Correction: A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

[This corrects the article DOI: 10.1371/journal.pntd.0010611.].

A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus.

BACKGROUND: Assessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources. METHODOLOGY/PRINCIPLE FINDINGS: We optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-γ, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients. CONCLUSIONS/SIGNIFICANCE: In conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level.

A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

BACKGROUND: Scrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses. Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function. CONCLUSIONS/SIGNIFICANCE: This study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.

Correction: Yuhana et al. Rickettsial Infections Are Neglected Causes of Acute Febrile Illness in Teluk Intan, Peninsular Malaysia. Trop. Med. Infect. Dis. 2022, 7, 77.

The authors wish to make the following correction to this paper [...].

Rickettsial Infections Are Neglected Causes of Acute Febrile Illness in Teluk Intan, Peninsular Malaysia.

Rickettsial infections are among the leading etiologies of acute febrile illness in Southeast Asia. However, recent data from Malaysia are limited. This prospective study was conducted in Teluk Intan, Peninsular Malaysia, during January to December 2016. We recruited 309 hospitalized adult patients with acute febrile illness. Clinical and biochemistry data were obtained, and patients were stratified into mild and severe infections based on the sepsis-related organ failure (qSOFA) scoring system. Diagnostic assays including blood cultures, real-time PCR, and serology (IFA and MAT) were performed. In this study, pathogens were identified in 214 (69%) patients, of which 199 (93%) patients had a single etiology, and 15 (5%) patients had >1 etiologies. The top three causes of febrile illness requiring hospitalization in this Malaysian study were leptospirosis (68 (32%)), dengue (58 (27%)), and rickettsioses (42 (19%)). Fifty-five (18%) patients presented with severe disease with a qSOFA score of ≥2. Mortality was documented in 38 (12%) patients, with the highest seen in leptospirosis (16 (42%)) followed by rickettsiosis (4 (11%)). While the significance of leptospirosis and dengue are recognized, the impact of rickettsial infections in Peninsular Malaysia remains under appreciated. Management guidelines for in-patient care with acute febrile illness in Peninsular Malaysia are needed.

Antimicrobial Peptide Modifications against Clinically Isolated Antibiotic-Resistant Salmonella.

Antimicrobial peptides are promising molecules to address the global antibiotic resistance problem, however, optimization to achieve favorable potency and safety is required. Here, a peptide-template modification approach was employed to design physicochemical variants based on net charge, hydrophobicity, enantiomer, and terminal group. All variants of the scorpion venom peptide BmKn-2 with amphipathic α-helical cationic structure exhibited an increased antibacterial potency when evaluated against multidrug-resistant Salmonella isolates at a MIC range of 4-8 µM. They revealed antibiofilm activity in a dose-dependent manner. Sheep red blood cells were used to evaluate hemolytic and cell selectivity properties. Peptide Kn2-5R-NH2, dKn2-5R-NH2, and 2F-Kn2-5R-NH2 (variants with +6 charges carrying amidated C-terminus) showed stronger antibacterial activity than Kn2-5R (a variant with +5 charges bearing free-carboxyl group at C-terminus). Peptide dKn2-5R-NH2 (d-enantiomer) exhibited slightly weaker antibacterial activity with much less hemolytic activity (higher hemolytic concentration 50) than Kn2-5R-NH2 (l-enantiomer). Furthermore, peptide Kn2-5R with the least hydrophobicity had the lowest hemolytic activity and showed the highest specificity to Salmonella (the highest selectivity index). This study also explained the relationship of peptide physicochemical properties and bioactivities that would fulfill and accelerate progress in peptide antibiotic research and development.

Characterization of a Novel Peptide from Pathogenic Leptospira and Its Cytotoxic Effect.

Leptospirosis is a zoonotic infectious disease caused by pathogenic Leptospira species. Virulence proteins have been shown to be key determinants of the pathogenesis of pathogenic Leptospira. A specific peptide at a mass-to-charge ratio of 7000 Da was identified in Leptospira whole cells using matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. This peptide was specifically present in pathogenic Leptospira and in clinical isolates. We report here the characterization of this specific peptide using a proteomics approach. This peptide was significantly matched to a hypothetical conserved L. interrogans protein (LA2458) with a calculated molecular weight of 7140.136 Da containing a tellurite-resistance domain at its C terminus (TerB-C). The amino acid sequences revealed the presence of hydrophobic transmembrane portions and two linear B-cell epitopes. Despite its low abundance, this synthetic peptide demonstrated dose-dependent cytotoxicity toward African green monkey kidney (Vero) cells via the apoptosis pathway. The concentration of the peptide 100 µM induced about 50% of cell death after a 24 h exposure. This peptide could be useful for the diagnosis of leptospirosis and the study of pathogenesis.

Helminth fauna of small mammals from public parks and urban areas in Bangkok Metropolitan with emphasis on community ecology of infection in synanthropic rodents.

In 2018, extensive field studies of diversity and prevalence of helminth infection in synanthropic rodents and non-rodent small mammals from public parks and citified areas in the Bangkok Metropolitan were conducted. Rattus rattus complex was the dominant small mammal in public parks. Of the 197 animals, 147 individuals were infected with one or more species of helminths, yielding an infection prevalence of 74.6%. Twenty-five species of helminths were recovered during necropsy. Pterygodermatites tani was the most prevalent (36.2%); other encountered species included Raillietina celebensis, Hydatigera taeniaformis (metacestode in liver tissue), Gongylonema neoplasticum and Hymenolepis diminuta. Different helminth assemblages infected three different host taxa, i.e. synanthropic Rattus spp., Tupaia belangeri (Northern treeshrew) and Suncus murinus (Asian house shrew). Nine species of possible zoonotic helminths were identified. The focus on synanthropic rats influenced the findings of helminth diversity by either host intrinsic or extrinsic factors. A significant positive correlation was found between host body mass and helminth species richness. Greater helminth species richness was found in rats from public parks compared with animals from citified areas (e.g. inside buildings or offices). Also, helminth species richness was negatively correlated with the proportion of post-flooding/rain-fed land. These results provide essential information for assessing the incidence of potential zoonotic health threats in Bangkok and updating research in parasite ecology.

Loop-mediated Isothermal Amplification (LAMP) for Identification of Pythium insidiosum.

OBJECTIVE: Pythium insidiosum causes a life-threatening condition called pythiosis. High morbidity and mortality of pythiosis are consequences of delayed diagnosis. We aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of P. insidiosum for use in remote areas, where pythiosis is prevalent. METHODS: We designed four LAMP primers to amplify the rDNA sequence. A side-by-side comparison evaluated performances of LAMP and the previously-established multiplex PCR (M-PCR), using gDNA samples extracted from colonies of P. insidiosum (n = 28) and other fungi (n = 54), and tissues of animals with (n = 16) or without (n = 13) pythiosis. RESULTS: LAMP demonstrated a 50% shorter assay duration (1.5 h) and a 10-fold lower limit of detection (10-4 ng) than did M-PCR. Based on colony-extracted gDNAs, LAMP and M-PCR correctly reported P. insidiosum in all 28 samples, providing 100% sensitivity. While M-PCR did not amplify all fungal controls (100% specificity), LAMP falsely detected one organism (98% specificity). Based on the clinical samples, LAMP and M-PCR provided an equivalently-high specificity (100%). However, LAMP showed a markedly-higher sensitivity than that of M-PCR (88% vs. 56%). CONCLUSIONS: LAMP is a simple, useful, efficient assay for the detection of P. insidiosum in clinical specimens and pure cultures in resource-limited laboratories.

Lipid profile of Trichinella papuae muscle-stage larvae.

Outbreaks of trichinellosis caused by Trichinella papuae have been reported in South-East Asia. Mebendazole and thiabendazole are the treatments of choice for trichinellosis; however, both drugs result in significant side effects and are less effective for muscle-stage larvae (L1). An alternative therapeutic agent is needed to improve treatment. Information on lipid composition and metabolic pathways may bridge gaps in our knowledge and lead to new antiparasitics. The T. papuae L1 lipidome was analysed using a mass spectrometry-based approach, and 403 lipid components were identified. Eight lipid classes were found and glycerophospholipids were dominant, corresponding to 63% of total lipids, of which the glycerolipid DG (20:1[11Z]/22:4[7Z,10Z,13Z,16Z]/0:0) (iso2) was the most abundant. Overall, 57% of T. papuae lipids were absent in humans; therefore, lipid metabolism may be dissimilar in the two species. Proteins involved T. papuae lipid metabolism were explored using bioinformatics. We found that 4-hydroxybutyrate coenzyme A transferase, uncharacterized protein (A0A0V1MCB5) and ML-domain-containing protein are not present in humans. T. papuae glycerophospholipid metabolic and phosphatidylinositol dephosphorylation processes contain several proteins that are dissimilar to those in humans. These findings provide insights into T. papuae lipid composition and metabolism, which may facilitate the development of novel trichinellosis treatments.

Whole cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of Leptospira spp. in Thailand and Lao PDR.

Leptospirosis is a zoonosis with a worldwide distribution, caused by pathogenic spirochetes of the genus Leptospira. The classification and identification of leptospires can be conducted by both genotyping and serotyping which are time-consuming and established in few reference laboratories. This study used matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as rapid and accurate tool for the identification of leptospires. The whole cell protein spectra of 116 Leptospira isolates including 15 references Leptospira spp. (pathogenic, n = 8; intermediate, n = 2; non-pathogenic, n = 5) and 101 Leptospira spp. clinical isolates was created as an in-house MALDI-TOF MS database. Ninety-seven clinical isolates from Thailand and Laos was validated with these protein spectra and revealed 98.9% correct identification when compared with 16S rRNA gene sequences method. Moreover, MALDI-TOF MS could identify spiked leptospires whole cell in urine. Biomarkers for differentiation of leptospires phylogeny and specific protein spectra for most found Leptospira spp. in this area (L. interrogans, L. kirschneri, L. borgpetersenii) based on MALDI-MS algorithm were demonstrated.

Diagnosis of Murine Typhus by Serology in Peninsular Malaysia: A Case Report Where Rickettsial Illnesses, Leptospirosis and Dengue Co-Circulate.

Murine typhus is a rarely diagnosed cause of acute febrile illness in Malaysia, and its true disease burden is unknown. We report a case of an acute murine typhus infection in a patient living in a small city in Peninsular Malaysia, presenting with fever, rash, and headache. Unresponsive to the initial empirical treatment for leptospirosis, he showed a rapid response to doxycycline when murine typhus was diagnosed later. This case highlights the importance of considering murine typhus as a diagnostic in cases of acute febrile illness in urban and sub-urban areas, such as that of in Peninsular Malaysia.

Geometric morphometrics of the scutum for differentiation of trombiculid mites within the genus Walchia (Acariformes: Prostigmata: Trombiculidae), a probable vector of scrub typhus.

The vectors of scrub typhus are the larval stage of trombiculid mites, termed "chiggers". These vectors are very small - the larvae are approximately 0.2 mm in size - and therefore their morphological identification is difficult. Trombiculid mites are widely distributed across Asia and they can be identified at the genus level by the shape, size and setae/sensilla distribution of their dorsal chitin plate (scutum = shield), while morphological identification at the species level requires more mite characteristics. We recently developed a methodology to ascertain paired matched genotype and morphotype of individual chiggers, based on autofluorescence and brightfield microscopy with subsequent molecular identification using the COI gene (approximately 640bp length). However, based on 20 chigger specimens characterised by paired genotypic and morphological data consisting of the four species [Walchia ewingi with 2 subspecies]: Walchia ewingi lupella (n = 9), W. ewingi ewingi (n = 2), W. alpestris (n = 2), W. kritochaeta (n = 5) and W. minuscuta (n = 2) we found evidence of genetic polymorphism and morphological plasticity within the genus Walchia. The phylogenetic inference of the intra-genus relationships within the Walchia spp., based on COI gene (Blankaartia spp. served as outgroup), delineated the five included species by an average interspecific divergence of mean distance 0.218 (0.126 - 0.323). We therefore applied landmark-based and outline-based geometric morphometric (GM) approaches to differentiate Walchia species using scutum measurements. A total of 261 scutum images of Walchia spp. were examined by landmark-based GM (140 chigger specimens) and outline-based GM (121 specimens) techniques. All Walchia spp. showed significant differences in scutum size and shape. W. minuscuta showed the smallest mean scutum size in both techniques. The largest scutum was found in W. ewingi lupella and W. ewingi ewingi by landmark-based and outline-based GM analysis, respectively. The scutum shapes of W. alpestris and W. minuscuta were clearly distinguished from the other species. Cross-validated classification scores were different depending on species and digitizing techniques and landmark-based GM showed better scores than outline-based GM. We conclude that the morphologically closely-related trombiculid mite species can be further differentiated by their scutum features alone, using GM approaches. This technique is a promising tool for the much-needed characterization studies of chiggers and needs evaluation using matched morphometric and genotyping data for other genera of trombiculids.

Novel high-throughput screening method using quantitative PCR to determine the antimicrobial susceptibility of Orientia tsutsugamushi clinical isolates.

Objectives: To develop a method to enable the large-scale antimicrobial susceptibility screening of Orientia tsutsugamushi clinical isolates, using one timepoint and one concentration of antibiotics to considerably speed up the time to result. Methods: Growth, harvesting, multiplicity of infection (moi) and the day to determine the MICs were optimized using five O. tsutsugamushi reference strains [susceptible (Karp, Kato and Gilliam) and putatively resistant (AFC-3 and AFSC-4)], one clinical isolate (UT76) and one rodent isolate (TA763). Subsequently, the MICs of azithromycin, chloramphenicol and doxycycline for these strains and 51 clinical isolates including AFSC-7 were determined. An optimal concentration was calculated using the epidemiological cut-off value. Results: The conditions for O. tsutsugamushi infection, growth and harvesting were determined to be an moi of 100:1 and trypsinization with the peak growth on day 10. The resulting MICs were in line with previously published susceptibility data for all reference strains, except for Karp and AFSC-4, which showed azithromycin MICs of 0.0156 and 0.0313 mg/L, compared with 0.0078 and 0.0156 mg/L, respectively, in previous reports. The MIC of doxycycline for AFC-3 was 0.125 mg/L compared with >4 mg/L in earlier reports. The final single screening concentrations were identified as: azithromycin, 0.125 mg/L; chloramphenicol, 8 mg/L; and doxycycline, 1 mg/L. Conclusions: This simplified procedure facilitates the simultaneous screening of 48 isolates for actively monitoring potential resistance of this important fever pathogen, with an 8-fold throughput improvement over early methods. The data do not support the existence of doxycycline- and chloramphenicol-resistant scrub typhus.

Autochthonous lactic acid bacteria isolated from pig faeces in Thailand show probiotic properties and antibacterial activity against enteric pathogenic bacteria.

Lactic acid bacteria (LAB) play an important role in pig health and performance that arises from their beneficial impacts on the balance of gastrointestinal microbes, ability to fight enteric pathogens, and capacity to support the immune system. The aim of this study was to evaluate the functional and safety aspects of five previously isolated autochthonous LAB strains, (Lactobacillus plantarum 22F, 25F and 31F, Pediococcus acidilactici 72N and Pediococcus pentosaceus 77F) from pig faeces as potential probiotics for a pig feed supplement. The functional and safety properties of the strains were assessed by in vitro tests. The functional properties tested were their abilities in tolerating low pH values under simulated gastric conditions, their cell surface properties (hydrophobicity, auto- and co-aggregation), antibacterial activity against the common enteric pathogenic bacteria in pigs (such as pathogenic Escherichia coli, Salmonella Choleraesuis and Streptococcus suis), and diacetyl production. The safety of the strains was analyzed based on the absent of haemolysis on blood and bile salt hydrolase activity. Although all strains demonstrated diacetyl production, good survivability and antibacterial activities, L. plantarum 22F and 25F showed the best performance with the strongest antibacterial actions against the indicator pathogens. Of the strains, only P. pentosaceus 77F exhibited haemolysis or bile salt hydrolase activity. Furthermore, a principal component analysis revealed that L. plantarum 22F possessed superior functional and safety aspects compared to the other four autochthonous strains and to reference strains L. plantarum JCM 1149 and P. acidilactici DSM 20284. Further in vivo studies using oral administration of the strains are justified to assess their effectiveness as feed supplements for pigs.

Correction: The Diversity and Geographical Structure of Orientia tsutsugamushi Strains from Scrub Typhus Patients in Laos.

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

MULTILOCUS SEQUENCE TYPING OF BRUCELLA ISOLATES FROM THAILAND.

Although brucellosis outbreaks in Thailand are rare, they cause abortions and infertility in animals, resulting in significant economic loss. Because Brucella spp display > 90% DNA homology, multilocus sequence typing (MLST) was employed to categorize local Brucella isolates into sequence types (STs) and to determine their genetic relatedness. Brucella samples were isolated from vaginal secretion of cows and goats, and from blood cultures of infected individuals. Brucella species were determined by multiplex PCR of eight loci, in addition to MLST based on partial DNA sequences of nine house-keeping genes. MLST analysis of 36 isolates revealed 78 distinct novel allele types and 34 novel STs, while two isolates possessed the known ST8. Sequence alignments identified polymorphic sites in each allele, ranging from 2-6%, while overall genetic diversity was 3.6%. MLST analysis of the 36 Brucella isolates classified them into three species, namely, B. melitensis, B. abortus and B. suis, in agreement with multiplex PCR results. Genetic relatedness among ST members of B. melitensis and B. abortus determined by eBURST program revealed ST2 as founder of B. abortus isolates and ST8 the founder of B. melitensis isolates. ST 36, 41 and 50 of Thai Brucella isolates were identified as single locus variants of clonal cluster (CC) 8, while the majority of STs were diverse. The genetic diversity and relatedness identified using MLST revealed hitherto unexpected diversity among Thai Brucella isolates. Genetic classification of isolates could reveal the route of brucellosis transmission among humans and farm animals and also reveal their relationship with other isolates in the region and other parts of the world.

The Diversity and Geographical Structure of Orientia tsutsugamushi Strains from Scrub Typhus Patients in Laos.

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.

Molecular confirmation of co-infection by pathogenic Leptospira spp. and Orientia tsutsugamushi in patients with acute febrile illness in Thailand.

Leptospirosis and scrub typhus are major causes of acute febrile illness in rural Asia, where co-infection is reported to occur based on serologic evidence. We re-examined whether co-infection occurs by using a molecular approach. A duplex real-time polymerase chain reaction was developed that targeted a specific 16S ribosomal RNA gene of pathogenic Leptospira spp. and Orientia tsutsugamushi. Of 82 patients with an acute febrile illness who had dual infection on the basis of serologic tests, 5 (6%) had polymerase chain reaction results positive for both pathogens. We conclude that dual infection occurs, but that serologic tests may overestimate the frequency of co-infections.

Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand.

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case-control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0-52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0-89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5-94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.