A pan-molecular diagnostic method such as TAC produces quantifiable and comparable results of all tested pathogens, thereby reducing the variability associated with multiple conventional methods. This allows better determination of the clinical relevance of each diarrhea etiologic agent, as well as their geographical relevance in Thailand.

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.

Aims:  To develop and validate assays based on real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) for rapid detection and strain identification (European and North American strains) of porcine reproductive and respiratory syndrome virus (PRRSV) by using SYBR Green I and TaqMan probe chemistries. Methods and Results:  This study describes two alternative assays based on real‐time RT‐PCR for rapid detection and strain identification of PRRSV in comparison with conventional RT‐PCR. The first assay utilized SYBR Green I with melting curve analysis; another assay was performed using strain‐specific TaqMan probes. Primers were selected from the conserved regions within ORF7 (N) of both strains whereas two TaqMan probes labelled with different fluorescent dyes were specifically designed for each strain. The result of strain identification was confirmed by direct sequencing. Both assays can be used for rapid detection and strain identification of PRRSV with a sensitivity of 104 and 103 copies μl−1 for SYBR Green and TaqMan probe, respectively. Conclusions:  Real‐time RT‐PCR is a powerful method combining rapidity, specificity and efficiency for large‐scale screening and strain identification of PRRSV. Significance and Impact of the Study:  The data indicate that the methods developed are invaluable for detecting low levels of PRRSV infection in swine.

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.

Objective: 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. Method: Our group analyzed 9 samples of whole-genome HPV18 in infected women ranging from normal to cervical cancer by PCR, a sequencing method and bioinformatics programs. Results: Phylogenetic analysis based on the whole genome showed that HPV18 samples were more closely related to the European and Asian-American type than the African type. The vaccine strain’s L1 nucleotide (US patent 5820870) showed a close relationship to the African type. However, our data cannot indicate the correlation between cytological data and nucleotide or amino acid variation. Conclusion: Our group cannot draw any inference between the clinical stage of disease progression and amino acid alterations as there were only 1 or 2 samples available for each clinical trial. However, we hope that these new data on the HPV genome, which are representative of the entire genome of HPV in Southeast Asia, can serve as basis data for future research on the pathogenesis of cervical cancer. Additionally, the second-generation HPV18 vaccines should be tested on both HPV18-L1 and HPV18-L2 for increasing potential protection.

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 strain's 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.

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 Diarrhea remains a major public health problem for both civilian and military populations. This study describes the prevalence of acute diarrheal illness etiological agents, their antibiotic resistance distribution patterns, the resulting impact upon military force health protection, and potential prevention and treatment strategies. Results Forty-eight acute diarrhea stool samples from US military personnel deployed to Thailand from 2013–2017 were screened for enteric pathogens using ELISA, the TaqMan Array Card (TAC), and conventional microbiological methods. These isolates were also evaluated using antimicrobial susceptibility testing (AST) against ampicillin (AMP), azithromycin (AZM), ceftriaxone (CRO), ciprofloxacin (CIP), nalidixic acid (NA), erythromycin (ERY), and trimethoprim-sulfamethoxazole (SXT) using commercial methodology. Susceptibility results were interpreted following the CLSI and NARM guidelines. Questionnaire data obtained from 47/48 volunteers indicated that 89.4% (42/47) reported eating local food and the most common clinical symptoms were nausea and abdominal pain (51%; 24/47). Multiple bacterial species were identified from the 48 stool samples with diarrhea etiological agents being detected in 79% (38/48) of the samples distributed as follows: 43.8% (21/48) Campylobacter jejuni and Campylobacter species, 42% (20/48) diarrheagenic Escherichia coli, and 23% (11/48) Salmonella. Co-infections were detected in 46% (22/48) of the samples. All C. jejuni isolates were resistant to CIP and NA. One C. jejuni isolate exhibited resistance to both AZM and ERY. Lastly, an association between exposure to poultry and subsequent detection of the diarrhea-associated pathogens E. coli and P. shigelloides was significant (p < 0.05). Conclusion The detection of Campylobacter isolates with CIP, AZM and ERY resistance has critical force health protection and public health implications, as these data should guide effective Campylobacteriosis treatment options for deployed military members and travelers to Southeast Asia. Additional research efforts are recommended to determine the association of pathogen co-infections and/or other contributing factors towards diarrheal disease in military and traveler populations. Ongoing surveillance and AST profiling of potential disease-causing bacteria is required for effective disease prevention efforts and treatment strategies.