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

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

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

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

Sensitive leptospira DNA detection using tapered optical fiber sensor

This paper presents the development of tapered optical fiber sensor to detect a specific Leptospira bacteria DNA. The bacteria causes Leptospirosis, a deadly disease but with common early flu-like symptoms. Optical single mode fiber (SMF) of 125 μm diameter is tapered to produce 12 μm waist diameter and 15 cm length. The novel DNA-based optical fiber sensor is functionalized by incubating the tapered region with sodium hydroxide (NaOH), (3-Aminopropyl) triethoxysilane and glutaraldehyde. Probe DNA is immobilized onto the tapered region and subsequently hybridized by its complementary DNA (cDNA). The transmission spectra of the DNA-based optical fiber sensor are measured in the 1500 to 1600 nm wavelength range. It is discovered that the shift of the wavelength in the SMF sensor is linearly proportional with the increase in the cDNA concentrations from 0.1 to 1.0 nM. The sensitivity of the sensor toward DNA is measured to be 1.2862 nm/nM and able to detect as low as 0.1 fM. The sensor indicates high specificity when only minimal shift is detected for non-cDNA testing. The developed sensor is able to distinguish between actual DNA of Leptospira serovars (Canicola and Copenhageni) against Clostridium difficile (control sample) at very low (femtomolar) target concentrations

The utility of an AMR dictionary as an educational tool to improve public understanding of antimicrobial resistance

Background: Communicating about antimicrobial resistance (AMR) to the public is challenging.   Methods: We developed a dictionary of terms commonly used to communicate about AMR. For each term, we developed learning points to explain AMR and related concepts in plain language.  We conducted a pilot evaluation in 374 high school students in Ubon Ratchathani, Thailand. In three 50-minute sessions, students were asked to answer five true/false questions using a paper-based questionnaire. The first session assessed their understanding of AMR at baseline, the second after searching the internet, and the third after the provision of the printed AMR dictionary and its web address.    Results: We developed the AMR dictionary as a web-based application (www.amrdictionary.net). The Thai version of the AMR dictionary included 35 terms and associated learning points, seven figures displaying posters promoting AMR awareness in Thailand, and 66 recommended online videos. In the pretest, the proportion of correct responses to each question ranged from 10% to 57%; 10% of the students correctly answered that antibiotics cannot kill viruses and 57% correctly answered that unnecessary use of antibiotics makes them ineffective. After the internet searches, the proportions of correct answers increased, ranging from 62% to 89% (all p&lt;0.001). After providing the AMR dictionary, the proportions of correct answers increased further, ranging from 79% to 89% for three questions (p&lt;0.001), and did not change for one question (p=0.15). Correct responses as to whether taking antibiotics often has side-effects such as diarrhoea reduced from 85% to 74% (p&lt;0.001). The dictionary was revised based on the findings and comments received. Conclusions: Understanding of AMR among Thai high school students is limited. The AMR dictionary can be a useful supportive tool to increase awareness and improve understanding of AMR. Our findings support the need to evaluate the effectiveness of communication tools in the real-world setting.</ns3:p