Identification of Bacteria and Viruses Associated with Patients with Acute Febrile Illness in Khon Kaen Province, Thailand.

The majority of cases of undifferentiated acute febrile illness (AFI) in the tropics have an undefined etiology. In Thailand, AFI accounts for two-thirds of illnesses reported to the Ministry of Public Health. To characterize the bacterial and viral causes of these AFIs, we conducted molecular pathogen screening and serological analyses in patients who sought treatment in Chum Phae Hospital, Khon Kaen province, during the period from 2015 to 2016. Through integrated approaches, we successfully identified the etiology in 25.5% of cases, with dengue virus infection being the most common cause, noted in 17% of the study population, followed by scrub typhus in 3.8% and rickettsioses in 6.8%. Further investigations targeting viruses in patients revealed the presence of Guadeloupe mosquito virus (GMV) in four patients without other pathogen co-infections. The characterization of four complete genome sequences of GMV amplified from AFI patients showed a 93-97% nucleotide sequence identity with GMV previously reported in mosquitoes. Nucleotide substitutions resulted in amino acid differences between GMV amplified from AFI patients and mosquitoes, observed in 37 positions. However, these changes had undergone purifying selection pressure and potentially had a minimal impact on protein function. Our study suggests that the GMV strains identified in the AFI patients are relatively similar to those previously reported in mosquitoes, highlighting their potential role associated with febrile illness.

First report of three novel Bartonella species isolated in rodents and shrews from nine provinces of Thailand.

Background and Aim: Bartonella spp. are Gram-negative zoonotic bacteria that are transmitted to humans by several types of animal hosts, including rodents. Several studies have been conducted on the prevalence of Bartonella infections in rodents. However, the risk of rodent-associated Bartonella spp. infection in humans remains unclear. This study aimed to estimate the prevalence and genetic heterogeneity of Bartonella spp. in rodents and shrews from nine provinces of Thailand using culture and molecular techniques. Materials and Methods: A total of 860 blood samples from rodents and shrews across nine provinces of Thailand were collected from January 2013 to June 2016. Bartonella spp. were isolated from all samples using conventional culture techniques and polymerase chain reaction. Phylogenetic tree analysis was used to align the Bartonella sequences obtained from this study. Results: The prevalence of Bartonella spp. in rodents and shrews was 11.5% (99/860, 95% confidence interval: 9.38-13.64%). The following nine species of Bartonella were detected: Bartonella tribocorum, Bartonella rattimassiliensis, Bartonella queenslandensis, Bartonella elizabethae, Bartonella chanthaburi spp. nov., Bartonella satun spp. nov., Bartonella coopersplainsensis, Bartonella ranong spp. nov., and Bartonella henselae. The prevalence of Bartonella-positive animals differed significantly among provinces. Conclusion: To the best of our knowledge, the three novel Bartonella spp. isolated from rodents and shrews across Thailand were detected for the first time in this study. Further studies on the epidemiology of Bartonella infection in rodents and its interaction with human health should be conducted in accordance with the Thai government's "One Health" approach to humans, animals, and the environment.

Plasmodium falciparum phenotypic and genotypic resistance profile during the emergence of Piperaquine resistance in Northeastern Thailand.

Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013-2015. The majority of parasites harbored a pfmdr1-Y184F mutation. A single pfmdr1 copy number had CVIET haplotype of amino acids 72-76 of pfcrt and no pfcytb mutations. All isolates had a single pfk13 point mutation (R539T, R539I, or C580Y), and increased % survival in the ring-stage survival assay (except for R539I). Multiple copies of pfpm2 and pfcrt-F145I were detected in 2014 (12.8%) and increased to 30.4% in 2015. Parasites containing either multiple pfpm2 copies with and without pfcrt-F145I or a single pfpm2 copy with pfcrt-F145I exhibited elevated IC90 values of piperaquine. Collectively, the emergence of these resistance patterns in Thailand near Cambodia border mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions need to coordinate the proposed interventions.

Rickettsiae exposure related to habitats of the oriental house rat (Rattus tanezumi, Temminck, 1844) in Salaya suburb, Thailand.

Rickettsial zoonotic diseases, in particular scrub typhus, murine typhus, and tick typhus, are caused by Orientia tsutsugamushi, Rickettsia typhi, and Rickettsia honei infections. Rickettsiae exposure is typically related to a rodent host in various habitats of marginal regions, including between rural areas and communities such as the Salaya suburb. This allows the oriental house rat (OHR), a highly adaptive species, to live in close proximity to the community and possibly introduce rickettsial diseases. To understand rickettsial exposure in the OHR from different habitats, knowledge of disease exposure can serve as baseline information for disease management and prevention. A total of 185 OHRs from three unrelated habitats were assessed using a standard indirect immunofluorescence assay (IFA) for specific antibody reaction to O. tsutsugamushi, R. typhi, and R. honei. The presence of antibody in the OHR to rickettsiae, either scrub or murine typhus, was associated with the habitat, whereas tick typhus had general exposure. This finding shows the OHR to be a potential reservoir host for rickettsial diseases along the borders of geographic regions in the suburban environment.

Development of multiplex PCR for neglected infectious diseases.

Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi, Rickettsia typhi, and pathogenic Leptospira spp., respectively. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality. Antibody-based serological test currently used as gold standard has limitations due to insufficient antibody titers, especially in the early phase of infection. In this study, we aimed to develop multiplex PCR to combine 3 primer pairs that target specific genes encoding 56-kDa TSA of O. tsutsugamushi, 17-kDa antigen of R. typhi, and LipL32 of L. Interrogans and evaluate its performance in comparison to the standard serological tests. Using EDTA blood samples of known patients, the sensitivity and specificity of our multiplex PCR was 100% and 70%, respectively. In addition, the assay was able to diagnose the co-infection of scrub typhus and leptospirosis. The assay may be useful in identifying causative agents during the early phase of these diseases, enabling prompt and appropriate treatment.

Survey of Rickettsia spp. and Orientia tsutsugamushi Pathogens Found in Animal Vectors (Ticks, Fleas, Chiggers) in Bangkaew District, Phatthalung Province, Thailand.

Rickettsial infections (Rickettsioses) are the causes of acute fever found in Thailand. It is classified as acute febrile illnesses transmitted by bloodsucking arthropod vectors (tick, flea, and chigger). This research investigated pathogens of scrub typhus in vectors from Bangkaew District, Phatthalung Province. A total of 303 pools of vector samples were ticks (Rhipicephalus sanguineus, R. microplus, and Haemaphysalis sp.), fleas (Ctenocephalides felis orientis, C. f. felis, and C. canis), and chiggers (Leptotrombidium deliense, Aschoschoengastia indica, Blankaartia acuscutellaris and Walchia disparunguis pingue) collected from reservoir hosts (dogs and rodents). The 17 and 56 kDa gene of Rickettsia causing scrub typhus were found in 29% of ticks and 98% of flea. DNA sequence analysis reveeled the detected strains were R. asembonensis and Rickettsia sp. cf1 and 5.The chiggers, 1%, were infected with Rickettsia strain TA763, a pathogen of scrub typhus.

Surveillance for Scrub Typhus, Rickettsial Diseases, and Leptospirosis in US and Multinational Military Training Exercise Cobra Gold Sites in Thailand.

We report findings of field surveillance for disease vectors and the prevalence of Orientia tsutsugamushi, the causative agent for scrub typhus, and other Rickettsial species that cause murine typhus and spotted fever group rickettsioses, in chigger mites and small rodents; and Leptospira in rodent kidney, urine, and environmental water samples. The study sites included various Royal Thai Army military installations and other training sites, and surrounding areas where the multinational military training exercise Cobra Gold was conducted in Thailand in 2017 and 2018. The overall prevalence of O. tsutsugamushi and Rickettsia infection in chiggers was 1.3% (20/1,594) and 7.5% (119/1,594), respectively. Serum samples of the captured rodents indicated previous exposure to O. tsutsugamushi infection with a seropositive rate of 12.2%. Leptospira species were isolated from rodent kidneys and water samples collected from catchment areas as well as tap water used for hand washing. Findings from this surveillance are important in determining the potential for scrub typhus, rickettsioses, and leptospirosis risk to military and US government personnel, as well as for informing regional and combatant commanders for prevention, correct diagnosis, prompt treatment, and timely and focused implementation of vector control and personal protective measures.

Scrub Typhus Outbreak in Chonburi Province, Central Thailand, 2013.

Investigation of a scrub typhus outbreak in Thailand during September 2013 found that 9.1% of Thai soldiers and 11.1% of residents living in areas surrounding training sites had antibodies against the causative agent, Orientia tsutsugamushi. Sequence analysis of O. tsutsugamushi from rodents and chiggers identified 7 genogroups and 3 genotypes.

Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus.

We developed a rapid dot-enzyme-linked immunosorbent assay (dot-ELISA) using the combination of recombinant 56-kDa protein antigens that exhibited broad reactivity with serum antibodies against the four most prevalent strains (Karp, Kato, Gilliam, and TA763) of Orientia tsutsugamushi. The assay is rapid (30 minutes), and can be done at room temperature, and results can be read by the naked eye. Only a simple shaker is required to wash the membrane. Sera from 338 patients suspected of being ill with scrub typhus from rural hospitals around Thailand were tested using this dot-ELISA. Seventy-five (22.2%) patients were found to be positive. The sensitivity and specificity of dot-ELISA were determined using the indirect immunofluorescent assay (IFA) test as the gold standard, with the cutoff titer of immunoglobulin peroxidase conjugate M (IgM)/G (IgG) greater than 1:400/1:400. The dot-ELISA had a sensitivity of 98.5%, a specificity of 96.3%, a positive predictive value of 86.7%, and a negative predictive value of 99.6% for the acute-phase specimens. The results indicate that dot-ELISA rapid test using recombinant 56-kDa protein antigen was comparable with the IFA test and may be very useful for the diagnosis of scrub typhus in rural hospitals, where IFA is not available.

Scrub typhus outbreak, northern Thailand, 2006-2007.

During a scrub typhus outbreak investigation in Thailand, 4 isolates of O. tsutsugamushi were obtained and established in culture. Phylogenetic analysis based on the 56-kDa type-specific antigen gene demonstrated that the isolates fell into 4 genetic clusters, 3 of which had been previously reported and 1 that represents a new genotype.

Efficacy of plant essential oils for the repellents against chiggers (Leptotrombidium imphalum) vector of scrub typhus.

Scrub typhus caused by the Orientia tsutsugamushi. Rodents, particularly rats, serve as principal reservoir hosts. Infection in man is transmitted by the, chigger bite. Repellents provide an effective agent of protecting individuals from chigger. In the present study 6 plant essential oils were tested for evaluation of their repellent activity against the chigger, Leptotrombidium imphalum. The results showed that Clove oil was significantly more effective than others with ED50 and EC50 of 0.420 mg and 2.3%, followed by Zingiber oil (8.458 mg and 42.3%), Vetiver oil (19.582 mg and 97.9%), Turmeric oil (24.343 mg and 121.7%), Orange oil (27.310 mg and 136.6%) and Boesenbergia oil (30.486 mg and 152.4%). These results suggested that Clove oil was the most efficient repellent against chigger which is the vector for scrub typhus.

Isolation and characterization of Orientia tsutsugamushi from rodents captured following a scrub typhus outbreak at a military training base, Bothong district, Chonburi province, central Thailand.

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.

Incidence of rickettsial infection in patients with acute fever in provincial Thai army hospitals.

Scrub typhus is common among patients with acute fever in rural areas of Thailand. The authors prospectively recruited patients with acute fever from provincial Thai army hospitals. Dot-ELISA test for scrub typhus was done in hospitals and then compared with standard immunofluorescent assay for diagnosis of scrub typhus. Among 178 patients, scrub typhus was diagnosed by immunofluorescent assay in 10 patients (5.61%). The incidence was high in the northeastern and northern regions. Dot-ELISA gave positive results in 4 of 115 patients, while immunofluorescent assay gave positive results in 6 patients (sensitivity = 66.7%). No false positive results of Dot-ELISA were found among 109 patients (specificity = 100%). All patients gave negative results for murine typhus and Thai tick typhus using immunofluorescent assay. Regarding this present study, Dot-ELISA for scrub typhus has a good sensitivity and specificity and can be used in rural hospitals. This test could be useful for diagnosis of scrub typhus in hospitals where immunofluorescent assay is not available.

Hepatitis E virus infection in Thai troops deployed with U.N. peacekeeping forces.

The hepatitis E virus (HEV) is thought to be endemic throughout much of the world, particularly where sanitary infrastructure remains inadequate. HEV has been considered a military health threat and has been reported in several military environments. This study determined HEV seroconversion (defined by a 4-fold increase in antibody titers) occurring in Thai soldiers deployed to the HEV-endemic areas of East Timor, Afghanistan, and Iraq, as part of the U. N. multinational forces. With an average deployment of 6.4 months, the annualized seroconversion rates after deployments to East Timor, Afghanistan, Burundi, and Iraq were 1.9%, 4.6%, 4.6%, and 3.9%, respectively.