Professor Daniel H Paris

Research Area: Microbiology
Technology Exchange: Cellular immunology, Immunohistochemistry, Microscopy (Confocal) and Vaccine production and evaluation
Scientific Themes: Tropical Medicine & Global Health and Immunology & Infectious Disease
Keywords: Rickettsia, Scrub typhus, Murine Typhus, Pathophysiology, Vaccine, Host response and Immune response
Web Links:
Figure 1. Immune cells of the skin fighting a typhus infection.

Orientia tsutsugamushi is a bacterium that causes scrub typhus, a common infectious disease in Asia. Chigger mites deposit the bacteria (small bright green spots) into the skin during the bite, which attract specialised immune cells (in red and blue) to the site of infection. Red blood cells are colored in golden-yellow.

Double-immunofluorescence staining with T-cell marker CD3 (red) and anti-Orientia tsutsugamushi (green) antibodies, nuclear counterstain with DAPI (blue). Magnification x400.
Image by Daniel H. Paris.

Figure 1. Immune cells of the skin fighting a typhus infection. Orientia tsutsugamushi is a ...

Figure 2. Typhus bacteria invading blood vessels of the skin.
Orientia tsutsugamushi is a bacterium transmitted by chigger mites and causes scrub typhus, a tropical infectious disease of Asia. These specialised bacteria (in green) can infect immune cells and use them as ‘taxis’ to escape towards lymph nodes and through blood vessels (red cells) in the topmost layer of the skin (blue).

Double-immunofluorescence staining with endothelial surface marker CD31 (red) and anti-Orientia tsutsugamushi (green) antibodies, nuclear counterstain with DAPI (blue). Magnification x400.
Image by Daniel H. Paris.

Figure 2. Typhus bacteria invading blood vessels of the skin. Orientia tsutsugamushi is a ...

Fig 3. Typhus bacteria invading the skin.
An activated immune cell (red, centre) takes a turn as it targets three small bacteria called Orientia tsutsugamushi (in green), in the skin of a patient from the Lao PDR, who suffered from a tropical infectious disease called scrub typhus.

Double-immunofluorescence staining, using LSP-1 (red) and anti-Orientia tsutsugamushi (green) antibodies. Nuclear counterstain with DAPI (blue). Magnification x400.
Image by Daniel H. Paris.

Fig 3. Typhus bacteria invading the skin. An activated immune cell (red, centre) takes a turn as ...

Figure 4. Morphological characteristics of chigger mites. 
Panel A represents the unfed larval stage of a Leptotrombidium mite typically termed ‘chigger’. The scutum and mouthparts play an important role in the identification of genus and species. Panel B shows the mouthparts (termed gnathosoma) of Schoengastia sp. with the cheliceral blade in the detailed insert. Variations of hairs and sensillory antennae in the scutum, a characteristic dorsal plate, that enables differentiation between genera and subgenera (Panel C depicts a scutum of the subgenera Leptotrombidium, while panel D is from Ascoschoengastia sp.).

Image compilation from Daniel H. Paris et al., Molecular Medical Microbiology. All rights reserved. Special thanks go to Dr. Benjamin Makepeace and Kittipong Chaisiri, University of Liverpool, UK.

Figure 4. Morphological characteristics of chigger mites. Panel A represents the unfed larval ...

Figure 5. The rodent hunting team sets out to place traps at dusk, Laos.
Capturing rodents and their ectoparasites (especially mites and fleas) to study the bacteria they transmit and harbour within, is an important aspect of research into dissecting the natural history of scrub typhus. 
Photo by Daniel H. Paris

Figure 5. The rodent hunting team sets out to place traps at dusk, Laos. Capturing rodents and ...

Figure 6. A small cluster of mites in a rodents ear.
This rodent was captured in Chiangrai, N-Thailand, where severe clinical manifestations of scrub typhus occur and potential antibiotic resistance has been reported. We are studying Orientia tsutsugamushi, the causative bacteria of scrub typhus, isolated from patients, rodents and from mites (collected from soil and rodents) to better understand the bacterial virulence factors and the population genetic structure. The orange spots within this ear represent a cluster of Leptotrombidium mites, that transmit scrub typhus in this region. 
Photo by Daniel H. Paris

Figure 6. A small cluster of mites in a rodents ear. This rodent was captured in Chiangrai, ...

Daniel H. Paris co-ordinates the rickettsial research performed in the Thailand and Laos MOUs. His major interests include the epidemiology, diagnostics, pathophysiology and immune response of rickettsial infections and typhus-like illnesses (Leptospirosis, Typhoid and Dengue) afflicting rural populations throughout Asia and beyond.

His particular research focus encompasses clinical studies on the immune response, the characterisation of an animal model and various immuno-pathophysiology studies in scrub typhus, building towards improving diagnostics and vaccine development.

Name Department Institution Country
Professor Nicholas PJ Day FMedSci FRCP Tropical Medicine Oxford University, Bangkok Thailand
Professor Sir Nicholas J White FRS Tropical Medicine Oxford University, Bangkok Thailand
Professor Paul Newton Tropical Medicine Oxford University, Vientiane Laos
Professor Derek Smith Pathogen Evolution Group Cambridge University United Kingdom
Dr Allen L Richards Viral and Rickettsial Diseases Naval Medical Research Centre (NMRC) United States
Professor Susanna J Dunachie FRCP FRCPath Tropical Medicine Oxford University, Peter Medawar Building United Kingdom
Professor Phil L Felgner Microbiology University California Irvine (UCI) United States
Dr Pacharee Kantipong Chiangrai Prachanukroh Hospital Thailand
Professor Jeremy Day Tropical Medicine Oxford University, Ho Chi Minh City Vietnam
Professor David H Walker Pathology University Texas Medical Branch (UTMB) United States
Dr Jeanne Salje Tropical Medicine Oxford University, Bangkok Thailand
Professor Yoel Lubell Tropical Medicine Oxford University, Bangkok Thailand
Dr Sabine Dittrich Tropical Medicine Oxford University, Vientiane Laos
Paris DH, Dumler JS. 2016. State of the art of diagnosis of rickettsial diseases: the use of blood specimens for diagnosis of scrub typhus, spotted fever group rickettsiosis, and murine typhus. Curr Opin Infect Dis, 29 (5), pp. 433-439. | Show Abstract | Read more

PURPOSE OF REVIEW: With improved malaria control, acute undifferentiated febrile illness studies in tropical regions reveal a startling proportion of rickettsial illnesses, especially scrub typhus, murine typhus, and spotted fever group rickettsioses. Laboratory diagnosis of these infections evolved little over the past 40 years, but combinations of technologies like PCR and loop-mediated isothermal amplification, with refined rapid diagnostic tests and/or ELISA, are promising for guidance for early antirickettsial treatment. RECENT FINDINGS: The long-term reliance on serological tests - useful only late in rickettsial infections - has led to underdiagnosis, inappropriate therapies, and undocumented morbidity and mortality. Recent approaches integrate nucleic acid amplification and recombinant protein-based serological tests for diagnosing scrub typhus. Optimized using Bayesian latent class analyses, this strategy increases diagnostic confidence and enables early accurate diagnosis and treatment - a model to follow for lagging progress in murine typhus and spotted fever. SUMMARY: A laboratory diagnostic paradigm shift in rickettsial infections is evolving, with replacement of indirect immunofluorescence assay by the more objective ELISA coupled with nucleic acid amplification assays to expand the diagnostic window toward early infection intervals. This approach supports targeted antirickettsial therapy, reduces morbidity and mortality, and provides a robust evidence base for further development of diagnostics and vaccines.

Weitzel T, Dittrich S, López J, Phuklia W, Martinez-Valdebenito C, Velásquez K, Blacksell SD, Paris DH, Abarca K. 2016. Endemic Scrub Typhus in South America. N Engl J Med, 375 (10), pp. 954-961. | Show Abstract | Read more

Scrub typhus is a life-threatening zoonosis caused by Orientia tsutsugamushi organisms that are transmitted by the larvae of trombiculid mites. Endemic scrub typhus was originally thought to be confined to the so called "tsutsugamushi triangle" within the Asia-Pacific region. In 2006, however, two individual cases were detected in the Middle East and South America, which suggested that the pathogen was present farther afield. Here, we report three autochthonous cases of scrub typhus caused by O. tsutsugamushi acquired on Chiloé Island in southern Chile, which suggests the existence of an endemic focus in South America. (Funded by the Chilean Comisión Nacional de Investigación Científica y Tecnológica and the Wellcome Trust.).

James SL, Blacksell SD, Nawtaisong P, Tanganuchitcharnchai A, Smith DJ, Day NPJ, Paris DH. 2016. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand. PLoS Negl Trop Dis, 10 (6), pp. e0004723. | Show Abstract | Read more

BACKGROUND: Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. CONCLUSIONS/SIGNIFICANCE: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.

Phetsouvanh R, Sonthayanon P, Pukrittayakamee S, Paris DH, Newton PN, Feil EJ, Day NPJ. 2016. Correction: The Diversity and Geographical Structure of Orientia tsutsugamushi Strains from Scrub Typhus Patients in Laos. PLoS Negl Trop Dis, 10 (5), pp. e0004742. | Show Abstract | Read more

[This corrects the article DOI: 10.1371/journal.pntd.0004024.].

Blacksell SD, Lim C, Tanganuchitcharnchai A, Jintaworn S, Kantipong P, Richards AL, Paris DH, Limmathurotsakul D, Day NPJ. 2016. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay. J Clin Microbiol, 54 (6), pp. 1472-1478. | Show Abstract | Read more

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.

Lubell Y, Althaus T, Blacksell SD, Paris DH, Mayxay M, Pan-Ngum W, White LJ, Day NPJ, Newton PN. 2016. Modelling the Impact and Cost-Effectiveness of Biomarker Tests as Compared with Pathogen-Specific Diagnostics in the Management of Undifferentiated Fever in Remote Tropical Settings. PLoS One, 11 (3), pp. e0152420. | Show Abstract | Read more

BACKGROUND: Malaria accounts for a small fraction of febrile cases in increasingly large areas of the malaria endemic world. Point-of-care tests to improve the management of non-malarial fevers appropriate for primary care are few, consisting of either diagnostic tests for specific pathogens or testing for biomarkers of host response that indicate whether antibiotics might be required. The impact and cost-effectiveness of these approaches are relatively unexplored and methods to do so are not well-developed. METHODS: We model the ability of dengue and scrub typhus rapid tests to inform antibiotic treatment, as compared with testing for elevated C-Reactive Protein (CRP), a biomarker of host-inflammation. Using data on causes of fever in rural Laos, we estimate the proportion of outpatients that would be correctly classified as requiring an antibiotic and the likely cost-effectiveness of the approaches. RESULTS: Use of either pathogen-specific test slightly increased the proportion of patients correctly classified as requiring antibiotics. CRP testing was consistently superior to the pathogen-specific tests, despite heterogeneity in causes of fever. All testing strategies are likely to result in higher average costs, but only the scrub typhus and CRP tests are likely to be cost-effective when considering direct health benefits, with median cost per disability adjusted life year averted of approximately $48 USD and $94 USD, respectively. CONCLUSIONS: Testing for viral infections is unlikely to be cost-effective when considering only direct health benefits to patients. Testing for prevalent bacterial pathogens can be cost-effective, having the benefit of informing not only whether treatment is required, but also as to the most appropriate antibiotic; this advantage, however, varies widely in response to heterogeneity in causes of fever. Testing for biomarkers of host inflammation is likely to be consistently cost-effective despite high heterogeneity, and can also offer substantial reductions in over-use of antimicrobials in viral infections.

Phommasone K, Althaus T, Souvanthong P, Phakhounthong K, Soyvienvong L, Malapheth P, Mayxay M, Pavlicek RL, Paris DH, Dance D et al. 2016. Accuracy of commercially available c-reactive protein rapid tests in the context of undifferentiated fevers in rural Laos. BMC Infect Dis, 16 (1), pp. 61. | Show Abstract | Read more

BACKGROUND: C-Reactive Protein (CRP) has been shown to be an accurate biomarker for discriminating bacterial from viral infections in febrile patients in Southeast Asia. Here we investigate the accuracy of existing rapid qualitative and semi-quantitative tests as compared with a quantitative reference test to assess their potential for use in remote tropical settings. METHODS: Blood samples were obtained from consecutive patients recruited to a prospective fever study at three sites in rural Laos. At each site, one of three rapid qualitative or semi-quantitative tests was performed, as well as a corresponding quantitative NycoCard Reader II as a reference test. We estimate the sensitivity and specificity of the three tests against a threshold of 10 mg/L and kappa values for the agreement of the two semi-quantitative tests with the results of the reference test. RESULTS: All three tests showed high sensitivity, specificity and kappa values as compared with the NycoCard Reader II. With a threshold of 10 mg/L the sensitivity of the tests ranged from 87-98 % and the specificity from 91-98 %. The weighted kappa values for the semi-quantitative tests were 0.7 and 0.8. CONCLUSION: The use of CRP rapid tests could offer an inexpensive and effective approach to improve the targeting of antibiotics in remote settings where health facilities are basic and laboratories are absent. This study demonstrates that accurate CRP rapid tests are commercially available; evaluations of their clinical impact and cost-effectiveness at point of care is warranted.

Cross R, Ling C, Day NPJ, McGready R, Paris DH. 2016. Revisiting doxycycline in pregnancy and early childhood--time to rebuild its reputation? Expert Opin Drug Saf, 15 (3), pp. 367-382. | Show Abstract | Read more

INTRODUCTION: Doxycycline is highly effective, inexpensive with a broad therapeutic spectrum and exceptional bioavailability. However these benefits have been overshadowed by its classification alongside the tetracyclines - class D drugs, contraindicated in pregnancy and in children under 8 years of age. Doxycycline-treatable diseases are emerging as leading causes of undifferentiated febrile illness in Southeast Asia. For example scrub typhus and murine typhus have an unusually severe impact on pregnancy outcomes, and current mortality rates for scrub typhus reach 12-13% in India and Thailand. The emerging evidence for these important doxycycline-treatable diseases prompted us to revisit doxycycline usage in pregnancy and childhood. AREAS COVERED: A systematic review of the available literature on doxycycline use in pregnant women and children revealed a safety profile of doxycycline that differed significantly from that of tetracycline; no correlation between the use of doxycycline and teratogenic effects during pregnancy or dental staining in children was found. EXPERT OPINION: The change of the US FDA pregnancy classification scheme to an evidence-based approach will enable adequate evaluation of doxycycline in common tropical illnesses and in vulnerable populations in clinical treatment trials, dosage-optimization pharmacokinetic studies and for the empirical treatment of undifferentiated febrile illnesses, especially in pregnant women and children.

Blacksell SD, Tanganuchitcharnchai A, Nawtaisong P, Kantipong P, Laongnualpanich A, Day NPJ, Paris DH. 2015. Diagnostic Accuracy of the InBios Scrub Typhus Detect Enzyme-Linked Immunoassay for the Detection of IgM Antibodies in Northern Thailand. Clin Vaccine Immunol, 23 (2), pp. 148-154. | Show Abstract | Read more

In this study, we examined the diagnostic accuracy of the InBios Scrub Typhus Detect IgM enzyme-linked immunosorbent assay (ELISA) and determined the optimal diagnostic optical density (OD) cutoffs for screening and diagnostic applications based on prospectively collected, characterized samples from undifferentiated febrile illness patients in northern Thailand. Direct comparisons with the serological gold standard, indirect immunofluorescence assay (IFA), revealed strong statistical correlation of ELISA OD values and IFA IgM titers. Determination of the optimal ELISA cutoff for seroepidemiology or screening purposes compared to the corresponding IFA reciprocal titer of 400 as previously described for Thailand was 0.60 OD, which corresponded to a sensitivity (Sn) of 84% and a specificity (Sp) of 98%. The diagnostic performance against the improved and more-stringent scrub typhus infection criteria (STIC), correcting for low false-positive IFA titers, resulted in an Sn of 93% and an Sp of 91% at an ELISA cutoff of 0.5 OD. This diagnostic ELISA cutoff corresponds to IFA reciprocal titers of 1,600 to 3,200, which greatly reduces the false-positive rates associated with low-positive IFA titers. These data are in congruence with the recently improved serodiagnostic positivity criteria using the Bayesian latent class modeling approach. In summary, the InBios Scrub Typhus Detect IgM ELISA is affordable and easy-to-use, with adequate diagnostic accuracy for screening and diagnostic purposes, and should be considered an improved alternative to the gold standard IFA for acute diagnosis. For broader application, regional cutoff validation and antigenic composition for consistent diagnostic accuracy should be considered.

Slesak G, Inthalath S, Dittrich S, Paris DH, Newton PN. 2015. Leeches as further potential vectors for rickettsial infections. Proc Natl Acad Sci U S A, 112 (48), pp. E6593-E6594. | Read more

Lubell Y, Blacksell SD, Dunachie S, Tanganuchitcharnchai A, Althaus T, Watthanaworawit W, Paris DH, Mayxay M, Peto TJ, Dondorp AM et al. 2015. Performance of C-reactive protein and procalcitonin to distinguish viral from bacterial and malarial causes of fever in Southeast Asia. BMC Infect Dis, 15 (1), pp. 511. | Show Abstract | Read more

BACKGROUND: Poor targeting of antimicrobial drugs contributes to the millions of deaths each year from malaria, pneumonia, and other tropical infectious diseases. While malaria rapid diagnostic tests have improved use of antimalarial drugs, there are no similar tests to guide the use of antibiotics in undifferentiated fevers. In this study we estimate the diagnostic accuracy of two well established biomarkers of bacterial infection, procalcitonin and C-reactive protein (CRP) in discriminating between common viral and bacterial infections in malaria endemic settings of Southeast Asia. METHODS: Serum procalcitonin and CRP levels were measured in stored serum samples from febrile patients enrolled in three prospective studies conducted in Cambodia, Laos and, Thailand. Of the 1372 patients with a microbiologically confirmed diagnosis, 1105 had a single viral, bacterial or malarial infection. Procalcitonin and CRP levels were compared amongst these aetiological groups and their sensitivity and specificity in distinguishing bacterial infections and bacteraemias from viral infections were estimated using standard thresholds. RESULTS: Serum concentrations of both biomarkers were significantly higher in bacterial infections and malaria than in viral infections. The AUROC for CRP in discriminating between bacterial and viral infections was 0.83 (0.81-0.86) compared with 0.74 (0.71-0.77) for procalcitonin (p < 0.0001). This relative advantage was evident in all sites and when stratifying patients by age and admission status. For CRP at a threshold of 10 mg/L, the sensitivity of detecting bacterial infections was 95% with a specificity of 49%. At a threshold of 20 mg/L sensitivity was 86% with a specificity of 67%. For procalcitonin at a low threshold of 0.1 ng/mL the sensitivity was 90% with a specificity of 39%. At a higher threshold of 0.5 ng/ul sensitivity was 60% with a specificity of 76%. CONCLUSION: In samples from febrile patients with mono-infections from rural settings in Southeast Asia, CRP was a highly sensitive and moderately specific biomarker for discriminating between viral and bacterial infections. Use of a CRP rapid test in peripheral health settings could potentially be a simple and affordable measure to better identify patients in need of antibacterial treatment and part of a global strategy to combat the emergence of antibiotic resistance.

Kingston HWF, Blacksell SD, Tanganuchitcharnchai A, Laongnualpanich A, Basnyat B, Day NPJ, Paris DH. 2015. Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology. Clin Vaccine Immunol, 22 (10), pp. 1130-1132. | Show Abstract | Read more

This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ≥1:1,600 versus 92% and 95% at ≥1:6,400, respectively.

Taylor AJ, Paris DH, Newton PN. 2015. A Systematic Review of Mortality from Untreated Scrub Typhus (Orientia tsutsugamushi). PLoS Negl Trop Dis, 9 (8), pp. e0003971. | Show Abstract | Read more

BACKGROUND: Scrub typhus, a bacterial infection caused by Orientia tsutsugamushi, is increasingly recognized as an important cause of fever in Asia, with an estimated one million infections occurring each year. Limited access to health care and the disease's non-specific symptoms mean that many patients are undiagnosed and untreated, but the mortality from untreated scrub typhus is unknown. This review systematically summarizes the literature on the untreated mortality from scrub typhus and disease outcomes. METHODOLOGY/PRINCIPAL FINDINGS: A literature search was performed to identify patient series containing untreated patients. Patients were included if they were symptomatic and had a clinical or laboratory diagnosis of scrub typhus and excluded if they were treated with antibiotics. The primary outcome was mortality from untreated scrub typhus and secondary outcomes were total days of fever, clinical symptoms, and laboratory results. A total of 76 studies containing 89 patient series and 19,644 patients were included in the final analysis. The median mortality of all patient series was 6.0% with a wide range (min-max) of 0-70%. Many studies used clinical diagnosis alone and had incomplete data on secondary outcomes. Mortality varied by location and increased with age and in patients with myocarditis, delirium, pneumonitis, or signs of hemorrhage, but not according to sex or the presence of an eschar or meningitis. Duration of fever was shown to be long (median 14.4 days Range (9-19)). CONCLUSIONS: Results show that the untreated mortality from scrub typhus appears lower than previously reported estimates. More data are required to clarify mortality according to location and host factors, clinical syndromes including myocarditis and central nervous system disease, and in vulnerable mother-child populations. Increased surveillance and improved access to diagnostic tests are required to accurately estimate the untreated mortality of scrub typhus. This information would facilitate reliable quantification of DALYs and guide empirical treatment strategies.

Giengkam S, Blakes A, Utsahajit P, Chaemchuen S, Atwal S, Blacksell SD, Paris DH, Day NPJ, Salje J. 2015. Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis, 9 (8), pp. e0004009. | Show Abstract | Read more

BACKGROUND: Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families. METHODOLOGY: A clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells. KEY RESULTS: Development of a standardised growth assay to quantify bacterial replication and viability in vitro. Quantitative comparison of different DNA extraction methods. Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement. Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds. Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing. CONCLUSIONS: Here we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.

Paris DH, Stephan F, Bulder I, Wouters D, van der Poll T, Newton PN, Day NPJ, Zeerleder S. 2015. Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos. PLoS Negl Trop Dis, 9 (8), pp. e0003990. | Show Abstract | Read more

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus.

Phetsouvanh R, Sonthayanon P, Pukrittayakamee S, Paris DH, Newton PN, Feil EJ, Day NPJ. 2015. The Diversity and Geographical Structure of Orientia tsutsugamushi Strains from Scrub Typhus Patients in Laos. PLoS Negl Trop Dis, 9 (8), pp. e0004024. | Show Abstract | Read more

Orientia tsutsugamushi is the causative agent of scrub typhus, a disease transmitted by Leptotrombidium mites which is responsible for a severe and under-reported public health burden throughout Southeast Asia. Here we use multilocus sequence typing (MLST) to characterize 74 clinical isolates from three geographic locations in the Lao PDR (Laos), and compare them with isolates described from Udon Thani, northeast Thailand. The data confirm high levels of diversity and recombination within the natural O. tsutsugamushi population, and a rate of mixed infection of ~8%. We compared the relationships and geographical structuring of the strains and populations using allele based approaches (eBURST), phylogenetic approaches, and by calculating F-statistics (FST). These analyses all point towards low levels of population differentiation between isolates from Vientiane and Udon Thani, cities which straddle the Mekong River which defines the Lao/Thai border, but with a very distinct population in Salavan, southern Laos. These data highlight how land use, as well as the movement of hosts and vectors, may impact on the epidemiology of zoonotic infections.

Dittrich S, Phuklia W, Turner GDH, Rattanavong S, Chansamouth V, Dumler SJ, Ferguson DJP, Paris DH, Newton PN. 2015. Neorickettsia sennetsu as a Neglected Cause of Fever in South-East Asia. PLoS Negl Trop Dis, 9 (7), pp. e0003908. | Show Abstract | Read more

Neorickettsia sennetsu infection is rarely recognized, with less than 100 globally reported patients over the last 50 years. The disease is thought to be contracted by eating raw fish, a staple of many South-East Asian cuisines. In 2009, the first patient with sennetsu was identified in the Lao PDR (Laos), raising the question as to how common this organism and related species are in patients presenting with fever. We investigated the frequency of N. sennetsu infection at hospitals in diverse areas of Laos. Consenting febrile hospital inpatients from central (Vientiane: n = 1,013), northern (Luang Namtha: n = 453) and southern (Salavan: n = 171) Laos were screened by PCR for N. sennetsu, if no previous positive direct diagnostic test was available. A PCR-restriction fragment length polymorphism assay was developed to differentiate between N. sennetsu, Ehrlichia chaffeensis and Anaplasma phagocytophilum. To allow more detailed studies of N. sennetsu, culture was successfully established using a reference strain (ATCC VR-367), identifying a canine-macrophage cell line (DH82) to be most suitable to visually identify infection. After screening, N. sennetsu was identified and sequence confirmed in four (4/1,637; 0.2%) Lao patients. Despite the previously identified high seroprevalence of N. sennetsu antibodies in the Lao population (~17%), acute N. sennetsu infection with sufficient clinical signs to prompt hospitalization appears to be rare. The reservoir, zoonotic cycle and pathogenicity of N. sennetsu remain unclear and require further investigations.

Dittrich S, Sunyakumthorn P, Rattanavong S, Phetsouvanh R, Panyanivong P, Sengduangphachanh A, Phouminh P, Anantatat T, Chanthongthip A, Lee SJ et al. 2015. Blood-Brain Barrier Function and Biomarkers of Central Nervous System Injury in Rickettsial Versus Other Neurological Infections in Laos. Am J Trop Med Hyg, 93 (2), pp. 232-237. | Show Abstract | Read more

Blood-brain barrier (BBB) function and cerebrospinal fluid (CSF) biomarkers were measured in patients admitted to hospital with severe neurological infections in the Lao People's Democratic Republic (N = 66), including bacterial meningitis (BM; N = 9) or tuberculosis meningitis (TBM; N = 11), Japanese encephalitis virus (JEV; N = 25), and rickettsial infections (N = 21) including murine and scrub typhus patients. The albumin index (AI) and glial fibrillary acidic protein (GFAP) levels were significantly higher in BM and TBM than other diseases but were also raised in individual rickettsial patients. Total tau protein was significantly raised in the CSF of JEV patients. No differences were found between clinical or neurological symptoms, AI, or biomarker levels that allowed distinction between severe neurological involvement by Orientia tsutsugamushi compared with Rickettsia species.

Taylor AJ, Paris DH, Newton PN. 2015. A Systematic Review of the Mortality from Untreated Leptospirosis. PLoS Negl Trop Dis, 9 (6), pp. e0003866. | Show Abstract | Read more

BACKGROUND: Leptospirosis occurs worldwide, but the global incidence of human disease and its mortality are not well understood. Many patients are undiagnosed and untreated due to its non-specific symptoms and a lack of access to diagnostics. This study systematically reviews the literature to clarify the mortality from untreated leptospirosis. Results will help quantify the global burden of disease and guide health policies. METHODOLOGY/PRINCIPAL FINDINGS: A comprehensive literature search was performed to identify untreated patient series. Included patients were symptomatic, but asymptomatic patients and those who had received antibiotics, dialysis or who were treated on Intensive Care Units were excluded. Included patients had a confirmed laboratory diagnosis by culture, PCR, or serological tests. Data was extracted and individual patient series were assessed for bias. Thirty-five studies, comprising 41 patient series and 3,390 patients, were included in the study. A high degree of bias within studies was shown due to limitations in study design, diagnostic tests and missing data. Median series mortality was 2.2% (Range 0.0-39.7%), but mortality was high in jaundiced patients (19.1%) (Range 0.0-39.7%), those with renal failure 12.1% (Range 0-25.0%) and in patients aged over 60 (60%) (Range 33.3-60%), but low in anicteric patients (0%) (Range 0-1.7%). CONCLUSIONS: This systematic review contributes to our understanding of the mortality of untreated leptospirosis and provides data for the estimation of DALYs attributable to this disease. We show that mortality is significantly higher in older patients with icteric disease or renal failure but is lower in younger, anicteric patients. Increased surveillance and accurate point-of-care diagnostics are required to better understand the incidence and improve diagnosis of disease. Empirical treatment strategies should prioritize early treatment to improve outcomes from leptospirosis.

Blacksell SD, Kantipong P, Watthanaworawit W, Turner C, Tanganuchitcharnchai A, Jintawon S, Laongnuanutit A, Nosten FH, Day NPJ, Paris DH, Richards AL. 2015. Underrecognized arthropod-borne and zoonotic pathogens in northern and northwestern Thailand: serological evidence and opportunities for awareness. Vector Borne Zoonotic Dis, 15 (5), pp. 285-290. | Show Abstract | Read more

Although scrub typhus and murine typhus are well-described tropical rickettsial illnesses, especially in Southeast Asia, only limited evidence is available for rickettsia-like pathogens contributing to the burden of undifferentiated febrile illness. Using commercially available kits, this study measured immunoglobulin G (IgG) antibody seroprevalence for Coxiella burnetii, Ehrlichia chaffeensis, Bartonella henselae, Anaplasma phagocytophilum, and spotted fever group rickettsiae (SFGR) in 375 patients enrolled in undifferentiated febrile illness studies at Chiangrai (northern Thailand) and Mae Sot (Thai-Myanmar border). Ehrlichia and SFGR were the most common causes of IgG seropositivity. A distinct relationship between age and seropositivity was found in Chiangrai with acquisition of IgG titers against Ehrlichia, Bartonella, Anaplasma, and SFGR in young adulthood, suggesting cumulative exposure to these pathogens. At Mae Sot, high early IgG titers against Ehrlichia and SFGR were common, whereas Anaplasma and Bartonella IgG titers increased at 50-60 years. Q fever associated with low IgG positivity at both study sites, with significantly higher prevalence at 30 years of age in Chiangrai. These data suggest that other rickettsial illnesses could contribute to the burden of febrile illness in Thailand and possibly adjacent regions. Improved diagnostics and better understanding of antibody longevity and cross-reactivity will improve identification and management of these easily treatable infectious diseases.

Thompson CN, Blacksell SD, Paris DH, Arjyal A, Karkey A, Dongol S, Giri A, Dolecek C, Day N, Baker S et al. 2015. Undifferentiated febrile illness in Kathmandu, Nepal. Am J Trop Med Hyg, 92 (4), pp. 875-878. | Show Abstract | Read more

Undifferentiated febrile illnesses (UFIs) are common in low- and middle-income countries. We prospectively investigated the causes of UFIs in 627 patients presenting to a tertiary referral hospital in Kathmandu, Nepal. Patients with microbiologically confirmed enteric fever (218 of 627; 34.8%) randomized to gatifloxacin or ofloxacin treatment were previously reported. We randomly selected 125 of 627 (20%) of these UFI patients, consisting of 96 of 409 (23%) cases with sterile blood cultures and 29 of 218 (13%) cases with enteric fever, for additional diagnostic investigations. We found serological evidence of acute murine typhus in 21 of 125 (17%) patients, with 12 of 21 (57%) patients polymerase chain reaction (PCR)-positive for Rickettsia typhi. Three UFI cases were quantitative PCR-positive for Rickettsia spp., two UFI cases were seropositive for Hantavirus, and one UFI case was seropositive for Q fever. Fever clearance time (FCT) for rickettsial infection was 44.5 hours (interquartile range = 26-66 hours), and there was no difference in FCT between ofloxacin or gatifloxacin. Murine typhus represents an important cause of predominantly urban UFIs in Nepal, and fluoroquinolones seem to be an effective empirical treatment.

Dittrich S, Rattanavong S, Lee SJ, Panyanivong P, Craig SB, Tulsiani SM, Blacksell SD, Dance DAB, Dubot-Pérès A, Sengduangphachanh A et al. 2015. Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos: a prospective study. Lancet Glob Health, 3 (2), pp. e104-e112. | Show Abstract | Read more

BACKGROUND: Scrub typhus (caused by Orientia tsutsugamushi), murine typhus (caused by Rickettsia typhi), and leptospirosis are common causes of febrile illness in Asia; meningitis and meningoencephalitis are severe complications. However, scarce data exist for the burden of these pathogens in patients with CNS disease in endemic countries. Laos is representative of vast economically poor rural areas in Asia with little medical information to guide public health policy. We assessed whether these pathogens are important causes of CNS infections in Laos. METHODS: Between Jan 10, 2003, and Nov 25, 2011, we enrolled 1112 consecutive patients of all ages admitted with CNS symptoms or signs requiring a lumbar puncture at Mahosot Hospital, Vientiane, Laos. Microbiological examinations (culture, PCR, and serology) targeted so-called conventional bacterial infections (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, S suis) and O tsutsugamushi, Rickettsia typhi/Rickettsia spp, and Leptospira spp infections in blood or cerebrospinal fluid (CSF). We analysed and compared causes and clinical and CSF characteristics between patient groups. FINDINGS: 1051 (95%) of 1112 patients who presented had CSF available for analysis, of whom 254 (24%) had a CNS infection attributable to a bacterial or fungal pathogen. 90 (35%) of these 254 infections were caused by O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp. These pathogens were significantly more frequent than conventional bacterial infections (90/1051 [9%] vs 42/1051 [4%]; p<0·0001) by use of conservative diagnostic definitions. CNS infections had a high mortality (236/876 [27%]), with 18% (13/71) for R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp combined, and 33% (13/39) for conventional bacterial infections (p=0·076). INTERPRETATION: Our data suggest that R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp infections are important causes of CNS infections in Laos. Antibiotics, such as tetracyclines, needed for the treatment of murine typhus and scrub typhus, are not routinely advised for empirical treatment of CNS infections. These severely neglected infections represent a potentially large proportion of treatable CNS disease burden across vast endemic areas and need more attention. FUNDING: Wellcome Trust UK.

Paris DH, Chattopadhyay S, Jiang J, Nawtaisong P, Lee JS, Tan E, Dela Cruz E, Burgos J, Abalos R, Blacksell SD et al. 2015. A nonhuman primate scrub typhus model: protective immune responses induced by pKarp47 DNA vaccination in cynomolgus macaques. J Immunol, 194 (4), pp. 1702-1716. | Show Abstract | Read more

We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi-specific, IFN-γ-producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine-induced immune responses and correlates of immunity for scrub typhus.

Lim C, Blacksell SD, Laongnualpanich A, Kantipong P, Day NPJ, Paris DH, Limmathurotsakul D. 2015. Optimal Cutoff Titers for Indirect Immunofluorescence Assay for Diagnosis of Scrub Typhus. J Clin Microbiol, 53 (11), pp. 3663-3666. | Show Abstract | Read more

We determined the optimal cutoff titers in admission and convalescent-phase samples for scrub typhus indirect immunofluorescence assay using Bayesian latent class models. Cutoff titers of ≥1:3,200 in an admission sample or of a ≥4-fold rise to ≥1:3,200 in a convalescent-phase sample provided the highest accuracy (sensitivity, 81.6%; specificity, 100%).

Lim C, Paris DH, Blacksell SD, Laongnualpanich A, Kantipong P, Chierakul W, Wuthiekanun V, Day NPJ, Cooper BS, Limmathurotsakul D. 2015. How to Determine the Accuracy of an Alternative Diagnostic Test when It Is Actually Better than the Reference Tests: A Re-Evaluation of Diagnostic Tests for Scrub Typhus Using Bayesian LCMs. PLoS One, 10 (5), pp. e0114930. | Show Abstract | Read more

BACKGROUND: The indirect immunofluorescence assay (IFA) is considered a reference test for scrub typhus. Recently, the Scrub Typhus Infection Criteria (STIC; a combination of culture, PCR assays and IFA IgM) were proposed as a reference standard for evaluating alternative diagnostic tests. Here, we use Bayesian latent class models (LCMs) to estimate the true accuracy of each diagnostic test, and of STIC, for diagnosing scrub typhus. METHODS/PRINCIPAL FINDINGS: Data from 161 patients with undifferentiated fever were re-evaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar, and tested with blood culture for Orientia tsutsugamushi, three different PCR assays, IFA IgM, and the Panbio IgM immunochromatographic test (ICT). True sensitivity and specificity of culture (24.4% and 100%), 56kDa PCR assay (56.8% and 98.4%), 47kDa PCR assay (63.2% and 96.1%), groEL PCR assay (71.4% and 93.0%), IFA IgM (70.0% and 83.8%), PanBio IgM ICT (72.8% and 96.8%), presence of eschar (42.7% and 98.9%) and STIC (90.5% and 82.5%) estimated by Bayesian LCM were considerably different from those obtained when using STIC as a reference standard. The IgM ICT had comparable sensitivity and significantly higher specificity compared to IFA (p=0.34 and p<0.001, respectively). CONCLUSIONS: The low specificity of STIC was caused by the low specificity of IFA IgM. Neither STIC nor IFA IgM can be used as reference standards against which to evaluate alternative diagnostic tests. Further evaluation of new diagnostic tests should be done with a carefully selected set of diagnostic tests and appropriate statistical models.

McGready R, Prakash JAJ, Benjamin SJ, Watthanaworawit W, Anantatat T, Tanganuchitcharnchai A, Ling CL, Tan SO, Ashley EA, Pimanpanarak M et al. 2014. Pregnancy outcome in relation to treatment of murine typhus and scrub typhus infection: a fever cohort and a case series analysis. PLoS Negl Trop Dis, 8 (11), pp. e3327. | Show Abstract | Read more

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.

Maude RR, Maude RJ, Ghose A, Amin MR, Islam MB, Ali M, Bari MS, Majumder MI, Tanganuchitcharnchai A, Dondorp AM et al. 2014. Serosurveillance of Orientia tsutsugamushi and Rickettsia typhi in Bangladesh. Am J Trop Med Hyg, 91 (3), pp. 580-583. | Show Abstract | Read more

Scrub and murine typhus infections are under-diagnosed causes of febrile illness across the tropics, and it is not known how common they are in Bangladesh. We conducted a prospective seroepidemiologic survey across six major teaching hospitals in Bangladesh by using an IgM enzyme-linked immunosorbent assay. Results indicated recent exposure (287 of 1,209, 23.7% seropositive for Orientia tsutsugamushi and 805 of 1,209, 66.6% seropositive for Rickettsia typhi). Seropositive rates were different in each region. However, there was no geographic clustering of seropositive results for both organisms. There was no difference between those from rural or urban areas. Rickettsia typhi seroreactivity was positively correlated with age. Scrub typhus and murine typhus should be considered as possible causes of infection in Bangladesh.

Dittrich S, Phommasone K, Anantatat T, Panyanivong P, Slesak G, Blacksell SD, Dubot-Pérès A, Castonguay-Vanier J, Stenos J, Newton PN, Paris DH. 2014. Rickettsia felis Infections and comorbid conditions, Laos, 2003-2011. Emerg Infect Dis, 20 (8), pp. 1402-1404. | Read more

Dittrich S, Castonguay-Vanier J, Moore CE, Thongyoo N, Newton PN, Paris DH. 2014. Loop-mediated isothermal amplification for Rickettsia typhi (the causal agent of murine typhus): Problems with diagnosis at the limit of detection Journal of Clinical Microbiology, 52 (3), pp. 832-838. | Show Abstract | Read more

Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria (affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who ne ed them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates (n=41), buffy coat specimens from R. typhi PCR-positive Lao patients (n=42), and diverse negative controls (n=47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus (n=266). The limit of detection was ∼40 DNA copies/LAMP reaction, with an analytical sensitivity of < 10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%] ) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies. Copyright © 2014 Dittrich et al.

Dittrich S, Castonguay-Vanier J, Moore CE, Thongyoo N, Newton PN, Paris DH. 2014. Loop-mediated isothermal amplification for Rickettsia typhi (the causal agent of murine typhus): problems with diagnosis at the limit of detection. J Clin Microbiol, 52 (3), pp. 832-838. | Show Abstract | Read more

Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria (affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who need them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates (n=41), buffy coat specimens from R. typhi PCR-positive Lao patients (n=42), and diverse negative controls (n=47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus (n=266). The limit of detection was ∼40 DNA copies/LAMP reaction, with an analytical sensitivity of <10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%]) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies.

Takhampunya R, Tippayachai B, Promsathaporn S, Leepitakrat S, Monkanna T, Schuster AL, Melendrez MC, Paris DH, Richards AL, Richardson JH. 2014. Characterization based on the 56-Kda type-specific antigen gene of Orientia tsutsugamushi genotypes isolated from Leptotrombidium mites and the rodent host post-infection. Am J Trop Med Hyg, 90 (1), pp. 139-146. | Show Abstract | Read more

Abstract. Characterization of the 56-kDa type-specific antigen (TSA) genes of Orientia tsutsugamushi (OT) from three naturally infected, laboratory-reared mite colonies comprising three species (Leptotrombidium deliense [Ld], Leptotrombidium imphalum [Li], and Leptotrombidium chiangraiensis [Lc]) has revealed the presence of single and coexisting OT genotypes found in individual chiggers. The Karp genotype was found in all of the chiggers examined, whereas Gilliam and UT302 genotypes were only observed in combination with the Karp genotype. From analysis of these OT genotypes after transmission from chiggers to mice it was determined that with the Lc and Li mites, the OT genotype composition in the rodent spleens post-infection had not changed and therefore resembled that observed in the feeding chiggers. However, only the Karp genotype was found in rodents after feeding by Ld chiggers carrying Karp and Gilliam genotypes. The current findings reveal a complex association among the host, pathogen, and vector.

Mayxay M, Castonguay-Vanier J, Chansamouth V, Dubot-Pérès A, Paris DH, Phetsouvanh R, Tangkhabuanbutra J, Douangdala P, Inthalath S, Souvannasing P et al. 2013. Causes of non-malarial fever in Laos: a prospective study. Lancet Glob Health, 1 (1), pp. e46-e54. | Show Abstract | Read more

BACKGROUND: Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. METHODS: For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5-49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. FINDINGS: With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. INTERPRETATION: Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos. FUNDING: Wellcome Trust, WHO-Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention

Paris DH, Shelite TR, Day NP, Walker DH. 2013. Unresolved problems related to scrub typhus: a seriously neglected life-threatening disease. Am J Trop Med Hyg, 89 (2), pp. 301-307. | Read more

Phommasone K, Paris DH, Anantatat T, Castonguay-Vanier J, Keomany S, Souvannasing P, Blacksell SD, Mayxay M, Newton PN. 2013. Concurrent Infection with murine typhus and scrub typhus in southern Laos--the mixed and the unmixed. PLoS Negl Trop Dis, 7 (8), pp. e2163. | Read more

Elliott I, Dittrich S, Paris D, Sengduanphachanh A, Phoumin P, Newton PN. 2013. The use of dried cerebrospinal fluid filter paper spots as a substrate for PCR diagnosis of the aetiology of bacterial meningitis in the Lao PDR. Clin Microbiol Infect, 19 (10), pp. E466-E472. | Show Abstract | Read more

We investigated whether dried cerebrospinal fluid (CSF) conserved on filter paper can be used as a substrate for accurate PCR diagnosis of important causes of bacterial meningitis in the Lao PDR. Using mock CSF, we investigated and optimized filter paper varieties, paper punch sizes, elution volumes and quantities of DNA template to achieve sensitive and reliable detection of bacterial DNA from filter paper specimens. FTA Elute Micro Card™ (Whatman, Maidstone, UK) was the most sensitive, consistent and practical variety of filter paper. Following optimization, the lower limit of detection for Streptococcus pneumoniae from dried mock CSF spots was 14 genomic equivalents (GE)/μL (interquartile range 5.5 GE/μL) or 230 (IQR 65) colony forming units/mL. A prospective clinical evaluation for S. pneumoniae, S. suis and Neisseria meningitidis was performed. Culture and PCR performed on fresh liquid CSF from patients admitted with a clinical diagnosis of meningitis (n = 73) were compared with results derived from dried CSF spots. Four of five fresh PCR-positive CSF samples also tested PCR positive from dried CSF spots, with one patient under the limit of detection. In a retrospective study of S. pneumoniae samples (n = 20), the median (IQR; range) CSF S. pneumoniae bacterial load was 1.1 × 10(4) GE/μL (1.2 × 10(5) ; 1 to 6.1 × 10(6) DNA GE/μL). Utilizing the optimized methodology, we estimate an extrapolated sensitivity of 90%, based on the range of CSF genome counts found in Laos. Dried CSF filter paper spots could potentially help us to better understand the epidemiology of bacterial meningitis in resource-poor settings and guide empirical treatments and vaccination policies.

Jiang J, Paris DH, Blacksell SD, Aukkanit N, Newton PN, Phetsouvanh R, Izzard L, Stenos J, Graves SR, Day NPJ, Richards AL. 2013. Diversity of the 47-kD HtrA nucleic acid and translated amino acid sequences from 17 recent human isolates of Orientia. Vector Borne Zoonotic Dis, 13 (6), pp. 367-375. | Show Abstract | Read more

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.

Chheng K, Carter MJ, Emary K, Chanpheaktra N, Moore CE, Stoesser N, Putchhat H, Sona S, Reaksmey S, Kitsutani P et al. 2013. A prospective study of the causes of febrile illness requiring hospitalization in children in Cambodia. PLoS One, 8 (4), pp. e60634. | Show Abstract | Read more

BACKGROUND: Febrile illnesses are pre-eminent contributors to morbidity and mortality among children in South-East Asia but the causes are poorly understood. We determined the causes of fever in children hospitalised in Siem Reap province, Cambodia. METHODS AND FINDINGS: A one-year prospective study of febrile children admitted to Angkor Hospital for Children, Siem Reap. Demographic, clinical, laboratory and outcome data were comprehensively analysed. Between October 12(th) 2009 and October 12(th) 2010 there were 1225 episodes of febrile illness in 1180 children. Median (IQR) age was 2.0 (0.8-6.4) years, with 850 (69%) episodes in children <5 years. Common microbiological diagnoses were dengue virus (16.2%), scrub typhus (7.8%), and Japanese encephalitis virus (5.8%). 76 (6.3%) episodes had culture-proven bloodstream infection, including Salmonella enterica serovar Typhi (22 isolates, 1.8%), Streptococcus pneumoniae (13, 1.1%), Escherichia coli (8, 0.7%), Haemophilus influenzae (7, 0.6%), Staphylococcus aureus (6, 0.5%) and Burkholderia pseudomallei (6, 0.5%). There were 69 deaths (5.6%), including those due to clinically diagnosed pneumonia (19), dengue virus (5), and melioidosis (4). 10 of 69 (14.5%) deaths were associated with culture-proven bloodstream infection in logistic regression analyses (odds ratio for mortality 3.4, 95% CI 1.6-6.9). Antimicrobial resistance was prevalent, particularly in S. enterica Typhi, (where 90% of isolates were resistant to ciprofloxacin, and 86% were multi-drug resistant). Comorbid undernutrition was present in 44% of episodes and a major risk factor for acute mortality (OR 2.1, 95% CI 1.1-4.2), as were HIV infection and cardiac disease. CONCLUSION: We identified a microbiological cause of fever in almost 50% of episodes in this large study of community-acquired febrile illness in hospitalized children in Cambodia. The range of pathogens, antimicrobial susceptibility, and co-morbidities associated with mortality described will be of use in the development of rational guidelines for infectious disease treatment and control in Cambodia and South-East Asia.

Sunyakumthorn P, Paris DH, Chan T-C, Jones M, Luce-Fedrow A, Chattopadhyay S, Jiang J, Anantatat T, Turner GDH, Day NPJ, Richards AL. 2013. An intradermal inoculation model of scrub typhus in Swiss CD-1 mice demonstrates more rapid dissemination of virulent strains of Orientia tsutsugamushi. PLoS One, 8 (1), pp. e54570. | Show Abstract | Read more

Scrub typhus is an important endemic disease of the Asia-Pacific region caused by Orientia tsutsugamushi. To develop an effective vaccine to prevent scrub typhus infection, a better understanding of the initial host-pathogen interaction is needed. The objective of this study was to investigate early bacterial dissemination in a CD-1 Swiss outbred mouse model after intradermal injection of O. tsutsugamushi. Three human pathogenic strains of O. tsutsugamushi (Karp, Gilliam, and Woods) were chosen to investigate the early infection characteristics associated with bacterial virulence. Tissue biopsies of the intradermal injection site and draining lymph nodes were examined using histology and immunohistochemistry to characterize bacterial dissemination, and correlated with quantitative real-time PCR for O. tsutsugamushi in blood and tissue from major organs. Soluble adhesion molecules were measured to examine cellular activation in response to infection. No eschar formation was seen at the inoculation site and no clinical disease developed within the 7 day period of observation. However, O. tsutsugamushi was localized at the injection site and in the draining lymph nodes by day 7 post inoculation. Evidence of leukocyte and endothelial activation was present by day 7 with significantly raised levels of sL-selectin, sICAM-1 and sVCAM-1. Infection with the Karp strain was associated with earlier and higher bacterial loads and more extensive dissemination in various tissues than the less pathogenic Gilliam and Woods strains. The bacterial loads of O. tsutsugamushi were highest in the lungs and spleens of mice inoculated with Karp and Gilliam, but not Woods strains. Strains of higher virulence resulted in more rapid systemic infection and dissemination in this model. The CD-1 mouse intradermal inoculation model demonstrates features relevant to early scrub typhus infection in humans, including the development of regional lymphadenopathy, leukocyte activation and distant organ dissemination after low-dose intradermal injection with O. tsutsugamushi.

Wuthiekanun V, Amornchai P, Paris DH, Langla S, Thaipadunpanit J, Chierakul W, Smythe LD, White NJ, Day NPJ, Limmathurotsakul D, Peacock SJ. 2013. Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar. Antimicrob Agents Chemother, 57 (1), pp. 297-302. | Show Abstract | Read more

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

Siritantikorn S, Jintaworn S, Noisakran S, Suputtamongkol Y, Paris DH, Blacksell SD. 2012. Application of ImageJ program to the enumeration of Orientia tsutsugamushi organisms cultured in vitro. Trans R Soc Trop Med Hyg, 106 (10), pp. 632-635. | Show Abstract | Read more

The ImageJ program was applied to the enumeration of Orientia tsutsugamushi organisms in cell culture using indirect immunofluorescence assay (IFA). The highest correlation (r=0.984) was observed between manual counting methods and the ImageJ program (MaxEntropy threshold algorithm). This software-based methodology is cheaper, more standardised and better reproducible than a manual-based approach.

Blacksell S, Richards A, Paris DH, Tanganuchitcharnchai A, Day N. 2012. Validation of a semi-quantitative IgM ELISA for the detection of Orientia tsutsugamushi IgM antibodies for use in seroepidemiology studies in Thailand INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E390-E390. | Read more

Blacksell SD, Paris DH, Chierakul W, Wuthiekanun V, Teeratakul A, Kantipong P, Day NPJ. 2012. Prospective evaluation of commercial antibody-based rapid tests in combination with a loop-mediated isothermal amplification PCR assay for detection of Orientia tsutsugamushi during the acute phase of scrub typhus infection. Clin Vaccine Immunol, 19 (3), pp. 391-395. | Show Abstract | Read more

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.

Siritantikorn S, Jintaworn S, Noisakran S, Suputtamongkol Y, Paris DH, Blacksell SD. 2012. Application of ImageJ program to the enumeration of Orientia tsutsugamushi organisms cultured in vitro Transactions of the Royal Society of Tropical Medicine and Hygiene, 106 (10), pp. 632-635. | Show Abstract | Read more

The ImageJ program was applied to the enumeration of Orientia tsutsugamushi organisms in cell culture using indirect immunofluorescence assay (IFA). The highest correlation (r = 0.984) was observed between manual counting methods and the ImageJ program (MaxEntropy threshold algorithm). This software-based methodology is cheaper, more standardised and better reproducible than a manual-based approach. © 2012 Royal Society of Tropical Medicine and Hygiene.

Paris DH, Phetsouvanh R, Tanganuchitcharnchai A, Jones M, Jenjaroen K, Vongsouvath M, Ferguson DPJ, Blacksell SD, Newton PN, Day NPJ, Turner GDH. 2012. Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium. PLoS Negl Trop Dis, 6 (1), pp. e1466. | Show Abstract | Read more

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an "inflammatory" monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.

Paris DH, Chansamouth V, Nawtaisong P, Löwenberg EC, Phetsouvanh R, Blacksell SD, Lee SJ, Dondorp AM, van der Poll T, Newton PN et al. 2012. Coagulation and inflammation in scrub typhus and murine typhus-a prospective comparative study from Laos Clinical Microbiology and Infection, 18 (12), pp. 1221-1228. | Show Abstract | Read more

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Paris DH, Chansamouth V, Nawtaisong P, Löwenberg EC, Phetsouvanh R, Blacksell SD, Lee SJ, Dondorp AM, van der Poll T, Newton PN et al. 2012. Coagulation and inflammation in scrub typhus and murine typhus--a prospective comparative study from Laos. Clin Microbiol Infect, 18 (12), pp. 1221-1228. | Show Abstract | Read more

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation.

Blacksell SD, Jarman RG, Bailey MS, Tanganuchitcharnchai A, Jenjaroen K, Gibbons RV, Paris DH, Premaratna R, de Silva HJ, Lalloo DG, Day NPJ. 2011. Evaluation of six commercial point-of-care tests for diagnosis of acute dengue infections: the need for combining NS1 antigen and IgM/IgG antibody detection to achieve acceptable levels of accuracy. Clin Vaccine Immunol, 18 (12), pp. 2095-2101. | Show Abstract | Read more

Six assays were evaluated in this study to determine their suitability for the diagnosis of acute dengue infection using samples from 259 Sri Lankan patients with acute fevers (99 confirmed dengue cases and 160 patients with other confirmed acute febrile illnesses): (i) the Merlin dengue fever IgG & IgM combo device (Merlin), (ii) the Standard Diagnostics Dengue Duo nonstructural 1 (NS1) antigen and IgG/IgM combo device (Standard Diagnostics, South Korea), (iii) the Biosynex Immunoquick dengue fever IgG and IgM (Biosynex, France) assay, (iv) the Bio-Rad NS1 antigen strip (Bio-Rad, France), (v) the Panbio Dengue Duo IgG/IgM Cassette (Inverness, Australia), and (vi) the Panbio dengue NS1 antigen strip (Inverness, Australia). The median number of days of fever prior to admission sample collection was 5 days (interquartile range, 3 to 7 days). Sensitivity and specificity of the NS1 antigen tests ranged from 49 to 59% and from 93 to 99%, respectively, and sensitivity and sensitivity of the IgM antibody test ranged from 71 to 80% and from 46 to 90%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics Dengue Duo test gave the best compromise of sensitivity and specificity (93% and 89%, respectively) and provided the best sensitivity in patients presenting at different times after fever onset. The Merlin IgM/IgG antibody tests correctly classified 64% and 86% of the primary and secondary dengue infection cases, respectively, and the Standard Diagnostics IgM/IgG antibody tests correctly classified 71% and 83% of the primary and secondary dengue infection cases, respectively. This study provides strong evidence of the value of combining dengue antigen- and antibody-based test results in the rapid diagnostic test (RDT) format for the acute diagnosis of dengue.

Paris DH, Blacksell SD, Nawtaisong P, Jenjaroen K, Teeraratkul A, Chierakul W, Wuthiekanun V, Kantipong P, Day NPJ. 2011. Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection. PLoS Negl Trop Dis, 5 (9), pp. e1307. | Show Abstract | Read more

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.

Blacksell SD, Tanganuchitcharnchai A, Jarman RG, Gibbons RV, Paris DH, Bailey MS, Day NPJ, Premaratna R, Lalloo DG, de Silva HJ. 2011. Poor diagnostic accuracy of commercial antibody-based assays for the diagnosis of acute Chikungunya infection. Clin Vaccine Immunol, 18 (10), pp. 1773-1775. | Show Abstract | Read more

A Sri Lankan fever cohort (n = 292 patients; 17.8% prevalence) was used to assess two standard diagnostic Chikungunya IgM tests. The immunochromatographic test (ICT) acute sample sensitivity (SN) was 1.9 to 3.9%, and specificity (SP) was 92.5 to 95.0%. The enzyme-linked immunosorbent assay (ELISA) gave an acute sample SN of 3.9% and an SP of 92.5% and a convalescent sample SN of 84% and an SP of 91%. These assays are not suitable for the acute diagnosis of Chikungunya virus infection.

Cited:

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Paris DH, Phetsouvanh R, Tanganuchitcharnchai A, Jones M, Jenjaroen K, Vongsouvath M, Ferguson DPJ, Blacksell SD, Newton PN, Day NPJ, Turner GDH. 2012. Orientia tsutsugamushi in Human Scrub Typhus Eschars Shows Tropism for Dendritic Cells and Monocytes Rather than Endothelium PLOS NEGLECTED TROPICAL DISEASES, 6 (1), pp. e1466-e1466. | Read more

Izzard L, Fuller A, Blacksell SD, Paris DH, Richards AL, Aukkanit N, Nguyen C, Jiang J, Fenwick S, Day NPJ et al. 2010. Isolation of a novel Orientia species (O. chuto sp. nov.) from a patient infected in Dubai. J Clin Microbiol, 48 (12), pp. 4404-4409. | Show Abstract | Read more

In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patient's acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene (rrs), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name "Orientia chuto," and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.

Koh GCKW, Maude RJ, Paris DH, Newton PN, Blacksell SD. 2010. Diagnosis of scrub typhus. Am J Trop Med Hyg, 82 (3), pp. 368-370. | Show Abstract | Read more

Scrub typhus is transmitted by trombiculid mites and is endemic to East and Southeast Asia and Northern Australia. The clinical syndrome classically consists of a fever, rash, and eschar, but scrub typhus also commonly presents as an undifferentiated fever that requires laboratory confirmation of the diagnosis, usually by indirect fluorescent antibody (IFA) assay. We discuss the limitations of IFA, debate the value of other methods based on antigen detection and nucleic acid amplification, and outline recommendations for future study.

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Koh GCKW, Maude RJ, Paris DH, Newton PN, Blacksell SD. 2010. Review: Diagnosis of scrub typhus American Journal of Tropical Medicine and Hygiene, 82 (3), pp. 368-370. | Show Abstract | Read more

Scrub typhus is transmitted by trombiculid mites and is endemic to East and Southeast Asia and Northern Australia. The clinical syndrome classically consists of a fever, rash, and eschar, but scrub typhus also commonly presents as an undifferentiated fever that requires laboratory confirmation of the diagnosis, usually by indirect fluorescent antibody (IFA) assay. We discuss the limitations of IFA, debate the value of other methods based on antigen detection and nucleic acid amplification, and outline recommendations for future study. Copyright © 2010 by The American Society of Tropical Medicine and Hygiene.

McGready R, Ashley EA, Wuthiekanun V, Tan SO, Pimanpanarak M, Viladpai-Nguen SJ, Jesadapanpong W, Blacksell SD, Peacock SJ, Paris DH et al. 2010. Arthropod borne disease: the leading cause of fever in pregnancy on the Thai-Burmese border. PLoS Negl Trop Dis, 4 (11), pp. e888. | Show Abstract | Read more

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.

Paris DH, Aukkanit N, Jenjaroen K, Blacksell SD, Day NPJ. 2009. A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi. Clin Microbiol Infect, 15 (5), pp. 488-495. | Show Abstract | Read more

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.

Paris DH, Blacksell SD, Newton PN, Day NPJ. 2008. Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification. Trans R Soc Trop Med Hyg, 102 (12), pp. 1239-1246. | Show Abstract | Read more

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.

Blacksell SD, Luksameetanasan R, Kalambaheti T, Wutheikanun V, Cheirakul W, Paris DH, Chueasuwanchai S, McGready R, Peacock SJ, Day NPJ. 2008. Phylogenetic and antigenic analysis of Orientia tsutsugamushi isolated from scrub typhus patients in Thailand INFECTION GENETICS AND EVOLUTION, 8 (4), pp. S11-S11.

Paris DH, Jenjaroen K, Blacksell SD, Phetsouvanh R, Wuthiekanun V, Newton PN, Day NPJ, Turner GDH. 2008. Differential patterns of endothelial and leucocyte activation in 'typhus-like' illnesses in Laos and Thailand. Clin Exp Immunol, 153 (1), pp. 63-67. | Show Abstract | Read more

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.

Blacksell SD, Luksameetanasan R, Kalambaheti T, Aukkanit N, Paris DH, McGready R, Nosten F, Peacock SJ, Day NPJ. 2008. Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand. FEMS Immunol Med Microbiol, 52 (3), pp. 335-342. | Show Abstract | Read more

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.

Paris DH, Blacksell SD, Stenos J, Graves SR, Unsworth NB, Phetsouvanh R, Newton PN, Day NPJ. 2008. Real-time multiplex PCR assay for detection and differentiation of rickettsiae and orientiae. Trans R Soc Trop Med Hyg, 102 (2), pp. 186-193. | Show Abstract | Read more

The high incidence of rickettsial diseases in Southeast Asia necessitates rapid and accurate diagnostic tools for a broad range of rickettsial agents, including Orientia tsutsugamushi (scrub typhus) and Rickettsia typhi (murine typhus), but also spotted fever group infections, which are increasingly reported. We present an SYBR-Green-based, real-time multiplex PCR assay for rapid identification and differentiation of scrub typhus group, typhus group and spotted fever group rickettsiae using 47kDa, gltA and ompB gene targets. Detection limits for amplification of these genes in reference strains ranged from 24 copies/microl, 5 copies/microl and 1 copy/microl in multiplex and 2 copies/microl, 1 copy/microl and 1 copy/microl in single template format, respectively. Differentiation by melt-curve analysis led to distinct melt temperatures for each group-specific amplicon. The assay was subjected to 54 samples, of which all cell-culture and 75% of characterised clinical buffy coat samples were correctly identified. Real-time PCR has the advantage of reliably detecting and differentiating rickettsial and orientia cell-culture isolates in a single-template assay, compared with the more time-consuming and laborious immunofluorescence assay. However, further optimisation and validation on samples taken directly from patients to assess its clinical diagnostic utility is required.

Paris DH, Blacksell SD, Newton PN, Day NPJ. 2008. Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification Transactions of the Royal Society of Tropical Medicine and Hygiene,

Paris DH, Imwong M, Faiz AM, Hasan M, Yunus EB, Silamut K, Lee SJ, Day NPJ, Dondorp AM. 2007. Loop-mediated isothermal PCR (LAMP) for the diagnosis of falciparum malaria. Am J Trop Med Hyg, 77 (5), pp. 972-976. | Show Abstract

A recently described loop-mediated isothermal polymerase chain reaction (LAMP) for molecular detection of Plasmodium falciparum was compared with microscopy, PfHRP2-based rapid diagnostic test (RDT), and nested polymerase chain reaction (PCR) as the "gold standard" in 115 Bangladeshi in-patients with fever. DNA extraction for LAMP was conducted by conventional methods or simple heating of the sample; test results were either assessed visually or by gel electrophoresis. Conventional DNA extraction followed by gel electrophoresis had the highest agreement with the reference method (81.7%, kappa = 0.64), with a sensitivity (95% CI) of 76.1% (68.3-83.9%), comparable to RDT and microscopy, but a specificity of 89.6% (84.0-95.2%) compared with 100% for RDT and microscopy. DNA extraction by heat treatment deteriorated specificity to unacceptable levels. LAMP enables molecular diagnosis of falciparum malaria in settings with limited technical resources but will need further optimization. The results are in contrast with a higher accuracy reported in an earlier study comparing LAMP with a non-validated PCR method.

Paris DH, Jenjaroen K, Blacksell SD, Rattanaphone P, Newton PN, Turner GD, Day NP. 2007. Differential patterns of endothelial activation in 'typhus-like' illness TROPICAL MEDICINE & INTERNATIONAL HEALTH, 12 pp. 189-189.

Blacksell SD, Bryant NJ, Paris DH, Doust JA, Sakoda Y, Day NPJ. 2007. Scrub typhus serologic testing with the indirect immunofluorescence method as a diagnostic gold standard: a lack of consensus leads to a lot of confusion. Clin Infect Dis, 44 (3), pp. 391-401. | Show Abstract | Read more

A review was performed to determine the evidence base for scrub typhus indirect immunofluorescence assay (IFA) methodologies and the criteria for positive results. This review included a total of 109 publications, which comprised 123 eligible studies for analysis (14 publications included 2 substudies). There was considerable underreporting of the IFA methodology and seropositivity criteria used, with most studies using a defined cutoff titer rather than an increase in the titer in paired samples. The choice of positivity cutoff titer varied by country and purpose of the IFA test. This variation limits the comparability of seroprevalence rates between studies and, more seriously, raises questions about the appropriateness of the cutoffs for positive IFA results chosen for diagnosis of acute scrub typhus infection. We suggest that the diagnosis of scrub typhus using IFA should be based on a > or =4-fold increase in the titer in paired serum samples and should only be based on a single sample titer when there is an adequate local evidence base.

Paris D, Ledergerber B, Weber R, Jost J, Flepp M, Opravil M, Ruef C, Zimmerli S. 1999. Incidence and predictors of virologic failure of antiretroviral triple-drug therapy in a community-based cohort. AIDS Res Hum Retroviruses, 15 (18), pp. 1631-1638. | Show Abstract | Read more

Highly active antiretroviral therapy fails to reach its recommended goal of sustained suppression of viral replication in a substantial proportion of patients. We analyzed incidence and predictors of virologic failure of the first regimen of a triple-drug combination therapy, including a protease inhibitor and two nucleoside analog reverse transcriptase inhibitors (NRTIs), in 274 HIV-infected patients. Long-term virologic response to combination therapy including salvage regimens was assessed 2.5 years after treatment initiation. During an initial observation period of up to 1.8 years (median, 0.8 years) 152 patients (55%) experienced sustained suppression of HIV-1 RNA to <500 copies/ml. Failure to reduce viral load to <500 copies/ml within 6 months (initial failure) was observed in 51 patients (19%). Independent risk factors for initial failure included higher baseline viral load; addition of a protease inhibitor to an unchanged NRTI regimen; use of saquinavir hard-gel capsules; and longer duration of prior NRTI treatment. Within a median of 7 months viral load rebound above 500 copies/ml occurred in 71 of 223 patients (32%) whose viral load had initially decreased below this threshold. In proportional hazard analysis none of the potential risk factors was significantly associated with viral load rebound. However, in 40 patients (56%) with viral load rebound, incomplete adherence to therapy or treatment interruptions preceded the rebound. Virologic outcome after 2.5 years correlated with initial response to the first regimen: viral load was <500 copies/ml in 88, 55, and 21% of patients with sustained suppression, viral load rebound, and initial failure, respectively.

Cross R, Ling C, Day NPJ, McGready R, Paris DH. 2016. Revisiting doxycycline in pregnancy and early childhood--time to rebuild its reputation? Expert Opin Drug Saf, 15 (3), pp. 367-382. | Show Abstract | Read more

INTRODUCTION: Doxycycline is highly effective, inexpensive with a broad therapeutic spectrum and exceptional bioavailability. However these benefits have been overshadowed by its classification alongside the tetracyclines - class D drugs, contraindicated in pregnancy and in children under 8 years of age. Doxycycline-treatable diseases are emerging as leading causes of undifferentiated febrile illness in Southeast Asia. For example scrub typhus and murine typhus have an unusually severe impact on pregnancy outcomes, and current mortality rates for scrub typhus reach 12-13% in India and Thailand. The emerging evidence for these important doxycycline-treatable diseases prompted us to revisit doxycycline usage in pregnancy and childhood. AREAS COVERED: A systematic review of the available literature on doxycycline use in pregnant women and children revealed a safety profile of doxycycline that differed significantly from that of tetracycline; no correlation between the use of doxycycline and teratogenic effects during pregnancy or dental staining in children was found. EXPERT OPINION: The change of the US FDA pregnancy classification scheme to an evidence-based approach will enable adequate evaluation of doxycycline in common tropical illnesses and in vulnerable populations in clinical treatment trials, dosage-optimization pharmacokinetic studies and for the empirical treatment of undifferentiated febrile illnesses, especially in pregnant women and children.

Slesak G, Inthalath S, Dittrich S, Paris DH, Newton PN. 2015. Leeches as further potential vectors for rickettsial infections. Proc Natl Acad Sci U S A, 112 (48), pp. E6593-E6594. | Read more

Dittrich S, Rattanavong S, Lee SJ, Panyanivong P, Craig SB, Tulsiani SM, Blacksell SD, Dance DAB, Dubot-Pérès A, Sengduangphachanh A et al. 2015. Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos: a prospective study. Lancet Glob Health, 3 (2), pp. e104-e112. | Show Abstract | Read more

BACKGROUND: Scrub typhus (caused by Orientia tsutsugamushi), murine typhus (caused by Rickettsia typhi), and leptospirosis are common causes of febrile illness in Asia; meningitis and meningoencephalitis are severe complications. However, scarce data exist for the burden of these pathogens in patients with CNS disease in endemic countries. Laos is representative of vast economically poor rural areas in Asia with little medical information to guide public health policy. We assessed whether these pathogens are important causes of CNS infections in Laos. METHODS: Between Jan 10, 2003, and Nov 25, 2011, we enrolled 1112 consecutive patients of all ages admitted with CNS symptoms or signs requiring a lumbar puncture at Mahosot Hospital, Vientiane, Laos. Microbiological examinations (culture, PCR, and serology) targeted so-called conventional bacterial infections (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, S suis) and O tsutsugamushi, Rickettsia typhi/Rickettsia spp, and Leptospira spp infections in blood or cerebrospinal fluid (CSF). We analysed and compared causes and clinical and CSF characteristics between patient groups. FINDINGS: 1051 (95%) of 1112 patients who presented had CSF available for analysis, of whom 254 (24%) had a CNS infection attributable to a bacterial or fungal pathogen. 90 (35%) of these 254 infections were caused by O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp. These pathogens were significantly more frequent than conventional bacterial infections (90/1051 [9%] vs 42/1051 [4%]; p<0·0001) by use of conservative diagnostic definitions. CNS infections had a high mortality (236/876 [27%]), with 18% (13/71) for R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp combined, and 33% (13/39) for conventional bacterial infections (p=0·076). INTERPRETATION: Our data suggest that R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp infections are important causes of CNS infections in Laos. Antibiotics, such as tetracyclines, needed for the treatment of murine typhus and scrub typhus, are not routinely advised for empirical treatment of CNS infections. These severely neglected infections represent a potentially large proportion of treatable CNS disease burden across vast endemic areas and need more attention. FUNDING: Wellcome Trust UK.

Paris DH, Chattopadhyay S, Jiang J, Nawtaisong P, Lee JS, Tan E, Dela Cruz E, Burgos J, Abalos R, Blacksell SD et al. 2015. A nonhuman primate scrub typhus model: protective immune responses induced by pKarp47 DNA vaccination in cynomolgus macaques. J Immunol, 194 (4), pp. 1702-1716. | Show Abstract | Read more

We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi-specific, IFN-γ-producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine-induced immune responses and correlates of immunity for scrub typhus.

McGready R, Prakash JAJ, Benjamin SJ, Watthanaworawit W, Anantatat T, Tanganuchitcharnchai A, Ling CL, Tan SO, Ashley EA, Pimanpanarak M et al. 2014. Pregnancy outcome in relation to treatment of murine typhus and scrub typhus infection: a fever cohort and a case series analysis. PLoS Negl Trop Dis, 8 (11), pp. e3327. | Show Abstract | Read more

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.

Paris DH, Shelite TR, Day NP, Walker DH. 2013. Unresolved problems related to scrub typhus: a seriously neglected life-threatening disease. Am J Trop Med Hyg, 89 (2), pp. 301-307. | Read more

Paris DH, Phetsouvanh R, Tanganuchitcharnchai A, Jones M, Jenjaroen K, Vongsouvath M, Ferguson DPJ, Blacksell SD, Newton PN, Day NPJ, Turner GDH. 2012. Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium. PLoS Negl Trop Dis, 6 (1), pp. e1466. | Show Abstract | Read more

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an "inflammatory" monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.

Paris DH, Chansamouth V, Nawtaisong P, Löwenberg EC, Phetsouvanh R, Blacksell SD, Lee SJ, Dondorp AM, van der Poll T, Newton PN et al. 2012. Coagulation and inflammation in scrub typhus and murine typhus--a prospective comparative study from Laos. Clin Microbiol Infect, 18 (12), pp. 1221-1228. | Show Abstract | Read more

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation.

Paris DH, Blacksell SD, Nawtaisong P, Jenjaroen K, Teeraratkul A, Chierakul W, Wuthiekanun V, Kantipong P, Day NPJ. 2011. Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection. PLoS Negl Trop Dis, 5 (9), pp. e1307. | Show Abstract | Read more

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.

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Paris DH, Phetsouvanh R, Tanganuchitcharnchai A, Jones M, Jenjaroen K, Vongsouvath M, Ferguson DPJ, Blacksell SD, Newton PN, Day NPJ, Turner GDH. 2012. Orientia tsutsugamushi in Human Scrub Typhus Eschars Shows Tropism for Dendritic Cells and Monocytes Rather than Endothelium PLOS NEGLECTED TROPICAL DISEASES, 6 (1), pp. e1466-e1466. | Read more

Izzard L, Fuller A, Blacksell SD, Paris DH, Richards AL, Aukkanit N, Nguyen C, Jiang J, Fenwick S, Day NPJ et al. 2010. Isolation of a novel Orientia species (O. chuto sp. nov.) from a patient infected in Dubai. J Clin Microbiol, 48 (12), pp. 4404-4409. | Show Abstract | Read more

In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patient's acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene (rrs), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name "Orientia chuto," and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.

McGready R, Ashley EA, Wuthiekanun V, Tan SO, Pimanpanarak M, Viladpai-Nguen SJ, Jesadapanpong W, Blacksell SD, Peacock SJ, Paris DH et al. 2010. Arthropod borne disease: the leading cause of fever in pregnancy on the Thai-Burmese border. PLoS Negl Trop Dis, 4 (11), pp. e888. | Show Abstract | Read more

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.

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