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

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

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-gamma, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-gamma/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-gamma, 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

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

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.; 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.; 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.; 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

Diagnostic Accuracy of Recombinant Immunoglobulin-like Protein A-Based IgM ELISA for the Early Diagnosis of Leptospirosis in the Philippines

Background Leptospirosis is an important but largely under-recognized public health problem in the tropics. Establishment of highly sensitive and specific laboratory diagnosis is essential to reveal the magnitude of problem and to improve treatment. This study aimed to evaluate the diagnostic accuracy of a recombinant LigA protein based IgM ELISA during outbreaks in the clinical-setting of a highly endemic country. Methodology/Principal Findings A prospective study was conducted from October 2011 to September 2013 at a national referral hospital for infectious diseases in Manila, Philippines. Patients who were hospitalized with clinically suspected leptospirosis were enrolled. Plasma and urine were collected on admission and/or at discharge and tested using the LigA-IgM ELISA and a whole cellbased IgM ELISA. Sensitivity and specificity of these tests were evaluated with cases diagnosed by microscopic agglutination test (MAT), culture and LAMP as the composite reference standard and blood bank donors as healthy controls: the mean+3 standard deviation optical density value of healthy controls was used as the cut-off limit (0.062 for the LigA-IgM ELISA and 0.691 for the whole cell-based IgM ELISA). Of 304 patients enrolled in the study, 270 (89.1%) were male and the median age was 30.5 years; 167 (54.9%) were laboratory confirmed. The sensitivity and ROC curve AUC for the LigA-IgM ELISA was significantly greater than the whole cell-based IgM ELISA (69.5% vs. 54.3%, p<0.01; 0.90 vs. 0.82, p<0.01) on admission, but not at discharge. The specificity of LigA-IgM ELISA and whole cell-based IgM ELISA were not significantly different (98% vs. 97%). Among 158 MAT negative patients, 53 and 28 were positive by LigA- and whole cell-based IgM ELISA, respectively; if the laboratory confirmation was re-defined by LigA-IgM ELISA and LAMP, the clinical findings were more characteristic of leptospirosis than the diagnosis based on MAT/ culture/LAMP. Conclusions/Significance The newly developed LigA-IgM ELISA is more sensitive than the whole cell-based IgM based ELISA. Although the final diagnosis must be validated by more specific tests, LigAIgM ELISA could be a useful diagnostic test in a real clinical-setting, where diagnosis is needed in the early phase of infection

Scrub typhus ecology: a systematic review of Orientia in vectors and hosts

Abstract Scrub typhus, caused by Orientia tsutsugamushi, is an important and neglected vector-borne zoonotic disease with an expanding known distribution. The ecology of the disease is complex and poorly understood, impairing discussion of public health interventions. To highlight what we know and the themes of our ignorance, we conducted a systematic review of all studies investigating the pathogen in vectors and non-human hosts. A total of 276 articles in 7 languages were included, with 793 study sites across 30 countries. There was no time restriction for article inclusion, with the oldest published in 1924. Seventy-six potential vector species and 234 vertebrate host species were tested, accounting for over one million trombiculid mites (‘chiggers’) and 83,000 vertebrates. The proportion of O. tsutsugamushi positivity was recorded for different categories of laboratory test and host species. Vector and host collection sites were geocoded and mapped. Ecological data associated with these sites were summarised. A further 145 articles encompassing general themes of scrub typhus ecology were reviewed. These topics range from the life-cycle to transmission, habitats, seasonality and human risks. Important gaps in our understanding are highlighted together with possible tools to begin to unravel these. Many of the data reported are highly variable and inconsistent and minimum data reporting standards are proposed. With more recent reports of human Orientia sp. infection in the Middle East and South America and enormous advances in research technology over recent decades, this comprehensive review provides a detailed summary of work investigating this pathogen in vectors and non-human hosts and updates current understanding of the complex ecology of scrub typhus. A better understanding of scrub typhus ecology has important relevance to ongoing research into improving diagnostics, developing vaccines and identifying useful public health interventions to reduce the burden of the disease.</jats:p

High Rates of Homologous Recombination in the Mite Endosymbiont and Opportunistic Human Pathogen Orientia tsutsugamushi

The study was funded by the Wellcome Trust. PS was supported by grants from the Wellcome Trust (073135) and British Infection Society.Orientia tsutsugamushi is an intracellular alpha-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 alpha-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.Publisher PDFPeer reviewe