Jintana Pradutkanchana

Comparative evaluation of four serodiagnostic tests for scrub typhus in Thailand

The commercial dot-blot enzyme-linked immunosorbent assay Dip-S-Ticks dipstick test was compared with the indirect immunoperoxidase (IIP) and Weil-Felix (WF) tests for the diagnosis of scrub typhus, using the indirect immunofluorescent antibody test (IFA) as the reference standard. With a panel of 117 positive and 75 negative sera, the dipstick test was 94% sensitive and 98.7% specific at a cut-off value of one or more positive dots. The IIP was 90.6% sensitive and 100% specific at a cut-off titre of 1:400, and was more sensitive than the IFA with acute sera (79.6% vs. 68.5% at a titre > or = 1:400). All 3 were superior to the WF, which lacked sensitivity. The dipstick assay was easy to perform and did not require sophisticated electrical equipment, and the results were available within one hour. It is therefore suitable for use in rural Thailand, where scrub typhus is common.

High tumor necrosis factor-α levels in the patients with Epstein–Barr virus-associated peripheral T-cell proliferative disease/lymphoma

We have attempted to find out any relationships between circulating tumor necrosis factor (TNF)-alpha levels and Epstein-Barr virus (EBV) associated peripheral T-cell and NK-cell proliferative disease/lymphoma (PTPD/L) status. The distribution of TNF-alpha level was significantly higher (P<0.05) in patients than in controls. Patients carrying EBV genome in their peripheral T-cells showed higher TNF-alpha levels than the patients with EBV negative peripheral T-cells (P<0.001). Among patients whose peripheral T-cells were positive for EBV genome, TNF-alpha levels between the wild type LMP-1 gene carriers and the 30-bp deletion type LMP-1 gene carriers were compared and the wild type LMP-1 gene carrier group showed significantly higher TNF-alpha levels (P<0.01). As for the outcome of the patients and TNF-alpha levels, significant differences were observed between dead and alive with disease group (P<0.001), and dead and alive with complete remission group (P<0.01). Since circulating TNF-alpha levels in PTPD/L patients correlate with the disease and EBV infection status, it may be possible that monitoring of the TNF-alpha levels will be a useful prognostic marker.

Randomized study of antiinflammatory and immune-modulatory effects of enteral immunonutrition during concurrent chemoradiotherapy for esophageal cancer.

Concurrent chemoradiotherapy (CCRT) induces toxicities from inflammation and immunological suppression. Omega-3 fatty acids, glutamine, and arginine are therapeutic factors that can attenuate such inflammation and promote cellular immunity. The question is whether immunonutrition (IN) during CCRT reduces inflammation and improves the immune function in patients with esophageal squamous cell carcinoma (ESCC). Seventy-one locally advanced ESCC patients being treated with CCRT (5-FU and cisplatin) were randomized into 2 groups. The IN group received a combination of omega-3 fatty acids, glutamine, and arginine, whereas the control group received standard formula. The levels of C-reactive protein (CRP), tumor necrosis factor (TNF), interferon-gamma (IFN), interleukin (IL-6, IL-10), CD3, CD4, CD8, white blood cells, neutrophils, and total lymphocytes were measured before and during treatment. The levels of CRP (P = 0.001) and TNF (P = 0.014) increased more during treatment in the control group than the treatment group, whereas IFN, IL-6, and IL-10 were similar but not significantly. CD3, CD4, CD8, white blood cells, neutrophils, and total lymphocytes decreased more in the control group than in the treatment group, but not significantly. Enteral IN during CCRT reduced the increase of inflammatory cytokine levels.

Hepatic cytotoxic T-cell infiltrates in patients with peripheral T-cell proliferative diseases/lymphomas: Clinicopathological and molecular analysis

Seventy patients with various types of peripheral T‐cell proliferative disease/lymphoma who manifested with prolonged fever, weight loss, anemia, lymphadenopathy, hepatosplenomegaly and elevated serum levels of alkaline phosphatase and/or lactate dehydrogenase were evaluated. Histopathological examination of the livers revealed T‐cell infiltration into the hepatic sinusoids and portal tracts. The morphology of the infiltrated T cells varied from mature small lymphocytes to malignant lymphoid cells. The liver pathology was classified into four groups on the basis of cellular atypia. Group A and group B showed mature lymphoid cell infiltration; however, only group B had multiple large areas of hepatocellular necrosis. Group C showed atypical lymphoid cell infiltration and in group D malignant lymphoid cell infiltrates were demonstrated. The majority of the antigenic phenotypes of these T‐cell infiltrates were CD3+, CD4–, CD8+, CD20–, CD45RO+, CD56–, CD57–, TIA‐1+ and βF1–. Epstein–Barr virus RNA in the nuclei of the infiltrated T cells was recorded in 38.6% of the patients and was more common in groups C and D. Patients in groups B, C and D had a very poor prognosis, median survival was only 1 month, whereas median survival in group A patients was 36 months. Chemotherapy was not effective in improving survival.  Monoclonal  band/s  of  T‐cell  receptors  (TCR) β and/or γ gene rearrangements were detected in 88.6% of patients, and DNA‐sequence analysis showed high identity to the human TCR germline gene.

Direct-STR typing from presumptively-tested and untreated body fluids

Body fluids provide key pieces of information for a forensic investigation. However, sometimes only a small amount of body fluids is found and/or DNA are also degraded by environmental factors at the crime scene. In extreme cases, a forensic analyst may have to decide whether to perform a presumptive test on the stains or proceed straightaway to DNA profiling, which could be wasteful for non-biological stains. Additionally, due to the inefficient DNA extraction process, the amount of DNA may not be enough for STR typing, especially if parts of the evidence had been subjected to presumptive testing. To overcome these problems, we developed a direct PCR method for STR profiling of stains (blood, saliva, and semen) that had been subjected to presumptive tests and also those that had not undergone presumptive tests. Using the optimized protocols, 86 of 90 untreated samples (95.6%) resulted in a full DNA profile. For presumptively-tested samples, both the type of presumptive test used and the surfaces where the stains are deposited affected the quality of the STR profiles. With blood, we obtained full STR profiles from 88% of samples tested with luminol and 78% with Hemastix. The acid phosphatase test for semen and Phadebas test for saliva resulted in full STR profiles from 85% and 73% of samples, respectively. Different substrates also affected the resulting STR profiles, but there was no clear trend based on absorbency or texture. The interactions of types of body fluids, presumptive tests, and substrates must be considered together. Our direct PCR protocol can be used to detect DNA even with 6 months-old biological samples. The benefits of the developed protocol include increasing amount of DNA obtained from evidence, decreasing chances of DNA contamination from complex or lengthy extraction steps, using minimal sample amount for analysis, and most importantly, improving STR profiles. Also, the process could save analysis time and cost due to the omission of DNA extraction and quantification. Our developed method could be beneficial to cases with limited stains available, as forensic analysts can perform indirect presumptive testing on the suspected stains and direct PCR could be carried out from the filter paper used, thus leaving the original stain for subsequent DNA extraction or re-analysis.

Lytic replication of Epstein-Barr virus in human peripheral T-lymphocytes

Abstract Background: There are few reports about the interactions of EBV with peripheral T-cells, especially during the early phase of infection. Objective: Demonstrate the capability of EBV to infect and replicate in human peripheral T-cells in vitro. Methods: After treating with EBV, the susceptibility of in vitro EBV infection into T-cells was confirmed using electron microscopy, the expression of EBV mRNA using RT-PCR, and the expression of EBV proteins using Western blot analysis. The expression of CD19 and CD21 mRNA was determined using RT-PCR. The induction of cell death was measured using trypan blue exclusion assay. Results: The susceptibility of in vitro EBV infection was confirmed by the presence of virus particles in the cytoplasm. The entering to lytic infection was confirmed by detection the expression of EBV lytic (BZLF1) mRNA, and the expression of late lytic proteins (VCA and gp350/220). The expression of CD19 and CD21 were not observed using RT-PCR. The interactions of EBV with T-cells leaded to induction of T-cell death. Conclusion: Peripheral T-cells are a direct target of EBV infection. At the beginning of infection by EBV, EBV infection of T-cells leads to the entering into lytic virus replication. EBV binds to these cells through a receptor distinct from the CD21.

Cytokine induction after Epstein-Barr virus infection of peripheral T-lymphocytes in vitro

Abstract Background: Although the presence of Epstein-Barr virus (EBV) in different T-cell malignancies has been widely reported, there is very few data available for EBV infection of normal T cells. This leads to the lack of knowledge on the early events after T cell infection. Objective: Investigate the early events occurring after normal human peripheral T-cells are infected with EBV in vitro. Methods: T-cells were treated with EBV in vitro. The expression of tumor necrosis factor- α (TNF-α) mRNA were determined using reverse-transcription (RT)-PCR, and the level of TNF-α and interferon- γ (IFN-γ) in the culture supernatant were measured using ELISA. The effect of virus inactivation on cytokine induction from T-cells was also determined. Results: At the beginning of T cell infection by EBV, the expression of several lytic EBV transcripts (BALF5, BcLF1, and BLLF1) were observed using RT-PCR. This indicated the susceptibility of in vitro EBV infection and the entering lytic cycle of EBV-infected T-cells. The interactions of EBV with T-cells lead to induction of inflammatory cytokines, tumour necrosis factor- α (TNF-α) and interferon- γ (IFN-γ), production from the T-cells. Inactivation of the virus by UV irradiation eliminated the TNF-α and IFN-γ induction by EBV, suggesting the involvement in the expression of viral gene(s). Conclusion: This in vitro analysis demonstrated the cytokine induction by EBV after primary infection of T-cells.