Evaluation of direct immunofluorescence test for diagnosis of upper respiratory tract infection by Chlamydia pneumoniae.

BACKGROUND: Chlamydia pneumoniae causes a variety of respiratory infections and is involved in cardiovascular diseases. Diagnosis of C. pneumoniae infection currently relies on antibody detection by microimmunofluorescence (MIF), which has limited use, and is the retrospective diagnosis for acute infection. OBJECTIVE: Find an effective early diagnosis of acute upper respiratory infection, or use in combination with MIF to accurately diagnose the infection by C. pneumoniae. MATERIAL AND METHOD: Direct immunofluorescence (DIF) was developed to detect C. pneumoniae in nasopharyngeal specimens obtained from patients with upper respiratory tract infection, and normal individuals. IgM and IgG antibodies against C. pneumoniae by MIF were determined for evaluation of the detected C. pneumoniae and seroconversion. RESULTS: DIF gave positive results in 29 of 37 (78.4%) samples from 31 patients. Fifteen samples positive by DIF illustrated antibody titers interpreted as acute C. pneumoniae infection, and eight DIF positive samples showed antibody titers of chronic infection. Negative results by both DIF and MIF were found in two patients and 23 of 25 by DIF but 20 of 25 by MIF in normal subjects. Five paired sera subsequently collected from three of the 31 patients illustrated seroconversion 2-4 months after the primary specimen collection, which gave positive results by DIF but negative for antibodies. Significant association was found between C. pneumoniae detection by DIF and antibodies by MIF when analysis was done in the group of patients and normal subjects (p < 0.001; Pearson chi-square test). CONCLUSION: DIF could be an alternative assay for early diagnosis of C. pneumoniae infection, and may be used in combination with MIF for accurate diagnosis of acute C. pneumoniae infection.

Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr virus (EBV) is documented as the important etiologic agent of nasopharyngeal carcinoma (NPC) but the mechanism of development and pathogenesis induced by EBV is presently unclear. Hypermethylation of epithelial-cadherin (E-cadherin) promoter has been shown to be induced in NPC cell line by EBV LMP1 via DNA methyltransferase activation. EBV genomes and hypermethylation of E-cadherin promoter were investigated in NPC tissues to evaluate the role of EBV in the hypermethylation and pathogenesis of NPC. METHODS: Methylation-specific polymerase chain reaction (MSP) was performed to detect E-cadherin promoter hypermethylation in paraffin embedded tissues from patients with NPC and normal nasopharyngeal tissues. EBV genomes were detected by PCR in the tissue samples. RESULTS: Hypermethylation of E-cadherin promoter and EBV were predominantly detected in undifferentiated and non-keratinizing NPC compared to those in squamous cell NPC. Hypermethylation of E-cadherin was found in 28 of 38 (73.7%) patient samples. EBV was detected in 22 of the 28 (78.6%) NPC samples demonstrating E-cadherin hypermethylation. EBV genomes and hypermethylation were not detected in normal nasopharyngeal tissues. Significant association was found between E-cadherin hypermethylation and EBV genomes (p<0.001; Fisher's exact test). Hypermethylation of E-cadherin was more frequently detected in advanced stages compared to those in early stages of NPC (p=0.036; Fisher's exact test). CONCLUSIONS: The high incidence of EBV with the consistency of E-cadherin hypermethylation, particularly in undifferentiated and non-keratinizing NPC suggests the role of EBV in the hypermethylation. EBV exists at early stage of NPC that induces the hypermethylation and contributes to progression of the disease to the advanced stage of NPC.

Development and evaluation of an in-house enzyme-linked immunosorbent assay for early diagnosis and monitoring of human pythiosis.

Human pythiosis is an emerging, fatal, infectious disease caused by Pythium insidiosum and occurs in both tropical and subtropical countries. Thalassemic patients, farmers, and aquatic-habitat residents are predisposed to this disease. Delayed treatment due to the long time required for isolation and identification of the causative organism, as well as the difficulty in obtaining internal organ specimens, results in high morbidity and mortality. To facilitate rapid diagnosis, an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against P. insidiosum was developed and evaluated for the diagnosis and monitoring of human pythiosis. Sixteen sera were collected from seven culture-proven human pythiosis cases. A total of 142 sera from thalassemic patients, from patients with other infectious diseases, and from healthy blood donors served as controls. All sera were tested in duplicate. By choosing a suitable cutoff point to maximize sensitivity and specificity, sera from pythiosis cases were all determined to be positive, whereas sera from control groups were all determined to be negative. ELISA signals from serial samples of sera taken from treated patients showed gradually declining levels of antibodies to P. insidiosum. The ELISA test was highly sensitive (100%) and specific (100%) and was useful for early diagnosis and for monitoring the treatment for pythiosis.