Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler(® )Real-time PCR

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART. In the present study, a relative quantification assay of HIV-1 proviral DNA by LightCycler(® )real-time PCR based on SYBR Green I detection was developed in comparison to the number of purified CD4+ cells as estimated by the quantification of the β-globin gene. METHODS: The ability of the designed gag primers to quantify HIV-1 Group M and the PCR efficiency were assessed on HIV-1 reference isolate subtypes A, B, C and D. The 8E5 cell line containing a single defective copy of HIV-1 proviral DNA was used as a standard for both the HIV-1 target gene and the β-globin reference gene. The assay was applied on thirty consecutive patient samples received for RNA viral load determinations and on retrospective samples from fifteen patients undergoing 2 years of structured treatment interruption (STI). RESULTS: The lower limit of quantification was 50 HIV-1 DNA proviral copies per CD4+ cell sample. The dynamic range was from 50 to 10(6 )HIV-1 DNA copies per CD4+ cell sample with intra- and inter-assay coefficients of variability ranging from 3.1% to 37.1%. The β-globin reference gene was quantified down to a limit of 1.5 pg of DNA/μl (approximately 5 cells) with intra- and interassay coefficients of variability ranging from 1.8% to 21%. DNA proviral load varies widely among HIV-1 infected patients. Proviral load and plasma viral load rebound were high in STI patients who took longer to achieve an undetectable plasma viral load under therapy. A statistically significant correlation was observed between DNA proviral load and RNA steady state viral load in STI patients (p-value = 0,012). CONCLUSION: We have developed a fast, sensitive and specific relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells. The assay enables the monitoring of HIV-1 proviral load, which may be useful to monitor therapy efficacy especially in patients with undetectable plasma RNA viral load, and allows the exploration of viral reservoirs

Stable hepatitis C virus RNA detection by RT-PCR during four days storage

BACKGROUND: Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV) RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4°C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples. METHODS: Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 – 25.4°C) or at 4°C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems). RESULTS: The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4°C). CONCLUSIONS: We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4°C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results

Study of structure and function relationship in HIV-1 proteins

The past decade has been a rapid evolution of knowledge and technology in virology, both fundamental and applied. The advances in the diagnosis of viral infections and in research on the viral pathogenic mechanisms and the relationship between virus and host cells, owe much to the rapid development in the fields of immunology and molecular biology. The work we present here has evolved together with the availability of new techniques and illustrates how, starting from very practical questions on the diagnosis and the epidemiology of human immunodeficiency virus (HIV), one arrives at more fundamental questions on viral diversity and the characteristics of viral proteins. In our studies in 1990, we were interested in the epidemiology and the clinical correlates of HIV infections in Benin, where our institution has longstanding contacts with des University of Cotonou. Serological and virological studies indicated a much wider diversity of HIV-1 in part of Africa than what is seen in industrialized countries. This diversity has been further described by different groups and led to the categorization of HIV-1 in subgroups and subtypes. Our approach, which is described in the second part, was to generate monoclonal antibodies against the Gag (“group specific antigen”)-encoded proteins with the aim of better defining the antigenic structure and diversity of these conserved genome products. Through mapping of the antigenic sites recognized on the HIV-1 protein Cap24 (the major capsid protein) we were able to describe a previously unrecognized epitope and to identify one monoclonal against a putative conformational epitope. The availability of monoclonals allowed us to go beyond the purely descriptive and to investigate the structure-function relationships, particularly with the anti-nucleocapsid (NCp15 and NCp7) mAbs. These functional studies led us to the third part of our work, where we applied recombinant technology to produce and characterize regulatory proteins of HIV-1. The interaction of these proteins with cellular elements could thus be studied, with particular attention for Nef, whose role is still debated, but which seems to be essential for disease progression. Finally, difficulties in the production of recombinant Vpr led us to investigate its role in the inhibition of cell growthThèse d'agrégation de l'enseignement supérieur -- UCL, 199

Predictive value of maternal-IgG avidity for congenital human cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) is now the most common cause of viral intrauterine infection. Fetal damage is mostly linked to maternal primary infection. It is therefore important to differentiate primary from recurrent or persistent HCMV infection in pregnant females. For this purpose, IgM tests are not reliable enough and the measurement of the IgG avidity appears to be presently the best method. OBJECTIVE: To evaluate the performance of the measurement of HCMV-IgG avidity by a 8 M urea denaturation assay in predicting congenital infection in the offspring. STUDY DESIGN: Seventy-eight women were included in this study on the basis of a HCMV IgM positive or equivocal result on a first serum during pregnancy, but without a documented seroconversion history. The IgG avidity was measured and correlated with the outcome of the pregnancy. RESULTS: In eight cases of HCMV in utero infection the maternal HCMV-IgG avidity index was below 50%. One case of HCMV in utero infection was observed despite a high avidity index during the second trimester of the pregnancy. High or intermediate HCMV-IgG avidity indexes during the first trimester of pregnancy were not associated with a congenital infection. CONCLUSIONS: Even in the presence of an IgM positive result, an HCMV IgG avidity index above 65% on a serum obtained during the first trimester of pregnancy could reasonably be considered as a good indicator of past HCMV infection. In these conditions invasive prenatal diagnosis is not necessary

L'infection congénitale à cytomégalovirus : rôle du laboratoire de virologie

L'infection à CMV est l'infection congénitale la plus fréquente, avec une incidence moyenne de 1 %. L'infection peut se transmettre in utero lors d'une primo-infection maternelle ou lors d'une réactivation de l'infection chez une mère séropositive avant grossesse. La fréquence et la gravité de l'infection congénitale sont très différentes suivant le cas et il est donc essentiel de pouvoir faire le diagnostic différentiel entre infection primaire et réactivation. Dans ce contexte, il n'existe aucun test de référence et la présence d'IgM est encore trop souvent considérée comme un critère d'infection récente. Différentes techniques ont été développées afin d'améliorer le diagnostic. Parmi ces nouvelles approches, la plus utilisée est la mesure de l'avidité des IgG qui permet d'exclure une infection récente dans bon nombre de cas. Si le risque est élevé pour le foetus (séroconversion maternelle ou IgG de faible avidité) le diagnostic de l'infection congénitale est réalisé par la recherche de CMV dans le liquide amniotique en culture et/ou en PCR ; les performances de ces 2 techniques en termes de sensibilité et de spécificité sont comparables. Il faut cependant garder à l'esprit, que si la mise en évidence de virus dans le liquide amniotique signe une infection congénitale, elle ne permet pas d'en évaluer la gravité

Increased risk of cytomegalovirus transmission in utero during late gestation.

OBJECTIVE: To determine whether the rate of human cytomegalovirus transmission in utero is related to the gestational age at the time of maternal infection. METHODS: One hundred twenty-three pregnant women followed in our units between 1988 and 1998 were studied retrospectively. Each had developed a primary infection with cytomegalovirus evidenced by a seroconversion, confirmed by specific enzyme immunoassays. Infants were diagnosed by urine culture. RESULTS: Regardless of gestational age at the time of maternal cytomegalovirus seroconversion, the mean rate of intrauterine transmission was 57.5%. There was a statistically significant difference between early seroconversion (during the first trimester) and late seroconversion (during the third trimester) (36.0% versus 77.6%; P < .001). The risk of transmission calculated for seroconversion during the second trimester was intermediate (44.9%). CONCLUSION: A statistically significant difference in the rate of intrauterine cytomegalovirus transmission was observed according to the duration of pregnancy at which primary infection occurred. The rate of transmission increased with gestational age

Ability of three IgG-avidity assays to exclude recent cytomegalovirus infection.

At present, the measurement of IgG avidity appears to be the best method for differentiating primary from nonprimary cytomegalovirus (CMV) infection. This study compared the performances of three denaturation assays for the measurement of CMV IgG avidity: an in-house method and the commercially available assays Enzygnost (Dade-Behring, Germany) and Vidas (bioMérieux, France). The ability of these assays to exclude or to detect a recent CMV infection were calculated according to the results obtained in two control groups of pregnant women: 49 who had seroconverted and 80 with past infections. All three assays demonstrated a good ability to detect a recent infection (98-100%). The Dade-Behring test, in its present form, appears to be ineffective in excluding a recent CMV infection (exclusion ability: 30%), while the in-house method (exclusion ability: 96%) and the bioMérieux method (exclusion ability: 82%) performed better. The practical use of the in-house and the bioMérieux assays was evaluated in 80 women with CMV-specific IgG and a positive IgM result but without documented seroconversion. At the recommended diagnostic thresholds, the concordance between these two tests was 70%. Larger studies will allow more precise determination of the capacities of both assays and specification of the diagnostic thresholds or grey areas to be used

Avidity of IgG antibodies distinguishes primary from non-primary cytomegalovirus infection in pregnant women.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Fetal damage is mostly linked to maternal primary infection. It is therefore important to differentiate primary from non-primary infection in pregnant females. IgM tests often used for this purpose are not reliable enough. OBJECTIVE: To evaluate an HCMV-IgG urea-elution assay for its ability to distinguish primary from non-primary infection. In this assay, soaking the antigen-antibody complex with an urea containing solution frees antibodies with low avidity but has no influence on those with high avidity. An avidity index (AI) was calculated: AI = (OD with urea/OD without urea) x 100. STUDY DESIGN: HCMV-IgG avidity was measured on a single serum of 79 patients with past infection (pregnant women, graft recipients and blood donors) and of 63 patients (78 sera) with documented seroconversion (pregnant women and graft recipients). Sixty-one pregnant women positive or equivocal for HCMV-IgM but without a documented seroconversion were included in this study. RESULTS: Most (72/79) of the patients with past infection had an AI > 65% and all but one had an AI > 50%. In pregnant women, in the case of a primary infection within the past 3 months, AI are usually (51/53) 65%. Among the IgM positive pregnant women who lack a seroconversion history, 38 had AI > 65% suggestive of an infection that had occurred at least 3 months earlier, 11 had an AI in a grey area between 50 and 65% and 12 had an AI 65% highly suggests a past infection while an AI < 50% corresponds to a recent primary infection