Introduction
Dengue is a common febrile illness, and also one of the leading cause of morbidity and mortality particularly in developing countries. Dengue virus, an arthropod-borne, RNA virus that infects humans by Aedes mosquitoes and cause of major disease burden in tropical and subtropical countries worldwide.1 Dengue Virus seems to have hepatotoxic effects.2 DENV, is a major human pathogen, and are of four antigenically different serotypes.(although there is a report of 2013 that claims a fifth serotype has been found). These four subtypes are different strains of dengue virus that have 60-80% homology but which is most hepatotoxic is not focus of the researchers. In India, dengue is an endemic, and begins after 2 to 14 days incubation period from the bite of the mosquito, with high fever, headache, retroocular pain, lumbosacral pain, conjunctival congestion, and/or facial flushing with biphasic fever etc. The major difference is in the surface proteins of the different dengue subtypes. Infection induces immunity against the infecting serotype, but it gives only a short time cross protective immunity against the other types, which explains the phenomenon of Antibody-Dependent Enhancement.3 So we conduct this study to estimate the hepatotoxic effects of different dengue serotypes on the basis of liver transaminases, ALP and GGT.
Materials and Methods
The study design does not include the direct intervention to the patients as we collected the ELISA confirmed dengue patients sample for the study, we get the ethical clearance for the same. After taking the ethical clearance from the ethical committee, A hospital based prospective study was conducted from August 2017 to November 2018, on patients attending OPD and IPD, of Medicine Department at Shri Mahant Indiresh Hospital Dehradun. Informed consent was taken by the patients for the same. 60 ELISA confirmed Dengue Positive patients samples were selected.
Method for RNA isolation for serotyping
In 140µl of serum sample, 560µl of Lysis buffer and 5.4µl carrier RNA added, and incubated with intermittent vortex for 10 minutes. Then 560µl of absolute ethanol was added and vortexed. Out of this MCT (micro centrifugation tube) 700 µl of sample was transferred to the silica column and centrifuged at 10000 rpm for 1 minute in cooled centrifuge and decant the supernatant. Column dry washed and transferred to the new freshly labeled MCT. 60µl of elusion buffer was added and incubated at room temperature for 4 minutes. MCT with the column was centrifuged at 10000 rpm for 1 minute in cooling centrifuge. The silica column was discarded, and the extracted nucleic acid was used as a template for detection. The extracted template was utilized using Reverse Transcriptase PCR, Conventional PCR and Real Time PCR techniques and further visualized using Agarose gel electrophoresis techniques at the CMRL and liver Enzymes SGPT,4 SGOT.5 ALP,6 GGT7 were analyzed at Central Lab of SMI Hospital using the vitros 5600 fully autoanlyzer of the Orthoclinical-diagnostics.
Results
Out of 60 samples DENV-1 was 15, DENV-2 was 29 and DENV-3 was 16.
Well 1: 100 bp DNA difference ladder
Well 2: PCR amplicon of 482 for DENV-1 serotype
Well 3: PCR amplicon of 119 for DENV-2 serotype
Well 4 and 5: PCR amplicon of 482 for DENV-1 serotype
Well 6: PCR amplicon of 482 for DENV-1 serotype
Well 7: PCR amplicon of 119 for DENV-2 serotype
Well 8: PCR amplicon of 290 for DENV-3 serotype
The results of serotyping are described above in Figure 2 and Figure 1.
The results of the involvement of the Liver enzymes are tabulated below in Table 1 and represented graphically below in Figure 3, Figure 4, Figure 5.
In our results the DENV-3 was found to be most hepatotoxic among the different dengue viruses.
Discussion
Till date there are two hypothesis that explains the damage of liver in dengue patients. The first is immune enhancement hypothesis. In 2004, a strong correlation was found between T-cell activation and hepatic cellular infiltration in immune competent mice infected with dengue virus. It was noted that kinetics of liver enzymes elevation was also correlated with that of T-cell activation, which suggest a relationship between T cell activation and elevations of liver enzymes.8 One of the study detected the appearance of different helper cells and cytokines in human WBC's cultures, infected invitro with dengue virus. They reported that during infection WBC's (monocytes, B cells, T cells and mast cells) produced a large amount of cytokines.9 The second hypothesis relates the damage in the liver to direct virulence of the virus.10 According to these studies, we hypothesized the same mechanism responsible for the liver damage which occurred in our study. The aspartate aminotransferase (AST) levels in dengue infection tend to be greater than alanine aminotransferase (ALT) levels.11, 12
