The development of clone-unspecific micropropagation protocols for three commercially important Eucalyptus hybrids.

Thesis (M.Sc.)-University of Natal, Durban, 2001.Micropropagation methods are often used to supplement existing clonal programmes for Eucalyptus species. However, genotypic differences among clones require the implementation of clone-specific protocols, an expensive and labour-intensive exercise. Hence, this study aimed at determining high-yielding hybrid-specific rather than clone-specific, micropropagation protocols for E. grandis x nitens (GN), E. grandis x nitens (NH), and E. grandis x urophylla (GU). Different conditions for surface sterilisation, bud-break (3 protocols, 2 media), multiplication (4 media), elongation (2 protocols) and rooting (4 media) were tested. A single successful surface sterilisation approach was possible for all clones of the tested hybrids (0.0-11.8% contamination, 0.0-22.9% necrosis). It involved rinsing nodal explants in a fungicide mixture (lg/l Benlate, 1g/1 boric acid, 0.5ml/1 Bravo, Tween 20) for 15 minutes followed by calcium hypochlorite (10g/l with Tween 20) for three minutes. Results at each culture stage were dependent on genotypes, and results indicated here represent ranges in values among the clones of each hybrid. The highest bud-break values for GN clones (87-90%) and NH clones (17-75%) were achieved on a medium containing MS, 0.1mg/1 biotin, 0.1mg/l calcium pantothenate, 0.04mg/1 NAA, 0.11mg/l BAP and 0.05mg/1 kinetin. In GU clones, bud-break values on this medium (84-97%) were not significantly different to those achieved directly on a multiplication medium (80-91%) (MS, 0.1 mg/l biotin, 0.1 mg/l calcium pantothenate, 0.2mg/l BAP, 0.01mg/1 NAA). Shoot multiplication yields for GN clones (4-13 shoots/bud) and GU clones (2-6 shoots/bud) were achieved on a medium consisting of MS, 0.1mg/1 biotin, 0.1 mg/l calcium pantothenate, 0.2mg/1 BAP and 0.01 mg/l NAA. As genotypic effects were highly significant among NH clones, a single multiplication medium for all clones of this hybrid could not be determined. The best method of elongation for clones of all three hybrids involved culturing shoots on MS, 0.1 mg/l calcium pantothenate, 0.1mg/1 biotin, 0.35mg/1 NAA, 0.1mg/l kinetin and 0.1mg/1 IBA, under photoperiod conditions, rather than total darkness, for 6 weeks. This resulted in 82.3-86.6% elongation and shoot lengths increasing by 22.9-35.2 mm for GN clones, 80.2-82.3 % elongation and an increase in length of 24.7-32.2 mm for NH clones and 70.8-78.1 % elongation, and shoot elongation of 21.6-29.3 mm for GU clones from passage 1-2. For all the above stages, media contained 20/25 g/l sucrose and 3.5g/l Gelrite, and cultures were maintained at 25°C ± 2°C day/ 21°C night with a 16 h light/ 8 h dark photoperiod (PPFD 66µmol/m2/s). In terms of rooting, cultures on different media were initially subjected to a 72 hour period of total darkness at room temperature, then a 16 h light/8 h dark photoperiod (PPFD 37µmol/m2 /s) at 24°C day/ 21°C night for 7 days. This was followed by a 16 h light/ 8 h dark photoperiod (PPFD 66µmol/m2/s) at 25°C ± 2°C day/ 21°C night for 21 days. Tested clones of the three hybrids were all rooted successfully (56-93% rooting in GN clones, 36-76% rooting in NH clones and 46-96% rooting in GU clones) on a medium containing ¼ MS, 0.1 mg/l biotin, 0.1 mg/l calcium pantothenate, 0.1mg/l IBA, 0.22g/1 CaCI2 .2H20, 0.185g/l MgS04.7H2O, 15g/l sucrose and 3.5g/1 Gelrite. Predicted yields from the established protocol are also presented (168-667 plants of E. grandis x nitens (GN), 35- 854 plants of E. grandis x nitens (NH) and 54-349 plants of E. grandis x urophylla from 100 initial nodal explants, depending on the clone). Hence, the established protocols can be used successfully for some of the clones, but the implementation of specific media and methods to obtain high yields may still be necessary for certain clones

The "ART" of Linkage: Pre-Treatment Loss to Care after HIV Diagnosis at Two PEPFAR Sites in Durban, South Africa

BACKGROUND. Although loss to follow-up after antiretroviral therapy (ART) initiation is increasingly recognized, little is known about pre-treatment losses to care (PTLC) after an initial positive HIV test. Our objective was to determine PTLC in newly identified HIV-infected individuals in South Africa. METHODOLOGY/PRINCIPAL FINDINGS. We assembled the South African Test, Identify and Link (STIAL) Cohort of persons presenting for HIV testing at two sites offering HIV and CD4 count testing and HIV care in Durban, South Africa. We defined PTLC as failure to have a CD4 count within 8 weeks of HIV diagnosis. We performed multivariate analysis to identify factors associated with PTLC. From November 2006 to May 2007, of 712 persons who underwent HIV testing and received their test result, 454 (64%) were HIV-positive. Of those, 206 (45%) had PTLC. Infected patients were significantly more likely to have PTLC if they lived =10 kilometers from the testing center (RR=1.37; 95% CI: 1.11-1.71), had a history of tuberculosis treatment (RR=1.26; 95% CI: 1.00-1.58), or were referred for testing by a health care provider rather than self-referred (RR=1.61; 95% CI: 1.22-2.13). Patients with one, two or three of these risks for PTLC were 1.88, 2.50 and 3.84 times more likely to have PTLC compared to those with no risk factors. CONCLUSIONS/SIGNIFICANCE. Nearly half of HIV-infected persons at two high prevalence sites in Durban, South Africa, failed to have CD4 counts following HIV diagnosis. These high rates of pre-treatment loss to care highlight the urgent need to improve rates of linkage to HIV care after an initial positive HIV test.US National Institute of Allergy and Infectious Diseases (R01 AI058736, K24 AI062476, K23 AI068458); the Harvard University Center for AIDS Research (P30 AI42851); National Institutes of Health (K24 AR 02123); the Doris Duke Charitable Foundation (Clinical Scientist Development Award); the Harvard University Program on AID

Drug-Resistant Tuberculosis among HIV-Infected Patients Starting Antiretroviral Therapy in Durban, South Africa

Objective: To estimate the prevalence of drug-resistant tuberculosis (TB) and describe the resistance patterns in patients commencing antiretroviral therapy (ART) in an HIV clinic in Durban, South Africa. Design Cross-sectional cohort study. Methods Consecutive HIV-infected adults (≥18y/o) initiating HIV care were enrolled from May 2007–May 2008, regardless of signs or symptoms of active TB. Prior TB history and current TB treatment status were self-reported. Subjects expectorated sputum for culture (MGIT liquid and 7H11 solid medium). Positive cultures were tested for susceptibility to first- and second-line anti-tuberculous drugs. The prevalence of drug-resistant TB, stratified by prior TB history and current TB treatment status, was assessed. Results: 1,035 subjects had complete culture results. Median CD4 count was 92/µl (IQR 42–150/µl). 267 subjects (26%) reported a prior history of TB and 210 (20%) were receiving TB treatment at enrollment; 191 (18%) subjects had positive sputum cultures, among whom the estimated prevalence of resistance to any antituberculous drug was 7.4% (95% CI 4.0–12.4). Among those with prior TB, the prevalence of resistance was 15.4% (95% CI 5.9–30.5) compared to 5.2% (95% CI 2.1–8.9) among those with no prior TB. 5.1% (95% CI 2.4–9.5) had rifampin or rifampin plus INH resistance. Conclusions: The prevalence of TB resistance to at least one drug was 7.4% among adults with positive TB cultures initiating ART in Durban, South Africa, with 5.1% having rifampin or rifampin plus INH resistance. Improved tools for diagnosing TB and drug resistance are urgently needed in areas of high HIV/TB prevalence

Factors Associated with Self-Reported Repeat HIV Testing after a Negative Result in Durban, South Africa

Background: Routine screening for HIV infection leads to early detection and treatment. We examined patient characteristics associated with repeated screening in a high prevalence country. Methods: We analyzed data from a cohort of 5,229 adults presenting for rapid HIV testing in the outpatient departments of 2 South African hospitals from November 2006 to August 2010. Patients were eligible if they were ≥18 years, reported no previous diagnosis with HIV infection, and not pregnant. Before testing, participants completed a questionnaire including gender, age, HIV testing history, health status, and knowledge about HIV and acquaintances with HIV. Enrollment HIV test results and CD4 counts were abstracted from the medical record. We present prevalence of HIV infection and median CD4 counts by HIV testing history (first-time vs. repeat). We estimated adjusted relative risks (ARR’s) for repeat testing by demographics, health status, and knowledge of HIV and others with HIV in a generalized linear model. Results: Of 4,877 participants with HIV test results available, 26% (N = 1258) were repeat testers. Repeat testers were less likely than first-time testers to be HIV-infected (34% vs. 54%, p<0.001). Median CD4 count was higher among repeat than first-time testers (201/uL vs. 147/uL, p<0.001). Among those HIV negative at enrollment (N = 2,499), repeat testing was more common among those with family or friends living with HIV (ARR 1.50, 95% CI: 1.33–1.68), women (ARR: 1.24, 95% CI: 1.11–1.40), and those self-reporting very good health (ARR: 1.28, 95% CI: 1.12–1.45). Conclusions: In this high prevalence setting, repeat testing was common among those undergoing HIV screening, and was associated with female sex, lower prevalence of HIV infection, and higher CD4 counts at diagnosis

Extensive Intrasubtype Recombination in South African Human Immunodeficiency Virus Type 1 Subtype C Infections▿

Recombinant human immunodeficiency virus type 1 (HIV-1) strains containing sequences from different viral genetic subtypes (intersubtype) and different lineages from within the same subtype (intrasubtype) have been observed. A consequence of recombination can be the distortion of the phylogenetic signal. Several intersubtype recombinants have been identified; however, less is known about the frequency of intrasubtype recombination. For this study, near-full-length HIV-1 subtype C genomes from 270 individuals were evaluated for the presence of intrasubtype recombination. A sliding window schema (window, 2 kb; step, 385 bp) was used to partition the aligned sequences. The Shimodaira-Hasegawa test detected significant topological incongruence in 99.6% of the comparisons of the maximum-likelihood trees generated from each sequence partition, a result that could be explained by recombination. Using RECOMBINE, we detected significant levels of recombination using five random subsets of the sequences. With a set of 23 topologically consistent sequences used as references, bootscanning followed by the interactive informative site test defined recombination breakpoints. Using two multiple-comparison correction methods, 47% of the sequences showed significant evidence of recombination in both analyses. Estimated evolutionary rates were revised from 0.51%/year (95% confidence interval [CI], 0.39 to 0.53%) with all sequences to 0.46%/year (95% CI, 0.38 to 0.48%) with the putative recombinants removed. The timing of the subtype C epidemic origin was revised from 1961 (95% CI, 1947 to 1962) with all sequences to 1958 (95% CI, 1949 to 1960) with the putative recombinants removed. Thus, intrasubtype recombinants are common within the subtype C epidemic and these impact analyses of HIV-1 evolution