Large scale study of HPV genotypes in cervical cancer and different cytological cervical specimens in Thailand.

Identification of high-risk HPV genotypes in patients is essential for vaccination and prevention programs while the geographic distribution of cervical cancer varies widely. HPV 16 is the major cause of cervical cancer followed by HPV 18, HPV 31, HPV 52, or HPV 58 depending on geographic area. In this study, the distribution of HPV genotypes in cervical specimens from women living in Thailand was analyzed by HPV testing with electrochemical DNA chip and PCR direct sequencing. The 716 specimens were grouped according to their cytological grades; 100 normal, 100 low-grade squamous intraepithelial lesions, 100 high grade squamous intraepithelial lesions, and 416 specimens of cervical cancer. The results showed that HPV 16, HPV 18, HPV 52, and HPV 58 are the most common HPV genotypes in Thailand, respectively. With respect to age, women below the age of 26 years were almost negative for high-risk HPV DNA exclusively. Conversely, high prevalence of high-risk HPV DNA and abnormal cytology were usually found in women between 26 and 45 years while cervical cancer was detected mainly in women above the age of 45 years. To increase protection efficiency, a vaccine including HPV 52 and HPV 58 should be offered to Asian women, and primary HPV screening should start at 26-30 years of age.

Prevalence of human papillomavirus genotypes in cervical cancer.

BACKGROUND AND OBJECTIVE: Cervical cancer is the second most common female genital cancer worldwide. There is strong epidemiological and molecular evidence indicating that human papillomavirus (HPV) infection is a necessary event in the development of cervical intraepithelial lesion and subsequent invasive carcinoma. The aim of this study was to investigate the HPV genotype distribution and prevalence in cervical cancer of Thai women. MATERIALS AND METHODS: One hundred fifty-five cervical cancer specimens were enrolled in this study. The HPV genotypes were determined by means of the combined use of a line probe assay (INNO-LiPA) and DNA chip methods. RESULTS: Of the overall prevalence of HPV in the study group, 83.2% and 11.6% of the cases had single and multiple genotype infections, respectively. The most prevalent genotypes were HPV 16 (51%), followed by HPV 18 (20%), HPV 52 (10.3%), HPV 58 (5.8%), and HPV 33 (4.5%). All HPV genotypes found in this study could be classified as 13 high-risk HPV, 2 low-risk HPV, and 2 additional types. Of the specimens, 94.8% had at least one high-risk HPV genotype infection. CONCLUSION: As for the potential benefits of commercially available prophylactic vaccines to prevent HPV infection in Thailand, both vaccines (bivalent and quadrivalent) can protect from HPV-related cervical cancer in only approximately 71%. Therefore, screening programs such as routine Papanicolaou test, cytology, and HPV DNA detection are still essential for cervical cancer prevention. Moreover, future generations of HPV vaccines should also include the other most common genotypes and decrease the severe adverse effects reported at the present time.

Seroprevalence of an antibody against diphtheria, tetanus, and pertussis among the elderly in Khon Kaen, Thailand.

BACKGROUND: Owing to a declining birth rate and longer lifespan, the number of elderly people (≥ 60 years) in Thailand has grown rapidly. However, the elderly are at significant risk of infectious diseases because they have never been immunized, because they have not been completely immunized, or because their immunity has waned. Immunity against infectious diseases in the elderly is an important means of controlling diseases in the community. Our objective was to evaluate the seroprotective rate against diphtheria, tetanus, and pertussis in the elderly Thai population. METHODS: In total, 430 healthy individuals from the northeastern region of Thailand were enrolled in this study and stratified into five age groups: 60-65, 66-70, 71-75, 76-80, and > 80 years. Serum samples were collected and quantitatively analyzed for diphtheria, tetanus, and pertussis IgG antibody by using commercial ELISA kits. For anti-diphtheria toxoid and anti-tetanus toxoid ELISA, values < 0.01 IU/ml were interpreted as seronegative, and for anti-Bordetella pertussis toxin ELISA, values < 5 IU/ml were interpreted as seronegative; these definitions were in accord with previous studies. RESULTS: For diphtheria toxoid Ab, the majority of the population had antibody levels > 0.01 IU/ml. For tetanus anti-toxoid Ab, the majority of the population had antibody levels of > 0.01 IU/ml, of which approximately 34% had durable antibody protection levels (DAPL) of ≥ 1 IU/ml. Meanwhile, nearly 45% of the population had an Ab level against pertussis lower than the protectivity level. CONCLUSIONS: In total, 97.2%, 83.5%, and 55.8% of the population had a higher antibody level than the minimal protective level for diphtheria, tetanus, and pertussis, respectively. In order to prevent an outbreak of these diseases in the future, the elderly should be administered with Tdap revaccination to provide diphtheria herd immunity in the population; this will increase cocoon phenomenon for pertussis and protect the population from tetanus-prone injury.

Serological evidence of hepatitis A, B, and C virus infection in older adults in Khon Kaen, Thailand and the estimated rates of chronic hepatitis B and C virus infection in Thais, 2017.

Hepatitis A (HAV), hepatitis B (HBV), and hepatitis C (HCV) viruses are hepatotropic viruses responsible for acute/chronic hepatitis associated with liver failure, cirrhosis, and hepatocellular carcinoma. Due to the limited data on the prevalence of hepatitis in the older population in Thailand, this study aimed to evaluate the seroprevalence of these viruses in elderly Thais. Using an automated immunoassay, serum samples from individuals older than 60 years of age in Chum Phae district of Khon Kaen province in northeast Thailand were analyzed for anti-HAV (n = 93), HBV markers (n = 460, HBsAg, anti-HBs, and anti-HBc), and anti-HCV (n = 460). Samples were classified into five age groups (61-65, 66-70, 71-75, 76-80, and >80 years). The overall seroprevalence of anti-HAV, HBsAg, anti-HBc, anti-HBs, and anti-HCV was 98.9%, 4.6%, 51.5%, 32.4%, and 1.3%, respectively. When samples were stratified into three groups representing three generations (children/young adults aged 6 months-30 years and middle-aged adults between 31-60 years old from a previous survey, and older adults aged >60 years from the current study), the highest levels of anti-HAV and anti-HBc were found in older adults. Children/young adults had the lowest levels of HBsAg and anti-HCV, and the highest level of anti-HBs. These findings are consistent with the integration of HBV vaccination into the Expanded Program on Immunization (EPI) in 1992 and coincide with increased awareness of blood-borne viral transmission in Thailand. Extrapolating from our data, the estimated numbers of cases of chronic HBV and HCV infection in Thailand in 2017 were 2.2 and 0.79 million, respectively. Thus, effective treatments for viral hepatitis B and C for middle-aged and elderly Thais are needed. This seroprevalence survey could be used to help formulate policies and possible guidelines for treatment and prevention in specific age groups, which is recommended to facilitate the elimination of viral hepatitis by 2030.

Lack of Associations between TNF-αPolymorphisms and Cervical Cancer in Thai women.

The risk of developing cervical cancer in women infected with human papillomavirus (HPV) may be influenced by an individual's genetic susceptibility. Published data linking single nucleotide polymorphisms (SNPs) in the tumor necrosis factor-alpha (TNF-?) promoter region at positions -308G>A (rs1800629) and -238G>A (rs361525) to cervical cancer risk have been inconclusive. In this study, we examined 251 cervical specimens and classified them into two groups according to their cytological findings: 121 cancer cases and 130 controls (low-grade squamous intraepithelial lesion and normal cytology). All specimens were typed by PCR and sequencing for TNF-αpromoter -308G>A (rs1800629) and -238G>A (rs361525). The genotype distribution of SNPs in either rs1800629 or rs361525 did not significantly demonstrate higher frequency in the cancer group (p=0.621 and p=0.68, respectively). Based on these results, neither the TNF-αpromoter -308G>A (rs1800629) nor the -238G>A (rs361525) polymorphism presents a major risk factor for cervical cancer among Thai women. Larger studies are necessary to elucidate possible genetic mechanisms influencing cervical cancer development.

Comparative study of different methods to genotype hepatitis C virus type 6 variants.

Hepatitis C virus (HCV) genotype 6a is found frequently in Southeast Asia. In Thailand, however, genotype 6 variants may exist which posses a genotype 1 like sequence in the 5' non-coding region. In order to genotype correctly these viruses, four different methods; INNO-LiPA assay, two RFLP assays on the core region (using different restriction enzymes) and phylogenetic analysis of the core sequences were compared. Samples from 17 chronic HCV patients from the Netherlands and Thailand and 18 anti-HCV positive blood donors recruited from Thailand were tested. The INNO-LiPA could not distinguish genotype 6 variants. The RFLP methods used could not, or only in combination with 5'NCR genotyping methods, identify type 6 variants. In conclusion, for identification of type 6 variants at least two different regions of the HCV genome have to be analyzed (both 5'NCR and core).

Interspousal transmission of hepatitis C in Thailand.

BACKGROUND: Previous studies evaluating the possibility of interspousal sexual transmission of hepatitis C virus (HCV) have yielded many conflicting results. Our study was carried out to determine the exact potential and risk factors of interspousal HCV transmission. METHODS: The spouses (54 men and 106 women; mean age +/-SD, 48 +/- 8 years) of 160 patients with HCV infection (106 men and 54 women) were serologically tested for HCV using a third-generation enzyme-linked immuno- sorbent assay (ELISA). Positive results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). For positive couples, the cluster nucleotides of the HCV gene and genotypes were compared on the basis of restriction fragment length polymorphism (RFLP), Innogenetic Line Probe Assay (INNO-LiPA), and direct sequencing. Similarly, phylogenetic tree and sequence homology analysis was performed in order to precisely verify interspousal transmission. Risk factors promoting interspousal HCV transmission were also identified. RESULTS: Throughout a mean duration of exposure of 23 + 5 years, most of the 160 partners had their usual and unprotected sexual relationships with the index patients. HCV-associated antibodies and HCV-RNA were detected in only 3 (1.88%) of the 160 spouses. Furthermore, homology and phylogenetic tree analysis could not clearly demonstrate that any one of these 3 positive spouses was infected with the same strain of HCV as that identified in the index cases. Because a positive group remained elusive, risk factors of interspousal HCV transmission could not be determined in this study. CONCLUSIONS: According to this study, interspousal transmission of HCV seems to be very rare. HCV-positive spouses should be firmly reassured that they can maintain their normal marital life.

Prevalence and genotypes of hepatitis C virus infection among drug addicts and blood donors in Thailand.

Hepatitis C virus (HCV) is an infectious agent that has the potential to cause chronic liver disease, cirrhosis and hepatocellular carcinoma. We determined the prevalence and genotypes of HCV infection among groups of drug addicts: intravenous drug users (n = 134), methamphetamine users (n = 100), inhaled-drugs users (n = 19) and alcoholics (n = 50); a group of blood donors acted as a control. The control group consisted of 179 randomly-selected anti-HCV positive samples: these were subjected to HCV RNA screening and genotyping. The anti-HCV test was performed by ELISA: HCV RNA screening was by nested RT-PCR that employed primers from the 5' noncoding region. The genotype assay was based upon analysis of the 5' NCR amplified sequences and RFLP. Hepatitis C virus was highly prevalent among all groups of drug addicts (12-70%). In 2000. among the new blood donors (n = 66,340) at the National Blood Center, Thai Red Cross, anti-HCV prevalence amounted to 0.98%. The HCV genotype distribution showed that the most prevalent genotype was 3a, followed by 1b and 6a. Our data demonstrated the very high prevalence of HCV infection in IVDUs, a finding that is consistent with the blood-borne nature of the virus. In order to curb HCV infection, a determined effort to educate both the general population and high-risk groups is required; such a program of education would address both general and particular methods of transmission, especially the use of non-sterile needles etc.

Polymorphisms in TP53 (rs1042522), p16 (rs11515 and rs3088440) and NQO1 (rs1800566) genes in Thai cervical cancer patients with HPV 16 infection.

The risk of cervical cancer development in women infected with HPV varies in relation to the individual host's genetic makeup. Many studies on polymorphisms as genetic factors have been aimed at analyzing associations with cervical cancer. In this study, single nucleotide polymorphisms (SNPs) in 3 genes were investigated in relation to cervical cancer progression in HPV16 infected women with lesions. Two thousand cervical specimens were typed by PCR sequencing methods for TP53 (rs1042522), p16 (rs11515 and rs3088440) and NQO1 (rs1800566). Ninety two HPV16 positive cases and thirty two normal cases were randomly selected. Analysis of TP53 (rs1042522) showed a significantly higher frequency in cancer samples (OR=1.22, 95%CI=1.004-1.481, p-value=0.016) while differences in frequency were not significant within each group (p-value=0.070). The genotype distributions of p16 (rs11515 and rs3088440) and NQO1 (rs1800566) did not show any significantly higher frequency in cancer samples (p-value=0.106, 0.675 and 0.132, respectively) or within each group (p-value=0.347, 0.939 and 0.111, respectively). The results indicated that the polymorphism in TP53 (rs1042522) might be associated with risk of cervical cancer development in HPV16 infected women. Further studies of possible mechanisms of influence on cervical cancer development would be useful to manage HPV infected patients.

High-risk human papillomavirus genotype detection by electrochemical DNA chip method.

High-risk human papillomavirus (HPV) genotypes are the major cause of cervical cancer. Hence, HPV genotype detection is a helpful preventive measure to combat cervical cancer. Recently, several HPV detection methods have been developed, each with different sensitivities and specificities. The objective of this study was to compare HPV high risk genotype detection by an electrochemical DNA chip system, a line probe assay (INNO- LiPA) and sequencing of the L1, E1 regions. A total of 361 cervical smears with different cytological findings were subjected to polymerase chain reaction-sequencing and electrochemical DNA chip assessment. Multiple infections were found in 21.9% (79/361) of the specimens, most prevalently in 20-29-year olds while the highest prevalence of HPV infection was found in the 30-39-year age group. The most prevalent genotype was HPV 16 at 28.2% (138/489) followed by HPV 52 at 9.6% (47/489), with the other types occurring at less than 9.0%. The electrochemical DNA chip results were compared with INNO-LiPA and sequencing (E1 and L1 regions) based on random selection of 273 specimens. The results obtained by the three methods were in agreement except for three cases. Direct sequencing detected only one predominant genotype including low risk HPV genotypes. INNO-LiPA identified multiple infections with various specific genotypes including some unclassified-risk genotypes. The electrochemical DNA chip was highly accurate, suitable for detection of single and multiple infections, allowed rapid detection, was less time-consuming and was easier to perform when compared with the other methods. It is concluded that for clinical and epidemiological studies, all genotyping methods are perfectly suitable and provide comparable results.

Comparison between direct sequencing and INNO-LiPA methods for HPV detection and genotyping in Thai Women.

Human papillomaviruses (HPVs) have been recognized as etiologic factors in cervical carcinoma and several other anogenital cancers in females and males. HPV are classified as low risk (LR), probable high risk and high risk (HR) on the basis of their oncogenic potential. HPV genotypes, which are crucial for diagnosis and relationship with carcinogenesis, have been determined by several genotyping methods. In this study, two genotyping methods were compared: direct sequencing and INNO-LiPA. In total, 2,494 cervical specimens were tested and 27.2 % of these were found to be HPV DNA positive with 24.5% showing normal cytology. Specimens were divided into four groups according to their pathological cytology as normal, LSIL, HSIL and cancer and 134 specimens were selected for HPV genotyping by both methods. HPV genotyping results showed 87.5% positive correlation. With 17 specimens, the results were discordant, 12 specimens showed different genotypes. Others had genotypes that could not be typed by the INNO-LiPA method. Neither did direct sequencing in 3 different regions yield unequivocal results. Both genotyping methods have advantages and disadvantages. Consequently, the method most suitable for the study objective, budget and predominance of HPV genotype in any given area should be selected.

Long-term humoral and cellular immune response to hepatitis B vaccine in high-risk children 18-20 years after neonatal immunization.

Eighty-seven high-risk individuals in Thailand who had received a complete course of recombinant HBV vaccine 18-20 y ago were investigated with regard to their immunological memory. To evaluate humoral immunity, anti-HBs antibody titers were measured. Cellular immunity was determined by ELISPOT to detect HBV-specific IFN-gamma-producing cells. Overall 83.9% of participants developed circulating anti-HBs (titer > or = 1 mIU/mL) and 58.6% were seroprotected (titer > or = 10 mIU/mL). As for cellular immunity, 50.6% were positive on ELISPOT. Moreover, there was no correlation between the level of anti-HBs and positive ELISPOT results. However, the majority of participants (81.8%) who were positive for IFN-gamma-producing cells were seropositive, but only 50% of seropositive participants were ELISPOT-positive. Thus, 18-20 y after immunization, it appears that a second booster dose should be considered, especially in high-risk groups.

222 base pairs in NS5B region and the determination of hepatitis C virus genotype 6.

OBJECTIVE: The present study was performed to genotype hepatitis C virus (HCV) by direct sequencing of a 222-bp nucleotide in the NS5B region and comparing the results with those of direct sequencing in the core region. We investigated a new region for HCV genotyping which gave the best performance to discriminate HCV genotype 6a, the unique genotype found in Southeast Asia. METHODS: Plasma samples taken from 57 HCV-infected blood donors were used in this study. RT-PCR products were amplified using primers located in the NS5B region. The 222-bp PCR products were purified and sequenced. The genotype of HCV isolates were obtained by phylogenetic analysis and compared with HCV reference strains stored in the GenBank database. The HCV sequences clustering in the same node were considered to be of the same genotype. RESULTS: Thirty-one, 22 and 4 samples of HCV genotype 3a, 1a and 1b, respectively, were analyzed by this method. Upon comparison with genotyping in the core region, 86 and 14% of the samples yielded concordant and discordant genotype results, respectively. The majority of discordant results (63%; 5 of 8) was observed with HCV genotype 6a which yielded 6a upon core sequencing as opposed to 1a or 3a upon NS5B sequencing. CONCLUSION: HCV genotype 6a obtained by direct sequencing in the core region could not be unequivocally arrived at by sequencing 222 bp in the NS5B region. Hence, sequencing in the core region is preferable for genotyping our specimens, even though longer PCR products are required as this method enables discrimination between genotype 6a and the remaining genotypes.