Identification of Concurrent Infection by Multiple Dengue Virus Serotypes During an Epidemic in 2011 in Pakistan

J Microbiol Exp 2016, 3(3): 00088 DENV-1 and DENV-2 [6]. In 1994 the first lab confirmed cases of DENV-2 was reported from Baluchistan Province [7]. The prevalence of dengue virus serotype 3 in Pakistan was detected during the 2005 dengue virus outbreak in Karachi [8]. The cocirculation of DENV-2 and DENV-3 was identified in 2006 during the outbreak of dengue virus in Karachi [9]. In 2008 the cocirculation of three serotypes DENV-2, DENV-3 and DENV-4 was also documented during the outbreak of dengue virus in Lahore [10]. Recently it is reported that the dominant serotypes of dengue virus circulating in Pakistan from 2007-2009 are DENV2 and DENV-3. The dengue is a major public health problem in Pakistan over the last 20-25 years. In last few years co-circulation of different dengue virus serotypes is identified with mixed infection. From Pakistan only one study in which they reported some cases of mixed infection with DENV-2 and DENV-3.In present study we for the first time reported many cases of mixed infection with DENV-1/DENV-2, DENV-1, DENV-2 and DENV-3 from different geographic areas of Pakistan. The laboratory detection of dengue virus infection is achieved by virus isolation in cell culture, Serologic diagnosis by detection of IgM/IgG antibodies and more sensitive and rapid technique is the Molecular detection. From August to October 2011 a total of 87 serum samples were received at the department of virology National Institute of Health Islamabad Pakistan during an outbreak. These samples were collected from suspected dengue virus cases along the complete history sheet including the date of onset, age; sex etc. All serum samples were stored at -70 until further processing. All serum samples were analyzed for the detection of dengue virus IgM antibodies by using IgM capture ELISA (Panbio Tech Co Brisbane, Australia) according to the instructions provided by the manufacturer. RNA was extracted from 140ul of ELISA Positive serum samples by using QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to manufacturer instructions. In the present study RNA samples were analyzed by the molecular detection by using the real time PCR assay. Real time PCR is a one step assay using Primer pair and Probes specific to dengue serotypes. In single plex reaction mixture 5ul of RNA template was added with 50pmol of each primer and 9pmol of probe in 50ul total iScript one-step RT-PCR kit for Probes. Each reaction tube contains primers and probe for only one dengue serotype. In this assay one RNA sample was tested against four dengue serotypes in four separate reactions tubes [11]. In this technique the use of fluorescent probe enables the detection of PCR reaction product without need of gel electrophoresis. Another advantage of this assay is the ability to determine viral load in the patient sample which is important to understand the severity of dengue infection [12].