HIV-1 proviral resistance mutations: usefulness in clinical practice.

OBJECTIVES: Transmitted HIV strains may harbour drug resistance mutations. HIV-1 drug resistance mutations are currently detected in plasma viral RNA. HIV-1 proviral DNA could be an alternative marker, as it persists in infected cells. METHODS: This was a prospective study assessing the prevalence and persistence of HIV-1 drug resistance mutations in DNA from CD4 cells before and after protease inhibitor (PI)- or nonnucleoside reverse transcriptase inhibitor (NNRTI)-based therapy initiation in 69 drug-naïve patients. RESULTS: Before therapy, 90 and 66% of detected mutations were present in CD4 cells and plasma, respectively. We detected seven key mutations, and four of these (M184M/V, M184M/I, K103K/N and M46M/I) were only found in the cells. When treatment was started, 40 patients were followed; the mutations detected at the naïve stage remained present for at least 1 year. Under successful treatment, new key mutations emerged in CD4 cells (M184I, M184M/I and Y188Y/H). CONCLUSIONS: The proportion of mutations detected in the DNA was statistically significantly higher than that detected in standard RNA genotyping, and these mutations persisted for at least 1 year irrespective of therapy. The pre-existence of resistance mutations did not jeopardise treatment outcome when the drug concerned was not included in the regimen. Analysis of HIV-1 DNA could be useful in chronic infections or when switching therapy in patients with undetectable viraemia.

Evaluation of the new architect cytomegalovirus immunoglobulin M (IgM), IgG, and IgG avidity assays.

A panel of new cytomegalovirus (CMV) assays for use on the Architect instrument has been developed, including a CMV avidity assay based on a new technology. The purpose of this study was to compare the performance characteristics of the fully automated CMV immunoglobulin M (IgM), IgG, and IgG avidity tests on the Architect instrument with those of other available assays. A total of 503 consecutive fresh patient serum specimens (routine serum specimens) and 96 serum specimens from 33 pregnant women with a recent CMV primary infection (seroconversion serum specimens) were tested for CMV IgM and IgG by the Architect (Abbott), Vidas (BioMérieux), and Enzygnost (Siemens) assays. The seroconversion sera and 100 preselected serum specimens IgM negative and IgG positive by the AxSYM assay were also tested by the IgG avidity tests on the Architect and Vidas instruments. The relative agreements for CMV IgM determination with routine sera between the Architect assay and the Vidas, Enzygnost, and AxSYM assays were 97%, 94%, and 93%, respectively, for the CMV IgM tests and 99%, 98%, and 98%, respectively, for the CMV IgG tests. The specificities of the CMV IgG avidity test were 98% for the Architect assay and 76% for the Vidas assay. No high CMV IgG avidity test results were found within the first 3 months after seroconversion by either of those assays. The correlation between the results of the newly developed CMV IgM and IgG tests on the Architect instrument with the Vidas and Enzygnost assays was excellent (> or = 94%). The CMV IgG avidity test reliably excluded patients with recent infections and showed an excellent specificity (98%).

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.

Hospital outbreak of gastroenteritis due to Norovirus in Belgium.

We report an outbreak of gastroenteritis due to Norovirus in a care unit in a Belgian hospital involving thirty-three people. The origin of the outbreak was traced to one nursing assistant. The virus strain identified by reverse transcription polymerase chain reaction and electron microscopy belonged to the genogroup II.

Seroepidemiology of herpes simplex type 2 in pregnant women in Belgium.

OBJECTIVE: The objective of this study was to study the prevalence of herpes simplex virus (HSV) type 2 in pregnant women in Belgium. STUDY DESIGN: The serum of 1000 consecutive women was collected. HSV-1 and HSV-2 control sera were added to the study. HSV-2 antibodies were tested with the HerpeSelect 2 enzyme-linked immunosorbent assay (ELISA; Focus) based on the use of the recombinant gG-2 antigen. RESULTS: The 21 HSV-2 control subjects were positive. Among the HSV-1 control subjects, 18 were negative and 4 were positive. Among the pregnant women, 80.3% were negative, 1.5% had equivocal results, and 18.2% were positive. No statistical difference was observed according to the origin (European or African) of the women. CONCLUSIONS: The results obtained with the control sera indicate a high sensitivity of the Focus ELISA, as well as a capacity to discriminate between HSV-1/HSV-2 infection. The HSV-2 prevalence in the studied population raises the question of the possible benefit of a specific preventive program in pregnant women.

Anticytomegalovirus IgG avidity in pregnancy: a 2-year prospective study.

OBJECTIVES: To analyze the practical use of the anticytomegalovirus IgG avidity and its impact on the follow-up of pregnancy. To evaluate the performance of IgG avidity to exclude the risk of congenital infection. METHODS: 409 IgM-positive women without a documented seroconversion were prospectively followed. Data concerning the follow-up of the pregnancies were collected (amniotic fluid puncture and samples from the offspring). These observations were compared to those of 76 seroconversions during the same period. RESULTS: High avidity excluding a primary infection within the past 3 months was observed in 270 women. As the gestational age was less than 3 months for 121 women, exclusion of a primary infection was achieved in 30% of the cases. The rate of amniotic fluid puncture was influenced by the serological result: high avidity (9%), low avidity (42%) and seroconversion (65%). CONCLUSIONS: A high avidity index during the first trimester of pregnancy could reasonably be considered as a good indicator of past infection and invasive prenatal diagnosis is not necessary. Nearly 70% of the IgM-positive women could be reassured if the first serology was systematically performed before 12 weeks of gestation.

Evaluation of a cytomegalovirus glycoprotein B recombinant enzyme immunoassay to discriminate between a recent and a past infection.

Fetal damage following cytomegalovirus (CMV) intrauterine infection is mostly linked to primary infection. To differentiate primary infection from nonprimary infection, immunoglobulin M (IgM) tests are not reliable enough, and measurement of the IgG avidity appears to be the method that is the most widely used at present. In the present study the performance of the Vidas (bioMérieux) avidity assay was compared with that of a new enzyme immunoassay based on the use of a recombinant CMV glycoprotein B protein (Biotest).

Age trend in hepatitis C virus genotype distribution as seen in a Brussels teaching hospital.

The hepatitis C virus genotype distribution was studied among age groups in 501 referred patients with chronic hepatitis C by INNO-LiPA HCV II (Innogenetics, Belgium). Ten patients had coinfection with several genotypes. Two hundred seventy of 491 singly infected individuals (57%) had 1b, 66 (13.4%) 3a, 57 (11.6%) 1a. HCV subtype 1b was predominant but its prevalence increased with age (76.5% of patients born in the '20s, 39.3% in the '70s) (P < 0.0001). Three possibilities could explain the shift towards a wider variety of genotypes in younger age. (1) 1b could be the original subtypes in this population, (2) the non-1b subtypes could give less chronic carriers, (3) the non-1b subtypes could have a higher mortality, which seems improbable. The 1b genotype seems the oldest subtype in our country while others were imported later through increased population movements and changing habits.

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.

False-positive IgM antibody tests for cytomegalovirus in patients with acute Epstein-Barr virus infection.

The diagnosis of acute cytomegalovirus (CMV) infection is frequently based on a positive IgM result. False-positive reactions due to interfering infections may exist. Between August 1998 and May 1999, 62 patients were found to be IgM positive and IgG negative with the Axsym assay (Abbott, Germany). Serological testing for Epstein-Barr virus (EBV) was performed in these patients to detect any cross-reactivity due to acute mononucleosis. Additionally, the results of the CMV Axsym was evaluated in 40 patients with acute EBV infection. The results suggest that the CMV-IgM Axsym assay shows a lack of specificity due to acute EBV infection. Precautions must be taken when CMV-IgM Axsym results are interpreted. It seems necessary to confirm equivocal results with another technique and to take into account other clinical and biological observations.

Human herpes virus 8 infection in kidney transplant patients in Belgium.

BACKGROUND: Kaposi sarcoma (KS) may arise as a complication of kidney transplantation. In the Saint Luc Teaching Hospital in Brussels, patients of both Belgian and foreign origin are treated. The prevalence of human herpes virus 8 (HHV-8) infection differs in different geographical settings. We wanted to estimate the background infection rate and the risk of infection in our transplant population: a first step towards evaluating the necessity of HHV-8 screening. METHODS: Serum samples were taken from 210 organ donors over a period of 7 years (30 per year) and from 200 kidney recipients from whom two sera were tested, one pre-transplant and the second 6-12 months post-transplant. All sera were screened for HHV-8 by an enzyme-linked immunosorbant assay using recombinant ORF 65 and ORF 73 antigens and an immunofluorescence assay for the latent antigen. Reactive samples were confirmed by western blotting. RESULTS: Seven donors (3.3%) were positive for HHV-8 antibodies. Of 198 pre-transplant sera available for evaluation, 15 were positive (7.6%). Post-transplantation 18/199 (9%) were positive: four (2.1% of negatives) had a documented seroconversion and one lost the antibodies. No patients developed KS. CONCLUSIONS: A substantial number of kidney transplant patients already had antibodies to HHV-8 at the time of transplantation. A further 2.1% of seronegative patients had seroconversion, which could have been acquired through the transplanted organ (3.3% of donors were positive) or through transfusion.

Reactivity to p52 and CM2 recombinant proteins in primary human cytomegalovirus infection with a microparticle agglutination assay.

We evaluated the reactivities of sera against p52 and CM2 recombinant antigens of human cytomegalovirus (HCMV), coated on microparticles, for the differentiation of primary HCMV infection from an established infection. Two different test formats of the CMV Multiplex Copalis assay were evaluated. The 214 serum samples tested were immunoglobulin M (IgM) positive or equivocal by our reference assay. Reactivities against p52 and CM2 antigens were tested for sera from 37 patients with a well-documented seroconversion within the preceding 3 months (119 serum specimens), 31 patients known to have had a seroconversion at least 8 months earlier (31 serum specimens), and 57 patients without a documented seroconversion (64 serum specimens). The assay had a sensitivity for the detection of a primary infection of 70 or 86% by the first test format and a sensitivity of 88 or 94% by the second test format, according to the criteria used to indicate a primary infection by this test. A good correlation of the results of the assay with our in-house avidity index was found. The specificity of the assay warrants further evaluation. With IgM-positive sera, the assay was not sufficiently specific to make a distinction between a primary infection and an established infection.

Characteristics of Epstein-Barr virus primary infection in pediatric liver transplant recipients.

BACKGROUND/AIM: Pediatric liver transplant recipients are at high risk of Epstein-Barr virus infection. However the incidence of clinical symptoms and the graft function at the time of acute infection remains poorly documented. The aim of this study was to monitor the clinical and biochemical events associated with primary Epstein-Barr virus infection. METHODS: Clinical and biological patterns associated with Epstein-Barr virus infection were prospectively searched in 38 liver transplanted children. Polymerase chain reaction and anti-Epstein-Barr virus IgM antibodies were used at regular intervals to detect the timing of primary infection. RESULTS: Five children (13%) had pretransplant immunity, 26 (68.5%) developed primary Epstein-Barr virus infection 15 to 90 days after transplantation and seven (18.5%) remained Epstein-Barr virus negative. The four patients with clinical symptoms at the time of infection subsequently developed post-transplant lymphoproliferative disease. A single post-transplant lymphoproliferative disease occurred in non-symptomatic patients (overall incidence 13%). No mortality was associated with post-transplant lymphoproliferative disease. Two asymptomatic patients had abnormal liver function tests possibly related to primary Epstein-Barr virus infection. CONCLUSION: Epstein-Barr virus primary infection occurs in 80% of seronegative patients within 3 months after OLT. Clinical symptoms are rare and closely associated with post-transplant lymphoproliferative disease. Outside post-transplant lymphoproliferative disease, the consequences of infection are marginal.

Epstein-Barr virus infection in sixty pediatric liver graft recipients: diagnosis of primary infection and virologic follow-up.

BACKGROUND: Epstein-Barr virus (EBV) is an important cause of infection after pediatric liver transplantation. Earlier detection of EBV could result in shortening the delay in diagnosis and allow better management of a pediatric high risk population. OBJECTIVES: To determine the timing of EBV primary infection after graft and to compare the performances of different assays for an early detection of the virus. METHODS: Sixty pediatric liver graft recipients were followed. Kinetics of appearance of different EBV serologic parameters (anti-EBV-IgG, -IgM and -IgA, and anti-EBV nuclear antigen-IgG) and of the viral DNA in peripheral blood lymphocytes by PCR were compared. RESULTS: Thirty-six patients had a primary EBV infection. The first PCR and IgM positive result appeared after a mean delay of 56 and 61 days, respectively, and preceded the IgG response (mean delay, 143 days). Most of the studied patients (13 of 16) developed anti-EBV-IgA and only 3 developed anti-EBNA-IgG during the follow-up period. CONCLUSIONS: EBV primary infection occurred in most cases during the first 2 months after graft. The IgG response was delayed. The best performance was obtained by PCR. However, the IgM test compared well with the PCR and could be a more widely accessible measure to follow regularly.

Prenatal diagnosis of human cytomegalovirus by culture and polymerase chain reaction: 98 pregnancies leading to congenital infection.

Human cytomegalovirus (HCMV) is the most common cause of viral intra-uterine infection. The experience with prenatal diagnosis remains limited and is based on few reports of small numbers of cases. It is thus difficult to compare the accuracy of the different tests because the groups studied were small and heterogeneous. We describe here our experience on a series of 98 pregnancies leading to HCMV congenital infection, among which 71 have been tested by amniotic fluid (AF) sampling followed by culture and/or polymerase chain reaction (PCR). Independently of the delay between AF sampling and the first HCMV IgM positive result, the mean sensitivity of both culture and PCR was around 70 per cent. The best sensitivity (95.5 per cent) was obtained after a delay > or = 6 weeks in late pregnancy (> or = 23 weeks). The present study demonstrated clearly that the delay between AF puncture and the presumed date of seroconversion is more important for sensitivity than the technique used for the diagnosis (PCR or culture). However, even in the best diagnostic conditions, negative results of HCMV culture or PCR in AF cannot formally exclude intra-uterine infection.

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.

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.

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) < 50% and never > 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 < 50%, suggestive of a recent primary infection. CONCLUSIONS: In pregnant women, measurement of the IgG avidity may help to date a HCMV infection, an AI > 65% highly suggests a past infection while an AI < 50% corresponds to a recent primary infection.

Early signs and risk factors for the increased incidence of Epstein-Barr virus-related posttransplant lymphoproliferative diseases in pediatric liver transplant recipients treated with tacrolimus.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a life-threatening condition the incidence of which in pediatric solid organ transplantation may be related to the immunosuppressive load. It has been suggested that tacrolimus, a new and potent immunosuppressor, causes an increased incidence of this syndrome. METHODS: The incidence, early signs, and risk factors for lymphoproliferative disease were reviewed in a cohort of 89 pediatric liver transplant recipients treated with tacrolimus. RESULTS: Eighteen patients (20%) developed a PTLD-16 concomitant to a primary Epstein-Barr virus (EBV) infection and 2 with previous immunity against EBV. Three additional patients had preliminary signs of PTLD concomitant to primary EBV infection, but did not develop individualized lymphoid masses. Six patients died (6.7% of all tacrolimus-treated patients). Mean tacrolimus blood level during the 3 months preceding EBV infection reached 11.8+/-1.8 ng/ml in PTLD patients versus 9.4+/-3.4 ng/ml in non-PTLD patients (0.05

Inhibition of prokaryotic cell growth by HIV1 Vpr.

We have cloned the nef, vif, vpr and vpu genes of HIV1 in the pGEX system to produce auxiliary proteins of HIV1 as N-terminal fusions with glutathione S-transferase (GST). Some GST proteins are difficult to obtain under standard conditions. The synthesis and solubility varied considerably from one protein to another. We investigated the reasons for the poor production of GST-Vpr, GST-Vpu and GST-Vif. Interestingly, using this GST prokaryotic model, we demonstrated that Vpr, which is known to block the cell cycle of mammalian and yeast cells at the G2 phase, is also bacteriostatic for Escherichia coli. The effect on E. coli was specific to Vpr, and was not linked to the expression of the other HIV1 proteins. This suggests that Vpr interferes with components of cell replication that are conserved from prokaryotes to eukaryotes. Thus, E. coli appears to be a convenient model system for studies on the function of Vpr.