Performance of molecular and serologic tests for the diagnosis of scrub typhus

Abstract Background Diagnosis of scrub typhus, caused by the bacterium Orientia tsutsugamushi, is challenging because of the overlap of its nonspecific symptoms with other infections coupled with the lack of sufficient data on the performance of diagnostic tests. Early diagnosis of scrub typhus is crucial to improve outcomes and this study evaluated the diagnostic performance of various tests. Methods Adult patients with acute febrile illness and manifestations suggestive of scrub typhus confirmed by positive PCR in the blood or eschar were characterized as cases. Patients with acute febrile illness and a confirmed alternate etiology such as culture-confirmed typhoid, smear/PCR positive for malaria, PCR/NS1 antigen positive for dengue, PCR positive for influenza, PCR/MAT positive for leptospirosis, PCR positive for spotted fever were characterized as controls with other infections. The healthy controls consisted of subjects from the same geographic region. We performed the following tests on blood samples for scrub typhus and calculated the sensitivity, specificity, positive predictive value, and negative predictive value: (1) Quantitative PCR using 47 kDa gene (qPCR); (2) Conventional PCR using 56kDa gene (cPCR); (3) Loop-mediated isothermal amplification assay (LAMP assay); (4) Immunofluorescence assay (IFA); (5) Enzyme-linked immunosorbent assay (ELISA); (6) Weil–Felix test (WF test); and (7) Immunochromatographic Rapid Diagnostic Test (RDT). Results Among the 302 participants, 152 had confirmed scrub typhus (cases) and 150 were controls. ELISA and RDT detecting IgM antibodies had excellent discriminative potential with sensitivities and specificities of 94%, 93% and 92%, 93%, respectively. False-positive IgM serology was observed with spotted fever and leptospirosis. The sensitivity and specificity of IFA were found to be 80% and 85%, respectively. qPCR exhibited excellent sensitivity (96%) and perfect specificity. Conclusion ELISA and RDT detecting IgM antibodies have excellent sensitivity and specificity while the sensitivity of IFA is suboptimal for the diagnosis of scrub typhus. Given its perfect specificity and superior sensitivity, qPCR is preferred for diagnostic confirmation in reference laboratories. Disclosures All authors: No reported disclosures.

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Evaluation of loop-mediated isothermal amplification assay for detection of scrub typhus in patients with acute febrile illness presenting to a Tertiary Care Center in Puducherry, India

Journal of Laboratory Physicians ◽

10.4103/jlp.jlp_148_18 ◽

2019 ◽

Vol 11 (01) ◽

pp. 082-086 ◽

Cited By ~ 2

Author(s):

Patricia Anitha Karthikeyan ◽

Sugeerappa Laxmanappa Hoti ◽

Reba Kanungo

Keyword(s):

Diagnostic Test ◽

Scrub Typhus ◽

Tertiary Care ◽

Lamp Assay ◽

Febrile Illness ◽

Isothermal Amplification ◽

Acute Febrile Illness ◽

Loop Mediated Isothermal Amplification ◽

Igm Antibodies ◽

Two Samples

Abstract PURPOSE: Scrub typhus an acute febrile illness has diverse clinical manifestations, which overlap with other febrile illnesses. Due to this reason, it is misdiagnosed, leading to inappropriate treatment, sometimes resulting in fatality. Thus, accurate diagnosis of scrub typhus is important for appropriate treatment. This study evaluated the loop-mediated isothermal amplification (LAMP) assay as a diagnostic test for scrub typhus among patients with fever. MATERIALS AND METHODS: A total of 50 cases of acute febrile illness clinically resembling scrub typhus, with or without an eschar, or cases of pyrexia of unknown origin were included in the study. Blood samples collected from these cases were subjected to detection of IgM antibodies to Orientia tsutsugamushi by ELISA, conventional groEL polymerase chain reaction (PCR), and the LAMP assay. RESULTS: Twelve cases had fever for less than a week, and two had fever for more than 3 weeks. IgM antibodies to O. tsutsugamushi were detected in 37 out of 50 samples (74%). LAMP assay was positive in 33 samples (66%). groEL gene-based PCR detected 35 (70%) samples as positive. Two samples negative by LAMP assay were positive by this PCR. Twenty samples collected from patients with dengue, typhoid, and malaria tested by the LAMP assay were negative, indicating its good specificity. LAMP assay and the conventional groEL-based PCR could detect 72.7% and 74.3% of the samples, respectively before the 10th day after onset of fever, whereas IgM ELISA could detect only 40.5% of the 37 samples. CONCLUSION: This study suggests that LAMP assay could be a useful diagnostic test for detecting scrub typhus in the acute phase of the illness and a cheaper alternative to other molecular methods in resource poor settings.

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

10.21203/rs.2.10024/v5 ◽

2020 ◽

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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Searching and Finding the Hidden Treasure: A Retrospective Analysis of Rickettsial Disease Among Dutch International Travelers

Clinical Infectious Diseases ◽

10.1093/cid/ciaa091 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sophia G de Vries ◽

Louise E van Eekeren ◽

Hans van der Linden ◽

Benjamin J Visser ◽

Martin P Grobusch ◽

...

Keyword(s):

Retrospective Analysis ◽

Medical Records ◽

Scrub Typhus ◽

Spotted Fever ◽

Febrile Illness ◽

Empiric Antibiotic Therapy ◽

Spotted Fever Group ◽

Orientia Tsutsugamushi ◽

Rickettsial Disease ◽

Empiric Antibiotic

Abstract Background Rickettsial disease (RD) is a prevalent and underestimated cause of febrile illness worldwide, especially in the absence of an inoculation eschar. We attempted to quantify this underestimation at our clinic, by investigating past cases of febrile illness in travelers who had tested negative for leptospirosis, a disease that can initially present similarly to non-eschar RD, and which we routinely consider when other important causes of unspecified febrile illness have tested negative. Methods We performed a retrospective analysis in febrile returned travelers from Asia, Africa, or the Americas between 2010 and 2017, who had tested negative for leptospirosis. Serologic immunofluorescence assays were performed for Orientia tsutsugamushi (scrub typhus), typhus group, and spotted fever group RD. We performed a medical records review of all patients who tested positive. In case of a fitting medical history, cases were deemed either confirmed (based on convalescent serology) or suspected (based on single serology). Results Among 97 patients, convalescent serology was available in 16 (16.5%) patients, and a single serology in 81 (83.5%) patients. RD was the likely diagnosis in 8 of 16 (50.0%) patients with convalescent serology, and in 8 of 81 (9.9%) with single serology. Of the 16 confirmed/suspected cases, 11 (69%) had been missed and 7 (44%) had not received adequate empiric antibiotic therapy. Conclusions This study shows that non-eschar RD is an important and poorly recognized cause of illness in travelers, even in a specialized travel clinic. A lower threshold to test and treat for RD is warranted in returning travelers with febrile illness.

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Scrub Typhus in Children

Journal of Nepal Paediatric Society ◽

10.3126/jnps.v38i1.18659 ◽

2018 ◽

Vol 38 (1) ◽

pp. 59-62

Author(s):

Madiha Zainab ◽

Atul Kumar Gupta ◽

Suparna Guha

Keyword(s):

Acute Kidney Injury ◽

Scrub Typhus ◽

Kidney Injury ◽

Febrile Illness ◽

Acute Febrile Illness ◽

Common Complication ◽

Orientia Tsutsugamushi ◽

Pain Abdomen ◽

One Year ◽

High Index Of Suspicion

Introduction: Scrub typhus is an acute febrile illness caused by infection with rickettsial bacilli Orientia tsutsugamushi. This was a retrospective observational study to study the clinical profile of paediatric scrub typhus, its associated complications and response to treatmentMaterial and Methods: Record files of all patients diagnosed with positive Weil felix (OXK>1:80) and Scrub IgM positive over a period of one year were analysed. Total of 10 cases were diagnosed as scrub with median age of presentation 4.1 years.Results: Fever was present in all followed by pain abdomen (50%), rash. Anaemia (90%), lymphadenopathy (70%) hepatomegaly (100%), Leukopenia was present in those cases with fever <1 week while leucocytosis was found thereafter. Most common complication were hepatitis (100%) shock (50%), acute kidney injury (AKI) 30%, DIC in 20% cases. Secondary HLH was found in 20% and pancarditis in one case. All the cases showed dramatic response to doxycycline.Conclusion: So a high index of suspicion is required to diagnose scrub and early initiation of treatment is essential to prevent mortality from the disease.

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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/v4 ◽

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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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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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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Spotted Fever Group Rickettsiae among Children with Acute Febrile Illness, in Gorakhpur, India

Journal of Tropical Pediatrics ◽

10.1093/tropej/fmaa031 ◽

2020 ◽

Author(s):

Siraj A Khan ◽

Trishna Bora ◽

Jeromie Wesley Vivian Thangaraj ◽

Manoj V Murhekar

Keyword(s):

Spotted Fever ◽

Uttar Pradesh ◽

Febrile Illness ◽

Health Facilities ◽

Spotted Fever Group ◽

Acute Febrile Illness ◽

Orientia Tsutsugamushi ◽

Spotted Fever Group Rickettsiae ◽

Acute Encephalitis ◽

Indian State

Abstract Seasonal outbreaks of acute encephalitis syndrome have been occurring in Gorakhpur division in the Indian state of Uttar Pradesh during monsoon and post-monsoon months. Orientia tsutsugamushi was identified as the major aetiology of these outbreaks. Orientia tsutsugamushi was also identified as one of the important aetiology of febrile illness among children attending peripheral health facilities. The present study was undertaken to detect antibodies against spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) among children with acute febrile illness presenting at peripheral health facilities in Gorakhpur district. Of the 224 blood samples tested, SFGR infection was detected in 13 (6%) patients. None of the samples tested positive for TGR.

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