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Rapid diagnostic tests (RDTs) are routinely used in African blood centres. We analysed data from two cross-sectional studies representing 95 blood centres in 29 African countries. Standardized panels of sera containing varying concentrations of anti-human immunodeficiency virus (HIV) antibodies (Ab), hepatitis B virus antigen (HBsAg) and antihepatitis C virus (HCV) Ab were screened using routine operational testing procedures at the centres. Sensitivity of detection using RDTs was high for HIV Ab-positive samples, but low for intermediately HBsAg (51·5%) and HCV Ab (40·6%)-positive samples. These findings suggest that current RDT use in Africa could pose a hazard to blood safety.

There has been little blood safety research in sub-Saharan Africa, often consisting of local efforts whose findings had limited impact The "Francophone Africa Transfusion Research Network" was created in May 2007 with the objective of developing common evidence-based blood safety policies that may be adapted to each country's situation. The Group's activities to date have focused mainly on obtaining epidemiological and laboratory data on blood transfusion and on suggesting blood safety strategies, particularly in the field of TTIs. To carry out such research activities, the group works closely with the National Blood Transfusion Services (NBTS), the Regional Blood Transfusion Services (RBTS), the hospital blood banks (HBB) and collection stations. For the first 5years, four research priorities were identified: (i) descriptive studies of the characteristics of francophone African blood donors and blood centers; (ii) estimation of the residual risk of transfusion-transmitted major viral infections; (iii) an analysis of blood donor deferral strategies; and (iv) a description of TTI screening strategies and an external quality assurance system (EQAS) project. During this period, seven projects have been implemented at the national level and published and five multicenter studies were conducted and published. The present review reports the main observations and recommendations from those studies that could improve blood safety statute in Africa.

International audience; Background. Characterisation of human-associated viral communities is essential for epidemiological surveillance and to be able to anticipate new potential threats for blood transfusion safety. In high-resource countries, the risk of blood-borne agent transmission of well-known viruses (HBV, HCV, HIV and HTLV) is currently considered to be under control. However, other unknown or unsuspected viruses may be transmitted to recipients by blood-derived products. To investigate this, the virome of plasma from individuals at high risk for parenterally and sexually transmitted infections was analysed by high throughput sequencing (HTS). Materials and methods. Purified nucleic acids from two pools of 50 samples from recipients of multiple transfusions, and three pools containing seven plasma samples from either HBV-, HCV- or HIV-infected blood donors, were submitted to HTS. Results. Sequences from resident anelloviruses and HPgV were evidenced in all pools. HBV and HCV sequences were detected in pools containing 3.8x10(3) IU/mL of HBV-DNA and 1.7x10(5) IU/mL of HCV-RNA, respectively, whereas no HIV sequence was found in a pool of 150 copies/mL of HIV-RNA. This suggests a lack of sensitivity in HTS performance in detecting low levels of virus. In addition, this study identified other issues, including laboratory contaminants and the uncertainty of taxonomic assignment of short sequence. No sequence suggestive of a new viral species was identified. Discussion. This study did not identify any new blood-borne virus in high-risk individuals. However, rare and/or viruses present at very low titre could have escaped our protocol. Our results demonstrate the positive contribution of HTS in the detection of viral sequences in blood donations.

INTRODUCTION: The World Health Organization (WHO) recommends the creation of national blood transfusion services. Burkina Faso has a CNTS (Centre National de Transfusion Sanguine—National Blood Transfusion Center) but it currently covers only 53% of the national blood supply versus 47% produced by independent hospital blood banks. STUDY DESIGN: To evaluate blood collection, testing, preparation, and prescription practices in the regions of Burkina Faso that are not covered by the CNTS, a cross-sectional survey was conducted. METHODS: Data were collected by trained professionals from May to June 2009 at 42 autonomous blood centers not covered by the CNTS. RESULTS: Blood collection was supervised in all sites by laboratory technicians without specific training. There was no marketing of community blood donation nor mobile collection. Donation was restricted to replacement (family) donors in 21.4% of sites. Predonation screening of donors was performed in 63.4% of sites, but some did not use written questionnaires. Testing for HIV, hepatitis B virus, and syphilis was universal, although some sites did not screen for hepatitis C virus. In 83.3% of the sites, blood typing was performed without reverse ABO typing. In 97.6% of the sites, nurses acted alone or in conjunction with a physician to order blood transfusions. CONCLUSION: Shortcomings in non-CNTS blood centers argue for the development of a truly national CNTS. Such a national center should coordinate and supervise all blood transfusion activities, and is the essential first step for improving and institutionalizing blood transfusion safety and efficacy in a developing country.

Since its discovery, human parvovirus B19 (B19V), now termed erythrovirus, has been associated with many clinical situations (neurological and myocardium infections, persistent B19V DNAemia) in addition to the prototype clinical manifestations, i.e., erythema infectiosum and erythroblastopenia crisis. In 2002, the use of new molecular tools led to the characterization of three different genotypes of human B19 erythrovirus. Although the genomic organization is conserved, the geographic distribution of the different genotypes varies worldwide, and the nucleotidic divergences can impact the molecular diagnosis of B19 virus infection. The cell cycle of the virus remains partially unresolved; however, recent studies have shed light on the mechanism of cell entry and the interactions of B19V proteins with apoptosis pathways.

Platelet concentrates account for near 10% of all labile blood components but are responsible for more than 25% of the reported adverse events. Besides factors related to patients themselves, who may be particularly at risk of side effects because of their underlying illness, there are aspects of platelet collection and storage that predispose to adverse events. Platelets for transfusion are strongly activated by collection through disposal equipment, which can stress the cells, and by preservation at 22 °C with rotation or rocking, which likewise leads to platelet activation, perhaps more so than storage at 4 °C. Lastly, platelets constitutively possess a very large number of bioactive components that may elicit pro-inflammatory reactions when infused into a patient. This review aims to describe approaches that may be crucial to minimising side effects while optimising safety and quality. We suggest that platelet transfusion is complex, in part because of the complexity of the “material” itself: platelets are highly versatile cells and the transfusion process adds a myriad of variables that present many challenges for preserving basal platelet function and preventing dysfunctional activation of the platelets. The review also presents information showing - after years of exhaustive haemovigilance - that whole blood buffy coat pooled platelet components are extremely safe compared to the gold standard (i.e. apheresis platelet components), both in terms of acquired infections and of immunological/inflammatory hazards.

In low-income-countries, screening for hepatitis C virus (HCV) infection is often based on rapid tests (RT). Their lower sensitivity compared to enzyme immunoassay (EIA) suggests that newer HCV Antigen/Antibody (Ag/Ab) combination assays might have a role in such countries. To test this idea, 1998 blood donors were tested at the University Teaching Hospital blood bank in Yaoundé, Cameroon simultaneously with a RT (HCV rapid test, Human Diagnostics, Berlin, Germany) according to standard practice (S1) and with an Ag/Ab assay (Monolisa HCV Ag/Ab Ultra, Biorad, France) (S2). All discordant, borderline and reactive samples were submitted to confirmatory testing by immunoblot and/or HCV-RNA. Of the 86 (4.3%) samples positive with one or both strategies, 29 were confirmed negative, 37 positive and 20 were false positive or resolved infection. There was a significant difference in test sensitivity (p= 0.01) between S1 (70.3%) and S2 (91.9%) but not in test specificity (99.4% and 98.6%, respectively). The benefit of the Ag/Ab assay in the detection of recent HCV seronegative infections could not be evaluated since no Antigen-only donations were identified. However, better Ag/Ab test sensitivity compared to RT supports the implementation of these newer immunoassays for HCV screening in the African blood bank setting. © 2014 Elsevier B.V.

This study, involving 20 laboratories and using currently available assays for hepatitis C virus RNA quantification, demonstrated that differences in viral load values are due not to interlaboratory variations but rather to the nature of the assay itself. This underlines the importance of using the same assay in multicenter studies or when monitoring antiviral therapy.

A cohort of 103 human immunodeficiency virus type 1 (HIV-1)-infected persons with well-defined dates of seroconversion were studied to determine whether baseline plasma HIV RNA loads 6-12 months after seroconversion have prognostic value. Baseline plasma virus loads had predictive value for the disease-free survival rate and for the survival rate. The level of baseline HIV RNA also had a strong negative predictive value for the CD4 + T cell count during the fifth year of infection: A baseline load >5 log was predictive of a CD4 + T cell count

The aims of this study were to determine the outcome and the natural history of GBV-C/hepatitis G virus (HGV) infection and to establish the frequency of acute or persistent infections in multiply-transfused individuals and blood donors. We used a GBV-C/HGV RNA polymerase chain reaction (PCR) and an assay evidencing antibodies to the envelop protein E2, which is considered a marker for virus clearance. Among 16 PCR-positive recipients, 11 were still positive for GBV-C/HGV RNA at the end of the study period; six of the 16 recipients were GBV-C/HGV infected during the study period and thus had a well-defined date of infection. The 16 patients were shown to carry GBV-C/HGV RNA over a mean period of 4.4 years, for a mean observational period (defined as the follow-up period since the first sample positive for GBV-C/HGV RNA) of 5.3 years. Within the limits of the study period, the patients with a well-defined date of infection were positive for GBV-C/HGV RNA during a mean period of 4.7 years. If defined by the presence of GBV-C/HGV RNA for at least 6 months, the persistent infection rate was 100% in this recipient cohort. Serum anti-E2 antibody was evidenced at least once in five (31.2%) recipients and, except in one case, became detectable after the loss of GBV-C/HGV RNA. Among the 11 blood donors, all were still positive for GBV-C/HGV RNA after a mean follow-up period of 7.7 months. The persistent infection rate was 100% in this donor cohort. Once acquired, the infection to GBV-C/HGV generally tends to persist in immunocompetent patients.

Despite a decade of human immunodeficiency virus (HIV) seropositivity, a few individuals termed as long-term nonprogressors (LTNPs) maintain a stable CD4+ T-cell count for a period of time. The aim of this study was to establish, through the sequential determination of all known predictors of HIV disease, the proportion of such patients having stringent criteria of true long-term nonprogression. Among 249 individuals who were HIV-infected and prospectively followed up over a 10-year period (1985 to 1995), 12 having a CD4+ T-cell count greater than 500/μL (LTNP I group) and 9 having a CD4+ T-cell count less than 500 but stable over time (LTNP II group) after at least 10 years of infection without intervention of antiviral therapy, were studied over the entire follow-up period. The plasma HIV RNA copy number and the serum concentrations of p24 antigen, each anti-HIV antibody, neopterin, β-2-microglobulin, Immunoglobulin (Ig) G and IgA were determined every 18 months over the study period. Cellular and plasma viremias were cross-sectionaly assayed in all 21 patients. Only two patients had strictly no marker of progression over the follow-up period. They were the only ones who had, over the 10-year period, a viral copy number too low to be detected. The other patients had a viral copy number higher than 400/mL at at least one visit and increasing over the follow-up period, and they evidenced one or more markers of virological or immunological deterioration. Cellular viremia was positive in all patients but two, while plasma viremia was negative in all but one. The population of individuals termed as LTNPs is not virologically and immunologically homogeneous. The majority present biological signs of HIV disease progression. A new pattern of true LTNP can be drawn through stringent criteria based on the whole known predictors. This pattern appears to be rare in HIV-positive population.

Les travaux de recherches sur la sécurité transfusionnelle en Afrique sub-saharienne sont peu nombreux, souvent limités à des initiatives locales avec des conclusions difficilement représentatives de cette région. Le Groupe de recherches transfusionnelles en Afrique sub-saharienne francophone a été créé en mai 2007 avec pour objectif de développer des stratégies globales d’amélioration de la sécurité transfusionnelle mais adaptables à la situation de chaque pays. Les activités du Groupe à ce jour ont porté essentiellement sur l’obtention de données épidémiologiques et de laboratoire sur la transfusion sanguine et à proposer des stratégies de sécurité transfusionnelle dans le domaine des infections transmissibles par la transfusion. Pour mener à bien ces activités de recherche, le Groupe travaille en étroite collaboration avec les Centres nationaux de transfusion sanguine (CNTS), les Centres régionaux de transfusion sanguine (CRTS), les banques de sang hospitalières (BSH) et les postes de collecte de sang. Pour les 5 premières années, quatre priorités de recherche ont été identifiées: (i) des études descriptives sur les caractéristiques des donneurs de sang et des centres de transfusion; (ii) une estimation du risque résiduel post-transfusionnel des principales infections virales transmissibles par la transfusion; (iii) une analyse des stratégies de sélection médicale des donneurs de sang; et (iv) une description des stratégies de dépistage des ITT et une description du système d’assurance qualité externe existant. Durant cette période, sept projets ont été mis en œuvre au niveau national et publiés et cinq études multicentriques ont été réalisées et publiées. La présente étude rapporte les principales observations et recommandations de ces études.

Lefrère Jean-Jacques. Edmond de Goncourt dans le Journal de Brinn'Gaubast. In: Cahiers Edmond et Jules de Goncourt n°4, 1995. Hommage à Edmond de Goncourt pour le centième anniversaire de sa mort. pp. 290-294.

Blood donor selection is important to ensure the safety of both donors and recipients. There is a paucity of data on reasons for blood donor deferral in Ivory Coast. The aim of this study was to identify the reasons for predonation deferral at a blood collection site at General Hospital, Yopougon Attié in Abidjan.The investigators conducted a retrospective audit of data pertaining to donor deferral for blood donors that presented to the general hospital of Yopugon Attié from January 1, 2006 to December 31, 2008.A total of 10,694 prospective blood donors, presented over the study period, and 24,363 attempts to donate were registered. The majority were repeat blood donors (77.4%). A total of 2618 (10.8%) donors were deferred. The most frequent reason for deferral was a low hemoglobin level (42.5%), with females constituting the majority of those deferred. The second most frequent reason for deferral was a reported change of or new sexual partner (34.3%); male donors were predominant in this group. Additional reasons for deferral included short interdonation interval (4.6%) and reactivity for a screened biomarker (2.3%).Although the rates for permanent and temporary deferral rates are similar between the Ivory Coast and high-middle income countries, the causes and demographics differ. The reasons for exclusion are preventable through awareness and education of prospective blood donors.

To evaluate the prognostic value of provirus copy number through quantitative DNA polymerase chain reaction (PCR) in early stages of human immunodeficiency virus type 1 (HIV-1) infection, 42 untreated and asymptomatic HIV-1-seropositive subjects with baseline CD4+ cell counts > 200 x 10(6)/L were included in a prospective study and followed over a median of 27 months. Disease progression was defined as decrease in CD4+ cells to < 200 (14 events). At enrollment, provirus copy number was associated with CD4+ cell count and percentage, serum IgA, and p24 antigenemia. Elevated provirus copy number above 20 allowed identification of patients at high risk of a subsequently decreasing CD4+ cell count, even after adjusting for baseline CD4+ cell count (P = .003). Measuring provirus copy number by PCR at early stages of HIV-1 infection could offer a useful early means to predict progression to AIDS.

Lefrère Jean-Jacques. Les Goncourt avant l'Académie et après. In: Cahiers Edmond et Jules de Goncourt n°3, 1994. pp. 122-129.

Hepatitis C virus RNA quantification results obtained in 18 laboratories using real-time PCR methods with 10 negative samples and 22 sample dilutions (viral loads of 0.5 to 500 IU/ml) showed a score of correct results of up to 93.5%. However, 55.6% of the laboratories did not follow the recommendations for the interpretation of their results, leading to ambiguous conclusions.

Human parvovirus 4 infections are primarily associated with parenteral exposure in western countries. By ELISA, we demonstrate frequent seropositivity for antibody to parvovirus 4 viral protein 2 among adult populations throughout sub-Saharan Africa (Burkina Faso, 37%; Cameroon, 25%; Democratic Republic of the Congo, 35%; South Africa, 20%), which implies existence of alternative transmission routes.

STUDY OBJECTIVE--The aim was to develop a new approach for estimating the incubation period of acquired immunodeficiency syndrome (AIDS), based on age distributions. DESIGN--Incubation period was expressed as the difference between age at time of diagnosis and age at time of contamination. Assuming independence between age at time of infection and incubation period, the age distribution of newly diagnosed AIDS cases is thus the convolution product between the distributions of the age of freshly infected patients and of the incubation times. AIDS incubation time can therefore be estimated from the age distribution of newly HIV infected subjects and newly diagnosed AIDS cases. SUBJECTS--Subjects were 2220 AIDS cases diagnosed until 1987, reported to the Ministry of Health, France, and 172 subjects discovered to be HIV-1 seropositive during a blood donation in Paris between August 1985 and July 1988. In both groups, the only known risk factor was homosexuality. MAIN RESULTS--The estimated median incubation time was 9.9 years (90% CI 9.0-10.9 years). Confidence intervals were narrow, even when taking into account the uncertainty in serodetection delay (90% CI 6.7-13.5 years). CONCLUSIONS--The incubation estimate is as accurate as previous estimates based on other models. This technique could therefore be applied to other risk groups.

Although HIV-1 Vpr displays several functions in vitro, limited information exists concerning their relevance during infection. Here, we characterized Vpr variants isolated from a rapid and a long-term non-progressor (LTNP). Interestingly, vpr alleles isolated from longitudinal samples of the LTNP revealed a dominant sequence that subsequently led to diversity similar to that observed in the progressor patient. Most of primary Vpr proteins accumulated at the nuclear envelope and interacted with host-cell partners of Vpr. They displayed cytostatic and proapoptotic activities, although a LTNP allele, harboring the Q65R substitution, failed to bind the DCAF1 subunit of the Cul4a/DDB1 E3 ligase and was inactive. This Q65R substitution correlated with impairment of Vpr docking at the nuclear envelope, raising the possibility of a functional link between this property and the Vpr cytostatic activity. In contradiction with published results, the R77Q substitution, found in LTNP alleles, did not influence Vpr proapoptotic activity.

A vital research enterprise has been essential to the development and maintenance of high levels of transfusion safety in developed countries. Virology, immunology and clinical pathology research contribute to the development of sensitive and specific assays for infectious disease, accurate matching of blood components to the recipient, and approaches for the production of high quality, sterile and contaminant-free blood components. Equally important are epidemiologic and clinical research efforts aimed at developing accurate statistics on transfusion related infections and other complications, to evaluate hypotheses on transfusion-related disease and implement clinical trials of new transfusion therapies. Developing countries often struggle with a lack of trained personnel and financial resources to carry out their routine operations, and are deprived of clinical research that could guide blood safety policy appropriate to their populations and resources. Even though there is often a willingness to perform research in these countries, the means are often lacking to start the process with precise research questions and continue it with rigourous study designs based upon sound epidemiological and logistical considerations. To help develop intellectual capital in transfusion medicine research in developing countries, Blood Systems Research Institute (BSRI) and the University of California San Francisco (UCSF), together with other blood centers in the USA and Europe, have long welcomed medium-term trainees from developing countries. However these six-week to six-month internships are relatively expensive, require the trainee to be absent from their blood center and family for long periods of time and for this reason are suited to only small numbers of trainees. Instead, over the past five years, we have developed a novel two-week research training course which is given in-country. The course is based upon the format of a six-week course directed by Professor Steven Hulley at UCSF[1] and is centered on the development of a feasible transfusion research protocol. Mornings are devoted to lectures devoted to specific topics in transfusion medicine research and clinical research methodology, including choice of the research question and hypotheses, study design, choice of subjects, definition and measurement of variables, data analysis and calculation of sample size. Lecture content is specifically adapted to issues in transfusion-related research with real-life examples and interaction with the participant projects. In the afternoons, practical workshops allow each trainee to develop a one paragraph research question into a 5 to 6 page research protocol under the direct guidance of the course faculty-preceptors. The expectation is that the protocol will be complete and ready for implementation in the participant’s country. Each afternoon’s writing assignment follows the morning lectures and readings from the course textbook[1]. Research questions are chosen mainly by the subject in consultation with their mentor or supervisor at their home blood center, but course faculty suggest modifications to assure that the protocol describes precise hypotheses, appropriate study design, adequate sample size and available resources. Trainees are generally MDs, PhD’s or senior technicians who work in blood centers in Latin America, Africa or Asia. We choose younger candidates whose careers will benefit most from the training and who have the potential for future leadership in transfusion medicine research in their home countries. Applicants are chosen on the basis of their curriculum vitae and letters of recommendation from their home country supervisors, with certification that adequate release time will be given to the trainee after their return from course so that they may accomplish their research. During the course, trainees are evaluated on the basis of their written research protocol, and their participation in a peer review session on the last day of the course during which each trainee is assigned to present a verbal critique of one of their colleague’s protocols in the group setting. This exercise, which simulates a protocol or a grant application review committee, reinforces the lessons of the course teaches the ability to give criticism constructively and to receive and benefit from peer feedback. Over the past four years, the course has been given eight times to a total of 84 trainees. The course was initially developed for Brazil and Latin America under seed grant funding from BSRI, and has been given in Sao Paulo (2004; 9 students), Buenos Aires (2005; 13 students), Tegucigalpa (2006; 7 students) and Belo Horizonte (2008; 16 students). Subsequently, it was incorporated into the curriculum of the Ecole Pasteurienne d’Infectiologie, a new master’s degree granting program in public health at the Institut Pasteur in Paris, France. That course, given in French to an average of 10 trainees annually in 2007, 2008 and planned for May 2009, is oriented primarily towards blood bankers from Francophone Africa. Finally, an unrestricted educational grant from Novartis Vaccines and Diagnostics (Chiron) and co-sponsorship by South African National Blood Service allowed the development of a South African venue for the course. Courses have been given in Johannesburg (2007; 12 students), Durban (2008; 10 students) and tentatively Cape Town (2009) with trainees drawn initially from South Africa and eventually from all of Anglophone sub-Saharan Africa. Another innovation is an annual competition for mini-grants of approximately $5000 apiece to allow alumni of that year’s training course to accomplish their newly-designed research projects. Applications consist of the research protocol developed by the trainee together with their curriculum vitae. A committee reviews the applications and awards grants according to the following criteria: scientific merit of the research protocol; promise of the candidate for a research career; and need for external funding to accomplish the research project in their blood center. Several alumni from the course have also participated in longer training experiences in San Francisco directed at specific research interests or laboratory technique, or at data analysis and manuscript preparation in a 6-week course developed by the Center for AIDS Prevention Studies (CAPS) at UCSF. Our aim is to assist the most promising candidates to develop independent research careers by mentoring them in the development of additional research projects of applications for external grant funding. To date, there have been a total of six published research articles [2-7], another four manuscripts in preparation, and more than 10 abstracts submitted to international scientific conferences. This issue contains two articles which exemplify a new approach to developing transfusion medicine research capacity in low and medium resource countries. Tayou-Tagny et al. present a cross-sectional survey of current blood collections, infectious disease testing and blood center organization in Francophone Africa. Most of the contributing authors were participants in the first Francophone Africa research training course described below. The article gives reason for hope: national blood policies adopted in five out of seven countries, systematic screening for infectious disease in all (at least most of the time). However much remains to be done: only a minority of blood center personnel are well trained in transfusion medicine, only two countries have 100 percent volunteer donors and component preparation remains the exception rather than the rule. Laperche et al. describe a collaborative approach to infectious disease marker test external quality assessment (EQAS). Starting from the same network described in the Tayou Tagny article, the French National Institute of Transfusion Safety (INTS) provided pedigreed serum aliquots in a blinded fashion to participating blood center laboratories. The results were provocative. Whereas sensitivity and specificity of traditional enzyme immunoassays was acceptable at most centers, the sensitivity of rapid tests was unacceptably low, particularly for HBsAg. The use of rapid tests in a pre-donation screening algorithm may still be defensible, but these tests ought not be used without subsequent enzyme immunoassay testing of the donated blood unit. The study is another outcome of our recent training activities in transfusion medicine research. Future plans for the course include the development of a two-week, in-country scientific manuscript writing course to help trainees publish and disseminate their research findings. We also hope to offer selected alumni of the 2-week course opportunities for longer internships or even Master’s degree education in San Francisco, Paris or Sao Paulo. Finally, a planned website will allow us to facilitate communication and networking between trainees and alumni of the program, to post announcements regarding future courses and conferences of interest to alumni, to aid in collaborative writing of manuscripts, and to post grant opportunities for which the alumni may be eligible. We are currently in the process of applying for grant funding to continue current training activities and to fund these future plans. In summary, we have developed a unique short course program directed at the development of transfusion research capability rather than operational skills. Initial results from the program are encouraging, and we hope that it will be successful in addressing critical needs for intellectual capital among blood bankers in developing countries. We are aware that other centers have developed training activities in developing countries and would encourage them to incorporate training in research in their outreach activities.

Hepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5′-untranslated region (5′UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5′UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5′UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5′UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.

A national evaluation study was performed in 14 specialized laboratories with the objective of assessing their capacities to provide (i) hepatitis B virus (HBV) viral loads (VL), (ii) HBV genotypes, and(iii) identification of precore/core mutants. The panel consisted of 12 HBV DNA-positive samples with VLs from 2.8 to 9.1 log10copies/ml, different HBV genotypes (A to F), and 3 mutant and 9 wild-type samples at nucleotide 1896. The coefficients of variation of the mean VLs ranged from 2.4% to 10.4% with the Cobas HBV Monitor assay, from 1.8% to 5.5% with the CobasTaqMan 48, from 1.5 to 26.2% with RealArt HBV PCR, and from 0 to 7% with branched DNA (bDNA). The Cobas Monitor assay underestimated the VLs of genotype F samples, with differences ranging from 1.4 to 2.4 log10copies/ml. The accuracies of genotype determinations ranged from 33% to 100%, and those of precore mutant determinations ranged from 25 to 100%. This study showed some drawbacks of two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping and the determination of precore/core mutants need to be clearly stated to justify the standardization of these methods.

A multicenter study of NS5b hepatitis C virus (HCV) genotype determination involving 12 laboratories demonstrates that any laboratory with expertise in sequencing techniques would be able to provide a reliable HCV genotype for clinical and epidemiological purposes as long as they are provided a consensus reference sequence database.

Zeta-associated protein 70 (ZAP-70), a member of the Syk family of protein tyrosine kinases, is normally expressed in T and NK cells. While little is known about ZAP-70 expression in normal human B cells, it has been reported that ZAP-70 is expressed in a subset of patients with chronic lymphocytic leukemia (CLL) with a poor prognosis. In this study, we examined the expression and phosphorylation status of ZAP-70 in B-lineage acute lymphoblastic leukemia (Blin-ALL).First, ZAP-70 protein expression was assessed by Western blotting and flow cytometry and ZAP-70 mRNA transcripts were analyzed by reverse transcription polymerase chain reaction (RT-PCR) on human precursor B cell lines. Experiments were then carried out on cells obtained from 18 patients with Blin-ALL and from normal human bone marrow.ZAP-70 was constitutively expressed and phosphorylated on tyr319 in human precursor Blin-ALL cell lines as well as in primary B leukemic cells from all examined Blin-ALL patients with pro-B, pre-B and B phenotypes, but not in malignant myeloid cells. Importantly, analysis of normal human bone marrow revealed expression of ZAP-70 transcripts only in the CD34+ cell fraction (either CD19-CD10- or CD19+CD10+) but not in the CD34- cell fraction (CD19+sIgM- pre-B cells or CD19+sIgM+ immature B cells).ZAP-70 was found to be expressed in the CD34+ normal bone marrow compartment including earlier B-cell progenitors, but not in CD34- pre-B and immature B cells. By contrast, ZAP-70 was consistently expressed and phosphorylated in Blin-ALL cells. Further studies are required to determine whether ZAP-70 may play a pathophysiological role in Blin-ALL.

A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5′ noncoding (5′ NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5′ NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5′ NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.

Before initiating new large-scale therapeutic trials for hepatitis C virus (HCV)-infected patients, the French Health Authorities for HCV research decided to organize an evaluation of the expertise of laboratories that could be engaged to undertake molecular biology assays in such trials; 21 experienced laboratories participated in this national evaluation of laboratory expertise, which was performed in two successive rounds. The first round evaluated the laboratories for their abilities to detect HCV RNA in serum, determine genotypes, and quantify HCV RNA loads. The results observed by qualitative assays for HCV RNA detection were 100% sensitivity and 100% specificity for all laboratories. The genotyping results were 100% concordant for 9 laboratories and greater than 90% for 10 laboratories. By contrast, large coefficients of variation were observed for quantitative determination of HCV RNA loads, leading to a second round with standardized quantitative assays only. The dispersion of the results was larger by the AMPLICOR HCV Monitor assay than by the branched-DNA assay (mean coefficients of variation, 57.4 and 16.9%, respectively). In the majority of cases, discrepancies between the results of the two tests were found for samples with high viral loads. These results indicate the usefulness of validating, by controlling for expertise, both the reliabilities of laboratories involved in multicenter work and the standardized assays chosen for use in the evaluation of the biological impacts of new therapies.

Little is known about the natural history and the pathogenicity of the TT virus (TTV). We present our findings of a cross-sectional study based on the TTV DNA screening of 173 multiple-transfused patients and a longitudinal study based on the follow-up of TTV DNA–positive patients. Overall, 48 patients (27.7%) tested positive for TTV DNA. The influence of the number of blood donor exposures on the prevalence of blood-borne viral infection indicates that TTV, hepatitis C virus (HCV), and an RNA virus known as GB virus C/hepatitis G virus (GBV-C/HGV) share a parenteral transmission, but that TTV, in contrast to the 2 other viruses, is also transmitted by at least another efficient means. The patients having a well-defined date of TTV infection were positive for TTV DNA during a mean period of 3.1 years. A chronic infection was observed in 31 cases (86%). TTV carriage appeared clinically benign in all patients. No clinical evidence of a disease potentially linked to the TTV infection was observed in patients with TTV DNA carriage over several years. The majority of TTV carriers had no biochemical evidence of liver disease. The prevalence of elevated serum alanine aminotransferase (ALT) level was higher in the TTV DNA–positive group, even in the absence of HCV infection, but the observed peaks of ALT level were most often transient and very mild. The prevalence of TTV DNA observed in blood recipients is consistent with that of TTV infection observed in blood donors. TTV infection frequently tends to persist. (Blood. 2000;95:347-351)

To clarify the frequency and prognostic significance of a plasma human immunodeficiency virus (HIV) RNA load below the detection threshold during the natural history of infection, an ultrasensitive assay was used to identify persons with low virus loads in a cohort of 111 untreated subjects with a known date of seroconversion. Six persons had HIV RNA loads

The prevalence of GB virus C (GBV-C) infection is high in human immunodeficiency virus (HIV)-infected persons. However, the long-term consequences of coinfection are unknown. HIV-positive persons with a well-defined duration of infection were screened on the basis of their GBV-C/hepatitis G virus (HGV) RNA status and studied. GBV-C/HGV viremia was observed in 23, who carried the virus over a mean of 7.7 years. All parameters (survival, CDC stage B/C, HIV RNA load, CD4 T cell count) showed significant differences in terms of the cumulative progression rate between persons positive and negative for GBV-C/HGV RNA. When GBV-C/HGV RNA-positive and -unexposed subjects were matched by age, sex, baseline HIV RNA load, and baseline CD4 T cell count, HIV disease progression appeared worse in GBV-C/HGV RNA-negative subjects. The carriage of GBV-C/HGV RNA is associated with a slower progression of HIV disease in coinfected persons.

Cases of partial seroreversion have been reported in hemodialyzed or immunodepressed patients, but spontaneous clearance of viremia associated with a disappearance of specific antibodies or clearance while receiving therapy has not been precisely documented in immunocompetent hepatitis C virus (HCV)-infected persons. A longitudinal study of markers of HCV infection in a cohort of 178 multitransfused patients followed over an 8-year period was done to establish well-documented cases of partial or full seroreversion. Thirty (16.8%) of 178 patients were HCV-infected; among them, 5 had partial or full seroreversion. Seroreversion to an anti-HCV-negative state is characterized by a quantitative decrease in antibody. A seroreversion may be observed in three circumstances: spontaneously, induced by therapy, and in conjunction with human immunodeficiency virus infection. Long-term follow-up of seroreverters will establish whether they have definitively eradicated HCV from their systems.

The presence of human herpesvirus 8 (HHV-8) DNA sequences was sought by polymerase chain reaction (PCR) in peripheral blood mononuclear cells of 4 groups : 6 human immunodeficiency virus (HIV)-infected persons with well-defined dates of seroconversion, during the period between the diagnosis of HIV infection and the appearance of Kaposi's sarcoma (KS) ; 45 HIV-positive persons with no symptoms of HIV infection ; 11 AIDS patients with KS ; and 14 AIDS patients without KS. HHV-8 DNA PCR was positive in 3 of the 6 patients during HIV infection preceding the appearance of KS and in all but 1 of 11 AIDS patients with KS. HHV-8 DNA PCR was negative in all but 1 of the 45 HIV-positive persons with no symptoms of infection and in all but 1 AIDS patient without KS. These results indicate that HHV-8 DNA may be detected several years before the occurrence of KS in HIV-infected subjects.

To assess the place of passive immunotherapy in the treatment of AIDS, a randomized study was conducted that evaluated the safety and short-term efficacy of serial transfusions of human immunodeficiency virus type 1 (HIV-1) seropositive plasma in 18 patients. Heat-inactivated anti-HIV antibody-rich plasma was compared with seronegative fresh-frozen seronegative plasma given in addition to zidovudine and other conventional prophylactic treatments. Seven transfusions every 2 weeks of immune plasma significantly reduced (2 vs. 8, P = .016) the number of opportunistic infections. Antigenemia became undetectable. When transfusions were stopped, positive p24 antigenemia returned at a level higher than before treatment and was correlated with a severe clinical deterioration, suggesting a rebound effect. This trial suggests that passive immunotherapy is promising in AIDS treatment. It confirms also that plasma donation does not affect donors' CD4 cell count over a 1-year period. In patients with severe immunodeficiency, special attention should be paid to withdrawal of an effective therapy as virologic relapse may be explosive and poorly tolerated.

To confirm the presence of DNA from human T lymphotropic virus type I (HTLV-I), HTLV-II, or both in individuals found HTLV-I/II-positive through systematic screening of blood donations in Guadeloupe (French West Indies), 42 blood donors repeatedly positive for HTLV-I/II by ELISA were studied by polymerase chain reaction (PCR). Three primer pairs (env, pol, tax) targeted on conserved regions of HTLV-I or -II sequences (or both) and six probes (two generic, two HTLV-I-specific, two HTLV-II-specific) were used in a multiplex PCR. HTLV-I sequences were detected in 31 individuals (74%). All 31 subjects positive by Western blot (WB) harbored HTLV-I sequences. Fifteen individuals (48%) were positive with the three primer pairs used, 10 (32%) with two, and 6 (20%) with one. Subjects indeterminate or negative by WB were all negative by PCR. No HTLV-II sequences were detected with specific probes. The results indicate the absence of HTLV-I and -II infection in individuals with indeterminate WB, the presence of HTLV-I DNA in individuals positive for WB in the French West Indies, and the absence of HTLV-II infection in the cohort.

Zeta-associated protein 70 (ZAP-70), a member of the Syk family of protein tyrosine kinases is normally expressed in T and NK cells. ZAP-70 plays a key role in the signaling pathway of the TCR. It has been recently shown that this protein is also expressed in mouse B lineage cells. While little is known about ZAP-70 expression in normal human B cells, it has been reported that ZAP-70 is expressed in a subset of chronic lymphocytic leukemia (CLL), with a poor prognosis, that have nonmutated immunoglobulin variable region genes. In this study, we examined the expression and phosphorylation status of ZAP-70 and Syk in B-lineage acute lymphoblastic leukemia (B-ALL) cells. ZAP-70 and Syk were constitutively expressed and phosphorylated (tyr319 for ZAP and tyr352 for Syk) in human precursor B-ALL cell lines as well as in B leukemic cells from all examined B-ALL patients (n=18). The expression of ZAP-70 was specific to these B-ALL cells as it was not detected in the K562 cell line or malignant myeloid cells. This expression was independant of maturation state of leukemic cells (pro-B or pre-B) and caryotypic status. The expression and phosphorylation of the T-cell “specific” ZAP-70 kinase in all B-ALL cells raises important questions concerning the contribution of this protein to the leukemogenesis process resulting in B-ALL.

Serum vitamin K concentrations and prothrombin induced by absence of vitamin K (PIVK-II) concentrations were assayed in 43 patients with cystic fibrosis. Twenty nine showed a normal PIVKA-II and vitamin K concentrations; 14 showed an increased PIVKA-II concentration, in one of whom serum vitamin K was decreased. Although their vitamin K concentrations were normal, some patients with cystic fibrosis still had an increased PIVKA-II. There was a significant correlation between PIVKA-II concentrations and the administration of antibiotics, a factor which has not previously been considered responsible for an increase in PIVKA-II.

A new method for assaying the activity of des-γ-carboxyprothrombin (DCP), using staphylocoagulase on undiluted adsorbed plasma, is described. The thrombin-coagulase formed is measured on a chromogenic substrate, and the results are expressed in milliunits per milliliter of increment of the optical density following the release of p-nitroaniline. Levels of DCP in 96 normal subjects were under 10 mU/ml (mean, 3.58 mU/ml). Of 70 nonhepatectomized patients with hepatocellular carcinomas, 74% had increased DCP levels of between 20 and 420 mU/ml (most of the values were between 20 and 100 mU/ml). Des-carboxyprothrombin and α-fetoprotein measurements gave complementary information, one marker or the other being positive in 87% of hepatocellular carcinoma. Fourteen of 15 patients with metastatic carcinoma of the liver had normal DCP levels, as did 95 patients with liver cirrhosis and 13 patients with chronic hepatitis. When the level of "total factor II" is below 40%, it is-recommended that a second determination of DCP be performed 5 days after the injection of vitamin K, to exclude any vitamin K deficiency (in the case of hepatocellular carcinoma the DCP level will remain elevated). The DCP assay appears more sensitive and more specific than the a-fetoprotein assay for the diagnosis of hepatocellular carcinoma; furthermore, both tests are complementary.

Three cases of acute myeloblastic leukemia of FAB-M2 type revealed by an isolated otitis media are reported. This mean of revelation is valuable for early diagnosis and treatment of the disease. A karyotype was performed in two cases and showed an 8;21 translocation. As far as we know, these are the first acute myeloblastic leukemia cases with otitis media reported in which an 8;21 translocation was detected.

From March 1984 through November 1985, 24 children and adults with aplastic crises were admitted in several Parisian hospitals. Twelve patients had known hemolytic anemia. Aplastic crisis revealed hemolytic anemia in the remaining patients. The detection of human parvovirus antigen was performed by counter-immunoelectrophoresis, and specific IgM antibodies were detected by IgM-antibody-capture-radioimmunoassay, in order to establish the incidence of human parvovirus infection in the genesis of the aplastic crisis. Twenty-one patients had acute infection with human parvovirus. In the three remaining patients, no marker of human parvovirus infection was found. The features of the human parvovirus linked aplastic crisis are described. We consider that human parvovirus infection, and unknown hemolytic anemia, must be systematically researched in any case of unexplained acute aplastic anemia.