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IntroductionLow or undetectable plasma viral load (VL) using current qPCR assays is common for HIV‐2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio‐Rad) underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results [1].Materials and MethodsA RT‐PCR protocol was set up using the One‐Step RT‐ddPCR Kit for Probes on the QX200 platform (Bio‐Rad, Hercules, CA) in an accredited environment (ISO15189:2012 norm). Parameters tested were in line with the digital MIQE guidelines [2]. Inter‐run coefficient of variation (CV) was established using synthetic RNA controls diluted in HIV‐negative plasma. The ddPCR assay was compared to a qRT‐PCR previously used in routine (LOQ 50 cop/mL [3]) using 46 clinical samples and the NIBSC international HIV‐2 RNA standard.ResultsThe optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5′FAM‐probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168) were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999) and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter‐run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5). Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both methods (respective means of 10,612 and 2,224 cop/mL, delta log=0.68) and 12 others were quantified by ddPCR only below 50 cop/mL (mean=16 cop/mL).ConclusionsWe validated a ddPCR HIV‐2 VL assay that is more sensitive and more reproducible than the qPCR assay used as comparator, with a limit of quantification of 7 cop/mL of plasma. A careful definition of the limit of blank allows the management of false positive droplets, but the variable user‐defined positive threshold may be an issue for compliance to the quality norms.

IntroductionWe studied factors associated with the continuum of HIV care in Belgium.MethodsData of the national registration of new HIV diagnosis and of the national cohort of HIV‐infected patients in care were combined to obtain estimates of and factors related with proportions of HIV‐infected patients in each step of the continuum of care from diagnosis to suppressed viral load (VL). Factors associated with ignorance of HIV seropositivity were analyzed among patients co‐infected with HIV and STI in the Belgian STI sentinel surveillance network. Associated factors were identified by multivariate logistic regression.ResultsAmong 4038 individuals diagnosed with HIV between 2007 and 2010, 90.3% were linked to care. Of 11684 patients in care in 2010, 90.8% were retained in care up to the following year, 88.3% of those were on ART, of whom 95.3% had suppressed VL (<500 cp/ml) (Figure 1). In multivariate analyses, factors associated with ignoring HIV+ status were being younger (p<0.001), being heterosexual compared to MSM, and of a region of origin other than Belgium, Sub‐Saharan Africa and Europe. Non‐Belgian regions of origin were associated with lower entry and retention in care (p<0.001 for both). Preoperative HIV testing was associated with lower entry in care (p=0.003). MSM had a higher retention in care (p<0.001), whilst IDU had lower retention (p=0.004). Low CD4 at first clinical contact and clinical reasons for HIV testing were independently associated with being on ART (p<0.001 for both); whilst prenatal HIV diagnosis was associated with lower proportion on ART (p=0.016) and lower proportion with suppressed VL among those on ART (p=0.005). Older age was associated with both being on ART and having suppressed VL among those on ART (p=0.007 and p<0.001 respectively), independently of time since HIV diagnosis (Table 1).ConclusionsRegions of origin and risk groups (MSM/heterosexual/IDU) are the main factors associated with ignorance of HIV seropositivity, entry and retention in care, but once the HIV patient is retained in care, no effect of these factors on the proportions on ART and with suppressed VL are observed. The association of prenatal HIV diagnosis and proportions on ART and with suppressed VL could be biased by transitory CD4 disturbances during pregnancy and ART discontinuation after pregnancy. The higher probabilities of older patients to be on ART and have suppressed VL once retained in care could be influenced by factors not studied here like comorbidities, adherence or duration on ART.