Recency of HIV infection, antiretroviral therapy use and viral loads among symptomatic sexually transmitted infection service attendees in South Africa.

BACKGROUND: Better integration of HIV and sexually transmitted infection (STI) prevention and treatment services is needed to accelerate progress towards the goal of zero new HIV infections. OBJECTIVES: To describe HIV positivity, antiretroviral therapy (ART) use, viral suppression and recency of HIV infection among symptomatic STI service attendees at two primary care clinics in South Africa. METHODS: In a cross-sectional study, male and female STI service attendees presenting with symptoms consistent with STI syndromes were enrolled following informed consent. An interviewer-administered questionnaire was completed and appropriate genital and blood specimens were collected for STI testing and HIV biomarker measurements including recency of infection and antiretroviral (ARV) drug levels. Descriptive statistics were used to describe enrolled attendees, and to determine the proportion of attendees who were HIV-positive, recently infected, taking ART and virally suppressed. HIV-positive attendees with detectable ARVs were considered to be on ART, while those with viral loads (VLs) ≤200 copies/mL were considered virally suppressed. RESULTS: Of 451 symptomatic attendees whose data were analysed, 93 (20.6%) were HIV-positive, with 15/93 (16.1%) being recently infected. Recent infection was independently associated with genital ulcer disease at presentation, especially ulcers with no detectable STI pathogens. Among the 78 (83.9%) with long-term infection, only 30 (38.5%) were on ART, with 23/30 (76.7%) virally suppressed. CONCLUSIONS: In a population at risk of HIV transmission, there was a high burden of recent infection and unsuppressed VLs. Incorporating pre-exposure prophylaxis, ART initiation and adherence support into STI services will be necessary for progress towards eliminating HIV transmission.

Recency of HIV infection, antiretroviral therapy use and viral loads among symptomatic sexually transmitted infection service attendees in South Africa

Background. Better integration of HIV and sexually transmitted infection (STI) prevention and treatment services is needed to accelerate progress towards the goal of zero new HIV infections. Objectives. To describe HIV positivity, antiretroviral therapy (ART) use, viral suppression and recency of HIV infection among symptomatic STI service attendees at two primary care clinics in South Africa. Methods. In a cross-sectional study, male and female STI service attendees presenting with symptoms consistent with STI syndromes were enrolled following informed consent. An interviewer-administered questionnaire was completed and appropriate genital and blood specimens were collected for STI testing and HIV biomarker measurements including recency of infection and antiretroviral (ARV) drug levels. Descriptive statistics were used to describe enrolled attendees, and to determine the proportion of attendees who were HIV-positive, recently infected, taking ART and virally suppressed. HIV-positive attendees with detectable ARVs were considered to be on ART, while those with viral loads (VLs) ≤200 copies/mL were considered virally suppressed. Results. Of 451 symptomatic attendees whose data were analysed, 93 (20.6%) were HIV-positive, with 15/93 (16.1%) being recently infected. Recent infection was independently associated with genital ulcer disease at presentation, especially ulcers with no detectable STI pathogens. Among the 78 (83.9%) with long-term infection, only 30 (38.5%) were on ART, with 23/30 (76.7%) virally suppressed. Conclusions. In a population at risk of HIV transmission, there was a high burden of recent infection and unsuppressed VLs. Incorporating pre-exposure prophylaxis, ART initiation and adherence support into STI services will be necessary for progress towards eliminating HIV transmission

Monitoring diagnosis, retention in care and viral load suppression in children testing HIV polymerase chain reaction-positive in two districts in South Africa.

BACKGROUND: Retention in care is associated with improved virological control and survival among HIV-infected children. However, retention of children in HIV care remains a challenge. OBJECTIVES: To describe, using routine laboratory HIV test data, the retention-in-care and virological outcomes of HIV-infected children aged <18 months in two districts in South Africa. METHODS: HIV polymerase chain reaction (PCR)-positive results of children from uMkhanyakude and Tshwane districts in KwaZulu-Natal and Gauteng provinces, respectively, tested between April 2015 and May 2016, were extracted from the National Health Laboratory Service's Corporate Data Warehouse (CDW). HIV-related tests (PCR, viral load (VL), CD4+) were documented longitudinally for each child for ≥13 months after the first positive PCR result by manually searching demographics within the CDW, supplemented by an automated patient-linking algorithm. Test sets were linked if two or more demographics (surname, name, date of birth, folder number) matched exactly. Programmatic indicators assessed included age at first positive PCR test, presumed confirmatory test rates, retention in care, and VL suppression at 6 and 12 months. RESULTS: Ninety-four and 304 children tested HIV PCR-positive in uMkhanyakude and Tshwane, respectively. The median age at diagnosis was 3.6 months (interquartile range (IQR) 1.4 - 7.1) for uMkhanyakude and 2.3 months (IQR 0.1 - 6.7) for Tshwane. In uMkhanyakude, confirmed in utero infections accounted for 18.1% of transmissions (n=17), compared with 29.6% (n=90) in Tshwane. Presumed confirmatory test rates following an initial positive PCR result were 77.7% and 71.7% for uMkhanyakude and Tshwane, respectively. Within 6 months of starting antiretroviral therapy, 43 children (58.9%) were lost to follow-up in uMkhanyakude compared with 160 (73.4%) in Tshwane. Of those retained in care at 6 months with a VL measurement, 15 (60.0%) from uMkhanyakude had a VL <1 000 copies/mL, compared with 24 (48.0%) in Tshwane. For both districts, a third of all HIV PCR-positive children were retained in care at the end of follow-up, with 29 (30.9%) in uMkhanyakude and 99 (32.5%) in Tshwane. Of these, 12 (41.4%) had a VL <1 000 copies/mL in uMkhanyakude compared with 28 (28.3%) in Tshwane. CONCLUSIONS: We demonstrate the value of routine laboratory data in monitoring diagnosis, retention and VL suppression in HIV-infected children. This approach is scalable, can be reported near real-time, is relatively inexpensive to implement, and provides a tool for improving paediatric HIV services until clinical databases can assume this role.

Monitoring diagnosis, retention in care and viral load suppression in children testing HIV polymerase chain reaction-positive in two districts in South Africa

Background. Retention in care is associated with improved virological control and survival among HIV-infected children. However, retention of children in HIV care remains a challenge.Objectives. To describe, using routine laboratory HIV test data, the retention-in-care and virological outcomes of HIV-infected children aged <18 months in two districts in South Africa.Methods. HIV polymerase chain reaction (PCR)-positive results of children from uMkhanyakude and Tshwane districts in KwaZulu-Natal and Gauteng provinces, respectively, tested between April 2015 and May 2016, were extracted from the National Health Laboratory Service's Corporate Data Warehouse (CDW). HIV-related tests (PCR, viral load (VL), CD4+) were documented longitudinally for each child for ≥13 months after the first positive PCR result by manually searching demographics within the CDW, supplemented by an automated patient-linking algorithm. Test sets were linked if two or more demographics (surname, name, date of birth, folder number) matched exactly. Programmatic indicators assessed included age at first positive PCR test, presumed confirmatory test rates, retention in care, and VL suppression at 6 and 12 months.Results. Ninety-four and 304 children tested HIV PCR-positive in uMkhanyakude and Tshwane, respectively. The median age at diagnosis was 3.6 months (interquartile range (IQR) 1.4 - 7.1) for uMkhanyakude and 2.3 months (IQR 0.1 - 6.7) for Tshwane. In uMkhanyakude, confirmed in utero infections accounted for 18.1% of transmissions (n=17), compared with 29.6% (n=90) in Tshwane. Presumed confirmatory test rates following an initial positive PCR result were 77.7% and 71.7% for uMkhanyakude and Tshwane, respectively. Within 6 months of starting antiretroviral therapy, 43 children (58.9%) were lost to follow-up in uMkhanyakude compared with 160 (73.4%) in Tshwane. Of those retained in care at 6 months with a VL measurement, 15 (60.0%) from uMkhanyakude had a VL <1 000 copies/mL, compared with 24 (48.0%) in Tshwane. For both districts, a third of all HIV PCR-positive children were retained in care at the end of follow-up, with 29 (30.9%) in uMkhanyakude and 99 (32.5%) in Tshwane. Of these, 12 (41.4%) had a VL <1 000 copies/mL in uMkhanyakude compared with 28 (28.3%) in Tshwane.Conclusions. We demonstrate the value of routine laboratory data in monitoring diagnosis, retention and VL suppression in HIV-infected children. This approach is scalable, can be reported near real-time, is relatively inexpensive to implement, and provides a tool for improving paediatric HIV services until clinical databases can assume this role

Hydroxyl radical generation: the effect of bicarbonate, dioxygen and buffer concentration on pH-dependent chemiluminescence.

Enhanced chemiluminescence (CL) induced by HO* was detected using the primary enhancers luminol (Lum), isoluminol (ILum) and lucigenin (Luc), in the absence of HCO3-, at pH 9.5, 10.0 and 10.5 but not between pH 4.0 and 9.0. This was confirmed using nine different HO* generators. FeCl3/NTA/H2O2 was the only HO* generator that was able to generate singular HO* which was obtained entirely from the Fenton reaction. However, this was so only at pH 10.0 since at all other pHs multiple ROS were produced. This was confirmed by the chemical detection of the fluorescent hydroxylated product of terephthalic acid in the absence of O2. No HO* Lum-CL pH optima coincided with the O2*- mediated Lum-CL optima found at pH 8.0 and 9.0. Bicarbonate had an enhancing effect on Lum-CL which was 70-2700% at pH 10.0 for the different HO* generators. This was due to the conversion of the radical-electron from HO* to CO3*-, making CL detection more efficient since less HO* were lost initially before detection. Methyl-cypridine-luciferin analogue (MCLA) elicited CL in the pH range 4.0-10.0 with the same set of generators in the absence of HCO3-. The iron-containing generators had their different MCLA-CL optima at pH 4.5, 5.0 or 6.0, excluding those overlapping with the O2(*-)-mediated CL optima. The two copper-containing generators had optima at the same pHs, viz, 7.0 and 10.5. Again, FeCl3/NTA/H2O2 was the only HO* generator able to produce singular HO* by the Fenton reaction. However, whereas Lum-CL was able to detect singular HO* only at pH 10.0, MCLA-CL detected it at pH 5.0 and 5.5. Therefore, MCLA is the most suitable CL enhancer for physiological assessments since it is the most sensitive enhancer and has HO* CL optima nearer to physiological pH than the other probes. The HCO3- enhancement of MCLA-CL was even greater than that of Lum-CL, since increases of 114-fold and 37-fold, respectively, were obtained at these HO*-specific pH optima for FeCl3/NTA/H2O2. Therefore, bicarbonate concentration is as important a parameter as pH when the enhanced CL of a non-cellular system is determined. Hydrogen peroxide was not able to elicit CL directly but, due to trace metal contamination, it produced artifactual CL due to HO* formation. High H2O2 levels, which prevent spontaneous O2*- dismutation, helped to establish the overlapping pH optima of CL mediated by O2*- and HO* which were artifactually produced either by O2*- via H2O2 and trace metals or by perferryl intermediates, respectively. Due to spontaneous dismutation to H2O2, only 22% of the O2*- produced by HX/XO could be detected by enhanced CL.

Heat shock treated oesophageal cancer cells become thermosensitized against anticancer drugs.

BACKGROUND: Cancer of the oesophagus is the most common gastrointestinal malignancy in South African blacks. The aim of this study was to determine whether repetitive heat-shock (HS) treatment of oesophageal cancer cells would induce multi-drug resistance (MDR) as was previously found with human renal carcinoma cells. METHODS: The oesophageal cancer-line WHCO-3 was heat-shocked in sequence, on five occasions, for 90 min each at 42 degrees C, followed by a recovery period of 24 h between the consecutive HSs. After each shock and recovery period the cells were divided; one part was used for the next shock treatment and the other, designated the HS fraction, was used for assessments of drug cytotoxicity, enhanced mdr-1 and mrp gene expression by RT-PCR, and enhanced isoenzyme GsT-P activity. The IC50s of the drugs doxorubicin, amsacrine, melphalan, and cisplatin were determined after each HS using the sulphorhodamine B(SRB) cell-proliferation assay which was able to assess cytotoxicity. A drug accumulation assay was conducted by measuring 3H-Vinblastin uptake in surviving cells using the SRB assay to quantitate the viable cells. RESULTS: Multiple heat-shocks did not introduce MDR via the MDR-1 or MRP mechanisms because these genes were not over-expressed after consecutive HS treatments. Deminished cytotoxicity of the drugs, as measured by increased IC50, did not occur, as it would have been if MDR was introduced. Therefore neither the topoisomerase drug resistant mechanism nor the enhanced GsT-P detoxification mechanism were introduced with repetitive heat-shocks. On the contrary the IC50s of doxorubucin and amsacrine decreased after five HSs, whereas melphalan and cisplatin, had no cytotoxic effects. The GsT-P levels were dramatically reduced by 90% after five HSs and an 8 day recovery period, indicating that the cancer cells became more sensitive towards toxic drugs and not drug resistant as expected. However, with the drug influx assay the uptake of 3H-vinblastin was reduced, with each consecutive HS, and not reversed by verapamil thereby confirming the finding that the efflux mechanisms of MDR-1 and MRP were not introduced. CONCLUSIONS: Repetitive heat shocks did not introduce multi-drug resistance, but on the contrary, sensitized the oesophageal cancer cells against toxic anticancer drugs and they therefore became thermosensitized.

Glutathione-linked enzymes in benign and malignant oesophageal tissue.

Oxyradicals are involved in multiple mutational events and can contribute to the conversion of healthy cells to cancer cells. Glutathione (GSH) and the GSH-replenishing enzymes keep the antioxidant status of normal cells at a level where they can avert oxyradical derived mutations. The aim of this study was to determine whether in cancer cells the GSH-replenishing, GSH antioxidant and GSH-depleting enzymes were not at appropriate levels and therefore not able to protect cancer cells adequately against oxyradical-induced mutations. Cancer of the oesophagus was chosen since it is the most common gastrointestinal malignancy in South African Blacks. Biopsies and blood from 31 patients with cancer of the oesophagus and 29 non-cancer patients were assessed for these enzymes. The mean activity of the antioxidant and depleting enzyme GSH-peroxidase was elevated significantly by twofold in the cancer tissue compared to normal tissue. However, the activity of the replenishing enzyme GSSG-reductase and the level of the depleting enzyme GSH-s-transferase P1-isoenzyme were significantly reduced by 23% and 33% respectively. As in a previous paper we found that GSH was depleted and gamma-glutamine transpeptidase was diminished in oesophageal cancer. There can be two reasons for GSH depletion. Firstly, elevated GSH-peroxidase will use more GSH in an attempt to cope with the excessive production of oxyradicals as revealed by elevated lipid peroxidation; this was, as shown by us before, elevated sixfold in oesophageal cancer. Secondly, if little replenishment of GSH occurred the level of GSH would become lower. This was confirmed by our findings that the activities of the replenishing enzymes were significantly diminished in oesophageal cancer tissue. Contrary to what was expected, the other depleting enzyme GSH-s-transferase P1 was not elevated in cancer tissue but was significantly lower. However, in the blood of the same patients it was significantly elevated. An explanation for this phenomenon is that, although the production of GST-P1 was enhanced in cancer, it did not show because it was rapidly extruded into the blood by an unknown mechanism operational only in cancer cells.

Antioxidants suitable for use with chemiluminescence to identify oxyradical species.

From a panel of 24 alleged antioxidants the most suitable antioxidants (AO) for use with chemiluminescence (CL) experiments were determined. Superoxide dismutase (SOD), using luminol as the chemiluminescence probe (Lum-CL), was inhibitory only towards O2*- and not HO* or (1)O2. SOD was thus a suitable antioxidant for O2*-, as was tiron. Tiron had advantages, however, since SOD acted as a pro-oxidant in the presence of H2O2 or H2O2/HO* generators. The two most suitable antioxidants for (1)O2 were diphenylisobenzofuran (DBF) and tryptophan, for both Lum and Lucigenin-CL (Luc-CL). Desferrioxamine, with both Lum and Luc-CL, was a very effective scavenger for HO*, but appeared to be an even more effective scavenger for (1)O2. Cysteamine showed the best discrimination between IC50s when the two (1)O2 generators NaOCl/H2O2 and NDPO2 were compared. Cysteamine was, therefore, the only scavenger that was appropriate for studies with hypochlorite. Melatonin, with Lum-CL, was found to be the most suitable scavenger for HO*. Mannitol, the classical AO for HO*, was not suitable when used with CL since it acted as a pro-oxidant. Some of the AOs revealed either calyx- or bell-shaped CL inhibition profiles and presumably, therefore, may act as both pro- or antioxidants at different concentrations. Antioxidants showing these kinds of dual activities should be used with caution in CL studies.

The effect of pH on chemiluminescence of different probes exposed to superoxide and singlet oxygen generators.

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO2 and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO2 and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H2O2 did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H2O2, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H2O2 also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H2O2 had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H2O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used.