Profile of Persons Recently Infected with HIV-1 in Belgium: New Insights to Tailor Prevention Efforts.

Despite wide availability of prevention and treatment services, including the ongoing roll-out of pre-exposure prophylaxis (PrEP), the HIV epidemic is not under control in Belgium. Hence, there is a recognized need to improve case finding and early diagnosis to curb the further spread of HIV more effectively. The objective of the present study was to improve insight into the profiles of persons recently infected with HIV-1 and on their prevention trajectory. Between May 2018 and December 2022, we selected persons diagnosed in Belgium within three months of the presumed infection date. We then analyzed information collected using a questionnaire covering topics on HIV testing, sexually transmitted infections (STIs), PrEP use, sexual behavior, partner notification and substance use. The data obtained were analyzed alongside information derived from phylogenetic cluster analysis of the viral source of infection. A total of 93 persons with a recent HIV-1 infection completed the questionnaire, the majority (74%) being MSM, 14% were heterosexual women and 12% were heterosexual men. Nearly one-third of participants engaged in sexual activity with an average of 2 to 5 casual partners around the presumed time of infection. A significant percentage reported frequent substance use during sexual activity (65%), being previously diagnosed with STI (65%) and using condoms infrequently (44%). 63% reported a testing frequency of at least one HIV test per year before being diagnosed and 46% notified their previous sex partner(s) after being diagnosed. Over 20% of respondents (including 11 MSM, 4 heterosexual men and 5 heterosexual women) reported exclusive sexual activity with their steady partner. Eight participants (9%, all MSM, 75% born outside of Belgium) reported PrEP use in the past. No significant differences in behavioral characteristics were found between persons who were part of a local transmission cluster (48%) and persons that were not part of a cluster (47%). The study results revealed that the majority of persons diagnosed early with HIV-1 infection in Belgium exhibited characteristics corresponding to a high-at-risk population and were aware of this risk, as evidenced by a high testing frequency. However, partner notification rates were low and use and awareness of PrEP limited. A notable group of persons not corresponding to the high-risk profiles was also identified. This information may help to expose missed opportunities for prevention and contribute to enhancing the implementation of future prevention measures.

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.

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.

Mixed phenotype murine leukemias.

Cell lines were derived from eight individual leukemias induced by X-rays in NFS mice. First typed as null cells (surface immunoglobulin negative, Thy-1 negative), they turned out to have a mixed phenotype with myeloid cytochemical markers, pre-B surface antigens and molecular markers of pro-B lymphocytes. They represent murine models for mixed phenotype (pro-pre-B-myeloid) leukemias.

Evaluation of proviral copy number and plasma RNA level as early indicators of progression in HIV-1 infection: correlation with virological and immunological markers of disease.

OBJECTIVE: We compared the proviral DNA level in peripheral blood mononuclear cells (PBMC), viral RNA level in plasma, presence of p24 antigen in serum, viral phenotype, and results of immunological markers of HIV-1 disease. METHODS: Consecutive samples of 62 HIV-1-infected patients, representing all stages of disease were tested for proviral DNA in PBMC and viral RNA in plasma using a semi-quantitative limiting dilution polymerase chain reaction (PCR). The presence of a syncytium-inducing (SI) phenotype was assessed after direct cocultivation of patient PBMC with MT-2 cells. Results of the quantitative PCR and the MT-2 coculture were correlated with the clinical stage of the disease, with the number of CD4+ T cells, and with the results of other virological and immunological markers, such as the level of p24 antigen, beta 2-microglobulin (beta 2M) and neopterin. RESULTS: Significant differences were observed between the results for asymptomatic and symptomatic patients for all markers under study. In the group of asymptomatic patients with a CD4+ T-cell count > 200 x 10(6)/l, patients with high amounts of proviral DNA had significantly higher amounts of beta 2M, neopterin and viral RNA, they were more frequently p24 antigen-positive and harboured more frequently SI strains than patients with low amounts of proviral DNA. A good correlation between the proviral DNA and the viral RNA levels was observed. Significant changes of viral RNA but not proviral DNA levels were observed after initiation of therapy or when therapy failed. CONCLUSIONS: We demonstrated the relationship between high proviral DNA level in PBMC, high viral load in plasma, elevated beta 2M and neopterin concentrations in serum, and the presence of p24 antigen in serum in a group of asymptomatic patients with a CD4+ T-cell count > 200 x 10(6)/l. We suggest the possible usefulness of proviral load as an early indicator of disease progression. The presence of SI strains is highly correlated with disease; however, SI strains were detected in only 46% of symptomatic patients. It also appeared that the measurement of viral RNA levels is a useful marker for therapy monitoring.

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.

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.

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.

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.

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.

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.

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.

Comparison of six commercial enzyme linked immunosorbent assays for detecting IgM antibodies against Toxoplasma gondii.

To evaluate the usefulness of different commercial enzyme linked immunosorbent assays (ELISAs) for the detection of IgM antibodies against Toxoplasma gondii the results of six of these assays for a panel of 81 sera were compared. The following tests were selected: Toxoplasma gondii IgM ELISA (Clark Laboratories), Toxoplasma IgM EIA (Labsystems), Toxo-M EIA (Abbott), Toxonostika M (Organon), Toxo M Enzyme Immunoassay (Hybritech) and Platelia Toxo IgM (Diagnostics Pasteur). An antibody capture ELISA developed at our laboratory was used as the reference test. An IgM immunoblotting assay was also performed. Four (Toxoplasma IgM EIA, Tox-M EIA, Toxonostika M, and Platelia Toxo IgM) of the commercial IgM ELISAs gave a high sensitivity and a high specificity. Toxo-M EIA, Toxonostika M, Toxoplasma IgM EIA and the Toxo M Enzyme Immunoassay were too insensitive, and the Toxoplasma gondii IgM ELISA was both insensitive and unspecific. No remarkable differences were observed between the results of indirect or antibody capture ELISAs, and between the results of ELISAs performed with polyclonal or monoclonal antibodies.

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%).

Standardisation of primers and an algorithm for HIV-1 diagnostic PCR evaluated in patients harbouring strains of diverse geographical origin. The Belgian AIDS Reference Laboratories.

Eight Belgian AIDS Reference Laboratories established a multicentre quality control to evaluate the performance of their diagnostic human immunodeficiency virus type 1 (HIV-1) DNA polymerase chain reaction (PCR). A set of Belgian and African HIV-1 seropositive and seronegative patient samples, collected in Belgium, and the British Medical Research Council (MRC) HIV-1 PCR reference reagent kit, containing plasmid HIV-1 DNA at several dilutions in human carrier DNA with appropriate negative controls, were tested by the laboratories. No false positive results were reported. All laboratories were able to detect one to two copies of HIV-1 DNA. Among the 17 Belgian and African HIV-1 seropositives, some laboratories reported up to four indeterminate results, mainly due to failure of the SK38-39, SK68-69 (Ou et al. (1988) Science 239, 295-297) and/or gag881-882 (Simmonds et al. (1990) J. Virol. 64, 864-872) primers and a poorly performing algorithm. Only the H1POL4235-4538 nested pol primer set, developed by one of the laboratories, correctly identified all the tested HIV-1 positive and negative samples. Consequently, the laboratories decided to evaluate these pol primers as a reference primer set and to standardise the testing algorithm. All laboratories achieved a sensitivity and specificity of 100% on testing 10 additional Belgian and African patient samples, when adapting a standardised algorithm based on three HIV-1 primer sets, one of which is the H1POL4235-4538 primer set.

Isolation of HIV-1 RNA from plasma: evaluation of eight different extraction methods.

The efficacy of eight different methods for the extraction of HIV-1 RNA from plasma was compared. The RNA preparation method that gave the best results by RT-PCR was the one described by Chomczynski and Sacchi (1987, Anal. Biochem. 162, 156-159). This method consists of a guanidine thiocyanate treatment followed by three phenol-chloroform-isoamylalcohol extractions and an ethanol precipitation. The disadvantage of this method is that it is time consuming and less suitable for the extraction of large series of samples. Moreover, due to the large number of procedural steps, there is a greater risk of sample mix-up or contamination. Of the single-step RNA purification methods, good results were obtained with the TRIzol method (Gibco Life Technologies, Paisley, UK) and with the extraction method offered by the NASBA kit (Organon Teknika, Turnhout, Belgium). The above single-step methods are recommended since both are sensitive enough to detect low copy numbers of HIV-RNA in the plasma of asymptomatic patients, and require only 2 h for completion. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extraction varied between 17.3 and 47.3%). Inter-laboratory reproducibility was evaluated only for the TRIzol-method and found to be low (mean variation coefficient 63.4).

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.

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.

Hepatitis C among risk groups for HIV and hepatitis B.

In order to obtain more information on sexual transmission of hepatitis C (HCV) we compared different high-risk groups for HIV and hepatitis B to see if they were seropositive for HCV. A high seroprevalence (38/81) of hepatitis C (HCV) was found among intravenous drug users. Nursing staff (n = 35) and patients of a dialysis unit (n = 57) had a low prevalence of anti-HCV antibodies (0% and 5%, respectively). Serology laboratory technicians also had a very low prevalence (0% out of 29). Among prostitutes (n = 114), healthy homosexual men (n = 132) and HIV-infected homosexual men (n = 31), we found a remarkably low seroprevalence of HCV (3.5%, 0.8% and 0.0% respectively). These data support the view that parenteral exposure to the virus is the most important way of acquiring the infection and that neither heterosexual nor homosexual promiscuity are associated with a high risk of transmission of hepatitis C.