Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus

Rodkvamtook, Wuttikon; Zhang, Zhiwen; Chao, Chien‐Chung; Huber, Erin; Bodhidatta, Dharadhida; Gaywee, Jariyanart; Grieco, John P.; Sirisopana, Narongrid; Kityapan, Manerat; Lewis, Michael D.; Ching, Wei‐Mei

doi:10.4269/ajtmh.14-0627

Cited by 21 publications

(17 citation statements)

References 24 publications

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“…This notion is supported by the results of a dot-ELISA assay based on the same three recombinant protein antigens. Rodkvamtook et al demonstrated that the dot-ELISA assay had very good sensitivity and specificity for ST diagnosis [30]. Our results concluded that the use of these recombinant proteins in ELISA also provided good sensitivity and specificity to diagnose scrub typhus.…”

Section: Discussionsupporting

confidence: 56%

An ELISA assay using a combination of recombinant proteins from multiple strains of Orientia tsutsugamushi offers an accurate diagnosis for scrub typhus

et al. 2017

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BackgroundScrub typhus (ST) is a disease caused by an obligate intracellular bacterium, Orientia tsutsugamushi, an organism that requires a BSL3 laboratory for propagation. The disease is hallmarked by an eschar at the site of the chigger bite, followed by the development of fever, malaise, myalgia, anorexia, and papulomacular rash. Indirect immunofluorescent assay (IFA) is the gold standard for scrub typhus diagnosis, however, the subjectivity of the assay, the need for a specialized laboratory and instruments has limited the wide use of the test in resource limited areas.MethodsA recombinant-protein based enzyme linked immunosorbent assay (ELISA) using the most abundant and immunodominant protein for the detection of Orientia specific antibodies in serum has been developed. The performance of the assay was evaluated using prospectively collected acute sera from 248 randomly selected patients in Thailand. The ELISA assay was evaluated using two different cutoff values.ResultsThe receiver operating characteristic (ROC) curve generated cutoff values gave slightly better consistency with diagnosis of ST than those cutoff values established by averaging ELISA optical density of known negatives at 99% confidence interval. Both cutoff values provided similar statistical parameters when compared with the diagnosis of ST, indicating the validity of both calculations to derive cutoff values. These results suggest that both IgG and IgM ELISA performed well to accurately diagnose scrub typhus cases in endemic areas using only acute serum samples.ConclusionsWe have successfully developed an ELISA assay for the detection of Orientia-specific antibodies in serum that could provide effective screening of acute sera under clinical setup and it is also a useful assay to estimate seroprevalence in various endemic areas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-017-2512-8) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 56%

An ELISA assay using a combination of recombinant proteins from multiple strains of Orientia tsutsugamushi offers an accurate diagnosis for scrub typhus

et al. 2017

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“…This is supported by our finding that some patients who had a firm diagnosis of scrub typhus were IgM ELISA negative, despite the time between the onset of fever and convalescent/discharge sampling being more than 10 days (see Table S1 in the supplemental material). An IgM ELISA using well-characterized contemporary local strain recombinant O. tsutsugamushi 56-kDa outer membrane antigens ( 38 , 39 ) could be developed and evaluated in clinical settings using appropriate statistical models. Third, the accuracy of diagnostic tests varies based on prevalence, clinical variability, and availability and timing of convalescent-phase samples ( 40 ).…”

Section: Discussionmentioning

confidence: 99%

Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay

et al. 2016

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The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.

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“…Once thought to be “junk DNA,” the intergenic noncoding regions account for transcription of 85% of cellular RNA vs. 3% for protein-encoding genes (Elgar and Vavouri, 2008 ; Pennisi, 2012 ). It has become clear that noncoding RNAs, such as lincRNAs, miRNAs, tRNAs among others likely play major roles in gene regulation (Chavali et al, 2011 ; Ching et al, 2015 ; Rodkvamtook et al, 2015 ; Eidem et al, 2016 ; Nam et al, 2016 ). That the majority of AnkA binding sites localize to such regions suggests the need to investigate their potential interactions with ncRNAs.…”

Section: Discussionmentioning

confidence: 99%

Genome-Wide Anaplasma phagocytophilum AnkA-DNA Interactions Are Enriched in Intergenic Regions and Gene Promoters and Correlate with Infection-Induced Differential Gene Expression

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Sinclair

Pappas-Brown

et al. 2016

Front. Cell. Infect. Microbiol.

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Anaplasma phagocytophilum, an obligate intracellular prokaryote, infects neutrophils, and alters cardinal functions via reprogrammed transcription. Large contiguous regions of neutrophil chromosomes are differentially expressed during infection. Secreted A. phagocytophilum effector AnkA transits into the neutrophil or granulocyte nucleus to complex with DNA in heterochromatin across all chromosomes. AnkA binds to gene promoters to dampen cis-transcription and also has features of matrix attachment region (MAR)-binding proteins that regulate three-dimensional chromatin architecture and coordinate transcriptional programs encoded in topologically-associated chromatin domains. We hypothesize that identification of additional AnkA binding sites will better delineate how A. phagocytophilum infection results in reprogramming of the neutrophil genome. Using AnkA-binding ChIP-seq, we showed that AnkA binds broadly throughout all chromosomes in a reproducible pattern, especially at: (i) intergenic regions predicted to be MARs; (ii) within predicted lamina-associated domains; and (iii) at promoters ≤ 3000 bp upstream of transcriptional start sites. These findings provide genome-wide support for AnkA as a regulator of cis-gene transcription. Moreover, the dominant mark of AnkA in distal intergenic regions known to be AT-enriched, coupled with frequent enrichment in the nuclear lamina, provides strong support for its role as a MAR-binding protein and genome “re-organizer.” AnkA must be considered a prime candidate to promote neutrophil reprogramming and subsequent functional changes that belie improved microbial fitness and pathogenicity.

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Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus

Cited by 21 publications

References 24 publications

An ELISA assay using a combination of recombinant proteins from multiple strains of Orientia tsutsugamushi offers an accurate diagnosis for scrub typhus

An ELISA assay using a combination of recombinant proteins from multiple strains of Orientia tsutsugamushi offers an accurate diagnosis for scrub typhus

Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay

Genome-Wide Anaplasma phagocytophilum AnkA-DNA Interactions Are Enriched in Intergenic Regions and Gene Promoters and Correlate with Infection-Induced Differential Gene Expression

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