Orientia tsutsugamushi , the cause of scrub typhus, is a major pathogen in the Asia-Pacific region. The severity of infection ranges from mild features to multiorgan failure and death. The aim of this prospective study was to define the O. tsutsugamushi loads in the blood samples of patients with scrub typhus on the day of hospital admission and to determine whether this was associated with disease severity. Quantitation was performed using a real-time PCR assay targeting the 16S rRNA gene of O. tsutsugamushi . A total of 155 patients with a confirmed diagnosis of scrub typhus had a median (interquartile range [IQR], range) O. tsutsugamushi DNA load in blood of 13 (0 to 334, 0 to 310,253) copies/ml. This included 74 patients who had undetectable bacterial loads. An analysis of bacterial load versus clinical features for all 155 patents demonstrated that duration of illness ( P < 0.001), presence of eschar ( P = 0.004), and concentrations of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase ( P < 0.001 for all three) were positively correlated with bacterial load. Patients who died had a significantly higher bacterial load than those who survived (mean [standard deviation] values: 17,154 [12.7] versus 281 [5.2] copies/ml; P < 0.001). This study has demonstrated a relationship between bacterial load and disease severity in adults with scrub typhus.

Orientia tsutsugamushi is an intracellular α-proteobacterium which resides in trombiculid mites, and is the causative agent of scrub typhus in East Asia. The genome sequence of this species has revealed an unprecedented number of repeat sequences, most notably of the genes encoding the conjugative properties of a type IV secretion system (T4SS). Although this observation is consistent with frequent intragenomic recombination, the extent of homologous recombination (gene conversion) in this species is unknown. To address this question, and to provide a protocol for the epidemiological surveillance of this important pathogen, we have developed a multilocus sequence typing (MLST) scheme based on 7 housekeeping genes (gpsA, mdh, nrdB, nuoF, ppdK, sucD, sucB). We applied this scheme to the two published genomes, and to DNA extracted from blood taken from 84 Thai scrub typhus patients, from 20 cultured Thai patient isolates, 1 Australian patient sample, and from 3 cultured type strains. These data demonstrated that the O. tsutsugamushi population was both highly diverse [Simpson's index (95% CI) = 0.95 (0.92–0.98)], and highly recombinogenic. These results are surprising given the intracellular life-style of this species, but are broadly consistent with results obtained for Wolbachia, which is an α-proteobacterial reproductive parasite of arthropods. We also compared the MLST data with ompA sequence data and noted low levels of consistency and much higher discrimination by MLST. Finally, twenty-five percent of patients in this study were simultaneously infected with multiple sequence types, suggesting multiple infection caused by either multiple mite bites, or multiple strains co-existing within individual mites. Scrub typhus, the rickettsial infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi, is endemic across the Asia Pacific region. The bacterium is transmitted by the bite of larval stages of trombiculid mites (“chiggers”; Leptotrombidium spp.), which more typically feed on small rodents. Clinical features include fever, headache, myalgia, lymphadenopathy and an eschar at the site of the bite. Despite the importance of this pathogen, little is known of the population diversity or the role of homologous recombination in driving the microevolution of this species. Here, we describe the development and application of a multilocus sequence typing (MLST) scheme that can be applied directly to blood samples, and that was applied to 108 O. tsutsugamushi isolates. We found that this organism demonstrated a high rate of homologous recombination, a surprising finding given the intracellular life-style of this species. We also found that 25% of patients in our study were simultaneously infected with multiple sequence types, suggesting multiple infection caused by either multiple mite bites, or multiple strains co-existing within individual mites.

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

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.

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.