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ORIGINAL ARTICLE  
Year : 2017  |  Volume : 60  |  Issue : 1  |  Page : 70-73
Molecular detection of Orientia tsutsugamushi from suspected scrub typhus cases


Department of Microbiology, Dr. A. L. Mudaliar PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, Tamil Nadu, India

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Date of Web Publication14-Feb-2017
 

   Abstract 

Context: Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi. The disease is under-diagnosed in India, because of low index of suspicion and also due to its nonspecific presentation, and lack of confirmatory diagnostic tests. Aims: This study was undertaken to diagnose scrub typhus in patients with undifferentiated fevers by serology and molecular methods. Materials and Methods: A total of 68 blood samples were collected from patients clinically suspected to have scrub typhus. After transportation to the laboratory, the serum was separated from the blood and subjected to rapid card test. The ethylenediaminetetraacetic acid blood samples were subjected to DNA extraction using QIAamp DNA Mini Kit followed by nested polymerase chain reaction (nPCR). Results: 24/68 (35.29%) cases showed the presence of antibody against scrub typhus by serology. 6/68 (8.8%) patients showed the presence of outer membrane protein antigen gene 56 kDa by nPCR. 5/24 serology positive cases showed the presence of 56 kDa outer membrane protein antigen gene by nPCR. A large number of cases positive by serology were negative by PCR which may indicate a low sensitivity of this test either due to low copy numbers or due to excess host DNA. Conclusion: Delay in treatment may increase disease severity and leads to higher mortality. Thus, molecular methods of diagnosis may aid in the early diagnosis of infection and enable prompt treatment. This is the first report on the diagnosis of scrub typhus in the suburbs of Chennai using molecular methods and reemphasizes the need for increased awareness of rickettsial infections in rural areas.

Keywords: Diagnosis, nested polymerase chain reaction, Orientia tsutsugamushi, rapid test, scrub typhus

How to cite this article:
Srinivasan S, Menon T. Molecular detection of Orientia tsutsugamushi from suspected scrub typhus cases. Indian J Pathol Microbiol 2017;60:70-3

How to cite this URL:
Srinivasan S, Menon T. Molecular detection of Orientia tsutsugamushi from suspected scrub typhus cases. Indian J Pathol Microbiol [serial online] 2017 [cited 2017 Oct 17];60:70-3. Available from: http://www.ijpmonline.org/text.asp?2017/60/1/70/200043



   Introduction Top


Scrub typhus is an acute febrile illness caused by the obligate intracellular organism Orientia tsutsugamushi. It is transmitted by trombiculid mites, which feed on human tissue fluid and is endemic to Southeast Asia and Northern Australia.[1] The disease is characterized by nonspecific clinical symptoms such as fever, breathlessness, myalgia, vomiting, and nausea. Organ dysfunctions such as acute renal failure, hepatitis, acute respiratory distress syndrome, meningitis, or circulatory collapse with hemorrhagic features may also occur.[2] The presence of eschar supports the diagnosis in occasional cases. Vasculitis is the main pathology which affects any part or organ of the body and has varied clinical manifestations.[3] However, scrub typhus also commonly presents as an undifferentiated fever which is difficult to diagnose. Scrub typhus is under-diagnosed in India due to its nonspecific presentations, low index of suspicion and the lack of confirmatory diagnostic tests.[4] Epidemics of scrub typhus have been reported from various parts of North, East, and South India.[2] Diagnosis of scrub typhus is difficult due to the limited access to definite laboratory tests in developing countries.[3],[5] Introduction of improved diagnostic methods would improve detection of the disease.[6] Thus, in this study, we have used nested polymerase chain reaction (nPCR) to confirm the diagnosis of scrub typhus in patients with undifferentiated fever in Chennai and its suburbs.


   Materials and Methods Top


A total of 68 blood samples were collected from three hospitals in and around Chennai from patients with undifferentiated fevers, clinically suspected to be scrub typhus. Ethics clearance was obtained from the Institutional Human Ethics Committee. Three milliliter of venous blood was collected from each of the 68 patients, of which 2 ml was in ethylenediaminetetraacetic acid (EDTA) and 1 ml in a plain tube. After transportation to the laboratory, the serum was separated from the blood sample and subjected to rapid card test (SD Bioline Tsutsugamushi test, SD Diagnostics, Korea), which is a solid phase immunochromatographic assay that detects IgG, IgM or IgA antibodies to O. tsutsugamushi.[7] The EDTA blood samples were subjected to DNA extraction using QIAamp DNA Mini Kit (Qiagen, GmBh, Germany) according to manufacturer's instructions. The purified DNA samples were stored at –20°C. The 626 bp segment gene encoding the 56 kDa protein antigen of O. tsutsugamushi was amplified by nPCR using modified protocol of Izzard et al.[8] The reaction mixture for first and second round included 2.5 µl of 10X Taq buffer, 0.8 µl of 10 mM dNTP's, 0.5 µl of 25 mM MgCl2, 0.5 µl of 10 picomoles forward and reverse primers [Table 1], 1.5 units Taq DNA polymerase, 1 µl of DNA template and volume adjusted to 25 µl with Milli-Q water. The cycling conditions standardized for the first round of nested nPCR was 95°C for 3 min, followed by 30 cycles of 94°C for 30 s, 55°C for 1.5 min, 72°C for 2 min, and a final extension at 72°C for 5 min. First round products (~800 bp) were considered template for second round with initial denaturation at 95°C for 3 min, followed by 25 cycles of 94°C for 30 s, 57°C for 1 min, 72°C for 1 min, and final extension at 72°C for 5 min. PCR products were analyzed by electrophoresis with 2% agarose gel in 1X Tris-borate EDTA buffer. The PCR products were visualized using the gel documentation system (Carestream Gel Logic 212 Pro, USA) and amplification of 626 bp was considered positive in the second round PCR.
Table 1: Primer sets used in the present study

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   Results Top


A total of 68 patients with undiagnosed fever were included in the study and 24/68 (35.29%) cases showed the presence of antibody against scrub typhus by serology. Six of the 68 (8.8%) patients showed the presence of outer membrane protein antigen gene 56 kDa by nPCR. Two of these six patients showed the presence of eschar. Majority of the samples (39/68, 57.35%) were females. Most of the cases were seen from October 2014 to January 2015.

The common symptoms noted were fever (68/68, 100%), myalgia (20/68, 29.41%), vomiting (8/68, 11.76%), abdominal pain (10/68, 14.71%), chills (11/68, 16.17%), rigors, and headache (9/68, 13.23%) cases. Other findings were thrombocytopenia (6/68, 8.82%), lymphadenopathy (4/68, 5.88%), eschar formation (8/68, 11.76%), diabetic ketoacidosis (1/68, 1.47%), hepatosplenomegaly (1/68, 1.47%), acute appendicitis (5/68, 7.35%), diabetes mellitus (5/68, 7.35%), ARDS (2/68, 2.94%), and jaundice (2/68, 2.94%) cases [Table 2].
Table 2: Clinical features of the patients included in the present study

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Five of 6 nPCR positive samples were from female patients and were distributed across all age groups [Table 3]. Out of 24 serology positive cases, 12 were males and 12 were females. The majority of cases were in the age group 11–20 years and 30–50 years [Table 4]. Nineteen of the 24 cases positive by serology failed to amplify outer membrane protein antigen gene (56 kDa) by nPCR. One patient who was negative by serology was positive by nPCR [Figure 1]. Five of the 24 serology positive cases showed the presence of 56 kDa outer membrane protein antigen gene by nPCR by amplifying a segment of 626 bp [Figure 2].
Table 3: Age and sex distribution of polymerase chain reaction positive cases

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Table 4: Age and sex distribution of serology positive cases

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Figure 1: Bar diagram shows cases positive by serology and nested polymerase chain reaction

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Figure 2: Representative gel picture of nested polymerase chain reaction targeting 56 kDa protein antigen gene, Lane M: 100 bp DNA ladder, Lane 1: First round negative control, Lane 2: Second round negative control, Lane 3: Sample OTS66, Lane 4: Positive control (Karp)

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   Discussion Top


Scrub typhus has been found to account for almost 50% of cases of undifferentiated febrile illness occurring in many parts of India, especially during cooler months.[3] Previous studies have reported the occurrence of scrub typhus in several states of India such as Tamil Nadu, Andhra Pradesh, Puducherry, Uttarakhand, and Shimla.[9],[10],[11],[12],[13]

Although serology remains the mainstay for diagnosing scrub typhus, it has certain limitations. The oldest test, the Weil–Felix test, is easy to perform but lacks sensitivity. The gold standard immunofluorescence assay (IFA) requires a fluorescence microscope and commercially available kits have antigens only for the prototype strains such as Gilliam, Karp, Kato, and Boryong, and hence cannot detect new strains. In addition, the interpretation of IFA test requires technical expertise. The culturing of the organism O. tsutsugamushi is a cumbersome procedure and requires not only experienced personnel but also biosafety Level III facility which is unavailable in most of the laboratories.[14] The serological tests may fail to diagnose scrub typhus in the early stages of the disease due to low antibody levels.[15] Wahid et al.[7] in 2014 had reported approximately 99% and 96% sensitivity and specificity, respectively, of antibody screening by rapid card test for diagnosis of scrub typhus, and emphasized that the rapid card test should be interpreted in combination with other diagnostic procedures and clinical findings. In the present study, the rapid card test was performed as a screening test whereas PCR targeting the 56 kDa gene was used as a confirmatory test. The 56 kDa gene is a type specific protein gene, which is unique to O. tsutsugamushi. This protein gene has four hypervariable domains which are responsible for antigenic variation.[16] It is a major outer membrane protein, containing both group-specific and type-specific epitopes and being located on the surface of the organism is involved in penetration and entry into the host cells.[17] Many recent studies in India [4],[18] and from other parts of the world [7],[19] demonstrated the usefulness of nPCR targeting 56 kDa type specific gene specific for O. tsutsugamushi.

In the present study, it is noted that the positive patients were located from the suburbs of Chennai and Villupuram districts. Fever was the most common symptom in our group of patients which is similar to a previous report.[20] Narvencar et al.[2] in 2012 reported fever with myalgia as the predominant symptom in 80% of the patients, but in the present study, myalgia was observed in only twenty cases (29.41%). Although eschar formation is a useful diagnostic tool for scrub typhus, we observed it in only 11.76% of patients (8/68). Only one patient was serology and PCR positive and showed eschar formation. Occasionally, the formation of eschar may go unnoticed in dark-skinned persons.[18] PCR of eschar material is considered more sensitive than blood but may not be useful in a setting where eschar formation itself is not common.[21] A large number of cases positive by serology were negative by PCR which may indicate a low sensitivity of this test either due to low copy numbers or due to excess host DNA.[22]


   Conclusion Top


Molecular diagnosis may aid in early diagnosis of infection and enable prompt treatment. This is the first study on diagnosis of scrub typhus in the suburbs of Chennai using molecular methods and reemphasizes the need for increased awareness of rickettsial infections in rural areas.

Acknowledgments

We acknowledge Dr. Daniel H. Paris, Head of Rickettsiology, MORU, Thailand who provided us the standard strains of Orientia tsutsugamushi. We also acknowledge Dr. Boopathy Raja, Dr. Esther Mary and Dr. Shankar P for providing the clinical samples used in this study.

Financial support and sponsorship

Indian Council of Medical Research (ICMR)

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Ha NY, Kim Y, Choi JH, Choi MS, Kim IS, Kim YS, et al. Detection of antibodies against Orientia tsutsugamushi Sca proteins in scrub typhus patients and genetic variation of sca genes of different strains. Clin Vaccine Immunol 2012;19:1442-51.  Back to cited text no. 1
    
2.
Narvencar KP, Rodrigues S, Nevrekar RP, Dias L, Dias A, Vaz M, et al. Scrub typhus in patients reporting with acute febrile illness at a tertiary health care institution in Goa. Indian J Med Res 2012;136:1020-4.  Back to cited text no. 2
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3.
Mathai E, Rolain JM, Verghese GM, Abraham OC, Mathai D, Mathai M, et al. Outbreak of scrub typhus in Southern India during the cooler months. Ann N Y Acad Sci 2003;990:359-64.  Back to cited text no. 3
    
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Sinha P, Gupta S, Dawra R, Rijhawan P. Recent outbreak of scrub typhus in North Western part of India. Indian J Med Microbiol 2014;32:247-50.  Back to cited text no. 4
[PUBMED]  Medknow Journal  
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Wahid A, Meena SR, Saluja M, Jain G, Pahadia M, Puneeth UM. Epidemic of scrub typhus in Haroti region of Rajasthan. Indian Med Gaz 2014;435-40.  Back to cited text no. 7
    
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Izzard L, Fuller A, Blacksell SD, Paris DH, Richards AL, Aukkanit N, et al. Isolation of a novel Orientia species (O. chuto sp. nov.) from a patient infected in Dubai. J Clin Microbiol 2010;48:4404-9.  Back to cited text no. 8
    
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Bharathi SL, Jayachandran S, Senthil N, Sujatha S. Scrub typhus causing myocarditis and ARDS: A case report. Heart India 2013;1:85-6.  Back to cited text no. 9
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Subbalaxmi MV, Madisetty MK, Prasad AK, Teja VD, Swaroopa K, Chandra N, et al. Outbreak of scrub typhus in Andhra Pradesh – Experience at a tertiary care hospital. J Assoc Physicians India 2014;62:490-6.  Back to cited text no. 10
    
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Viswanathan S, Muthu V, Iqbal N, Remalayam B, George T. Scrub typhus meningitis in South India – A retrospective study. PLoS One 2013;8:e66595.  Back to cited text no. 11
    
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Singh SP, Singh R, Ahmad N. A study of complications of scrub typhus in a tertiary health care institute of Uttarakhand, India. Int J Res Med Sci 2014;2:246-9.  Back to cited text no. 12
    
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Lal B, Minhas RS, Sharma DR, Kumar S. Scrub typhus presenting as acute mastoiditis. Online J Health Allied Sci 2013;12:14.  Back to cited text no. 13
    
14.
Roopa KS, Karthika K, Harish BN. Molecular diagnosis of scrub typhus: A preliminary report from Pondicherry. Int J Microbiol Res Rev 2015;4:158-60.  Back to cited text no. 14
    
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Huber E, Ji D, Howell L, Zhang Z, Chen HW, Ching WM, et al. Loop-mediated isothermal amplification assay targeting the 47-Kda gene of Orientia tsutsugamushi: A rapid and sensitive alternation to real-time PCR. J Med Microbiol Diagn 2012;1:112.  Back to cited text no. 15
    
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Varghese GM, Janardhanan J, Mahajan SK, Tariang D, Trowbridge P, Prakash JA, et al. Molecular epidemiology and genetic diversity of Orientia tsutsugamushi from patients with scrub typhus in 3 regions of India. Emerg Infect Dis 2015;21:64-9.  Back to cited text no. 16
    
17.
Lee YM, Kim DM, Lee SH, Jang MS, Neupane GP. Phylogenetic analysis of the 56 kDa protein genes of Orientia tsutsugamushi in Southwest Area of Korea. Am J Trop Med Hyg 2011;84:250-4.  Back to cited text no. 17
    
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Kumar V, Kumar V, Yadav AK, Iyengar S, Bhalla A, Sharma N, et al. Scrub typhus is an under-recognized cause of acute febrile illness with acute kidney injury in India. PLoS Negl Trop Dis 2014;8:e2605.  Back to cited text no. 18
    
19.
Ruang-Areerate T, Jeamwattanalert P, Rodkvamtook W, Richards AL, Sunyakumthorn P, Gaywee J. Genotype diversity and distribution of Orientia tsutsugamushi causing scrub typhus in Thailand. J Clin Microbiol 2011;49:2584-9.  Back to cited text no. 19
    
20.
Chrispal A, Boorugu H, Gopinath KG, Prakash JA, Chandy S, Abraham OC, et al. Scrub typhus: An unrecognized threat in South India – Clinical profile and predictors of mortality. Trop Doct 2010;40:129-33.  Back to cited text no. 20
    
21.
Koh GC, Maude RJ, Paris DH, Newton PN, Blacksell SD. Diagnosis of scrub typhus. Am J Trop Med Hyg 2010;82:368-70.  Back to cited text no. 21
    
22.
Sonthayanon P, Chierakul W, Wuthiekanun V, Blacksell SD, Pimda K, Suputtamongkol Y, et al. Rapid diagnosis of scrub typhus in rural Thailand using polymerase chain reaction. Am J Trop Med Hyg 2006;75:1099-102.  Back to cited text no. 22
    

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Correspondence Address:
Thangam Menon
Department of Microbiology, Dr. A. L. Mudaliar PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.200043

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