This present report was written as the result of completing the credited internship program organized by Indonesia International Institute of Life Sciences (I3L) in the Indonesian National Cancer Center, Dharmais Cancer Hospital. I did three tests to facilitate early HIV diagnosis through HIV rapid tests and regular HIV monitoring through HIV viral load test and CD4+ count test. Therefore, this report provided information on how Dharmais Cancer Hospital conducts the test, the test principle, cost, sample type, etc.
Finally, I would like to sincerely thank all the staff of the Clinical Pathology Laboratory Department of Dharmais Cancer Hospital for their guidance and support during my internship project. Dharmais Cancer Hospital participates in this strategy by offering three tests that make it easier to detect HIV at an early stage and to monitor HIV patients regularly. In addition, this study also evaluated the relationship between HIV viral load assay and CD4+ count test.
Four different relationship trends were observed, where HIV viral load assay showed to be indirectly or directly proportional to CD4+ count test. During this project, most research findings showed an inverse proportional relationship between the two tests, proving the efficacy of ART provided by Dharmais Cancer Hospital.
INTRODUCTION
Company
- Description About The Company
- Description of Department
- Product of The Company
All of these missions are necessary to achieve the hospital's vision as a compassionate and knowledgeable national cancer center. The internship project took place in the diagnostic departments, more precisely in the molecular diagnostics departments of clinical pathology laboratories. The goal of this department is to accurately diagnose cancer or other infectious pathogens (ie HIV, HBV, EBV, etc.) from samples of blood, urine, or other body fluids based on aspects of molecular biology.
This department is equipped with the latest diagnostic tools, distinct laboratory techniques (i.e. PCR, RT-PCR, immunophenotyping, karyotyping, etc.), and various human resource experts in their field. With the presence of this department, it is expected that each patient can receive personalized care that meets their needs. Dharmais Cancer Hospital offers various medical services including diagnostic tests, monitoring tests and therapy facilities.
In addition to services, this hospital also offers innovative products to support the internal and external needs of Dharmais Cancer Hospital, one of which is a radiopharmaceutical PET scan to determine the stage of cancer and evaluate the effectiveness of therapy in the patient. In addition, this hospital also supports many research projects related to cancer and other public health issues to further improve the future development of the medical field.
PROJECT DESCRIPTION
- Internship Project
- Project Background
- Scope of The Project
- Objectives / Aims
- Problem Formulation and Proposed Solutions
Three different molecular laboratory tests to diagnose and monitor HIV patients were covered during the two months of the internship project in the clinical pathology laboratory of Dharmais Cancer Hospital. Even the effectiveness of ART in suppressing the viral load itself is unknown or estimated to be only 1% (Gedela et al., 2008). A similar principle was also applied to the formation of the control band, which was composed of immobilized antibodies directed against immunoglobulin.
At the end of the test, excess specimens will then be absorbed into the absorbent pad. Only ten µL of samples were needed to perform the test with these sample types. Using this technique, HIV genetic material was amplified and the exponential phase of the amplification reaction could be observed, allowing quantitative detection of the initial viral RNA concentration (Fiscus et al., 2006).
The purification step was then performed using wash2 reagents, leading to an abundance of viral RNA at the bottom of the tube (Gupta et al., 2017). In general, a single RT-PCR reaction can run for 40 cycles, with each cycle doubling the initial amount of template DNA (Lorenz, 2012). In this study, the oligonucleotide reagent provided oligo dT primers that would bind to HIV-1 poly A RNA tail sequences, specifically in the pol integrase region of the HIV-1 genome (Mbisa et al., 2009).
During the amplification cycle analysis, the Abbott m2000rt machine further lowered the temperature to allow the detection of the amplification products based on the fluorescence signal. In general, HIV-1/IC probe fluorescence will be quenched by hybridization with the quencher oligonucleotide in the absence of the HIV-1/IC target. In contrast, the HIV-1/IC probe will prefer to hybridize with the target sequence in the presence of the HIV-1 target.
It used the principle of flow cytometry, in which the physical and chemical properties of cells are examined as they pass through a fluid stream through a flow cytometer (Adan et al., 2017). The laser beam is used as a light source that would provide signals to be detected by the detectors (Manohar et al., 2021). After the routine hematology test, 50 μL of whole blood sample was mixed with 5 μL of BD Tritest reagent CD3 fluorescein isothiocyanate (FITC) / CD4 phycoerythrin (PE) / CD45 peridinin protein chlorophyll (PerCP) (appendix 2).
FINDINGS
- Result
- Analysis/Discussion
Data of HIV viral load count and CD4+ absolute count of 20 patients were recorded (table 1) and were plotted in multiple line charts (appendices 4-7). Four types of result trends depicting the relationship between HIV viral load count and CD4+ absolute count were observed, and each of the types is shown in figure 6. About 65% of the patient showed that HIV viral load count was inversely proportional to the CD4+ absolute count (figure 6a).
In contrast, the HIV viral load count was directly proportional to the CD4+ absolute count based on the remaining 35% of the patient (Figure 6b). These graphs showed an increase in the absolute number of CD4s when the number of HIV viral loads increased or vice versa. On the other hand, the HIV viral load test confirmed the presence of HIV based on the amount of viral nucleic acid (Figure 4).
Compared to rapid HIV tests, HIV viral load testing may be less appropriate in resource-limited settings (WHO, 2014). This was possible because the HIV viral load test directly detected the presence of HIV antigen instead of HIV antibodies. About 65% of the results showed that the HIV viral load count was inversely proportional to the absolute CD4+ count (Figure 6a).
However, treatment-impotent cases were also found, which were indicated by an increase in the number of HIV viral loads, followed by a decrease in the absolute CD4+ count (appendix 4k). Different trends were also observed, where absolute CD4+ count and HIV viral load assay were directly proportional to each other (figure 6b). Assuming that these patients have not received any treatment properly, both the decrease in the absolute CD4+ count and the HIV viral load count may be due to the presence of other opportunistic infections.
Accordingly, the decline in absolute CD4+ counts after HIV viral load became undetectable (Figure 6d) indicated that patients were harboring additional opportunistic infections. If patients have received appropriate HIV treatment (ART), various factors can result in a directly proportional relationship between the HIV viral load count and the absolute CD4+ count. Nevertheless, some results showed that the absolute CD4+ count continued to decrease even though the HIV viral load count was reduced (Figure 6d).
In contrast, some trends showed that HIV viral load continued to increase as the absolute CD4+ count increased (Appendix 5g). In contrast, HIV viral load can increase significantly at this stage (Manoto et al., 2018).
CONCLUSION AND RECOMMENDATION
SELF REFLECTION
Abbott RealTime HIV-1 Internal Control (non-infectious shielded RNA with internal control sequences in negative human plasma). Abbott RealTime HIV-1 negative control (negative human plasma tested and found to be non-reactive for HIV-1 sequences). Abbott RealTime HIV-1 low positive control (non-infectious shielded RNA with HIV-1 sequences in negative human plasma).
Abbott RealTime HIV-1 High Positive Control (non-infectious shielded RNA with HIV-1 sequences in negative human plasma). The multiple line graph shows that the HIV viral load count was inversely proportional to the absolute CD4+ count. The multi-line graph shows that the HIV viral load count was directly proportional to the absolute CD4+ count.
Multiple line graph showing that CD4+ absolute count increased as HIV viral load count became undetectable. Multiple line graph showing that CD4+ absolute count decreased as HIV viral load count became undetectable. Viral load testing and the use of test results for clinical decision making for HIV treatment in Cameroon: An insight into the clinic-laboratory interface.
The missed potential of CD4 and viral load testing to improve clinical outcomes for people living with HIV in a less resourced environment.
