Detection of Burkholria pseudomallei DNA in whole blood samples of bacterial septicemia patients using conventional PCR and real-time PCR

Burkholderia pseudomallei is a bacterium that causes a disease known as melioidosis. Infections of B. pseudomallei appear in several organs including acute septicemia which showed high mortality rate. The rapid and high efficient diagnostic test may reduce the mortality rate of melioidosis patients. We performed conventional PCR using LPS primers and real-time PCR using 16S rDNA primers for detection of B. pseudomallei in clinical blood specimens. Bacterial culture was used as a gold standard. Blood specimens from 32 suspected bacterial septicemia patients were obtained from admitted patients in Khon Kaen Hospital and other hospitals in the region. These consist of the patients with 19 B. pseudomallei and 13 other bacterial infections including 1 Burkholderia cepacia, 1 Escherichia coli, 4 Klebsiella pneumoniae, 1 Enterobacter species, 3 Staphylococcus aureus, 1 Streptococcus pneumoniae and 2 group A Streptococcus. The sensitivity, specificity, positive and negative predictive values were determined. Conventional PCR showed low sensitivity of 37 % (95% CI:15-59) and high specificity of 92 % (95% CI:78-100) with 88 % (95% CI: 65-100) and 50 % (95% CI:30-70) of positive predictive value (PPV) and negative predictive value (NPV), respectively. In contrast, a real-time PCR and using of combination test showed higher sensitivity (63 %, 95% CI: 41-85) and lower specificity (69 %, 95% CI: 44-94) with 75 % (95% CI:54-96)  and 56 % (95% CI:32-81) of PPV and NPV, respectively. In 5 melioidosis patients who have died, both PCR methods showed rising of sensitivity [80 % (95 % CI: 45-100) and 100 % (95% CI: 100-100)], respectively. The lower detection limit of B. pseudomallei by conventional PCR was 103 cfu/ml. The conventional PCR and real-time PCR could detect B. pseudomallei DNA of 10 pg and 50 fg per PCR reaction, respectively. The higher sensitivity of real-time PCR may be useful for early screening test, whereas the conventional PCR with higher specificity and PPV may be used as a confirmatory test for diagnosis of B. pseudomallei. Therefore, the two assays may be used in combination as rapid molecular diagnostic for melioidosis which might lead to a reduction in the mortality rate of melioidosis patients. 

The alteration of MHC class I expression in cervical cancer

It was well established that HPV infection, especially type 16 and 18 is the major cause of cervical cancer. Immune evasion of infected cells or tumor cells is also known as one of contributing factors of tumor development caused by down-regulation of MHC class I expression. The aim of this study is to investigate the level and pattern of MHC class I expression in 182 cervical cancerous tissues and 57 normal cervix using immunoperoxidase staining technique. Of 182 cervical cancerous tissues, 50 cases were classified as pre-cancerous lesions whereas, 132 cases were invasive cervical carcinoma. No significant association in grading or intensity of MHC class I was shown between patient and control group. Interestingly, the distribution pattern of MHC class I was significantly shown as non-surface presenting molecules accumulating in cytoplasm of cancerous tissues compared to normal cervix tissues with p-value \< 0.001 χ² test .This result indicates that tumor cells have lost their immune surveillance by reducing their antigen presenting MHC class I molecules at cell surface. Moreover, this alteration can be used as an early marker for cancer prognosis in pre-cancerous group. Therefore, the mechanism of the impairment in antigen presenting of MHC class I molecules should be investigated in further study.

Tapasin expression in cervical carcinoma

Cervical cancer is the most common cancer among women worldwide. Even though HPV infection is known as the major risk factor of cervical carcinogenesis, immune evasion of infected cells or tumor cells is also well established as the major contributing factors. Down regulation of MHC class I as well as the presence of non-surface presenting MHC class I molecules have been previously reported. Our aim of this study is to investigate the expression level of tapasin protein which is one of the MHC class I antigen processing proteins in various stages of cancerous tissues. Seventy nine dysplasia and 49 cases of cervical cancer and 49 normal cervix from myoma patients as control were included in this study. The association between the expression level and disease progression was analyzed using χ2 for trend. The reduction of tapasin expression is significantly associated with respect to the progression of staging in both dysplasia and cervical cancer group (p=0.001; χ2 for trend). In conclusion, our study illustrates that the reduction in tapasin expression might exert as one of the mechanism leading to the impairment of antigen presenting of MHC class I in cancerous tissues. Therefore, tapasin staining might be useful as an early prognostic marker in dysplasia. More antigen processing proteins should be also further investigated.

Prevalence of hepatitis virus infections in the first donating blood donors at the Regional YALA Hospital during 2001-2005

The screening tests for donor blood samples in blood bank are important to prevent the recipients from blood transmitted diseases. The common hepatitis viruses that are transmitted through blood transfusion are hepatitis B and hepatitis C viruses. The objective of this study is to investigate the prevalence of hepatitis B and C virus infections in the first donating blood donor in the YALA province from January 2001 to December 2005 by retrospective analysis of data from laboratory record of the YALA regional hospital. The total number of the first time blood donors was 11,611. Amongst these, hepatitis B virus infected donors were 310 (2.67%) consisting of 262 males (2.78%) and 48 females (2.17%). The prevalence of hepatitis C virus infected donors was 183 (1.58%) with 164 males (1.74%) and 19 females (0.86%). Hepatitis B virus infections were mostly found in age ranging of 36-45 years old (2.92%) where as hepatitis C virus infections were mostly found in 26-35 years of age (2.21%). However, there were no statistically significant associations between the prevalences with the age groups and sexes of people who were infected with hepatitis B viruses but statistically significant associations between the prevalences with the age groups and sexes were found in the hepatitis C virus infections (P=0.003; chi-square test). These data form basic information on the prevalences of viral hepatitis infections in the YALA province would be useful for strategic planning in donor recruitment and blood transfusion.

Development of method for integrated HPV16 detection in cervical carcinoma using multiplex quantitative PCR

Infection with high-risk HPV has been implicated as one of the major risk factors of cervical cancer. Integration of viral DNA into host DNA is essential for cervical carcinogenesis. This study was aimed to develop a multiplex real-time PCR to quantify Early gene 2 (E2 gene) and Early gene 6 (E6 gene) of HPV16 using Taqman probe. The ratio of E2 and E6 copy number was calculated to determine physical status of HPV16. The pure episomal form was expected to have an equivalent copy number of E2 and E6 gene giving rise to E2/E6 ratio of 1 whereas, viral integration resulted in less E2 than E6. The detection limit as well as precision of this developed method were obtained at 103 copies with CV of less than 10%. Cut off value of E2/E6 ratio for complete episomal form was found more than 0.83, whereas complete integration was expected to 0. This method was further analyzed with DNA from 15 pre-invasive or dysplasia lesions and 15 invasive cervical carcinoma tissues. The percentage of total integration form in invasive cases showed significantly higher than pre-invasive cervical lesions which obtained about 93%(14/15) and 66%(10/15), respectively (p value \< 0.05). The method described here is sensitive to assess the physical state as well as viral copy numbers, which suggests as a potential marker for disease progression. Furthermore, followed-up cases should be studied in large scale sample sizes in order to evaluate its potential as prognostic marker in cervical cancer.

Expression pattern of p63 and p73 isoforms in cervical carcinoma

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Detection of HLA-B*27 un Thais, Burmese and Karens

Human Leukocyte Antigen (HLA) is an antigen system found on surface of white blood cells and body tissues. Their functions are related to immune response to recognize and eliminate foreign antigens. Many diseases are associated with HLA alleles, especially HLA-B*27 which is strongly associated with ankylosing spondylitis (AS). In this study, the high resolution PCR-SSP has been developed to detect HLA-B*27 by 2 primer mixtures (Sc1 and Sc2). The HLA-B*27 group specific primers have been tested in 846 unrelated healthy northeastern Thais (NET), 338 northern Thais (NT), 271 Karens and 308 Burmese. Sixty-three NET (7.4 %), 24 NT (7.1 %), 5 Karens (1.8 %), and 12 Burmese (3.9 %) were positive for HLA-B*27. This study established a simple technology for HLA-B*27 testing and provided basic information for assessing the risk of AS and further study in disease associations.