Edouard Tuaillon

Dried blood spot for hepatitis C virus serology and molecular testing

We investigated the performance of dried blood spots (DBS) in hepatitis C virus (HCV) diagnosis using modified commercial tests. Paired DBS and serum samples were collected from 200 patients: 100 patients with anti‐HCV antibodies (anti‐HCV), including 62 patients with detectable serum HCV RNA, and 100 patients without anti‐HCV. The DBS sample consisted of three drops of approximately 50 μL of whole blood applied to a paper card, which was then stored at −20°C within 48 hours of collection. Using the Ortho HCV 3.0 enzyme‐linked immunosorbent assay kit on DBS, we observed both a specificity and sensitivity of 99% in detecting anti‐HCV. HCV RNA was detected on DBS in 60/62 (97%) patients with detectable serum HCV RNA, which was then successfully quantified in 55 samples (89%) using the Cobas TaqMan HCV test. A good correlation was observed between the DBS HCV RNA concentration and the serum level (r2 = 0.95; P < 0.001). HCV genotyping was successfully performed on DBS samples, with a full concordance between the 14 paired DBS and serum samples (genotypes 1‐4). Conclusion: This study presents DBS as a reliable alternative to serum specimens for detecting anti‐HCV, quantifying HCV RNA and genotyping HCV. DBS may increase the opportunities for HCV testing and treatment follow‐up in hard‐to‐reach individuals. (HEPATOLOGY 2010.)

Phenotypic susceptibility to nonnucleoside inhibitors of virion-associated reverse transcriptase from different HIV types and groups.

Objectives: To evaluate a phenotype assay based on plasma reverse transcriptase (RT) to assess HIV susceptibility to nonnucleoside RT inhibitors (NNRTIs). To compare RT-based phenotype with recombinant virus assay (RVA) phenotype- and genotype-based analysis. To assess group O and HIV-2 susceptibility to NNRTIs in correlation with genotype polymorphisms. Methods: RT activity was quantified and its susceptibility to efavirenz, nevirapine, and delavirdine measured as drug concentration resulting in 50% inhibition. RT phenotype was compared with genotype analysis. Eighteen plasma samples from 14 group M- and culture supernatants from 4 group M-, 9 group O-, and 7 HIV-2-infected patients were investigated. RT-based and RVA-based phenotypes were compared for identical plasma from 9 group M-infected patients. Results: RT-based and RVA-based phenotypes were in complete agreement. RT-based phenotype- and genotype-predicted susceptibility were concordant for all but 1 group M samples. One plasma showed susceptibility to 3 NNRTIs by phenotypes, despite the presence of 101E and 106I/V residues. The HIV-2 RTs were totally resistant to the NNRTIs tested. Among HIV-1 group O, 6 were totally resistant to NNRTIs independently of the presence of the 181C mutation and 3 were susceptible to some NNRTIs. Conclusion: Plasma RT-based phenotype could be useful as a simple alternative for monitoring resistance to NNRTIs. This assay is suitable for highly divergent strains. It would be particularly useful for large epidemiologic survey of the natural HIV polymorphism and the potential impact in emergence of drug resistance, particularly to nevirapine, widely used to prevent mother-to-child transmission.

Human Milk-Derived B Cells: A Highly Activated Switched Memory Cell Population Primed to Secrete Antibodies

While secretory Abs have been extensively explored in human breast milk, the existence, features, and functions of B lymphocytes remain largely unexplored in this compartment. We analyzed breast milk and blood lymphocytes from 21 lactating women, including 12 HIV-1-infected mothers. Breast milk B cells displayed a phenotype of class-switched memory B cells, with few IgD+ memory and naive B cells. We observed that breast milk B lymphocytes bore a unique profile of adhesion molecules (CD44+, CD62L−, α4β7+/−, α4β1+). Higher percentages of activated B cells (CD38+), large-sized B cells, plasmablasts, and plasma cells (CD19+, CD20low/−, CD27high, CD138+) were found as compared with blood. This indicates that a significant proportion of breast milk B cells underwent terminal plasma cell differentiation. We also observed a higher frequency of cells secreting Ig spontaneously in breast milk. Among these cells, IgG-secreting cells predominated over IgA-secreting cells as measured by Ig ELISPOT assays. Specific Ab-secreting cells were investigated following polyclonal activation using the CD40L ligation. Finally, the detection of anti-HIV-1-secreting cells demonstrates the existence of B cells specific to HIV-1 Ag in breast milk from HIV-1-infected women. Breast milk B cells display a phenotype strikingly different from blood, are primed to secrete Abs, and have a mucosal homing profile similar to B cells located in gut-associated lymphoid tissue.

HIV-1 Reservoirs in Breast Milk and Challenges to Elimination of Breast-Feeding Transmission of HIV-1

The breast milk of HIV-infected mothers contains reservoirs of HIV, even when they are successfully treated with antiretroviral therapy; new approaches to prophylactic therapy are needed to prevent HIV transmission to their infants through breast-feeding. The breast milk of HIV-infected mothers contains reservoirs of HIV, even when they are successfully treated with antiretroviral therapy; new approaches to prophylactic therapy are needed to prevent HIV transmission to their infants through breast-feeding. By compensating for the relative immaturity of the neonatal immune system, breast milk and breast-feeding prevent deaths in children. Nevertheless, transmission of HIV-1 through breast-feeding is responsible for more than half of new pediatric HIV infections. Recent studies of possible HIV-1 reservoirs in breast milk shed new light on features that influence HIV-1 transmission through breast-feeding. The particular characteristics of breast milk CD4+ T cells that distinguish them from circulating blood lymphocytes (high frequency of cell activation and expression of memory and mucosal homing markers) facilitate the establishment of HIV-1 replication. Breast milk also contains a plethora of factors with anti-infectious, immunomodulatory, or anti-inflammatory properties that can regulate both viral replication and infant susceptibility. In addition, CD8+ T lymphocytes, macrophages, and epithelial cells in breast milk can alter the dynamics of HIV-1 transmission. Even during efficient antiretroviral therapy, a residual stable, CD4+ T cell–associated reservoir of HIV-1 is persistently present in breast milk, a likely source of infection. Only prophylactic treatment in infants—ideally with a long-acting drug, administered for the entire duration of breast-feeding—is likely to protect HIV-exposed babies against all forms of HIV transmission from breast milk, including cell-to-cell viral transfer.

Thalidomide for steroid-dependent immune reconstitution inflammatory syndromes during AIDS.

Management of relapsing or refractory immune reconstitution inflammatory syndromes (IRISs) despite corticosteroid therapy is yet to be defined. We describe three HIV-infected patients with corticosteroid-dependent and life-threatening paradoxical immune reconstitution inflammatory syndrome for whom thalidomide treatment induced rapid clinical remission and permitted complete corticosteroid withdrawal without clinical relapse.

Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB.

Comprehensive proteomic analysis of the human milk proteome: Contribution of protein fractionation

In-depth analysis of the milk proteome by mass spectrometry is challenged by the presence of few high-abundance proteins that interfere with the detection of lower-abundance proteins. Here, we evaluated the proteomic analysis of milk samples following a strong anion exchange fractionation procedure using denaturating conditions ensuring the disruption of protein-protein interactions. Crude whey or skim milk and their different resulting fractions were analyzed by protein chip array mass spectrometry. Using protein chip array mass spectrometry, several high-abundance proteins were localized in distinct fractions increasing the total number of unique peptides and proteins detected. This total number increased by about 20-30% by combining different chromatographic surface arrays used for capture. Reproducible results were obtained in human skim milk and whey; however this approach was not successful with milk fat globule membrane and required refinement. Hence, milk profiling by anion exchange fractionation combined to protein chip array mass spectrometry represents a promising tool to detect unknown low-abundance milk proteins that may ultimately prove useful as biomarkers of diseases transmitted by breastfeeding.

Comparison of Serum HBsAg Quantitation by Four Immunoassays, and Relationships of HBsAg Level with HBV Replication and HBV Genotypes

Background The decline in hepatitis B virus surface antigen (HBsAg) may be an early predictor of the viral efficacy of Hepatitis B virus (HBV) therapy. The HBsAg levels obtained by different immunoassays now need comparing and the relationships between levels of HBsAg and HBV DNA alongside HBsAg and genotype must be evaluated. Methodology/Principal Findings HBsAg levels were compared among 80 patients using the Abbott Architect assay, a commercial immunoassay approved for HBsAg detection and quantitation, and three other assays derived from immunoassays approved for HBsAg detection (manufactured by Diasorin, Bio-Rad and Roche). Good correlation was found between the Abbot vs. Diasorin, Bio-Rad and Roche assays with narrow 95% limits of agreement and small mean differences: −0.06 to 0.11, −0.09 log10 IU/mL; −0.57 to 0.64, −0.04 log10 IU/mL; −0.09 to 0.45, −0.27 log10 IU/mL, respectively. These agreements were not affected by genotypes A or D. HBsAg was weakly correlated with HBV DNA, whatever the HBsAg assay used: Abbott, ρ = 0.36 p = 0.001, Diasorin ρ = 0.34, p = 0.002; Bio-Rad ρ = 0.37, p<0.001; or Roche ρ = 0.41, p<0.001. This relationship between levels of HBsAg and HBV DNA seemed to depend on genotypes. Whereas HBsAg (Abbott assay) tended to correlate with HBV DNA for genotype A (ρ = 0.44, p = 0.02), no such correlation was significant for genotypes D (ρ = 0.29, p = 0.15). Conclusion/Significance The quantitation of HBsAg in routine clinical samples is comparable between the reference assay and the adapted assays with acceptable accuracy limits, low levels of variability and minimum discrepancy. While HBsAg quantitation is not affected by HBV genotype, the observed association between levels of HBsAg and HBV DNA seems genotype dependent.

Combining rapid diagnostic tests and dried blood spot assays for point-of-care testing of human immunodeficiency virus, hepatitis B and hepatitis C infections in Burkina Faso, West Africa

People screened for human immunodeficiency virus (HIV) using rapid diagnostic tests (RDTs) in Africa remain generally unaware of their status for hepatitis B (HBV) and hepatitis C (HCV) infections. We evaluated a two-step screening strategy in Burkina Faso, using both HIV RDTs and Dried Blood Spot (DBS) assays to confirm an HIV-positive test, and to test for HBV and HCV infections. HIV counselling and point-of-care testing were performed at a voluntary counselling and testing centre with HBV, HCV status and HIV confirmation using DBS specimens, being assessed at a central laboratory. Serological testing on plasma was used as the reference standard assay to control for the performance of DBS assays. Nineteen out of 218 participants included in the study were positive for HIV using RDTs. A fourth-generation HIV ELISA and immunoblot assays on DBS confirmed HIV status. Twenty-four out of 25 participants infected with HBV were found positive for hepatitis B surface antigen (HBsAg) using DBS. One sample with a low HBsAg concentration on plasma was not detected on DBS. Five participants tested positive for HCV antibodies were confirmed positive with an immunoblot assay using DBS specimens. Laboratory results were communicated within 7 days to participants with no loss to follow up of participants between the first and second post-test counselling sessions. In conclusion, DBS collection during HIV point-of-care testing enables screening and confirmation of HBV, HCV and HIV infections. Diagnosis using DBS may assist with implementation of national programmes for HBV, HCV and HIV screening and clinical care in middle- to low-income countries.

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

BackgroundTransmission 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.MethodsThe ex vivo 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.ResultsAmong 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.ConclusionsActivated 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.