Multiplexed immunoassay for detection of rheumatoid factors by FIDISTM technology.

Our objective was to (i) compare FIDIS Rheuma, a new multiplexed immunoassay designed for simultaneous detection of IgM class rheumatoid factors (RF) directed against Fc determinants of IgG from humans and animals, with agglutination and ELISA (conventional methods) and (ii) evaluate the clinical sensitivity and specificity of biological markers for rheumatoid arthritis (RA). To do this, FIDIS technology was employed using the Luminex system. It consists of distinct color-coded microsphere sets, a flow cytometer, and digital signal processing hardware and software. Agglutination and ELISA tests were performed with commercial kits. The study included 134 samples from RA patients and 105 from healthy blood donors. For human specificity, we compared FIDIS with latex agglutination and ELISA. Relative sensitivities were 98.9% and 88.5% and specificities were 90.2% and 94.6%, respectively. For animal specificity, we compared FIDIS with Waaler-Rose and ELISA. The results were 84.9% and 71.9% for the sensitivities and 97.5% and 98.4% for the specificities, respectively. Detection of IgG anti-CCP by ELISA and IgG antikeratin by immunofluorescence was also determined in order to compare their clinical sensitivity and specificity with IgM-RF, according to the method used. The results were: IgG anti-CCP 72.3%, 97.2%; IgG antikeratin 36.6%, 100%; latex agglutination 66.4%, 97.2%; Waaler-Rose 55.9%, 96.3%; FIDIS human 73.9%, 92.1%; FIDIS animal 49.2%, 97.2%; ELISA human 93.2%, 95.5%; and ELISA animal 74.6%, 91.3%. The results showed the efficiency of FIDIS with analytical performance equivalent to the conventional methods, but having the advantage of giving quantitative results (IU/mL).

Detection of hepatitis B virus core mutants by PCR-RFLP in chronically infected patients.

HBV chronic infection is an important health problem. The HBV core antigen carries several epitopes for T and B cell recognition and the immune response is crucial for determining the outcome of viral infection. Using PCR-RFLP several point mutations were detected in the HBV core ORF of HBV extracted from the serum of 140 chronically infected patients and 86 samples from another 37 patients followed-up in a longitudinal study. Mutations at position 2248 and 2147 (A3) and at 2038 (M2) were found most frequently. The wild type core genotype was found in about 50% of the samples. PCR-RFLP results were confirmed by direct sequencing of amplified products from HBV DNA present in chronically infected patients. The method is rapid and reliable and may be particularly useful for a rapid detection of viral mutants in a large number of patients.

Filtration induces changes in activity states and leucocyte populations.

The distribution of leucocyte subpopulations in platelet concentrates (PC) derived from pre-storage filtered platelet-rich plasma (PRP), the cell suspension obtained by reverse filter washing and the post-filtered PC, were monitored by immunophenotyping analysis using CD3, CD20 and CD33. Leucocyte activation analysis with the CD11b marker revealed that this molecule is up regulated in neutrophils taken from the filter. This, together with the loss of cell viability during the enrichment process, suggests that contact with the filter matrix and processing and storage of samples containing leucocytes may lead to activation and loss of leucocyte viability. These changes were found to be more pronounced in less stable myeloid cells and account for the differences reported among various authors which in some cases related to operational conditions such as the enrichment process used and the length of time between filtration and analysis of samples. Finally, statistical analysis of the results obtained by immunophenotypic studies indicate that post-filter samples (S) contain significantly higher numbers of CD33+ myeloid cells when compared to (PF) the pre-filter samples (65.03%+/-12.6 and 24.56%+/-14.73, p<0.0000), with a decrease in T cells (50.72%+/-14.80 in PF and 24.05+/-9.48 in the cell suspension (S), p<0.0007) and B cells (14.96+/-9.31 in PF and 9.9+/-5.22 in S, p<0.201). A new strategy for assessing the influence of the filtration process on residual leucocyte activation and viability is described. This has direct relevance to collection, processing, storage and quality monitoring of PC.

Cross-reactivity between Chlamydia trachomatis heat shock protein 10 and early pregnancy factor.

Chlamydia trachomatis heat shock protein 10 (Chsp10) is associated with chronic genital tract infection with C. trachomatis. Chsp10 is homologous to human chaperonin 10 (Cpn10) and early pregnancy factor (EPF), a form of human Cpn10 that is specifically secreted at the start of pregnancy. We investigated cross-reactions between serum anti-Chsp10 antibodies and anti-EPF antibodies in pregnant and nonpregnant patients. Pregnancy was found to be associated with the presence of anti-EPF antibodies, which are specifically induced in pregnant women with a history of C. trachomatis infection, and with the presence of serum anti-Chsp10 antibodies. We also found that infertility was associated with the presence of anti-Chsp10 and anti-EPF antibodies. The HLA class II haplotype DR8 DQ4 was associated with the presence of anti-Chsp10 antibodies but not of anti-EPF antibodies.

Active replication of hepatitis B virus (HBV) in HIV type 1 and in HIV type 2 infected patients

A fim de avaliar as consequencias da infeccao por HIV no curso da infeccao por HBV, ou na imunidade anteriormente adquirida, estudamos um grupo de 66 doentes Caucasoides HIV1+ sintomaticos e outro de 38 individuos seropositivos para HIV2 e provenientes da Africa, quanto a marcadores serologicos de infeccao por HBV e quanto a presenca de DNA viral circulante, tomada como sinal de replicacao do virus da hepatite. Os grupos HIV+ foram comparados com controles seronegativos adequados tendo-se verificado que 7,6 por cento dos doentes HIV1+ eram tambem HBV-DNA+ (versus 3,2 por cento nos seronegativos) bem como 2,6 por cento dos HIV2+ (versus 2,9 por cento nos controles seronegativos), nao sendo as diferencas estatisticamente significativas em qualquer um dos casos e nao tendo sido encontrada correlacao entre infeccao por HIV e replicacao ativa de HBV...

Active replication of hepatitis B virus (HBV) in HIV type 1 and in HIV type 2 infected patients.

To evaluate the effect of concurrent infection by HIV on HBV infection or immunity, we have studied a group of 66 HIV1+ symptomatic Caucasian patients and another of 38 African HIV2+ asymptomatic individuals, concerning their HBV status: serological markers of infection and presence of HBV-DNA in serum, the last taken as sign of hepatitis B virus active replication, were monitored. HIV+ groups were compared with seronegative controls, adequately matched for age, sex and ethnological background. HBV DNA was found in 7.6% of HIV1+ Caucasian patients and 3.2% of seronegative controls; in African HIV2+ individuals 2.6% were also HBV DNA+, a percentage close to that found in HIV2 seronegative controls (2.9%). No correlation was found between HIV infection and HBV active replication. Immunodepression that follows HIV infection over time may be compatible with a degree of T cell function capable of avoiding reinfection with or reactivation of HBV, even in symptomatic stages of acquired immunodeficiency syndrome. Our findings are relevant to the choice of preventive strategies in populations at risk for HIV and HBV infection.