Orientia tsutsugamushi is a pathogen transmitted by Leptotrombidium that causes scrub typhus. To develop an infection mouse model, a mite-derived isolate of O. tsutsugamushi was established from a laboratory-maintained colony of Leptotrombidium chiangraiensis (O. tsutsugamushi Lc-1). This Lc-1 isolate was initially presented to ICR (CD-1) mice by feeding an infected Lc chigger on the ear of a mouse. Once the Lc-1 was adapted to the ICR mice, quantitative real-time polymerase chain reaction was used to investigate O. tsutsugamushi genomic equivalent copies in tissues and sera. Furthermore, times to onset of the signs of infection are reported in this study. This study provides information useful for future research on this host-pathogen interaction and the associated vaccine efficacy trials.
Background. Oral lichen planus (OLP) is a common chronic inflammatory immune-mediated disease, with an etiopathogenesis associated with cell-mediated immunological dysfunction. Viral infection has been hypothesized as a predisposing factor in the pathogenesis of this disease. Viruses may alter host cell function by inducing the abnormal expression of cellular proteins leading to disease development. However, reports on the relationship between human papillomavirus (HPV) and OLP are inconclusive. Objective. To explore the association between HPV and OLP in Thai patients. Materials and Methods. DNA was extracted from thirty-seven fresh-frozen tissue biopsy specimens from OLP lesions, and polymerase chain reaction assay for the L1 and E1 genes covering 32 types of high- and low-risk HPV was performed. Results. HPV DNA was detected in one tissue biopsy from an atrophic-type OLP lesion. All control samples were negative. Genomic sequencing of the E1 gene PCR product demonstrated that the HPV-type 16 found in the lesion is closely related to the East Asian type. Conclusion. Our data indicate a low prevalence of HPV infection in OLP lesions in Thai patients.
Cervical cytological data may not be sufficient for cervical cancer screening and prevention. In this project, we determined HPV genotype among infected Thai women with different cytological findings by characterization of E1 genes. Five hundred and thirty-five specimens were tested by PCR amplification of the E1 genes. HPV genotypes were determined by sequencing, comparison with the GenBank database and were analyzed in relation to different cytological findings. HPV-DNA by PCR were typed and revealed 32 different genotypes. HR-HPV (HPV16, 18 or 52) was detected in all samples with cervical cancer cytology. HPV16 was most prevalent irrespective of cervical cytology. Moreover, HPV31 and 52 were most prevalent in the HSIL and LSIL groups whereas HPV66 was found mostly in the LSIL group. The LSIL group displayed the highest variation of HPV genotypes. Moreover, HPV31 and 52 predominated in the HSIL and LSIL groups especially HPV52 which was found in cancer samples. We hoped that these data of HPV genotypes can be used as preliminary data of HPV in Thailand and can serve as basic data for future research into the HPV genotype in south-east Asia.
One of the most common cancers in women worldwide is cervical cancer, with death rates highest in less developed countries, including Thailand. This study was conducted to explore the prevalence of human papillomavirus (HPV) and its related cytological abnormalities among women attending cervical screening clinics in Thailand using the polymerase chain reaction (PCR). LBC specimens (ThinPrep, Hologic, West Sussex, UK) were subjected to PCR of the E1 region to identify the most prevalent HPV types. Information on age and cytology grade was also collected. Among a total of 1,662 women, 29 different HPV types were found and the overall HPV prevalence was 8.7%. HPV prevalence among the general population amounted to 7.8%. The following HPV types were identified: HPV16 (17.9%), HPV90 (16.6%) and HPV71 (10.3%). The rates of other types were as follows; HPV66 (6.9%), HPV52 (6.2%), HPV34 (5.5%), HPV31 (5.3%), HPV42 (4.8%) and HPV39 (3.4%). HPV infection peaked in women aged around 20-39 years and thereafter gradually declined. As expected, HPV DNA can be found in normal cytology specimens. These results which elucidate HPV distribution in Thailand could be useful for vaccine development and the national cervical cancer prevention program.
The aim of this study was to attain molecular knowledge of human papillomavirus type 18 (HPV18) by sequencing the whole genome of HPV18 isolated from Thai women at various clinical stages of disease progression.
The hybrid capture II (HCII) assay is widely used in the detection of human papillomavirus virus (HPV). However, due to the limited number of HPV genotypes, it does not permit a comprehensive typing of viruses and "grey zone" (borderline negative or positive results) are often difficult to interpret. As such, polymerase chain reaction (PCR) should be used in parallel with HCII assays, and consensus PCR detection is capable of covering a wider detection range than with the HCII method. We examined the relationship between HCII relative light unit/cutoff (RLU/CO) ratios and PCR amplification results. This was done using previously described primer sets (MY/GP) as well as with our primers for HPV E1, L1 and E6 gene amplification, and performed on samples exhibiting different cytological findings. Together, 243 samples were divided into three groups having RLU/CO ratios of < 0.4 (n = 21), 0.4-4 (n = 64) and > or = 4 (n = 158), respectively. All samples were subjected to PCR amplification using MY/GP and the newly designed E1, L1 and E6 primers. Results were verified by direct sequencing. PCR amplification sensitivities were higher when using the E1 primers than for the MY/GP, E6 or L1 primers. The E1 assay can be used for HPV detection with a sensitivity of 10(2) copies microl(-1). Samples with RLU/CO ratios exceeding 4, and grey zone samples of 0.4-4, were amplified using E1 primers in 79.74% and 26.56% of the total cases, respectively. Cytological data of grey zone samples were primarily found to be normal (77%) whereas those with RLU/CO ratios > 4 were found in any of the cytological data categories. We concluded that HPV screening by HCII for grey zone samples should be analyzed together with cytological data, as well as with a PCR screening tool that incorporates the E1 primers.
Global prevalence of human papillomavirus type 16 (HPV16) exceeds that of other types. This project has been aimed at attaining basic molecular knowledge of HPV16 by sequencing the whole genome of HPV16 isolated from Thai women at various clinical stages of disease progression. Our group analyzed seven samples of HPV16 in infected women ranging from normal to cervical cancer and discovered two critical non-synonymous changes within the coding region converting the E2-219P prototype to E2-219T in cervical cancer and the L2-269S prototype to L2-269D in CIN III, respectively. Phylogenetic analysis based on the whole genome with special emphasis on the genes E2, E6, L1, and L2 showed the Thai samples to be more closely related to the European than the non-European strains. The vaccine strains L1 polypeptides showed close relationship to our samples. The results provide basic data for future research on cervical cancer pathogenesis and representative data of HPV16 genome in Southeast Asia.
Rodents are the natural hosts for Leptotrombidium mites that transmit Orientia tsutsugamushi, the causative agent of scrub typhus, a potentially fatal febrile human disease. Utilizing mite lines that included O. tsutsugamushi infected and non-infected Leptotrombidium species we investigated the varied infection response of outbred mice (ICR) exposed to L. chiangraiensis (Lc), L. imphalum (Li) and L. deliense (Ld). Each of six mite lines (Lc1, Lc5, Li3, Li4, Li7 and Ld) was separately placed in the inner ears of ICR mice either as a single individual (individual feeding, IF) or as a group of 2-4 individuals (pool feeding, PF). The species of infected chigger feeding on mice significantly affected mortality rates of the mice, with mite lines of Lc causing higher mean (±SE) mortality (90.7 ± 3.6 %) than mite lines of Li (62.9 ± 5.6 %) or Ld (53.6 ± 5.8 %). Mouse responses which included time to death, food consumption and total mice weight change depended on mite species and their O. tsutsugamushi genotype, more than on feeding procedure (IF vs. PF) except for mite lines within the Lc. Infected mite lines of Lc were the most virulent infected mites assessed whereas the infected Ld species was the least virulent for the ICR. Mice killed by various mite lines showed enlarged spleens and produced ascites. The results of this investigation of the clinical responses of ICR mice to feeding by various infected mite lines indicated that the different species of infected mites and their O. tsutsugamushi genotype produced different clinical presentations in ICR mice, a scrub typhus mouse model which mimics the natural transmission of O. tsutsugamushi that is critical for understanding scrub typhus disease in terms of natural transmission, host-pathogen-vector interaction and vaccine development.
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