Inter- and Intra-Operator Variability in the Reading of Indirect Immunofluorescence Assays for the Serological Diagnosis of Scrub Typhus and Murine Typhus

Phetsouvanh, Rattanaphone; Thojaikong, Thaksinaporn; Phoumin, Phonlavanh; Sibounheuang, Bountoy; Phommasone, Koukeo; Chansamouth, Vilada; Lee, Sue J.; Newton, Paul N.; Blacksell, Stuart D.

doi:10.4269/ajtmh.12-0325

Cited by 44 publications

(33 citation statements)

References 11 publications

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“…Febrile controls, positive by LAMP but previously negative by R. typhi qPCR, were further evaluated by IFA, as described previously (27). These samples were from patients presenting at Mahosot Hospital with suspected typhus ( n = 67) and Salavan Provincial Hospital with fever ( n = 10) (6).…”

Section: Methodsmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

et al. 2014

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Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria (affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who need them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates (n = 41), buffy coat specimens from R. typhi PCR-positive Lao patients (n = 42), and diverse negative controls (n = 47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus (n = 266). The limit of detection was ∼40 DNA copies/LAMP reaction, with an analytical sensitivity of <10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%]) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies.

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Section: Methodsmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

et al. 2014

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“…IFA has additional limitations as this depends on the microscopist who reads the slides and determines the end-point titer. There might be inter-and intra-operator variability so microscopists must be supervised by more experienced laboratory professional and undergo several months of training before their results can be considered reliable [18]. Requirements of a trained personal, fluorescent microscope, validation of appropriate diagnostic cut offs and relatively high cost is the main drawback of the IFA.…”

Section: Discussionmentioning

confidence: 99%

Diagnostic evaluation of IgM ELISA and IgM Immunofluorescence assay for the diagnosis of Acute Scrub Typhus in central Nepal

Gautam

Parajuli

Tshokey³

et al. 2020

BMC Infect Dis

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Background: Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium, Orientia tsutsugamushi. Immunochromatography (ICT) and IgM ELISA are two of the routinely employed antibody based assays for diagnosis of Scrub typhus fever in Nepal, although the recommended gold standard diagnostic test is IgM Immunofluorescence assay (IFA). This study evaluated InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. Method: Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal. Blood sample was collected from the suspected patients of scrub typhus, presenting with acute febrile illness. IgM antibody to Orientia tsusugamushi was detected by using Scrub Typhus Detect™ Kit and an in-house IgM IFA. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. Result: Statistical analysis of IgM ELISA results when compared to reference test, IgM IFA results demonstrated the following characteristics, sensitivity 84.0% (95%CI: 79.73-87.68%), specificity 94.82% (95% CI: 93.43-95.99%), positive likelihood ratio 16.21% (95% CI: 12.71-20.67%), negative likelihood ratio 0.17% (95% CI: 0.13-0.21%), disease prevalence 22.08% (95% CI: 20.06-24.21%), positive predictive value 82.12% (95% CI: 78.28-85.42%) and negative predictive value 95.44% (95% CI: 94.27-96.38%) respectively. Conclusion: Although IgM IFA is considered the gold standard test for the diagnosis of scrub typhus cases, it is relatively expensive, requires trained personal and a microscope with fluorescence filters. Scrub typhus IgM ELISA may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

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“…Diagnosis of E. chaffeensis infection with a twostep method has some disadvantages. Due to the low specificity of the peptide ELISA screening stage, confirmation is still reliant on IFA, a technique that requires extensive training to read and interpret test results and is dependent on the availability of a fluorescence microscope, requirements which are impractical in resource-limited regions (22). However, screening with peptide ELISA vastly reduces the workload of IFA.…”

Section: Discussionmentioning

confidence: 99%

Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua

et al. 2016

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e Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to <20%. Using a two-step diagnostic approach (IFA is performed if the ELISA is positive), we identified E. chaffeensis or a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies. Rickettsiae are frequently the cause of acute febrile illness (AFI) in the tropics when rigorously sought; however, they are infrequently identified because of limited diagnostic tools to identify and confirm infections (1, 2). Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States (3). Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date (4). Serologic diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity (5, 6). However, the technique is too time-consuming to be practical for large epidemiologic studies, requires the use of a fluorescence microscope, and requires highly experienced operators for reproducibility. Hence, clinical diagnosis and epidemiologic studies of Ehrlichia chaffeensis are not readily done in resource-poor regions. A peptide-based enzyme-linked immunosorbent assay (ELISA) has promise as a high-throughput, objective, and inexpensive diagnostic tool (7). We refined and optimized a peptide-based ELISA for E. chaffeensis infection and applied it to ascertain whether E. chaffeensis is an unrecognized cause of acute febrile illness in humans in Nicaragua. MATERIALS AND METHODSEhrlichia chaffeensis peptide ELISA cutoff optimization. Convalescentphase serum samples from patients with PCR-, culture-, and/or blood smear-proven infections with E. chaffeensis (n ϭ 16; E. chaffeensis geometric mean titer [GMT] by IFA of 1,503 [range, 256 to 16,384] and Anaplasma phagocytophilum GMT by I...

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Inter- and Intra-Operator Variability in the Reading of Indirect Immunofluorescence Assays for the Serological Diagnosis of Scrub Typhus and Murine Typhus

Cited by 44 publications

References 11 publications

Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

Diagnostic evaluation of IgM ELISA and IgM Immunofluorescence assay for the diagnosis of Acute Scrub Typhus in central Nepal

Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua

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