Development of a method for direct quantification of cytomegalovirus antigenemia by flow cytometry.

Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.     

HHV-6 cell receptor CD46 expression on various cell subsets of six blood and graft sources: A prospective series

BackgroundCord Blood (CB) are increasingly used as an alternative stem cells source in adults for allogeneic Stem Cell Transplantation (allo-SCT). The risk of human herpesvirus (HHV-6) reactivation is significantly higher after CB transplant vs unrelated peripheral blood stem cells (PBSC) allo-SCT. Higher HHV-6 cell receptor CD46 expression on progenitor cells in CB may explain this difference.ObjectivesTo prospectively compare the HHV-6 cell receptor CD46 expression on various cell subsets of three freshly harvested blood sources on one hand and of three graft sources on the other hand.Study design52 samples were used for the purpose of this study. They were issued from peripheral blood (PB, n = 10), G-CSF mobilised PB (GCSF-PB, n = 10), cord blood (CB, n = 10), unmanipulated bone marrow (uBM, n = 5), leukapheresis product (LP, n = 10) and thawed CB graft (n = 7). CD46 expression was assessed by FACS analysis on total lymphocytes, monocytes, NK cells, T and B cells subsets, plasmacytoid (pDCs) dendritic cells and stem cells.ResultsAs all cell subsets were found CD46 positive, CD46 mean fluorescence intensity (MFI) was then considered for comparison between the three blood sources and the three graft sources. The most impressive result observed was that HHV-6 cell receptor CD46 expression was significantly reduced in almost all cell components of thawed CB graft compared to other graft sources.ConclusionsThis original study shows strong differences in term of quantitative CD46 expression between several blood and grafts samples. Our results suggest that other factors than the qualitative CD46 expression play a role in the higher HHV-6 reactivation observed after CB transplant in adults.     

The epidemiology of Neisseria meningitidis meningitis in Togo during 2003-2005

Few reports documenting the epidemiology of Neisseria meningitidis (Nm) serogroup W135 exist, and none from Togo. During 2003-2005, we conducted acute bacterial meningitis surveillance at three major reference hospitals in Togo. Of 116 Nm identified, 83 (71%) were NmA, 23 (20%) were NmW135, and 10 (9%) did not have a serogroup identified. Nine percent of NmW135 cases and 35% of NmA cases occurred among those aged 15 years or older. The two hospitals in central Togo reported 23% of all Nm cases and 78% of NmW135 cases. Twelve of the 23 NmW135 cases occurred during February-March 2003, while the remaining 11 occurred sporadically over the remaining 18 months of the study. NmW135 meningitis showed pronounced temporal and geographic clustering and occurred almost exclusively among those younger than 15 years old. By the 2004-2005 epidemic season, NmW135 had largely disappeared from Togo for unknown reasons.     

Sequential use of paraformaldehyde and methanol as optimal conditions for the direct quantification of ZEBRA and rta antigens by flow cytometry

A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixation-permeabilization procedures were compared. The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies, were found to be the optimal conditions in these cell lines. Our procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.     

Whole-Genome Characterization of Epidemic Neisseria meningitidis Serogroup C and Resurgence of Serogroup W,Niger, 2015

In 2015, Niger reported the largest epidemic of Neisseria meningitidis serogroup C (NmC) meningitis in sub-Saharan Africa. The NmC epidemic coincided with serogroup W (NmW) cases during the epidemic season, resulting in a total of 9,367 meningococcal cases through June 2015. To clarify the phylogenetic association, genetic evolution, and antibiotic determinants of the meningococcal strains in Niger, we sequenced the genomes of 102 isolates from this epidemic, comprising 81 NmC and 21 NmW isolates. The genomes of 82 isolates were completed, and all 102 were included in the analysis. All NmC isolates had sequence type 10217, which caused the outbreaks in Nigeria during 2013–2014 and for which a clonal complex has not yet been defined. The NmC isolates from Niger were substantially different from other NmC isolates collected globally. All NmW isolates belonged to clonal complex 11 and were closely related to the isolates causing recent outbreaks in Africa.Key words: Meningococcal meningitis, Neisseria meningitidis serogroup C, whole-genome sequencing, Niger, meningitis belt, bacteriaNeisseria meningitidis commonly causes meningitis in the African meningitis belt, where periodic meningococcal epidemics have contributed to the highest reported incidence of meningococcal meningitis in the world (1). Most meningococcal disease historically has been caused by N. meningitidis serogroup A (NmA); however, NmA disease dramatically decreased after the preventative MenAfriVac vaccination campaign was initiated in 2010 (2). Serogroup W (NmW) has been the major cause of meningococcal disease in the region since then (2).N. meningitidis serogroup C (NmC) disease has rarely been reported in the meningitis belt; it has not been detected in many molecular studies of invasive isolates (3,4) and is rarely found in carriage studies (5,6). The last large NmC epidemic in Africa occurred in Burkina Faso (then Upper Volta) in 1979 (7). During 2013 and 2014, NmC outbreaks were reported in Nigeria (8). The Nigerian outbreaks were caused by a novel NmC strain with a previously undescribed sequence type, 10217 (ST-10217), which does not belong to a defined clonal complex. In 2015, an epidemic of 9,367 meningococcal meningitis cases occurred in Niger, with NmC disease comprising most laboratory-confirmed cases (9).NmW disease has been reported in the meningitis belt since the 1980s (10,11), and NmW from clonal complex 11 (CC11) has been a major concern in the region since 2001 (12). The first large epidemic of disease caused by CC11 NmW occurred during 2002 in Burkina Faso (13). Subsequently, NmW disease outbreaks were reported in Niger during 2010 and 2011, both involving CC11 (14). These outbreaks were followed by another large epidemic caused by CC11 NmW in Burkina Faso during 2012 (15). Whole-genome sequencing (WGS) analysis of diverse NmW isolates from around the world has demonstrated that a clone within CC11, commonly associated with NmC, became globally dispersed after it switched to serogroup W (16,17). WGS analyses also provided sufficient resolution to assign isolates from the meningitis belt to a long-standing regional population and to a clone that became globally dispersed after an outbreak during the 2000 Hajj pilgrimage (16,17; A. Retchless, unpub. data).In addition to distinguishing among closely related strains, WGS provides information about allelic variation in genes that may affect antibiotic susceptibility and the coverage of protein-based vaccines. Two vaccines designed for serogroup B meningococcus have been approved for use in the United States and Europe: Trumenba and Bexsero. Trumenba targets the factor H–binding protein (FHbp), and includes components belonging to FHbp subfamilies A and B (18). Bexsero includes 4 different components: an FHbp of variant 1 (subfamily B); a Neisseria adhesion A protein (NadA); a neisserial heparin-binding antigen (NhbA); and outer membrane vesicles from a serogroup B strain containing PorA P1.4 (19). Recognizing the diversity of these genes among strains can aid in evaluating whether these vaccines may provide protection. Likewise, whole-genome sequences can be rapidly screened for indications of antibiotic resistance when the genetic determinants are well characterized, as with genes penA, gyrA, and rpoB, which are involved in reduced susceptibility to penicillin, ciprofloxacin, and rifampin, respectively. To clarify the meningococcal population in Niger during the 2015 epidemic season, we completed genomic analysis on the 102 NmC and NmW invasive isolates collected during this period.     

Three cases of invasive meningococcal disease caused by a capsule null locus strain circulating among healthy carriers in Burkina Faso

During reinforced surveillance of acute bacterial meningitis in Burkina Faso, meningococcal strains of phenotype NG:NT:NST were isolated from cerebrospinal fluid samples from 3 patients. The strains were negative for the ctrA gene but were positive for the crgA gene. Molecular typing revealed that the strains harbored the capsule null locus (cnl) and belonged to the multilocus sequence type (ST)-192. PorA sequencing showed that all strains were either P1.18-11,42; P1.18,42-1; P1.18-11,42-1; P1.18-11,42-3; or P1.18-12,42-1. Sequencing also showed that all strains were negative for the FetA receptor gene. Serum killing assays showed these strains to be resistant, with the resistance comparable with that of a fully capsular serogroup B strain, MC58. The same strains were found in 14 healthy carriers in the general population of Bobo-Dioulasso (100% of ST-192 isolates tested for cnl). The presence of cnl meningococci that can escape serum killing and cause invasive disease is of concern for future vaccination strategies and should promote rigorous surveillance of cnl meningococcal disease.     

Modulation of associated ovarian carcinoma antigens by 5 cytokines used as single agents or in combination

Optimization of intraperitoneal radioimmunotherapy of ovarian cancer depends on increasing the antigenic expression of tumor cells. For this purpose, we studied the effect of 5 cytokines (IFN-α, IFN-β, IFN-γ, TNF-α and TGF-β), used as single agents or in combination, on 4 ovarian cancer cell lines which present different antigenic profiles with the monoclonal antibodies (MAbs) tested (OC125, OVTL-3, MOv 18 and MOv 19). Analyses were performed by flow cytometry and the Scatchard technique in order to study antigenic modulation. The effect on proliferation was determined by cell counting. Expression of O3 antigen, recognized by the OVTL3 MAb, was increased up to 2.5 times after IFNs and TNF-α (used as single agent) on the 2 lines presenting low basal expression (SHIN-3 and IGROVI). The expression of CAI25 antigen and the antigens recognized by MOv 18 and MOv 19 MAbs was not increased by any of the cytokines tested. The combination IFN-γ + TNF-α was synergistic on cytotoxicity and enhanced O3 expression, providing 10 times as many sites per cell on the SHIN-3 line. For 3 other associations (IFN-α + IFN-γ, IFN-β + IFN-γ and IFN-α + TNF-α), there was an additive effect on O3 expression and on cell cytotoxicity. © 1994 Wiley-Liss, Inc.     

Clinical relevance of direct quantification of pp65 antigenemia using flow cytometry in solid organ and stem cell transplant recipients

A total of 1,305 blood samples from 85 solid organ transplant (SOT) recipients and 25 stem cell transplant (SCT) recipients at risk for cytomegalovirus (CMV) infection were prospectively collected and tested using the shell vial assay (SVA) and a leukocytic qualitative PCR (q-PCR). Of these, 462 specimens were further tested by direct quantification of CMV antigenemia by flow cytometry (FC-Ag), 125 were tested with a quantitative competitive PCR, and 200 were tested for pp65 antigenemia using the slide method (S-Ag). Laboratory data were statistically analyzed according to the presence of CMV-related symptoms. In SOT and SCT recipients, active CMV infection occurred in 63.5 and 36%, respectively, and CMV disease occurred in 53 and 24%, respectively. FC-Ag results correlated better with q-PCR and S-Ag than with SVA. The first test found to be positive during follow-up was FC-Ag in 73% of cases. In SOT recipients, FC-Ag showed the highest sensitivity and negative predictive value for the diagnosis of any grade of CMV disease. For FC-Ag, the threshold beyond which CMV disease was highly probable seemed to lie at 0.20% positive polymorphonuclear leukocytes. FC-Ag appears to be a useful test for the early detection of CMV infection and the prediction of CMV disease.