Due to the lack of viral load monitoring in developing areas, WHO has recommended CD4+ cell count guidelines for when to initiate ART in resource-limited settings. In this dissertation, we review HIV/AIDS and HIV-1 viral load tests in developed and developing countries.
Literature Review
HIV/AIDS
Both this membrane and the matrix protein house the viral capsid, which consists of polymers of the core antigen (p24). This then allows the fusion of membranes and subsequent entry of the viral capsid into the host cell25.
HIV Disease Burden and the Motivation for more Effective Viral load Testing
Finally, there are chemokine coreceptor antagonists that disrupt the initial binding of HIV-1 to the CD4+ cell. Viral load can be defined as the level of HIV-1 RNA in plasma (copies/ml)2.
Viral load monitoring
Viral load monitoring tests often use one of the following scientific techniques to determine viral load. PCR has proven to be one of the most powerful tools for amplifying genes or their RNA transcripts66. The higher the initial copy number of the sample, the earlier the fluorescent signal is detected.
The measurement of this higher wavelength is analyzed to provide a count of the number of CD4+ cells present in the sample77. The sample is then loaded into the collection stage of the flow cytometer, drawn up into the fluidic system and then pumped into the flow chamber. The sample is then loaded onto the collection stage of the flow cytometer, drawn up into the fluidic system and pumped into the flow chamber.
The excitation of the cells results in an emitted fluorescence, which is converted into an electrical signal via photodetectors and analyzed.
Review of Current HIV-1 Viral Load Assays for Developed Countries
When the TaqMan probe is cleaved, the distance between the quencher and the TaqMan probe increases, resulting in detectable fluorescence emission, see Figure 15A. When the fluorescence intensity exceeds the background signal, the cycle number of the PCR is recorded at this time and used for quantification. Due to the close proximity of the TaqMan probe and quenchers, the fluorescence of the TaqMan probes is negatively suppressed by the quencher.
During the amplification step, Taq DNA polymerase extends the forward primer and cleaves the TaqMan probe, which then hybridizes to the amplified HIV-1 cDNA. When the TaqMan probe is split, the distance between the quencher and the TaqMan probe increases, resulting in detectable fluorescence emission2. When the fluorescence intensity exceeds the background signal, the cycle number of PCR at this point is recorded and used for quantification.
The cycle number is compared to a standard curve, whereby the HIV-1 RNA viral load is extrapolated from the cycle number 2.81.
Alternative HIV-1 Screening methods and approaches in Resource-Constrained
The low-cost alternative surveillance tools developed for viral load monitoring in resource-limited settings include; Cavidi ExaVir™ RT assay, PerkinElmer Ultrasensitive p24 assay and finally in-house RT-qPCR assays2. In addition, the Ultrasensitive p24 assay and the ExaVir™ assay are both technologically simpler compared to commercial RNA virus load assays and therefore do not require highly skilled operators2. Although the in-house RT-qPCR method is highly correlated with commercial viral load assays, it still requires expensive thermal cyclers and trained operators, hindering their use in decentralized laboratories and clinics2.
This assay also may not reflect response to ART and nucleic acid viral load assays, so further refinement and evaluation are needed so that this assay can be used for viral load monitoring in resource-limited settings2. RT-qPCR is commonly used in commercial viral load assays, but recently there has been the development of low-cost in-house RT-qPCR for HIV-1 RNA quantification. Although these inexpensive alternative viral load assays can be performed in centralized laboratories, they would still be very difficult to implement within rural areas.
HIV-1 POC viral load assays should be performed using a readily available low-cost device, less than USD 10 (ZAR 156).
Microfluidic Technology
Microfluidic Fabrication Process
At the end of the photolithography process, the resultant patterned silicon wafer is known as the master mold. From the exposure, a post-exposure bake is completed, which is then followed by development of the pattern that is transferred to the wafer. Briefly, the typical photolithography process includes the following steps; first, silicon wafers are prepared by dehydration annealing and photoresist coating on the wafer.
After the wafer is dried, it is aligned with the photomask in a mask alignment and exposed using UV light in the 350 – 450 nanometer (nm) range. After exposure, a post-exposure bake is completed, which is then followed by development of the pattern that is transferred to the wafer. The development process removes any photoresist that was not exposed to the pattern, creating etching within the mass.
The final step in photolithography involves stripping, a process that removes the remaining photoresist on the wafer using a phenol-based organic solvent94.
Advantages and Limitations of Microfluidics
The process of MSL for the thesis can be found in more detail in Appendix A. Both the thick layer and flow layer molds were placed in a convection oven and baked. Limitations of the platform are mainly related to the properties of PDMS, for example, chemicals to which the elastomer is not inert cannot be processed88.
Increasing temperatures is not feasible for the PDMS chip as this results in expansion of the PDMS chip. The softness of the PDMS elastomer limits the aspect ratio (height, length, and width) of the microstructures in the PDMS. If the aspect ratio of the height and length is too high or too low, it causes the microstructures in the PDMS to deform, generating defects in the resulting pattern.
Other limitations include; for now, problems with the implementation of microfluidic technology in the field of POC, as hand-held devices are required.
Current Applications of Microfluidic Technology
As a result, FITC cannot be detected by the antibodies due to the absence of the microsphere-HIV-1 complex. A graphical user interface (Figure 28) was created, which enables the control of the microfluidic chip and the microscope. An image of the microsphere before and after UV exposure is shown in Figure 33.
The result of a microsphere that had been previously photobleached (Figure 32) and then subjected to multiple incubations of FITC detection antibodies at 4 mg/ml was that with each incubation with FITC detection antibodies, the fluorescence intensity increased (Figure 34). An image of a microsphere becoming fluorescent after 1 incubation of FITC detection antibodies can be seen in Figure 35. The reason why objective (D) could not be well met is the detection limit of the microfluidic system.
Preparation of polystyrene goat anti-HIV-1 conjugated gp120 microspheres 1–10 µl of the microsphere solution was placed in 90 µl of PBS.
HIV-1 POC Viral Load Assays In Resource-Limited Settings
Dissertation Aim and Objectives
Monitoring a patient's viral load in developing areas still poses many hurdles to this day. Most rural areas rely on CD4+ cell counts or send samples to other laboratories that can perform the necessary tests to determine a patient's viral load. This remains largely unaffordable due to transportation costs and often leads to loss of patient follow-up due to the turnaround time of several weeks. 1.11.2.
To design, develop, and investigate the utility of an automated microfluidic device system that can capture, concentrate, and quantify whole HIV-1 particles via an ELISA approach. 1.11.3.
Materials and Methods
- Reagents
- Microfluidic Device Fabrication
- Experimental Setup
- Experimental Assays
Next, to determine whether the FITC detection antibody could be detected by the microscope filter cube set, a channel was filled with goat anti-HIV-1 FITC and imaged. The next experiment to analyze the microsphere was to test whether there was non-specific binding between the microsphere and the FITC detection antibody. A microsphere, previously treated with photobleach, was captured in the microfluidic chip and exposed to multiple FITC detection antibody incubations at 4 mg/ml.
After every 15 min of incubation, the FITC detection antibody was washed away with PBS and the fluorescence signal was measured. The identification of non-specific binding that occurs between the microsphere and the FITC detection antibody is important. The first experiment performed involved recording the natural autofluorescence of a microsphere and subsequently the fluorescence signal when the microsphere had been incubated with FITC detection antibody.
To try to avoid the presence of residues, PBS and FITC detection antibodies were filtered through a cellulose syringe filter.
Results
- Initial Stage of Microfluidic Designing
- Chip Fabrication by MSL
- Automation of Microfluidic Device
- Testing Experimental Setup and Reagents
- Photobleaching Autofluorescence
- Detection Antibody and gp120 Conjugated Microsphere Non-Specific Binding
- Evaluation of Efficient Blocking Agents
- Removing Debris in the System
- Off-Chip Viral Stock Validation
- On-Chip Detection of Virus Particle Concentrations
Each reactor contains three channels that can be supplied by any of the input ports (̴ 1000 µm in diameter) with desired reagents. The primary function of the LabVIEW script was to relay a recipe that was provided to the graphical user interface prior to the start of an analysis that controlled the analysis. This occurred despite removal of microsphere autofluorescence through UV photobleaching before and removal of excess FITC detection antibody via PBS wash before each incubation cycle.
The diamond points on the graph represent the raw fluorescence intensity values every 15 min during 1 of 4 experiments and not the mean values of all replicates. The first experiment performed involved recording the natural auto-fluorescence of the microsphere and then the fluorescence signal when the microsphere was incubated with an antibody to detect FITC (Figure 36). The mean fluorescence intensity for each of the concentrations was derived as follows; for each concentration, the experiment was repeated a total of 3 times.
Finally, the mean value from each of the three experiments was averaged together, resulting in an overall average for each of the three experiments for each viral concentration.
Discussion and Conclusion
Within this thesis, goals are to (A) design and create a microfluidic device in-house, (B) automate operations of the microfluidic device, whereby the experiment will proceed with minimal human involvement and (C) to improve the capability of the microfluidic system assay to quantify whole HIV-1 virus particles has been completed. However, due to the low limit of detection of the system, the final goal (D) of creating a standard curve of known HIV-1 viral particle concentrations could not be very well accomplished. Low-cost tools for diagnosing and monitoring HIV infection in low-resource settings
48 Børn og HIV/AIDS,
Optimized infectivity of the cell-free single-cycle human immunodeficiency viruses type 1 (HIV-1) and its restriction by host cells. Thus, the mean value for 500,000 vpc/µl is the average of the means for the 3 experiments. Therefore, the average value for 1 million vpc/µl is the average of the averages for the 3 experiments.
Assessing Different Blocking Agents
