The prevalence of dengue virus in Brunei Darussalam during January-November 2010.

The aim of this study was to retrospectively determine the prevalence of dengue virus from April 2010 to November 2010 in Brunei Darussalam. A total of 250 serum samples from dengue diagnosed patients were examined. All serum samples were tested for dengue IgM and IgG antibodies and dengue NS1 antigen using the PanBio dengue ELISA commercial kit. To determine the prevalence of dengue virus serotype in the country, serotyping was performed for the 14 samples that were positive for NS1 antigen. Dengue virus serotyping was carried out using the conventional reverse transcriptase-polymerase chain reaction (RT-PCR). Of the 250 serum samples included in the study, 196 were laboratory dengue confirmed cases. Dengue virus serotype 1 (DENV-1) was the predominant circulating serotype, followed by DENV-3 and DENV-2. This is the first report of DENV-3 isolation in Brunei Darussalam.

Methicillin-resistant Staphylococcus aureus colonization among healthcare workers in Oman.

INTRODUCTION: Methicillin-Resistant Staphylococcus aureus (MRSA) is a S. aureus strain characterized by resistance to cloxacillin. Healthcare workers (HCWs), are recognized for their heightened risk for MRSA acquisition and possibly for MRSA nosocomial transmission. This cross-sectional study aimed to determine the prevalence and the associated risk factors of MRSA colonization among healthcare workers at Sultan Qaboos University Hospital (SQUH) in Oman. METHODOLOGY: A total of 200 nasal swab samples were collected from the healthcare workers at SQUH during the period October 2nd 2018 to January 7th 2019. All nasal swab samples were examined microbiologically for the presence of MRSA using the standard method and the results were confirmed by detection of the mecA product (PBP2a). Data on associated risk factors for MRSA colonization was collected and analyzed. RESULTS: Forty-one of the 200 screened healthcare workers (20.5%) were found to have nasal carriage of Staphylococcus aureus of which 63.4% were Methicillin Sensitive and 36.6% were Methicillin-Resistant (MRSA). Methicillin-Resistant Staphylococcus aureus (MRSA) was isolated from fifteen of the 200 screened healthcare workers giving a prevalence rate of nasal colonization with MRSA of 7.5%. We found no statistical association between healthcare worker MRSA nasal colonization and age, gender, HCWs specialty, hand hygiene practices, skin condition, previous MRSA infection, and previous exposure to antibiotics. CONCLUSIONS: Identification of the prevalence and the associated risk factors of MRSA colonization in healthcare workers mandates continuous surveillance and the implementation of all possible preventive measures to reduce re-occurrences.

Investigation of the effect of UV irradiation on DNA damage: comparison between skin cancer patients and normal volunteers.

BACKGROUND: Susceptibility to environmental carcinogenesis is the consequence of a complex interplay between intrinsic hereditary factors and actual exposure to potential carcinogenic agents. Exposure to sunlight is the primary etiological agent for basal cell carcinoma (BCC). AIM: The aim of this study was to determine the effects of different ultraviolet (UV) doses on DNA damage in epidermal keratinocytes in vivo and to elucidate if patients with BCC are more susceptible to UV-induced DNA damage in comparison with normal healthy volunteers in response to solar simulator radiation (SSR). MATERIALS AND METHODS: Skin biopsies obtained post-UV irradiation from both normal healthy volunteers and BCC patients were analyzed for DNA damage, using immunohistochemical approach with TDM-2 antibody, which binds specifically to cyclobutane pyrimidine dimmers (CPDs). RESULTS: In both normal volunteers and BCC patients, the peak of CPD-positive cells occurred at 4.5 h post-SSR. There was a statistically significant difference in CPD positivity between BCC patients and normal volunteers, at time points (from 4.5 h to 48 h post-SSR). For a given dose of SSR based on each individual minimal erythema dose (MED), a greater number of CPD-positive cells could be shown. CONCLUSIONS: This study has shown for the first time and in vivo in human volunteers that BCC patients are more susceptible to UV-induced DNA damage in comparison with normal healthy volunteers.

Immunohistochemical determination of the P15 protein expression in cutaneous squamous cell carcinoma.

The tumor suppressor gene p15(INK4b) is a cyclin-dependent kinase inhibitor, in which its inactivation has been determined in primary tumors and in several tumor-derived cell lines. The precise role of p15(INK4b) protein expression in cutaneous squamous cell carcinoma (SCC) is currently not known. In a previous study, we have shown the frequent occurrence of allelic imbalance/loss of heterozygosity in cutaneous SCC using two microsatellite markers flanking the p15(INK4b) gene. This study is a continuation of our previous study and aims to determine the possible role of p15(INK4b) protein expression in the genesis of cutaneous SCC. P15(INK4b) protein expression was determined using immunohistochemical approach in 107 cases of cutaneous SCC tissue arrays and 19 cases of normal human skin tissues. The expression of p15(INK4b) was significantly reduced in the cutaneous SCC cases as compared with normal human skin (p = 0.017 and p < 0.05). However, there were no significant relationship between clinicopathologic variables of the patients (age, sex and tumor grade) and p15(INK4b) protein expression. The absence of p15(INK4b) expression in the majority of tissue microarray cores of cutaneous SCC indicated that p15(INK4b) could possibly be involved in the pathogenesis of cutaneous SCC.

Molecular genetic analysis of the BRCA2 tumor suppressor gene region in cutaneous squamous cell carcinomas.

BACKGROUND: Germ line mutations of the BRCA2 tumor suppressor gene with subsequent loss of the remaining wild-type BRCA2 allele have been identified in up to 35% of familial breast cancer cases. A high frequency of allelic loss at the BRCA2 gene locus has also been reported in a variety of sporadic epithelial tumors including oesophageal squamous cell carcinomas (SCC), and sporadic head and neck SCC. AIM: The present study aimed to examine the integrity of the BRCA2 gene in cutaneous SCC. MATERIALS AND METHODS: Allelic imbalance/loss of heterozygosity (AI/LOH) was examined in 22 histologically confirmed cutaneous SCC at two microsatellite markers, D13S260 (centromeric to the BRCA2 gene) and D13S267 (telomeric to the BRCA2 gene). Immunohistochemical analysis of BRCA2 protein expression was also examined in the cutaneous SCC. RESULTS: AI/LOH at the D13S260 locus was found in eight of the 19 informative SCC, and AI/LOH at the D13S267 locus was found in 12 of the 18 informative SCC. Seven SCC showed allelic loss at both markers, and six SCC showed retention of heterozygosity at both markers. Expression of BRCA2 protein was only detected in six of the normal epidermises and three of the 21 SCC examined. CONCLUSION: AI/LOH of the BRCA2 gene region was found to be common in the cutaneous SCC.

Analysis of FHIT allelic imbalance/loss of heterozygosity and FHIT expression in cutaneous squamous cell carcinomas.

Aberrations of the tumor suppressor gene, the fragile histidine triad gene (FHIT), have been reported in a wide variety of human cancers including cervical carcinoma, oral squamous cell carcinoma (SCC) and oesophageal SCC. This study aimed to examine the integrity of the FHIT gene in cutaneous SCC. Allelic imbalance/loss of heterozygosity (AI/LOH) was examined in 21 histologically confirmed cutaneous SCC at two microsatellite markers, D3S1300 and D3S4103, both of which are located within intron 5 of the FHIT gene. Immunohistochemical analysis of FHIT protein expression was also examined in the cutaneous SCC. Ten of the 16 informative cutaneous SCC samples (63%) displayed AI/LOH at the D3S1300 locus, and 13 of the 17 informative cutaneous SCC samples (76%) displayed AI/LOH at the D3S4103 locus. Nine of the 21 SCC samples were found to be positive for FHIT protein expression, however, a further nine SCC samples showed reduced FHIT expression, and three SCC samples did not express the FHIT protein. No correlation between AI/LOH of the FHIT gene and reduced/absent FHIT expression was detected in the cutaneous SCC samples. Further investigation into the role of the FHIT protein in the epidermis is warranted to determine if the reduced/lost expression of FHIT observed in a subset of the cutaneous SCC is contributing to tumorigenesis.

The effect of infection of hatched blastocyst bovine embryos with border disease and bovine viral diarrhea viruses.

The effect of infection with teratogenic viruses at early stages of pregnancy is not fully understood. This study aimed to look at the effect of infection with teratogenic viruses such as bovine viral diarrhea virus (BVDV) and border disease virus (BDV), on early stage embryos at the hatched blastocyst stage. BVDV and BDV are known to cross the placenta of infected mothers and lead to congenital defects and death of developing fetuses. This study can be a good model for better understanding the effects of other teratogenic viruses such as Rubella virus in humans.

Overexpression of EGFR protein in Bruneian lung cancer patients.

BACKGROUND: Lung cancer is the leading cause of cancer death in Brunei Darussalam, accounting for almost 20% of the total. The epidermal growth factor receptor (EGFR) is a member of the erbB family of tyrosine kinase receptor proteins, which includes c-erbb2(HER2/neu), erb-B3, and erb-B4. EGFR overexpression is found in a third of all epithelial cancers, often associated with a poor prognosis. MATERIALS AND METHODS: Protein expression of EGFR in 27 cases of lung cancer tissue samples and 9 cases of normal lung tissue samples was evaluated using an immunohistochemical approach. RESULTS: The results demonstrated significant increase and overexpression of EGFR in Bruneian lung cancer tissue samples in comparison to normal lung tissue. However, there was no significant relationship between clinicopathologic variables (age and sex) of patients and EGFR protein expression. CONCLUSIONS: EGFR is overexpressed in Bruneian lung cancer patient tissue samples in comparison to normal lung tissue samples. This may indicate that EGFR protein over expression plays an important role in the genesis of this type of cancer in Brunei Darussalam.

In situ hybridization: detecting viral nucleic acid in formalin-fixed, paraffin-embedded tissue samples.

In situ hybridization is a method for detecting specific nucleic acid sequences within individual cells. This technique permits visualization of viral nucleic acid or gene expression in individual cells within their histologic context. In situ hybridization is based on the complementary binding of a labeled nucleic acid probe to complementary sequences in cells or tissue sections, followed by visualization of target sequences within the cells. It has been used widely for the detection of viral nucleic acid sequences within individual cells. This review will define the technical approaches of in situ hybridization and its current application to detect viral nucleic acids within formalin-fixed, paraffin-embedded tissue samples, with special reference to the Epstein-Barr virus.

Lack of association between hepatitis C viral RNA in serum and liver and histologic gradings: a study on Irish anti-D-treated patients.

In this study the authors applied a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect hepatitis C virus (HCV) RNA in 15 frozen liver biopsy samples from anti-D-treated patients. They also correlated the presence or absence of HCV RNA in the serum and liver of each patient with their histologic gradings. RNA was extracted from 36 frozen liver biopsy samples. These included 15 liver biopsy samples from patients infected with HCV through contamination of anti-D blood products. Three of these 15 anti-D-treated patients were receiving alpha-interferon treatment at the time of liver biopsy. Nine frozen liver biopsy samples from patients with a history of intravenous drug abuse were included as positive controls. HCV-negative frozen liver biopsy samples from 12 noninfected patients were used as negative controls. RNA was also extracted from six frozen skin biopsy specimens to check for cross-contamination of samples. Eleven of 15 anti-D-treated patients were HCV RNA positive by RT-PCR, with 100% correlation between HCV RNA in the serum and liver. The nine frozen liver biopsy samples from the intravenous drug abuse patients (positive controls) were also RT-PCR positive for HCV RNA. The 12 noninfected samples and the negative control biopsy samples were negative for HCV. Twenty-seven percent of the recombinant immunoblot assay-positive patients were serum and liver HCV RNA negative. HCV-positive patients receiving alpha-interferon therapy at the time of biopsy had cleared the virus from the serum and the liver. There was no correlation between the presence or absence of serum and liver HCV RNA with the histologic grading. This lack of correlation shows clearly the importance of histopathologic evaluation of liver biopsy samples in monitoring HCV-associated liver disease progression. In addition, this finding indicates that one cannot rely only on the presence or absence of HCV RNA in either serum or liver tissue as a parameter in monitoring HCV-associated liver disease progression in this unique cohort of anti-D-treated patients.

Investigation into a possible association between oral lichen planus, the human herpesviruses, and the human papillomaviruses.

BACKGROUND: Oral lichen planus (OLP) is a chronic relapsing cell-mediated condition of unknown etiology. The purpose of this study was to ascertain if the human herpesviruses (HHVs) or human papillomaviruses (HPVs) act as possible factors or co-factors in the pathogenesis of OLP. METHODS: Thirty-eight histologically confirmed OLP and 20 normal control buccal mucosa tissue samples were analyzed. Polymerase chain reaction analysis was employed to detect members of the HHV and HPV families. RESULTS: The Epstein-Barr virus and HHV-7 were detected in a small percentage of tissue samples. However, HPV-16 was detected in 26.3% of OLP samples and 0% of the normal control tissues. The epidermodysplasia verruciformis-related HPV types were detected in 42% of OLP samples and 45% of normal control samples. CONCLUSION: The results of this study do not suggest a causative role for members of the HHV family in the pathology of OLP. However, a statistical association was found between HPV-16 presence and OLP.

Lack of increased P15INK4B protein expression in basal cell carcinomas.

BACKGROUND: The basal cell carcinoma (BCC) is the most common non-melanoma skin cancer (NMSK). BCC might develop because of the faulty cell cycle arrest. P15INK4b is a tumor suppressor gene, involved in cell cycle arrest and inactivated in most human cancers. The role of p15INK4b protein expression in the genesis of BCC is as yet unknown. In a previous study we showed the absence of p15INK4b expression in the majority of tissue microarray cores of cutaneous squamous cell carcinoma (SCCs), another type of non-melanoma skin cancer, indicating that p15INK4b could possibly be involved in the pathogenesis of cutaneous SCC. The aim of this study was to investigate p15INK4b protein expression in BCCs. MATERIALS AND METHOD: Protein expression of p15INK4b in 35 cases of BCC tissue arrays and 19 cases of normal human skin tissue was studied using an immunohistochemical approach. RESULTS: The expression of p15INK4b was not significantly different in the BCC cases as compared with normal human skin (p=0.356; p>0.05). In addition, there were no significant relationship between clinicopathologic variables of patients (age and sex) and p15INK4b protein expression. CONCLUSIONS: Our finding may indicate that p15INK4b protein expression does not play a role in the genesis of BCC.

Analysis of APC allelic imbalance/loss of heterozygosity and APC protein expression in cutaneous squamous cell carcinomas.

BACKGROUND: The adenomatous polyposis coli (APC) gene is a tumor suppressor gene which is mutated in the hereditary disease, familial adenomatous polyposis (FAP). Somatic mutations of the APC gene have also been identified in the majority of sporadic colorectal carcinomas, and mutation of the APC gene appears to be an early step in the initiation of colon cancer. Loss of heterozygosity (LOH) of APC has been described in a variety of other cancer types, including renal cell carcinoma, gastric cancer, non-small cell lung cancer, endometrial cancer and oral squamous cell carcinomas (SCC). AIM: To determine the role played by APC gene in the genesis of cutaneous SCC. MATERIALS AND METHODS: Allelic imbalance/loss of heterozygosity (AI/LOH) was examined in twenty-two histologically confirmed cutaneous squamous cell carcinomas (SCC) using microsatellite markers, proximal to the APC gene. Immunohistochemical analysis of APC protein expression was also examined in the cutaneous SCC. RESULTS: AI/LOH was detected in 60% of the SCC samples using D5S346 marker (proximal to the APC gene). Ninty-five percent of the SCC samples showed positive reduced APC expression, however the localization of the APC protein was abnormal. CONCLUSION: The abnormal expression of APC suggests that APC gene may play a role in cutaneous SCC development.

hTR RNA component as a marker of cellular proliferation in oral lichen planus.

Previously, we have shown that the telomerase RNA component hTR is highly expressed in the epithelium of non-dysplastic Oral Lichen Planus (OLP) lesions (11). We concluded that it is possible that this high expression might be related to the increased cellular proliferation seen in OLP rather than being an indicator of potential malignant transformation. In the present study, and in order to confirm our finding in the previous study that hTR might be a marker for cellular proliferation in OLP, we analysed OLP biopsies known to be positive for RNA component of Telomerase (hTR) for the expression of Ki-67 as a marker for cellular proliferation. Fourteen OLP tissue biopsies known to be positive for telomerase RNA component hTR, were investigated using an immunohistochemical approach to determine the rate of cellular proliferation in OLP, looking at the expression of Ki-67 protein as a marker for cellular proliferation. A statistically significant increase was found between Ki-67 expression in OLP in comparison to normal control buccal mucosa samples. The expression of hTR component in OLP might thus be a marker for cellular proliferation.

Methylthioadenosine phosphorylase expression in cutaneous squamous cell carcinoma.

The methylthioadenosine phosphorylase (MTAP) gene is a tumour suppressor gene, located on chromosome 9p21, 100 kb telomeric of the p15 and p16 genes, which are often deleted in tumor cells. The role of MTAP protein expression in the genesis of cutaneous squamous cell carcinoma (SCC) is currently not known. In a previous study we have shown the frequent occurrence of allelic imbalance/loss of heterozygosity (AI/LOH) in cutaneous SCCs using AI/LOH markers flanking the p15, p16, and MTAP genes and demonstrated reduction in p15 and p16 protein expression in comparison to normal human skin. The present study is a continuation to our previous studies, aimed at determining possible roles played by MTAP protein expression in the genesis of cutaneous SCC. The expression of MTAP protein was detected using immunohistochemical approach in 109 micro array cutaneous SCC and 20 normal human skin tissue samples. The expression of MTAP was not significantly different in the cutaneous SCC cases as compared with normal human skin. This may indicate that MTAP protein expression does not contribute to the genesis of cutaneous SCC.

Detection of Epstein-Barr virus in lower gastrointestinal tract lymphomas: a study in Malaysian patients.

BACKGROUND: Many studies in the literature have shown that Epstein-Barr virus (EBV) is associated with several human lymphoid and epithelial malignancies. However, the prevalence of EBV in non-Hodgkin lymphoma (NHL) of the lower gastrointestinal (GI) tract has not been fully elucidated. AIM: The aim of this study was to determine the presence and distribution of EBV in formalin-fixed paraffin-embedded tissue samples obtained from 18 Malaysian patients diagnosed with NHL of the lower GI tract. METHODS: The GI tract lymphoma tissue samples analyzed for the presence of EBV were divided into the following groups: NHL of the small intestine (seven cases); NHL of the ileocecum (ten cases); and NHL of the rectum (one case). The presence of EBV-encoded RNA (EBER) in all of the above tissue samples was tested for using conventional in situ hybridization technology. RESULTS: Two of 18 cases (11.1%) of NHL of the lower GI tract demonstrated positive signals for EBV/EBER. In the first positive case, EBV/EBER signals were located in lymphoma cells in the serosa layer of the small intestine. In the second EBV/EBER-positive case, EBV/EBER signals were detected in diffuse B-cell lymphoma of the ileocecum. CONCLUSION: These findings demonstrate a rare association between EBV and lower GI tract lymphomas in this group of Malaysian patients.

Enhanced detection of microsatellite instability and mismatch repair gene expression in cutaneous squamous cell carcinomas.

BACKGROUND: Microsatellite instability (MSI) is a phenotypic characteristic of tumors with biallelic inactivation of mismatch repair genes, such as MSH2 or MLH1, and contributes to malignant transformation. AIMS: The aim of this study was to examine the prevalence of MSI in cutaneous squamous cell carcinoma (SCC) using a PCR and fluorescent-based detection system. These methods of analysis offer several advantages over the use of silver staining and autoradiographic techniques. We also aimed to determine if MSI status correlated with expression of the MSH2 and MLH1 mismatch repair proteins in these cutaneous SCC samples. METHODS: The MSI status of 22 histologically confirmed invasive cutaneous SCC samples were analyzed at five microsatellite markers (the National Cancer Institute's Bethesda panel of two mononucleotide and three dinucleotide markers) using a PCR and fluorescent-based detection system. Immunohistochemical analysis of MSH2 and MLH1 protein expression was also carried out on the SCC samples. RESULTS: Only one case of cutaneous SCC displayed MSI. This was found at just one of five markers, and thus was low frequency MSI. All 22 cutaneous SCC cases strongly expressed MSH2 protein. Eighteen (82%) of the cutaneous SCC cases showed moderate to strong expression of MLH1 protein. The remaining four cases of cutaneous SCC were negative for MLH1 protein. Therefore, the majority of the SCC patients analyzed showed a correlation between absence of MSI and expression of MSH2 and MLH1 proteins. CONCLUSIONS: MSI is uncommon in cutaneous SCC. In addition, MSH2 was strongly expressed in all SCC samples analyzed and appeared to be upregulated when compared with the corresponding normal tissue. MLH1 protein was not detected in 4 of 22 SCC cases, although it was expressed in the corresponding normal tissue, suggesting that inactivation of MLH1 may be a late event in a subset of invasive SCC cases.

Telomerase: is it the future diagnostic and prognostic tool in human cancer?

A number of methods exist to detect levels of telomerase activity and the presence of telomerase subunits in a variety of tissues. As telomerase activation seems to be an important step in tumorigenesis, accurate detection of the presence and activity of the enzyme and its subunits is vital. The original method of detecting telomerase activity was developed by Kim and coworkers in 1994, and was termed the telomeric repeat amplification protocol. This assay led to a staggering increase in the number of telomerase-associated publications in scientific journals (85 publications from 1974-1994, 5063 publications from 1994-2004). A number of methods have been described to detect telomeres and to measure their length, with the standard measurement of telomere length performed using a modification of the Southern blot protocol. RNA in situ hybridization can be performed to detect levels of the RNA component of telomerase, and standard in situ hybridization and immunohistochemistry can be applied to examine expression levels and localization of the catalytic subunit of the enzyme. Reverse transcriptase PCR has also been applied to assess expression levels of the telomerase components in various tissues. This review provides a synopsis of telomeres, telomerase, telomerase and cancer, and finally, methods for the detection of telomerase in cancer.

Loss of Fhit expression as a potential marker of malignant progression in preinvasive squamous cervical cancer.

OBJECTIVE: In a previous study using the same cases of squamous cervical neoplasia and microinvasive carcinoma (MICA) we found an association between FHIT gene deletion and infection with high-risk HPV (HR HPV). The purpose of this study was to evaluate Fhit protein expression by immunohistochemistry in order to determine whether FHIT gene deletion or infection with HR HPV correlated with aberrant protein expression and grade of lesion. METHODS: A total of 74 archival LLETZ biopsy cases consisting of 23 cervical intraepithelial neoplasia grade 1 (CIN1), 28 CIN3, and 23 MICA cases were selected for Fhit immunostaining. The results of this study on Fhit immunostaining were analyzed in relation to our previous findings using Epi-Info and SPSS-PC statistical analysis software. RESULTS: Fifty percent (14/28) of CIN3 lesions and 78% (18/23) of MICA lesions had a marked reduction or absence of Fhit protein expression (P = <0.001, strength of association, Cramers' V, 0.632). CIN1 lesions were found to have moderate to strong cytoplasmic expression of Fhit. Seventy percent of cases in this study with reduced/absent Fhit protein expression were also positive for FHIT gene loss of heterozygosity (LOH) (P = 0.04, strength of association, phi, 0.254). A significant statistical relationship was found between Fhit protein expression and HPV 16 infection in combined CIN1, CIN3, and MICA cases (P = <0.001). Eighty-seven percent of cases with reduced/absent Fhit protein expression were positive for HPV 16 (strength of association, phi, 0.552). Ninety percent of HPV 16 and 31 positive cases had reduced/absent Fhit expression. CONCLUSION: Our findings suggest an association between HPV infection and FHIT gene abnormalities raising the possibility of a mechanistic role for the FHIT gene as a cofactor with HPV in triggering the development of cervical cancer.