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Scrub typhus, a vector-borne bacterial infection, is an important but neglected disease globally. Accurately characterizing the burden is challenging because of nonspecific symptoms and limited diagnostics. Prior seroepidemiology studies have struggled to find consensus cutoffs that permit comparisons of estimates across contexts and time. In this study, we present a novel approach that does not require a cutoff and instead uses information about antibody kinetics after infection to estimate seroincidence. We use data from three cohorts of scrub typhus patients in Chiang Rai, Thailand, and Vellore, India, to characterize antibody kinetics after infection and two population serosurveys in the Kathmandu Valley, Nepal, and Tamil Nadu, India, to estimate seroincidence. The samples were tested for IgM and IgG responses to Orientia tsutsugamushi-derived recombinant 56-kDa antigen using commercial enzyme-linked immunosorbent assay kits. We used Bayesian hierarchical models to characterize antibody responses after scrub typhus infection and used the joint distributions of the peak antibody titers and decay rates to estimate population-level incidence rates in the cross-sectional serosurveys. Median responses persisted above an optical density (OD) of 1.8 for 23.6 months for IgG and an OD of 1 for 4.5 months for IgM. Among 18- to 29-year-olds, the seroincidence was 10 per 1,000 person-years (95% CI, 5-19) in Tamil Nadu, India, and 14 per 1,000 person-years (95% CI: 10-20) in the Kathmandu Valley, Nepal. When seroincidence was calculated with antibody decay ignored, the disease burden was underestimated by more than 50%. The approach can be deployed prospectively, coupled with existing serosurveys, or leverage banked samples to efficiently generate scrub typhus seroincidence estimates.

Rickettsiosis is caused by Orientia spp. and Rickettsia spp., arthropod-borne zoonotic intracellular bacteria. The close relationships between pet dogs, cats and owners increase the risk of rickettsial transmission, with limited studies on the seroprevalence in pets. This study investigated the prevalence of rickettsia exposure among dogs and cats in Bangkok and neighboring provinces. The samples from 367 dogs and 187 cats used in this study were leftover serum samples from routine laboratory testing stored at the Veterinary Teaching Hospital. In-house Enzyme-linked immunosorbent assay (ELISA) tests included IgG against the scrub typhus group (STG), typhus group (TG), and spotted fever group (SFG). The seroprevalence in pet dogs was 30.25% (111/367), including 21.53% for STG, 4.36% for TG, and 1.09% for SFG. Co-seroprevalence consisted of 2.72% for STG and TG, 0.27% for STG and SFG, and 0.27% for pangroup infection. The prevalence in cats was 62.56% (117/187), including 28.34% for STG, 4.28% for TG, and 6.42% for STG. Co-seroprevalence in cats consisted of STG and TG (4.28%), STG and SFG (5.35%), TG and SFG (3.21%), and three-group infection (10.69%). No significant difference in seroprevalence for the three serogroups was observed in any of the 64 districts sampled. The mean hematocrit level significantly decreased in seropositive dogs (P<0.05). Seropositive dogs and cats were detected in significantly greater numbers of anemia cases than nonanemia cases (P<0.05) (odds ratio: 7.93, 0.44, p = 0.00, p = 0.01). A significantly higher number of seropositive cats had decreased hemoglobin levels (P<0.05) (odds ratio: 3.63, p = 0.00). The seropositive samples significantly differed among older cats (P<0.05). These high exposures in pet dogs and cats could constitute important relationship dynamics between companion animals and rickettsial vectors. Significantly decreased hematocrit and hemoglobin levels indicated anemia in the exposed dogs and cats. The study findings will raise awareness of this neglected disease among pet owners and veterinary hospital personnel and aid in future public health preventative planning., Competing Interests: Professor Stuart Blacksell has a conflict-of-interest in that he is a section editor of the PLoS Neglected Tropical Diseases journal. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Fa-ngoen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Scrub typhus is a significant tropical disease, occurring in rural settings and therefore usually afflicting remote agricultural populations who have lower socioeconomic status and limited access to medical care. A large proportion of the hill tribe people in Thailand are financially poor, have limited education, and do not have adequate health care access. This study aimed to estimate the prevalence of and determine factors associated with scrub typhus exposure among the hill tribe population living in high-incidence areas in northern Thailand., Methods: A cross-sectional study design was used to gather information from hill tribe people aged 18 years and over living in ten hill tribe villages in Mae Fah Luang, Chiang Rai Province, Thailand. Participants who met the inclusion criteria were invited to participate in the study. A validated questionnaire was used as the research instrument, and 5 mL blood samples were taken. Orientia tsutsugamushi IgM and IgG antibodies were detected by enzyme-linked immunosorbent assay (ELISA) and then confirmed by immunofluorescence assay (IFA). Logistic regression was used to detect associations between variables at a significance level of α = 0.05., Results: A total of 485 hill tribe people participated in the study; 57.1% were female, 29.9% were over 60 years of age, 46.4% were from the Akha tribe, and 74.2% had never attended school. The overall prevalence of scrub typhus exposure was 48.0%. In the multivariate model, five variables were found to be associated with scrub typhus exposure. Participants aged over 60 years had a 4.31-fold increased risk (95% CI = 1.73-10.72) of scrub typhus exposure compared to those who were younger than 30 years. Those who were illiterate had a 3.46-fold increased risk (95% CI = 1.93-6.21) of scrub typhus exposure than those who had at least a primary education level. Participants from the Akha tribe had a 2.20-fold increased risk (95% CI = 1.31-3.72) of scrub typhus exposure than those from the Lahu tribe. Subjects who had a history of cutting grass had a 1.85-fold increased risk (95% CI = 1.20-2.84) of scrub typhus exposure. Those who never wore gloves for farming had a 2.12-fold increased risk (95% CI = 1.28-3.49) of scrub typhus exposure than those who wore gloves daily., Conclusions: There is a high prevalence of scrub typhus exposure among the hill tribe in Thailand. Effective public health interventions to promote scrub typhus awareness and prevention are urgently needed in these populations., (© 2023. The Author(s).)

Introduction. Rapid diagnostic tests (RDTs) that can facilitate the diagnosis of a panel of tropical infectious diseases are critically needed. DPP® Fever Panel II Asia is a multiplex lateral flow immunoassay comprising antigen and IgM panels for the diagnosis of pathogens that commonly cause febrile illness in Southeast Asia. Hypothesis/Gap Statement. Accuracy of DPP® Fever Panel II Asia has not been evaluated in clinical studies. Aim. To evaluate the sensitivity and specificity of DPP® Fever Panel II Asia for malaria, dengue and melioidosis. Methodology. We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon sepsis). We included 143 and 98 patients diagnosed with malaria or dengue based on a positive PCR assay and 177 patients with melioidosis based on a culture positive for Burkholderia pseudomallei . Controls included 200 patients who were blood culture-positive for Staphylococcus aureus , Escherichia coli or Klebsiella pneumoniae , and cases of the other diseases. Serum samples collected from all patients within 24 h of admission were stored and tested using the DPP® Fever Panel II Asia antigen and IgM multiplex assays. We selected cutoff values for each individual assay corresponding to a specificity of ≥95 %. When assessing diagnostic tests in combination, results were considered positive if either individual test was positive. Results. Within the DPP® Fever Panel II Asia antigen assay, a combination of pLDH and HRPII for malaria had a sensitivity of 91 % and a specificity of 97 %. The combination of dengue NS1 antigen and dengue antibody tests had a sensitivity of 61 % and a specificity of 91 %. The B. pseudomallei CPS antigen test had a sensitivity of 27 % and a specificity of 97 %. An odds ratio of 2.34 (95 % CI 1.16-4.72, P =0.02) was observed for the association between CPS positivity and mortality among melioidosis patients. Conclusion. The performance of the DPP® Fever Panel II Asia for diagnosis of malaria was high and that for dengue and melioidosis was relatively limited. For all three diseases, performance was comparable to that of other established RDTs. The potential operational advantages of a multiplex and quantitative point-of-care assay are substantial and warrant further investigation.

In this study, we estimated exposure for Scrub typhus (STG), Typhus (TG) and Spotted fever groups (SFG) rickettsia using serology at a fine scale (a whole sub-district administration level) of local communities in Nan Province, Thailand. Geographical characteristics of the sub-district were divided into two landscape types: lowland agricultural area in an urbanized setting (lowland-urbanized area) and upland agricultural area located close to a protected area of National Park (upland-forested area). This provided an ideal contrast between the two landscapes with low and high levels of human-altered habitats to study in differences in disease ecology. In total, 824 serum samples of participants residing in the eight villages were tested by screening IgG ELISA, and subsequently confirmed by the gold standard IgG Immunofluorescent Assay (IFA). STG and TG IgG positivity were highest with seroprevalence of 9.8% and 9.0%, respectively; whereas SFG positivity was lower at 6.9%. Inhabitants from the villages located in upland-forested area demonstrated significantly higher STG exposure, compared to those villages in the lowland-urbanized area (chi-square = 51.97, p < 0.0001). In contrast, TG exposure was significantly higher in those villagers living in lowland-urbanized area (chi-square = 28.26, p < 0.0001). In addition to the effect of landscape types, generalized linear model (GLM) analysis identified socio-demographic parameters, i.e., gender, occupation, age, educational level, domestic animal ownership (dog, cattle and poultry) as influential factors to explain the level of rickettsial exposure (antibody titers) in the communities. Our findings raise the public health awareness of rickettsiosis as a cause of undiagnosed febrile illness in the communities., Competing Interests: The authors have declared that no competing interests exist.

Background: Key infection prevention and control measures to limit transmission of COVID-19 include social distancing, hand hygiene, use of facemasks and personal protective equipment. However, these have limited or no impact if not applied correctly through lack of knowledge, inappropriate attitude or incorrect practice. In order to maximise the impact of infection prevention and control measures on COVID-19 spread, we undertook a study to assess and improve knowledge, attitudes and practice among 119 healthcare workers and 100 general public in Thailand. The study setting was two inpatient hospitals providing COVID-19 testing and treatment. Detailed information on knowledge, attitudes and practice among the general public and healthcare workers regarding COVID-19 transmission and its prevention were obtained from a combination of questionnaires and observations., Results: Knowledge of the main transmission routes, commonest symptoms and recommended prevention methods was mostly very high (> 80%) in both groups. There was lower awareness of aerosols, food and drink and pets as sources of transmission; of the correct duration for handwashing; recommended distance for social/physical distancing; and about recommended types of face coverings. Information sources most used and most trusted were the workplace, work colleagues, health workers and television. The results were used to produce a set of targeted educational videos which addressed many of these gaps with subsequent improvements on retesting in a number of areas. This included improvements in handwashing practice with an increase in the number of areas correctly washed in 65.5% of the public, and 57.9% of healthcare workers. The videos were then further optimized with feedback from participants followed by another round of retesting., Conclusions: Detailed information on gaps in knowledge, attitudes and practice among the general public and healthcare workers regarding COVID-19 transmission and its prevention were obtained from a combination of questionnaires and observations. This was used to produce targeted educational videos which addressed these gaps with subsequent improvements on retesting. The resulting videos were then disseminated as a resource to aid in efforts to fight COVID-19 in Thailand and worldwide.

Despite estimates suggesting Leptospira spp. being endemic in Southeast Asia, evidence remains limited. Diagnostic accuracy evaluations based on Leptospira ELISA mainly rely on hospitalized and severe patients; therefore, studies measuring the pathogen burden may be inaccurate in the community. We evaluated the Panbio Leptospira ELISA IgM among 656 febrile outpatients attending primary care in Chiangrai, Thailand, and Hlaing Tha Yar, Yangon, Myanmar. ELISA demonstrated limited diagnostic accuracy for the detection of acute leptospiral infection using the manufacturer recommended cutoff, with a sensitivity of 71.4% and specificity of 36.4%, and an area under the receiver operator characteristic curve value of 0.65 (95% CI: 0.41-0.89), compared with our reference test, the PCR assay. ELISA also performed poorly as a screening tool for detecting recent exposure to Leptospira spp. compared with the "gold-standard" microscopic agglutination test, with a specificity of 42.7%. We conclude that the utility of the Leptospira IgM ELISA for both serodiagnosis and seroprevalence is limited in our setting.

Background: Scrub typhus, caused by Orientia tsutsugamushi, is a major cause of acute febrile illness in children in the rural tropics., Methods: We recruited 60 febrile pediatric patients with a positive scrub typhus rapid diagnostic test result and 40 healthy controls from Chiang Rai Province in northern Thailand. Diagnosis was confirmed by the detection of (1) O. tsutsugamushi-specific DNA in blood or eschar samples with a polymerase chain reaction assay, (2) a fourfold rise in immunoglobulin M (IgM) titer to ≥1:3200 in paired plasma samples with an indirect immunofluorescence assay (IFA), or (3) a single IgM titer of ≥1:3200 in an acute plasma sample with an IFA. Demographic, clinical, and laboratory data were collected, and patients were followed up for 1 year., Results: Diagnosis was confirmed in 35 (58%) of 60 patients, and all controls tested negative for scrub typhus. Patients with confirmed scrub typhus had clinical symptoms, including fever (35 of 35 [100%]), eschar (21 of 35 [60%]), cough (21 of 35 [60%]), tachypnea (16 of 35 [46%]), lymphadenopathy (15 of 35 [43%]), and headache (14 of 35 [40%]). Only 4 (11%) of 35 patients received appropriate antibiotic treatment for scrub typhus before admission. The median fever-clearance time was 36 hours (interquartile range, 24-53 hours). Complications observed include hepatitis (9 of 35 [26%]), severe thrombocytopenia (7 of 35 [20%]), pneumonitis (5 of 35 [14%]), circulatory shock (4 of 35 [11%]), and acute respiratory distress syndrome (3 of 35 [9%]). Treatment failure, defined by failure to defervesce within 72 hours of antibiotic treatment initiation, was noted in 8 (23%) of 35 patients, and 1 (3%) of the 35 patients died. No evidence of relapse or reinfection was found., Conclusion: Pediatric scrub typhus in northern Thailand is often severe and potentially fatal with delays in treatment a likely contributing factor. Additional studies to investigate the bacterial, pharmacologic, and immunologic factors related to treatment outcome along with measures to improve public awareness should be prioritized., (© The Author(s) 2019. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society.)

In this diagnostic accuracy study, we evaluated data from 135 febrile patients from Chiang Rai, to determine the optimal optical density (OD) cutoffs for an in-house scrub typhus IgM ELISA. Receiver operating characteristic curves were generated using a panel of reference assays, including an IgM immunofluorescence assay (IFA), PCR, in vitro isolation, presence of an eschar, or a combination of these. Altogether, 33 patients (24.4%) were diagnosed as having scrub typhus. Correlation between positivity by IFA and increasing OD values peaked at a cutoff of 2.0, whereas there was little association between positivity by culture or eschar with increasing ELISA cutoffs-cutoffs of 3.0 and 4.0 were demonstrated to be optimal for the total absorbance of the OD at dilutions 1:100, 1:400, 1:1,600, and 1:6,400, for admission and convalescent samples, respectively. The optimal cutoff at a 1:100 dilution was found to be between 1.85 and 2.22 for admission samples and convalescent-phase samples, respectively. Sensitivities for the cutoffs varied from 57.1% to 90.0% depending on the reference test and sample timing, whereas specificities ranged from 85.2% to 99.0%. We therefore recommend a cutoff of around 2.0, depending on the sensitivity and specificity desired in clinical or epidemiological settings. The results demonstrate the ELISA to be a valuable diagnostic tool, suitable for use in resource-limited endemic regions, especially when used in combination with other diagnostic modalities such as the presence of an eschar.

The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus., (Copyright © 2016 Blacksell et al.)

Background: There is a paucity of published reports on pregnancy outcome following scrub and murine typhus despite these infections being leading causes of undifferentiated fever in Asia. This study aimed to relate pregnancy outcome with treatment of typhus., Methodology/principal Findings: Data were analyzed from: i) pregnant women with a diagnosis of scrub and/or murine typhus from a fever cohort studies; ii) case series of published studies in PubMed using the search terms "scrub typhus" (ST), "murine typhus" (MT), "Orientia tsutsugamushi", "Rickettsia tsutsugamushi", "Rickettsia typhi", "rickettsiae", "typhus", or "rickettsiosis"; and "pregnancy", until February 2014 and iii) an unpublished case series. Fever clearance time (FCT) and pregnancy outcome (miscarriage and delivery) were compared to treatment. Poor neonatal outcome was a composite measure for pregnancies sustained to 28 weeks or more of gestation ending in stillbirth, preterm birth, or delivery of a growth restricted or low birth weight newborn., Results: There were 26 women in the fever cohort. MT and ST were clinically indistinguishable apart from two ST patients with eschars. FCTs (median [range] hours) were 25 [16-42] for azithromycin (n=5), 34 [20-53] for antimalarials (n=5) and 92 [6-260] for other antibiotics/supportive therapy (n=16). There were 36.4% (8/22) with a poor neonatal outcome. In 18 years, 97 pregnancies were collated, 82 with known outcomes, including two maternal deaths. Proportions of miscarriage 17.3% (14/81) and poor neonatal outcomes 41.8% (28/67) were high, increasing with longer FCTs (p=0.050, linear trend). Use of azithromycin was not significantly associated with improved neonatal outcomes (p=0.610)., Conclusion: The published ST and MT world literature amounts to less than 100 pregnancies due to under recognition and under diagnosis. Evidence supporting the most commonly used treatment, azithromycin, is weak. Collaborative, prospective clinical trials in pregnant women are urgently required to reduce the burden of adverse maternal and newborn outcomes and to determine the safety and efficacy of antimicrobial treatment.

Orientia tsutsugamushi, the etiologic agent of potentially fatal scrub typhus, is characterized by a high antigenic diversity, which complicates the development of a broadly protective vaccine. Efficacy studies in murine and nonhuman primate models demonstrated the DNA vaccine candidate pKarp47, based upon the O. tsutsugamushi Karp 47-kD HtrA protein gene, to be a successful immunoprophylactic against scrub typhus. To characterize 47-kD HtrA protein diversity among human isolates of Orientia, we sequenced the full open reading frame (ORF) of the 47-kD HtrA gene and analyzed the translated amino acid sequences of 17 patient isolates from Thailand (n=13), Laos (n=2), Australia (n=1), and the United Arab Emirates (UAE) (n=1) and 9 reference strains: Karp (New Guinea), Kato (Japan), Ikeda (Japan), Gilliam (Burma), Boryong (Korea), TA763, TH1811 and TH1817 (Thailand), and MAK243 (China). The percentage identity (similarity) of translated amino acid sequences between 16 new isolates and 9 reference strains of O. tsutsugamushi ranged from 96.4% to 100% (97.4% to 100%). However, inclusion of the recently identified Orientia chuto sp. nov. reduced identity (similarity) values to 82.2% to 83.3% (90.4% to 91.4%). These results demonstrate the diversity of Orientia 47-kD HtrA among isolates encountered by humans and therefore provide support for the necessity of developing a broadly protective scrub typhus vaccine that takes this diversity into account.

A questionnaire was used to collect data on small poultry farm management and wild bird observed in poultry keeping areas to identify putative risk factors for infection with HPAI H5N1. The study was conducted in 2008 in four subdistricts of central Thailand that had experienced outbreaks of HPAI H5N1 in poultry. Descriptive and inferential analyses including univariable analyses and multivariable logistic regression were used to identify putative risk factors. Risk factors included purchasing native chickens/fighting cocks from commercial hatcheries, replacing or restocking birds individually, and observing lesser whistling ducks (Dendrocygna javanica) on the farm daily. Selecting healthy animals when purchasing animals to ensure that they were disease free was a protective factor. To fully understand the epidemiology of infection of small poultry farms with HPAI H5N1, control of movement of domestic poultry and serological and virological testing of the poultry population should be applied., (© 2012 Blackwell Verlag GmbH.)

Samples from 160 prospectively recruited febrile patients with typhus-like illness in an area of Thailand (Chiang Rai, northern Thailand) where scrub typhus is endemic were used to evaluate the diagnostic capabilities of four rapid immunochromatographic tests (ICTs) for the detection of Orientia tsutsugamushi IgM and total antibodies during acute scrub typhus infection. Of the 160 cases, 54 (34%) had been confirmed to have scrub typhus using the reference scrub typhus infection criteria (STIC), i.e., positive cell culture isolation, an admission IgM antibody titer of ≥1:12,800, a 4-fold rising IgM antibody titer, and/or positivity for ≥2 out of 3 PCR gene targets). The ICTs gave the following sensitivities and specificities: the Panbio IgM ICT, 46% (95% confidence interval [CI], 33 to 60) and 95% (95% CI, 89 to 98), respectively; the Standard Diagnostics IgM ICT, 68% (95% CI, 60 to 75) and 73% (95% CI, 68 to 78), respectively; the AccessBio IgM ICT, 56% (95% CI, 48 to 63) and 90% (95% CI, 87 to 94), respectively; and the AccessBio total antibody ABt ICT, 61% (95% CI, 53 to 68) and 68% (95% CI, 63 to 73), respectively. An isothermal loop amplification (LAMP) PCR assay for scrub typhus demonstrated a sensitivity of 52% (95% CI, 38 to 66) and a specificity of 94% (95% CI, 88 to 98). This study has revealed the diagnostic limitations of antibody-based assays in an acute care setting. However, the combination of ICTs with LAMP usually increased sensitivity with a minimal reduction in specificity. The best combination, the Panbio IgM ICT and LAMP, resulted in a sensitivity of 67% (95% CI, 53 to 79) and a specificity of 91% (95% CI, 83 to 95). The combination of antibody-based assays with DNA- or antigen-based tests shows promise for improved diagnostic sensitivity.

Background: There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy., Methodology/principal Findings: In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand. A robust reference comparator set comprising following 'scrub typhus infection criteria' (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the 'gold standard' indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays. Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96-99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%., Conclusions/significance: The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus.

Clinically useful diagnostic tests of dengue virus infection are lacking. We prospectively evaluated the performance of real-time reverse transcriptase (rRT)-PCR, NS-1 antigen and IgM antibody tests to confirm dengue virus infection in acute blood specimens from 162 patients presenting with undifferentiated febrile illness compatible with dengue infection. rRT-PCR was the most sensitive test (89%) and potentially could be used as a single test for confirmation of dengue infection. NS-1 antigen and IgM antibody were not sufficiently sensitive to be used as a single confirmatory test with sensitivities of 54% and 17% respectively. The specificities of rRT-PCR, NS-1 antigen and IgM antibody tests were 96%, 100% and 88% respectively. Combining NS-1 and rRT-PCR or the combination of all three tests resulted in the highest sensitivity (93%) but specificities dropped to 96% and 83% respectively. We conclude that at least the combination of two tests, either agent detection (rRT-PCR) or antigen detection (NS-1) plus IgM antibody detection should be used for laboratory confirmation of dengue infection., (Copyright © 2010 Royal Society of Tropical Medicine and Hygiene. All rights reserved.)

Limited microbiology services impede adequate diagnosis and treatment of common infections such as pyelonephritis in resource-limited settings. Febrile pregnant women attending antenatal clinics at Shoklo Malaria Research Unit were offered urine dipstick, sediment microscopy, urine culture, and a 5-mL blood culture. The incidence of pyelonephritis was 11/1,000 deliveries (N = 53 in 4,819 pregnancies) between January 7, 2004 and May 17, 2006. Pyelonephritis accounted for 20.2% (41/203) of fever cases in pregnancy. Escherichia coli was the most commonly isolated pathogen: 87.5% (28/32) of organisms cultured. Susceptibility of E. coli to ampicillin (14%), cotrimoxazole (21%), and amoxicillin-clavulanic acid (48%) was very low. E. coli was susceptible to ceftriaxone and ciprofloxacin. The rate of extended spectrum β-lactamase (4.2%; 95% confidence interval = 0.7-19.5) was low. The rate and causes of pyelonephritis in pregnant refugee and migrant women were comparable with those described in developed countries. Diagnostic innovation in microbiology that permits affordable access is a high priority for resource-poor settings.

Background: Fever in pregnancy is dangerous for both mother and foetus. In the 1980's malaria was the leading cause of death in pregnant women in refugee camps on the Thai-Burmese border. Artemisinin combination therapy has significantly reduced the incidence of malaria in the population. The remaining causes of fever in pregnancy are not well documented., Methodology: Pregnant women attending antenatal care, where weekly screening for malaria is routine, were invited to have a comprehensive clinical and laboratory screen if they had fever. Women were admitted to hospital, treated and followed up weekly until delivery. A convalescent serum was collected on day 21. Delivery outcomes were recorded., Principal Findings: Febrile episodes (n = 438) occurred in 5.0% (409/8,117) of pregnant women attending antenatal clinics from 7-Jan-2004 to 17-May-2006. The main cause was malaria in 55.5% (227/409). A cohort of 203 (49.6% of 409) women had detailed fever investigations and follow up. Arthropod-borne (malaria, rickettsial infections, and dengue) and zoonotic disease (leptospirosis) accounted for nearly half of all febrile illnesses, 47.3% (96/203). Coinfection was observed in 3.9% (8/203) of women, mostly malaria and rickettsia. Pyelonephritis, 19.7% (40/203), was also a common cause of fever. Once malaria, pyelonephritis and acute respiratory illness are excluded by microscopy and/or clinical findings, one-third of the remaining febrile infections will be caused by rickettsia or leptospirosis. Scrub and murine typhus were associated with poor pregnancy outcomes including stillbirth and low birth weight. One woman died (no positive laboratory tests)., Conclusion/significance: Malaria remains the leading cause of fever in pregnancy on the Thai-Burmese border. Scrub and murine typhus were also important causes of fever associated with poor pregnancy outcomes. Febrile pregnant women on the Thai-Burmese border who do not have malaria, pyelonephritis or respiratory tract infection should be treated with azithromycin, effective for typhus and leptospirosis.

Wild bird surveillance for highly pathogenic avian influenza (HPAI) H5N1 virus from 2004 to 2007 in Thailand indicated that the prevalence of infection with avian influenza H5N1 virus in wild birds was low (1.0%, 95% confidence interval [CI]: 0.7-1.2, 60/6,263 pooled samples). However, the annual prevalence varied considerably over this period, with a peak of 2.7% (95% CI: 1.4, 4.1) in 2004. Prevalence dropped to 0.5% (95% CI: 0.3, 0.8]) and 0.6% (95% CI: 0.3, 1.0) in 2005 and 2006, respectively, and then increased to 1.8% (95% CI: 1.0, 2.6) in 2007. During this period, 16 species from 12 families of wild birds tested positive for H5N1 virus infection. All samples from juvenile birds were negative for H5N1 virus, whereas 0.6% (95% CI: 0.4, 0.9) of pooled samples from adult birds were positive. Most positive samples originated from peridomestic resident species. Infected wild bird samples were only found in provinces where poultry outbreaks had occurred. Detection of H5N1 virus infection in wild birds was reported up to 3 yr after eradication of the poultry outbreaks in those provinces. As observed with outbreaks in poultry, the frequencies of H5N1 outbreaks in wild birds were significantly higher in winter. Further understanding of the mechanisms of persistence and ongoing HPAI H5N1 transmission between wild birds and domestic poultry is needed.

Partial nucleotide sequences (459 bp) of the groEL gene (encoding the 60-kDa heat shock protein, HSP60) from 23 contemporary isolates of Orientia tsutsugamushi isolated from patients with acute scrub typhus in Thailand were compared with 16 reference strain sequences to evaluate the potential of groEL as a conserved and representative target for molecular diagnostics.. Overall nucleotide identity within all available O. tsutsugamushi isolates (n = 39) was 98.8% (range: 95.0-100), reflecting a high degree of conservation; nucleotide identities were 67.5% and 65.6%, respectively, when typhus and spotted fever group rickettsiae were included.. A highly sensitive and quantitative real-time PCR assay was designed and evaluated using 61 samples, including buffy coats from patients in Thailand and Laos. Reliable and accurate quantitation of bacterial loads allows further investigation of other diagnostic methods and may lead to an improved understanding of the pathophysiology of acute scrub typhus, an important but under-recognized disease.

We present a loop-mediated isothermal PCR assay (LAMP) targeting the groEL gene, which encodes the 60kDa heat shock protein of Orientia tsutsugamushi. Evaluation included testing of 63 samples of contemporary in vitro isolates, buffy coats and whole blood samples from patients with fever. Detection limits for LAMP were assessed by serial dilutions and quantitation by real-time PCR assay based on the same target gene: three copies/microl for linearized plasmids, 26 copies/microl for VERO cell culture isolates, 14 copies/microl for full blood samples and 41 copies/microl for clinical buffy coats. Based on a limited sample number, the LAMP assay is comparable in sensitivity with conventional nested PCR (56kDa gene), with limits of detection well below the range of known admission bacterial loads of patients with scrub typhus. This inexpensive method requires no sophisticated equipment or sample preparation, and may prove useful as a diagnostic assay in financially poor settings; however, it requires further prospective validation in the field setting.

Scrub typhus is responsible for a large proportion of undifferentiated fevers in south-east Asia. The cellular tropism and pathophysiology of the causative agent, Orientia tsutsugamushi, remain poorly understood. We measured endothelial and leucocyte activation by soluble cell adhesion molecule enzyme-linked immunosorbent assays in 242 Lao and Thai patients with scrub or murine typhus, leptospirosis, dengue, typhoid and uncomplicated falciparum malaria on admission to hospital. Soluble E-selectin (sE-selectin) levels were lowest in dengue, sL-selectin highest in scrub typhus with a high sE-selectin to sL-selectin ratio in leptospirosis patients. In scrub typhus patients elevated sL-selectin levels correlated with the duration of skin rash (P = 0.03) and the presence of eschar (P = 0.03), elevated white blood cell (WBC) count (P = 0.007), elevated lymphocyte (P = 0.007) and neutrophil counts (P = 0.015) and elevated levels of sE-selectin correlated with the duration of illness before admission (P = 0.03), the presence of lymphadenopathy (P = 0.033) and eschar (P = 0.03), elevated WBC (P = 0.005) and neutrophil counts (P = 0.0003). In comparison, soluble selectin levels in murine typhus patients correlated only with elevated WBC counts (P = 0.03 for sE-selectin and sL-selectin). Soluble intercellular adhesion molecule-1 and soluble vascular adhesion molecule-1 levels were not associated significantly with any clinical parameters in scrub or murine typhus patients. The data presented suggest mononuclear cell activation in scrub typhus. As adhesion molecules direct leucocyte migration and induce inflammatory and immune responses, this may represent O. tsutsugamushi tropism during early dissemination, or local immune activation within the eschar.

Orientia tsutsugamushi is the causative agent of scrub typhus, a major cause of febrile illness in the rural areas of Southeast Asia. Twenty-three strains of O. tsutsugamushi were isolated from patients with scrub typhus in north-east (Udorn Thani province) and western Thailand (Tak province) between 2003 and 2005. The isolates were characterized by sequencing the entire ORF of the 56-kDa-type-specific antigen gene, followed by phylogenetic analysis. The majority (15/23) of isolates clustered with the Karp-type strain, six with a Gilliam-type strain and one each with the TA716- and TA763-type strains. Overall, there was considerable diversity in sequence, comparable to that seen in strains from across the rest of the scrub typhus-endemic world. There was no significant difference in the distributions of strains between the two provinces (P=0.08, Fisher's exact) nor a temporal change in distribution with year of isolation (P=0.80, Fisher's exact). Within this diversity there were also examples of isolates with identical 56-kDa genotypes that were cultured from patients from the same geographical areas.

Here we describe the diagnostic utility of the indirect immunofluorescence assay (IFA) during a recent outbreak of highly pathogenic avian influenza (HPAI) subtype H5N1 virus in southern Thailand and demonstrate the usefulness of the cardiac tissue from infected chickens, quail, and ducks for diagnosis. The most reliable sample for IFA diagnosis of influenza A virus was cardiac tissue (83.0%; 44/53) which when divided by species (chicken, quail and duck cardiac tissues) gave respective positivity rates of 88% (22/25), 88.9% (16/18) and 60.0% (6/10). Cardiac tissue also gave the highest IFA intensity for the three species. We believe that the IFA method has wide applicability in developing countries or remote settings where clinically similar avian diseases with high morbidity and mortality such as Newcastle disease and fowl cholera are common and could be rapidly excluded thereby conserving valuable reference laboratory capacity for true HPAI outbreaks.

Molecular typing of leptospiral strains based on variation within putative O-antigen polymerase gene (wzy) was determined among reference strains and those isolated from patients. Using the PCR primers designed from the flanking gene of wzy derived from Leptospira interrogans serovar Copenhageni, all L. interrogans serovars as well as human and rodent leptospiral isolates from Thailand could be amplified. The size of PCR product ranged from 1 to 1.5 kb. The limitation of these primer pairs was the inability to amplify those strains whose sequences differ in the region of the primers, these included Leptospira biflexa (serovar Patoc), Leptospira borgpetersenii (serovar Tarassovi) and Leptospira kirschneri (serovar Bim, Bulgarica, Butembo). Notably, amplification was not limited to L. interrogans as demonstrated by the amplification of some strains from L. kirschneri, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, L. borgpetersenii and Leptospira weilii. The phylogenetic tree of wzy sequence, inferred by posterior probability of the Bayesian, enabled the categorization of leptospiral serovars into seven genetically related group, of which its differentiation power was better than that of the more highly conserved 16S rRNA gene, which is used extensively for genotyping.

Orientia tsutsugamushi is the causative agent of scrub typhus infection, a major cause of human disease in rural areas of Southeast Asia. Twenty-six blood samples collected from patients with serologically proven scrub typhus during a six month period were sent to Bangkok (535 km from the clinical site) by road at ambient temperature (average daily temperature range: 27.1-29.1 degrees C) for attempted in vitro isolation in Vero cells. O. tsutsugamushi was isolated from 12 samples (sensitivity 46.7%) with the time to isolation ranging from 16 to 37 days [median 27 days, inter-quartile range (IQR) 22.5-33.5 days]. Patient factors such as days of fever and O. tsutsugamushi IgM antibody titer, transport factors such as transit time, and isolate genotype (Karp and Gilliam/Kawasaki) were assessed to determine their influence on the outcome of in vitro isolation. None of the factors significantly influenced the isolation outcome. This study demonstrates that O. tsutsugamushi can often be isolated in vitro from the blood of scrub typhus patients when transported at ambient tropical temperatures for many days.

The aim of this study was to evaluate the use of polymerase chain reaction (PCR) amplification of the O. tsutsugamushi 16S rRNA gene for the diagnosis of scrub typhus in rural Thailand. A prospective study of acute febrile illness in Udon Thani, northeast Thailand, identified 183 patients as having scrub typhus on the basis of immunofluorescent antibody testing (IFA) of paired sera. A further 366 febrile patients admitted concurrently with a range of other diagnoses acted as negative controls. Diagnostic sensitivity and specificity of 16S rRNA PCR was 44.8% and 99.7%, respectively, compared with IFA. PCR positivity was related to duration of symptoms and presence of eschar (P < 0.001, both cases). PCR using primers to amplify a fragment of the 56-kd gene had a sensitivity and specificity of 29.0% and 99.2%, respectively. PCR has a high specificity but low sensitivity for the rapid diagnosis of scrub typhus in this endemic setting.

The tropism of a Thailand strain of highly pathogenic avian influenza H5N1 virus was demonstrated on tissues (lung, trachea, heart, liver, spleen, pancreas, rectum, kidney, brain, skeletal muscle, duodenum, and oviduct) from naturally infected native chickens (Gallus gallus), Japanese quail (Coturnix coturnix japonica) and ducks (Anas spp.) by indirect immunofluorescence assay. In chickens and quail, the distribution and localization of nucleoprotein viral antigen was similar and detected at the highest level in cardiac myocytes, at 88% (chickens) and 89% (quail), and respiratory, digestive and urinary systems all showed high levels of antigen. Antigen in duck tissues were detected at significantly lower levels (P < 0.05) with the exception of brain and skeletal muscle samples. In most cases, antigen in duck tissue was absent in the digestive organs but present in respiratory organs, which supports the hypothesis that aerosol and oral-oral transmission are the main method of highly pathogenic avian influenza virus transmission from this species.

Antisera were produced at a central laboratory in Thailand against the endemic serotypes (O, A and Asia 1) of foot and mouth disease (FMD) virus. At a regional veterinary laboratory, these antisera were used in an indirect sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and serotyping of FMD virus (FMDV) antigen. ELISA readings of < 0.10 optical density (OD) units were considered negative. This was verified using fifty tissue samples which were known to be negative for FMDV. The highest mean sample value for three different dilutions was 0.02 OD units. Of a total of 93 samples submitted for antigen typing, 80 (86%) tested positive by ELISA and 13 (14%) were negative. No FMDV was detected in ELISA-negative samples following attempted tissue-culture virus isolation.

A survey of type O foot and mouth disease (FMD) virus isolates from northern Thailand was undertaken to determine the relationship between field viruses and the vaccine in use, and to gauge the range of antigenic variation among field viruses. Isolates were collected from the two most recent epizootics, 1986-1987 and 1989-1990, and assessed using a two-dimensional neutralisation test to determine their relationship to FMD type O1 Bangkok 1960 (O BKK/60) reference (vaccine challenge) virus. The critical r value for the survey was 0.259 and all isolates tested were found to have an r value considerably greater than this (range 0.66 to 0.80). The results showed close antigenic relationships between the isolates and the reference virus, and indicated a relatively small range of antigenic variation between the isolates.