Summary
In “STANDUP,” we have been developing two testing methods, CFNT and 3D-PCR, to address the lack of surveillance for dengue fever. These methods are designed to be high-throughput, cost-effective, and highly sensitive. We are working to demonstrate the functionality of “STANDUP” through experimental results and by assessing factors such as sensitivity, specificity, efficiency, and cost-effectiveness.
CFNT
CFNT, or Cell Fluorescence Neutralization Test, is a testing method designed to measure the levels of neutralizing antibodies against different serotypes of Dengue virus (DENV) in a subject's serum. CFNT is composed of infection detection cells that respond to antibodies and modify the fluorescence pattern when infected with SRIP. Furthermore, CFNT is characterized by being high-throughput, cost-effective, and having high sensitivity, similar to PRNT (Plaque Reduction Neutralization Test), while maintaining an equivalent level of sensitivity. We will now proceed to demonstrate this.
Demonstration of infection detection cells
In the CFNT (Cell Fluorescence Neutralization Test), the measurement of neutralizing antibodies in the subject's serum is based on the fluorescence ratio emitted by infection detection cells. In other words, the functionality of these infection-detection cells is a crucial element. We needed assurance that the infection detection cells were functioning correctly. As shown in the and below, the infection detection cells transfected with the synthesized pCMV N-Cre exhibit green fluorescence, indicating that they are producing EGFP exclusively. For more detailed information, please refer to the DesignResults, pages.
Analysis by agarose gel electrophoresis about pCMV N-Cre construction A: 0.6% agarose gel image, loaded with extracted pCMV N-Cre from E.coli and pCMV N-Cre digested with Afl II B: pCMV N-Cre plasmid map
SRIP infection detecting cells Vero transfected with pCMV N-Cre
Furthermore, in parallel with the transfection process, the results of electrophoresis confirmed that the positions of the observed bands match the designed sequence length.
These findings indicate that our designed sequence will not produce any fluorescent protein other than EGFP within Vero cells unless the switch is turned on.
Demonstration that infection detecting cells can be used without an incubator
In CFNT, the interpretation of results relies on the ratio of fluorescence in infection detecting cells, making test sensitivity dependent on the activity of these cells. However, mammalian cells, including Vero cells, require the use of an incubator to maintain their growth rate. As mentioned in the Design, if we can use mosquito cells for infection detection cells instead of mammalian cells with strict culture conditions, it increases the potential for conducting CFNT tests without the need for an incubator. We have obtained experimental results that support this possibility, showing that C6/36 cells, mosquito cells, can be cultured without the need to maintain a 5% carbon dioxide concentration. The addition of HEPES to the culture medium allows C6/36 cells to be cultured at room temperature (28°C) without the requirement for a 5% carbon dioxide concentration, as shown in the figure below ().
C6/36 cell growth curve C6/36 cells is cultured under optimal conditions (28℃ and 5% CO_2), and not optimal conditons (28℃, 0% CO_2 and HEPES 10, 20 or 30 mM)
Regarding sensitivity and specificity
The evaluation results for the D1-SRIP antigen neutralization assay are shown in the figure below. The dose-dependent inhibition curves for DENV-1 and D1-SRIP antigens were found to be equivalent. These curves represent dose-dependent inhibition percentage curves obtained using two types of serum and two types of MAbs (D1-IV-7F4 and D1-4G2) in the absence of complement in virus-antibody mixtures. (A)Furthermore, the results of neutralization assays conducted using DENV-1 antigen and D1-SRIP antigen with eight serum samples and six MAbs, both in the presence and absence of complement, were plotted on a scatter chart. As shown in the figure below, a significant correlation coefficient (R=0.919; P<0.001) was obtained between the two antigens. (B)This indicates that D1-SRIP antigen could serve as a substitute antigen for the authentic DENV-1 antigen in neutralization assays using Vero cells. It is conceivable that D2-SRIP, D3-SRIP, and D4-SRIP also exhibit a high degree of correlation with authentic viral antigens [1].
Evaluation of D1-SRIP antigen performance in a conventional Vero-cell neutralization test.
(A) Dose-dependent percent inhibition curves. Curves were obtained using D1-SRIP (closed circles) and DENV-1 (open circles) antigens with two indicated human serum samples (no. 551 and no. 1412), two mouse MAbs (D1-IV-7F4 and D1-4G2), and a human negative control serum (Negative control). All displayed results were obtained in the absence of a complement. Each datum represents an average of values obtained in two separate assays, with SDs indicated by error bars.
(B)Correlation between D1-SRIP and DENV-1 antigens. Individual PRNT50s obtained from eight human sera (open and closed circles) and six MAbs (open and closed triangles) in the absence (closed circles and triangles) or presence (open circles and triangles) of complement are plotted on the graph, with ordinate and abscissa indicating data obtained using D1-SRIP and DENV-1 antigens, respectively. The solid line is a linear regression line, with the R-value corresponding to the correlation coefficient.
There are several types of Dengue PRNT (Plaque Reduction Neutralization Test), and different assays are used by research institutions and vaccine development efforts. Dengue PRNT50, based on the guidance provided by the World Health Organization (WHO), involves some modifications to the traditional method. This assay is conducted in 24-well plates using a monolayer of Vero cells to visualize virus infection foci. Specific immunostaining with monoclonal antibodies (MAbs) against Dengue virus serotype-specific envelope proteins are employed to measure levels of Dengue virus serotype-specific neutralizing antibodies.
Validation results of the Dengue PRNT50 with optimized conditions have shown that both sensitivity and specificity are within an acceptable range[2].
Based on the information presented, SRIP serves as an excellent substitute antigen for authentic Dengue virus antigen. PRNT, known for its high sensitivity and specificity, suggests that CFNT using SRIP is expected to exhibit a comparable level of sensitivity and specificity to PRNT.
Regarding efficiency and price
CFNT demonstrates its superiority over the golden standard PRNT (Plaque Reduction Neutralization Test) in terms of efficiency for detecting neutralizing antibodies against the dengue virus. While PRNT typically takes approximately 7 days from the mixture of serum and virus intake to obtain results, CFNT can accomplish the same in just 3 days.
CFNT also proves its cost-effectiveness compared to the golden standard PRNT for detecting neutralizing antibodies against the dengue virus. According to our estimates, CFNT can conduct tests at approximately 16% of the cost of PRNT, as shown in the following graph.
Furthermore, CFNT requires fewer materials. For instance, PRNT necessitates eight 6-well plates per individual to examine the neutralizing antibody titers against the four dengue virus serotypes. In contrast, CFNT requires only one 96-well plate per individual, making it more efficient and cost-effective.
Efficiency of CFNT
CFNT demonstrates its superiority over the golden standard PRNT in terms of efficiency. While PRNT takes approximately 7 days from mixing serum and virus to obtaining results, CFNT can be completed in just 3 days ().
In addition, CFNT proves to be cost-effective compared to PRNT. According to our estimation, CFNT allows for testing at only 16% of the cost of PRNT (), as depicted in the graph below. Furthermore, CFNT requires fewer materials. For instance, PRNT necessitates 8 six-well plates per person to determine neutralizing antibody titers for all four Dengue virus serotypes. In contrast, CFNT only requires a single 96-well plate per person, making it more efficient and cost-effective.
Comparison of the time required for dengue virus neutralization testing CFNT: Cell Fluorescence Neutralization Test, PRNT: Plaque Reduction Neutralization Test. “The required time means the Time from inoculation of each cell with the virus-serum mixture until the results are known.
Comparison of per capita costs of Neutralization Test for dengue virus CFNT: Cell Fluorescence Neutralization Test, PRNT: Plaque Reduction Neutralization Test.
3D-PCR
Demonstration of 3D-PCR
3D-PCR is a method, as demonstrated in the Design section, where specific sample mixing is carried out to detect the dengue virus genome via PCR. We conducted a pseudo-experiment envisioning the use of this technique to detect virus genomes and obtained results that support the effectiveness of 3D-PCR for dengue fever surveillance.
The following electrophoresis results are for samples compiled using 3D-PCR, where DNA (pUC19) was placed in two wells at positions (x,y,z)=(8,7,3) and (4,3,6) (, ). In this experiment, you can observe DNA amplification at the positions corresponding to the components of coordinates where DNA was added on each face. These results demonstrate the functionality of the 3D-PCR method we devised.
3D-PCR where (8, 7, 3) and (4, 3, 6) is positive In this case, you can see that Sample x-4, x-8, y-4, y-7, z-3 and z-7 will be positive.
Analysis by agarose gel electrophoresis about 3D-PCR where (8, 7, 3) and (4, 3, 6) are positive 1.5% agarose gel image, from left to right X coordinate (x-1, x-2, …, x-12, P, N, M), Y coordinate (y-1, y-2, …, y-8, P, N, M), Z coordinate (z-1, z-2, …, z-10, P, N, M). P, N, and M means Positive Control, Negative Control, and Marker.
Regarding sensitivity and specificity
Currently, RT-qPCR, used in COVID-19 testing, is considered to have the highest sensitivity and specificity among various testing methods. It is a highly accurate method, but it may take some time to produce results.
Antigen and antibody test kits, on the other hand, offer the advantage of rapid testing at the point of care. Antigen tests detect specific proteins (antigens), while antibody tests identify pathogen-specific antibodies produced by the host's immune system.
However, antigen tests may not be suitable for diagnosing cases with low antigen levels, such as asymptomatic individuals with a low pre-test probability of infection. Additionally, antibody tests are less useful in diagnosing acute-phase COVID-19, as it takes approximately 1-3 weeks after the onset of symptoms for antibodies to develop. Furthermore, asymptomatic individuals may not mount a strong immune response, leading to low or absent antibody levels.
Both antigen and antibody test kits tend to have lower sensitivity compared to PCR, particularly when the quantity of antigen or antibodies is low.
In contrast, RT-qPCR can amplify viral RNA even when present in small quantities and maintain specificity through the design of virus-specific primers.
The High-Throughput PCR method we have devised operates on a similar principle to RT-qPCR. However, there are differences in the sample preparation phase. One notable distinction is that the mixing of samples in each tube dilutes the RNA. The degree of dilution depends on the number of wells in each direction (a, b, c for x, y, and z-axes, respectively). The dilution factors are bc for the x-axis, ac for the y-axis, and a*b for the z-axis. This could be a concern, especially for patients with low viral RNA levels. Nevertheless, we believe that this issue can be addressed by adjusting the scale based on the extent of the outbreak.
Conversely, combining samples into tubes could result in an excess of RNA when there are many positive cases. However, this concern can also be managed by adjusting the scale based on the prevalence of the disease.
Another potential concern is that the process of combining samples in each axis may be prone to errors by laboratory personnel. Yet, this issue can be resolved by adjusting the scale or introducing automated machines.
Therefore, while High-Throughput PCR has some concerns in the sample preparation phase, we believe that these issues are manageable. With proper adjustments and precautions, it can maintain the same level of accuracy as RT-qPCR while being an efficient PCR method.
Regarding efficiency
Time Efficiency of PCR
In terms of time efficiency for PCR, there is an improvement in the stages of RNA extraction and PCR reaction when compared to conventional methods(). The specific reaction times have been calculated based on the following assumptions:
- Total number of samples: 960
- Time required for mastermix adjustment: 10 minutes
- Time for reaction mixture combination: 0.5 minutes per sample
- Duration for RT-qPCR: 120 minutes
- Only one PCR machine available, with a maximum capacity (of the Thermal Cycler) for 96 samples
- The ISOGEN-LS kit is used for RNA extraction.
- For serum separation: 0.5 minutes per sample for reagent mixing and 10 minutes for centrifugation.
- For RNA separation: 0.5 minutes per sample for reagent mixing and 15 minutes for centrifugation.
- The centrifuge has a capacity of 30 samples, leading to a total of 32 centrifugation rounds (calculated as 960/30 = 32).
Conventional PCR
Given there's only one PCR machine:
The PCR reaction will be carried out 10 times due to 960 samples divided by the Thermal Cyclere's maximum capacity of 96.
Time for serum separation + RNA separation + mastermix preparation + reaction mixture combination + PCR reaction:
= (0.5 x 960 + 10 x 32) + (0.5 x 960 + 15 x 32) + 10 + 960 x 0.5 + 120 x 10
= 3450 minutes
3D-PCR (Positive 1)
Time for serum separation + mastermix preparation + sample compression + RNA separation + reaction mixture combination + PCR reaction:
= (0.5 x 960 + 10 x 32) + 10 + 120 + (0.5 x 30 + 15 x 1) + 30 x 0.5 + 120 = 1095 minutes
3D-PCR (Positive 2)
Time for serum separation + mastermix preparation + sample compression + RNA separation + reaction mixture combination + PCR reaction + specific PCR operations to identify 8 samples:
= (0.5 x 960 + 10 x 32) + 10 + 120 + (0.5 x 30 + 15 x 1) + 30 x 0.5 + 120 + (8 x 0.5 + 120)
= 1219 minutes
3D-PCR (Positive 3)
Time for serum separation + mastermix preparation + sample compression + RNA separation + reaction mixture combination + PCR reaction + specific PCR operations to identify 27 samples:
= (0.5 x 960 + 10 x 32) + 10 + 120 + (0.5 x 30 + 15 x 1) + 7.5 + 120 + (27 x 0.5 + 120)
= 1228.5 minutes
Time efficiency of 3D-PCR was graphed. The following shows the time required for each method and its breakdown when compared to conventional PCR.
Regarding price
3D-PCR demonstrates its cost-effectiveness compared to other dengue virus detection methods. Methods such as “RDT (Rapid Diagnostic Test)” and RT-qPCR are effective for detecting dengue virus in patient serum, but 3D-PCR can be conducted at a cost that is less than 10% of the price of these two methods ().
Comparison of per capita costs of dengue virus detection (3D-PCR at a scale of 960 people)
Reference
- Yamanaka, A., Suzuki, R., & Konishi, E. (2014). Evaluation of single-round infectious, chimeric dengue type 1 virus as an antigen for dengue func- tional antibody assays. Vaccine, 32(34), 4289-4295. Retrieved from https://www.sciencedirect.com/science/article/pii/S0264410X14008068 doi: https://doi.org/10.1016/j.vaccine.2014.06.017 1
- Timiryasova, T. M., Bonaparte, M. I., Luo, P., Zedar, R., Hu, B. T., & Hildreth, S. W. (2013). Optimization and validation of a plaque reduction neutralization test for the detection of neu- tralizing antibodies to four serotypes of dengue virus used in support of dengue vaccine development. The American Society of Tropical Medicine and Hygiene, 88 (5), 962 - 970. Retrieved from https://www.ajtmh.org/view/journals/tpmd/88/5/article-p962.xml doi: 10.4269/ajtmh.12-0461 1