1. CD4, CD8 and CD4/CD8 T-Cells Dynamics in HIV-1C Infected Patients on Truvada Based cART in Botswana
Prisca K Thami, Bonolo B Phinius, Sikhulile Moyo, Rosemary Musonda, Joseph Makhema, Vlad Novitsky, Max Essex, Richard Marlink, Simani Gaseitsiwe
Background: HIV attacks and depletes CD4 T cells hence the basis for CD4 cell counts as markers for HIV/AIDS disease progression. Antiretroviral therapy has significantly reduced CD4 T cell recovery however not significantly normalizing CD4/CD8 ratio. This neglects the function of CD4/CD8 ratio as a probable marker for HIV disease progression and ART efficacy as an inverted ratio increases the chances of progression to AIDS and the development of non-AIDS defining conditions. The aim of the study was to determine CD4 T-cell count, CD8 T-cell count and CD4/CD8 T-cell ratio dynamics in a retrospective study of 300 HIV positive adult participants on Truvada based cART.
Methods: A normal CD4/CD8 T-cell ratio was defined as ≥ 1.0. Median CD4/CD8 T-cell ratios were compared using Mann-Whitney test between age groups, gender, HBV status, HIV viral load category and CD4 T-cell. Linear regression was performed to explore the relationship between CD4/CD8 T-cell ratios and CD4 and CD8 T-cells in 24 months. All Statistical analysis was done by use of STATA.
2. Cost effective Real-Time PCR Assay for the detection of Occult Hepatitis B
Motswedi Anderson, Bonolo Phinius, Ntansode Mlopo, Tapiwa Nkhisang, Sikhulile Moyo, Theresa Sebunya, Rosemary Musonda, Simani Gaseitsiwe
Background: Occult Hepatitis B (OBI); the presence of HBV DNA with undetectable surface antigen (HBsAg) is undetectable using standard serological assays and can be missed during routine blood donations screening. Molecular OBI screening is critical, however expensive when using commercial HBV viral load assays which is not practical in most low resource settings. We evaluated a less expensive in-house real time PCR assay for detecting OBI.
Methods: We tested 80 samples from 54 OBI positive and 26 HBsAg positive individuals from a cohort of HIV infected adults initiating HAART in Botswana. HBV DNA levels were determined using COBAS® AmpliPrep/COBAS® TaqMan®HBV Test, version 2.0 (Roche Diagnostics, Mannheim, Germany) with a limit of detection of 20 IU/ml. For the in-house (IH) assay, samples were extracted using QIAamp® DNA Kit (Qiagen) and detected using ABI 7500 Real Time PCR System using TaqMan® Universal PCR Master mix (IH-TaqMan) and the Kapa Probe Force qPCR Kit Master mix (IH-Kapa). We estimated agreement and compared sensitivity of the IH assay to the commercial assay in detecting HBV DNA.
CD4, CD8 and CD4/CD8 T-Cells Dynamics in HIV-1C Infected Patients on Truvada Based cART in Botswana
Results: In subjects ≤ 35 years old at 24 months, median CD4/CD8 T-cell ratio was 0.6 (IQR: 0.4-0.7) vs. 0.5 (IQR: 0.3-0.7) in > 35 years old subjects, p-value=0.006. Baseline CD4/CD8 T-cell ratio was 0.3 (IQR: 0.2-0.4) in subjects with CD4 T-cells > 250 cells/ml and 0.2 (IQR: 0.09-0.3) in subjects with CD4 T-cells ≤ 250 cells/ml, p-value <0.001. CD4 T-cell count trajectory over 24 months showed a progressive increase (R2=0.26, p < 0.001), while CD8 T-cell count was steady (R2=0.00, p < 0.296). CD4 T-cell count correlated positively with CD4/CD8 T-cell ratio over 24 months (R2=0.38, p < 0.001). CD8 T-cell count had a negative correlation with CD4/CD8 T-cell ratio (R2=-0.21, p < 0.001).
Conclusion: CD4/CD8 T-cell ratios were significantly different between age groups, gender and CD4 T-cell counts. CD4 T-cell count increase over 24 months was the strongest predictor of CD4/CD8 T-cell ratio normalization.
Throughout this project I have got to learn data analysis using STATA and I have also been able to be exposed to Flow cytometry because of the interest I had in this. I have therefore become part of the Lymphoma Diagnostics by use of Flow Cytometry team here in Botswana. I have also been awarded an opportunity to further this knowledge at the Ragon Institute where I would train on more Immunological techniques.
Cost effective Real-Time PCR Assay for the detection of Occult Hepatitis B
Results: The amplification rate using IH-Kapa was 83.8% (67), compared to 58.8% (46) using the IH-TaqMan. A total 51.2% (27/54) of the occults were undetected with the IH-TaqMan whereas 13% were undetected using IH-Kapa. Of the 8 samples undetected by the commercial assay, IH-Kapa detected (6/8, 75%) and the IH-TaqMan detected (3/8, 37.5%). Agreement between IH-Kapa and the commercial assay was 78.8%. IH-Kapa assay had a sensitivity of 87% whereas the IH-TaqMan assay had 59.7%. The estimated cost for the commercial assay ($65/test) is at least twice the price of IH-TaqMan and at least three times the cost of IH-Kappa ($20/test).
Conclusion: The IH-Kapa is more accurate and less expensive for OBI detection compared to IH-TaqMan and commercial assays. The potential use of the IH-Kapa method for OBI detection warrants further investigation.
In this project I have been able to learn extensively the use the 7500 Real Time PCR System. I have also learnt DNA sequencing through the use of the Genetic Analyzer 3130xl. Throughout my internship I have also learnt abstract submission and improved a lot on my presentation skills.