Scrub typhus (ST) caused by Orientia tsutsugamushi is now drawing the attention of clinicians and health au- thorities in view of its high morbidity and serious com- plications in untreated patients1. Scrub typhus, known for its endemicity in the so called “tsutsugamushi triangle”
spreading across Japan, Taiwan, China, and South Korea is an emerging infection in different parts of India in- volving several states and union territories, viz. Delhi, Tamil Nadu, Puducherry, Karnataka, Manipur, Maharashtra, Uttarakhand, Haryana, Jammu & Kashmir, Andhra Pradesh, West Bengal, Himachal Pradesh, Goa, Kerala, Rajasthan and Chandigarh including Himalayan region2–7. The early diagnosis would help initiating prompt treatment and preventing various complications.
In view of the highly infective nature of this chigger-borne rickettsiosis, which demands CDC category B/Bio-safety level 3 containment facilities, isolation by the culture is restricted to research/reference laboratories only. Sero- logical diagnosis is the preferred option.
Our objective was early and prompt laboratory diag- nosis of scrub typhus (ST) by rapid diagnostic test kits. A new scrub typhus test kit, viz. ImmuneMed Scrub Ty- phus Rapid, Gangwon-do, South Korea, which simulta- neously detects both IgM and IgG antibodies to O. tsut- sugamushi, has been evaluated in ST patients, non-ST patients and normal healthy controls. The performance of this kit, which is not yet marketed in India, has been compared against existing ST IgM and ST IgG ELISA (Scrub Typhus Detect IgM/IgG ELISA System, InBios International, Washington, USA). A total of 20 represen- tative sera were subjected to the “gold standard” indirect fluorescent antibody (IFA) test1 blindly at the Department of Microbiology and Immunology, School of Medicine, Hallym University, South Korea. The results are presented in this communication.
The satisfactory performance of a rapid test kit, viz.
SD Bioline tsutsugamushi, South Korea, which detects total antibody (IgM/IgG/IgA) has been reported by us earlier3. A new rapid ST test kit which discriminates IgM and IgG simultaneously in the same cassette ELISA has been validated by us for the first time in India. This research work has been approved by the institutional human ethical committee (IHEC) of Mahatma Gandhi Medical College and Research Institute (MGMCRI), Puducherry and the written informed and free consent was obtained from the participants/patients/relatives of the patients/parents or legal guardians of minors, prior to collection of blood samples for ST investigation.
This is an observational and investigative study based on diagnostic accuracy of tests. The study was carried out in the Department of Microbiology, MGMCRI, Puducherry, India. The IFA test was performed blindly at the Department of Microbiology and Immunology, School of Medicine, Hallym University, South Korea.
During a 16 months period from August 2013 to Novem- ber 2014, blood samples were collected from Puducherry and neighbouring districts of Tamil Nadu like Villupuram, Cuddalore, Ariyalur and Nagapattinam from three differ- ent population categories: Clinically suspected ST cases (87), non-ST patients with fever of unknown origin (FUO) (30), and healthy voluntary blood donors (10). About 21 ST and eight non-ST patients provided paired serum samples, taken at intervals of 10–14 days. Of 87 ST and 11 FUO patients gave only single (acute) samples. Thus, a total of 156 serum samples were included in the study.
Inclusion and exclusion criteria were set as per our ear- lier study4.As per the policy of the MGMCRI hospital, the clinically suspected ST patients were first screened by ST Rapid test kit; and the following investigations were performed: Total WBC count, platelet count, haemoglo- Short Research Communication
Evaluation of ImmuneMed scrub typhus rapid test kit, for diagnosis of scrub typhus
Selvaraj Stephen
1, Seung-Han Kim
2, Jothimani Pradeep
1, Young Jin Kim
2, Eun-Ye Kim
2, Sungman Park
2, Min-Woo Kim
3& Yoon-Won Kim
31Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Puducherry, India; 2ImmuneMed Inc., Gangwon- do; 3Department of Microbiology and Immunology, School of Medicine, Hallym University, Gangwon-do, South Korea
Key words ImmuneMed scrub typhus test kit; scrub typhus; south India
bin, serum bilirubin, alkaline phosphatase (ALP)/
glutamate oxaloacetate aminotransferase/alanine trans- ferase (SGOT/ALT)/glutamate pyruvate aminotrans- ferase/aspartate transferase (SGPT/AST), urea, serum creatinine and albumin. Depending upon the clinical pre- sentation, microbiological investigations like peripheral blood examination for malarial parasite and serology for typhoid (Widal-Span Diagnostics, Gujarat, India), den- gue (SD Bioline Dengue Duo kit, South Korea) and lep- tospirosis (SD Bioline Leptospira IgM/IgG, Korea) were also performed. Our ST panel comprised of ImmuneMed Rapid ST Immunochromatographic Test (ICT), ST IgM and ST IgG ELISA, InBios and Weil-Felix (WF) test for Proteus mirabilis OXK antibody detection. The tests were performed in accordance with procedures outlined by kits’
manufacturers.
Weil-Felix test: Proteus OXK coloured antigen (Plasmatech, South El Monte, California, USA) was used to screen the serum samples at dilutions ranging from 1:20 to 1:640 and up to their end points.
ST ImmuneMed test kit—Rapid diagnostic test (RDT):
A mixture of cr56, kr56 and r21 of O. tsutsugamushi as antigen was added to RDT. This RDT was manufactured by ImmuneMed. In brief, the test procedure was as fol- lows: 300 μl diluent buffer and 3 μl serum were applied to the sample port of the test kit. The result was read within 10 min. The red band appearing on control line (C) and test line (T) concurrently or either way was regarded as positive. The test was considered as negative when only control band appeared as red. And if, no hand appeared in the control line, then the test was considered invalid (Fig. 1).
ST IgM and IgG InBios ELISA: Both ELISA plates were coated with 10 recombinant antigens of O. tsutsuga- mushi targeting antibodies to 56-kDa antigen. The proce- dure is common for both kits except for the step involv- ing rheumatoid factor (RF) sorbent use for IgM ELISA.
The initial serum dilution was 1:100. After incubation and washing of ELISA plates, optical density (OD) read-
ings were taken at 450 nm in iMark Microplate Reader (Bio-Rad, Japan). A total of 20 samples were collected from healthy volunteers from ST endemic area of Kurinjipadi taluk, Cuddalore district, Tamil Nadu, and used in the calculation of the cut-off value in both IgM and IgG ELISA tests. The cut-off values were calculated as follows:
Fig. 1:ImmuneMed Scrub Typhus Rapid test kit—Positive and negative results.
The samples with OD values above the cut-off were considered positive and those below the cut-off were taken as negative. Borderline samples were tested in triplicate.
Indirect immunofluorescent antibody (IFA) assay:
Serological diagnosis of scrub typhus was performed by the IFA using O. tsutsugamushi IFA IgM antibody test kit (OTM-120, Fuller Laboratories, Fullerton California, USA) and O. tsutsugamushi IFA IgG antibody test kit (OTG-120, Fuller Laboratories, Fullerton, California, USA) as described in the manufacturer’s instruction.
In case of O. tsutsugamushi strain Boryong, the test was carried out as per Kim et al8. Endpoint titer was serum dilution at which rickettsiae exhibited clear positive fluorescence.
Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calcu- lated considering InBios ST IgM and IgG ELISA as gold standard against ImmuneMed and WF. The sensitivity and specificity were calculated as TP/(TP+FN) and TN/
(TN+FP), respectively, where, TP stands for true posi- tive; FN—False negative; TN—True negative; and FP—
False positive. PPV was calculated as = a/a+b = a (true positive)/a+b (true positive + false positive); while NPV was calculated as = d/c+d = d (true negative)/c+d (false negative + true negative). For other parameters (Spearman’s correlation and Kappa) statistical analyses were performed using IBM SPSS Statistics 17 for Win- dows (SPSS Inc., Chicago, USA). The chi-square test with Yates correction (Fisher’s test) was employed for small number of samples.
Randomly selected serum samples of 18 ST positive and two negative patients were blinded and sent to Hallym University, Korea, to carry out ST IFA and four geno- types were included in this IFA, viz. Kato, Karp, Gilliam and Boryong. A titre of ≥ 1:40 for IgM and ≥ 1:80 for IgG was considered suggestive of ST infection (Fig. 1)
In ImmuneMed Rapid test kit, IgM was observed only in 15 samples, IgG in nine whereas, 73 were positive for both IgM and IgG (Fig. 2). The kit did not show any in- valid result. In InBios ELISA, 11, 9 and 88 sera were positive for IgM, IgG and for both IgM and IgG respec-
[Average of the normal human serum samples (NHS)]+
[Three times of standard deviation (SD) from NHS]
Cut-off value =
tively. Varying levels of OXK agglutinin titres were ob- served in 37 patients: 320 (9); 640 (18); 1280 (1); 2560 (7); 5120 (1) and 10240 (1). The results of serological tests with 16 different combinations are presented in Table 1. Out of 20 sera subjected to ST IFA, 11 were positive for both IgM and IgG antibodies, four for IgM only, and two for IgG only. Results of ST IFA in comparison with other serological tests for 20 serum samples are presented in Table 2.
The sensitivity, specificity, PPV and NPV for ImmuneMed IgM were 87, 94.64, 96.67 and 80.3% re- spectively. Similarly for ImmuneMed IgG, the sensi- tivity, specificity, PPV and NPV were 77.32, 86.44, 90.36 and 69.86% respectively.
Minimum OXK titre ≥ 1:320 is recommended as sig- nificant, since lower titres are present even in normal population3–4, 7, 9. Statistical analyses of WF against InBios IgM revealed that the sensitivity, specificity, PPV and NPV were 59, 92.98, 93.65 and 56.38% respectively. For WF test against InBios IgG, the above values were 59.79,
Table 1. Results of ST serological tests in various combinations (n = 156 sera)
Category Combination of seropositivity Total % positive
for ST positive
A InBios IgM + IgG & ImmuneMed 40 37.04 IgM + IgG & WF*
B InBios IgM + IgG & ImmuneMed 24 22.22 IgM + IgG
C InBios IgM + IgG & ImmuneMed 8 7.41 IgM + WF
D InBios IgM + IgG & ImmuneMed 5 4.63 IgM
E InBios IgM + IgG & ImmuneMed 4 3.70 IgG + WF
F InBios IgM + IgG & ImmuneMed 1 0.93 IgG
G InBios IgM + ImmuneMed IgM 2 1.85
H InBios IgG + ImmuneMed IgG 3 2.78
I InBios IgM + ImmuneMed 3 2.78
IgM + IgG + WF
J InBios IgM + ImmuneMed 4 3.70
IgM + IgG
K InBios IgG + ImmuneMed 2 1.85
IgM + IgG
L InBios IgM + IgG & WF 2 1.85
M InBios IgM + IgG 4 3.70
N InBios IgM only 1 0.93
O InBios IgG & WF 4 3.70
P InBios IgM + ImmuneMed IgG 1 0.93
Sub Total 108
All tests negative* 48
Total tested 156
*Only the serum samples with OXK titres ≥1:320 are considered.
Table 2. Comparison of IFA results with other serological tests of 20 selected samples
S. Scrub typhus Scrub typhus WF (OXK) Scrub typhus IFA
No. InBios ELISA ImmuneMed
IgM IgG IgM IgG IgM IgG
1. + – + – 1: 320 640↑(Karp) 80 (Karp)
2. + – + – 1: 80 640↑(Gilliam) 80 (Karp)
3. + + + + >1: 640 640↑(Karp) 1280↑(Karp)
4. + + + + >1: 640 640↑(Karp) 80 (Karp)
5. + + + + 1: 80 – (1:40) 1280↑(Gilliam)
6. + – – – 1: 160 – (1:40) – (1:80)
7. + – + – 1: 160 160 (Gilliam) 80 (Kato)
8. + + + + 1: 640 640↑(Karp, Boryong) 80 (Karp)
9. + + + + >1: 640 640↑(Karp) 1280↑(Karp)
10. + + + + 1: 320 640↑(Karp) 1280↑(Karp)
11. + + + + >1: 640 640↑(Karp) 1280↑(Karp)
12. + + + + 1: 320 640↑(Karp) 1280↑(Karp)
13. + + + + > 640 640↑(Karp) 1280↑(Karp)
14. + + + + 1: 20 640↑(Karp) – (1:80)
15. + + + + 1: 40 160 (Gilliam) – (1:80)
16. + + + + 1: 320 640↑(Karp) – (1:80)
17. + + + + 1: 160 640↑(Karp) – (1:80)
18. + + – + 1: 80 – (1:40) 1280↑(Karp, Boryong)
19. – – – – 1: 20 – (1:40) – (1:80)
20. – – – – 1: 20 – (1:40) – (1:80)
(+)Positive; (–) Negative.
Fig. 2: IFA–IgM and IgG positive/negative images.
93.22, 93.55 and 58.51% respectively.
Patients’ age varied from six months to 70 yr. The predominant clinical presentations were fever (≥ 7 days), chills/rigor, headache, cough with expectoration, vomiting, abdominal pain and myalgia. Eschar was observed only in five patients (5.75%), and similar low to moderate/very high percentage of eschar positivity in ST patients was reported by different workers3, 7, 10. Increased serum protein (≥ 6.2 mg/dl), elevated levels of liver enzymes and thrombocytopenia (platelets ≤ 1 lakh/mm3) were observed in 68.75, 68.57 and 14.08% respectively, which is comparable to the earlier reports3–10.
ImmuneMed ST Rapid test kit is not yet available in Indian market. Our investigation was carried out with the free test kits very kindly provided by ImmuneMed, South Korea. InBios IgM and IgG ELISA, which have been evaluated by us and others2–4 were considered as refer- ence standard to compare the performance of ImmuneMed test kit. Concordance between ImmuneMed and InBios for IgM ELISA was 79.6% while for IgG antibody, it was 68.5%. OXK titre of ≥ 1:320 in acute/convalescent sera was considered suggestive of ST3–4, 7–9. However, being a non-specific test, WF can be considered as an supplementary test only and not as the sole criterion to decide ST infection status of an individual. Occurrence of several genotypes of O. tsutsugamushi makes it highly challenging to screen all suspected patients of ST by IFA.
The IFA results of 20 sera indirectly point to circulating genotypes of Karp, Kato, Gilliam and Boryong in and around Puducherry (Table 2). Reports of O. tsutsugamushi genotypes prevalent in India are very few11–12. Identifi- cation of the genotypes circulating in India and incorpo- rating local strains in IFA test kits might help in identify- ing more number of ST cases.
In conclusion, India as a whole has been included as a ST endemic region of the “tsutsugamushi triangle” mostly based on non-specific Weil-Felix test. In the recent past, many states from south, north, northeast and a few parts of western India have emerged as ST endemic foci on spe- cific serological tests like IFA/ELISA2–7. It would be ap- propriate to look for both IgM and IgG antibodies, since IgM points to recent infection but IgG might indicate chronic infection/past exposure. A combination test kit which rapidly discriminates both IgM and IgG class of immunoglobulins at the same time fulfills this criterion.
Conflict of interest
ImmuneMed Scrub Typhus Rapid test kits were pro- vided by ImmuneMed, Gangwon-do, South Korea. InBios International IgM and IgG ELISA test kits were purchased
from ICMR fund. The IFA test was performed by the Department of Microbiology and Immunology, School of Medicine, Hallym University South Korea, on the blind samples sent to them. However, the planning, execution and interpretation of results were carried out by the first author (SS) independently. There is no conflict of inter- est by any author.
ACKNOWLEDGEMENTS
First author (SS) is thankful to the Indian Council of Medical Research, New Delhi for providing research grant for this Rickettsial Project (Iris id No. 2008–08180; File No. 30/3/41/2008/ECD-II). The authors (SS and JP) wish to express their sincere gratitude to the Chairman, Vice- Chancellor, Dean (Faculty of Medicine and PG) and Dean (Research and Allied Health Sciences) of Mahatma Gandhi Medical College and Research Institute, Puducherry, for their encouragement and financial sup- port provided for this work.
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Correspondence to: Dr Selvaraj Stephen, Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Puducherry– 607 403, India.
E-mail: [email protected]
Received: 4 April 2016 Accepted in revised form: 2 June 2016
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