Systematic review of the scrub typhus treatment landscape: Assessing the feasibility of an individual participant-level data (IPD) platform.

BACKGROUND: Scrub typhus is an acute febrile illness caused by intracellular bacteria from the genus Orientia. It is estimated that one billion people are at risk, with one million cases annually mainly affecting rural areas in Asia-Oceania. Relative to its burden, scrub typhus is understudied, and treatment recommendations vary with poor evidence base. These knowledge gaps could be addressed by establishing an individual participant-level data (IPD) platform, which would enable pooled, more detailed and statistically powered analyses to be conducted. This study aims to assess the characteristics of scrub typhus treatment studies and explore the feasibility and potential value of developing a scrub typhus IPD platform to address unanswered research questions. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a systematic literature review looking for prospective scrub typhus clinical treatment studies published from 1998 to 2020. Six electronic databases (Ovid Embase, Ovid Medline, Ovid Global Health, Cochrane Library, Scopus, Global Index Medicus), ClinicalTrials.gov, and WHO ICTRP were searched. We extracted data on study design, treatment tested, patient characteristics, diagnostic methods, geographical location, outcome measures, and statistical methodology. Among 3,100 articles screened, 127 were included in the analysis. 12,079 participants from 12 countries were enrolled in the identified studies. ELISA, PCR, and eschar presence were the most commonly used diagnostic methods. Doxycycline, azithromycin, and chloramphenicol were the most commonly administered antibiotics. Mortality, complications, adverse events, and clinical response were assessed in most studies. There was substantial heterogeneity in the diagnostic methods used, treatment administered (including dosing and duration), and outcome assessed across studies. There were few interventional studies and limited data collected on specific groups such as children and pregnant women. CONCLUSIONS/SIGNIFICANCE: There were a limited number of interventional trials, highlighting that scrub typhus remains a neglected disease. The heterogeneous nature of the available data reflects the absence of consensus in treatment and research methodologies and poses a significant barrier to aggregating information across available published data without access to the underlying IPD. There is likely to be a substantial amount of data available to address knowledge gaps. Therefore, there is value for an IPD platform that will facilitate pooling and harmonisation of currently scattered data and enable in-depth investigation of priority research questions that can, ultimately, inform clinical practice and improve health outcomes for scrub typhus patients.

Clinical Profile and Therapeutic Response of Scrub Typhus in Children: A Recent Trend from Eastern India.

OBJECTIVE: The aim of this study was to assess the clinico-laboratory parameters, complications and therapeutic responses in children with scrub typhus in Eastern India. MATERIALS AND METHODS: In this prospective, observational study, all children (age, <12 years) with suspected scrub typhus with a compatible clinical scenario were enrolled consecutively over six months. Cases confirmed by means of a positive IgM serology or a positive Weil-Felix reaction (OXK = 1/80 or above) were administered enteral doxycycline (4.5 mg/kg/day). RESULTS: Out of 94 recruited children, 61 had confirmed scrub typhus (mean age = 6.1 years, M:F = 1.1:1) with or without complications and having a considerably higher incidence of neurological presentation (meningoencephalistis n = 21, 34.4%). The most frequent manifestations included vomiting (n = 39, 63.9%), abdominal pain (n = 33, 54.1%), lymphadenopathy (n = 36, 59%), hepatosplenomegaly (n = 32, 52.5%), pedal edema (n = 32, 52.5%) and eschar formation (n = 30, 49.2%). Low hemoglobin levels, leukocytosis, thrombocytopenia, hypoalbuminemia, hyponatremia, increased liver enzymes and increased C-reactive protein were associated with delayed defervescence (>48 h). CONCLUSION: Scrub meningoencephalitis, with a notably higher incidence, showed favorable therapeutic response. Prompt and empiric doxycycline therapy could be lifesaving.

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.

Prolonged coma in a scrub typhus patient.

Central nervous system symptoms occur in more than 80% of patients with scrub typhus infection; however, the entity of central nervous system involvement is still not fully understood. We present the case of a patient with fulminant scrub typhus with multiple organ failure, including prolonged deep coma, and detail the sequential neurological symptoms, signs, laboratory data, and neuroradiological findings.

Dual Genotype Orientia tsutsugamushi Infection in Patient with Rash and Eschar, Vietnam, 2016.

We report a dual genotype Orientia tsutsugamushi infection in Vietnam in 2016. The patient had fever, rash, and an eschar. The Kawasaki genotype was identified in the eschar specimen and Karp genotype in the whole blood specimen. The genotype co-infection rate for scrub typhus is unknown and should be further evaluated.

Mixed Infection with Severe Fever with Thrombocytopenia Syndrome Virus and Two Genotypes of Scrub Typhus in a Patient, South Korea, 2017.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne viral disease with a high mortality rate. Infection can also occur through close contact with an infected patient. Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, a bacterium transmitted to humans through chigger mite bites. South Korea is an endemic region of SFTS and scrub typhus. In this study, we confirmed that a patient was coinfected with SFTS virus and two (Boryong and Taguchi) genotypes of O. tsutsugamushi.

Orientia tsutsugamushi Is Highly Susceptible to the RNA Polymerase Switch Region Inhibitor Corallopyronin A In Vitro and In Vivo.

Scrub typhus is a potentially lethal infection caused by the obligate intracellular bacterium Orientia tsutsugamushi Reports on the emergence of doxycycline-resistant strains highlight the urgent need to develop novel antiinfectives against scrub typhus. Corallopyronin A (CorA) is a novel α-pyrone compound synthesized by the myxobacterium Corallococcus coralloides that was characterized as a noncompetitive inhibitor of the switch region of the bacterial RNA polymerase (RNAP). We investigated the antimicrobial action of CorA against the human-pathogenic Karp strain of O. tsutsugamushiin vitro and in vivo The MIC of CorA against O. tsutsugamushi was remarkably low (0.0078 µg/ml), 16-fold lower than that against Rickettsia typhi In the lethal intraperitoneal O. tsutsugamushi mouse infection model, a minimum daily dose of 100 µg CorA protected 100% of infected mice. Two days of treatment were sufficient to confer protection. In contrast to BALB/c mice, SCID mice succumbed to the infection despite treatment with CorA or tetracycline, suggesting that antimicrobial treatment required synergistic action of the adaptive immune response. Similar to tetracycline, CorA did not prevent latent infection of O. tsutsugamushiin vivo However, latency was not caused by acquisition of antimicrobial resistance, since O. tsutsugamushi reisolated from latently infected BALB/c mice remained fully susceptible to CorA. No mutations were found in the CorA-binding regions of the ß and ß' RNAP subunit genes rpoB and rpoC Inhibition of the RNAP switch region of O. tsutsugamushi by CorA is therefore a novel and highly potent target for antimicrobial therapy for scrub typhus.

Central Nervous System Infection Associated with Orientia tsutsugamushi in South Korea.

Orientia tsutsugamushi is a major cause of vector-borne infection in Asia. Prompt recognition and appropriate treatment are crucial because of its potentially fatal complications and lack of response to beta-lactam antibiotics. The present study retrospectively evaluated the clinical characteristics and laboratory findings of 16 patients with scrub typhus-related central nervous system (CNS) infections. Single titers ≥ 1:40 of total serum antibodies against O. tsutsugamushi detected by an indirect immunofluorescent assay were considered as positive results. The median age was 35.5 (range, 14-72) years, and 10 (62.5%) patients were female. The most common symptoms were headache (81.3%) and fever (81.3%). Eschar formation was found in three (18.8%) patients. Among patients with encephalitis, seizures and altered consciousness occurred in five (83.3%) and four (66.7%) patients, respectively. An abnormal liver function was noted in seven (43.8%) patients. The median antibody titer was 1:120 (range, 1:40-1:2,560). Typical cerebrospinal fluid profiles were lymphocytic pleocytosis, mild protein elevations, and normal glucose levels. All patients received an empirical treatment with doxycycline and most (93.8%) of them recovered without neurological sequelae. None of the patients reported side effects of the doxycycline treatment. An empirical treatment with doxycycline is needed in patients with CNS infections in scrub typhus endemic areas.

Nitric oxide enhanced the growth of an obligated intracellular bacterium Orientia tsutsugamushi in murine macrophages.

Orientia tsutsugamushi is the causative agent of scrub typhus. It is an obligate intracellular bacterium that grows only in eukaryotic cells. Macrophages play an important role in innate immunity by surveilling the human body for pathogens. In present study, it was demonstrated that O. tsutsugamushi propagated well in LPS-activated RAW 264.7 macrophages, but not in non-activated macrophages. In LPS-activated macrophages, the expression of Nos2, which encodes the inducible nitric oxide (NO) synthase (iNOS), was highly upregulated compared to those in non-activated macrophages. Parallel to this upregulation, high NO production was observed in LPS-activated macrophages. Transmissible electron microscopy showed that O. tsutsugamushi replicated in the cytosol of macrophages. Thus, O. tsutsugamushi was thought to escape the phagosomes at an early stage of phagosome maturation to avoid the bactericidal effect of NO. Furthermore, O. tsutsugamushi growth was enhanced in NO donor-supplied RAW 264.7 macrophages, as well as in LPS-activated, but not in non-activated macrophages. Consequently, these results suggested that NO was rather essential for enhancing the replication of O. tsutsugamushi in RAW 264.7 macrophages, despite the typically detrimental effects of NO against intracellular pathogens. In the present study, NO was suggested to activate specific pathways to enhance the growth of O. tsutsugamushi.

In Vitro Activity of Tigecycline Against Orientia tsutsugamushi.

Scrub typhus is a zoonosis caused by Orientia tsutsugamushi (O. tsutsugamushi) occurring mainly in autumn in Korea. The need of new antibiotics has arisen with a report on strains resistant to antibiotics and chronic infection. This study aims to identify susceptibility of tigecycline in-vitro as a new therapeutic option for O. tsutsugamushi. Antibacterial activity of tigecycline against the O. tsutsugamushi was compared with doxycycline using flow cytometry assay. The inhibitory concentration 50 (IC50) was 3.59×10⁻³ µg/mL in doxycycline-treated group. Whereas in 0.71×10⁻³ µg/mL tigecycline-treated group. These findings indicate that tigecycline may be a therapeutic option for the treatment of scrub typhus.

An outbreak of scrub typhus in military personnel despite protocols for antibiotic prophylaxis: doxycycline resistance excluded by a quantitative PCR-based susceptibility assay.

Scrub typhus is caused by the obligate intracellular bacterium Orientia tsutsugamushi and is endemic to many countries in the Asia-Pacific region, including tropical Australia. We describe a recent large outbreak amongst military personnel in north Queensland. A total of 45 clinical cases were identified (36% of all potentially exposed individuals). This occurred despite existing military protocols stipulating the provision of doxycycline prophylaxis. Doxycycline resistance in O. tsutsugamushi has been described in South-East Asia, but not Australia. In one case, O. tsutsugamushi was cultured from eschar tissue and blood. Using quantitative real-time PCR to determine susceptibility to doxycycline for the outbreak strain, a minimum inhibitory concentration (MIC) of ≤0.04 µg/mL was found, indicating susceptibility to this agent. It seems most probable that failure to adhere to adequate prophylaxis over the duration of the military exercise accounted for the large number of cases encountered rather than doxycycline resistance.

In Vitro Activity of Tigecycline Against Orientia tsutsugamushi

Scrub typhus is a zoonosis caused by Orientia tsutsugamushi (O. tsutsugamushi) occurring mainly in autumn in Korea. The need of new antibiotics has arisen with a report on strains resistant to antibiotics and chronic infection. This study aims to identify susceptibility of tigecycline in-vitro as a new therapeutic option for O. tsutsugamushi. Antibacterial activity of tigecycline against the O. tsutsugamushi was compared with doxycycline using flow cytometry assay. The inhibitory concentration 50 (IC50) was 3.59×10(-3) µg/mL in doxycycline-treated group. Whereas in 0.71×10(-3) µg/mL tigecycline-treated group. These findings indicate that tigecycline may be a therapeutic option for the treatment of scrub typhus.

In vitro bacteriostatic effects of rifampin on Orientia tsutsugamushi.

We performed an in vitro cell culture experiment to ascertain whether rifampin exhibits bactericidal effects against Orientia tsutsugamushi, the causative agent of scrub typhus. ECV304 cells were infected with the Boryong or AFSC-4 strain of O. tsutsugamushi and then, the cultures were maintained in media with increasing concentrations of rifampin, azithromycin, doxycycline, or chloramphenicol for 4 days. On day 5, the media were replaced with fresh antibiotic-free medium and the cultures were maintained until day 28. On days 5, 13, and 28, immunofluorescence (IF) staining of O. tsutsugamushi was performed. IF staining on days 13 and 28 revealed increasing numbers of IF-positive foci in all cultures, even in cultures initially exposed to the highest concentration of rifampin (80 µg/mL), azithromycin (80 µg/mL), doxycycline (20 µg/mL), or chloramphenicol (100 µg/mL). The present study reveals that rifampin has no bactericidal effect against O. tsutsugamushi as observed for azithromycin, doxycycline, and chloramphenicol. A subpopulation of the bacteria that are not killed by high concentrations of the antibiotics may explain the persistence of O. tsutsugamushi in humans even after complete recovery from scrub typhus with antibiotic therapy.

In Vitro Bacteriostatic Effects of Rifampin on Orientia tsutsugamushi

We performed an in vitro cell culture experiment to ascertain whether rifampin exhibits bactericidal effects against Orientia tsutsugamushi, the causative agent of scrub typhus. ECV304 cells were infected with the Boryong or AFSC-4 strain of O. tsutsugamushi and then, the cultures were maintained in media with increasing concentrations of rifampin, azithromycin, doxycycline, or chloramphenicol for 4 days. On day 5, the media were replaced with fresh antibiotic-free medium and the cultures were maintained until day 28. On days 5, 13, and 28, immunofluorescence (IF) staining of O. tsutsugamushi was performed. IF staining on days 13 and 28 revealed increasing numbers of IF-positive foci in all cultures, even in cultures initially exposed to the highest concentration of rifampin (80 microg/mL), azithromycin (80 microg/mL), doxycycline (20 microg/mL), or chloramphenicol (100 microg/mL). The present study reveals that rifampin has no bactericidal effect against O. tsutsugamushi as observed for azithromycin, doxycycline, and chloramphenicol. A subpopulation of the bacteria that are not killed by high concentrations of the antibiotics may explain the persistence of O. tsutsugamushi in humans even after complete recovery from scrub typhus with antibiotic therapy.

Active escape of Orientia tsutsugamushi from cellular autophagy.

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. After entry into host cells, the bacterium rapidly escapes from the endosomal pathway and replicates in the cytosol of eukaryotic host cells. Here we show that O. tsutsugamushi infection efficiently promotes cellular autophagy, a cell-autonomous defense mechanism of innate immunity. However, most of the internalized bacteria barely colocalized with the induced autophagosomes, even when stimulated with rapamycin, a chemical inducer of autophagy. Treatment of infected cells with tetracycline suppressed bacterial evasion from autophagy and facilitated O. tsutsugamushi targeting to autophagosomes, suggesting that the intracellular pathogen may be equipped with a bacterial factor or factors that block autophagic recognition. Finally, we also found that chemical modulators of cellular autophagy or genetic knockout of the atg3 gene does not significantly affect the intracellular growth of O. tsutsugamushi in vitro. These results suggest that O. tsutsugamushi has evolved to block autophagic microbicidal defense by evading autophagic recognition even though it activates the autophagy pathway during the early phase of infection.

Differences in clinical features according to Boryoung and Karp genotypes of Orientia tsutsugamushi.

BACKGROUND: Scrub typhus is an infectious disease caused by Orientia tsutsugamushi. The differences in virulence of O. tsutsugamushi prototypes in humans are still unknown. We investigated whether there are any differences in the clinical features of the Boryoung and Karp genotypes. METHODOLOGY/PRINCIPAL FINDINGS: Patients infected with O. tsutsugamushi, as Boryoung and Karp clusters, who had visited 6 different hospitals in southwestern Korea were prospectively compared for clinical features, complications, laboratory parameters, and treatment responses. Infected patients in the Boryoung cluster had significantly more generalized weakness, eschars, skin rashes, conjunctival injection, high albumin levels, and greater ESR and fibrinogen levels compared to the Karp cluster. The treatment response to current antibiotics was significantly slower in the Karp cluster as compared to the Boryoung cluster. CONCLUSION: The frequency of occurrence of eschars and rashes may depend on the genotype of O. tsutsugamushi.

Lack of effect of dexamethasone on growth of Orientia tsutsugamushi Gilliam in mouse L929 cells.

PURPOSE: Previous studies and our own clinical experience suggest that concurrent corticosteroid treatment for severe rickettsial disease with multiorgan failure may improve the clinical course or reduce mortality. However, the use of corticosteroids as adjunctive treatment for rickettsial diseases is controversial. We attempted to determine the influences of corticosteroid on the growth of Orientia tsutsugamushi in vitro to justify and evaluate the clinical applicability of corticosteroid in rickettsial disease. MATERIALS AND METHODS: L929 cells were infected with Orientia tsutsugamushi Gilliam. Dexamethasone was added to the cells at final concentrations of 10¹ and 107 pg/mL. Cultures were incubated at 35°C and processed for flow cytometry on the 6th day after addition of dexamethasone. RESULTS: Observation on the 6th day after treatment with dexamethasone in infected cultures revealed that there was no difference in fluorescence intensity among the treatment wells. Treatment of the cells with dexamethasone at concentrations of 10¹ and 107 pg/mL showed no influence on the growth of Orientia tsutsugamushi. CONCLUSION: Our results to show that isolated corticosteroid does not enhance the replication of Orientia tsutsugamushi in vitro. Concurrent use of anti-inflammatory or immunosuppressive doses of corticosteroids in conjunction with antibiotics may not have detrimental effects on the course of scrub typhus.

Involvement of Ca²+ signaling in intracellular invasion of non-phagocytic host cells by Orientia tsutsugamushi.

Calcium signaling has been implicated in various steps in bacterial pathogenesis. Here, we investigated the role of Ca(2+) signaling in intracellular invasion of non-phagocytic host cells infected with Orientia tsutsugamushi, the causative agent of scrub typhus. The bacteria induced a transient Ca(2+) increase in HeLa cells immediately after infection and the source of the mobilized Ca(2+) appears to be intracellular stores, not the extracellular milieu, as determined using extracellular (EGTA) or intracellular (BAPTA-AM) Ca(2+) chelators. O. tsutsugamushi rapidly induced activation of PLC-γ1, as indicated by tyrosine phosphorylation. PLC-γ1 activity increased within 1 min of infection and returned to the basal level by 5 min. Pretreatment of host cells with inhibitors of PLC-γ1 (U73122) or IP3R channel activity (2-APB) significantly blocked bacterial entry without affecting bacterial attachment. In addition, these chemical inhibitors were effective in suppressing not only the activation of ERK MAP kinase but also the expression of the chemokine MCP-1. Taken together, Ca(2+) signaling induced by O. tsutsugamushi may play a crucial role in bacterial pathogenesis including inflammatory reactions as well as intracellular invasion into non-phagocytic host cells.