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

Abstract Introduction Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium,Orientia tsutsugamushi. Given their affordability and ease of use, antibody based diagnostic assays can be important diagnostic tools for early detection of scrub typhus fever in resource poor countries like Nepal. 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 the InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. Methodology Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal from April 2017 to March 2018. 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 (In Bios International, USA) and an in-house IgM IFA (Australian Rickettsial Reference Laboratory, Geelong, Australia. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. Result Statistical analysis of ELISA IgM 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 The study indicated that the IgM ELISA has the sensitivity 84.0% (95% CI: 79.73%-87.68%) and specificity 94.82% (95% CI: 93.43%-95.99%). 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 with appropriate OD cut–off values may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

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

10.21203/rs.2.10024/v2 ◽

2019 ◽

Author(s):

Rajendra Gautam ◽

Keshab Parajuli ◽

Tshokey Tshokey ◽

John Stenos ◽

Jeevan Bahadur Sherchand

Keyword(s):

Likelihood Ratio ◽

Predictive Value ◽

Gold Standard ◽

Scrub Typhus ◽

Febrile Illness ◽

Immunofluorescence Assay ◽

Acute Febrile Illness ◽

Central Nepal ◽

Igm Elisa ◽

Typhus Fever

Abstract Introduction Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium,Orientia tsutsugamushi. Given their affordability and ease of use, antibody based diagnostic assays can be important diagnostic tools for early detection of scrub typhus fever in resource poor countries like Nepal. 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 the InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. Methodology Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal from April 2017 to March 2018. 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 (In Bios International, USA) and an in-house IgM IFA (Australian Rickettsial Reference Laboratory, Geelong, Australia. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. Result Statistical analysis of ELISA IgM 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 The study indicated that the IgM ELISA has the sensitivity 84.0% (95% CI: 79.73%-87.68%) and specificity 94.82% (95% CI: 93.43%-95.99%). 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 with appropriate OD cut–off values may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

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

10.21203/rs.2.10024/v1 ◽

2019 ◽

Author(s):

Rajendra Gautam ◽

Keshab Parajuli ◽

John Stenos ◽

Tshokey Tshokey ◽

Jeevan Bahadur Sherchand

Keyword(s):

Likelihood Ratio ◽

Predictive Value ◽

Gold Standard ◽

Scrub Typhus ◽

Febrile Illness ◽

Immunofluorescence Assay ◽

Acute Febrile Illness ◽

Central Nepal ◽

Igm Elisa ◽

Typhus Fever

Abstract Introduction Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium,Orientia tsutsugamushi. Given their affordability and ease of use, antibody based diagnostic assays can be important diagnostic tools for early detection of scrub typhus fever in resource poor countries like Nepal. 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 the InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. Methodology Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal from April 2017 to March 2018. 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 (In Bios International, USA) and an in-house IgM IFA (Australian Rickettsial Reference Laboratory, Geelong, Australia. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. Result Statistical analysis of ELISA IgM 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 The study indicated that the IgM ELISA has the sensitivity 84.0% (95% CI: 79.73%-87.68%) and specificity 94.82% (95% CI: 93.43%-95.99%). 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 with appropriate OD cut–off values may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

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

10.21203/rs.2.10024/v3 ◽

2019 ◽

Author(s):

Rajendra Gautam ◽

Keshab Parajuli ◽

Tshokey Tshokey ◽

John Stenos ◽

Jeevan Bahadur Sherchand

Keyword(s):

Likelihood Ratio ◽

Predictive Value ◽

Gold Standard ◽

Scrub Typhus ◽

Febrile Illness ◽

Immunofluorescence Assay ◽

Acute Febrile Illness ◽

Central Nepal ◽

Igm Elisa ◽

Typhus Fever

Abstract Introduction Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium,Orientia tsutsugamushi. Given their affordability and ease of use, antibody based diagnostic assays can be important diagnostic tools for early detection of scrub typhus fever in resource poor countries like Nepal. 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 the InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. Methodology Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal from April 2017 to March 2018. 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 (In Bios International, USA) and an in-house IgM IFA (Australian Rickettsial Reference Laboratory, Geelong, Australia. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. Result Statistical analysis of ELISA IgM 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 The study indicated that the IgM ELISA has the sensitivity 84.0% (95% CI: 79.73%-87.68%) and specificity 94.82% (95% CI: 93.43%-95.99%). 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 with appropriate OD cut–off values may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

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Scrub Typhus and Molecular Characterization of Orientia tsutsugamushi from Central Nepal

Pathogens ◽

10.3390/pathogens10040422 ◽

2021 ◽

Vol 10 (4) ◽

pp. 422

Author(s):

Rajendra Gautam ◽

Keshab Parajuli ◽

Mythili Tadepalli ◽

Stephen Graves ◽

John Stenos ◽

...

Keyword(s):

Genetic Diversity ◽

Scrub Typhus ◽

Febrile Illness ◽

Acute Febrile Illness ◽

Limited Information ◽

Orientia Tsutsugamushi ◽

Health Measures ◽

Central Nepal ◽

Vector Borne

Scrub typhus is a vector-borne, acute febrile illness caused by Orientia tsutsugamushi. Scrub typhus continues to be an important but neglected tropical disease in Nepal. Information on this pathogen in Nepal is limited to serological surveys with little information available on molecular methods to detect O. tsutsugamushi. Limited information exists on the genetic diversity of this pathogen. A total of 282 blood samples were obtained from patients with suspected scrub typhus from central Nepal and 84 (30%) were positive for O. tsutsugamushi by 16S rRNA qPCR. Positive samples were further subjected to 56 kDa and 47 kDa molecular typing and molecularly compared to other O. tsutsugamushi strains. Phylogenetic analysis revealed that Nepalese O. tsutsugamushi strains largely cluster together and cluster away from other O. tsutsugamushi strains from Asia and elsewhere. One exception was the sample of Nepal_1, with its partial 56 kDa sequence clustering more closely with non-Nepalese O. tsutsugamushi 56 kDa sequences, potentially indicating that homologous recombination may influence the genetic diversity of strains in this region. Knowledge on the circulating strains in Nepal is important to the development of diagnostic tests and vaccines to support public health measures to control scrub typhus in this country.

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Diagnostic Accuracy of the InBios Scrub Typhus Detect™ ELISA for the Detection of IgM Antibodies in Chittagong, Bangladesh

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed3030095 ◽

2018 ◽

Vol 3 (3) ◽

pp. 95 ◽

Cited By ~ 7

Author(s):

Stuart Blacksell ◽

Hugh Kingston ◽

Ampai Tanganuchitcharnchai ◽

Meghna Phanichkrivalkosil ◽

Mosharraf Hossain ◽

...

Keyword(s):

General Population ◽

Diagnostic Accuracy ◽

Diagnostic Tests ◽

Scrub Typhus ◽

Febrile Illness ◽

High Sensitivity ◽

Immunofluorescence Assay ◽

Immunoglobulin M ◽

Regional Assessment ◽

Igm Elisa

Here we estimated the accuracy of the InBios Scrub Typhus Detect™ immunoglobulin M (IgM) ELISA to determine the optimal optical density (OD) cut-off values for the diagnosis of scrub typhus. Patients with undifferentiated febrile illness from Chittagong, Bangladesh, provided samples for reference testing using (i) qPCR using the Orientia spp. 47-kDa htra gene, (ii) IFA ≥1:3200 on admission, (iii) immunofluorescence assay (IFA) ≥1:3200 on admission or 4-fold rise to ≥3200, and (iv) combination of PCR and IFA positivity. For sero-epidemiological purposes (ELISA vs. IFA ≥1:3200 on admission or 4-fold rise to ≥3200), the OD cut-off for admission samples was ≥1.25, resulting in a sensitivity (Sn) of 91.5 (95% confidence interval (95% CI: 96.8–82.5) and a specificity (Sp) of 92.4 (95% CI: 95.0–89.0), while for convalescent samples the OD cut-off was ≥1.50 with Sn of 66.0 (95% CI: 78.5–51.7) and Sp of 96.0 (95% CI: 98.3–92.3). Comparisons against comparator reference tests (ELISA vs. all tests including PCR) indicated the most appropriate cut-off OD to be within the range of 0.75–1.25. For admission samples, the best Sn/Sp compromise was at 1.25 OD (Sn 91.5%, Sp 92.4%) and for convalescent samples at 0.75 OD (Sn 69.8%, Sp 89.5%). A relatively high (stringent) diagnostic cut-off value provides increased diagnostic accuracy with high sensitivity and specificity in the majority of cases, while lowering the cut-off runs the risk of false positivity. This study underlines the need for regional assessment of new diagnostic tests according to the level of endemicity of the disease given the high levels of residual or cross-reacting antibodies in the general population.

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Diagnostic Accuracy of the InBios Scrub Typhus Detect Enzyme-Linked Immunoassay for the Detection of IgM Antibodies in Northern Thailand

Clinical and Vaccine Immunology ◽

10.1128/cvi.00553-15 ◽

2015 ◽

Vol 23 (2) ◽

pp. 148-154 ◽

Cited By ~ 38

Author(s):

Stuart D. Blacksell ◽

Ampai Tanganuchitcharnchai ◽

Pruksa Nawtaisong ◽

Pacharee Kantipong ◽

Achara Laongnualpanich ◽

...

Keyword(s):

Diagnostic Accuracy ◽

False Positive ◽

Gold Standard ◽

Latent Class ◽

Scrub Typhus ◽

Febrile Illness ◽

Statistical Correlation ◽

Immunofluorescence Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Northern Thailand

ABSTRACTIn this study, we examined the diagnostic accuracy of the InBios Scrub Typhus Detect IgM enzyme-linked immunosorbent assay (ELISA) and determined the optimal diagnostic optical density (OD) cutoffs for screening and diagnostic applications based on prospectively collected, characterized samples from undifferentiated febrile illness patients in northern Thailand. Direct comparisons with the serological gold standard, indirect immunofluorescence assay (IFA), revealed strong statistical correlation of ELISA OD values and IFA IgM titers. Determination of the optimal ELISA cutoff for seroepidemiology or screening purposes compared to the corresponding IFA reciprocal titer of 400 as previously described for Thailand was 0.60 OD, which corresponded to a sensitivity (Sn) of 84% and a specificity (Sp) of 98%. The diagnostic performance against the improved and more-stringent scrub typhus infection criteria (STIC), correcting for low false-positive IFA titers, resulted in an Sn of 93% and an Sp of 91% at an ELISA cutoff of 0.5 OD. This diagnostic ELISA cutoff corresponds to IFA reciprocal titers of 1,600 to 3,200, which greatly reduces the false-positive rates associated with low-positive IFA titers. These data are in congruence with the recently improved serodiagnostic positivity criteria using the Bayesian latent class modeling approach. In summary, the InBios Scrub Typhus Detect IgM ELISA is affordable and easy-to-use, with adequate diagnostic accuracy for screening and diagnostic purposes, and should be considered an improved alternative to the gold standard IFA for acute diagnosis. For broader application, regional cutoff validation and antigenic composition for consistent diagnostic accuracy should be considered.

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Epidemiology, Risk Factors and Seasonal Variation of Scrub Typhus Fever in Central Nepal

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed4010027 ◽

2019 ◽

Vol 4 (1) ◽

pp. 27 ◽

Cited By ~ 4

Author(s):

Rajendra Gautam ◽

Keshab Parajuli ◽

Jeevan Bahadur Sherchand

Keyword(s):

Risk Factors ◽

Seasonal Variation ◽

Confidence Interval ◽

Odds Ratio ◽

Scrub Typhus ◽

Public Awareness ◽

Febrile Illness ◽

Acute Febrile Illness ◽

Orientia Tsutsugamushi ◽

Central Nepal

Orientia. tsutsugamushi, a zoonotic bacterial infection common in the region known as the tsutsugamushi triangle. This study aims to determine the seroprevalence, seasonal variation, and risk factors of scrub typhus among the acute febrile illness patients attending different hospitals of central Nepal. Blood samples were collected from hospitalized patients of acute febrile illness suspected of scrub typhus infection attending different hospitals of central Nepal from April 2017 to March 2018. The IgM antibody to Orientia tsutsugamushi was detected by using the Scrub Typhus Detect™ Kit. Among the total cases (1585), 358 (22.58%) were positive for IgM Antibodies. Multivariate analysis identified several risks factors to be significantly associated with the scrub typhus infection, including gender (female) (odds ratio [OR] = 1.976, p ≤ 0.001, confidence interval [CI] = 1.417–2.756), rural residential location (odds ratio [OR] = 0.431, p = 0.001, confidence interval [CI] = 0.260–0.715), house near grassland (odds ratio [OR] = 3.288, p ≤ 0.001, confidence interval [CI] = 1.935–5.587), and working in the field (odds ratio [OR] = 9.764, p = 0.004, confidence interval [CI] = 2.059–46.315). The study findings indicate scrub typhus infection to be a significant health problem in Nepal. The proper diagnosis of infection cases, timely institution of therapy, public awareness, and vector control are important measures to be taken for the prevention and management of scrub typhus.

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Scrub typhus in patients with acute febrile illness: a 5-year study from India

QJM ◽

10.1093/qjmed/hcz308 ◽

2019 ◽

Vol 113 (6) ◽

pp. 404-410

Author(s):

C K Thakur ◽

R Chaudhry ◽

N Gupta ◽

E V Vinayaraj ◽

V Singh ◽

...

Keyword(s):

Scrub Typhus ◽

Monsoon Season ◽

Geographical Location ◽

Febrile Illness ◽

Immunofluorescence Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Acute Febrile Illness ◽

Nested Polymerase Chain Reaction ◽

Pulmonary Manifestations ◽

Post Monsoon Season

Abstract Background Scrub typhus was once thought to be a disease of rural origin and was confined to specific pockets in South Asia. Early diagnosis and treatment is extremely important as it is associated with high mortality if left untreated. Aim To delineate the clinical and molecular epidemiology of scrub typhus in patients presenting with acute febrile illness from various parts of India. Methods During the study period of 5 years (October 2013 to October 2018), a total of 1742 patients with acute febrile illness <15 days were enrolled after taking informed consent. Patients were diagnosed using IgM Enzyme-linked immunosorbent assay (ELISA) based on the pre-determined region specific cut offs. Patients with positive IgM ELISA were also subjected to IgM Immunofluorescence assay and nested polymerase chain reaction (PCR) assay. The demographic and relevant clinical details of the patients were documented and analyzed. Results A total of 210 (12.1%) patients were diagnosed with scrub typhus. Of these, nested PCR was positive in only 85 patients. Sequencing and phylogenetic analysis showed that the predominant circulating genotypes were Gilliam and Karp. On multivariate analysis, acute respiratory distress syndrome, myocarditis, encephalitis/encephalopathy, jaundice and splenomegaly were significantly more common in those patients who were diagnosed with scrub typhus. A total of 14 patients diagnosed with scrub typhus succumbed to the illness. Conclusion Patients with fever, headache, pulmonary manifestations, CNS manifestations, myocarditis, transaminitis or thrombocytopenia presenting in the monsoon and post-monsoon season should be evaluated for scrub typhus irrespective of the geographical location in India.

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