ETD Collection

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Author: search.filters.author.Bronze, Michelle SaltaoHas files: Yes

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    In vitro HIV-1 drug resistance phenotyping, genotyping and novel virological failure detection tools for clinical patient management.
    (2014-03-28) Bronze, Michelle Saltao
    Of the 22.5 million individuals infected with the human immunodeficiency virus (HIV) in sub-Saharan Africa, 62% of patients requiring treatment had access to highly active antiretroviral therapy (HAART) in 2011. The delivery of HAART and the appropriate laboratory monitoring of HIV positive individuals in sub-Saharan African countries has become a public health priority, an intervention which has and will continue to dramatically reduce HIV-related morbidity and mortality. Routine laboratory monitoring of HIV infected individuals should ideally include CD4+ T cell testing to assess when to start ART, viral load monitoring to assess virological failure on ART and when indicated, HIVDR genotyping.However, this is often not implemented in resource limited settings due to challenges such as inadequate infrastructure and laboratory capacity, amongst others. Thus the Affordable Resistance Testing for Africa (ART-A) initiative was established to develop an affordable HIV drug resistance testing (HIVDR) algorithm applicable to Africa. The objective of this study was to evaluate the role of in vitro HIVDR phenotyping in the context of HIV-1 subtype C (the most prevalent circulating subtype in sub-Saharan Africa), genotyping and genotypic interpretation tools using existing algorithms, as well as novel virological failure detection tools for clinical patient management. Current gold standard HIVDR phenotyping technologies use an HIV-1 subtype B backbone to create recombinant viruses with patient-derived polymerase (protease and partial reverse transcriptase). This backbone could impact on the in vitro phenotyping results of non-B subtypes, and therefore it was deemed necessary to establish the applicability of HIVDR phenotypic testing of subtype C polymerase when a commercially available subtype B backbone is used. One hundred and fourteen HIV-1 subtype C samples were HIVDR phenotyped against 17 antiretroviral drugs using both subtype B and C backbones and showed a high level of concordance between the two backbone phenotypic resistance profiles (95.8%; 1590 of 1660 fold change comparisons). Natural assay variability was largely responsible for discordant results. Results confirmed that HIV-1 phenotypic reverse transcriptase inhibitor drug resistance test interpretation is independent of the virus backbone subtype. No conclusions could be made for protease inhibitor resistance since limited samples from 2nd line failure were available. Subsequently, the HIVDR genotypic and phenotypic results of the 114 patient samples were compared to determine whether genotyping is a viable alternative to phenotyping. Results showed a 92.3% concordance between genotyping and phenotyping of individual drug comparisons for a number of HIVDR profiles. Discrepancies were attributed to phenotypic assay variability in addition to the role of mutation mixtures, which impacted genotypic interpretations. Overall, HIVDR genotyping is a reliable tool to detect and interpret antiretroviral drug resistance in HIV-1 subtype C infected patients, and can thus be used for clinical patient management. Once the accuracy of HIVDR genotyping was established, the development, validation and evaluation of a potential virological failure assay (ARTA-VFA) and a simplified HIVDR (ARTA-HIVDRultralight) assay was undertaken. A simplified and conceptually novel approach using a qualitative viral load assay with a pre-determined cut-off that gives a threshold above which virological failure (VF) could be confirmed and below which treatment success was likely, was tested. A real-time PCR (ARTA-VFA) assay was developed which involved the amplification of a short sequence of the HIV-1 LTR region from RNA extracted either from plasma and/or dried blood spots (DBS). The ARTA-VFA was tested on 409 patient samples,and successfully amplified samples from all major HIV-1 group M subtypes with equal specificity. The VF was qualitatively classified as a viral load >1000 RNA copies/ml in plasma samples, and >5000 RNA copies/ml in DBS samples. Comparative testing yielded accurate VF determination for therapy-switching in approximately 93% of clinical cases tested, compared to current gold standard quantitative viral load assays. A simplified HIVDR genotyping assay (ARTA-HIVDRultralight) targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line regimen failures was developed and assessed. The ARTAHIVDRultralight assay was designed to be practical, faster, and more affordable, show flexibility with respect to equipment (open platform), use DBS or plasma as starting material and amplify and sequence a smaller amplicon (RT). The assay performed well when compared to the in-house assay used in the laboratory at the time for both 212 plasma and 25 DBS samples, yielding identical mutations and subsequent resistant profiles. Furthermore, a theoretical in silico exercise to investigate the consequences of using 125,329 shortened RT genotype (ARTA-HIVDRultralight) as compared to full-length RT sequences showed >95% and >90% concordance when using the Stanford HIVdb algorithm and the virco®TYPE tool, respectively. Differences noted were minor and unlikely to have any impact on clinical decision-making. Overall, this study illustrated that the short RT sequences can be reliably used to generate HIVDR genotypes using the Stanford HIVdb and virco®TYPE algorithms and reduce sequencing costs substantially. A field evaluation using the ARTA-VFA and ARTA-HIVDRultralight on 288 clinical samples was conducted, showing that the accuracy and precision of both assays (using 248 plasma or 40 DBS sampling methods) compared well to the reference methodology, thereby extending access of testing to more remote settings.These assays were designed to either be used as a testing strategy of initially assessing VF,and once confirmed performing an HIVDR assay, or alternatively to be used separately as stand-alone, or within different laboratory tiers in resource limited settings. It is envisaged that the ARTA-VFA could be used in the middle laboratory tier, and if confirmatory, patient samples can be referred to a reference laboratory with the available infrastructure for HIVDR testing using the ARTA-HIVDRultralight. Lastly, an automated sequence analysis and editing software for use in correct base calling of nucleotide/mutation mixtures in HIVDR genotyping was validated on 1624 sequences. Compared to reference software, where interpretation is often operator dependent, this software performed extremely well, with minor discrepancies noted. The automated software can be used to reduce subjectivity, time taken for analysis which is often the rate-limiting step and thus improving the turn-around time and clinical relevance of HIVDR genotyping. Overall, the results obtained describe the validation of using HIVDR genotyping as an alternative tool to phenotyping, and the subsequent development and validation of simple, affordable, "open-platform" alternatives to currently used methods for virological failure monitoring, and accommodate a centralized approach to HIVDR with DBS testing in resource limited settings.
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    Interaction between dietary iron overload and aflatoxin B1 in hepatocarcinogenesis using an experimental rat model
    (2007-02-22T13:16:03Z) Bronze, Michelle Saltao
    Hepatocellular carcinoma (HCC) is the most common primary malignant tumour of the liver. Aflatoxin B1 (AFB1) is a potent hepatocarcinogen, and dietary iron overload has been shown to contribute to HCC development in black africans. Both are well studied hepatotoxins. The aim of this study was to use a Wistar rat model over a 12 month period to investigate synergy and the extent thereof between AFB1 ingestion and dietary iron overload. 25ug/day of AFB1, reconstituted in DMSO, was administered by gavaging the animals, over a period of 10 days with a 2 day interval in between. The chow diet was supplemented with 0.75% (w/w) ferrocene iron. Experimental subjects were divided into 4 groups. Group 1 was fed the normal chow diet. Group 2 was fed 0.75% (w/w) ferrocene iron alone. Group 3 was gavaged 250μg AFB1 alone. Group 4 was fed the 0.75% (w/w) ferrocene iron and gavaged 250μg AFB1. A number of assays were conducted to investigate synergy. Colorimetric assays were used to measure serum iron, total-iron binding capacity, ALT, AST, GGT, nitrite production, lipid peroxidation and hydroxyproline concentrations. ELISA’s were used to determine ferritin, 8-isoprostane and 8-hydroxyguanosine concentrations. Nontransferrin bound iron was measured using an HPLC method. A chemiluminescent assay was used to measure superoxide anion production. Cytokines were measured using a suspension array system. Mutagenicity was assessed using the Ames mutagenicity assay using salmonella typhimirium strains TA97, TA98, TA100 and TA102. Iron profiling indicated that iron overloading occurred with the ingestion of the ferrocene diet. Biomarkers of oxidative stress, as illustrated by the measurement of 8-hydroxyguanosine and lipid peroxidation, showed additive synergistic effects between the two carcinogens. The anti-inflammatory interleukin-10 was shown to be markedly elevated with the co-administration of the two carcinogens, indicating the elevated inflammatory processes. Additive synergistic effects were noted in terms of the liver disease marker ALT. The salmonella typhimirium strain TA102 used in the Ames mutagenicity test showed increased colony counts with respect to the coadministration of carcinogens (P<0.05), although no synergistic effect was noted. In a few of the presented parameters, the AFB1 group was not significantly different to the control group, although significant differences between the Fe group and the Fe + AFB1 groups were noted. The implication of which is that the presence of AFB1 is increasing the activity of Fe as a carcinogen, thereby acting as a co-carcinogen. Examples of such parameters illustrating this are presented in the results section including serum ALT, serum nitrite, liver and serum lipid peroxidation, liver and serum 8-hydroxyguanosine, some of the mutagenicity assays, and interleukin-10. The conclusion of this study suggests that AFB1 acts as a co-carcinogen in the presence of iron overloading, implying that a synergistic relationship between these two toxins exists.