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Table of Contents
SHORT RESEARCH COMMUNICATION
Year : 2018  |  Volume : 55  |  Issue : 2  |  Page : 165-167

Diagnostic validation of IgM and IgG ELISA and real-time PCR in detecting scrub typhus infection in endemic regions


1 Department of Microbiology, Government Medical College, Haldwani, India
2 Department of Community Medicine, Government Medical College, Haldwani, India
3 Department of Medicine, Government Medical College, Haldwani, India

Date of Submission19-Sep-2017
Date of Acceptance23-Feb-2018
Date of Web Publication1-Oct-2018

Correspondence Address:
Vinita Rawat
Associate Professor, Department of Microbiology, Government Medical College, Haldwani, District Nainital, Uttarakhand
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.242565

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  Abstract 


Keywords: Diagnosis; ELISA; real-time PCR; scrub typhus


How to cite this article:
Rawat V, Singh RK, Kumar A, Singh Y, Chaturvedi P, Saxena SR, Varshney U. Diagnostic validation of IgM and IgG ELISA and real-time PCR in detecting scrub typhus infection in endemic regions. J Vector Borne Dis 2018;55:165-7

How to cite this URL:
Rawat V, Singh RK, Kumar A, Singh Y, Chaturvedi P, Saxena SR, Varshney U. Diagnostic validation of IgM and IgG ELISA and real-time PCR in detecting scrub typhus infection in endemic regions. J Vector Borne Dis [serial online] 2018 [cited 2018 Oct 21];55:165-7. Available from: http://www.jvbd.org/text.asp?2018/55/2/165/242565



Scrub typhus is an important cause of acute undifferentiated fever and is still underreported in many parts of India due to low index of clinical suspicion[1]. The disease is caused by Orientia tsutsugamushi, an obligate intracellular bacterium which is transmitted through the bite of trombiculid mites[1],[2]. Reliable laboratory testing is of great importance to detect scrub typhus infection. Serological diagnosis usually miss the early rickettesemic period of the disease[3]. Molecular assays are useful in detecting O. tsutsugamushi DNA even before antibody responses occur. Therefore, the combination of molecular and antibody based tests provide a strong diagnostic advantage in detecting the disease in early and late phase of infection[4].

The objective of this study was to evaluate the performance of commercial IgM ELISA, IgG ELISA and realtime PCR in diagnosing acute cases of scrub typhus; and to determine the optimal cutoff optical density (OD) of ELISA for its diagnosis in the study area. The study was carried out in Dr. Susheela Tiwari Hospital affiliated to Government Medical College, Haldwani which is a tertiary care hospital catering to Kumaon region, in Uttarakhand state of India. During the study period (April 2015 to March 2016) blood samples were collected in plain and EDTA vials from suspected scrub typhus cases. Cases of dengue, malaria and enteric fever were excluded by appropiate tests as these diseases share the same epidemiological and clinical features with scrub typhus. A total of 450 suspected scrub typhus cases enrolled in the study were subjected to IgM and IgG ELISA. Real-time PCR was conducted in 273 suspected scrub typhus patients and in twenty healthy blood donors' samples. A total of 186 and 244 sera were tested by gold standard IgM and IgG immuno fluorescence assay (IFA), respectively. The ELISA tests were performed through commercially available IgM ELISA and IgG ELISA kits (Scrub Typhus Detect IgM and IgG ELISA System, InBios International, USA) as per the manufacturer's instructions.

Commercially available scrub typhus IFA kits were obtained from Fuller Labs, USA. Each well of the IFA slide contained four O. tsutsugamushi strains, viz. Karp, Kato, Boryong and Glilliam strains. The tests were carried out as per the manufacturer's instructions. Baseline cutoff titers of IgM and IgG IFA were calculated by testing the sera of 100 healthy blood donors. Less than 5% of healthy blood donors had IgM IFA titer of 512 and IgG IFA titer of 2048. Hence, the diagnostic cutoff for IgM and IgG IFA were taken as ≥512 and ≥2048, respectively, .

Real-time PCR kit—O. tsutsugamushi membrane protease (htrA) gene (genesig® Advanced kit) for scrub typhus was obtained from Primerdesign™ Ltd, UK. All the lyophilized components were reconstituted according to kit recommendations and stored at –20 °C. Real-time PCR was carried out as manufacturer's instructions. In brief, for each well, 10 μ1 reaction mixture was prepared by mixing 5 μ1 PrecisionPLUS™ 2×qPCR Master mix, 0.5 μ1 O. tsutsugamushi specific primer/probe mix, 2 μ1 of RNAse/DNAse free water and 2.5 μ1 of DNA template. Amplification conditions included enzyme activation at 95 °C for 2 min followed by 50 cycles of denaturation at 95 °C for 10 sec and data collection at 60 °C for 60 sec. With each run, positive and negative controls were used. For negative control, 2.5 μl of DNA template was replaced with RNAse/DNAse free water.

Results were interpreted as per manufacturer's interpretative criteria, i.e. positive control template must amplify between Cq 16 and 23. Failure to satisfy this quality control criterion was considered as a compromised experiment.

Of the total sera tested, 40% (76/186) were positive by IgM IFA and 33% (81/244) by IgG IFA while 36 sera were positive by both IgG and IgM IFA. To determine the optimal cutoff OD for the ELISA assay, a receiver operating characteristic (ROC) curve was plotted by using SPSS version 16, displaying every possible cutoff value for a positive OD generated [Figure 1] and [Figure 2]. The cutoff value of the IgG and IgM ELISA assays were selected, by using Youden's index (J = Sensitivity + specificity–1). The cutoff OD of IgM ELISA at 0.6 gave sensitivity and specificity of 96.4 and 82.7%, respectively. The cutoff OD calculated as per the manufacturer's formula [average OD of the normal human serum samples+three times the standard deviation (SD) of normal healthy serum] corresponded to 0.492; and the sensitivity and specificity were found to be 97.1 and 79.1%, respectively.
Figure 1: Receiver operating characteristic (ROC) curve of IgM ELISA with an area under curve (AUC) of 0.934 (95% CI, 0.898–0.970).

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Figure 2: ROC curve of IgG ELISA with an AUC of 0.883 (95% CI, 0.842–0.924).

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The cutoff value of IgG ELISA at 1.6, produced sensitivity and specificity of 91 and 75%, respectively while at the cutoff OD of 1, sensitivity and specificity were 97.5 and 60% respectively.

Out of 273 blood samples tested by real-time PCR, 102 were found positive for scrub typhus. When compared with gold standard IgM IFA, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of real-time PCR were found to be 69.7, 70.5, 63 and 76.3% respectively. Real-time PCR detected O. tsutsugamushi DNA in 31 IgM IFA and 59 IgG IFA negative cases. DNA of O. tsutsugamushi could not be amplified in 23 IgM IFA and 45 IgG IFA positive cases. Blood samples of 20 healthy blood donors tested by real-time PCR were found negative.

Scrub typhus infections are notoriously difficult to diagnose due to low index of suspicion, nonspecific sign and symptoms, and lack of readily available and validated assays[5],[6]. Failure of timely diagnosis of scrub typhus might lead to significant morbidity and mortality[2]. Treatment of this disease is easy, affordable and often successful with dramatic response with antimicrobials, if diagnosed timely.

In the present study, IgM ELISA at cutoff OD of 0.6 yielded sensitivity, specificity, PPV and NPV of 96.4, 82.7 79.3 and 96.8% respectively, which were close to that calculated by manufacturer's cutoff formula (0.492). Hence, the kit recommendation appears acceptable and matches with the study finding. A study from south India[7], reported sensitivity of 85% and specificity of 95% by IgM ELISA at diagnostic cutoff OD of 1. Similarly, in a study carried out in Delhi[8], sensitivity and specificity of IgM ELISA was found to be 100 and 97 %, respectively at cutoff OD of 0.87. The gross difference in diagnostic parameters in different studies may be attributed to the endemic disease pattern and their respective background cutoff levels in different regions.

Scrub typhus is endemic in various regions of India[1],[2], [6],[7]. In endemic areas secondary infections with different serotypes are very common. It has been observed that secondary infection with a different variant of O. tsutsugamushi produces sharp rise in IgG level with variable IgM response[4]. At present, ELISA for scrub typhus available in market are essentially qualitative in nature. In the present study, IgG ELISA at cutoff OD of 1.6 had >90% sensitivity and reasonably good specificity. However, it is not possible to interpret the qualitative ELISA results when cutoff OD is above 1. At cutoff OD of 1, the sensitivity (97.5%) observed was excellent, but specificity (60%) was compromised. Based on these observations, it was perceived that qualitative IgG ELISA is not an appropriate testing modality to differentiate secondary scrub typhus infection from the past infection in endemic regions.

Most hospital based Indian studies have used IgM ELISA or Weil-Felix test for diagnostic confirmation of this disease[2], [9]. Few studies have also used 56 kDa based nested PCR for diagnostic confirmation[10],[11]. Real-time PCR has been found to be more sensitive and specific than nested PCR[12].

In the present study, real-time PCR could detect O. tsutsugamushi DNA in 31 IgM IFA and 59 IgG IFA negative cases. As per gold standard IFA, these cases were false positive. However, more correct interpretation probably would be that real-time PCR being extremely sensitive test and when performed in early course of disease with no antibody response, can pick up the truly infected cases. So, these false real-time PCR positive cases may actually be true positive infection detected in early course of disease. These patients might have attended the health care facility before the antibodies response.

DNA of O. tsutsugamushi could not be amplified in 23 IgM IFA and 45 IgG IFA positive cases. This might be due to delayed presentation; since the DNA in blood is limited to the time window of rickettsemia. However, further work is required to define the extent and pattern of shedding of O. tsutsugamushi in blood stream.

Hence, IgM ELISA in conjunction with real-time PCR may provide a strong diagnostic advantage and can be employed as specific diagnostic tool for diagnosis of scrub typhus in endemic areas. In the present study area, IgM ELISA at cutoff OD of 0.6 could be used for diagnosis of scrub typhus infection. There is also need for development of point of care tests as available for dengue, which detects antigen and antibody simultaneously in one cassette.

Ethical statement

Ethical approval for this study was granted by local ethics committee of the Government Medical College, Haldwani, Uttarakhand, India.

Conflict of interest : Nil.


  Acknowledgements Top


The work was supported by the Indian Council of Medical Research, New Delhi, India (Reference No. GIA/34/2014-DHR). Authors acknowledge the technical assistance of Neeraj Arya.



 
  References Top

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Rathi N, Rathi A. Rickettsial infections: Indian perspective. Indian Paediatr 2010; 47(2): 157-61.  Back to cited text no. 1
    
2.
Mittal V, Gupta N, Bhattacharya D, Kumar K, Ichhpujani RL, Singh S, et al. Serological evidence of rickettsial infections in Delhi. Indian J Med Res 2012; 135(4): 538-41.  Back to cited text no. 2
    
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Paris DH, Shelite TR, Day NP, Walker DH. Unresolved problems related to scrub typhus: A seriously neglected life-threatening disease. Am J Trop Med Hyg 2013; 89(2): 301-7.  Back to cited text no. 3
    
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Land MV, Ching WM, Dasch GA, Zhang Z, Kelly DJ, Graves SR, et al. Evaluation of commercially available recombinant-protien enzyme linked immunosorbant assay for detection of antibodies produced in scrub typhus rickettsial infections. J Clin Microbiol 2000; 38(7): 2701-5.  Back to cited text no. 4
    
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Blacksell SD, Tanganuchitcharnchi A, Nwataisong P, Kantipong P, Laongnualpanich A, Day NP, et al. Diagnostic accuracy of the In-Bios scrub typhus detect enzyme linked-immunoassay for detection of IgM antibodies in northern Thailand. Clin Vaccine Immunol 2015; 23(2): 148-54.  Back to cited text no. 5
    
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Issac R, Varghese GM, Mathai E, JM, Mathai I. Scrub typhus: Prevalence and diagnostic issues in rural southern India. Clin Infect Dis 2004; 39(9): 1395-6.  Back to cited text no. 6
    
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Koraluru M, Bairy I, Varma M, Vidhyasagar S. Diagnostic validation of selected serological tests for detecting scrub typhus. Microbiol Immunol 2015; 59(7): 371-4.  Back to cited text no. 7
    
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Gupta N, Chaudhry R, Thakur CK. Determination of cutoff of ELISA and immunofluorescence assay for scrub typhus. J Glob Infect Dis 2016; 8(3): 97-9.  Back to cited text no. 8
    
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Bhargava A, Kaushik R, Kaushik RM, Sharma A, Ahmad S, Dhar M et al. Scrub typhus in Uttarakhand and adjoining Uttar Pradesh: Seasonality, clinical presentation and predictors of mortality. Indian J Med Res 2016; 144(6): 901-9.  Back to cited text no. 9
    
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Mathai E, Rolain JM, Verghese GM, Abraham OC, Mathai D, Mathai M, et al. Outbreak of scrub typhus in southern India during cooler months. Ann N Y Acad Sci 2003; 990: 359-64.  Back to cited text no. 10
    
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Usha K, Kumar E, Kalawat U, Kumar BS, Chadhury A, Gopal DV. Molecular detection of scrub in Tirupati, Andhra Pradesh, India. J Vector Borne Dis 2015; 52(2): 171-4.  Back to cited text no. 11
    
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Jeang J, Chan TC, Temenak JJ, Dasch GA, Ching WM, et al. Development of quantitative real-time PCR. Am J Trop Med Hyg 2004; 70(4): 351-6.  Back to cited text no. 12
    


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