Intra- and intergenotypic variations among human cytomegalovirus gB genotypes.

OBJECTIVES: To study genotypic variations among human cytomegalovirus (HCMV) gB genotypes in clinical samples of Thai patients. METHODS: gB genotyping of 31 HCMV-DNA-positive clinical samples were determined by PCR-RFLP, gene cloning and DNA sequencing methods. RESULTS: Eight gB1, 7 gB2, 7 gB3, 6 gB untype (UT1 and UT2) and 3 mixed gB genotypes were first identified by PCR-RFLP. All 3 mixed gB genotype samples and 1 gB2 sample were cloned and confirmed gB genotype by PCR-RFLP. Altogether, 57 strains (27 unique types and 30 transformants) were further analyzed by DNA sequencing method. Discordant results between PCR-RFLP and DNA sequencing methods were demonstrated in 5 samples and 1 clone. The gB UT1 and UT2 were all classified as gB1 except 1 sample of UT1 (9D), suggesting a new variant. Each genotype had a similarity of more than 97%, whereas strains of different genotypes had 77.71-92.75% homology. The most divergent type was gB3. CONCLUSIONS: Intra- and intergenotypic variations among strains were demonstrated either in individual or distinct patients. Intragenotypic variation in a person occurs possibly due mainly to a point mutation mechanism rather than reinfection of a new gB genotype.

Antiangiogenic activity of curcumin in hepatocellular carcinoma cells implanted nude mice.

Antiangiogenic activity of curcumin on the tumor neogenesis was investigated by evaluating the density of neocapillaries induced by Hepatocellular carcinoma cells (HepG2) in mice, using intravital fluorescence videomicroscopy. Male BALB/c nude mice (20-25 g) were used, and a dorsal skin-fold chamber was implanted. HepG2 (30 microl of 2 x 10(6) cells) were inoculated on the upper surface of the skin within the chamber. The mice were divided into two groups as follows. Dimethyl sulfoxide solution (0.1%) was fed (HepG2 group, n=5) or curcumin solution (3000 mg/kg bw) was fed oral daily (HepG2-Cur group, n=5), one day after the inoculation of HepG. On days 7 and 14 post-tumor-inoculation, the tumor microvasculature was visualized by injecting 0.1 ml of 0.5% rhodamine B isothiocyanate-labeled dextran intravenously, and observed under an intravital fluorescence videomicroscope. Based on the recorded videoimage, the tumor neocapillary density and microvasculature were evaluated using a digital image analysis and correlated with the tumor area. The image analysis demonstrated that in the HepG2-group the neocapillary densities were significantly increased on day 7, and day 14, compared to the aged-matched Sham-group (P<0.05). In the HepG2-Cur group, the increase of tumor neocapillary density was attenuated significantly. It was suggested that high dose of curcumin might be an effective anti-angiogenic drug in the treatment against tumor.

Risk factors for conjunctival squamous cell neoplasia: a matched case-control study.

AIMS: To identify roles of human papillomavirus (HPV) infection and solar elastosis as the risk factors for conjunctival squamous cell neoplasia (CSCN). METHODS: 30 consecutive pathological specimens, ranging from conjunctival intraepithelial neoplasia, carcinoma in situ, to invasive squamous cell carcinoma were retrieved from tissue archives. 30 controls were disease free conjunctiva from age and sex matched patients undergoing extracapsular cataract extraction. Two masked pathologists studied haematoxylin and eosin stains on paraffin embedded conjunctival tissues. Elastic stain for solar elastosis was blindly interpreted in comparison with negative and positive controls. HPV infection was studied by polymerase chain reaction and dot hybridisation. RESULTS: The mean age of CSCN patients was 54.9 years. The male to female ratio was 1:1. Solar elastosis was seen in 53.3% of CSCN and in 3.3% of controls with an odds ratio of 16.0 (95% CI, 2.49 to 670.96; p value = 0.0003). HPV DNA were not detected in any of the specimens. CONCLUSION: Solar elastosis is much more frequently found in CSCN cases than in their matched controls and is a risk factor for CSCN. These data are insufficient to conclude that HPV infection is a risk factor for CSCN.

Endothelial nitric oxide synthase expression in systemic and pulmonary circulation of streptozotocin induced diabetic rats: comparison using image analysis.

To compare the level of endothelial nitric oxide synthase (eNOS) expression produced in heart and lung vascular tissue, the protein content was determined using Western blot analysis with the enhancement of image processing. Heart and lung extracts from 12 and 24 weeks from control (CON) and streptozotocin-induced diabetic (DM) rats were collected for Western blot analysis. Using monoclonal antibody against rat eNOS protein (140 kDa), the eNOS-protein bands were detected with enhanced chemiluminescence (ECL; Amersham) and exposured to film (Hyperfilm-ECL; Amersham). Images of eNOS bands on each film were then scanned and saved to digital files. Using Global Lab Image software, the number of pixels in each digital file was counted and calibrated for eNOS-protein content. For the CON and DM groups, the mean values of eNOS-protein contents were calculated and expressed as a percentage of total protein content, 5 micrograms. It was found that the eNOS level in DM hearts was significantly decreased, as compared to age-matched CON hearts. On the other hand, eNOS levels in DM lungs was increased, compared to CON lungs. Therefore, it may be concluded that high, not low, flow-mediated eNOS expression is a good measure of hyperglycemic-induced endothelial dysfunction.

Human herpes virus 6 antibodies in beta-thalassemia/hemoglobin E pediatric patients.

Human herpesvirus 6 (HHV-6) is a viral pathogen that causes exanthem subitum in children. It has also been identified as the cause of life-threatening illness in immunocompromised pediatric patients and transplant recipients. We undertook a serological study of HHV-6 IgM and IgG antibody among 29 children (12 females and 17 males) with beta-thalassemia/HbE disease. The rate of infection was 86.2%; the rates of early recent infection (IgM positive only), recent infection (both IgM and IgG positive) and past infection (IgG positive only) were 13.8%, 41.4% and 31.0%, respectively. The geometric means of the IgM and IgG titers of the splenectomy group (9 cases) were 10.15 units and 11.18 units, respectively. The geometric means of the IgM and IgG titers of the non-splenectomy group (20 cases) were 10.10 units and 12.84 units, respectively. According to this study, the prevalence of HHV6 infection among pediatric patients with beta-thalassemia/HbE is very high; morever, the significantly higher titer among these patients may imply a high risk for further possible bone marrow transplantation. Increased awareness of HHV-6 infection among this population is necessary.

Seroprevalence of Epstein-Barr virus antibody among children in various age groups in Bangkok, Thailand.

There are no current data on previous Epstein-Barr virus (EBV) infections in different age groups of Thai children. This study was conducted to determine the prevalence of anti-EBV IgG antibody in healthy children of various age ranges in Bangkok, Thailand. Between June and December 1998, blood samples were collected from 425 volunteers aged 6 months to 15 years who attended a well baby clinic in the northern suburban part of Bangkok, Thailand. Serum samples were assayed for specific anti-EBV IgG antibodies using a commercial enzyme-linked immunosorbent assay kit. The percentage of children with positive anti-EBV IgG antibody increased with advancing age. The overall seropositivity rate was 72.7%. Children with anti-EBV IgG antibody were significantly older than those without the antibody. Seronegative children were reared at home significantly more frequently than seropositive children. These seroopidemiologic data will guide calculation of the appropriate age for administration of an EBV vaccine to children, when it becomes available.

Detection of dengue HI and IgM antibody: is it diagnostically useful? when and how?

The incidence of dengue infection at King Chulalongkorn Memorial Hospital from 1997 to 2000 was reviewed from laboratory information. The highest incidence of the disease occurred from the rainy season to the winter. Most cases were aged below 15 years (87.89%) and no sexual preponderance was observed. The majority of cases were secondary infection (62.09%). Detection of HI titer is still useful for diagnosis although paired serum has to be taken. The time interval between sera should be at least 7 days for suspected primary infection but a shorter time interval can be considered for suspected secondary cases. Rapid and accurate detection of dengue IgM has become more useful because only single serum is required. A request for dengue IgM should be done after day 5 of illness. However, interpretation of the result should be done carefully according to the timing of serum collection.

Necrotizing ileitis caused by cytomegalovirus in patient with systemic lupus erythematosus: case report.

We report a systemic lupus erythematosus (SLE) patient with necrotizing ileitis diagnosed at a tertially care centre in Thailand. The patient was surgically explored because peritonitis was suspected and segmental gangrenous and perforation of the terminal iliem were found. The pathological finding was necrotizing ileitis with appearance of cytomegalic intranuclear inclusion body. The presence of cytomegalovirus (CMV) infection in tissue was confirmed by CMV-DNA detection using polymerase chain reaction and ELISA probe hybridization method. The hemoculture and peritoneal fluid culture results revealed no pathogenic organisms. Postoperatively, the clinical course of the patient deteriorated and she developed hypotension. Vasopressive drugs were administered without clinical improvement. She expired on day 5 postoperation. Regarding CMV infection, the organism involves the small bowel in only 4.3 per cent of all CMV infections of the gastrointestinal tract. Isolated cases of ileal perforation due to CMV infection have never been reported in a SLE patient. Thus, chronic right lower abdominal pain, fever with or without diarrhea in immunocompromised patients should cause clinicians to consider CMV ileitis in the differential diagnosis. Immediate surgical resection and prompt antiviral therapy lead to successful treatment.

Seroprevalence of anti-human herpes virus-6 IgG antibody in children of Bangkok, Thailand.

The prevalence of HHV-6 infection was surveyed by determining the presence of anti-human herpesvirus-6 IgG (Anti-HHV-6 IgG) using an ELISA method. Two hundred and ten sera collected from healthy Thai children aged between 0 to 12 years (mean +/- standard deviation = 3.35+/-3.33) indicated the prevalence of HHV-6 infection was 88.10% (185/210). Samples were classified into 7 groups, 30 samples each, according to their ages, ie, group 1: 0 - < 6 months; group 2: 6 - < 12 months; group 3: 12 - < 18 months; group 4; 18 - < 24 months; group 5: 2 - < 5 years; group 6: 5 - < 8 years and group 7: 8-12 years. The prevalence of HHV-6 infection was 63.33%, 70%, 96.67%, 93.33%, 100%, 100% and 93.33%, respectively. The mean level of anti-HHV-6 IgG among those positive for HHV-6 infection (185 samples) increased from 0 < 6 months old (17.47+/-6.32 units) to 27.57+/-8.42 units in 6 - < 12 months old, with the highest value found in the 18 - < 24 months old group (33.08+/-8.64 units). The level declined thereafter. A statistically significant difference of the mean level of anti-HHV-6 IgG among positive groups was found (p-value < 0.05). The important factor associated with HHV-6 infection was age (p = 0.002), while sex, socioeconomic status, number of children in the family and child rearing place did not show any association.

p53 status and human papillomavirus infection in Thai women with cervical carcinoma.

Loss of p53 function has been implicated in a wide variety of human malignacies. Many studies suggest that in cervical carcinoma p53 function is inactivated either by gene mutation or by complex formation with E6 oncoprotein product of high-risk human papillomavirus (HPV). The aim of this study was to determine the status of HPV infection and p53 gene mutation as well as their correlation in cervical carcinomas. Formalin-fixed paraffin-embedded tissues of 12 cervicitis, 21 cervical intraepithelial neoplasia grade 3 (CIN 3) and 17 squamous cell carcinomas were determined for the presence of HPV using polymerase chain reaction (PCR) amplification and dot blot hybridization. The status of p53 mutations in exons 5-8 was evaluated by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and confirmed by direct nucleotide sequencing. HPV infections were detected in all CIN 3 and squamous cell carcinomas (100%). Mutations of p53 were present in 3 of 38 HPV-positive samples: one with an ATG-->TTG transversion (Met-->Leu) in codon 237 of exon 7; and the others with a TGC-->TGG transversion (Cys-->Trp) in codon 242 of exon 7, and a CGT-->CCT transversion (Arg-->Pro) in codon 273 of exon 8, respectively. Our findings show that the frequency of p53 mutation is low in primary cervical carcinoma and that the p53 gene mutation and HPV infection are not mutually exclusive events in the development of cervical cancer. Thus, other genetic events independent of p53 inactivation may also significantly contribute to the carcinogenesis of the uterine cervix.

Causes of fever in children with first febrile seizures: how common are human herpesvirus-6 and dengue virus infections?

This study was conducted to evaluate the etiologies of pyrexia in children with first febrile seizures using a prospectively recorded medical protocol, bacterial culture, and serologic tests for human herpesvirus-6 (HHV-6), dengue virus and Japanese B encephalitis (JE) virus. Of 82 children with first febrile seizures, who were between 3 months and 3 years old and had been admitted to Bhumibol Adulyadej Hospital between January 1997 and December 1998, 41 were boys and 41 were girls, with a mean age of 14.7 months. The average maximal body temperature was 39.7 degrees C. Approximately 70% of the children developed seizures on the first day of fever and the duration of the seizures varied from 1 to 30 minutes. In addition to fever and seizure, common symptoms and signs included coryza, diarrhea, vomiting, inflamed tympanic membranes and rash. The causes of fever documented upon discharge were, in order of frequency, upper respiratory tract infection, nonspecific febrile illness, diarrhea, urinary tract infection, viral infection, pneumonia, herpangina, measles, pneumococcal bacteremia and dengue fever. Serologic tests for HHV-6 IgM were positive in seven children (8.5%), and serologic tests for dengue and JE viruses were negative in all cases.

Detection and typing of human papillomavirus in cervical intraepithelial neoplasia grade III in Thai women.

Infection of human papillomaviruses (HPVs) has been shown to play an important role in the development of cervical cancer from precancerous lesions known as cervical intraepithelial neoplasia (CIN-I, CIN-II and CIN-III). In Thailand, cervical cancer is the most common cancer in women. Fifty tissue samples diagnosed as CIN-III and 50 tissues of normal histopathologic appearance as controls were examined for the presence of HPV-DNA and HPV typing using PCR and dot hybridization (DH) methods. All specimens used in this study were formalin-fixed paraffin embedded tissues. HPV-DNA was detected in 74% (37/50) of CIN-III and 6% (3/50) of the control group giving a crude odd ratio of 44.58 (95% confidence interval of 15.2-130). Among the CIN-III group, the most prevalent type was HPV-16; 48.65% (18/37) followed by HPV-18; 16.2% (6/37) and HPV-33; 10.8% (4/37). Mixed infection was identified in 4 specimens, ie HPV-6/16, HPV-16/18, HPV-16/33, and HPV-16/18/33. Twelve samples were untyped. In the control group, only one sample (33.3%) was detected to contain HPV-6 DNA and the remaining ones were untyped. Our results revealed infection with HPV, especially HPV-16 and HPV-18, to be strongly associated with CIN-III in Thai women.

The studies of HLA class II (HLA-DR) system by PCR-SSO typing and the relationship with serological method.

By using two methods together, namely, the conventional serological typing and the new method which is oligonucleotide typing, in order to study the HLA system, it was concluded that the new method was better. Some subtypes were detectable from the new method, whereas, the serological typing yielded negative results such as DRB1 subtype "0103". This method, therefore, provided more information in order to study the HLA system. However, the new method was twice as expensive as the conventional serological method.

Detection and typing of human papillomavirus in cervical cancer in the Thai.

One hundred formalin-fixed paraffin embedded tissues with histopathologic diagnosed invasive cervical cancer (squamous cell carcinoma) were examined for the presence of HPV-DNA by polymerase chain reaction (PCR) using L1-consensus primers. The results indicated that 82 out of 100 (82%) samples were positive for HPV-DNA. Among the positive samples, 50 samples (61%) were typed by dot hybridization technique (DH). HPV-16 was the dominant type (42.68%), followed by HPV-18 (20.73%) and HPV-33 (3.66%). There were double infection of HPV-16 and 18 in 5 (6.1%) samples. None of HPV-6 and 11 were detected in this study. This finding suggests that HPV infection is an important etiologic factor for the development of cervical cancer especially the infection with high risk types, i.e., HPV-16 and 18.

Improvement of PCR detection of HPV-DNA using enhanced chemiluminescence system and dot hybridization.

Infection with human papillomavirus (HPV) is an important etiological factor in the development of cervical cancer; detection of the viral genome is of prognostic importance, particularly for preneoplastic lesions. Nowadays, polymerase chain reaction (PCR) technique is a method of choice for HPV detection because of its sensitivity. However, providing a reliable diagnostic test, both sensitivity and specificity of the test should be evaluated. In the present study, purified plasmid HPV-16 DNA and HeLa-DNA, containing HPV-18 DNA, were amplified by PCR using L1 consensus primers specific for HPV. The amplified product was then analysed by gel electrophoresis (GE) and dot hybridization (DH). The generic oligonucleotide probes (GPs) labelled with Enhanced Chemiluminescence 3' labelling kit (ECL) were used in DH. The sensitivity of PCR reaction after determining by GE was 1 copy per cell for purified plasmid HPV-16 DNA and 20-80 copies per cell for HeLa-DNA while determining by DH was only 0.1 and 2-8 copies per cell, respectively. Thus, the detection of amplified product by DH using enhanced chemiluminescence system improved not only the specificity but also the sensitivity of HPV detection at least 10-fold.

Prevalence of anti-varicella zoster IgG antibody in undergraduate students.

Sera from 74 healthy Thai undergraduate students, mean age 21 + 1.7 years, were tested for the presence of IgG antibody against varicella zoster virus (anti-VZV IgG) by ELISA. Fifty-five of 74 (74.3%) individuals possessed anti-VZV IgG antibody. The presence of anti-VZV IgG was associated with a past history of varicella (p < 0.005, X2 = 33.4989). No sexual preponderance was observed. We therefore found that 1 of 4 Thai young adults was susceptible to VZV infection.

Encapsidation defectiveness of herpes simplex virus type 2 during replication at acid pH condition.

The maximal yield of herpes simplex virus type 2 (HSV-2) grown at pH 6.5 decreased 10(2)-10(3) fold compared to that recovered at pH 7.5. Electron microscopic observation of the infected cells maintained at these 2 pH conditions indicated that approximately equal amounts of immature virions were synthesized 6 hours after infection. However, at 18 hours post infection the majority of viruses present in the nucleus of infected cells maintained at pH 6.5 were empty or partially cored capsids with some particles enveloped and present in the cytoplasm, whereas at pH 7.5 mature virions already appeared at the cytoplasmic membrane. Analysis of viral polypeptides by radioimmunoprecipitation indicated that the synthesis of p40, a family of polypeptides closely involved in viral DNA encapsidation, was significantly impaired in infected cells maintained at pH 6.5.

Intratypic variations in neutralizable epitopes among herpes simplex virus type 2 isolates.

Intratypic variation among 94 isolates of herpes simplex virus type 2 (HSV-2) was investigated using 4 different monoclonal antibodies (MAbs). By neutralization test, these MAbs appeared to be directed to at least 2 distinct epitopes on the viral glycoprotein D (gD), i.e., 6G6.G9 and 6E8.F11 which did not require complement (C-MAb) and gD-105 and gD-110 whose neutralizing activities could be enhanced by complement (C+MAb). The C-MAb pairs each separately could detect significant intratypic variations among the isolates. Whether these variations also existed in the gD epitope(s) recognized by C+MAbs remains to be elucidated. The results suggested that intratypic variation occurred on at least one of the neutralizable (thus related to protective immunity) epitopes on gD of HSV-2.

Intratypic variation of herpes simplex virus type 2 isolates detected by monoclonal antibodies against viral glycoproteins.

Monoclonal antibodies (MAbs) to herpes simplex virus (HSV) glycoproteins gD, gG, gB, and gE were used to analyze antigenic variations of 128 genital HSV-2 isolates by an indirect enzyme-linked immunosorbent assay (ELISA). Isolates were considered significantly different from the standard HSV-2 strain 186 when their optical density (OD) in ELISA was less than half that of strain 186. This criterion gave 30 patterns of reactivity among the genital HSV-2 isolates. The MAbs to gB, gG, and 2 of the gD antibodies reacted with more than 90% of the isolates, suggesting that these MAbs recognized highly conserved epitopes. However, the gE MAb reacted with only 47% of the isolates, and one of the gD antibodies with only 39%. Thus, HSV-2 can readily tolerate modifications in some parts of the gD and gE molecules while remaining infectious.

Evaluation of biotin-streptavidin enzyme-linked immunosorbent assay for detection of genital herpes simplex virus infection.

A biotin-streptavidin enzyme-linked immunosorbent assay (B-SA ELISA) was evaluated for detection of herpes simplex virus (HSV) in clinical specimens which were cervico-vaginal swabs from 205 asymptomatic women and swabs from the genital lesions of 163 suspected patients. All specimens were also subjected to a conventional virus isolation in cell culture. A blocking B-SA ELISA had 100% specificity and 98% sensitivity compared with viral isolation from patients, but had only 40% sensitivity using specimens from asymptomatics. The conventional B-SA ELISA might also be used; it gave results corresponding to B-SA ELISA blocking test except for a single specimen which was considered a false positive.