Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples.

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0% for sensitivity and 91.2% for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0% and 91.2%, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0%, 89.0%, 84.0% and 99.0%, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5% for sensitivity and 95.4% for specificity, as well as PPV and NPV of 92.9% and 86.0%, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0 percent for sensitivity and 91.2 percent for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0 percent and 91.2 percent, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0 percent, 89.0 percent, 84.0 percent and 99.0 percent, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5 percent for sensitivity and 95.4 percent for specificity, as well as PPV and NPV of 92.9 percent and 86.0 percent, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Two variants of HIV-1 B serotype are transmitted heterosexually in Säo Paulo, Brazil

HIV-1 variability may have an important impact on transmission and pathogenicity. Better characterization of the HIV epidemic in Brazil is necessary for the development of vaccine trials in this country. We analyzed sera from 108 HIV-1-infected volunteers from Säo Paulo City to determine serotype and reactivity for V3 motifs of HIV in this population, and the relationship to transmission mode. We concluded that the HIV-1 B serotype is frequent among heterosexually infected women, even in the absence of anal sex, and that two major V3 motifs, GPGR and GWGR, had similar prevalence among women (48 per cent and 52 per cent, respectively) and men (56 per cent and 44 per cent, respectively). We also observed an equal distribution of these strains regardless of their CD4+ T cell counts, clinical status, and mode of transmission. Even though V3 serology for HIV-1 subtyping is an inexpensive tool for use in developing countries, additional methods, such as heteroduplex mobility assay and direct DNA sequencing, should be included to determine HIV-1 genetic diversity.

Two variants of HIV-1 B serotype are transmitted heterosexually in São Paulo, Brazil.

HIV-1 variability may have an important impact on transmission and pathogenicity. Better characterization of the HIV epidemic in Brazil is necessary for the development of vaccine trials in this country. We analyzed sera from 108 HIV-1-infected volunteers from São Paulo City to determine serotype and reactivity for V3 motifs of HIV in this population, and the relationship to transmission mode. We concluded that the HIV-1 B serotype is frequent among heterosexually infected women, even in the absence of anal sex, and that two major V3 motifs, GPGR and GWGR, had similar prevalence among women (48% and 52%, respectively) and men (56% and 44%, respectively). We also observed an equal distribution of these strains regardless of their CD4+ T cell counts, clinical status, and mode of transmission. Even though V3 serology for HIV-1 subtyping is an inexpensive tool for use in developing countries, additional methods, such as heteroduplex mobility assay and direct DNA sequencing, should be included to determine HIV-1 genetic diversity.

IL-2 and IFN-gamma, but not IL-4 secretion by peripheral blood mononuclear cells (PBMC) are related to CD4+ T cells and clinical status in Brazilian HIV-1-infected subjects.

It has been reported that production of IL-2 and IFN-gamma, known as T-helper type 1 cytokines, by peripheral mononuclear cells (PBMC) decreases with progression of HIV infection. In contrast, IL-4 and IL-10 production, Th2 cytokine profile, increases with HIV disease progression. PBMC were evaluated from 55 HIV-infected subjects from Divisão de Imunologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, to "in vitro" cytokines production after 24 hours of stimulation with PHA. Low levels of IL-4 production in both HIV-infected patients and normal subjects, were detected. The patients with CD4+ T cell counts < 200 showed a significant decrease of IL-2 and IFN-gamma production compared to controls. Patients with higher counts of CD4+ T cells (either between 200-500 or > 500 cells/mm3) also showed decreased production of IL-2 that was not statistically significant. There was a correlation between IL-2 and IFN-gamma release with CD4+ T cells counts. HIV-1-infected individuals with CD4+ T cells > 500 cells/mm3 showed increased levels of IL-2 and IFN-gamma, than individuals with CD4+ T cells < 500 cells/mm3. In conclusion, we observed a decline of IL-2 and IFN-gamma production at advanced HIV disease. IL-4 production was not affected during HIV infection. Taken together, these findings suggest that the cytokine profile might be influenced by the HIV infection rather than the cause of disease progression.

In vitro inhibitory activity of tumor necrosis factor alpha and interleukin-2 of human immunoglobulin preparations.

Human immunoglobulin preparations have been used in a number of clinical settings with good results, although in many of them the mechanism of action is not yet known. One possible mechanism is the modulation of cytokine activity. This study investigated the presence of inhibitory activity in intravenous immunoglobulin (IVIg) and F(ab')2 fragment preparations to two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2). Cytotoxic activity of human recombinant TNF-alpha or TNF-alpha secreted by peripheral blood mononuclear cells (PBMC) on L929 cells and the proliferative activity of the IL-2 on CTLL-2 cells were examined. Human serum albumin (HSA) was used as control. F(ab')2 inhibited, in a dose-dependent fashion, the TNF-alpha activity secreted by PBMC serial dilutions or, at the higher concentrations (25 and 10 mg/ml), recombinant TNF-alpha activity. In contrast, IVIg was able to inhibit only at 25 and 10 mg/ml the TNF-alpha activity secreted by any PBMC dilution tested, and did not inhibit the recombinant TNF-alpha activity. With IL-2, however, even HSA was able to inhibit its proliferative activity, possibly through a carrier effect. The IVIg inhibition of IL-2 activity was not different from that of HSA, but F(ab')2, at 12.5 mg/ml, was capable of inhibiting significantly more the IL-2 activity than HSA. Our results suggest an anticytokine effect of the immunoglobulin preparations that this activity may be mainly mediated by variable regions of the immunoglobulins, and that the more pronounced effect of F(ab')2 may be due to its greater molar concentration compared to intact IgG molecules.

Prevalence of HTLV-I and HTLV-II infections among HIV-1-infected asymptomatic individuals in São Paulo, Brazil.

Human immunodeficiency virus (HIV-1)-infected subjects with acquired immunodeficiency syndrome (AIDS) are often infected with multiple pathogens. In particular, HTLV-I and HTLV-II infections have been found more frequently in AIDS patients than in asymptomatic individuals in Europe and Japan. We carried out a serosurvey among asymptomatic HIV-1-infected subjects in São Paulo, Brazil and compared our results with those of other investigators. In this study, we found HTLV infection in 1.5% of 266 asymptomatic and 14% of 28 AIDS patients. Epidemiological data obtained from patients pointed out the use of intravenous drugs as the principal risk factor for acquiring retroviruses. In conclusion, our results are in accordance with other studies done in Brazil and elsewhere where the principal risk group for HIV/HTLV-I/II coinfection was IDU.

Antigen-specific immunosuppression in paracoccidioidomycosis.

To characterize the immune dysfunction associated with paracoccidioidomycosis, we studied the in vitro lymphocyte reactivity to phytohemagglutinin (PHA), pokeweed mitogen (PWM), a Candida albicans antigen (CMA), and a Paracoccidioides brasiliensis antigen (PbAg) in 32 patients with the acute and the chronic form of the disease before or during the initial phase of treatment and after clinical cure. We also studied, as controls, 30 healthy individuals, 15 of them immune to P. brasiliensis. Results showed a strong hyporesponsiveness to the PbAg while responses to mitogens and CMA were comparable with those of controls. Patients with the acute form of the disease (usually more severe) had more marked PbAg hyporesponsiveness than those with the chronic form. After patients' clinical cure, PbAg proliferative responses were similar to controls and greater than those seen before pretreatment. Changes in other parameters were also seen in the treated patients; skin test anergy to paracoccidioidin, high levels of anti-P. brasiliensis antibodies, leukocytosis, and eosinophilia. These changes were usually more intense in patients with the acute form of the disease. The post-treatment CD4+, CD8+, and total lymphocyte counts were similar to those of controls. Correlation between these parameters and the lymphoproliferative responses to the various stimuli was only found with PbAg: PbAg responses correlated inversely with eosinophil and anti P. brasiliensis antibody levels. Overall, our results demonstrate an antigen-specific-cellular immunity defect, which is reversible with treatment and possibly related to a T helper cell-2 pattern of immune response during active disease.

Responses of T and B lymphocytes to a Paracoccidioides brasiliensis cell wall extract in healthy sensitized and nonsensitized subjects.

Antigen-specific cellular immunity in paracoccidioidomycosis (PCM) has been poorly studied due to lack of standard in vitro lymphocyte proliferation assays. To standardize such an assay, we studied T and B cell responses to a Paracoccidioides brasiliensis cell wall extract (PbAg) in healthy subjects sensitized to either P. brasiliensis [Pb(+)Hc(-)] or to Histoplasma capsulatum [Hc(+)Pb(-)], and in nonsensitized persons. All subjects showed, as expected, a vigorous proliferative response to a control fungal antigen obtained from Candida albicans. Lymphocytes from Pb(+)Hc(-) donors, but not from Pb(-)Hc(-) donors, reacted to PbAg by proliferating in a dose-dependent manner with a maximum reaction after 6-9 days, suggesting a secondary specific immune response. Most activated cells were CD+CD4+ lymphocytes. However, Hc(+)Pb(-) donors' cells reacted with PbAg. Cross-reactivity with H. capsulatum was not unexpected, since both fungi, but not C. albicans, share cell wall immunogenic compounds. An enzyme-linked immunosorbent assay to detect human immunoglobulins (Ig) demonstrated that B cells from Pb(+)Hc(-) donors, but not from Pb(-)Hc(-) ones, reacted with PbAg by secreting high levels of IgG and IgM in 12-day culture supernatants. This secretion was possibly mediated by PbAg-activated CD4+ cells. We believe that analysis of T and B lymphocyte responses to PbAg will be useful in the investigation of the infection-associated immune impairment seen in some PCM patients.