scholarly journals Prospective Evaluation of Commercial Antibody-Based Rapid Tests in Combination with a Loop-Mediated Isothermal Amplification PCR Assay for Detection of Orientia tsutsugamushi during the Acute Phase of Scrub Typhus Infection

2012 ◽  
Vol 19 (3) ◽  
pp. 391-395 ◽  
Author(s):  
Stuart D. Blacksell ◽  
Daniel H. Paris ◽  
Wirongrong Chierakul ◽  
Vanaporn Wuthiekanun ◽  
Achara Teeratakul ◽  
...  
Keyword(s):  
Antibody Titer ◽  
Scrub Typhus ◽  
Orientia Tsutsugamushi ◽  
Pcr Assay ◽  
Content Type ◽  
Igm Antibody ◽  
Rapid Tests ◽  
Increased Sensitivity ◽  
Minimal Reduction ◽  
Diagnostic Capabilities

ABSTRACTSamples from 160 prospectively recruited febrile patients with typhus-like illness in an area of Thailand (Chiang Rai, northern Thailand) where scrub typhus is endemic were used to evaluate the diagnostic capabilities of four rapid immunochromatographic tests (ICTs) for the detection ofOrientia tsutsugamushiIgM and total antibodies during acute scrub typhus infection. Of the 160 cases, 54 (34%) had been confirmed to have scrub typhus using the reference scrub typhus infection criteria (STIC), i.e., positive cell culture isolation, an admission IgM antibody titer of ≥1:12,800, a 4-fold rising IgM antibody titer, and/or positivity for ≥2 out of 3 PCR gene targets). The ICTs gave the following sensitivities and specificities: the Panbio IgM ICT, 46% (95% confidence interval [CI], 33 to 60) and 95% (95% CI, 89 to 98), respectively; the Standard Diagnostics IgM ICT, 68% (95% CI, 60 to 75) and 73% (95% CI, 68 to 78), respectively; the AccessBio IgM ICT, 56% (95% CI, 48 to 63) and 90% (95% CI, 87 to 94), respectively; and the AccessBio total antibody ABt ICT, 61% (95% CI, 53 to 68) and 68% (95% CI, 63 to 73), respectively. An isothermal loop amplification (LAMP) PCR assay for scrub typhus demonstrated a sensitivity of 52% (95% CI, 38 to 66) and a specificity of 94% (95% CI, 88 to 98). This study has revealed the diagnostic limitations of antibody-based assays in an acute care setting. However, the combination of ICTs with LAMP usually increased sensitivity with a minimal reduction in specificity. The best combination, the Panbio IgM ICT and LAMP, resulted in a sensitivity of 67% (95% CI, 53 to 79) and a specificity of 91% (95% CI, 83 to 95). The combination of antibody-based assays with DNA- or antigen-based tests shows promise for improved diagnostic sensitivity.

scholarly journals Diagnostic Accuracy of a Loop-Mediated Isothermal PCR Assay for Detection of Orientia tsutsugamushi during Acute Scrub Typhus Infection

2011 ◽  
Vol 5 (9) ◽  
pp. e1307 ◽  
Author(s):  
Daniel H. Paris ◽  
Stuart D. Blacksell ◽  
Pruksa Nawtaisong ◽  
Kemajittra Jenjaroen ◽  
Achara Teeraratkul ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Scrub Typhus ◽  
Orientia Tsutsugamushi ◽  
Pcr Assay

scholarly journals Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

2017 ◽  
Vol 55 (5) ◽  
pp. 1377-1387 ◽  
Author(s):  
Wiwit Tantibhedhyangkul ◽  
Ekkarat Wongsawat ◽  
Saowaluk Silpasakorn ◽  
Duangdao Waywa ◽  
Nuttawut Saenyasiri ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Scrub Typhus ◽  
Febrile Illness ◽  
Asia Pacific ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Content Type ◽  
Serologic Tests

ABSTRACTScrub typhus, caused byOrientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targetingO. tsutsugamushi47-kDa,groEL, and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.

scholarly journals Kinetics and Magnitude of Antibody Responses against the Conserved 47-Kilodalton Antigen and the Variable 56-Kilodalton Antigen in Scrub Typhus Patients

2011 ◽  
Vol 18 (6) ◽  
pp. 1021-1027 ◽  
Author(s):  
Hua-Wei Chen ◽  
Zhiwen Zhang ◽  
Erin Huber ◽  
Elissa Mutumanje ◽  
Chien-Chung Chao ◽  
...  
Keyword(s):  
Scrub Typhus ◽  
Secondary Infection ◽  
Systematic Investigation ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Orientia Tsutsugamushi ◽  
Protein Antigens ◽  
Content Type ◽  
Molecular Sizes ◽  
Kinetics Of

ABSTRACTWestern blot analysis ofOrientia tsutsugamushiwhole-cell lysates with scrub typhus patient sera has identified at least five protein antigens ofO. tsutsugamushiwith molecular sizes of 22 kDa, 47 kDa, 56 kDa, 58 kDa, and 110 kDa. In this study, sera from serial bleedings of 108 patients were used to study the kinetics and the magnitude of specific antibody responses against the 47-kDa and 56-kDa antigens. Recombinant protein of the conserved 47-kDa antigen (r47b) or a mixture of truncated 56-kDa antigen (r56s) from three prototype strains was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). Our results showed that 76% and 93% of these patients had elevated IgM and IgG against r47b, respectively, and 98% and 100% had elevated IgM and IgG against r56s, respectively. The kinetics of antibody responses against r47b and r56s can be grouped into three patterns. In the first type of response, IgM and IgG against r47b and r56s appeared about the same time. The IgM and IgG titers against r56s were much higher than those against r47b. In the second type of response, induction of IgM appeared to be similar to that in the first type. The major difference to the first type is that the IgG titers against r47b were induced at least 1 week later than those against the r56s. The third type showed strong IgG responses against both r47b and r56s, and low or no IgM responses indicated a secondary infection. This is the first systematic investigation of antibody response kinetics against the conserved 47-kDa antigen versus the variable 56-kDa antigen in scrub typhus patients.

scholarly journals Orientia tsutsugamushi Nucleomodulin Ank13 Exploits the RaDAR Nuclear Import Pathway To Modulate Host Cell Transcription

mBio ◽  
2021 ◽  
Author(s):  
Haley E. Adcox ◽  
Amanda L. Hatke ◽  
Shelby E. Andersen ◽  
Sarika Gupta ◽  
Nathan B. Otto ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Scrub Typhus ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Intracellular Bacteria ◽  
Orientia Tsutsugamushi ◽  
Microbial Protein ◽  
Content Type ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene

Nucleomodulins are recently defined effectors used by diverse intracellular bacteria to manipulate eukaryotic gene expression and convert host cells into hospitable niches. How nucleomodulins enter the nucleus, their functional domains, and the genes that they modulate are incompletely characterized. Orientia tsutsugamushi is an intracellular bacterial pathogen that causes scrub typhus, which can be fatal. O. tsutsugamushi Ank13 is the first example of a microbial protein that coopts eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import.

scholarly journals Detection of Antibodies against Orientia tsutsugamushi Sca Proteins in Scrub Typhus Patients and Genetic Variation ofscaGenes of Different Strains

2012 ◽  
Vol 19 (9) ◽  
pp. 1442-1451 ◽  
Author(s):  
Na-Young Ha ◽  
Yuri Kim ◽  
Ji-Hye Choi ◽  
Myung-Sik Choi ◽  
Ik-Sang Kim ◽  
...  
Keyword(s):  
Scrub Typhus ◽  
Pcr Amplification ◽  
Febrile Illness ◽  
Immunofluorescence Assay ◽  
Antibody Responses ◽  
Diagnostic Methods ◽  
Orientia Tsutsugamushi ◽  
Content Type ◽  
Asian Pacific Region ◽  
Different Strains

ABSTRACTScrub typhus, caused byOrientia tsutsugamushiinfection, is one of the main causes of acute febrile illness in the Asian-Pacific region. Although early diagnosis and immediate antibiotic treatment are critical for reducing disease severity and mortality, current diagnostic methods using serological and molecular approaches have some limitations in sensitivity and applicability in clinical laboratories. In this study, we identified and characterizedO. tsutsugamushisurface cell antigen (sca) family genes encoding autotransporter proteins in order to test them as novel diagnostic targets. We evaluated antibody responses against the Sca proteins in scrub typhus patient sera and examined the genetic diversity of these genes in different strains after PCR amplification. Specific antibody responses against ScaA and ScaC were observed in patients with high indirect immunofluorescence assay titers (≥1:640), whereas specific responses against ScaB and ScaE were relatively low. Genetic analysis using genomic DNAs revealed thescagenes to be quite variable among the different strains. In contrast toscaA,scaC, andscaD, which were detected in all of the tested strains,scaBandscaEwere amplified differentially from the different strains, suggesting a differential presence of the genes in the genomes. Among the members of the gene family, the sequence ofscaCis the most highly conserved between the different strains, and the size ofscaDis the most variable due to the presence of different numbers of internal repeat sequences. These results suggest that thescagenes ofO. tsutsugamushimay be valuable targets for use in combination with classical assay methods for scrub typhus diagnosis.

scholarly journals Accuracy of Conventional PCR Targeting the 16S rRNA Gene with the Ot-16sRF1 and Ot-16sRR1 Primers for Diagnosis of Scrub Typhus: a Case-Control Study: TABLE 1

2015 ◽  
Vol 54 (1) ◽  
pp. 178-179 ◽  
Author(s):  
Choon-Mee Kim ◽  
Min Keun Cho ◽  
Dong-Min Kim ◽  
Na-Ra Yun ◽  
Seok Won Kim ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Scrub Typhus ◽  
Rrna Gene ◽  
Conventional Pcr ◽  
Content Type ◽  
Increased Sensitivity ◽  
Good Diagnostic Accuracy ◽  
Pcr Targeting ◽  
The 16S Rrna Gene

We retrospectively evaluated the accuracy of conventional PCR targeting the 16S rRNA gene (16S C-PCR) using the Ot-16sRF1/Ot-16sRR1 primers for diagnosing scrub typhus. The diagnosis ofOrientia tsutsugamushiinfection by 16S C-PCR presented an increased sensitivity of 87.0% and specificity of 100% compared with those obtained with other targets and is thus a simple and clinically useful method with good diagnostic accuracy.

scholarly journals Genetic Typing, Based on the 56-Kilodalton Type-Specific Antigen Gene, of Orientia tsutsugamushi Strains Isolated from Chiggers Collected from Wild-Caught Rodents in Taiwan

2011 ◽  
Vol 77 (10) ◽  
pp. 3398-3405 ◽  
Author(s):  
Pey-Ru Lin ◽  
Hui-Ping Tsai ◽  
Pei-Yi Tsui ◽  
Ming-Hui Weng ◽  
Ming-Der Kuo ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Scrub Typhus ◽  
Sequence Similarity ◽  
Specific Antigen ◽  
Febrile Illness ◽  
Prototype Strain ◽  
Asia Pacific ◽  
Orientia Tsutsugamushi ◽  
Content Type ◽  
Strain Characterization

ABSTRACTOrientia tsutsugamushiis the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization ofO. tsutsugamushiisolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, theO. tsutsugamushigenotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity betweenO. tsutsugamushiisolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.

scholarly journals Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus

2016 ◽  
Vol 84 (12) ◽  
pp. 3379-3387 ◽  
Author(s):  
Mohammad Gharaibeh ◽  
Monica Hagedorn ◽  
Stefanie Lilla ◽  
Matthias Hauptmann ◽  
Holger Heine ◽  
...  
Keyword(s):  
Scrub Typhus ◽  
Orientia Tsutsugamushi ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Content Type ◽  
Lethal Infection ◽  
Tnf Α ◽  
Toll Like Receptor 2 ◽  
Deficient Mice ◽  
Increased Susceptibility

Scrub typhus is a potentially lethal infection that is caused by the obligate intracellular bacteriumOrientia tsutsugamushi. The roles of Toll-like receptor 2 (TLR2) and TLR4 in innate recognition ofO. tsutsugamushihave not been elucidated. By overexpression of TLR2 or TLR4 in HEK293 cells, we demonstrated that TLR2, but not TLR4, recognizes heat-stable compounds ofO. tsutsugamushithat were sensitive to treatment with sodium hydroxide, hydrogen peroxide, and proteinase K. TLR2 was required for the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by dendritic cells. In an intradermal mouse infection model, TLR2-deficient mice did not show impaired control of bacterial growth or reduced survival. Moreover, after intraperitoneal infection, TLR2-deficient mice were even more resistant to lethal infection than C57BL/6 wild-type mice, which showed stronger symptoms and lower survival rates during the convalescent phase. Compared to the time of reduction of bacterial loads in TLR2-deficient mice, the reduction of bacterial loads in infected organs was accelerated in wild-type mice. The higher mortality of wild-type mice was associated with increased concentrations of serum alkaline phosphatase but not aspartate aminotransferase. The transcription of mRNA for TNF-α and IL-6 decreased more rapidly in peritoneum samples from wild-type mice than in those from TLR2-deficient mice and was therefore not a correlate of increased susceptibility. Thus, although TLR2 is an important mediator of the early inflammatory response, it is dispensable for protective immunity againstO. tsutsugamushi. Increased susceptibility toO. tsutsugamushiinfection in TLR2-competent mice rather suggests a TLR2-related immunopathologic effect.

scholarly journals Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

2015 ◽  
Vol 197 (19) ◽  
pp. 3097-3109 ◽  
Author(s):  
Andrea R. Beyer ◽  
Lauren VieBrock ◽  
Kyle G. Rodino ◽  
Daniel P. Miller ◽  
Brittney K. Tegels ◽  
...  
Keyword(s):  
Host Cell ◽  
Ubiquitin Ligase ◽  
Scrub Typhus ◽  
Ankyrin Repeat ◽  
Intracellular Bacterium ◽  
Host Cells ◽  
Orientia Tsutsugamushi ◽  
Ligand Interaction ◽  
Content Type ◽  
Cell Functions

ABSTRACTA rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions.Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here,in silicoand manual sequence analyses revealed that a large proportion ofO. tsutsugamushistrain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathioneS-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitatedO. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. AligningO. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate thatO. tsutsugamushistrain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that anO. tsutsugamushiAnk does so during infection, and advance overall understanding of microbial F-box proteins.IMPORTANCEAnkyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets.Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates thatO. tsutsugamushistrain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that anOrientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fideOrientiaeffector and the first example of an endogenous F-box-containing Ank–mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.

scholarly journals Misdiagnosis of scrub typhus as hemorrhagic fever with renal syndrome and potential co-infection of both diseases in patients in Shandong Province, China, 2013–2014

2021 ◽  
Vol 15 (3) ◽  
pp. e0009270
Author(s):  
Xiao-lan Gu ◽  
Rui Qi ◽  
Wen-qian Li ◽  
Yong-jun Jiao ◽  
Hao Yu ◽  
...  
Keyword(s):  
Scrub Typhus ◽  
Clinical Manifestations ◽  
Hemorrhagic Fever ◽  
Shandong Province ◽  
Enzyme Linked Immunosorbent Assay ◽  
Orientia Tsutsugamushi ◽  
Natural Focus ◽  
Serum Samples ◽  
Igm Antibody ◽  
Disease Stages

Background Scrub typhus, caused by Orientia tsutsugamushi, an obligate intracellular gram-negative bacterium, along with hemorrhagic fever with renal syndrome (HFRS), caused by hantaviruses, are natural-focus infectious diseases prevalent in Shandong Province, China. Both diseases have similar clinical manifestations in certain disease stages and similar epidemic seasons, which has caused difficulties for physicians in distinguishing them. The aim of this study was to investigate whether misdiagnosis of scrub typhus as HFRS occurred in patients in Shandong Province. Methods Serum samples (N = 112) of clinically suspected HFRS patients from 2013 to 2014 in Shandong Province were analyzed with enzyme-linked immunosorbent assay (ELISA) for antibodies to both hantavirus and Orientia tsutsugamushi. Results ELISA showed that 56.3% (63/112) and 8.0% (9/112) of clinically suspected HFRS patients were IgM antibody positive to hantavirus and O. tsutsugamushi, respectively. Among the hantavirus IgM antibody positive patients, 7.9% (5/63) were also IgM antibody positive to O. tsutsugamushi. Among the hantavirus IgM antibody negative sera, 8.2% (4/49) of sera were positive to O. tsutsugamushi. Conclusions We concluded that some scrub typhus patients were misdiagnosed as HFRS and co-infection of scrub typhus and HFRS might exist in China. Due to the different treatments for scrub typhus and HFRS, physicians should carefully differentiate between scrub typhus and HFRS and consider administering anti-rickettsia antibiotics if treatment for HFRS alone does not work.

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