Improved Detection of Dengue-1 Virus from IgM-Positive Serum Samples Using C6/36 Cell Cultures in Association with RT-PCR

Abstract

Dengue is the most important arboviral disease in the world, and its diagnosis is primarily made by serology. Virus isolation has been successful mainly in clinical samples obtained during the acute phase of illness, and is carried out through inoculation of clinical samples into C6/36 cell monolayers followed by the detection of infection by indirect immunofluorescence assay (IFA). We compared the efficiency of RT-PCR and IFA in the detection of dengue-1 virus after inoculation of C6/36 cells with samples obtained in the convalescent period of dengue infection. Out of 75 IgM-positive samples inoculated into C6/36 cells, 2 were positive by IFA while 17 were positive by RT-PCR. The 2 IFA-positive samples were collected during the acute phase of the illness; 17 positive samples were found by RT-PCR, including the 2 detected by IFA. For both methods, we also investigated the time necessary for viral detection using a fixed dose of 1 × 10⁴ viruses/ml. RT-PCR and IFA detected the dengue virus 1 and 4 days after virus inoculation, respectively. The results obtained here indicate that RT-PCR is the most sensitive method in the detection of dengue viruses using C6/36 cells for viral isolation.

Factors Influencing Dengue Virus Isolation by C6/36 Cell Culture and Mosquito Inoculation of Nested PCR-Positive Clinical Samples

“…However, for samples that could not be amplified in C6/36 cells the likelihood of amplifying first round PCR-positive samples in mosquitoes was modest with only 45% of these samples isolated in culture. These data are in agreement with the work of Oliveira De Paula and others 27 who showed that 78% of clinical samples could be isolated by C6/36 cells with positive first round (35 cycle) RT-PCR.…”

A Novel Semiquantitative Fluorescence-Based Multiplex Polymerase Chain Reaction Assay for Rapid Simultaneous Detection of Bacterial and Parasitic Pathogens from Blood

“…Some of them are gram-positive spore-forming bacteria, Bacillus anthracis and Clostridium botulinum that cause anthrax and botulism, respectively, 2 gram-negative bacterium Yersinia pestis that causes plague, 3 and viruses responsible for various hemorrhagic fevers. 4,5 Death due to anthrax occurs when the bacteremia reaches 10 7 to 10 8 bacilli/ml of blood. 6 Y. enterocolitica, a common contaminant of packed red blood cells, was responsible for 50% of all clinical sepsis episodes associated with the transfusion of red blood cells, of which 61% were fatal.…”

First evidence of dengue infection in domestic dogs living in different ecological settings in Thailand

“…This might be the result of low level of virus replicated in C6/36 cell line or the variation of virus strain or technical error due to handling with low viral load in the serum sample. Additionally, we used a combination of virus isolation and RT-PCR to confirm DENV genome in filtered supernatants of inoculated cell lines rather than to observe CPE or IFA as RT-PCR is more precise and sensitive [ 46 – 51 ], especially when detection of the low viral load present in the serum sample [ 52 ]. Although, Indirect immunofluorescence assay (IFA) is routinely used in the detection of DENV antigen in infected cell lines, however, this technique is time consuming and required experienced laboratory professionals.…”

“…At the time cell culture was performed, inoculation with low viral load present in dog serum samples, the concentration of DENV replicating inside the cells might be below the detection limit of IFA. RT-PCR is high sensitivity in detecting RNA replication in low viral load in the sample [ 46 , 52 ]. However, potential contamination during all steps of RT-PCR should be avoided.…”