CD4+ T cells spontaneously producing human immunodeficiency virus type I in breast milk from women with or without antiretroviral drugs

<p>Abstract</p> <p>Background</p> <p>Transmission of human immunodeficiency virus type 1 (HIV-1) through breast-feeding may involve both cell-free and cell-associated virus. This latter viral reservoir remains, however, to be fully explored. CD4⁺T cell-associated virus production in breast milk was therefore investigated.</p> <p>Methods</p> <p>The <it>ex vivo </it>spontaneous production of HIV-1 antigen and HIV-1 RNA by CD4⁺T cells was measured in paired blood and breast milk samples from 15 HIV-1 infected women treated or not with antiretroviral drugs. Spontaneous antigen secreting cells (HIV-1-AgSCs) from breast milk and blood were enumerated by an ELISpot assay, and cell-associated HIV-1 RNA was quantified by real-time PCR in supernatants of CD4⁺T cells cultured for 18 hours without addition of polyclonal activators.</p> <p>Results</p> <p>Among the CD4⁺T cells present in breast milk, memory cells expressing high levels of cell-surface activation markers were predominant. Spontaneous HIV-1-AgSCs were detected and enumerated in the breast milk of all 15 women, with a median number of 13.0 and 9.5 HIV-1- AgSCs/106 CD4⁺T cells in aviremic (n = 7) and viremic (n = 8) women, respectively. Cell- associated HIV-1 RNA was detected in cell-free supernatants from 4/7 aviremic and 5/8 viremic individuals at median levels of 190 and 245 copies/ml, respectively.</p> <p>Conclusions</p> <p>Activated CD4⁺T cells producing HIV-1 are detected in the breast milk of untreated individuals as well as those receiving highly active antiretroviral therapy. This finding strongly suggests that HIV-1 replication occurs in latently infected CD4⁺T cells that, upon spontaneous activation, revert to productively infected cells. These cells might be responsible for a residual breast milk transmission despite maternal highly active antiretroviral therapy.</p

The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification.

Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples.Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples.BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays.Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load

Case Report: Persistency Pneumococcal Polysaccharide in Cerebrospinal Fluid During a Post Pneumococcal Chronic Aseptic Meningitis: Coincidental or (Auto-)Inflammatory Embers

International audienceWe report the case of a 9-months-old boy that has presented a steroid-dependent post-pneumococcal chronic aseptic meningitis was associated with persistence of pneumococcal cell wall components in cerebrospinal fluid during more than 20 months. Suggesting that this antigenic persistence could be involved in post-infectious manifestations through innate immunity response

Biases, standard deviations and comparison of biases (P values) of Bland-Altman curves on different EBV strains.

<p>Biases, standard deviations and comparison of biases (P values) of Bland-Altman curves on different EBV strains.</p

Increased Epstein-Barr virus in breast milk occurs with subclinical mastitis and HIV shedding

Epstein–Barr virus (EBV) in breast milk and subclinical mastitis (SCM) are both associated with human immunodeficiency virus (HIV) shedding and possibly with postnatal HIV transmission. The objective of this nested case–control study was to investigate the interplay between SCM and EBV replication in breast milk of HIV-infected mothers. The relationships between EBV deoxyribonucleic acid (DNA) shedding, HIV-1 ribonucleic acid (RNA) level, and SCM were explored in breast milk samples of Zambian mothers participating in the ANRS 12174 trial. Mammary gland inflammation was defined as a breast milk sodium to potassium ratio (Na+/K+) greater than 0.6 and further subclassified as either “possible SCM” (Na+/K+ ratio 0.6–1.0) or SCM (Na+/K+ ratio ≥ 1.0). Breast milk interleukin 8 (IL-8) was measured as a surrogate marker of mammary gland inflammation. EBV DNA was detected in breast milk samples from 42 out of 83 (51%) participants and was associated with HIV-1 shedding in breast milk (P = 0.006). EBV DNA levels were higher in samples with SCM and “possible SCM” compared to non-SCM breast milk samples (P = 0.06; P = 0.007). An EBV DNA level of >200 copies/mL was independently associated with SCM and “possible SCM” (OR: 2.62; 95%: 1.13–6.10). In patients with SCM, higher EBV replication in the mammary gland was associated with a lower induction of IL-8 (P = 0.013). Resistance to DNase treatment suggests that EBV DNA in lactoserum is encapsidated. SCM and decreased IL-8 responses are associated with an increased EBV shedding in breast milk which may in turn facilitate HIV replication in the mammary gland

Bland-Altman bias plots comparing Bam-W repeated- and LMP2 single- sequence qPCRs on whole blood extracts of clinical samples.

<p>Values having EBV DNA load equal or higher than LOD of LMP2 (A) and Bam-W (B) in-house qPCR assays were compared with the initial clinical values (equal or higher than LOD) performed by EBV R-gene commercial kit, and with each other (C). Dotted lines represent the borders of 95% CI and 0 point.</p

Bland-Altman bias plots for three different quantitative EBV DNA real-time PCR assays.

<p>Ten serial dilutions of Raji and B95.8 cell line supernatants were tested for EBV DNA quantification by LMP2 and EBV R-gene qPCRs (A). The same samples were tested by Bam-W qPCR compared with R-gene (B) and LMP2 qPCRs (C).</p