Late presentation to HIV testing is overestimated when based on the consensus definition.

OBJECTIVES: In 2011, a consensus was reached defining "late presenters" (LPs) as individuals presenting for care with a CD4 count < 350 cells/µL or with an AIDS-defining event, regardless of CD4 count. However, a transient low CD4 count is not uncommon in recent infections. The objective of this study was to investigate how measurements of late presentation change if the clinical stage at the time of diagnosis is taken into account. METHODS: Case surveillance data for newly diagnosed patients in Belgium in 1998-2012 were analysed, including CD4 count at diagnosis, the presence of AIDS-defining events, and recent infections (< 6 months) as reported by clinicians in the case of acute illness or a recent negative test. First, proportions of LPs were calculated according to the consensus definition. Secondly, LPs were reclassified as "nonlate" if infections were reported as recent. RESULTS: A total of 7949 HIV diagnoses were included in the study. Recent infections were increasingly reported over time, accounting for 8.2% of new infections in 1998 and 37.5% in 2012. The consideration of clinical stage significantly modified the proportion of LPs: 18.2% of men who have sex with men (MSM) diagnosed in 2012 would be classified as LPs instead of 30.9% using the consensus definition (P < 0.001). The proportion of patients misclassified as LPs increased significantly over time: 5% in MSM in 1998 vs. 41% in 2012. CONCLUSIONS: This study suggests that low CD4 counts in recent infections may lead to overestimation of late presentation when applying the consensus definition. The impact of transient CD4 count on late presentation estimates should be assessed and, if relevant, the introduction of clinical stage in the definition of late presentation should be considered.

Good continuum of HIV care in Belgium despite weaknesses in retention and linkage to care among migrants.

BACKGROUND: The Belgian HIV epidemic is largely concentrated among men who have sex with men and Sub-Saharan Africans. We studied the continuum of HIV care of those diagnosed with HIV living in Belgium and its associated factors. METHODS: Data on new HIV diagnoses 2007-2010 and HIV-infected patients in care in 2010-2011 were analysed. Proportions were estimated for each sequential stage of the continuum of HIV care and factors associated with attrition at each stage were studied. RESULTS: Of all HIV diagnosed patients living in Belgium in 2011, an estimated 98.2% were linked to HIV care, 90.8% were retained in care, 83.3% received antiretroviral therapy and 69.5% had an undetectable viral load (<50 copies/ml). After adjustment for sex, age at diagnosis, nationality and mode of transmission, we found lower entry into care in non-Belgians and after preoperative HIV diagnoses; lower retention in non-Belgians and injecting drug users; higher retention in men who have sex with men and among those on ART. Younger patients had lower antiretroviral therapy uptake and less viral suppression; those with longer time from diagnosis had higher ART uptake and more viral suppression; Sub-Saharan Africans on ART had slightly less viral suppression. CONCLUSIONS: The continuum of HIV care in Belgium presents low attrition rates over all stages. The undiagnosed HIV-infected population, although not precisely estimated, but probably close to 20% based on available survey and surveillance results, could be the weakest stage of the continuum of HIV care. Its identification is a priority along with improving the HIV care continuum of migrants.

Determinants of adherence in a cohort of Belgian HIV patients: a pilot study.

Since the era of highly active antiretroviral therapy (HAART), HIV is considered a chronic disease. Adherence to HAART is crucial for effectiveness. Non-adherence negatively impacts patient outcome and the larger economy. However, data on adherence among the Belgian HIV cohort are scarce. Therefore, the purpose of this pilot study was to identify determinants of adherence among HIV patients treated in Belgium. The study was conducted at the Aids Reference Centre of Ghent University Hospital between 1 January and 31 December 2012. Sociodemographic data were collected, along with the Simplified Medication Adherence Questionnaire (SMAQ), the Center for Adherence Support Evaluation (CASE) Adherence Index, the EuroQol-6D, the Medical Outcomes Study-HIV (MOS-HIV), the Beck Depression Inventory-II, and three neurocognitive complaints screening questions. To date, 218 patients participated in the study, among whom 173 (79·4%) were male. Mean age was 46·0±10·6 years and 133 patients (63·9%) were homosexual. According to the SMAQ and the CASE, 78·5% and 93·5% of the patients were adherent to antiretroviral therapy. Logistic regression analysis revealed that smoking, neurocognitive complaints, and female sex were independent determinants of non-adherence. In conclusion, there is an elevated risk for non-adherence in smokers, people experiencing neurocognitive problems, and women in our sample. The latter could reflect differences between male and female HIV patients in Belgium. Adherence improving initiatives should be tailored to these three risk groups.

Impact of triplicate testing on HIV genotypic tropism prediction in routine clinical practice.

Guidelines state that the CCR5-inhibitor Maraviroc should be prescribed to patients infected with R5-tropic HIV-1 only. Therefore, viral tropism needs to be assessed phenotypically or genotypically. Preliminary clinical trial data suggest that genotypic analysis in triplicate is associated with improved prediction of virological response by increasing the detection of X4-tropic variants. Our objective was to evaluate the impact of triplicate genotypic analysis on prediction of co-receptor usage in routine clinical practice. Samples from therapy-naive and therapy-experienced patients were collected for routine tropism testing at three European clinical centres. Viral RNA was isolated from plasma and proviral DNA from peripheral blood mononuclear cells. Gp120-V3 was amplified in a triplicate nested RT-PCR procedure and sequenced. Co-receptor usage was predicted using the Geno2Pheno([coreceptor]) algorithm and analysed with a false-positive rate (FPR) of 5.75%, 10%, or an FPR of 20% and according to the current European guidelines on the clinical management of HIV-1 tropism testing. A total of 266 sequences were obtained from 101 patient samples. Discordance in tropism prediction for the triplicates was observed in ten samples using an FPR of 10%. Triplicate testing resulted in a 16.7% increase in X4-predicted samples and to reclassification from R5 to X4 tropism for four cases rendering these patients ineligible for Maraviroc treatment. In conclusion, triplicate genotypic tropism testing increases X4 tropism detection in individual cases, which may prove to be pivotal when CCR5-inhibitor therapy is applied.

Concordance between HIV-1 genotypic coreceptor tropism predictions based on plasma RNA and proviral DNA.

OBJECTIVE: The aim of the study was to evaluate the use of proviral DNA as a source of viral genetic material for genotypic coreceptor tropism testing (GTT). METHODS: GTT consisted of bulk V3 sequencing followed by geno2pheno interpretation with the interpretative cut-off [false positive rate (FPR)] set at 5 and 10%. GTT was performed for 165 patients with a viral load of >500 HIV-1 RNA copies/mL on simultaneously collected plasma RNA and proviral DNA, and for 126 patients with a viral load of <500 copies/mL on current proviral DNA and pretreatment plasma RNA. Phenotypic tropism testing (PTT) results were available for 142 samples. RESULTS: In the simultaneous RNA/DNA comparison, concordance in prediction was 95.2% (at FPR 10%) and 96.4% (at FPR 5%). Six RNA-R5/DNA-X4 and two RNA-X4/DNA-R5 discordances were observed at an FPR of 10%, and six RNA-R5/DNA-X4 discordances were observed at an FPR of 5%. In the longitudinal RNA/DNA comparison, concordance was 88.1% (at FPR 10%) and 90.5% (at FPR 5%). Eight RNA-X4/DNA-R5 and seven RNA-R5/DNA-X4 discordances were seen at an FPR of 10%, and 10 RNA-R5/DNA-X4 and two RNA-X4/DNA-R5 discordances at an FPR of 5%. The overall concordance of RNA GTT with PTT was 82% (at FPR 10%) and 83% (at FPR 5%). The overall concordance of DNA GTT with PTT was 85% (at both 10 and 5% FPRs). CONCLUSIONS: GTT produced highly concordant tropism predictions for proviral DNA and plasma RNA. GTT on proviral DNA offers a promising approach for tropism prediction in clinical practice, particularly for the assessment of treated patients with low or suppressed viraemia.

European guidelines on the clinical management of HIV-1 tropism testing.

Viral tropism is the ability of viruses to enter and infect specific host cells and is based on the ability of viruses to bind to receptors on those cells. Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. In most European countries, HIV tropism is identified with tropism phenotype testing. New data support genotype analysis of the HIV third hypervariable loop (V3) for the identification of tropism. The European Consensus Group on clinical management of tropism testing was established to make recommendations to clinicians and clinical virologists. The panel recommends HIV-tropism testing for the following groups: drug-naive patients in whom toxic effects are anticipated or for whom few treatment options are available; patients who have poor tolerability to or toxic effects from current treatment or who have CNS pathology; and patients for whom therapy has failed and a change in treatment is considered. In general, an enhanced sensitivity Trofile assay and V3 population genotyping are the recommended methods. Genotypic methods are anticipated to be used more frequently in the clinical setting because of their greater accessibility, lower cost, and faster turnaround time than other methods. For the interpretation of V3 loop genotyping, clinically validated systems should be used when possible. Laboratories doing HIV tropism tests should have adequate quality assurance measures. Similarly, close collaboration between HIV clinicians and virologists is needed to ensure adequate diagnostic and treatment decisions.

The AIDS epidemic: the spread of a deadly disease in the biotech era.

In the last two decades we have witnessed the progression of a newly introduced infection in humans. It is sobering that despite a world-wide effort and the tremendous progression of technical capabilities and scientific knowledge we are still not able to control the global epidemic of HIV. In 2004 more than 40 million people were infected. Educational approaches to modify risk-taking behavior is still the most critical component of prevention and the most important measure to limit the spread of the infection. Vaccine development, which is still far from promising, is probably the only way to control the disease in the future.

Prevalence of genotypic resistance among antiretroviral drug-naive HIV-1-infected patients in Belgium.

OBJECTIVES: To estimate the prevalence and the evolution over time (1995-1998) of genotypic resistance to antiviral drugs in antiretroviral drug-naive HIV-1-infected patients in Belgium. DESIGN: Belgian Aids Reference Laboratories provided retrospective samples and clinical data from antiretroviral drug-naive HIV-1-infected patients who visited the hospital for the first time in 1995 (n=45), 1997 (n=75) and 1998 (n=111). Genotypic resistance to the three available classes of drugs was monitored using the Line Probe Assay (Innogenetics, Gent, Belgium). Additionally, ARMS-151 was performed for scoring multinucleoside resistance. RESULTS: The prevalence of genotypic resistance at baseline to nucleoside analogue reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were each between 10% and 20% for 1995, 1997 and 1998 without an increasing trend over time. For NRTIs, resistance mutations were mainly related to zidovudine in 1995, whereas in 1997 and 1998 baseline resistance was scored for zidovudine, lamivudine or for both drugs simultaneously. No patients displayed the multi-nucleoside resistance Q151M mutation. Baseline resistance mutations to protease inhibitors (PIs) did not rise significantly: 4.4% in 1995, 8% in 1997 and 9.9% in 1998. When scoring any resistance-related mutation, 26.6% displayed genotypic baseline resistance in 1995, 26.6% in 1997 and 31.5% in 1998. DISCUSSION: The prevalence of genotypic baseline resistance to any drug, as scored with LiPA, in naive HIV-1 patients in Belgium is 29%, with baseline resistance mutations to one or several drugs from all available classes of antiviral drugs. The ability of LiPA to pick up minor variants could be an explanation for the higher overall prevalence we observe, when compared to recent estimates in other countries of 16.3% and 22%, which were based on sequencing methods. According to the European guidelines for resistance testing, resistance testing in Belgium before starting antiviral therapy should be considered.

Vaginal lavage with chlorhexidine during labour to reduce mother-to-child HIV transmission: clinical trial in Mombasa, Kenya.

OBJECTIVES: To evaluate the effect of vaginal lavage with diluted chlorhexidine on mother-to child transmission of HIV (MTCT) in a breastfeeding population. METHODS: This prospective clinical trial was conducted in a governmental hospital in Mombasa, Kenya. On alternating weeks, women were allocated to non-intervention or to intervention consisting of vaginal lavage with 120 ml 0.2% chlorhexidine, later increased to 0.4%, repeated every 3 h from admission to delivery. Infants were tested for HIV by DNA polymerase chain reaction within 48 h and at 6 and 14 weeks of life. RESULTS: Enrolment and follow-up data were available for 297 and 309 HIV-positive women, respectively, in the non-lavage and the lavage groups. There was no evidence of a difference in intrapartum MTCT (17.2 versus 15.9%, OR 0.9, 95% CI 0.6-1.4) between the groups. Lavage solely before rupture of the membranes tended towards lower MTCT with chlorhexidine 0.2% (OR 0.6, 95% CI 0.3-1.1), and even more with chlorhexidine 0.4% (OR 0.1, 95% CI 0.0-0.9). CONCLUSION: The need remains for interventions reducing MTCT without HIV testing, often unavailable in countries with a high prevalence of HIV. Vaginal lavage with diluted chlorhexidine during delivery did not show a global effect on MTCT in our study. However, the data suggest that lavage before the membranes are ruptured might be associated with a reduction of MTCT, especially with higher concentrations of chlorhexidine.

Correlation between maternal and infant HIV infection and low birth weight: a study in Mombasa, Kenya.

This article aimed to examine the association between maternal and infant HIV infection and low birth weight (LBW <2500 grams). Data from 8563 singleton liveborns in Mombasa, Kenya, were analysed. Maternal HIV infection was found in 14.1% of the women and 9.6% of neonates had a birth weight of <2500 grams. In multivariate analysis, maternal HIV infection was independently associated with LBW (RR=1.46, 95% CI=1.20-1.79, P =0.0002). Maternal age, primiparity, sex of the baby, religion, syphilis infection, anaemia and previous history of stillbirth were also independently associated with LBW (RR: 1.32, 2.19, 1.44, 1.56, 1.61, 1.31 and 1.69, respectively). The rate of intra-uterine HIV transmission was 5.1% and 20.1% of the exposed infants were infected during the intrapartum period. Intrapartum infected infants had a relative risk of LBW of 1.95 (95% CI=1.18-2.87, P <0.01) compared to uninfected children, whereas the birth weight of infants infected in utero was not different from uninfected infants (RR=1.18, 95% CI=0.56-2.60, P=0.630). HIV infected mothers are more likely to have small babies, even after controlling for possible confounding factors. Low birth weight babies were more at risk for peripartum HIV transmission, but further research is needed to study mechanisms of transmission in relation to birth weight.

Exposure to HIV-1 during delivery and mother-to-child transmission.

BACKGROUND: The correlation between the presence of HIV-1 in maternal cervicovaginal secretions and in the infant's oro-pharyngal secretions at birth, and mother-to-child HIV transmission (MTCT) were examined to obtain a better understanding of its mechanism. METHODS: Women without medical and obstetrical complications, living within a reasonable distance of the government hospital in Mombasa, Kenya, were recruited after informed consent. Maternal and infant characteristics were collected. Polymerase chain reaction was used to detect HIV-1 in cervico-vaginal and oro-pharyngal secretions. Infants were tested for HIV-1 by polymerase chain reaction within 48 h and at 6 weeks after delivery. RESULTS: Between April 1998 and April 1999, 228 woman-infant pairs were included in the study. HIV-1 DNA in cervico-vaginal secretions was independently associated with HIV-1 maternal viral load and with infant birth-weight, whereas HIV-1 RNA was associated with maternal viral load and maternal age. HIV-1 DNA in the oropharyngal secretions was also independently associated with maternal viral load. MTCT rate at the age of 6 weeks was 23.6%. Intrapartum and early postpartum HIV transmission was independently associated with maternal viral load [adjusted odds ratio (OR), 1.6; 95% confidence interval (CI),1.0-2.7], detection of HIV-1 RNA in cervico-vaginal secretions (adjusted OR, 3.2; 95% CI, 1.5-7.3) and of HIV-1 DNA in oro-pharyngeal secretions (adjusted OR, 3.2; 95% CI, 1.1-9.0). DISCUSSION: As far as is known, this is the first study showing that infant exposure to HIV-1 in the birth canal and the presence of HIV-infected cells in the infant's oropharyngeal cavity are independently associated with intrapartum and early postpartum MTCT. It supports the hypothesis that MTCT could occur through the oral route.

Baseline HIV type 1 genotypic resistance to a newly added nucleoside analog is predictive of virologic failure of the new therapy.

We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.

Blinded, multicenter quality control study for the quantification of human immunodeficiency virus type 1 RNA in plasma by the Belgian AIDS reference laboratories.

OBJECTIVE: In order to evaluate the interlaboratory variation of HIV-1 RNA measurements in plasma, the Belgian AIDS reference laboratories organized a blinded multicenter quality control study. METHODS: Atest panel of coded spiked HIV-1 plasma samples reflecting the dynamic range of the assay was composed and distributed. The HIV-1 RNA concentration of these samples was determined by the eight Belgian AIDS reference laboratories by means of the Amplicor HIV-1 Monitor version 1.5 assay. RESULTS: Analysis of the results demonstrated that there was little interlaboratory variation for the high concentration range (4.0-5.7 log10 copies/mL), never exceeding 0.2 log10 copies/mL. However the standard deviation for the low concentration range (2.6-3.9 log10 copies/mL) reached up to 0.22 log10 copies/mL. CONCLUSIONS: Since interlaboratory variability never reached 0.5 log10 copies/mL and each of the laboratories was able to detect four-fold differences in plasma HIV-1 RNA levels, the Amplicor assay can be used in multicenter studies without a centralized analysis of samples. Furthermore, this well-characterized proficiency panel of spiked plasma samples could be used as a standard in the study of interassay comparisons.

Human immunodeficiency virus nef gene expression affects generation and function of human T cells, but not dendritic cells.

Human immunodeficiency virus (HIV)-infected individuals develop an acquired immune deficiency syndrome (AIDS) due to loss in their lymphocyte numbers and cellular defects in T cells and antigen-presenting cells (APC). HIV infection of the thymus results in deficient replenishment of the peripheral naive T-cell pool. The HIV nef gene was shown to be important for progression towards AIDS and cellular depletion of the infected thymus. Here, we demonstrate by retroviral gene transfer that nef expression, in the absence of other HIV genes, impaired human thymic T-cell development. Thymocytes were generated in reduced numbers and downmodulated CD4 and CD8beta cell surface expression. T cells grown from nef-expressing thymocytes were hyperproliferative in vitro upon T-cell receptor triggering. Mature dendritic cells (DC) were functional and had normal surface CD4 levels despite nef expression. Thus, nef expression alone may contribute to AIDS development by reduced T-cell generation and T-cell hyperresponsiveness.

Interruption of reverse transcriptase inhibitors or a switch from reverse transcriptase to protease inhibitors resulted in a fast reappearance of virus strains with a reverse transcriptase inhibitor-sensitive genotype.

OBJECTIVES: To study the effect of the interruption of reverse transriptase inhibitor (RTI) therapy or a switch from RTI to protease inhibitors, on the genotypic drug-resistance pattern of plasma HIV-1. METHODS: Nine patients who completely stopped all medication, and five patients who switched from a treatment with RTI to a regimen containing protease inhibitors only, were studied. Direct sequencing of the plasma HIV-1 RT and protease gene was performed on follow-up samples taken before and after the interruption of treatment. RESULTS: Comparison of the amino acid sequence of the RT and protease genes in successive samples showed the rapid reappearance of wild-type viral variants in 12 of the 14 patients studied. Wild-type virus replaced the mutant strains 14 days to 2 months after the interruption of therapy, even in patients with a long treatment history. CONCLUSION: The results of this study indicate the sustained lower fitness of mutant strains in vivo. As a result, wild-type virus remains capable of outcompeting the RT or protease mutant strains very fast after removal of the drug. These findings highlight the importance of 'treatment history' in addition to genotypic and phenotypic markers determined at one time-point, when making therapeutic decisions for patients.

Haptoglobin polymorphism, iron metabolism and mortality in HIV infection.

BACKGROUND: Three phenotypes of the antioxidant protein haptoglobin are known: Hp 1-1, Hp 2-1 and Hp 2-2. OBJECTIVES: To investigate the outcome of HIV infection according to haptoglobin type. DESIGN AND METHODS: Haptoglobin phenotypes were determined using starch gel electrophoresis in serum obtained from 653 HIV-infected Caucasians in the AIDS reference centers of Gent (n = 184), Antwerp (n = 309), and Luxembourg (n = 160). Survival was compared between haptoglobin types using Kaplan-Meier curves. Plasma HIV-1 RNA was quantified by reverse transcriptase PCR. Serum iron, transferrin saturation, ferritin, and vitamin C were assayed to evaluate iron-driven oxidative stress in 184 HIV-infected patients and 204 controls. RESULTS: The haptoglobin type distribution amongst the patients (17.6% Hp 1-1, 49.9% Hp 2-1, 32.5% Hp 2-2) corresponded to that of the controls. Kaplan-Meier curves showed a higher mortality for the Hp 2-2 group (P = 0.0001; adjusted mortality risk ratio, 1.78; 95% confidence interval, 1.25-2.54). Median survival time was 11.0 years (Hp 1-1 and Hp 2-1) versus 7.33 years (Hp 2-2). Plasma HIV-1 RNA levels prior to antiviral therapy and their increase over 1 year were highest in Hp 2-2 patients (P = 0.03 and 0.003, respectively). The Hp 2-2 type was associated with higher serum iron, transferrin saturation, and ferritin levels and with low vitamin C concentrations. Furthermore, ferritin concentrations were higher in HIV-infected patients than in controls (P < 0.0001). CONCLUSION: HIV-infected patients carrying the Hp 2-2 phenotype show a worse prognosis, which is reflected by a more rapid rate of viral replication (in the absence of antiviral treatment). They also accumulate more iron and oxidize more vitamin C, suggesting that less efficient protection against haemoglobin/iron-driven oxidative stress may be a direct mechanism for stimulating viral replication.

Isolation of HIV-1 RNA from plasma: evaluation of seven different methods for extraction (part two).

Some new commercial methods for the extraction of viral RNA have been introduced recently. In addition to the study published previously (Verhofstede, C., Reniers, S., Van Wanzeele. F., Plum J., 1996. AIDS 8, 1421-1427), seven different methods (four newly developed and three reference methods) for extraction of HIV-1 RNA from plasma have been evaluated. The RNA preparation method that gave the best results (acceptable reproducibility, highest sensitivity, reasonable price, fast and easy to perform), was the QIAamp Viral RNA kit from QIAgen. The High Pure Viral RNA Kit (Boehringer Mannheim) as well as the non-commercialised extraction kits were also very sensitive. The non-commercial tests seem less suitable for routine use and for the processing of large number of samples. Two methods, RNA Insta-Pure LS (Eurogentec) and PANext RNA extraction kit 1 (NTL, PANsystems GmbH) are not adapted for HIV plasma extraction. The single step methods using glass fibre or silica column are rapid (from 60 to 75 min depending on the number of wash steps) and although the price is high they are cheaper than the Boom extraction methods: High Pure Viral RNA Kit (Boehringer Mannheim) ($3.3/sample), QIAamp Viral RNA Kit (Qiagen) ($3.6/sample), Boom extraction ($5/sample). The Qiagen kit is the only kit that combines sensitivity with reproducibility, it is commercialised, rapid and affordable in price and can be automated. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extractions varied between 26.4 and 48.6%).

Prevention of congenital toxoplasmosis in Szeged, Hungary.

BACKGROUND: Toxoplasma gondii infection of the fetus can only be discovered or prevented by the appropriate serological screening and subsequent treatment of the mother and her offspring. In Hungary, there is no obligatory toxoplasma screening for pregnant women and both the reporting and follow-up of congenital toxoplasmosis cases is limited. In 1987 we started a systematic study in the Szeged region of Hungary, in which all pregnant women were screened and appropriate treatment given to all mothers and their offspring where congenital toxoplasmosis was suspected. METHODS: All pregnant women were routinely screened within the first 16 weeks of gestation for toxoplasma antibodies by complement fixation test (CFT). Seronegative cases were retested for possible seroconversion every second month. Patients with CFT titres > or = 1:256 were retested for anti-P30 immunoglobulin A (IgA), IgM and IgG antibodies by ELISA and/or SDS-PAGE-Western immunoblot in order to distinguish the acute and chronic phases of the infection. RESULTS: Up to the end of 1994, the sera of 17,735 gravidae were screened. Ten women were found to have seroconverted during pregnancy and 78 had high initial antibody levels accompanied by anti-P30 IgA antibodies at the very first screening. These two groups together were considered as definitely (10) or possibly (78) infected with Toxoplasma during pregnancy and were treated with Spiramycin. All of their offspring were also treated for one month and followed-up by systematic serological and clinical screening for 2 years. No congenital toxoplasmosis was found in any of the offspring. CONCLUSIONS: Antenatal, early diagnosis and treatment of toxoplasmosis in mothers, together with treatment and follow-up of their offspring, may considerably reduce the incidence of the disease in the offspring.

Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene.

Upon prolonged treatment with various antiretroviral nucleoside analogs such as 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, (-)- beta-L-2', 3'dideoxy-3'thiacytidine and 2',3'-didehydro-3'-deoxythymidine, selection of human immunodeficiency virus type 1 (HIV-1) strains with mutations in the reverse transcriptase (RT) gene has been reported. We designed a reverse hybridization line probe assay (LiPA) for the rapid and simultaneous characterization of the following variations in the RT gene: M41 or L41; T69, N69, A69, or D69; K70 or R70; L74 or V74; V75 or T75; M184, I184, or V184; T215, Y215, or F215; and K219, Q219, or E219. Nucleotide polymorphisms for codon L41 (TTG or CTG), T69 (ACT or ACA), V75 (GTA or GTG), T215 (ACC or ACT), and Y215 (TAC or TAT) could be detected. In addition to the codons mentioned above, several third-letter polymorphisms in the direct vicinity of the target codons (E40, E42, K43, K73, D76, Q182, Y183, D185, G213, F214, and L214) were found, and specific probes were selected. In total, 48 probes were designed and applied to the LiPA test strips and optimized with a well-characterized and representative reference panel. Plasma samples from 358 HIV-infected patients were analyzed with all 48 probes. The amino acid profiles could be deduced by LiPA hybridization in an average of 92.7% of the samples for each individual codon. When combined with changes in viral load and CD4+ T-cell count, this LiPA approach proved to be useful in studying genetic resistance in follow-up samples from antiretroviral agent-treated HIV-1-infected individuals.