Comparison of Droplet Digital PCR and Real Time PCR Method for HBV DNA Quantification.

BACKGROUND: HBV infection causes a potential serious public health problem. The ability to detect HBV DNA concentration is an important issue that had been continuously improved. When using quantitative polymerase chain reaction (qPCR), several factors are of concern, for example, sources of material, standard curve calibration, and PCR efficiency. Digital PCR (dPCR) is an alternative PCR-based technique for absolute quantification using Poisson's statistics without requiring a standard curve. OBJECTIVE: Compare the data set of HBV DNA generated between dPCR and qPCR methods. MATERIAL AND METHODS: Fifty-four samples were quantified by Abbot's real time PCR and with 2-6 log10 HBV DNA were selected for comparison with dPCR. RESULTS: Of these 54 samples, there were two outlier samples defined as negative by dPCR, whereas 52 samples were positive by both of these assays. The difference between two assays was less than 0.25 log IU/mL in 24/52 samples (46%) of paired samples; less than 0.5 log IU/mL in 46/52 samples (88%) and less than 1 log in 50/52 samples (96%). The correlation coefficient (r) was 0.788 (p-value < 0.0001). Comparison with qPCR method, data generated by dPCR tend to be an overestimation in the sample with the low level ofHBVDNA concentration and underestimated in the sample with high viral load. The variation of DNA by dPCR measurement might be due to the pre-amplification procedure and PCR template. CONCLUSION: Measurement of HBV DNA by using dPCR, the results ofthe HBV DNA copy number tended to be deviated by over- or under-estimated when comparison to real time PCR method. In addition, a large quantity of DNA was used when compared to qPCR. However, the optimum processes of this assay have to be further investigated.

Lack of Association between IL-10 Gene Promoter Polymorphisms and Susceptible to Tuberculosis in Thai Patients.

BACKGROUND: Cytokines play a major role in defense against Mycobacterium tuberculosis infection. Polymorphisms in the genes encoding various cytokines have been associated with tuberculosis susceptibility. Polymorphisms of the regulatoy cytokine gene, the interleukin (IL)-10 is associated with the risk of tuberculosis (TB) in different populations. However IL-10 gene polymorphism and susceptibility to TB in Thai is still unknown. OBJECTIVE: The purpose of this study was to evaluate whether the common IL-10 promoter gene polymorphisms are associated with TB in Thai population. MATERIAL AND METHOD: Forty-eight patients with newly diagnosed pulmonary tuberculosis were studied. DNA samples were extracted from leukocytes and used to investigate -1087A/G -819C/T -252C/A (rs1800896, rs1800871, rs1800872) in IL-10 gene using restriction fragment length polymorphism (PCR-RFLP) methods. RESULTS: In this study, the genotype and allele frequencies of IL-10-1087A/G, -819C/T -252C/A polymorphism did not significantly different between TB patients and healthy controls ((genotype: p = 0.38, p = 0.92,p = 1; allele: p = 0.57, p = 0.77, p = 0.89, respectively). CONCLUSION: The lack of association between common IL-10 promoter polymorphisms and TB susceptibility in this study may provide clue for better understanding of IL-10-1087A/G -819C/T -252C/A polymorphism and TB susceptibility in Thai population, which might facilitate the rationale design of vaccines. However further studies in large scales population are required for confirmation.

CTLA-4 and its ligands on the surface of T- and B-lymphocyte subsets in chronic hepatitis B virus infection.

BACKGROUND: During chronic hepatitis B virus (CHB) infection, a number of co-stimulatory, co-inhibitory molecules and theirs ligands play a prominent role in the immune-regulation. OBJECTIVE: To compare the number of peripheral-blood mononuclear cells expressing co-inhibitory marker, cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and program cell death ligand-1 (PD-L1) between CHB infected patients and healthy controls. MATERIAL AND METHOD: Peripheral-blood mononuclear cells (PBMCs) from 19 CHB-infected patients and nine healthy controls were stained with specific combinations of the following monoclonal antibodies: CD3-PE/cy5, CD4-APC, CD8-APC, CD152-PE (CTLA-4), CD19PE/Cy5, CD80-FITC (B7-1), CD86-PE (B7-2) and CD274-FITC (B 7-H1) according to standard protocol. RESULTS: The frequencies of B-lymphocyte expressing B7-1, B7-2 and B7-H1 of CHB-infected patients and healthy controls were not shown any statistical differences. The mean percentage of B-lymphocyte with B7-2 molecule was higher than those with B7-1 molecules in both infected- and non-infected groups. In contrast, the frequencies of T-lymphocyte subsets, CD3+, CD4+ and CD8+ expressing CTLA-4 molecules in CHB-infected patients were significantly higher than those in healthy controls with p = 0.04, 0.01 and 0.04 respectively. CONCLUSION: An increase in percentage of circulating CD4+/CD152+ (T-cell) was observed in CHB-infected patients. A small but significant increase in percentage of CD8+/CD152+ T-cells raises the possibility that CTLA-4 are involved in the development of HBV-specific CD8+ T-cell exhaustion. Overall, CD4+ and CD8+ T-cells presenting CTLA-4 might contribute to the impaired immune response and likely to be a factor influencing in failure of immunological control of the persisting pathogens.

Cytokine profiles in HIV-1 subtype CRF01_AE infected individuals with different rates of diseases progression: a multiplex immunoassay.

OBJECTIVE: Cytokines play an important role in controlling the homeostasis of the immune system and contribute to the pathogenesis of HIV infection. The measurement soluble cytokines in plasma of HIV-1 infected individuals with different rates of disease progression may provide additional information to complement prognostic markers and understand disease process. The aim of the present study was to determine the cytokine profiles in plasma of Thai HIV-1 CRFO1_AE infected individuals with different rates of disease progression by using a multiplex system for simultaneous detection of 7 cytokines. MATERIAL AND METHOD: The authors used a multiplex immunoassay method to measure 7 cytokines (IL-2, IL-4, IL-6, IL-7, IL-10, IL-15 and IFN-gamma) in plasma of 23 progressors (PRs; symptomatic or AIDS within 5 years and CD4+ < 200/mm3), 23 slower progressors (SPs; asymptomatic more than 5 years and CD4+ > 350/mm3) and 23 normal healthy individuals. RESULTS: Both PRs and SPs demonstrated significantly higher levels of IL-7, IL-10 and IFN-gamma than healthy controls (p < 0.05). No significant difference in IL-6 between SPs and healthy controls but significant difference between RPs and controls were found. Furthermore, PRs showed significantly higher levels of plasma IL-6 (p = 0.001), IL-7 (p = 0.016), IL-10 (p < 0.001) and IFN-gamma (p = 0.026) than SPs. No significant difference in IL-2, IL-4 and IL-15 was found among 3 groups (PRs, SPs and healthy control). CONCLUSION: These results suggested that a Th1 to Th2 cytokine switch did not occur. However, the measurements of plasma levels of cytokines could be used for predicting disease progression.

ZAP-70 positive cells in treated and untreated HIV-1 infected patients.

ZAP-70 is a critical protein tyrosine kinase in T-cell activation and proliferation processes. Defective recruitment of ZAP-70 molecules results in termination of the T-cell receptor (TCR) signal transduction pathway. Impairment of this pathway is one of the early markers of disease progression in HIV-1 infected individuals. T-cell dysfunction in HIV infected patients may be connected to a defect in the proximal TCR signaling cascade. To evaluate this presumption, the numbers and mean fluorescence intensity (MFI) of ZAP-70 positive cells in patients with treated and untreated HIV-1 infection and healthy controls were analyzed by flow cytometry. A correlation between the MFI in ZAP 70 molecules and the viral load was evaluated. A total of 41 HIV-1 infected patients, 30 patients on HAART and 11 untreated patients, and 11 healthy controls were enrolled. The data show ZAP-70+/CD4+ cells in treated and untreated HIV-1 infected individuals had a greater MFI of ZAP-70 molecules than those from healthy controls (p < 0.001). The inverse correlation between the percentage of CD4+cells and the MFI of ZAP-70+/CD4+ T-cells was significant (r = -0.5; p < 0.01). A stronger correlation between the percentage of CD4+/CD25+ cells and the MFI of ZAP-70+/CD4+ cells was observed (r =-0.6; p < 0.01). However, no significant correlation was seen between the MFI of the ZAP-70+/CD4+cells and the viral load in patients with untreated HIV-1 infection (r = -0.4, p = 0.16). For HIV-1 treated patients, the viral loads were too low to detect so it was not possible to calculate the correlation. Elevated MFI levels of ZAP-70 molecules in CD4+ cells in HIV infected patients may be associated with an inability to further activate T-cells.

A whole blood lymphocyte proliferation assay in healthy Thais: comparison of heparinized blood and acid citrate dextrose blood.

The lymphocyte proliferation assay (LPA) is a technique to determine T-lymphocyte functions in vitro. The standard LPA using peripheral blood mononuclear cells (PBMC) separated from heparinized blood requires a large blood sample, time consuming and expensive. It is more useful if acid citrate dextrose (ACD) blood could be used not only for LPA but also for other purposes. To determine whether whole blood composing between heparinized blood and ACD blood could be substituted for standard LPA using PBMC. Heparinized and ACD blood of 35 healthy Thai blood donors were studied herein. PBMC separated by density gradient centrifugation and diluted heparinized and ACD blood were used to test and compare for lymphoproliferative responses to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and tetanus toxoid. A stimulation index (SI) for each mitogen or antigen was calculated. All Thai blood donors demonstrated positive proliferative responses to PHA and PWM by using PBMC and whole blood culture assays from both heparinized and ACD blood. However, the difference in the frequency of positive proliferative responses to tetanus toxoid by using PBMC and whole blood culture assays was significant. Nevertheless, no significant difference in frequency of positive responses to tetanus toxoid between heparinized and ACD blood was observed. This results suggested that no significant difference between using heparinized and ACD blood in standard LPA using PBMC. However, the whole blood LPA for measuring mitogen induced lymphoproliferation could be substituted for standard LPA from heparinized andACD blood. Whole blood LPA is easy, rapid, and more cost effective than PBMC culture assay. Thus, it would be applicable in a clinical laboratory as well as in research setting.

Structure and function of HIV-1 CRF01_AE envelope proteins from blood and genital fluid isolates.

The recombinant envelope protein (gp120) of the human immunodeficiency virus type 1 (HIV-1) CRF01_AE env gene isolated from the corresponding blood (rgp120-F36PC) and genital fluid (rgp120-F36VC) specimens obtained from HIV infected individuals was successfully produced in both prokaryote and eukaryote cells. The yields of HIV-1 recombinant envelope proteins rgp120-F36PC and rgp120-F36VC produced in E. coli and in mammalian cells were 1.0 and 1.2, and 0.3 and 0.5 mg/ml, respectively. Antibody responses in mice immunized with rgp120-F36VC protein were not significantly higher than those with rgp120-F36PC protein. The level of antibody response in mice immunized with V3 deleted recombinant gp120 proteins from rgp120-F36VC and rgp120-F36PC was not significantly different from wild type rgp120 proteins. beta-strands at the tip of the V3 loop of the HIV-1 envelope protein were predicted for the wild type genital fluid isolate but not for the wild type blood isolate. The replication capacity of both F36PC and F36VC was quite efficient. The infectivity assay of the epithelial cell line for pNL4-3/gp120F36VC was better than for pNL4-3/gp120F36PC. The extra beta-strands in the V3 loop may be involved in cell tropism.

The prevalence of anti-hepatitis E in occupational risk groups.

The seroprevalence of anti-hepatitis E virus (HEV) IgG was investigated by using ELISA commercial anti HEV test kit in 408 healthy adults who lived in central part of Thailand, 168 of which were swine workers, 102 were poultry farmers and 138 were government officers. The overall rate of seroprevalence of IgG anti-HEV was 23.3 % (range 16.7-27.9%). The prevalence of anti-HEV antibodies in government officers was 16.7 % and in subjects from swine workers and poultry farmers who worked in farms for more than 2 years were 27.9 % and 24.5%, respectively. Although there was no difference in anti-HEV prevalence according to three job categories (p = 0.06) and to age groups (p = 0.4), but seroreactivity of anti-HEV in swine and poultry farmers were statistically significantly higher than those in officers (p < 0.01). From this preliminary study, HEV is supposed to be circulating in the central area of Thailand. It appeared that the probability of exposure and reinfection to HEV are higher in farmers than that in government officers. Poor environmental conditions in farms, occupation and low socioeconomic status might be risk factors in HEV infection.

Cytokine production in NK and NKT cells from Mycobacterium tuberculosis infected patients.

Tuberculosis, a major health problem in developing countries, has re-emerged in recent years in many countries. While it is accepted that various lymphocyte subsets are important responses to mycobacterial infection, the roles of NK and NKT cells in producing cytokines are still unclear. Thus we have evaluated, in Mycobacterium tuberculosis infection, the frequency of cytokine producing cells by flow cytometry. Of 30 individuals examined, 17 had clinical evidence of pulmonary tuberculosis while the rest showed no evidence of infection. Patients had a significantly higher number of IFN-gamma and IL-4-producing T cells compared to control subjects, but the ratio of IFN-gamma to IL-4-producing T cells was similar in both groups. There were no differences between cytokine profiles of NK cells in patients and control subjects. A significant increase in the number of NKT cells was observed in patients. A striking finding was the higher frequency of IL-4-producing NKT cells compared to IFN-gamma-producing cells. Moreover, individual NKT cell produced both IFN-gamma and IL-4. The preferential type of Thl or Th2 cells is due to mycobacterial strain, type of antigen presenting cells and stage of disease, all of which can lead to different patterns of cytokine production by variety of lymphocyte subsets.

Binding antibody to neutralizing epitope gp41 in HIV-1 subtype CRF 01_AE infection related to stage of disease.

The responsiveness of gp41 antibody against epitope ELDKWA in HIV-1 infected subjects is of importance in neutralizing viral infectivity and for being related to disease progression. In this study, antibody titers to this neutralizing epitope from HIV-1 infected subjects at asymptomatic and AIDS stages in Thailand were investigated by peptide ELISA. The results showed that the frequency of antibody production against this neutralizing epitope was low (15-35%). Moreover, antibody titers to this epitope in sera from AIDS patients were significantly lower than those in sera from asymptomatic subjects which were collected in the same year (p=0.001). Comparison between the past (1992-1994) and present (2002) sera from asymptomatic infected individuals revealed that the earlier panel contained lower antibody titers than the later panel did (p = 0.05). In addition, random sera for HIV-1 infected subjects who were infected by diverse genetic subtypes, (A through G) including CRF 01_AE, had low titers of antibody to this region as well. It is assumed that antibody production to this epitope is low and related to the stage of HIV-1 infection.

Distribution of CD38 molecules on CD3+ and CD8+ T- lymphocyte in adulthood HIV-1-uninfected Thais.

The expression of CD38 on CD8+ T-lymphocyte is a significant predictive value in disease progression of HIV infected individuals and in monitoring a response to therapy. CD38 molecules expressing on CD3+ and CD8+ T-cells were measured quantitatively by flow cytometry in 30 healthy Thai adults. In each experiment, the known amount of fluorochrome in CD38 antibodies bound per cell of QuantiBRITE PE beads was plotted, and set a regression line. With this line, the amount of CD38 molecules bound to CD3 and CD8 target cells was estimated. The aim of this study was to determine the reference value of CD38 molecules on CD8+ T-lymphocyte, which is the baseline in comparison to the CD38 molecule expressing on CD8+ T-lymphocyte in HIV-infected individuals. The present results showed that the amount of CD38 expressions on CD8+ T-lymphocyte in HIV negative Thai adults was about 2 times higher than those from Caucasian's lymphocyte. The reference range of CD38 molecules in the present study would best be used as baseline in prognosis and drug monitoring of HIV-1 infection in Thailand.

Conserved neutralizing epitopes of HIV type 1 CRF01_AE against primary isolates in long-term nonprogressors.

Linear conserved B cell epitopes in envelope glycoprotein of long-term nonprogressors (LTNPs) HIV-1 CRF01_AE were determined. The envelope sequences of HIV-1 subtype E from Thailand were aligned to define consensus sequences. Then the peptides corresponding to these predicted regions were synthesized as peptides represent C1, C2, C3, C5, V2, V3, and gp41 regions. After that, the neutralizing B cell epitopes were determined by neutralized competitive assay with pool sera of typical progressor and LTNP HIV-1 CRF01_AE patients against HIV-1 CRF01_AE 24 primary isolates (PI) and laboratory strains (TCLA). We found that the strength and breadth of neutralization were greater for sera from LTNPs compared with sera from typical progressors. Peptides C1E and C2E could inhibit primary isolates but not the TCLA strain in LTNP sera. The new B cell epitopes, which were located in the C1 and C2 regions of CRF01_AE against primary HIV-1 isolates, were identified in HIV-1 CRF01_AE LTNPs. This may be important in HIV-1 vaccine development and trial.