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Leishmaniasis poses a significant health burden, particularly among immunocompromised patients. In Thailand, Leishmania infection caused by Leishmania martiniquensis and Leishmania orientalis lacks information about the incidence and risk factors among HIV-infected populations. This longitudinal cohort study aimed to investigate the incidence and persistence of Leishmania infection among HIV-infected individuals in an affected area, Trang Province, Southern Thailand. The study also identified risk factors associated with the incidence of Leishmania infection. The study enrolled 373 participants in the HIV clinic, Trang Hospital, who initially tested negative for Leishmania infection during 2015–2016, and 133 individuals initially tested positive for Leishmania infection. Thus, follow-up visits of 506 participants occurred during 2018–2019. Direct Agglutination Test (DAT) and nested PCR (nPCR) identified incidents and persistent cases of Leishmania infection. Cox proportional-hazards regression analyses were performed to assess risk factors for the incidence of Leishmania infection. Among the initially negative group, 12 incident cases comprised one L. orientalis infection and 11 seropositive cases using DAT, resulting in a cumulative incidence of 3.2% and an incidence density of 10.38 per 1000 person-years. Increasing age was a significant predictor of the incidence of Leishmania infection. Five persistent cases comprised one Leishmania donovani complex and four seropositive cases using DAT in the initially positive group, with a cumulative persistence rate of 3.7% and a persistence density of 12.85 per 1000 person-years. All patients were asymptomatic. This study sheds light on the incidence and persistence of Leishmania infection among HIV-infected individuals in Trang Province, Southern Thailand, underscoring the importance of continued monitoring and tailored interventions to mitigate the impact of this co-infection. Author summary: In Thailand, Leishmania martiniquensis and Leishmania orientalis are the causative agents of cutaneous (CL) and visceral leishmaniasis (VL) reported in immunocompetent and immunocompromised individuals. VL poses a significant health threat, especially to those with HIV infection. Our study conducted a cohort study among HIV-infected patients in Trang Hospital, Trang Province, Southern Thailand, to explore the incidence and persistence of Leishmania infection, a topic not extensively studied in this region. Our findings are crucial for multiple reasons. Firstly, this study reveals a significant incidence rate of 3.2% among HIV patients, and increasing age was a significant predictor of incident Leishmania infection. Secondly, the persistence of Leishmania infection in 3.7% of initially positive cases underscores the challenges in managing this co-infection. It has immediate implications for healthcare providers, policymakers, and public health officials, aiding them in implementing targeted interventions and preventive measures to reduce the impact of leishmaniasis among HIV-infected individuals. By shedding light on this understudied issue, we hope to contribute to better health outcomes for this vulnerable population. [ABSTRACT FROM AUTHOR]

Leishmaniasis, a neglected tropical disease caused by parasitic protozoa of the Leishmania genus, remains a global health concern with significant morbidity and mortality. In Thailand, the rising incidence of autochthonous leishmaniasis cases involving Leishmania (Mundinia) martiniquensis and novel Leishmania (Mundinia) orientalis underscores the critical need for accurate diagnosis and effective control strategies. This study presents a sensitive and specific nucleic acid lateral flow immunoassay (NALFIA) that integrates a duplex PCR assay with a lateral flow device (LFD) strip format. Targeting the internal transcribed spacer 1 (ITS1) region, known for its unique combination of conserved and variable sequences, this assay employs primers labeled with biotin, digoxigenin, and fluorescein isothiocyanate (FITC) markers, enabling precise species identification and differentiation of these two Leishmania species. Remarkably, the assay achieves a sensitivity that surpasses agarose gel electrophoresis, detecting as few as 10−2 parasite/μL for L. martiniquensis and 10−4 parasite/μL for L. orientalis. Notably, the assay exhibited reliable specificity, revealing no cross-amplification with other major viscerotropic Leishmania species or reference organisms. Evaluation using 62 clinical samples further confirms the effectiveness of the PCR-LFD assay, with a sensitivity of 100% for L. martiniquensis and 83.3% for L. orientalis, and an excellent agreement (κ value = 0.948) with nested PCR. This integrated assay represents a promising advancement in diagnostic tools, offering rapid and accurate results that can significantly contribute to effective disease management and control. Given the increasing relevance of these Leishmania species in current public health scenarios, this assay serves as a valuable tool for both diagnostic and research applications. [ABSTRACT FROM AUTHOR]

Opisthorchis viverrini infection is the major parasitic infection problem in Southeast Asian countries, and long-term infection will lead to cholangiocarcinoma (CCA), the bile duct cancer. The early diagnosis of O. viverrini infection may interrupt the progression of the opisthorchiasis and other related illnesses, especially CCA. The current diagnostic procedure is stool examination by microscope-based methods such as direct smear and concentration techniques but it is limited by low parasite egg numbers. The molecular diagnosis prompts the chance to evaluate the light infection with low number of parasite eggs but is currently inconvenient for routine use due to special equipment requirement and unstable sensitivities. Our present study aims to establish the efficiency of OvNad subunits, the mitochondrial gene, for introducing as a potential diagnostic target by conventional PCR, the cheapest and easiest molecular procedure. A total of 166 stool samples were investigated microscopically by the PBS-ethyl acetate concentration technique (PECT); 75 samples were O. viverrini positive with 28 samples that were positive with single parasite (hookworm, A. lumbricoides, S. stercoralis, Taenia spp., and T. trichiura), 11 samples were with mixed infection, and 52 samples were without parasite detection. The detection limits of OvNad subunits were evaluated in artificially spiked samples containing 0, 1, 5, 10, 20, 50, and 100 Ov-eggs. The result suggested that the best detection efficacy was of OvNad5 that had exact detection limits at only 5 eggs. In the PCR amplification of OvNad subunits, there exist 100% specificities with varied sensitivities from 64%, 88%, 80%, and 100% of OvNad1, OvNad2, OvNad4, and OvNad5, respectively. OvNad subunits were amplified specifically without cross reactivity with the other collected parasites. Our study established that OvNad subunits, especially OvNad5, are the potent candidates for PCR amplification of stool containing Ov-eggs with high confidential sensitivity, specificity, PPV, and NPV even in the light infection that would be a benefit for developing as a routine diagnosis of O. viverrini infection. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is an emerging infectious disease reported in the north and south of Thailand of which patients with HIV/AIDS are a high risk group for acquiring the infection. A lack of information regarding prevalence, and the risk association of Leishmania infection among asymptomatic immunocompetent hosts needs further investigation. Information on potential vectors and animal reservoirs in the affected areas is also important to control disease transmission. Methods: An outbreak investigation and a cross-sectional study were conducted following one index case of cutaneous leishmaniasis (CL) caused by L. martiniquensis in an immunocompetent male patient reported in August 2015, Chiang Rai Province, Thailand. From September to November 2015, a total of 392 participants at two study areas who were related to the index case, 130 students at a semi-boarding vocational school and 262 hill tribe villagers in the patient's hometown, were recruited in this study. The nested internal transcribed spacer 1-PCR (ITS1-PCR) was performed to detect Leishmania DNA in buffy coat, and nucleotide sequencing was used to identify species. Antibody screening in plasma was performed using the Direct Agglutination Test (DAT), and associated risk factors were analyzed using a standardized questionnaire. Captured sandflies within the study areas were identified and detected for Leishmania DNA using nested ITS1-PCR. Moreover, the animal reservoirs in the study areas were also explored for Leishmania infection. Results: Of 392 participants, 28 (7.1%) were positive for Leishmania infection of which 1 (4.8%) was L. martiniquensis, 12 (57.1%) were L. orientalis and 8 (38.1%) were Leishmania spp. Of 28, 15 (53.6%) were DAT positive. None showed any symptoms of CL or visceral leishmaniasis. Risk factors were associated with being female (adjusted odds ratio, AOR 2.52, 95%CI 1.01–6.26), increasing age (AOR 1.05, 95%CI 1.02–1.08), having an animal enclosure in a housing area (AOR 3.04, 95%CI 1.13–8.22), being exposed to termite mounds (AOR 3.74, 95%CI 1.11–12.58) and having domestic animals in a housing area (AOR 7.11, 95%CI 2.08–24.37). At the semi-boarding vocational school, six Sergentomyia gemmea samples were PCR positive for DNA of L. orientalis and one S. gemmea was PCR positive for DNA of L. donovani/L. infantum. Additionally, one Phlebotomus stantoni was PCR positive for DNA of L. martiniquensis, and one black rat (Rattus rattus) was PCR positive for DNA of L. martiniquensis. Conclusion: This information could be useful for monitoring Leishmania infection among immunocompetent hosts in affected areas and also setting up strategies for prevention and control. A follow-up study of asymptomatic individuals with seropositive results as well as those with positive PCR results is recommended. Author summary: This is the first community-based study investigating the prevalence and associated risk factors of Leishmania infection among immunocompetent individuals, demonstrating the situation of Leishmania infection in endemic areas of Thailand. An outbreak investigation and a cross-sectional study were conducted following one index case of cutaneous leishmaniasis (CL) caused by L. martiniquensis in an immunocompetent male patient reported in August 2015, Chiang Rai Province, Thailand. Of 392 participants, 28 (7.1%) were positive for Leishmania infection of which 1 (4.8%) was L. martiniquensis, 12 (57.1%) were L. orientalis and 8 (38.1%) were Leishmania spp. The factors associated with Leishmania infection included being female, increasing age, having an animal enclosure in a housing area, being exposed to termite mounds and having domestic animals in a housing area. This information revealed the first burden and risk factors of Leishmania infection among immunocompetent individuals in Thailand and highlighted the urgent need for effective interventions to prevent and control the disease in high risk populations in endemic areas. [ABSTRACT FROM AUTHOR]

To the Editor: Although rickettsioses caused by scrub typhus and typhus group rickettsiae are well recognized in Thailand, few spotted-fever group rickettsiae (SFGR), including Rickettsia honei TT118 and R. felis, [...]

The highly sensitive and selective determination of Escherichia coli (E. coli) in urine was achieved using a SYBR™ safe loop-mediated isothermal amplification (LAMP) method with a distance-based paper device. New primers set specific to multi-copy the 16s rRNA gene of E. coli were designed and used in this study. The detection sensitivity of these primers was higher than in related work and they could be incorporated with a low-cost paper-based device to quantify E. coli in urine at a concentration lower than 101 CFU mL−1. Regarding standard artificial urine, a linear range of a 10-fold dilution of E. coli concentration (105–100 CFU mL−1) with an R-square value (R2) = 0.9823 was observed directly using a fluorescent migratory distance of the 4 μL reaction mixture in the detection zone under blue light without the need for postreaction staining process. Based on the device, E. coli infection could be significantly categorized into 3 groups; none, light, and heavy levels, which is beneficial for UTI diagnosis. Hence, this paper-based device is suitable for use with the SYBR™ Safe-LAMP assay to semi-quantify E. coli, especially in resource-limited settings due to advantages of low cost, simple fabrication and operation, and no requirement for sophisticated instruments, as well as its disposability and portability. [ABSTRACT FROM AUTHOR]

Objective: To investigate the incidence, spontaneous clearance, persistence and risk factors of Blastocystis infection in a rural community in Thailand. Methods: In February 2016, a cohort study was conducted in a rural community of Thailand. Baseline information of intestinal parasitic infections and follow-up visits were conducted. Blastocystis infection was detected in stool samples examined with short-term in vitro cultivation using Jones' medium supplemented with 10% horse serum. Participants were tested for Blastocystis infection at every study visit. Cox proportional hazard regression analysis was used to evaluate co-variates to determine the incidence of Blastocystis infection. Results: Of 733 enrolled participants, 57 (7.78%) were positive and 676 (92.22%) were negative for Blastocystis infection. All 676 participants were followed-up for Blastocystis infection between December 2016 and January 2018. In all, 52 of 482 cases (10.79%) comprised individual Blastocystis infection. The incidence density rates of Blastocystis infection, of spontaneous clearance of the disease and of persistent infection were 8.13, 63.14 and 38.70 per 1 000 person-months of follow-up, respectively. Our multivariate analysis revealed that opened defecation in fields or bushes (HR=4.8; 95% CI =2.1-10.4, P <0.001), raising cows (HR=2.8; 95% CI =1.1-7.1, P =0.032), not washing hands after animal contact (HR=2.1; 95% CI =1.0-4.2, P =0.044) and presenting symptoms of nausea or vomiting (HR=2.3; 95% C/=1.0-5.3, P =0.047) were predictive associated risks for the incidence of Blastocystis infection. Conclusions: Our study indicates that the best way to prevent Blastocystis infection can be by maintaining good personal hygiene, hand washing after animal contact and improving sanitary facilities. [ABSTRACT FROM AUTHOR]

Background: Opisthorchis viverrini infection is a major public health problem in northern and northeastern Thailand. The chronic infection of O. viverrini is related to cholangiocarcinoma which causes high mortality in endemic areas. Therefore, the diagnosis, treatment, control and prevention of O. viverrini infection are necessary. The morphology of the egg is very similar to that of other species of human liver flukes (Opisthorchis felineus and Clonorchis sinensis) as well as that of small intestinal flukes in the family Heterophyidae. Thus, molecular characterization is crucially required to discriminate species of Opisthorchis-like eggs in fecal examination. Methodology/Principal findings: We aimed to determine the prevalence of O. viverrini infection among villagers living in Sanamchaikate District, Chachoengsao Province, in central Thailand, where O. viverrini infection has previously been reported. A total of 2,609 fecal samples were examined for Opisthorchis-like eggs using microscopic examination. PCR-RFLP analysis of the ITS2 region was used to discriminate Opisthorchis-like eggs. The genetic structure of O. viverrini infection was demonstrated using nucleotide sequencing of cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 1 (nad1). Testing of evolutionary neutrality of the cox1 and nad1 sequences of O. viverrini was performed using Tajima's D tests and Fu's Fs tests. Moreover, the haplotype networks and phylogenetic trees were constructed to study the relationships of O. viverrini isolated from different endemic areas. A high prevalence of O. viverrini infection is still observed in a rural community of Chachoengsao Province, central Thailand. The overall prevalence of Opisthorchis-like eggs using microscopic examination was 16.8%. PCR-RFLP profiles showed the predominant infection of O. viverrini (9.6%) including very low infections of other small intestinal flukes, Haplorchis taichui (0.08%) and Euparyphium albuferensis (0.08%). The genetic structure of O. viverrini populations in central Thailand was also described and revealed a non-significant difference in genetic diversity. In addition, the genetic background of the O. viverrini populations was closely related to the isolate from Lao PDR. Conclusions/Significance: Our study highlighted the prevalence of O. viverrini infection in central Thailand indicating that control programs and health education regarding opisthorchiasis is still required in this endemic area. Additionally, the study demonstrated the genetic structure of O. viverrini, in central Thailand which could provide information on the molecular epidemiology of this parasite. [ABSTRACT FROM AUTHOR]

Background: In Thailand, artemisinin-based combination therapy (ACT) has been used to treat uncomplicated falciparum malaria since 1995. Unfortunately, artemisinin resistance has been reported from Thailand and other Southeast Asian countries since 2003. Malarone®, a combination of atovaquone–proguanil (ATQ–PG), has been used to cease artemisinin pressure in some areas along Thai–Cambodia border, as part of an artemisinin resistance containment project since 2009. This study aimed to determine genotypes and phenotypes of Plasmodium falciparum isolates collected from the Thai–Cambodia border after the artemisinin resistance containment project compared with those collected before. Results: One hundred and nine of P. falciparum isolates collected from Thai–Cambodia border from Chanthaburi and Trat provinces during 1988–2016 were used in this study. Of these, 58 isolates were collected after the containment. These parasite isolates were characterized for in vitro antimalarial sensitivities including chloroquine (CQ), quinine (QN), mefloquine (MQ), piperaquine (PPQ), artesunate (AS), dihydroartemisinin (DHA), ATQ and PG and genetic markers for drug resistance including the Kelch13 (k13), Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1) and cytochrome b (cytb) genes. Mean CQ, QN, MQ, PPQ and AS IC50s of the parasite isolates collected from 2009 to 2016 exhibited significantly higher than those of parasites collected before 2009. Approximately 57% exhibited in vitro MQ resistance. Approximately 94% of the isolates collected from 2009 to 2016 contained the pfmdr1 184F allele. Mutations of the k13 gene were detected in approximately 90% of the parasites collected from 2009 to 2016 which were significantly higher than the parasite isolates collected before. No ATQ-resistant genotype and phenotype of P. falciparum were found among the isolates collected after the containment project. Conclusions: Although the containment project had been implemented in this area, the expansion of artemisinin-resistant parasites did not decline. In addition, reduced sensitivity of the partner drugs of ACT including MQ and PPQ were identified. [ABSTRACT FROM AUTHOR]

• Simplified closed tube LAMP assay can detect Leishmania in HIV patients. • Detection limits was as low as 102 parasites/mL. • The results could be visually observed by naked eyes without unambiguous. • Closed tube LAMP assay showed 94.4% sensitivity and 96.6% specificity. • The assay is easily delivered to end-users for asymptomatic leishmanasis diagnosis. In Thailand, asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, earlier diagnosis using a simple, sensitive and reliable diagnostic tool is needed because populations at risk mostly reside in rural communities where only basic laboratory equipment is available for health care services. In this present study, a closed tube loop mediated isothermal amplification (LAMP) was developed using a piece of parafilm placed between the dye and LAMP reaction mixture to form semi-layer that partially secured SYBR green I from spilling during amplification. No post-amplification preparation was required and accidental spill of the dye during LAMP amplification was prevented. The result could be visually interpreted under visible and UV lights after dye spinning down. The semi-layer modification of a closed tube LAMP showed successful amplification of Leishmania DNA with clear interpretation using both color and fluorescence dyes when observing by the naked eye. The sensitivity and specificity were as high as 94.4 and 96.9%, respectively whereas detection limits were 102 parasites/mL being ten fold more sensitive than other related studies. This user-friendly inexpensive approach is affordable and suitable for empowering leishmaniasis surveillance without the need of expensive devices in all levels of hospitals, including health services, as well as fieldwork, especially in low income countries. [ABSTRACT FROM AUTHOR]

Blastocystis sp., the most common intestinal protozoa, remains a public health problem among people in many countries, particularly in rural areas of developing countries. The infection usually reflects poor sanitation in communities by waterborne, zoonotic, and person-to-person transmission. Interestingly, at least 17 subtypes (STs) have been reported and are associated with a broad range of animal hosts, including humans. In this study, we reported potential evidence of zoonotic transmission of Blastocystis ST1 in rural communities of eastern Thailand where the overall prevalence of Blastocystis infection was 15.7%. Two major and three minor subtypes were found to be distributed unequally in this region. Of 5 STs, only ST1 was found to be associated with pig feces in an open farm system that produced organic fertilizer for agriculture uses in the community. This finding suggests that properly protective contact and standard production of organic fertilizer from pig feces by-products could be key factors for reducing the prevalence of Blastocystis infection and prevent Blastocystis reinfection among people in the community. IMPORTANCE Blastocystis sp. remains a public health problem among people, particularly in rural areas of many developing countries. The infection usually reflects poor sanitation in communities by waterborne, zoonotic, and person-to-person transmission. In this study, we reported potential evidence of zoonotic transmission of Blastocystis subtype 1 (ST1) in rural communities of eastern Thailand. Two major and three minor subtypes were found to be unequally distributed in this region. Interestingly, only ST1 was found to be associated with pig feces in an open farm system that produced organic fertilizer for agriculture uses in the community. The finding makes significant contributions to genetic and molecular investigations of microbial topics of practical value and suggest that properly protective contact and standard production of organic fertilizer from pig feces by-products could be key factors for reducing the prevalence of Blastocystis infection and prevent Blastocystis reinfection among people in the community.

The highly sensitive and selective determination of Escherichia coli (E. coli) in urine was achieved using a SYBR™ safe loop-mediated isothermal amplification (LAMP) method with a distance-based paper device. New primers set specific to multi-copy the 16s rRNA gene of E. coli were designed and used in this study. The detection sensitivity of these primers was higher than in related work and they could be incorporated with a low-cost paper-based device to quantify E. coli in urine at a concentration lower than 101 CFU mL-1. Regarding standard artificial urine, a linear range of a 10-fold dilution of E. coli concentration (105-100 CFU mL-1) with an R-square value (R2) = 0.9823 was observed directly using a fluorescent migratory distance of the 4 μL reaction mixture in the detection zone under blue light without the need for postreaction staining process. Based on the device, E. coli infection could be significantly categorized into 3 groups; none, light, and heavy levels, which is beneficial for UTI diagnosis. Hence, this paper-based device is suitable for use with the SYBR™ Safe-LAMP assay to semi-quantify E. coli, especially in resource-limited settings due to advantages of low cost, simple fabrication and operation, and no requirement for sophisticated instruments, as well as its disposability and portability.

Human liver fluke infection caused by Opisthorchis viverrini increases the risk of cholangiocarcinoma (CCA) reported along the Mekong basin including Thailand, Lao People's Democratic Republic (PDR), Cambodia, and Vietnam. The highest incidence of CCA has been reported in northeastern Thailand where liver fluke infection is prevalent. This study aimed to investigate the prevalence of O. viverrini infection in a northeastern-descendent community in rural Sa Kaeo Province, eastern Thailand, using stool examination and molecular technique. The Kato-Katz method was performed to determine eggs per gram (EPG) for infection intensity. Phosphate-buffered saline-ethyl acetate concentration was used to prepare specimens for polymerase chain reaction (PCR) and restriction fragment length polymorphism of the internal transcribed spacer 2 (ITS2) region of the ribosomal RNA. From 1,245 specimens, 105 (8.4%) samples were identified as Opisthorchis -like eggs from stool examination, and all positive specimens indicated light infection (< 1,000 EPG). From positive Opisthorchis -like egg samples, 55.2% (58/105) were identified as O. viverrini eggs from ITS2-PCR assay for which low infection intensity might result in a negative PCR result (44.8%). Using multiple logistic regression analysis, males were at 3.1 times higher risk of acquiring O. viverrini infection than females. From phylogenetic analysis, in eastern Thailand, nucleotide sequences of O. viverrini were grouped as a monoclade as those isolated from Greater Mekong, Vietnam, Myanmar, and west Siberia. The results revealed that the surveyed community is a low-grade endemic area of O. viverrini infection. Thus, data from this study can be used to improve health-promoting programs and activities to control the infection and its subsequent CCA.

Pyronaridine, a Mannich base antimalarial agent with a high activity against chloroquine-resistant Plasmodium falciparum , has been combined with artesunate as a new artemisinin based combination therapy (ACT). Pyronaridine-artesunate combination could be one of the choices for the treatment of uncomplicated falciparum malaria in multidrug-resistant areas including Thailand. The aim of this study was to determine in vitro sensitivity and cross-resistance pattern of pyronaridine in Thai isolates of P. falciparum . In addition, the influence of resistant genes concerning in vitro pyronaridine sensitivity was determined. The mean pyronaridine 50% inhibitory concentration (IC 50 ) of 118 parasite isolates was 5.6 ± 3.1 nM (range = 0.2-15.4 nM) with a significant positive correlation with artesunate IC 50 ( r = 0.246, P = 0.008) and amodiaquine IC 50 ( r = 0.220, P = 0.042) and a significant negative correlation with quinine IC 50 ( r = - 0.185, P = 0.047). Parasites containing the pfmdr1 86Y allele exhibited significantly reduced pyronaridine sensitivity compared with those with the pfmdr1 N86 allele (7.6 ± 3.3 nM and 5.4 ± 3.0 nM, respectively, P = 0.032, independent t test); however, the difference may not be clinically relevant. Pyronaridine-artesunate could be the candidate ACT to treat multidrug-resistant falciparum malaria in Thailand with careful monitoring.

Francisella is a genus of bacterial pathogens potentially lethal to humans. We report here for the first time a novel Francisella-like endosymbiont discovered in a hard-tick (Rhipicephalus sanguineus s.l.) obtained from a chicken (Gallus domesticus) in Thailand. The phylogenetic results indicate the 16S rDNA sequences of this Francisella bacterium form a unique clade with the Francisella-like endosymbiont of the tick species, Amblyomma varanense and Amblyomma helvolum, that have previously been found on snakes in Thailand. This species of Francisella is in a different group from the other Francisella-like endosymbionts previously reported from other countries. No Francisella was detected in Haemaphysalis wellingtoni ticks obtained from chickens in this study.

Scrub typhus, caused by antigenically disparate isolates of Orientia tsutsugamushi, is a widely distributed mite-borne human disease in the Asia Pacific region. Information regarding the heterogeneity of the immunodominant 56-kDa type-specific antigen (TSA) gene is crucial for the design and evaluation of scrub typhus-specific diagnostic assays and vaccines. Using indirect immunofluorescence assays (IFA) and PCR assays, O. tsutsugamushi was detected samples from rodents and patients with fever of unknown origin obtained from six provinces of Thailand during 2004 to 2007. Sequences were determined for a fragment of the 56-kDa TSA gene, and the relationship between these sequences and those previously determined were assessed. The phylogenetic analyses of partial 56-kDa TSA gene sequences demonstrated wide diversity and distribution of O. tsutsugamushi genotypes in Thailand. Furthermore, the genetic diversity grouped the scrub typhus agents into two commonly and five infrequently found genotypes within six provinces of Thailand. The two most commonly found genotypes of O. tsutsugamushi described in this study do not associate with the prototype strains that are widely used for the design and evaluation of diagnostic assays and vaccine candidates. Thus, these new genotypes should be considered for future scrub typhus assay and vaccine development.

Orientia tsutsugamushi, an obligate intracellular Gram-negative bacterium, is the causative agent of scrub typhus, a vector-borne disease transmitted by infected chiggers (trombiculid mite larvae). In 2002, an outbreak of scrub typhus occurred among Royal Thai Army troops during the annual field training at a military base in Bothong district, Chonburi province, central Thailand. This report describes the outbreak investigation including its transmission cycle. Results showed that 33.9% of 174 trained troops had scrub typhus-like signs and symptoms and 9.8% of those were positive for O. tsutsugamushi-specific antibodies by indirect fluorescence antibody assay. One hundred thirty-five rodents were captured from this training area, 43% of them had antibodies against O. tsutsugamushi. Six new O. tsutsugamushi isolates were obtained from captured rodent tissues and successfully established in cell culture. Phylogenetic studies showed that these six isolates were either unique or related to a native genotype of previously described isolates from Thailand.