Iranian Journal of Medical Microbiology

The present study was a hospital-based cross-sectional study conducted from July 2016 to September 2018 in a tertiary care center in Pune, India. The study was approved by Institutional Ethics Committee (Research Protocol No. IESC/PG/020/16) dated 10.11.2016.

Study design

Study design: - The present study was a hospital-based cross-sectional study.
The study period was Two years (July 2016 to September 2018).
The study population includes patients with a positive serological test for Dengue and healthy controls.
Thus, two groups were formed in the study
Study group: 150 cases of Dengue
Control group: 50 healthy controls
Following inclusion and exclusion criteria were used to select the study group subjects.
Inclusion criteria for study group:
All patients with a positive serological test for Dengue (Positive ELISA test; kit used: J. Mitra). The principle of NS1, IgM, and IgG ELISA was the “Direct Sandwich principle”, “MAC-capture ELISA”, and “GAC-capture ELISA,” respectively (12, 13). The manufacturer’s kit instructions were followed for interpretation of the result, which were as follows:

• If Dengue NS1/IgM/IgG units < 9, the tests were interpreted as “Negative”.

• If Dengue NS1/IgM/IgG units were between 9-11, the test was considered as “Equivocal”.

• If Dengue NS1/IgM/IgG units were > 11, then the test was interpreted as “Positive” (14-16).

The patients were classified as DF, DHF, and DSS according to WHO classification (17, 18).
All patients were within the age group of 15 – 65 yrs.
Exclusion criteria for study group:
a) Presence of other co-infections malaria, enteric fever, tuberculosis, HIV & HBsAg
b) Immuno-compromised patients
c) Pregnant females
Following inclusion and exclusion criteria were used to select the study group subjects.
Inclusion Criteria for Control Group:
Age & Sex matched healthy controls were considered for the Control Group.
Exclusion Criteria for Control Group:
Subjects having other co-morbidities like Hypertension, Diabetes Mellitus, HIV, and chronic kidney disease (CKD) were excluded from the studies.
All these patients were tested for Dengue serology by ELISA, and patients with positive serological tests were included in the study.
The presence of other co-infections, malaria, enteric fever, tuberculosis, HIV & HBsAg, Immuno-compromised patients, and pregnant females were excluded from the study.
Age & Sex matched healthy controls without co-morbidities were considered for the Control Group.
The Griess Nitrite Method measured serum NO levels (Cadmium granules modified) (5).

Test principle

The bio-molecule NO is short-lived, converting it to two stable products- nitrite (NO2) and nitrate (NO3). Cortas & Wakid invented the method, which includes the reduction of NO3 to NO2 by copper-coated cadmium granules (19). In this method, nitrate is first reduced to nitrite, then treated with sulfanilamide and N-1-naphthyl-ethylene di-amine. A red color compound is formed after the absorption spectrum, which is determined by spectrophotometry. The concentration of nitrite was determined by regression analysis. The NO was estimated in terms of total nitrites in all the study patients and controls included in the present study.
Normal levels of NO- 24.8-77.6µmol/L (20).

Data analysis
Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 16.0 software. Qualitative data: Frequencies were enlisted Quantitative data: - For continuous variables, Mean, Standard deviation.
 

The present study was conducted in the “Department of Central Clinical Laboratory (CCL), of Dr. D. Y. Patil Medical College and Hospital and Research Centre, Pimpri, Pune.” The study was conducted to assess the role of NO in the Pathogenesis of DF. A total of 150 patients with a positive serological test for Dengue were enrolled in the present study.
In the present study, it was observed that 64% of patients were male and 36% were female patients with a male: female ratio of 1.78:1. As men are more exposed to infected mosquitoes because of traveling time & working hours during daytime, there is slight preponderance compared to females in case of DENV infection. Our findings match with studies of Ashwini Kumar et al. (20) and Tejaswi et al. (21).
The majority of the patients in the present study (41.44%) were in the age group of 21-30 years of age, followed by <20yrs (30%) and 31-40yrs (12.67%). Our findings agree with Thai et al., who observed that the “median age for DENV infection in primary and secondary infection is 9-20 years and 14-31 years respectively” (19). Thus, it is observed that young males are affected more. This is because young males are exposed to different environments and are more likely to have mosquito bites than females and other age groups.
Thrombocytopenia was seen in 125(82.67%) cases in our study. (Table 4). Platelet count between 50000-100000/µL was observed in 24% of patients, while count between 20000-50000/µL was seen in 22.67% of patients. Platelet count was within normal limits in 25(17.33%) patients. Critically low platelet count (<20,000/µL) was observed in only 2% of patients, out of which 1(33%) patient suffered from DHF and the minimum platelet count noted in our study was 16,000/µL. Thrombocytopenia is caused by a virus's destruction of peripheral platelets or bone marrow megakaryocytes, which consequently reduces platelet production. The high DENV genome copies in platelets were directly correlated with the elevated platelet activation and the increased binding of complement factor C3 and IgG on their surface on day 4 (22). 
DF was diagnosed in 76.67% of patients, while DHF was diagnosed in 23.33% of patients, while no case of DSS was diagnosed. Our results are closer to the study by Ashwini Kumar et al. (20), who observed 83.9% with DF, 8.8% with DHF, and 7.3% with DSS. In a study conducted by Anjali S Raj et al. (23) study, DF patients were 49%, DHF patients were 40.8%, and DSS patients were10.2%. No case of DSS was diagnosed in our study. As the present study was conducted in the tertiary care institute, and the good patient care and adequate fluid replacement provided in the institute may be the possible reason for less detection rate of DSS.
Raised NO levels in 8% of cases of the control group may be because of some sub-clinical infections in apparently healthy subjects (Table 6). The difference observed was not statistically significant. It was seen that the rise in NO was observed in 24 (20.8%) patients with DF, while it was raised in only 1(2.8%) case of DHF, and the difference was observed as statistically significant (p=0.0247).
Rodhain and Rosen (1997) have discussed the clinical features of Dengue and the relation of NO with the severity of the disease (24). Activation of DV‐infected monocytes after stimulation of iNOS occurred both in vivo and in vitro, and the susceptibility of DV to NO production was also noted. NO inhibits aggregation, recruitment, and adhesion of platelets to the vascular endothelium.
Our study findings agreed with Valero et al. (25). NO and reactive oxygen species elicit modulating effects on inflammation and regulating immune responses. During inflammatory reactions, monocytes & macrophages produce a greater number of oxidants, including NO. The increased production of NO in patients with DF is an expected finding because DENV is capable of inducing NO in cultures of splenic cells and Kupffer cells (11). Platelets can generate NO by stimulating NO synthetase (26, 27).

 

Thus, regarding findings in the present study, it can be concluded that the rise in NO levels was statistically significant in DF patients as compared to DHF patients. NO plays a protective role in DF, while in DHF patients, the absence of rising NO levels may be one of the contributory factors leading to the progression of the disease and increased morbidity. NO levels can guide the physician about the evolution of a case of Dengue. The sample size of this study is small. Hence, a larger study and more investigations need to confirm the findings. It will also be interesting to study whether DHF patients will benefit from stimulating NO production in the future.

 

We sincerely acknowledge this study to Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune- 411018.
 

Conflicts of Interest

There are no conflicts of interest.