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

Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital

Background: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. Methods: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. Results: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5 UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. Conclusions: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular

The HUMTICK study: protocol for a prospective cohort study on post-treatment Lyme disease syndrome and the disease and cost burden of Lyme borreliosis in Belgium.

In Belgium, different routine surveillance systems are in place to follow-up Lyme borreliosis trends. However, accurate data on the disease and monetary burden for the different clinical manifestations are lacking. Despite recommended antibiotic treatment, a proportion of Lyme patients report persisting aspecific symptoms for six months or more (e.g. fatigue, widespread musculoskeletal pain, cognitive difficulties), a syndrome now named "post-treatment Lyme disease syndrome" (PTLDS). Controversy exists on the cause, incidence and severity of PTLDS. This study aims to estimate the incidence of PTLDS in patients with Lyme borreliosis and to quantify the disease burden and economic costs associated with the different clinical manifestations of Lyme borreliosis in Belgium

Pandemic A/H1N1v influenza 2009 in hospitalized children: a multicenter Belgian survey

<p>Abstract</p> <p>Background</p> <p>During the 2009 influenza A/H1N1v pandemic, children were identified as a specific "at risk" group. We conducted a multicentric study to describe pattern of influenza A/H1N1v infection among hospitalized children in Brussels, Belgium.</p> <p>Methods</p> <p>From July 1, 2009, to January 31, 2010, we collected epidemiological and clinical data of all proven (positive H1N1v PCR) and probable (positive influenza A antigen or culture) pediatric cases of influenza A/H1N1v infections, hospitalized in four tertiary centers.</p> <p>Results</p> <p>During the epidemic period, an excess of 18% of pediatric outpatients and emergency department visits was registered. 215 children were hospitalized with proven/probable influenza A/H1N1v infection. Median age was 31 months. 47% had ≥ 1 comorbid conditions. Febrile respiratory illness was the most common presentation. 36% presented with initial gastrointestinal symptoms and 10% with neurological manifestations. 34% had pneumonia. Only 24% of the patients received oseltamivir but 57% received antibiotics. 10% of children were admitted to PICU, seven of whom with ARDS. Case fatality-rate was 5/215 (2%), concerning only children suffering from chronic neurological disorders. Children over 2 years of age showed a higher propensity to be admitted to PICU (16% vs 1%, p = 0.002) and a higher mortality rate (4% vs 0%, p = 0.06). Infants less than 3 months old showed a milder course of infection, with few respiratory and neurological complications.</p> <p>Conclusion</p> <p>Although influenza A/H1N1v infections were generally self-limited, pediatric burden of disease was significant. Compared to other countries experiencing different health care systems, our Belgian cohort was younger and received less frequently antiviral therapy; disease course and mortality were however similar.</p

Usefulness of proviral DNA of human immunodeficiency virus type 1 in AIDS treatment decisions

1. Introduction: The project aims to establish the value of the quantification and characterization of the HIV-1 provirus. The assumption was that treatment that reduces the proviral load and the emergence of resistance has an impact on the reservoirs, thus on long-term effectiveness. 2. Development of the HIV-1 provirus DNA quantification The first objective of the project was to develop a real-time PCR assay to quantify the HIV-1 provirus present in subpopulations of circulating leukocytes, the CD4+ cells in comparison to a reference gene, in this case the beta-globin gene. The assay was also applied to a group of 15 patients undergoing a structured treatment interruption (STI) of at least 2 years. After interruption of treatment, 7 of 15 patients had a plasma viral load 2.5 DNA copies / 106 CD4 + cells. 3. Follow up of treated patients 3.1 Patients Between May 2002 and July 2007, 69 patients who had not yet received antiretroviral treatment were included. Thirty-two patients received a combination of 2 NRTIs+1 PI, 12 patients received 2 NRTIs+1 NNRTI and 25 patients remained naive. Fifty-eight percent of patients were Europeans and 42% from outside Europe, mainly from Central Africa. Thirty-nine percent of HIV-1 sequences were subtype B. 3.2 Evolution of the proviral load HIV-1 proviral DNA was successfully amplified for 28 patients in the PI group, 12 patients in the NNRTI group and 14 patients who remained naïve. The patients were followed for a mean of 29 months. No difference in mean changes in proviral load between the 3 groups of patients was observed while mean changes in CD4+ cell count were statistically different between the treated groups on the one hand and the naïve patients on the other hand. 3.3 Analysis of proviral DNA sequences compared to the viral RNA The virus was successfully sequenced for 63 of the 69 persons selected, in both plasma and cells. The results showed that before treatment, 90 and 66% of detected mutations were respectively present in CD4 cells and plasma. We detected seven key mutations, 4 of them (M184M / V, M184M / I K103K / N M46M / I) were only found in cells. In 40 followed patients, the mutations detected at the naive stage remained present for at least 1 year after initiation of treatment. New key mutations emerged in the CD4 (M184I, M184M / I and Y188Y / H) during treatment. 4. Conclusion: Although the present study has certain limitations, obviously the small number of patients and the heterogeneity of the study population, some interesting findings can be seen at least as directions for further research. In terms of proviral load, this work did not show a higher impact of certain regimens associating RTIs with or without PIs on the HIV-1 proviral load. The variations were not statistically different between treated and untreated patients, while the effect of treatment on plasma viral load and CD4+ cell count was obvious. However, the use of the HIV-1 proviral DNA load as a reflection of reservoirs is gaining a new interest in the assessment of new treatment strategies to eradicate HIV infection. In terms of proviral nucleotide sequencing, a significantly higher number of mutations was detected in proviral DNA than in plasma RNA, and these mutations persisted for at least 1 year, regardless of the type of combination therapy. Four new mutations were detected and these included three in the provirus. The DNA analysis of HIV-1 might be useful in chronic infections in which wild type virus might overgrow any resistant HIV-1 variant or during simplification of treatment in patients with undetectable viraemia, when treatment needs to be modified because of toxicity or intolerance.(FARM 3) -- UCL, 201

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

Genetic and phylogenic characterisation of Hepatitis B virus in the eastern part of Democratic Republic of Congo

Hepatitis B virus (HBV) genotypes show a distinctive geographical distribution worldwide and genotypes A, D and E are the most frequently found in Africa. There are only limited studies on HBV genotype distribution in Democratic Republic of Congo (DRC), all done in the western part showing a vast majority of genotype E. In our study, HBV strains from South Kivu, an eastern province of the DRC, were analysed. Sequencing of 41 serum samples from HBV infected patients revealed strains of genotype A in 40/41 (97.6%) and genotype E in 1/41 (2.4%). The phylogenetic analysis showed that nearly all genotypes A (38/40) were closely related to A1 subgenotype strains found in Rwanda, Haiti and Martinique while only two strains attached to the A2 subgenotype cluster were isolated. The remaining genotype E case was linked to the western African E crescent. Only the I169T nucleotide substitution was observed in 2 genotype A samples. In conclusion, the genotype A seems to be the most predominant genotype in eastern DRC with the majority belonging to the Afro-Asian subgenotype (A1). This contrasts with the western part of DRC where genotype E is predominant. These results support the hypothesis of an East-West genotypic demarcation. This article is protected by copyright. All rights reserved

Definition of clinical threshold for CMV real-time PCR after comparison with PP65 antigenaemia and clinical data.

BACKGROUND: pp65 antigenaemia and real-time PCR are two methods that are used to diagnose CMV infection in its early stages and, thereby, to facilitate initiation of pre-emptive therapy. OBJECTIVES: Firstly, to compare PCR with antigenaemia and clinical outcome in order to define a clinical threshold for starting pre-emptive therapy. Secondly, to study the impact of the transplant recipient's serological status on the viral load and on the cut-offs. STUDY DESIGN: Sixty-two patients were analysed using antigenaemia (APAAP method) and real-time PCR. ROC curves were established with antigenaemia or clinical outcome as reference. Patients were divided into primo-infection or reactivation on the basis of the serological status. RESULTS: PCR correlated better with the clinical data (AUC closer to 1 and best sensitivity, PPV and NPV) than antigenaemia. Furthermore, the performance of qPCR was even better in the reactivation patients. CONCLUSIONS: This work suggests that transplant recipients should be divided according to their serological status. Indeed, replacing antigenaemia by real-time PCR for decisions regarding initiation of pre-emptive therapy is of particular appeal in patients with positive serology. As a result of this work, we have set our clinical threshold at 1500 copies/ml for reactivation