Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays

In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capable of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6-H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10(3)copies/microl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.     

Diagnostic role of serum interleukin 6 and CA 19-9 in patients with cholangiocarcinoma

BACKGROUND/AIMS: Cholangiocarcinoma is particularly common in Northeastern Thailand where the liver fluke, O. viverrini, is endemic. Currently there is no sensitive and specific tumor marker for the diagnosis of this cancer. The aim of this study was to investigate the accuracy of carbohydrate antigen 19-9 and interleukin-6 in the diagnosis of cholangiocarcinoma. The first marker represents the commonly used serum marker in cholangiocarcinoma and the second marker is a multi-functional cytokine associated with cancer development. METHODOLOGY: The serum concentrations of carbohydrate antigen 19-9 and interleukin-6 were simultaneously determined in 45 patients with cholangiocarcinoma, 15 with hepatocellular carcinoma, 15 with metastatic liver cancers, 10 with benign biliary disease and 10 healthy controls. RESULTS: The sensitivity of carbohydrate antigen 19-9 value (> or = 37 U/mL) in diagnosing cholangiocarcinoma was 64.4%, while its specificity was 100% and 56.7% when compared with benign biliary disease and other liver cancers, respectively. The sensitivity and specificity of detectable interleukin-6 (> or = 0.18 ng/mL) for differentiating between cholangiocarcinoma and benign biliary disease was 71.1% and 90%, respectively, whereas the specificity for differentiating cholangiocarcinoma from other liver cancers was 26.7%. When higher concentrations of either carbohydrate antigen 19-9 (> or = 100 U/mL) or interleukin-6 (> or = 50 pg/mL) were used as the cut-off points, they provided additional diagnostic sensitivity and accuracy in differentiating between cholangiocarcinoma and other liver cancers to 80% and 76%, respectively. CONCLUSIONS: The combined assays of both serum carbohydrate antigen 19-9 and interleukin-6 could be useful in diagnosing cholangiocarcinoma, particularly in populations where this cancer is prevalent.     

Serum hepatocyte growth factor and clinical outcome in biliary atresia

Purpose

Biliary atresia (BA) remains one of the most intractable liver diseases leading to liver fibrosis. Serum hepatocyte growth factor (HGF) has been shown to increase in cirrhotic patients. The aim of this study was to investigate the possible role of HGF in BA.

Methods

Serum levels of HGF were determined using an enzyme-linked immunosorbent assay from 28 BA patients and 25 healthy children. The patients were categorized into 3 groups according to their clinical outcomes (good, fair, and poor): group A (good), jaundice-free patients (total bilirubin [TB] < 2.0 mg%); group B (fair), patients with mild to moderate jaundice (TB, 2 to 10 mg%); and group C (poor), patients with marked jaundice (TB > 10 mg%). Unpaired t test and analysis of variance (ANOVA) with post-hoc tests were used. Data were expressed as mean and SEM.

Results

Serum HGF levels in BA patients were higher than the controls (P = .02). Subgroup analysis found that there were 12 patients in group A, 8 patients in group B, and 8 patients in group C. The mean age of patients in groups A, B, and C were 5.34 ± 0.52, 7.45 ± 1.98, and 5.49 ± 1.57 years (P > .05). Serum HGF in controls and groups A, B, and C were 0.24 ± 0.03, 0.28 ± 0.04, 0.36 ± 0.09, and 0.56 ± 0.07 ng/mL, respectively. Serum HGF levels in BA patients with poor outcome were higher than patients with good outcome (P = .02). There was no difference in serum HGF of BA patients with fair outcome compared with other groups.

Conclusions

Serum HGF is elevated in BA. Furthermore, BA patients with poor outcome have significantly elevated HGF compared with patients with good outcome. Serum HGF levels may be predictive of prognosis with respect to the progression of liver dysfunction. However, the results of HGF in patients with fair outcome are inconclusive, probably because of the small sample size. Further studies are needed to elucidate the detailed mechanisms.     

Phase III randomized trial comparing LDR and HDR brachytherapy in treatment of cervical carcinoma

PURPOSE: Intracavitary brachytherapy plays an important role in the treatment of cervical carcinoma. Previous results have shown controversy between the effect of dose rate on tumor control and the occurrence of complications. We performed a prospective randomized clinical trial to compare the clinical outcomes between low-dose-rate (LDR) and high-dose-rate (HDR) intracavitary brachytherapy for treatment of invasive uterine cervical carcinoma. METHODS AND MATERIALS: A total of 237 patients with previously untreated invasive carcinoma of the uterine cervix treated at King Chulalongkorn Memorial Hospital were randomized between June 1995 and December 2001. Excluding ineligible, incomplete treatment, and incomplete data patients, 109 and 112 patients were in the LDR and HDR groups, respectively. All patients were treated with external beam radiotherapy and LDR or HDR intracavitary brachytherapy using the Chulalongkorn treatment schedule. RESULTS: The median follow-up for the LDR and HDR groups was 40.2 and 37.2 months, respectively. The actuarial 3-year overall and relapse-free survival rate for all patients was 69.6% and 70%, respectively. The 3-year overall survival rate in the LDR and HDR groups was 70.9% and 68.4% (p = 0.75) and the 3-year pelvic control rate was 89.1% and 86.4% (p = 0.51), respectively. The 3-year relapse-free survival rate in both groups was 69.9% (p = 0.35). Most recurrences were distant metastases, especially in Stage IIB and IIIB patients. Grade 3 and 4 complications were found in 2.8% and 7.1% of the LDR and HDR groups (p = 0.23). CONCLUSION: Comparable outcomes were demonstrated between LDR and HDR intracavitary brachytherapy. Concerning patient convenience, the lower number of medical personnel needed, and decreased radiation to health care workers, HDR intracavitary brachytherapy is an alternative to conventional LDR brachytherapy. The high number of distant failure suggests that other modalities such as systemic concurrent or adjuvant chemotherapy might lower this high recurrence, especially in Stage IIB and IIIB.     

The predominant genotypes of hepatitis B virus in Thailand

In Thailand, chronic liver disease (CLD) as a consequence of infection with hepatitis B virus (HBV) constitutes a public-health burden. Control and treatment are complicated by the virus exhibiting an unusually high mutation rate, with some genotypes apparently causing more severe disease than others. Restriction-fragment-length-polymorphism (RFLP) analysis of the pre-S region of the viral genome, amplified by PCR, was used to determine which genotypes were most prevalent among Thai patients chronically infected with the virus. The patients were chronic HBV carriers (40) or cases of chronic hepatitis (34), cirrhosis (14) or hepatocellular carcinoma (30). As indicated by the results of earlier studies on CLD patients in South-east Asia, genotype C (68.6%) was clearly predominant. RFLP patterns permitted the C1 (12.7%), C7 (45.7%), C8 (10.2%) and B1 (29.7%) subtypes to be identified. Two samples that could not be typed by RFLP were analysed by direct sequencing, categorized as type C, and tentatively designated as subtype C9. As comparison of the present data with those previously obtained by direct sequencing of PCR products indicates that RFLP analysis is as specific and reliable as sequencing and less expensive and time-consuming, RFLP analysis may be particularly useful for epidemiological studies.     

Characterization of the hemagglutinin and neuraminidase genes of recent influenza virus isolates from different avian species in Thailand

The hemagglutinin (HA) and neuraminidase (NA) genes of eight influenza A virus (H5N1) isolates obtained from various avian species in Thailand in 2003-2004 have been characterized in comparison with the Thai isolate A/Chicken/Nakorn-Pathom/Thailand/CU-K2/04(H5N1). Phylogenetic analyses of both genes revealed that all the eight avian isolates were closely related to the A/Chicken/Nakorn-Pathom/Thailand/CU-K2/ 04(H5N1). The amino acid sequence of the HA cleavage site revealed a common characteristic of a highly pathogenic virus strain. Moreover, a deletion of 20 amino acids in the NA stalk region was detected in all Thai isolates in contrast to the H5N1 strain that had caused outbreaks in eastern Asia in 1996-1997 and 2000-2001.     

TT virus infection in chronic liver disease.

BACKGROUND/AIMS: The exact role of the novel hepatotropic TT virus regarding the etiology of viral hepatitis, as well as the progression towards chronic liver disease has as yet not been defined. Moreover, the contribution of TTV infection to the course of chronic hepatitis B or C virus infections also still awaits clarification. Hence, the aim of our study was to investigate the impact of TTV infection on clinical severity and histology of chronic liver disease originating from HBV and/or HCV infections in Thai patients concomitant with the determination of TTV's association with non-B, non-C chronic liver disease and compared to its prevalence among voluntary blood donors. METHODOLOGY: DNA was extracted from the sera collected from 115 hepatitis B patients, 41 hepatitis C, and 48 negative for either viral marker, who had all been diagnosed with chronic liver disease ranging from chronic hepatitis over cirrhosis to hepatocellular carcinoma. The sera obtained from 200 voluntary blood donors served as controls. TTV DNA was amplified by seminested polymerase chain reaction (PCR) employing primers derived from the genome's most conserved region. The PCR products were analyzed by gel electrophoresis. Liver function tests were performed by means of a chemical analyzer. RESULTS: TTV DNA was detected in 20% of the HBV-positive and 19.5% of the HCV-positive chronic liver disease patients. Within the group of patients seronegative for both viral markers, TTV was detected in 8.3%. Furthermore, its DNA was identified in 6.8% of the HCC patients and finally, in 7% of the blood donors. Yet, no significant differences between TTV infected and non-infected patients were found as to demographic data, assumed source of infection, biochemical abnormalities, or severity of liver histology. CONCLUSIONS: TTV appears to be highly prevalent on a worldwide scale but regarding etiology of and progression towards serious liver disease, its contribution seems to be minor if not altogether non-existent. Hence, regarding clarification of its clinical significance, further studies are certainly required.     

Molecular characterization and complete genome analysis of human enterovirus 71 and coxsackievirus A16 from children with hand, foot and mouth disease in Thailand during 2008-2011

Hand, foot and mouth disease (HFMD) has mostly been caused by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). CA 16 was the most common cause of HFMD in 2010. EV71 had a high prevalence in 2008-2009 and has been identified with a higher frequency since 2011. Nearly complete genome sequences of three EV71 strains (2008-2009 strains) and two CA16 strains (2010 strains) obtained from outbreaks in Thailand in 2008 to 2010 were characterized. Based on a phylogenetic tree of the complete VP1 region, three EV71 strains grouped into the B5, C1 and C4 genotypes, and two CA16 strains grouped into the C genotype. Based on sequence analysis, nucleotide changes were found to cluster in the internal ribosome entry site (IRES) element of the 5′-untranslated region (5′-UTR). Amino acid differences identified in all strains were located in the non-structural protein. These data also provide the molecular epidemiology of EV71 and CA16 outbreaks in Thailand.     

Molecular epidemiology, genome characterization, and recombination event of human parechovirus

Human Parechovirus (HPeV), a member of the Picornaviridae family, is an infectious agent mostly affecting children. There are 16 recognized genotypes which have globally spread. This study incorporated a total of 2957 nasopharyngeal (NP) swab and 759 fecal samples that were collected from different parts of Thailand. The NP of HPeV was detected in 0.4% of NP swab and 6.1% of fecal samples. The majority of HPeV infections occur in infants below the age of 2 years, while infections were detected in children above the age of 10 years as well. Various genotypes comprising 1A, 1B, 2, 3, 4, 5, 6, 10 and 14 have been characterized. This study revealed recombination events in 16 samples in which HPeV1B was shown as the highest frequency. In conclusion, HPeV can be detected in both the respiratory and GI tract. Moreover, HPeV which circulates in Thailand is highly diverse and subject to recombination.     

Efficacy of pulsed dyed laser (585 nm) in the treatment of molluscum contagiosum subtype 1

Molluscum contagiosum is a common cutaneous disease that may be difficult to treat when there are multiple lesions; especially in children. This study was conducted to determine the efficacy of pulsed dye laser (585 nm) in the treatment of molluscum contagiosum in 20 children. In the treated group, 70.5% of lesions healed after the first treatment; the remaining 10.6% after the second treatment (2 weeks later). The overall cure rate was significantly different from the control group (p< 0.01). The therapy was also well tolerated. Only mild transient hypopigmentation and erythema were observed. None encountered infectious events. In conclusion, pulsed dye laser is a good alternative treatment for molluscum contagiosum due to high efficacy and mild transient side effects.