Priyadarshini, A Padaki (2014) An Evaluation of a new improved assay [ExaVir version 3.0 (CAVIDI, Uppsala, Sweden)] for the estimation of the plasma viral load in HIV-1 and HIV-2 infections. Masters thesis, Christian Medical College, Vellore.
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Abstract
OBJECTIVES: 1. To quantitate the plasma viral load using a new modified assay (ExaVir version 3.0) by estimating the Reverse Transcriptase activity in the plasma of individuals infected with HIV-1 and HIV-2 confirmed by serological methods. 2. To correlate the plasma Reverse Transcriptase activity of HIV-1 measured by the new assay with the viral load measured using Real time PCR. 3. To correlate the plasma Reverse Transcriptase level of HIV-1 and HIV-2 measured by the new assay with the CD4+ T Cell Count. 4. To assess the utility of the new assay to monitor response to ART by analyzing the Reverse Transcriptase activity prior to and after the initiation of ART in HIV-1 infected individuals. METHODS: Study period: May 2012 - November 2013 Samples from 75 HIV-1, 13 HIV-2, 20 HIV negative and an additional 50 HIV-1 treatment experienced individuals were tested. ExaVir is an ELISA format to quantitate the RT activity in plasma(fg/ml) and gives corresponding viral load (copies/ml) with the help of the software provided. The lower detection limit was 200 copies/ml. For HIV-1, values were compared with VL measured by real-time PCR Artus HIV-1 RG RT PCR(QIAGEN, Hilden, Germany) and discordant samples or the ones with log10 difference of >0.5 were tested with FDA approved assay Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A). For HIV-2 samples, ExaVir VL was correlated with CD4 counts. The ExaVir and Artus real time PCR VL values in HIV-1 infected treatment naïve individuals were compared with CDC staging for HIV. RESULTS and CONCLUSIONS: ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) was able to detect 97.87% of the samples in the treatment naïve group (n=47) which had a detectable viral load with Artus HIV-1 RG RT PCR(QIAGEN, Hilden, Germany) assay and 29.4% of the samples in the treatment experienced group (n=17). 11 samples which showed undetectable viral load with ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) but detectable viral load with Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany) were tested with Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A) and 10 of these had undetectable viral load with Abbott as well. Twenty five HIV-1 samples with viral load < 5000 copies/ml were tested and 72.7% of the samples > 200 copies/ml were detected by ExaVir version 3.0(CAVIDI, Uppsala, Sweden) as compared to Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A) assay. However all except one samples >1000 copies/ml were detected and thus the assay proved to be sensitive in detecting samples >1000 copies/ml. The overall sensitivity of the assay was 76.7% and 82.5% when compared to Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany) and Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A) respectively for samples with viral load >200copies/ml. The same for samples with viral load >1000copies/ml, was 86.8% and 94.1% respectively. The assay correlated well with Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany) and Abbott Real Time HIV-1 (ABBOTT, Illinois, U.S.A) real time PCR assays. However in our experience we found that the Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany) assay over estimated the viral load and ExaVir version 3.0(CAVIDI, Uppsala, Sweden) viral load values correlated more with Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A) than with Artus HIV-1 RG RT PCR(QIAGEN, Hilden, Germany) assay. The intraclass correlation was best between ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) and Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A)(ICC=0.783,p<0.0001) followed by ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) and Artus HIV-1 RG RT PCR(QIAGEN, Hilden, Germany)(ICC=0.219,p=0.03) and no significant correlation between Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany) and Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A)(ICC=0.0.7,p=0.314). The regression line graphs also showed similar results. The agreement was best(r=0.813, n=32) between ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) and Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A) followed by ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) and Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany) (r=0.445,n=32) and least (r=0.409, n=40) for Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany) and Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A). Bland Altman plot showed a mean difference of +0.1 log copies/ml between Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany) and ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) , +0.2 log copies/ml between Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A) and ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) and and 0.0 log copies/ml between Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A) and Artus HIV-1 RG RT PCR(QIAGEN, Hilden, Germany) assays. There was a significant negative correlation between viral load measured by ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) assay (rho= -0.5, p<0.0001, n=125) and the CD4+ T cell counts for all the HIV-1 samples. This was similar to the data available on viral load by molecular tests and CD4+ T cell count in literature from the same population. There was significant negative correlation between CD4 counts and HIV-2 viral load (rho= -0.8, p=0.0039, n=9). Comparison between CDC clinical staging and viral load measured by ExaVir version 3.0 (CAVIDI, Uppsala, Sweden) and Artus HIV-1 RG RT PCR(QIAGEN, Hilden, Germany) for HIV-1 treatment naïve individuals was not statistically significant. The WHO NIBSC standard was tested in dilutions (50 to 800 copies/ml) with all three assays. The Abbott Real time HIV-1 assay detected the sample even with a viral load of 50copies/ml. The Artus HIV-1 RG RT PCR detected samples above 100 copies/ml. However ExaVir version 3.0 assay detected only one sample with 800 copies/ml. So the lower detection limit is of the assay as assessed by us is close to 1000 copies/ml. There are a few disadvantages for this RT assay. The turn-around time for the assay is 48 hours which is longer when compared to PCR. The amount of plasma required for the test is 1ml which might be difficult in paediatric patients as against 200μl and 800μl required for Artus HIV-1 RG RT PCR(QIAGEN, Hilden, Germany) and Abbott Real Time HIV-1(ABBOTT, Illinois, U.S.A) assays respectively. The standard curve generated with each run is based on the dilution of the standard provided with the assay and the maximum limit of detection varies with each run. Batch testing needs to be done for the samples since each run of the assay can accommodate 30 samples and the reagents given can be reconstituted and used only once per run. The temperature for incubation required is 33ºC which might not be possible in all resource limited settings. The advantages include the following. The assay can be performed with minimal expertise. Clean separate rooms are not required as compared to PCR. The instrument consists of a suction apparatus with waste container which occupies a small work table. The ELISA plate can be washed in buckets and does not require manual or automated washers. However there is a protocol given by the manufacturers if automation is desired. The instrument can be easily disinfected with alcohol and reused. The assay can detect any subtype of HIV-1 and HIV-2 samples. Also measuring the RT enzyme rather than detecting the HIV gene has an advantage in situations where mutations could have taken place in the HIV genome particularly in patients on treatment. The hands on time was approximately 3 hours on the first day and 2 hours on the second day. The software provided with assay converts the RT activity into corresponding viral load. The software provided on a CD is user friendly and can be installed for use with any version of Windows. The cost of the reagent is only half the price (Rs. 1250) of the one required for molecular assays (Rs. 2500) per test was reduced to half when compared to the molecular assays. In conclusion, ExaVir version 3.0 (CAVIDI, Uppasala, Sweden) assay can be used as an alternative assay to PCR for monitoring therapy in HIV-1 and HIV-2 individuals. Based on the sensitivity (even with a lower detection limit of 1000 copies/ml) and specificity of this assay, National AIDS Control Organization (NACO, India) can establish these kind of viral load monitoring assays in all State Reference Laboratories with good quality control measures including EQAS from Apex and National Reference Laboratories.
| Item Type: | Thesis (Masters) |
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| Uncontrolled Keywords: | Viral load, Reverse Transcriptase activity, ELISA, PCR, ExaVir Version 3.0 (CAVIDI, Uppsala, Sweden), Artus HIV-1 RG RT PCR (QIAGEN, Hilden, Germany), Abbott Real Time HIV-1 (ABBOTT, Illinois, U.S.A). |
| Subjects: | MEDICAL > Microbiology |
| Depositing User: | Subramani R |
| Date Deposited: | 23 Mar 2020 16:35 |
| Last Modified: | 17 Aug 2020 09:11 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/12436 |
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