Browsing by Subject "Virology"

Now showing 1 - 13 of 13
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    Analysis of cytomegalovirus UL97 drug resistance mutations in patients receiving Ganciclovir
    (2018) Nkosi, Nokwazi Pearl; Hsiao, Nei-Yuan; Korsman, Stephen; Smuts, Heidi
    Introduction: Cytomegalovirus (CMV) drug resistance mutations, because of the widespread use of ganciclovir, have been widely reported in international literature, particularly in the post-transplant setting. However, a genotypic assay to detect CMV drug resistance is not available in South Africa and the prevalence of these mutations is therefore unknown. We aimed to document the prevalence and types of CMV UL97 mutations following exposure to ganciclovir in adult and paediatric oncology patients, transplant recipients and HIV-infected patients in the local tertiary level hospitals: Red Cross War Memorial Children's Hospital, Groote Schuur Hospital and Tygerberg Hospital. Methods: The study had two components, the first component being a retrospective cross-sectional study using stored extracted DNA from patients with serially elevated CMV viral load levels. Thirty-three samples were tested for this component. The second component was a prospective case series on patients who were referred by clinicians for genotypic testing in whom CMV drug resistance was suspected. Eight samples were tested for this component. The CMV UL97 gene was amplified by conventional nested polymerase chain reaction (PCR) and Sanger sequencing performed. Results: CMV UL97 mutations were identified in five of thirty-three (15%) retrospectively screened samples while the prospective testing of eight patient samples identified drug resistance mutations in three patients (38%). Overall 8/41 (20%) patients had CMV UL97 mutations. A trend of higher risk for development of drug resistance mutations among haematological oncology patients 7/23 (30%) compared to solid organ transplant recipients 1/10 (10%) was observed, however, this difference was not statistically significant (P=0.306). Conclusion: This study, the first of its nature in South Africa, identified the presence of CMV UL97 mutations conferring resistance to ganciclovir in the haematological oncology, primary immunodeficiency and solid organ transplant patients in the Western Cape. The assay successfully detected CMV UL97 drug resistance mutations in whole blood and cerebrospinal fluid clinical samples. Ongoing viral replication in the background of intensive immunosuppression and prolonged antiviral therapy selects for the emergence of CMV UL97 drug resistance mutations.
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    The association of Bromegrass mosaic virus with Puccinia graminis tritici
    (1982) Erasmus, Dimitri Spiro; Von Wechmar, M B
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    Classification of HIV virological failure using whole blood versus plasma viral load
    (2016) Khan, Aabida; Hsiao, Marvin
    Introduction: HIV viral load testing is the preferred monitoring approach for HIV infected patients on combination antiretroviral therapy (cART) as it is more sensitive than CD4 count and clinical monitoring. In resource limited settings, timely plasma separation and transportation to testing laboratories is a major barrier to the access of HIV viral load testing. The 2015 World Health Organisation guidelines recommend that cART should be initiated in all adults and children living with HIV regardless of disease stage or CD4 count, thereby escalating the demand for HIV viral load testing. Potential solutions to expand implementation and scale up of viral load testing in low and middle income countries are whole blood testing through point of care (POC) viral load assays or dried blood spots (DBS) collected at the health facility. Utilization of whole blood instead of plasma would simplify sample collection, storage and transportation requirements and be cost effective. However, the paucity of studies comparing whole blood HIV viral load across different test platforms, especially in the correct classification of virological failure, has resulted in the lack of a standardised programmatic approach to whole blood viral load testing. Methods: We evaluated four HIV whole blood viral load test methods namely Alere q HIV-1/2 POC, Abbott RealTime HIV-1 DBS original and updated protocols, and Roche CAP/CTM DBS free virus elution (FVE) protocol, against the standard of care, plasma viral load, on 299 samples across the viral load spectrum from South African patients on cART. Virological failure was defined at >1000 copies/ml. Proportions of correct classification of virological failure and overall correlation with plasma were used for evaluating each method's performance. Results: Alere q, Abbott original and updated, and Roche FVE correctly classified virological failure in 61%, 89%, 87% and 76% of all samples tested respectively. The performance varied across plasma viral load categories. Alere q showed good correlation above plasma viral load of 1000 copies/ml, with correct classification of virological failure in 100% of samples. However, below the plasma threshold of 1000 copies/ml, Alere q demonstrated significant over-quantification, resulting in reduced specificity and upward misclassification of virological failure in 39% of all samples tested. Abbott original and updated also had good sensitivity of 98% and 91% respectively and the best overall correlation with plasma (r² = 0.76 and 0.72 respectively), but there was upward misclassification in 10% and 8% of samples tested respectively. Roche FVE had the best specificity of 99% but with significantly reduced sensitivity of 53%, especially between 1000–10,000 copies/ml of plasma, resulting in downward misclassification in 24% of all samples tested. Greatest variability between the different testing methods was seen when plasma viral load was 40-1000 copies/ml. Correlation was best for all whole blood viral load assays at >10,000 copies/ml. Conclusion: The key finding highlighted by this study is the great variability between the different whole blood test methods. Various factors influence the ability to quantify whole blood HIV viral load such as input volume used in each assay vary, sample treatment/processing (DBS versus fresh blood samples versus FVE), extraction (RNA selective, total nucleic acid extraction), amplification target and detection methods are different for each of the platforms tested. Based on our study, Alere q and Abbott DBS need to raise their whole blood threshold for virological failure in order to reduce upward misclassification and Roche FVE needs to achieve better sensitivity around its limit of detection. Receiver operating characteristic curve analysis can be used to determine the optimum threshold of virological failure for each assay.
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    Comparing high-throughput methods to measure antibody dependent cellular cytotoxicity during HIV infection
    (2014) Mbodo, Iyaloo; Passmore, Jo-Ann; Gamieldien, Hoyam
    The prevalence of HIV-1 is highest in Sub-Saharan Africa. Protective immune responses directed against HIV are complex and involve both cellular and humoral immunity. Based on the recent finding that the best correlate of protection against the first protective prophylactic RV144 vaccine were HIV-specific antibody responses, including those mediating natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC), there has been considerable interest in measuring alternative roles for HIV-specific binding antibodies. The aim of this MSc dissertation was to optimise and compare two high-throughput flow cytometry based approaches - the GranToxilux and PanToxilux assays - to measure HIV-specific ADCC responses. To do this, NK cells from a panel of healthy HIV-negative individuals were screened for their ability to directly kill the tumour cell line K562, as a measure of direct NK cell cytotoxicity. The individual with the highest granzyme B and caspase activity against K562 cells was chosen as the universal NK cell donor for this study.
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    Epidemiology, molecular characterisation and tropism of the Hepatitis G Virus / GBV-C
    (1999) Tucker, Timothy Johan Paul; Kirsch, Ralph; Robson, Simon
    The hepatitis G virus and GBV-C are recently discovered variants of the same virus belonging to the family Flavivirus (HGV/GBV-C). Although initially thought to be a hepatitis virus, it has been shown to have no association with liver disease. No work has been performed on the prevalence or molecular characteristics of HGV/GBV-C in southern Africa. In addition, although it is clear that the liver is not the primary site of replication, there is no data on the sites of HGV/GBV-C replication in normal subjects. Thus, this study aimed to assess the prevalence of HGV/GBV-C carriage in the urban and rural adult Black communities of the Western and Eastern Cape Provinces of South Africa, and compare it to the prevalence of serological markers of the hepatitis viruses A-E. In addition, this study aimed to assess the molecular features of South African HGV/GBV-C isolates and demonstrate the organs where viral replication was present. The mean prevalences of antibodies to hepatitis A lgG, hepatitis B surface antigen and antibodies to hepatitis B surface antigen were 98%, 4.3% and 61.1 % respectively. The mean prevalence of antibodies to hepatitis C was 1.8%. No significant differences in prevalence were shown between the urban and rural regions for these viruses. The mean anti-hepatitis E prevalence varied from 5.8% to 19.1 % in the different regions. Those living in mud houses without access to chlorinated tap water had a significantly higher prevalence of antihepatitis E. No anti-hepatitis D positive samples were isolated. The overall prevalence of HGV/GBV-C was 26.9%, with rural communities having a significantly lower prevalence than urban communities. A significant relationship was observed between HGV/GBV-C infection with the use of illicit drugs, female gender, younger age and past blood transfusions. Phylogenetic analysis demonstrated a novel fourth South African HGV/GBV-C genotype, distinct from the previously described genotypes 1-3. In addition, certain isolates showed a major deletion in the highly conserved 5' non-coding region of HGV/GBV-C. Analysis of 23 tissue biopsies from infected cadavers suggested that the spleen and bone marrow were the primary sites of HGV/GBVC replication.
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    Genetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methods
    (2004) Loubser, A S
    Immune system pressure on HIV-1 replication drives the antigenic changes seen over time. The monitoring of changes in viral sequences can provide important information on the nature of the immune response and the correlates of protection. Viral diversification may also occur due to other selective pressures such as cell availability and differences in viral fitness. Information on the genetic characteristics of HIV-1 variants present in the mother and her infected infant are useful data for establishing whether any common features exist between source infection and transmitted genotypes. This helps in the understanding of the mechanism of transmission and the selective pressures occurring during and following transmission. The overall aim of this study was to explore alternative methods other than DNA sequencing for the monitoring of genetic diversity in the third variable region (V3) of the HIV-1 env gene of integrated HIV-I variants in peripheral blood mononuclear cells (PBMC's) derived from infected mother-child pairs. Two methods for displaying DNA differences were compared: I-leteroduplex Mobility Assay (I-IMA) and Base Excision Sequence Scanning (BESS). These methods were validated using sequence data. Extracted PBMC DNA from infected mother-child pairs were used to amplify the V3 region by nested PCR. DNA fragments were cloned into plasmid vectors and analyzed by HMA and BESS to establish subtype and intrasample genetic diversity. In addition, a PCR-ELISA quantitation system was developed to measure copy numbers of integrated HIV-1 genomes in order to confirm whether a sufficient number of template molecules were present to be representative of the total viral quasispecies. In conclusion, this study compared two methods (HMA and BESS) as cost-effective alternatives to DNA sequencing for HIV-1 diversity studies. in addition, a novel application of the BESS assay was demonstrated. Diversity studies are reliant on estimation of adequate input of amplifiable copies. The PCR-ELISA quantitation system developed provided an efficient and specific method for determining DNA copy number.
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    Immune correlates of HIV-associated cryptococcal meningitis
    (2017) Scriven, James E; Harrison, Thomas S; Jarvis, Joseph N
    The International Society for Burns Injuries (ISBI) has published guidelines for the management of multiple or mass burns casualties, and recommends that 'each country has or should have a disaster planning system that addresses its own particular needs.' The need for a national burns disaster plan integrated with national and provincial disaster planning was discussed at the South African Burns Society Congress in 2009, but there was no real involvement in the disaster planning prior to the 2010 World Cup; the country would have been poorly prepared had there been a burns disaster during the event. This article identifies some of the lessons learnt and strategies derived from major burns disasters and burns disaster planning from other regions. Members of the South African Burns Society are undertaking an audit of burns care in South Africa to investigate the feasibility of a national burns disaster plan. This audit (which is still under way) also aims to identify weaknesses of burns care in South Africa and implement improvements where necessary.
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    Independent predictors of tuberculosis mortality in a high HIV prevalence setting: a retrospective cohort study
    (2015) Pepper, Dominique J; Schomaker, Michael; Wilkinson, Robert J; de Azevedo, Virginia; Maartens, Gary
    BackgroundIdentifying those at increased risk of death during TB treatment is a priority in resource-constrained settings. We performed this study to determine predictors of mortality during TB treatment.MethodsWe performed a retrospective analysis of a TB surveillance population in a high HIV prevalence area that was recorded in ETR.net (Electronic Tuberculosis Register). Adult TB cases initiated TB treatment from 2007 through 2009 in Khayelitsha, South Africa. Cox proportional hazards models were used to identify risk factors for death (after multiple imputations for missing data). Model selection was performed using Akaike’s Information Criterion to obtain the most relevant predictors of death.ResultsOf 16,209 adult TB cases, 851 (5.3%) died during TB treatment. In all TB cases, advancing age, co-infection with HIV, a prior history of TB and the presence of both pulmonary and extra-pulmonary TB were independently associated with an increasing hazard of death. In HIV-infected TB cases, advancing age and female gender were independently associated with an increasing hazard of death. Increasing CD4 counts and antiretroviral treatment during TB treatment were protective against death. In HIV-uninfected TB cases, advancing age was independently associated with death, whereas smear-positive disease was protective.ConclusionWe identified several independent predictors of death during TB treatment in resource-constrained settings. Our findings inform resource-constrained settings about certain subgroups of TB patients that should be targeted to improve mortality during TB treatment.
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    Molecular investigations of subgroup I geminiviruses
    (1991) Hughes, Fiona Lesley; Rybicki, Edward P
    The diversity of Subgroup I geminiviruses causing streak disease in maize, sugarcane, and indigenous wild grasses was investigated. The virus. isolates studied originated from maize (several southern African isolates), two sugarcane cultivars (from Natal province, South Africa, and from Mauritius), wheat, and three grasses (Panicum, Setaria, and Eleusine spp. from South Africa). The following methods were used: analysis of restriction fragment length polymorphisms (RFLPs) between viral genomes in individual infected plants; DNA cross-hybridization between virus isolates; restriction endonuclease mapping of whole virus genomes; and nucleic acid sequencing. The complete genome of the Natal sugarcane streak virus isolate was sequenced. Partial sequences were obtained for other isolates, either by sequencing the ends of cloned viral genomes, or by sequencing a 250 base pair fragment of a highly conserved open reading frame that had been amplified using the polymerase chain reaction technique. The viruses being studied were compared both among themselves and with other Subgroup I geminiviruses of known DNA sequence, on the basis of sequence (nucleotide and amino acid) and restriction map data. Distance matrix methods were used to infer phylogenetic relationships between Subgroup I geminiviruses from restriction map and sequence data. Phylogenies deduced from sequence data were considered to be more accurate than those deduced from map data. Regardless of the method of analysis used, however, the relationships between the Subgroup I geminiviruses studied here remained constant. Thus, three strains of MSV (maize, Setaria, and Eleusine strains) were distinguished. Streak viruses distinct from MSV were also identified: panicum streak virus (PanSV), and two distantly related strains (Natal and Mauritius) of sugarcane streak virus (SSV). Restriction mapping of different geographical isolates of the maize strain of MSV demonstrated that variation existed within a single strain of virus. RFLP analysis indicated that minor variation existed between virus genomes within single diseased plants. Methods used to. type Subgroup I geminiviruses were evaluated, and discrepancies in the serological typing of geminiviruses from Subgroups I and III were pointed out. A unified scheme was proposed for distinguishing between distinct Subgroup I geminiviruses and strains of geminiviruses. The origins of maize and sugarcane streak viruses were speculated upon.
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    Sensory neuropathy and metabolic risk factors in human immune deficiency virus infected South Africans receiving protease inhibitors
    (2015) Vermaak, John-Randel; Dave, Joel A; Levitt, Naomi; Heckmann, Jeannine M
    BackgroundProtease inhibitors (PI)s have been associated with distal sensory polyneuropathy (DSP) and metabolic complications in high-income countries. No data exist in Africans where second-line antiretroviral therapy (ART) often include PIs.MethodWe performed a cross-sectional study to assess the DSP frequency and metabolic risk factors in community-based South Africans taking ritonavir-boosted lopinavir as PI. Examination findings categorized subjects as having DSP (≥1 neuropathic sign) or symptomatic DSP [DSP with symptom(s)]. Fasting-state glucose and lipid profiles were assessed. We compared the ritonavir/lopinavir-group to a nested group on first-line ART [dideoxy-nucleoside reverse transcriptase inhibitors (d-drugs)] selected from a dataset collected at the same time and matched for d-drug exposure.ResultsThe ritonavir/lopinavir-group (n=86) consisted predominantly of women (84%) with a median age of 36years (IQR 32–41). The median current CD4+ count was 489cells/μL (IQR 291–665). The median exposure time to ritonavir/lopinavir was 18months (IQR 10–26) and to d-drugs, 24months (IQR 16–38). DSP was present in 78% and symptomatic DSP in 48%; symptoms were most frequently of moderate intensity. Only age independently associated with DSP and symptomatic DSP (p=0.08 and p=0.04, respectively). None of the metabolic syndrome components showed associations with DSP or symptomatic DSP despite a trend towards hypertriglyceridemia overall. The ritonavir/lopinavir-group had less DSP compared to the d-drug only group (p=0.002) but the frequency of symptomatic DSP was similar (p=0.49).ConclusionRitonavir-boosted lopinavir did not add additional risk to developing DSP in this community-based African cohort after a median of 18months on second-line ART.Electronic supplementary materialThe online version of this article (doi:10.1186/s12981-015-0073-8) contains supplementary material, which is available to authorized users.
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    A study of the host-restricted lumpy skin disease virus as a vaccine vector using rabies babies virus as a model
    (2002) Aspden, Kate; Williamson, Anna-Lise
    Bibliography: leaves 155-183. Also available online.
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    Third-line antiretroviral therapy in Africa: effectiveness in a Southern African retrospective cohort study
    (2015) Meintjes, Graeme; Dunn, Liezl; Coetsee, Marla; Hislop, Michael; Leisegang, Rory; Regensberg, Leon; Maartens, Gary
    Background An increasing number of patients in Africa are experiencing virologic failure on second-line antiretroviral therapy (ART) and those who develop resistance to protease inhibitors (PI) will require third-line ART, but no data on the outcomes of third-line are available from the region. We assessed the virologic outcomes and survival of patients started on salvage ART in a Southern African private sector disease management programme.Methods Retrospective observational cohort study with linkage to the national death register. Adults (≥18years) who started salvage ART between July 2007 and December 2011 were included. Salvage ART was defined by inclusion of darunavir or tipranavir in an ART regimen after having failed another PI. For Kaplan–Meier (KM) analysis, patients were followed up until event, or censored at death (only for virologic outcomes), leaving the programme, or April 2014.Results152 patients were included. Subtype was known for 113 patients: 111 (98%) were infected with subtype C. All 152 had a genotype resistance test demonstrating major PI resistance mutations. Salvage drugs included were: darunavir/ritonavir (n=149), tipranavir/ritonavir (n=3), raltegravir (n=58), and etravirine (n=8). Median follow-up was 2.5years (IQR=1.5–3.3). 82.9% achieved a viral load ≤400 copies/ml and 71.1% ≤50 copies/ml. By the end of the study 17 (11.2%) of the patients had died. The KM estimate of cumulative survival was 87.2% at 2000days.ConclusionsVirologic suppression was comparable to that demonstrated in clinical trials and observational studies of salvage ART drugs conducted in other regions. Few deaths occurred during short term follow-up. Third-line regimens for patients with multidrug resistant subtype C HIV in Africa are virologically and clinically effective.
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    Utility of CD4 cell counts for early prediction of virological failure during antiretroviral therapy in a resource-limited setting
    (BioMed Central Ltd, 2008) Badri, Motasim; Lawn, Stephen D; Wood, Robin
    BACKGROUND:Viral load monitoring is not available for the vast majority of patients receiving antiretroviral therapy in resource-limited settings. However, the practical utility of CD4 cell count measurements as an alternative monitoring strategy has not been rigorously assessed. METHODS: In this study, we used a novel modelling approach that accounted for all CD4 cell count and VL values measured during follow-up from the first date that VL suppression was achieved. We determined the associations between CD4 counts (absolute values and changes during ART), VL measurements and risk of virological failure (VL > 1,000 copies/ml) following initial VL suppression in 330 patients in South Africa. CD4 count changes were modelled both as the difference from baseline (DeltaCD4 count) and the difference between consecutive values (CD4 count slope) using all 3-monthly CD4 count measurements during follow-up. RESULTS: During 7093.2 patient-months of observation 3756 paired CD4 count and VL measurements were made. In patients who developed virological failure (n = 179), VL correlated significantly with absolute CD4 counts (r = - 0.08, P = 0.003), DeltaCD4 counts (r = - 0.11, P < 0.01), and most strongly with CD4 count slopes (r = - 0.30, P < 0.001). However, the distributions of the absolute CD4 counts, DeltaCD4 counts and CD4 count slopes at the time of virological failure did not differ significantly from the corresponding distributions in those without virological failure (P = 0.99, P = 0.92 and P = 0.75, respectively). Moreover, in a receiver operating characteristic (ROC) curve, the association between a negative CD4 count slope and virological failure was poor (area under the curve = 0.59; sensitivity = 53.0%; specificity = 63.6%; positive predictive value = 10.9%). CONCLUSION: CD4 count changes correlated significantly with VL at group level but had very limited utility in identifying virological failure in individual patients. CD4 count is an inadequate alternative to VL measurement for early detection of virological failure.