Outcomes of an HIV cohort after a decade of comprehensive care at Newlands Clinic in Harare, Zimbabwe: TENART cohort.

BACKGROUND: Data on long-term outcomes of patients receiving antiretroviral therapy (ART) in sub-Saharan Africa are few. We describe outcomes of patients commenced on ART at Newlands Clinic between 2004 and 2006 after ≥10 years of comprehensive care including, psychosocial, adherence and food support. METHODS: In this retrospective cohort study, patient data from an electronic medical record collected during routine care were analysed. We describe baseline characteristics, virological and clinical outcomes, attrition rates, and treatment adverse effects until November 2016. We defined virological suppression as viral load <50 copies/ml and virological failure as >1000 copies/ml after ≥6 months of ART. RESULTS: We analysed data for 605 patients (67% female) who commenced ART, and were followed-up for 5819 person-years (median: 10.7 years, IQR: 10.1-11.4). Median age at ART initiation was 34 years (IQR: 17-42). Pre-ART, 129 (21.3%) patients had history of pulmonary tuberculosis (PTB). In care, 66 (11%) developed PTB, and 24 (4%) developed extrapulmonary tuberculosis. 385 (63.6%) patients experienced ≥1 adverse event, the most frequent being stavudine-induced peripheral neuropathy (n = 252, 41.7%). At database closure on 14 November 2016, 474 (78.3%) patients were still in care, 428 (90.3%) being virologically suppressed, and 21 (4.4%) failing. While 483 (79.8%) remained on first line, 122 (20.2%) were switched to second line ART. Fifty-nine patients (9.8%) were transferred to other ART facilities, 45 (7.4%) were lost to follow-up, 25 (4.1%) died, and two stopped ART. CONCLUSION: Comprehensive HIV care can result in low mortality, high retention in care and virologic suppression rates in resource limited settings.

Development and application of a universal Hemoplasma screening assay based on the SYBR green PCR principle.

Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.

Switching to second-line antiretroviral therapy in resource-limited settings: comparison of programmes with and without viral load monitoring.

BACKGROUND: In high-income countries, viral load is routinely measured to detect failure of antiretroviral therapy (ART) and guide switching to second-line ART. Viral load monitoring is not generally available in resource-limited settings. We examined switching from nonnucleoside reverse transcriptase inhibitor (NNRTI)-based first-line regimens to protease inhibitor-based regimens in Africa, South America and Asia. DESIGN AND METHODS: Multicohort study of 17 ART programmes. All sites monitored CD4 cell count and had access to second-line ART and 10 sites monitored viral load. We compared times to switching, CD4 cell counts at switching and obtained adjusted hazard ratios for switching (aHRs) with 95% confidence intervals (CIs) from random-effects Weibull models. RESULTS: A total of 20 113 patients, including 6369 (31.7%) patients from 10 programmes with access to viral load monitoring, were analysed; 576 patients (2.9%) switched. Low CD4 cell counts at ART initiation were associated with switching in all programmes. Median time to switching was 16.3 months [interquartile range (IQR) 10.1-26.6] in programmes with viral load monitoring and 21.8 months (IQR 14.0-21.8) in programmes without viral load monitoring (P < 0.001). Median CD4 cell counts at switching were 161 cells/microl (IQR 77-265) in programmes with viral load monitoring and 102 cells/microl (44-181) in programmes without viral load monitoring (P < 0.001). Switching was more common in programmes with viral load monitoring during months 7-18 after starting ART (aHR 1.38; 95% CI 0.97-1.98), similar during months 19-30 (aHR 0.97; 95% CI 0.58-1.60) and less common during months 31-42 (aHR 0.29; 95% CI 0.11-0.79). CONCLUSION: In resource-limited settings, switching to second-line regimens tends to occur earlier and at higher CD4 cell counts in ART programmes with viral load monitoring compared with programmes without viral load monitoring.

Evaluation of the Partec flow cytometer against the BD FACSCalibur system for monitoring immune responses of human immunodeficiency virus-infected patients in Zimbabwe.

A single-platform volumetric flow cytometer, the Partec Cyflow SL_3, was evaluated against a BD FACSCalibur/Sysmex XT1800i dual platform for measuring CD4(+) lymphocytes, total lymphocytes, and the percentage of CD4 lymphocytes in whole-blood samples for monitoring the immune systems of human immunodeficiency virus (HIV)/AIDS patients. Statistical analyses for precision, correlation, and agreement were performed. Coefficients of variation (CV) of 5.8, 4.6, and 3.9% were obtained for low, medium, and high CD4(+) cell counts, respectively, using the SL_3, and CV of 3.7, 4.0, and 0.94 were obtained for the same categories, using the BD FACSCalibur. Significant correlations (P < 0.005) between the two assays for CD4 counts, total lymphocyte counts, and percentages of CD4 were obtained, with correlation coefficients of 0.99, 0.96, and 0.99, respectively (n = 229). Using the Bland-Altman plot, mean biases of -18 cell/microl (95% confidence interval (CI); -91 to 54 cells/microl), -0.8% (95% CI; -3.6 to 2%), and -36.8 cells/microl (95% CI; -477 to 404 cells/microl) were obtained for comparisons of CD4 counts, percentages of CD4 cells, and total lymphocyte counts, respectively. The effects of the age of the samples on the three parameters were also analyzed by comparing results from the same samples analyzed at 6, 24, and 48 h after collection. The correlation coefficients for comparisons among different time points for the same machine and among all the time points for the two different machines were greater than 0.90. These data showed that the Partec Cyflow SL_3 assay is comparable to the BD FACSCalibur/Sysmex XT1800i dual-platform method for measuring the amount of CD4(+) cells and total lymphocytes and the percentages of CD4 cells in blood samples for the purpose of monitoring HIV/AIDS patients.

Ultrasensitive quantitative HIV-1 p24 antigen assay adapted to dried plasma spots to improve treatment monitoring in low-resource settings.

BACKGROUND: Our group has previously developed a quantitative and ultrasensitive HIV-1 p24 antigen assay that is inexpensive, easy-to-perform, and can be carried out in low-resource settings. Since antiretroviral therapies are becoming more accessible in resource-constrained countries, methods to assess HIV-1 viraemia are urgently needed to achieve a high standard of care in HIV-1 management. OBJECTIVES: To adapt our quantitative assay to dried plasma spots (DPS), in order to further simplify this test and make it more accessible to resource-constrained countries. STUDY DESIGN: DPS from 47 HIV-seropositive, treated or untreated adult individuals and 30 healthy individuals were examined. RESULTS: A specificity of 100% was observed when p24 antigen was measured using DPS, and no differences of p24 concentration could be seen between DPS and venous plasma. The correlation between DPS and venous plasma p24 was excellent (R=0.93, CI(95%)=0.88-0.96, p<0.0001). Similarly, p24 antigen concentrations using DPS were well correlated with RNA viral load (R=0.53, CI(95%)=0.27-0.72, p=0.0002). CONCLUSIONS: This quantitative p24 antigen test has similar sensitivity and specificity using DPS and venous plasma, and has the potential to improve health care delivery to HIV-affected individuals in resource-constrained countries.

Detection of low copy numbers of HIV-1 proviral DNA in patient PBMCs by a high-input, sequence-capture PCR (Mega-PCR).

An internally controlled high-input PCR method, termed HIV-1 Mega-PCR was developed to lower the detection limit of HIV-1 DNA polymerase chain reaction (PCR) and to improve its value as a complementary diagnostic test. It is based on PCR amplification of two target sequences in the gag gene of HIV-1 following the selective capture of the targeted sequence and removal of unselected DNA from up to 500 microg of DNA. Efficient selection and amplification was monitored by inclusion of two mimic plasmids. The method was evaluated with buffy coat cells from healthy blood donors which were spiked with blood from 106 different HIV-1-infected individuals, and with 107 HIV-1 seronegative control buffy coats. All specimens from HIV-infected individuals were positive by a PCR protocol using 1 microg of patient DNA. Amplification of 1 microg DNA of the 106 spiked, diluted samples resulted in 68 double positive, 14 single positive, and 24 double negative reactions. In the Mega-PCR, the average input was 260 +/- 84 microg DNA containing an estimated 1.1 +/- 0.6% of spiked patient DNA. Of the 106 samples tested by Mega-PCR, 102 were positive and three negative. One failed to select the mimic plasmid. Among the 107 negative buffy coat controls, none was false-positive and four exhibited a failure of the internal reaction control. Application of HIV-1 Mega-PCR to clinical specimens from seroreverting newborns of HIV-infected mothers and seroindeterminate, PCR-negative specimens revealed no indication for HIV infection, whereas three samples from confirmed, HIV-1-infected but PCR negative individuals showed evidence of the presence of HIV-1 DNA. Mega-PCR lowers the detection limit of an individual analysis to approximately 0.01 HIV-1 DNA copies/microg of applied DNA and may help to confirm or exclude HIV-1-infection in difficult situations diagnostic.

Combined effect of zidovudine (ZDV), lamivudine (3TC) and abacavir (ABC) antiretroviral therapy in suppressing in vitro FIV replication.

In view of close similarities at the molecular and clinical levels, feline immunodeficiency virus (FIV) infection of the domestic cat is subject of increasing attention as an animal model for human immunodeficiency virus (HIV) infection. A range of reverse transcriptase inhibitors effective against HIV are also active against FIV, allowing successful use of the cat model to investigate drug interactions and resistance development. Nevertheless, while combined nucleoside analog and protease inhibitor usage has proven remarkably effective in treating HIV infection, combination antiretroviral therapy of FIV infection has been hampered by lack of protease inhibitors specific for FIV. In an attempt to circumvent this problem, we have examined the feasibility of applying in the FIV system combination protocols lacking a protease inhibitor. We now report that, as observed during HIV infection, the nucleoside analog abacavir (ABC or 1592U89) is able to effectively block in vitro FIV-replication. Furthermore, we demonstrate that combined usage of ABC with the nucleoside analogs zidovudine (ZDV or AZT) and lamivudine (3TC) also blocks in vitro FIV replication in a synergistic manner. However, in contrast to its effect on HIV replication, the ribonucleotide reductase inhibitor hydroxyurea (HU) is unable to effectively control in vitro FIV replication.

Viracept (Nelfinavir): potência e baixa indução de resistência / Viracept (Nelfinavir): power and low resistance induction

Apresenta um estudo sobre o Nelfinavir como um inibidor altamente sletivo de enzima protease de HIV. Traz a farmacologia clínica e virologia, os testes clínicos, e a experiência pós-comercialização

Viracept (Nelfinavir): potência e baixa indução de resistência / Viracept (Nelfinavir): power and low resistance induction

Apresenta um estudo sobre o Nelfinavir como um inibidor altamente sletivo de enzima protease de HIV. Traz a farmacologia clínica e virologia, os testes clínicos, e a experiência pós-comercialização