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Examines the interlaboratory variation of HIV-1 RNA measurement on the plasma of Belgian patients. Prediction of the rate of disease progression; Observation on the antiretrovirus treatment; Use of Amplicor assay in detecting differences of HIV-1 RNA in plasma.

A spectral shift and new emission bands in the green and red regions have been observed in deep blue exciplex-based organic light-emitting diodes (OLEDs) using carbazole-based materials, namely, tris(4-carbazoyl-9-ylphenyl)amine (TCTA). To deeply understand the origin of these new bands, single-layer and bilayer TCTA-based OLEDs subjected to electrical and optical (ultraviolet (UV)) stresses were investigated by using various optical, electrical, morphological, and chemical measurements. The results showed that the stress-induced emission bands primarily originate from morphological changes rather than chemical changes. The accumulation of excitons in the TCTA layer induces molecular aggregation, leading to the formation of electrically active electronic states, namely, electroplexes and electromers, which lead to the appearance of additional emission bands in green and red regions. Impedance spectroscopy measurements on single-layer OLEDs complemented this study. The results showed that TCTA degradation affects charge injection and transport. It was concluded that the stress-induced emission bands are caused by aggregate domain formation and are closely linked to the formation of electrically active defects, which act as trap states for charge carriers in the TCTA band gap.

This study was undertaken within the framework of a surveillance project on the resistance of Mycobacterium tuberculosis to first-line antituberculosis drugs in four provinces of Rwanda with a high prevalence of tuberculosis (TB). The purpose was to determine the prevalence of primary and acquired resistance of M. tuberculosis to major antituberculosis drugs. A cohort of patients (n=710) with pulmonary TB documented by positive microscopic examinations of exhaustive samples was recruited at 7 treatment centers. Sputum samples were cultured on Löwenstein-Jensen and Coletsos media. Sensitivity to antituberculosis drugs was tested using a BACTEC 460 radiometric system. M. tuberculosis was isolated in 644 of the 710 patients (90.7%). A total of 296 out of 573 tested for HIV infection (51.7%) were positive. Primary resistance to one, two, three or four antituberculosis drugs was observed in 3.5%, 2.9%, 1.4% and 5.7% respectively. The prevalence of acquired resistance to antituberculosis drugs was 11.2%. Primary monoresistance to streptomycin was the most prevalent (2.3%) followed by resistance to ethambutol (1%). The combined rate of multiresistance was 11.6% with 7% involving new cases and 25.5% involving retreatment. This study showed that the rates of primary and acquired resistance to first-line antituberculosis drugs were high and that TB was associated with HIV infection. The National TB Control Program must implement measures to coordinate diagnosis and management of TB and HIV infection.

Objective: To evaluate the performance of the colorimetric resazurin microtiter assay (REMA) method for the detection of ofloxacin resistance., Methods: A panel of 120 multidrug-resistant Mycobacterium tuberculosis strains was tested blindly by the REMA method and compared with the results obtained using the BACTEC 460 method., Result: A very good correlation was observed between the two methods., Conclusion: The REMA method is simple, rapid and can be an inexpensive alternative procedure for the rapid detection of anti-tuberculosis drug resistance in laboratories with limited resources.

Objectives: To describe the clinical and microbiological features of Dientamoeba fragilis and Giardia lamblia infected patients, and to analyze the genetic variation of D. fragilis strains., Methods: For a period of two years, all stool samples collected from patients suspected of having a parasitic gastrointestinal infection were examined according to our specific triple feces test (TFT) protocol. A retrospective case-control study was performed on D. fragilis and G. lamblia infected patients. Furthermore, PCR and genotyping by restriction fragment length polymorphism (RFLP) were performed upon the former., Results: D. fragilis (6.3%) and G. lamblia (7.1%) were the most common pathogenic protozoa isolated out of 448 patients studied. Symptoms most frequently encountered with D. fragilis and G. lamblia infection were abdominal pain (69.2% and 72.4%, respectively) and diarrhea (61.5% and 79.3%, respectively). However, patients with D. fragilis infections suffered significantly less frequently from nausea and/or vomiting, anorexia and weight loss. After treatment, all D. fragilis and G. lamblia infected patients presenting a negative TFT follow-up also reported a complete resolution of their symptoms. Only genotype 1 could be detected in D. fragilis infected patients., Conclusions: D. fragilis and G. lamblia were the most frequently encountered parasites in our study population. Improved diagnostic tests are essential tools to study the prevalence and pathogenesis of D. fragilis.

Background: Resistance testing is increasingly accepted as a tool in guiding the selection of human immunodeficiency virus type 1 (HIV-1) antiretroviral therapy in HIV-1 infected individuals who fail their current regimen., Objectives: To descriptively compare the correlation between virologic treatment response and results using three genotypic HIV-1 drug resistance interpretation systems: the VERSANT HIV-1 Resistance Assay (LiPA) system and two sequence-based interpretation systems., Study Design: Specimens from 213 HIV-1-infected subjects, either starting (n=104) or switching to (n=109) a regimen of three or four antiretroviral drugs, were collected retrospectively at baseline and after 3 months of uninterrupted therapy. The correlation between viral load change and the number of predicted active drugs in the treatment regimen was assessed. An interpretation algorithm was recently developed to process VERSANT HIV-1 Resistance Assay (LiPA) data. The number of active drugs predicted using this algorithm was rank correlated with the viral load change over a 3-month treatment period. For comparison, a similar calculation was made using two sequence-based algorithms (REGA version 5.5 and VGI GuideLines Rules 4.0), both applied on the same sequences., Results: Statistically significant (p<0.05) correlation coefficients for each of the three HIV-1 drug resistance interpretation systems were observed in the treatment-experienced subjects on a 3-drug regimen (-0.39, -0.38, and -0.42, respectively) as well as on a 4-drug regimen (-0.33, -0.31, and -0.37, respectively). However, no significant correlation was observed in treatment-naive subjects, probably due to the very low frequency of drug resistance in these subjects., Conclusion: All three genotypic drug resistance interpretation systems (LiPA version 1, REGA version 5.5, and VGI GuideLines Rules 4.0) were statistically significantly correlated with virologic therapy response as measured by viral load testing.

The usefulness of employing Belgian selection criteria before performing nucleic acid amplification techniques (NAT) was evaluated. The results of this study show that for smear-negative patients with an abnormal chest radiology result in the absence of a respiratory tract infection by bacterial pathogens, testing with NAT is of major benefit.

In this study we evaluated the performance of the VERSANT HIV-1 Resistance Assays (LiPA) in detecting drug resistance in therapy-naive HIV-infected patients diagnosed in Belgium in 2000. We compared the results with population sequencing and found concordance to be in line with previous studies in treatment-experienced patients (86.87% for reverse transcriptase (RT); 92.77% for protease (PRO)). Discordance was mainly due to indeterminate reactions on LiPA (8.45% for RT; 6.85% for PRO) and minor discordances (4.13% for RT; 0.25% for PRO). Major discordances were rare (0.46% for RT; 0.12% for PRO). Indeterminate reactions were significantly associated with strains belonging to non-B subtypes.

Objectives: To evaluate the immune reconstitution in HIV-1-infected children in whom highly active antiretroviral therapy (HAART) controlled viral replication and to assess the existence of a relation between the magnitude of this restoration and age., Methods: All HIV-1-infected children in whom a new HAART decreased plasma viral load below 400 copies/ml after 3 months of therapy were prospectively enrolled in a study of their immune reconstitution. Viral load, lymphocyte phenotyping, determination of CD4+ and CD8+ T cell receptor repertoires and proliferative responses to mitogens and recall antigens were assessed every 3 months during 1 year., Results: Nineteen children were evaluated. Naive and memory CD4+ percentages were already significantly increased after 3 months of HAART. In contrast to memory CD4+ percentages, naive CD4+ percentages continued to rise until 12 months. Age at baseline was inversely correlated with the magnitude of the rise in naive CD4+ cells after 3, 6 and 9 months of therapy but not after 12 months. Although memory and activated CD8+ cells were already decreasing after 3 months, abnormalities of the CD8 T cell receptor repertoire and activation of CD8+ cells persisted at 1 year. HAART increased the response to mitogens as early as 3 months after starting therapy., Conclusions: In children the recovery of naive CD4+ cells occurs more rapidly if treatment is started at a younger age, but after 1 year of viral replication control, patients of all ages have achieved the same level of restoration. Markers of chronic activation in CD8+ cells persist after 1 year of HAART.

In a longitudinal study we address the hypothesis that resis tance to disease progression in lentivirus-infected chimpanzees is related to potent non-cytotoxic suppression of virus replication. In a long-term follow-up, the viral suppressive capacity in two simian immunodeficiency virus (SIV)cpz-infected chimpanzees was correlated with two polymerase chain reaction (PCR)- and two culture-based virus load measurements. In both animals, quantitative virus isolation (QVI) tended to decline slowly, whereas in vitro virus suppression was sustained or increased over time. In general, plasma virus loads in SIVcpz-infected animals were maintained for extended periods of time. Based on current assays that measure virus suppressive capacity in peripheral blood, it was not possible to conclude that virus suppression played a major role in the maintenance of the disease-free state in lentivirus-infected chimpanzees.

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.

Three commercial assays for quantifying plasma human immunodeficiency virus type 1 (HIV-1) RNA were evaluated. The assays differed in their sample volumes, the means of preparing samples, and methods of amplification and detection. Plasma samples were obtained from 36 HIV-1-infected patients representing all stages of HIV-1 infection and were analyzed as coded specimens. Measurement of HIV-1 RNA baseline levels revealed no significant difference in sensitivity between the three assays. The assays were also applied to the quantitation of HIV-1 RNA levels in the plasma of patients who were changing their antiretroviral therapy. The changes measured in HIV-1 RNA levels in plasma in response to therapy were comparable by the three assays. No close correlation was found between the amount of HIV-1 RNA and the CD4 T-cell count; HIV-1 RNA assays were more sensitive than p24 antigen assays as an indicator of plasma viremia. Overall, the study demonstrates that all three quantitative assays for HIV-1 RNA can be used to measure the HIV-1 RNA copy number representing the HIV-1 viremia status in patients with HIV-1 infection. Since this copy number is likely to be useful in monitoring the effectiveness of antiviral therapy, these quantitative assays for HIV-1 RNA are ready to be built into clinical trials.

Objective: To validate the technique of HIV-1 culture from whole blood for the quantitation of viral load in infected children., Patients: Forty-three HIV-1-infected children were followed in two paediatric centres., Methods: Quantitative HIV-1 cultures from unfractionated whole blood using an end-point dilution technique were compared with simultaneous quantitative cultures of peripheral blood mononuclear cells (PBMC) and plasma., Results: Good sensitivity (93%) of the methods used was confirmed. A close correlation (r = 0.80) was observed between HIV-1 titres measured directly from whole blood and those expected from PBMC and plasma titres. The mean whole blood viral load was higher in patients with more severe signs of disease, but the difference did not reach statistical significance. The whole blood viral titres measured sequentially at monthly intervals remained within one dilution of each other in 16 of the 22 patients studied., Conclusion: In this study, the quantitation of HIV-1 in unfractionated blood allowed for a reliable and sensitive measurement of the whole blood viral load in infected children.

A healthy 26-year-old patient presented a primary herpetic type 2 infection. The infection disseminated to the skin, the mucosa and the liver. The clinical course of the infection had a favorable issue.

We have developed a series of monoclonal antibodies against the calf strain RIT 4237 (subgroup 1) and the human strain 82-561 (subgroup 3) of rotavirus, both grown in tissue culture, and also against the human rotavirus 81-2162 (subgroup 2), extracted from a fecal specimen. A variety of different specificities was observed among these antibodies, namely, antibodies against group and subgroup determinants, as well as neutralizing antibodies. By using monoclonal antibodies against the subgroup antigen in an enzyme-linked immunoassay system, the constant predominance of subgroup 2 viruses in humans was confirmed in 74 stools collected from children in Brussels who suffered a diarrheal illness between July 1981 and June 1983. The availability of these antibodies also made it possible to improve the sensitivity and the specificity of the test system.

A randomised, double-blind, placebo-controlled trial was conducted to evaluate the ability of RIT 4237 live attenuated bovine rotavirus (subgroup 1) vaccine strain to protect against natural rotavirus infection in children. 178 infants aged 8 to 11 months received a single oral dose of RIT 4237 vaccine or placebo and were followed up serologically and clinically during a subgroup 2 rotavirus epidemic. No side-effects attributable to the vaccine were observed. During the 5 months' observation after vaccination 2 of the 86 vaccine recipients and 18 of 92 placebo recipients had rotavirus diarrhoea lasting more than 24 h (p less than 0.001). The vaccine-protection rate was thus 88%. The 2 children in the vaccine group with rotavirus diarrhoea were regarded as primary vaccine failures since they had no detectable serum antibody responses after vaccination. Vaccine prepared from RIT 4237 strain of attenuated bovine rotavirus thus seems to protect children against heterologous subgroup 2 rotavirus diarrhoea.

A candidate oral live rotavirus vaccine, strain RIT 4237, of bovine origin, was tested for immunogenicity and safety in man. In adults the vaccine did not cause clinical symptoms, and a booster response in rotavirus serum antibodies was seen in 2/20 subjects. In seronegative young children one oral dose induced seroconversion to homologous virus in 15/17 (88%) children seronegative by enzyme immunoassay and in 13/19 (68%) children seronegative by a neutralisation assay. The vaccine did not produce gastrointestinal or constitutional symptoms in the children, nor did it cause rotavirus excretion in the stools. The results suggest that the RIT 4237 strain is a promising candidate for a vaccine against human rotavirus, and the vaccine-induced immunity against natural human rotavirus infection should be evaluated in future trials.

Detection of HIV antibodies by means of an immunoenzymatic assay, an indirect immunofluorescence technique and Western blot was attempted on 375 serum samples collected in the Murunda area, a remote rural area situated in Rwanda, central Africa. Two out of 147 (1.4%) adults from a strict rural area, five out of 59 (8.5%) adults from an adjacent market place, and 49 out of 169 (30%) STD clinic attenders from the same area were HIV seropositive. In the first two groups, HIV seropositivity was associated with a history of sexually transmitted disease (STD) in the previous 2 years (P less than 0.001) and with a history of travel to a Rwandese urban centre in the previous 5 years (P less than 0.05). This study suggests that HIV seroprevalence is low in rural central Africa compared with urban centres. Risk factors for HIV seropositivity are similar in rural and urban-based adults in Rwanda, i.e. heterosexual promiscuity and STDs. Many HIV seropositive rural subjects from this study are likely to have acquired HIV infection through sexual contacts in Rwandese cities.

Because of the morbidity and mortality from infection with rotaviruses in children under 2 years, in many parts of the world but especially in developing countries, an urgent need for a vaccine has been recognized. At present, a vaccine candidate of bovine origin, the RIT 4237 strain vaccine developed from a strain received from Mebus et al., is available. This strain has been shown to induce cross protection by challenge of colostrum deprived piglets with human rotavirus strains. Before testing its protective effect in children, preliminary studies in adult volunteers are required to evaluate its degree attenuation and immunogenicity. With regard to the latter we have studied the antibody profile of 87 healthy adults, ages 25 to 40 years, from whom blood samples were taken between January and June 1982. Antibodies versus RIT 4237 were measured both by neutralization and ELISA techniques. Analysis of the results revealed a significant difference in antibody levels according to sex, since high neutralizing antibody titers (greater than 1:320) were observed about 7 times more frequently in women than in men. Furthermore, no seasonal variation in the level and the distribution of serum antibody titers was found when comparing blood specimens taken from male volunteers in winter and spring. As far as antibody levels obtained by neutralization and ELISA assays is concerned, a good correlations was noted in 75% of cases. However, in 22 out of 87 sera high levels of neutralizing antibodies were found in association with low ELISA antibody titers or vice versa. These results suggest that screening of adult volunteers should be performed by means of a neutralization assay prior to administration of a candidate vaccine. However, in the absence of such preliminary testing, male volunteers should be chosen since there is a higher probability of low pre-existing antibody levels among such subjects.

Electron microscopy, immune electron microscopy and complement fixation as methods of detecting rotavirus in the stools of young children with gastroenteritis were compared in a blind study during the winter of 1975-6. Complement fixation was the simplest to perform, was as sensitive as the other two, and allowed a quantitative measurement of viral excretion. Absorption of faecal extracts with fetal calf serum usually removed the anticomplementary activity of faecal extracts.

To determine the relative importance of two known serotypes of human rotavirus, we developed an enzyme-linked immunosorbent assay to differentiate serotype-specific rotavirus antigen and antibody. Using this technic, we studied the epidemiology of the two serotypes in acute gastroenteritis. Seventy-seven per cent of 414 rotavirus isolates were Type 2, and the remainder were Type 1. The serotype distribution was similar in specimens from children in Washington, D.C., and other parts of the world. Sero-epidemiologic studies revealed that most children living in the Washington, D.C., area acquired antibody to both types by the age of two years. An analysis of children who were reinfected indicated that sequential infections usually involved different serotypes and that illness caused by one serotype did not provide resistance to illness caused by the other serotype. These results suggest that, to be completely effective, a vaccine must provide resistance to both serotypes.

A seroepidemiological study was performed on HTLV-III, T. pallidum, C. trachomatis and Hepatitis B virus (HBV), in Butare, Rwanda, among 33 female prostitutes, 25 male customers of prostitutes, and 60 male and female controls. As compared with female controls the prostitutes had a higher prevalence of antibodies to HTLV-III (29/33 versus 4/33, p less than 0.001), T. pallidum (TPHA: 27/33 versus 6/33, p less than 0.001; RPR: 19/33 versus 2/33, p less than 0.001; FTA-Abs: 27/33 versus 5/33, p less than 0.001) and C. trachomatis (IgG IF: 31/33 versus 13/33, p less than 0.001). HBV serological markers were more often detected in the prostitutes than in the female controls (31/33 versus 18/33, p less than 0.001) although HBs antigen carriage rate was similar in both groups. As compared with male controls, the male customers of prostitutes had more frequently detectable antibodies to HTLV-III (7/25 versus 2/27, p = 0.05), and a positive RPR (10/25 versus 1/27, p less than 0.01). Among the 118 individuals studied, odds ratios and trend analysis disclosed a significant association between HTLV-III seropositivity and a positive TPHA, RPR, FTA-Abs, Chlamydia IgG IF test and serological markers to HBV. No association was found between HTLV-III seropositivity and HBs Ag carriage. This study suggests that HTLV-III has to be considered as an infectious agent transmitted among promiscuous Central African heterosexuals by sexual contact and/or parenteral contact with unsterile needles used for STD treatments.

The safety and immunogenicity of repeated doses of RIT 4237, an oral live attenuated rotavirus vaccine of bovine origin, as well as the influence of gastric acidity and maternal antibodies on the vaccine "take rate" were studied in newborns. The vaccine was given orally on the first day of life, at 1 month, and at 2 months of age. Fifty-seven newborns entered the study but only 36 completed the trial. No adverse reaction was observed after vaccination. Immune responses were evaluated by titrating pre- and postvaccination serum antibodies to RIT 4237 by a neutralization technique. Eight of 43 (18.6%) tested infants responded to the first dose of vaccine. Subsequently another six and 11 infants responded to the second and third dose, respectively. The total cumulative response after the third dose was 23 of 36 vaccinees (63.9%). The geometric mean neutralizing antibody titers was lower in responders than in nonresponders before the first dose of vaccine indicating that maternal antibodies interfered with vaccine "take." Since the RIT 4237 strain is acid sensitive, gastric pH was measured at the time of each vaccination. Mean pH values in responders were not significantly different than those found in nonresponders. Therefore, vaccine failures cannot be due to inactivation of the rotavirus vaccine in the stomach but instead they can be attributed to high levels of maternal antibodies. At each vaccination, additional subjects seroconverted. Therefore, a multiple dose scheme is advisable to obtain an optimal immune response.

In January 1987, HIV antibodies were detected by means of an immunoenzymatic assay, indirect immunofluorescence and Western blot in 52 out of 302 male urban-based professionals and in 28 out of 150 health workers in Kigali, Rwanda. Univariate analysis showed an association between HIV seropositivity and a history of sexually transmitted diseases (STD), blood transfusion, medical injections for treatment of STD, and medical injections for treatment of febrile illnesses. However, injection related to treatment of other conditions were not associated with HIV seropositivity. Among health workers, no association between HIV seropositivity and professional or accidental exposure to HIV-infected patients or to their body fluids was identified. Discriminant analysis showed that HIV seropositivity was associated only with a history of STD and with a history of blood transfusion. In central Africa, a history of STD and a history of blood transfusion should be considered as risk factors for HIV seropositivity. Medical or accidental injections do not seem to play a major role in the transmission of HIV among adults in central Africa.

The relative prevalence of human rotavirus subgroups was studied during a 3-year period (1978-1981) by means of a sensitive complement fixation technique. Among 93 rotavirus isolates from children with acute gastroenteritis in Brussels, the prevalence of subgroups 1, 2, and 3 was, respectively 24, 17, and 32%. The remaining 27% of strains could not be typed, but no evidence for the existence of any new subgroup was found. The proportion of strains belonging to the different subgroups remained roughly constant during the study period, showing the simultaneous occurrence of the various subgroups of viruses, even during the annual winter peak of rotavirus gastroenteritis.