Granulocyte contamination dramatically inhibits spot formation in AIDS virus-specific ELISpot assays: analysis and strategies to ameliorate.

The interferon-gamma (IFNgamma) ELISpot assay has become the most critical tool for HIV vaccine evaluation. External factors affecting ELISpot results must be minimized for the data to be reliably used in vaccine research and development processes. In pre-clinical pigtail macaque studies analyzing HIV/SIV vaccine studies, we detected a strong correlation between levels of granulocytes contaminating PBMC preparations and reduction in the quality and quantity of spots in the IFNgamma ELISpot assay. Acute SHIV infection of macaques worsened granulocyte contamination of PBMC fractions and made the assay much less reliable in detecting SIV-specific T cell immunity compared to intracellular cytokine staining (ICS). This problem could be ameliorated by using an F(ab)(2) form of the MD-1 IFNgamma capture antibody, presumably reflecting that activation of granulocytes in the well by the Fc portion of the standard capture antibody disrupts spot formation. Improving the standard ELISpot assay by using an F(ab)(2) capture antibody should make it more reliable for use in critical vaccine development studies.

Exploring the Benefits of Molecular Testing for Gonorrhoea Antibiotic Resistance Surveillance in Remote Settings.

BACKGROUND: Surveillance for gonorrhoea antimicrobial resistance (AMR) is compromised by a move away from culture-based testing in favour of more convenient nucleic acid amplification test (NAAT) tests. We assessed the potential benefit of a molecular resistance test in terms of the timeliness of detection of gonorrhoea AMR. METHODS AND FINDINGS: An individual-based mathematical model was developed to describe the transmission of gonorrhoea in a remote Indigenous population in Australia. We estimated the impact of the molecular test on the time delay between first importation and the first confirmation that the prevalence of gonorrhoea AMR (resistance proportion) has breached the WHO-recommended 5% threshold (when a change in antibiotic should occur). In the remote setting evaluated in this study, the model predicts that when culture is the only available means of testing for AMR, the breach will only be detected when the actual prevalence of AMR in the population has already reached 8 - 18%, with an associated delay of ~43 - 69 months between first importation and detection. With the addition of a molecular resistance test, the number of samples for which AMR can be determined increases facilitating earlier detection at a lower resistance proportion. For the best case scenario, where AMR can be determined for all diagnostic samples, the alert would be triggered at least 8 months earlier than using culture alone and the resistance proportion will have only slightly exceeded the 5% notification threshold. CONCLUSIONS: Molecular tests have the potential to provide more timely warning of the emergence of gonorrhoea AMR. This in turn will facilitate earlier treatment switching and more targeted treatment, which has the potential to reduce the population impact of gonorrhoea AMR.

Atazanavir is not associated with an increased risk of cardio- or cerebrovascular disease events.

OBJECTIVE: To investigate whether there is any association between exposure to atazanavir (ATV), either when boosted or unboosted by ritonavir, and myocardial infarction (MI) or stroke within the D:A:D: Study. DESIGN: Prospective cohort collaboration. METHODS: Poisson regression was used to investigate the association between cumulative exposure to ATV and MI/stroke risk after adjusting for known demographic and clinical confounders, as well as cumulative and recent exposure to specific antiretroviral drugs. Follow-up started on enrolment in the study and ended at the earliest of: a new MI/stroke event, death, 6 months after last clinic visit, or 1 February 2011. RESULTS: The incidence of MI varied from 0.28 [95% confidence interval (CI) 0.26-0.30)]/100 person-years of follow-up (PYFU) in those with no exposure to ATV to 0.20 (0.12-0.32)/100 PYFU in those with more than 3 years exposure. There was no evidence of an association between cumulative exposure to ATV and MI risk, either in univariate [relative rate/year 0.96 (95% CI 0.88-1.04)] or multivariable [0.95 (0.87-1.05)] analyses. The incidence of stroke was 0.17 (0.16-0.19)/100 PYFU in those with no exposure to ATV and 0.17 (0.10-0.27)/100 PYFU in those with more than 3 years exposure. As with the MI endpoint, there was no evidence of an association with ATV exposure in either univariate [1.02 (0.98-1.05)] or multivariable [0.95 (0.87-1.05)] analyses. CONCLUSION: These results argue against a class-wide association between exposure to HIV protease inhibitors and the risk of cardio/cerebrovascular events.