Development of shell vial culture assay for the rapid diagnosis of respiratory viruses using the human colorectal adenocarcinoma (CaCo2) cells

Abstract

Background: Respiratory diseases are common worldwide, which are caused by various respiratory viruses. As symptoms caused by these viruses are similar, laboratory diagnosis is essential to distinguish the virus. Conventionally, respiratory viruses are isolated by cell culture with a panel of cell lines. However, handling of several cell lines is labour intensive, and the turnaround time of conventional culture is long. In previous study, the use of human colon adeno-carcinoma (Caco-2) in conventional culture was investigated. The study has proven that Caco-2 is generally susceptible to the eight common respiratory viruses, i.e. Adenovirus, Influenza A and B, Respiratory Syncytial virus, Parainfluenza virus 1, 2,3 and 4. As turnaround time of conventional culture is long; therefore, in this study, rapid shell vial culture using Caco-2 cells were evaluated. Moreover, the application of Caco-2 shell vial culture on recovering human metapneumovirus (hMPV) was also investigated. Materials and methods: This study consisted of four stages. First, recovery of viruses by conventional culture and shell vial culture of Caco-2 were compared. Specimens were added to conventional culture and shell vial simultaneously. For conventional culture, formation of CPE was examined daily and IF staining was performed when CPE was indicated; meanwhile, shell vial culture were incubated for seven days and stained with IF to detect infected cells. In stage two, the effect of incubating shell vial culture in rolling drum was investigated. Shell vials inoculated with the same specimen in duplicate were incubated in rolling drum and without rolling drum simultaneously. IF staining was performed in day 2, and results were obtained. For those which are IF negative in day 2, second shell vial was further incubated to seven days before harvest. In the next stage, a large batch of samples was used to evaluate on the use of Caco-2 shell vial culture in day 2 and day 7. Lastly, Caco-2 shell vial and conventional culture and LLC-MK2 conventional culture were tested for isolation of hMPV. Results: Compared to Caco-2 conventional culture, recovery rate of shell vial culture was elevated slightly. When experimenting on the effect of incubation in rolling drum, results showed that recovery rate was raised in shell vial with rolling drum in day 2, moreover, the percentage of positive cells were increased significantly (p value < 0.05). Furthermore, in the evaluation of Caco-2 shell vial in day 2 and day 7, 75% of samples were isolated in day 2 while 85% were recovered in day 7. Lastly, in the investigation on recovery of hMPV, 53%, 42% and 17% hMPV positive cases were isolated by Caco-2 shell vial, Caco-2 conventional culture and LLC-MK2 conventional culture respectively. Conclusion: First, although recovery rate by shell vial and conventional culture were similar, turnaround time was reduced from a week to a few days by shell vial culture. Therefore, Caco-2 shell vial culture is a more efficient than Caco-2 conventional culture in isolating respiratory viruses. The study also showed that incubation of shell vial in rolling drum able to increase the number of positive cells. Furthermore, in this study, Caco-2 cells were also shown to be more efficient in isolating hMPV when compare to LLC-MK2 cells.