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  Table of Contents  
BRIEF COMMUNICATION
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 438-441
 

Phylogenetic diversity of Orientia tsutsugamushi isolates in patients with scrub typhus in Bengaluru, India


1 Department of Microbiology, Bangalore Medical College and Research Institute, Victoria Hospital, Bengaluru, Karnataka, India
2 Department of Paediatrics, Bangalore Medical College and Research Institute, Victoria Hospital, Bengaluru, Karnataka, India

Date of Submission31-Oct-2019
Date of Decision08-Nov-2019
Date of Acceptance25-Nov-2019
Date of Web Publication05-Dec-2019

Correspondence Address:
Dr. Sneha K Chunchanur
Department of Microbiology, Bangalore Medical College and Research Institute, Victoria Hospital, Fort, K. R. Road, Bengaluru - 560 002, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_267

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 ~ Abstract 


Scrub typhus has re-emerged as an important cause of acute febrile illness in India. There is a dearth of information on strain diversity of Orientia tsutsugamushi from Karnataka, India, hence the present study sought to address this issue. One hundred clinically suspected cases of scrub typhus/rickettsiosis (as per the DHR-ICMR guidelines) were included. Nested-polymerase chain reaction (PCR) for 56-kDa gene and phylogenetic analysis was performed. PCR was positive in 22 cases and phylogenetic analysis showed the presence of different strains, with predominance of clustering (57%) with Gilliam-type for the first time in Karnataka. Knowledge of genetic diversity has implications in development of diagnostics and vaccine.


Keywords: Orientia tsutsugamushi, phylogenetic analysis, polymerase chain reaction, scrub typhus


How to cite this article:
Chunchanur SK, Venugopal SJ, Ambica R, Dakshayani B. Phylogenetic diversity of Orientia tsutsugamushi isolates in patients with scrub typhus in Bengaluru, India. Indian J Med Microbiol 2019;37:438-41

How to cite this URL:
Chunchanur SK, Venugopal SJ, Ambica R, Dakshayani B. Phylogenetic diversity of Orientia tsutsugamushi isolates in patients with scrub typhus in Bengaluru, India. Indian J Med Microbiol [serial online] 2019 [cited 2020 Apr 2];37:438-41. Available from: http://www.ijmm.org/text.asp?2019/37/3/438/272347





 ~ Introduction Top


Scrub typhus is an important cause of acute febrile illness (AFI) in our country and outbreaks of scrub typhus have been reported from various states.[1] However, there is a paucity of studies with confirmed diagnosis by molecular methods and prevalent genotypes from Karnataka.[2]

Patients with scrub typhus often present with fever, headache, myalgia, malaise, rash and lymphadenopathy which are commonly seen in other AFI as well. Although the presence of an eschar can be characteristic of scrub typhus, its presence is highly variable. This makes laboratory confirmation of scrub typhus essential for diagnosis and treatment. Laboratory diagnosis can be done by serological and molecular assays.

Furthermore, clinical presentation in scrub typhus may vary depending on the type of strains prevalent in a particular geographic area. Apart from the prototype strains (Karp, Gilliam and Kato), newer recombinant strains are being reported such as one between the Karp and JP-1 types and another between Saitama and JG types.[3],[4]

Molecular methods like polymerase chain reaction (PCR) are very useful not only for early diagnosis but also for sequencing and phylogenetic analysis[5] As newer genotypes such as Kuroki, Shimokoshi, Irie, Hirano, Kawasaki, Boryong and other novel subtypes (including uncommon Ikeda like strains) continue to emerge,[3] sequencing and phylogenetic analysis has become crucial. Apart from evolving strain diversity, re-emergence of the disease has made it essential to study the prevalent genotypes in a particular geographical area and their association with clinical manifestations if any.[3],[4],[5]

Given this background, the present study sought to analyse the phylogenetic diversity of Orientia tsutsugamushi isolates in patients with scrub typhus in Bengaluru, India.


 ~ Materials and Methods Top


Prospective cross-sectional study was conducted in a tertiary care hospital in Bengaluru, Karnataka, from June 2015 to May 2017. Institutional ethical clearance (No: BMCRI/PS/415/2014-15) and written informed consent from the patients were obtained. One hundred consecutive patients presenting with AFI in whom scrub typhus (rickettsiosis) was clinically suspected (as per the DHR-ICMR guidelines 2015)[6] were enrolled, after ruling out other common causes of AFI.

EDTA blood (2–3 ml) was collected from patients for molecular diagnosis of scrub typhus by PCR. Bacterial DNA was extracted from EDTA blood, using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Nested PCR targeting the 56-kDa protein of O. tsutsugamushi was carried out on all 100 samples as reported previously.[7] Amplicons were electrophorosed using 1.5% agarose gel, stained with ethidium bromide and observed under ultraviolet transilluminator. Known-positive DNA of O. tsutusgamushi was used as positive control. The sample was considered positive when 487 bp specific band was detected.

For sequencing, amplicons (487 bp) obtained by nested PCR were purified using QIAquick PCR Purification Kit (Qiagen) as per the manufacturer's instructions. Sequencing was performed using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), followed by enumeration on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences obtained have been submitted to Genbank (Accession numbers MH508039-MH508044 and MH570187-MH570201). The sequences were edited using BioEdit Sequence alignment editor and subjected to phylogenetic tree construction. Phylogenetic analysis was carried out along with reference sequences obtained from GenBank using BLAST software (http://blast.ncbi.nlm.nih.gov). The evolutionary history was inferred by using the maximum likelihood method based on the Kimura two-parameter model.[8] Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms. Evolutionary analyses were conducted in MEGA7.[9]


 ~ Results Top


The study cohort consisted of 100 consecutive patients presenting with AFI, in whom scrub typhus (rickettsiosis) was clinically suspected and other common causes of fever were ruled out.[6]

Nested PCR for 56 kDa antigen of O. tsutsugamushi was positive in 22 cases [Figure 1]. Twenty-one selected amplicons were sequenced (one amplicon could not be sequenced due to technical reasons) and compared with O. tsutsugamushi prototype strains, as well as strains from India and neighbouring countries (retrieved from NCBI for phylogenetic tree construction) [Figure 2].
Figure 1: Nested polymerase chain reaction (positive band at 487 bp)

Click here to view
Figure 2: Phylogenetic analysis of 21 selected amplicons and comparison with O. tsutsugamushi prototype strains, as well as strains from India and neighbouring countries (retrieved from NCBI for phylogenetic tree construction). The evolutionary history was inferred by using the maximum likelihood method based on the Kimura two-parameter model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analysed. The analysis involved 49 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + non-coding. There were a total of 2224 positions in the final dataset. Evolutionary analyses were conducted in MEGA7

Click here to view


Phylogenetic and sequence analysis showed that 12 strains (57%) aligned with Gilliam prototype, 5 (24%) clustered with Kato prototype and 4 (19%) with Boryong-like isolates. Our study showed presence of O. tsutsugamushi of different prototypes/genotypes, most common (57%) being similar to Gilliam prototype. Blast analysis showed that 76% of the isolates showed 100% sequence similarity with isolates from Manipal, Karnataka, and 19% showed 99% homology with isolates from Bangladesh.


 ~ Discussion Top


Scrub typhus at present is known to be one of the predominant causes of AFI in India, with the reported prevalence of 10%–47%.[10] PCR positivity of 22% found in our study was more in comparison to another study (11.6%) from India.[11] PCR is useful for early and confirmed diagnosis, as follow-up testing is practically not feasible in clinical practice.

Sequencing and phylogenetic analysis in the present study showed predominance of Gilliam prototype for the first time in Karnataka, along with the presence of other strains. Gilliam-like strains have earlier been reported from Vellore, Shillong, Bihar and Jharkhand.[12] However, there are very few studies from Karnataka on phylogenetic analysis, and they have reported strains similar to Karp, Boryong and Ikeda but not Gilliam.[13],[14] Given this variation, there is a need for molecular studies from Karnataka on a large scale. Understanding of the circulating strains of O. tsutsugamushi in a particular geographic area is important for their inclusion in designing and assessing diagnostic assays[11] and vaccines. There have been failures of vaccine trials due to inability to protect against different prevalent strains.[15] Furthermore, short-lived immunity, lack of cross-protective immunity and variable virulence among different strains underscore the importance of strain typing in scrub typhus.[16]


 ~ Conclusion Top


Our study shows that scrub typhus is found in Karnataka and should be considered in the differential diagnosis of AFI. Knowledge of the genotype diversity of O. tsutsugamushi in a particular geographic area is important. Predominance of Gilliam strain has been reported for the first time from Karnataka in the present study.

Acknowledgement

This study was supported by Rajiv Gandhi University of Health Sciences, Bengaluru, under Advanced Research Project funding.

The authors would like to thank Dr. Harish B N and Dr. Madhan, Department of Microbiology, JIPMER, Puducherry, for providing positive DNA control for molecular work.

Financial support and sponsorship

This study was supported by Rajiv Gandhi University of Health Sciences, Bengaluru, under Advanced Research Project funding.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Peter JV, Sudarsan TI, Prakash JA, Varghese GM. Severe scrub typhus infection: Clinical features, diagnostic challenges and management. World J Crit Care Med 2015;4:244-50.  Back to cited text no. 1
    
2.
Kalal BS, Puranik P, Nagaraj S, Rego S, Shet A. Scrub typhus and spotted fever among hospitalised children in South India: Clinical profile and serological epidemiology. Indian J Med Microbiol 2016;34:293-8.  Back to cited text no. 2
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3.
Varghese GM, Janardhanan J, Trowbridge P, Peter JV, Prakash JA, Sathyendra S, et al. Scrub typhus in South India: Clinical and laboratory manifestations, genetic variability, and outcome. Int J Infect Dis 2013;17:e981-7.  Back to cited text no. 3
    
4.
Anitha PK, Hoti SL, Kanungo R, Jambulingam P, Nazeer Y, et al. Occurrence of Orientia Tsutsugamushi genotypes in areas of union territory of Puducherry and Tamil Nadu State, India. J Emerg Infect Dis 2017;2:1-7.  Back to cited text no. 4
    
5.
Kim DM, Yun NR, Neupane GP, Shin SH, Ryu SY, Yoon HJ, et al. Differences in clinical features according to Boryoung and Karp genotypes of Orientia tsutsugamushi. PLoS One 2011;6:e22731.  Back to cited text no. 5
    
6.
Rahi M, Gupte MD, Bhargava A, Varghese GM, Arora R. DHR-ICMR Guidelines for diagnosis management of Rickettsial diseases in India. Indian J Med Res 2015;141:417-22.  Back to cited text no. 6
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7.
Furuya Y, Yoshida Y, Katayama T, Yamamoto S, Kawamura A Jr. Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase chain reaction. J Clin Microbiol 1993;31:1637-40.  Back to cited text no. 7
    
8.
Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111-20.  Back to cited text no. 8
    
9.
Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870-4.  Back to cited text no. 9
    
10.
Shelke YP, Deotale VS, Maraskolhe DL. Spectrum of infections in acute febrile illness in central India. Indian J Med Microbiol 2017;35:480-4.  Back to cited text no. 10
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Kumar A, Biswal M, Zaman K, Sharma N, Suri V, Bhalla A. Genetic diversity of Orientia tsutsugamushi strains from patients in North India. Int J Infect Dis 2019;84:131-5.  Back to cited text no. 11
    
12.
Jain P, Prakash S, Tripathi PK, Chauhan A, Gupta S, Sharma U, et al. Emergence of Orientia tsutsugamushi as an important cause of acute encephalitis syndrome in India. PLoS Negl Trop Dis 2018;12:e0006346.  Back to cited text no. 12
    
13.
Gangannagari V, Beena P. Genotypes of Orientia Tsutsugamushi from patients with scrub typhus in Kolar, Karnataka. IJPHRD 2019;10:1559-62.  Back to cited text no. 13
    
14.
Koraluru M, Bairy I, Singh R, Varma M, Stenos J. Molecular confirmation of scrub typhus infection and characterization of Orientia tsutsugamushi genotype from Karnataka, India. J Vector Borne Dis 2016;53:185-7.  Back to cited text no. 14
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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. 15
    
16.
Valbuena G, Walker DH. Approaches to vaccines against Orientia tsutsugamushi. Front Cell Infect Microbiol 2012;2:170.  Back to cited text no. 16
    


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