Journal of Chemical Sciences|Lupine Publishers
Abstract
Aim: Molecular Characterization of Chikungunya Virus in Serum- Relevance for Disease Management.
Methods and Materials: 30 cases were considered having symptom of fever suspected to have chikungunya. RNA was isolated from magnetic beads methods which were further studied for Real Time-PCR (RT-PCR).
Results: Among the 30 cases suspected for chikungunya fever, only 18 were positive for the same. Hence, the prevalence was calculated to be 60%. About 39% of the positive cases were in the age range of 21-40 years, which is the active age group of the total positive cases, 66.66% were males and 33.33% were females. The virus can be even transmitted together as a co-infection. Patients infected with chikunguniya virus are more likely to experience symptoms of high fever, severe polyarthralgia and rash with lymphopenia.
Keywords: Chikunguniya; Real time polymerase chain reaction; Aedes aegypti; Aedes albopictus
Abbrevations: CHIK V: Chikungunya Virus; CMRL: Central Molecular Research Laboratory; SGRRIM & HS: Shri Guru Ram Rai Institute of Medical & Health Science; CDNA: Complementary DNA; MRNA: Messenger RNA
Introduction
Materials and Methods
Collection of EDTA Blood & Separation of Serum/Plasma
Whole blood samples were centrifuged at 5,000rpm for 10- 15 minutes at 4°C. The serum was separated from blood & then subjected for nucleic acid extraction. Then Isolation of RNA for the detection of chikungunya virus was done by magnetic beads method where Magnetic carriers bearing an immobilized affinity or hydrophobic ligand or ion exchange groups or magnetic biopolymer particles having affinity to the isolated structure are mixed with the sample containing target compounds. The specimens with target nucleic acid are processed by lysis buffer and then the nucleic acids are efficiently bound to the specifically modified magnetic beads. The beads were removed from the solution and attached to the tube wall manually using a magnetic separator stand. After washing, purification and elution, ultra-purified RNA can be obtained. After RNA was obtained master mix was prepared for chikungunya RNA for qualitative detection by Real Time PCR. Reverse transcription was done (RT-PCR) for the extracted RNA specimens. In this method, RNA was first transcribed into complementary DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA). The cDNA is then used as the template for the qPCR reaction.Results
Figure 1: Amplification curves in Real time PCR for Chikungunya virus.
Chikungunya (CHIKV) is a mosquito-borne disease caused by
an RNA alphavirus of the Togaviridae family. The main mosquito
vectors are the aggressive Aedesaegypti and Aedesalbopictus. The
most common presentation of CHIKV is acute onset of fever and
polyarthralgia, sometimes followed by a macupopular rash. Other
associated symptoms can include headache, myalgia, nausea and
vomiting. These symptoms typically occur 3 to 7 days after the
mosquito bite and generally last 7 to 10 days. Rare complication of
CHIKV can include uveitis, retinitis, myocarditis, hepatitis, nephritis,
bullos skin lesions, hemorrhage, meningoencephalitis. The clinical
manifestations of CHIKV can be very similar to dengue fever, which
is transmitted by the same species of the mosquito. The 2 viruses
can be even transmitted together as a co-infection. Patients infected
with CHIK are more likely to experience symptoms of high fever,
severe polyarthralgia and rash with lymphopenia, whereas patients
with dengue fever are more likely to have symptoms of hemorrhage,
shock and death with associated laboratory derangements of
neutropenia and thrombocytopenia. However, because of the
close similarities, dengue fever should be strongly considered
in the differential diagnosis of patients suspected with CHIK.
Conformational testing for CHIK can be performed in one of the three
ways. These methods include viral culture if tested within first three
days of illness, PCR in the first eight days, or antibody serology after
the first three days of illness. Other laboratory abnormalities may
include lymphopenia, thrombocytopenia, elevated creatinine and
elevated hepatic transaminases. Treatment is primarily supportive.
The preventive measure among the individual level included the use of
mosquito repellents like coils, body creams, mosquito nets,
etc. At the community level, it is important to ensure that there are
no collections of water in household vessels or around dwelling
places. The usual recommendation is that on every fifth day, all the
vessels, which contain water, should be emptied; this observance of
a dry day breaks the life cycle of the mosquito [10].
From above discussion, we conclude that, Chikungunya fever is self-limiting, the morbidity can be very high in major outbreaks, resulting in heavy social and economic tolls. The prevention of the disease requires a planned approach, besides knowledge and awareness on the early warning signs. An integrated vector management through the elimination of the breeding sites, the use of anti-adult and anti-larval measures and personal protection will contribute to the prevention of outbreaks. A community empowerment and mobilization is crucial for the prevention and control of Chikungunya.
The authors are grateful to Honorable Chairman, Shri Guru Ram Rai Education Mission for his kind support, guidance and favor.
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Discussion and Conclusion
From above discussion, we conclude that, Chikungunya fever is self-limiting, the morbidity can be very high in major outbreaks, resulting in heavy social and economic tolls. The prevention of the disease requires a planned approach, besides knowledge and awareness on the early warning signs. An integrated vector management through the elimination of the breeding sites, the use of anti-adult and anti-larval measures and personal protection will contribute to the prevention of outbreaks. A community empowerment and mobilization is crucial for the prevention and control of Chikungunya.
Acknowledgement
The authors are grateful to Honorable Chairman, Shri Guru Ram Rai Education Mission for his kind support, guidance and favor.
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