Burkholderia pseudomallei-specific recombinant protein and its potential in the diagnosis of melioidosis.

Melioidosis is an important public health problem in Southeast Asia and Northern Australia. This disease is caused by the gram-negative bacilli, Burkholderia pseudomallei. Wide spectra of clinical manifestations are observed in melioidosis ranging from asymptomatic to septicemic infection. Although serodiagnostic methods of melioidosis have been improved significantly in recent years, a highly specific diagnostic test that can differentiate asymptomatic seropositive individuals and melioidosis patients remains to be the subject of current investigations. In this study, a B. pseudomallei-specific gene, pBps-1, expressing a novel 18.7 kDa recombinant protein was selected from genomic libraries of two B. pseudomallei virulent isolates by using pooled sera from septicemic melioidosis patients. Nucleotide sequence analysis demonstrated that this gene is unique and does not show substantial similarity with any known genes in the Genbank database. The Bps-1 recombinant protein was evaluated for its potential in serodiagnosis of melioidosis by Western blot analysis. A high degree of specificity was demonstrated using sera from healthy individuals in the endemic (98.5%) and non-endemic areas (100%), with moderate sensitivity (69.7%) in melioidosis patients. The study demonstrated that this approach can be used to obtain highly specific recombinant antigens such as that described in the present report. A combination of such antigens should provide materials for successful serodiagnosis of melioidosis in the endemic areas.

Evaluation of monoclonal antibody reagents from three different manufacturers using flow cytometric two-color immunophenotyping.

We evaluated a flow cytometric (FCM) two-color immunophenotyping of CD3+/CD4+ T-helper and CD3+/CD8+ T-suppressor lymphocytes in whole blood samples from HIV-infected individuals using monoclonal antibody reagents from three different manufacturers. Lymphocytes were firstly determined using CD45/CD14 in association with a forward scatter/side scatter gating strategy. CD3+/CD4+ and CD3+/CD8+ were then determined and compared. Reagents from all manufacturers showed good separation of lymphocytes, monocytes and granulocytes with high purity and recovery. There was a good correlation of the percentage of CD3+/CD4+ and CD3+/CD8+ lymphocytes amongst each of the manufacturer's reagents, but the fluorescent intensities of positive cells were not the same. This difference can result in poor discrimination of positive and negative non CD3 cells leading to erroneous results.

The reference range of CD4+ and CD8+ T-lymphocytes in healthy non-infected infants born to HIV-1 seropositive mothers; a preliminary study at Siriraj Hospital.

The results of CD4+, CD8+ T-lymphocyte values as percentage, number, and ratio were studied in infants aged 1 to 29 months. The 283 subsequent blood samples from 89 infants born to HIV-1 seropositive mothers were investigated. From 208 sequential samples of 70 healthy non-infected infants, the reference values of CD4+ and CD8+ T-lymphocytes have been established and compared to Caucasian reference values. The results were analysed in 4 difference age groups (1-5, 6-11, 12-17 and > or = 18 months). At age 12 months, CD4 number and percentage declined significantly while CD8 percent increased. At age 6 months CD4/CD8 ratio decreased. Of 19 infected infants CD4+ percentage and number as well as CD4/CD8 ratio declined at age 6 months and showed significant differences from uninfected infants. A significantly elevated CD8 percentage was demonstrated in infected infants at age of 12 months. In 9 infants who showed symptoms at age 6-18 months, the CD4 and CD8 values were different from the reference range and 6 of 9 patients showed lower CD4 percentage, CD4 number and reversed CD4/CD8 ratio before the symptoms appeared. In 10 infants who were asymptomatic at age 18 months, there was no evidence of immunosuppression at age 6 months or before. After age 6 months, lymphocyte subset values of some asymptomatic infected children were beyond the reference range. These preliminary findings should be very useful for monitoring children born to HIV infected mothers. The results of CD4+ and CD8+ T-lymphocytes in uninfected infants could be used as reference values for the Thai and other Southeast Asian pediatric populations.

Molecular cloning and expression of hepatitis C virus core protein and production of monoclonal antibodies to the recombinant protein.

The gene encoding nucleocapsid (core) protein of hepatitis C virus (HCV) was isolated from a Thai blood donor infected with HCV genotype 1b. The nucleotide sequence of this clone showed a high degree of homology to that of 4 HCV strains isolated from other Thai blood donors as well as that of the HCV prototypes of genotypes 1a, 1b, 2a and 3a. The entire region of the core gene was cloned into an expression plasmid pGEX-3X to be expressed as a fusion protein with glutathione-S-transferase (GST). E. coli transformants containing this plasmid did not express the fusion protein. However, GST-HCV core fusion protein could be produced when the core gene was truncated at the 3' end resulting in a gene encoding only the first 123 amino acid residues of the core protein. This fusion protein was insoluble in standard buffers, but could be solubilized by sarkosyl and thus subsequently purified using glutathione-Sepharose 4B. The purified fusion protein was immunogenic and could react with antibodies from blood donors infected with all genotypes of HCV found in Thailand. In addition, two murine hybridoma clones secreting monoclonal antibodies specific to the recombinant HCV core protein were produced. The purified HCV core protein and the monoclonal antibodies to the recombinant protein will be useful for developing assay systems for detecting anti-HCV antibodies and HCV antigen, respectively.

Flow cytometric three-color determination of CD4 T-lymphocytes on blood specimens from AIDS patients who have a large number of contaminating non-lymphocytes.

A three-color flow cytometric determination of CD4 T-lymphocytes on whole blood specimens from AIDS patients which contain a high proportion of non-lymphocyte elements is described. Peripheral blood cells were stained by a three-color method using monoclonal antibodies conjugated respectively with fluorescein isothiocyanate (FITC)-CD3, phycoerythrin (PE)-CD4 and peridinin chlorophyll protein (PerCP)-CD45. CD45 stains all leukocytes with the highest fluorescence expression of CD45 antigen in lymphocytes. By combining light scatter with CD45 in the fluorescence 3 (FL3) channel, a light scattering window can be drawn to include almost all bright CD45 lymphocytes. This live gate of lymphocytes was then acquired and analysed simultaneously using other irrelevant two-color (FITC/PE) antibodies of CD3 and CD4 in the FITC and PE channels, respectively. This method is easy and straightforward, and gives successful analysis of CD4 T-lymphocytes in AIDS blood specimens contaminated with an unusually large number of non-lymphocytic cells.