,” Infection,
vol. 43, no. 1, pp. 13–19, 2015.
Acknowledgments [14] T.-S. Ho, S.-M. Wang, R. Anderson, and C.-C. Liu, “Anti-
bodies in dengue immunopathogenesis,” Journal of the For-
The study was partly funded by Kasturba Medical College, mosan Medical Association, vol. 112, no. 1, pp. 1-2, 2013.
Mangalore, MAHE. The authors are grateful to Kasturba [15] S. S. Md Sani, W. H. Han, M. A. Bujang, H. J. Ding, K. L. Ng,
Medical College, Mangalore, MAHE, for funding this study. and M. A. Amir Shariffuddin, “Evaluation of creatine kinase
The authors sincerely thank CDC, Atlanta, for providing the and liver enzymes in identification of severe dengue,” BMC
Infectious Diseases, vol. 17, no. 1, p. 505, 2017.
dengue primers and probes for conducting this study.

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