Chromosome Abnormalities and Absolute Telomere Lengths of Leukocytes from Silk Weavers with Emphasis on Potential Genotoxicity and Mutagenicity of Silk Dyes

Objectives: This study is aimed to assess the possible genotoxicity and mutagenicity of silk dyes on silk weavers. Methods: Peripheral blood leukocytes were obtained from 24 silk weavers and 24 age- and sex-matched controls in northeastern Thailand. After mitogen stimulation in culture, chromosome abnormalities were examined using Giemsa banding and the absolute telomere length (aTL) was measured with SYBR green qRT-PCR. To confirm genotoxic and mutagenic effects of silk dyes, leukocytes from one each of healthy male and female volunteers were cultured with various concentrations of 3 dark red silk dyes under the presence of mitogen. Chromosome abnormalities and the telomere length were determined as above. Results: The proportion of normal metaphase in the silk weaving workers was significantly lower than that in controls. The frequency of chromosome aberrations was higher in the silk weavers than in control group. Polyploidy was detected only in the silk weavers. The aTL was significantly shorter in the silk weavers than in control group (p < 0.05). When leukocytes from normal volunteers were stimulated with mitogen under the presence of various concentrations of 3 silk dyes, suppressed the mitotic index (MI) and normal metaphase, whereas the proportion of prophase and the incomplete chromosome forming increased significantly. All dyes induced polyploidy. Dye #CA5 induced structural changes in male leukocytes, whereas #30 induced the changes in female leukocytes. The #CA5 increased aTL of normal leukocytes in a dose-dependent manner. Conclusions: All dyes, especially #CA5, have high genotoxicity and mutagenicity to induce chromosome aberrations and telomeric instability. Taken all those results together, regular health checking of silk weavers who have been exposed to those dyes is critically necessary to prevent various chemical-induced carcinogenesis.

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax.

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380bp for An. minimus, 180bp for An. harrisoni, 150bp for An. aconitus, 310bp for An. varuna, 276bp for P. falciparum, and 300bp for P. vivax. The sensitivities were 0.5pg/µl (25sporozoites/µl) for P. falciparum DNA and between 0.5 and 5pg/µl (25-250sporozoites/µl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.

Comparison of Pap smear screening results between Akha hill tribe and urban women in Chiang Rai province, Thailand.

Cervical cancer is an important woman's health problems worldwide, especially in low socio-economic countries. The aim of this study was to compare the Pap smear screening results between Akha hill tribe and urban women who live in Chiang Rai province, Thailand. Screening was conducted for 1,100 Akha women and 1,100 urban women who came to have the Pap smear at Chiangrai Prachanukroh Hospital and 1 private cytology laboratory from January to June 2008. The demographic characteristics and factors related to abnormal Pap smears of these women were gathered using closed model questionnaires. Abnormal Rap smears were defined according to the Bethesda 2001 system. The results showed that the prevalence of abnormal Pap smears was 12.2% in Akha women and 4.5% in urban women. The highest prevalence of Pap abnormalities was found in the 41-50 years age group in both populations (4.5% in Akha and 1.7% in urban women). In both populations, abnormal Pap smears were found in <21 years age groups. From the questionnaires, the possible risk factors related to the higher prevalence of abnormal Pap smears in Akha women were early age at marriage (≤17 years), high frequency pregnacies and high parity and no/low education level. In conclusion, cervical cancer control by education and early detection by Pap smear screening is necessary for hill tribe women. More Pap smear screening service units should be set to improve the coverage for the risk group women who got married in young age, especial in ethnic groups.

Combined p16INK4a and human papillomavirus testing improves the prediction of cervical intraepithelial neoplasia (CIN II-III) in Thai patients with low-grade cytological abnormalities.

Thailand is in the process of developing a national cervical screening program. This study examined p16INK4a staining and HPV prevalence in abnormal cervical samples with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL), to evaluate the efficacy of combined HPV and p16INK4a detection to predict CIN II-III. Totals of 125 ASCUS and 87 LSIL cases were re-evaluated by Pap test and cervical cells of ASCUS and LSIL cases were prepared on slides for p16INK4a detection by immunocytochemistry. HPV genotyping of DNA extracts was performed by GP5+/6+ PCR and reverse line blot hybridization. Histopathologic tests were performed to identify cervical lesion. Total of 212 cases were diagnosed to normal (20), ASCUS (112), LSIL (78) and HSIL (2). HPV was detected in ASCUS (49/112, 43.8%), LSIL (60/78, 76.9%) and HSIL (2/2, 100%) cases. The majority of HPV positive samples typed for high-risk HPV. 55.7% (107/192) of abnormal cases (ASCUS, LSIL and HSIL) were positive p16INK4a. For the 111 HPV DNA positive cases, 34 of 49 (69.4%) ASCUS cases and 49 of 60 (81.7%) LSIL cases were p16INK4a positive. 140 biopsies were taken and histological classified: CIN negative (65 cases), CIN I (56 cases) and CIN II-III (19 cases). HPV DNA detection predicted CIN II-III with sensitivity and specificity of 84% and 49%, whereas p16INK4a staining showed higher sensitivity (89.5%) and specificity (56.2%). The prediction of CIN II-III was significantly better by combination of positive HPV DNA and p16INK4a with 93.8% sensitivity and 59.2% specificity. Detection of HPV DNA combined with p16INK4a in cervical cells can predict CIN II-III and may improve the screening diagnosis of Thai women at risk for CIN II-III or cancer.

Genetic analysis of the merozoite surface protein-1 block 2 allelic types in Plasmodium falciparum clinical isolates from Lao PDR.

BACKGROUND: MSP-1 is one of the potential malarial vaccine candidate antigens. However, extensive genetic polymorphism of this antigen in the field isolates of Plasmodium falciparum represents a major hindrance for the development of an effective vaccine. Therefore, this study aimed to establish the prevalence and genetic polymorphisms of K1, MAD20 and RO33 allelic types of msp-1 block 2 among P. falciparum clinical isolates from Lao PDR. METHODS: Plasmodium falciparum isolates were collected from 230 P. falciparum-infected blood samples from three regions of Lao PDR. K1, MAD20 and RO33 were detected by nested PCR; SSCP was used for polymorphism screening. The nested PCR products of each K1, MAD20 and RO33 allelic types that had different banding patterns by SSCP, were sequenced. RESULTS: The overall prevalence of K1, MAD20 and RO33 allelic types in P. falciparum isolates from Lao PDR were 66.95%, 46.52% and 31.30%, respectively, of samples under study. Single infections with K1, MAD20 and RO33 allelic types were 27.83%, 11.74% and 5.22%, respectively; the remainders were multiple clonal infections. Neither parasite density nor age was related to MOI. Sequence analysis revealed that there were 11 different types of K1, eight different types of MAD20, and 7 different types of RO33. Most of them were regional specific, except type 1 of each allelic type was common found in 3 regions under study. CONCLUSIONS: Genetic polymorphism with diverse allele types was identified in msp-1 block 2 among P. falciparum clinical isolates in Lao PDR. A rather high level of multiple clonal infections was also observed but the multiplicity of infection was rather low as not exceed 2.0. This basic data are useful for treatment and malaria control program in Lao PDR.

A survey of malarial infection in endemic areas of Savannakhet province, Lao PDR and comparative diagnostic efficiencies of Giemsa staining, acridine orange staining, and semi-nested multiplex PCR.

Malaria remains one of the most important parasitic diseases in Lao PDR, especially in forested rural areas. Knowing the rate of infection using highly sensitive and specific methods, and the factors related to malarial infection, may be helpful in reducing the infection and mortality rates. We aimed to study the malarial infection rate by comparing three detection methods, i.e., Giemsa staining, acridine orange (AO) staining and semi-nested multiplex PCR. The study also included some factors related to malarial infection in the endemic areas of Savannakhet province, Lao PDR. The respective malarial infection rates by Giemsa staining, AO staining and semi-nested multiplex PCR in Houy Jang vs. Keng Thong villages were 13.1 vs. 20.8, 16.2 vs. 25.4 and 20.8 vs. 30.8%. The infection rate among children not over 10 years of age was higher than infection rate among the older ages (p=0.002, Z-test for two proportions). The higher infection rates by semi-nested multiplex PCR over Giemsa and AO staining suggest the existence of many subclinical cases with low level parasitemia, undetected by microscopic techniques. We found no mixed infections using Giemsa or AO staining, but using semi-nested multiplex PCR we found 1.2% (3/260) mixed P. falciparum and P. vivax infections, suggesting that semi-nested multiplex PCR is suitable for detecting malarial infection from endemic areas whose cases may have low parasitemia and/or mixed infection. The factors significantly related to malarial infection from 260 questionnaires were: (1) children and young adults, (2) not having lived in the area more than 5 years, and (3) not using a mosquito net over the bed, indicating an increased risk of new residents of contracting malaria and a need to promote bed nets.

Pap smear, colposcopy and biopsy findings at follow-up of Pap smear positive women in North-east Thailand.

As part of an ongoing project involving a large cohort in the Khon Kaen Province in the North-east of Thailand, a total of 236 women who had tested positive for a Pap smear at the initial recruitment and advised to seek medical attention were followed up after a mean period of 3.1 years. The 204 individuals who could be contacted were interviewed to determine treatments received and underwent a further Pap smear as well as colposcopy in 179 of the cases. On clinical advice, biopsies were also taken from 32 of these. Only 15% of the total of 204 had actually received therapy, the majority undergoing surgery (self-reported). Possible positive Pap smear results were obtained for 23.5%, with 6.4% having high grade squamous intraepithelial lesions (HGSILs) or squamous cell carcinoma (SCC) (one case). Comparison of the different testing modalities demonstrated 5.6% false negatives and 16.2 false positives for the Pap smear with colposcopy as the gold standard. Compared with biopsy findings, there were 21.8% and 40.6% false positives with Pap and colposcopy, respectively, but no false negatives. The present results point to good efficacy for the initial screening, since only 0.5% of the total population developed an SCC. However, judgement as to therapy should depend on a biopsy since there were considerable false positives with the other two modalities employed.

Usefulness of combining testing for p16 protein and human papillomavirus (HPV) in cervical carcinoma screening.

OBJECTIVE: To evaluate the value of the combination of p16 and HPV detection in the screening for cervical cancer. METHODS: 186 patients with previous abnormal cervical lesion were studied. After colposcopic examination, two conventional Pap slides were prepared: the first was Papanicolaou-stained and examined by cytologist; the second was immunocytochemically stained for p16. Cervical cells were collected by brush using for HPV detection by Hybrid Capture II. Biopsy of any colposcopically abnormal lesions was performed. RESULTS: The 186 cervical samples were classified cytologically as normal (148), ASCUS (13), low-grade (11), high-grade (12) dysplasia and squamous cell carcinoma (2). P16 and HPV were found in all high-grade dysplasia and SCC, and in 64% and 27% of low-grade dysplasia, 62% and 0% of ASCUS and 7.4% and 3.4% of normal, respectively. 18 of p16-positive cases (11%) were HPV-negative, 14 of them in the ASCUS and normal group. Compared to histological results, all of the p16-positive cases of squamous metaplasia, CIN II/III and SCC were HR-HPV-positive. Therefore, the cases that were positive for both with normal cytology (5 cases) or low-grade dysplasia (3 cases) may comprise a high-risk group for neoplastic change. CONCLUSION: The combination of p16 and HPV detection may be useful in cervical cancer screening to identify high-risk patients requiring early and proper management.

Consistency of cytology diagnosis for cervical cancer between two laboratories.

The principal approach to the prevention of cancer of the cervix uteri has been through screening programmes, using the cervical smear (Pap test) to detect precursor lesions. The sensitivity and specificity of Pap smears depend on the skill of the observer in recognizing and classifying a variety of cellular abnormalities. We have studied the reproducibility of cytological diagnosis, according the Bethesda classification, made by cytologists in Khon Kaen, north-east Thailand, and in Helsinki, Finland, on smears taken from rural women undergoing screening during 1994-2001. A total of 313 slides were reviewed. The prevalence of abnormalities was relatively high, since the series included smears judged abnormal in Khon Kaen or from women who developed cancer during follow-up, as well as a group whose smears were negative. In general, the reviewing cytologist in Finland evaluated more slides as abnormal than in the initial report. The level of agreement between the two observers was evaluated by calculating the coefficient of concordance (Kappa). The kappa score depended upon the degree of detail in the diagnosis; it was 0.43 for the presence or not of an epithelial abnormality (the General Categorization of the Bethesda system), and rather higher (0.5) for separating low grade from high grade (HSIL or worse) abnormalities or glandular lesions. Agreement was only fair (0.37) when the more detailed Bethesda categories (seven) were used. The reproducibility of cervical cytology evaluations is critical to the success of screening programmes, and in this programme in a moderate-high risk population of women in rural Thailand, we found that agreement between skilled observers, at the level of tests requiring diagnostic follow-up or not, was only moderate. The women in this study are being traced to evaluate the true sensitivity of screening in terms of the lesions found on histology, during a prolonged follow up of 4 or more years.

Immunocytochemical staining of p16INK4a protein from conventional Pap test and its association with human papillomavirus infection.

The p16INK4a protein is immunocytochemically detected in liquid-based (LB) specimens as a diagnostic marker of cervical dysplasia and neoplasia. Its up-regulation is promoted by high-risk human papillomavirus (HR-HPV) infection. We aimed to detect p16INK4a on conventional Papanicolaou (Pap) test (CPT) slides and to determine the relationship between its overexpression and HR-HPV infection. CPT and LB Pap test (LBPT) slides (165 samples of each) were examined by immunocytochemical staining for p16INK4a. After polymerase chain reaction (PCR), HPV-DNA was genotyped by dot blot hybridization. The CPT slides displayed more numerous dispersed squamous cells and LBPT slides had a clearer background. Positive p16INK4a on CPT occurred in 0% (0/30), 52.5% (21/40), 54.3% (19/35), 100% (30/30), and 100% (30/30) in normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSILs), high-grade SILs (HSILs), and squamous cell carcinomas (SCCs) cases, respectively. LBPT slides showed comparable results but were less sensitive. HPV-DNA was detected in 86.7, 70, 45, 57.14, and 10% in SCCs, HSILs, ASCUS, LSILs, and normal cervical cells, respectively. Because HR-HPV was identified in all HPV+ samples of high-grade dysplasia (HSILs and SCCs) and all positive p16INK4a samples infected with HR-HPV, the association of p16INK4a overexpression with HR-HPV infection was confirmed. This study suggests that immunocytochemical staining of p16INK4a on CPT slides is convenient and cost-effective for cervical cancer screening by the detection of dysplastic cells infected with HR-HPV.

Experience with a self-administered device for cervical cancer screening by Thai women with different educational backgrounds.

BACKGROUND: This descriptive study was carried out to test the acceptability of a self-administered device for cervical cancer screening and assess certain risk factors in relation to the cancer in two districts of Khon Kaen province in Northeast Thailand. METHODS: A total of 354 women from the villages were selected (including 143 teachers from secondary and primary schools; 24 health officers from the rural health centres, and 37 nurses from the University Hospital, Khon Kaen University). The Kato device was introduced and used by the women, who were then asked to give their opinion on its usage. RESULTS: The Kato device was generally well accepted by the women. However, many of those with a higher educational background were more sceptical towards the device than their counterparts from the villages. CONCLUSIONS: A suitable approach to strengthen the control of cervical cancer in the rural areas of Thailand might be to introduce the Kato device as an integral part of primary health care. The introduction of the device should go along with health education on the importance of avoiding infection with the papilloma viruses. This message should be transmitted to both females and males.

Immunocytochemical detection of p16INK4a protein in scraped cervical cells.

OBJECTIVE: To develop an immunocytochemical technique for p16INK4a protein detection in scraped cervical cells for cancer screening. STUDY DESIGN: We took duplicate cervical scrapes from each participant, the first for a Pap smear and the second for p16INK4a protein detection. From a 50-microL cell suspension prepared from the scrape rinsing, a 10-microL aliquot was dropped in a 5-mm-diameter circle on a glass slide, air dried and fixed in 0.1% formal saline (1 hour) and in 95% ethanol (10 minutes). Using the immunocytochemical technique, slides from 30 samples of each Pap diagnosis class were stained sequentially with mouse monoclonal anti-p16INK4a (primary antibody), biotinylated goat antimouse IgG (secondary antibody), horse-radish peroxidase-labelled streptavidin and 3,3'-diaminobenzidine and mixed hydrogen peroxide, then counterstained with hematoxylin. A positive sample had to contain > or = 3 immunoreactive cells. Results were confirmed by western blot analysis of lysates from the remaining 40 microL of each cervical cell suspension. RESULTS: Samples were grouped as control (normal cervical cells), mild dysplasia (ASCUS, LSIL) and high abnormality (HSIL, SCC). Using the immunocytochemical technique, > 95% of the positive (SiHa cells) but 0% of the negative controls (human embryonic lung fibroblast cells) showed immunoreactive cells. All slides displayed a clear background without mucus, and positive cells were stained in both the cytoplasm and nucleus. p16INK4a Protein was detected in 17 of 30 (56.67%) ASCUS and 10 of 30 (33.33%) LSIL and increased with the degree of abnormality to 93.33% (28 of 30) and 96.67% (29 of 30) in the HSIL and SCC group, respectively. Normal cervical cells and degenerated malignant cells were nonimmunoreactive. Western blot analysis confirmed similar positive samples in the low-abnormality group, while the whole high-abnormality group was immunoreactive. A sampling error might have caused the 2 HSIL and 1 SCC sample to be negative using our immunocytochemical technique. CONCLUSION: p16INK4a Protein detection in scraped cervical cells using the immunocytochemical technique correlated with western blot analysis and was nontraumatic and precise. It offers a significant diagnostic adjunct to the Pap test for cervical cancer screening.

The coverage of cervical cancer screening in Khon Kaen, northeast Thailand.

Cervical cancer is a common cancer in Thai women and one of the only cancers that can be readily cured if early detection is successful. The Pap smear is an accepted as an appropriate means for cervical cancer screening at present. However, there are still some management problems with early detection programmes. Since data showing how many women have been screened by Pap smear are limited in Thailand, the present study was conducted with the aim of determining coverage in a defined population in the sample area, Thakaserm sub- district in Nampong district, Khon Kaen province, Thailand. The investigation was carried out during June-August 2000 to collect information on history of screening for cervical cancer using questionnaires. All women aged 20 and above were asked to answer the set of questionnaires a total of 1199 women responded. There were 66.9% that reported having received a Pap smear test for screening for cervical cancer at least once. However, there were 33.1 % that had never undergone a Pap smear in their life. It is important to find a strategy to increase the coverage of cervical cancer screening programme for this population.

A comparison of cytology with Pap smears taken by a gynecologist and with a self-sampling device.

Cervical cancer is the most common cancer in Thai women and as yet screening programmes are minimally effective. The Pap smear is the test accepted to be most appropriate for cervical cancer screening so far. One of the main reasons why women do not come to have Pap smear done is "shyness", which weakens compliance with recommendations to undergo Pap smear with pelvic examination. The self-administered device by the Kato method was established to overcome this problem and the present study was carried out to confirm the adequacy of the specimens obtained with this technique in comparison with specimens collected by gynecologists. Two hundred women were invited to participate in the study voluntarily. Each was allocated to have a Pap smear conducted by a gynecologist and then instructed to produce a self-obtained smear using Kato's device. The cytology results of Pap smears from both methods were compared to test for agreement using Kappa statistics. There was agreement between the adequacy of smears collected by gynecologists and those self-sampled with the percentage agreement of 96.5% and a Kappa score of 0.43 (95% CI 0.33-0.54, P<0.001). There were 8 cases detected as epithelial cell abnormalities from the cervical cells collected by gynecologists and also with the self-administered technique. Good agreement for detection of cellular changes was found with a percentage of 78.0% and the Kappa's score was 0.61 (95% CI 0.46-0.76, P<0.001). The results from this study provide convincing evidence that the self-administered device can be an alternative choice for women who are too shy to undergo pelvic examination or even for those who have limited time to visit health care centers or doctors to have a Pap smear test.