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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
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BRIEF COMMUNICATION
Year : 2018  |  Volume : 36  |  Issue : 3  |  Page : 422-425
 

Use of eschar for the molecular diagnosis and genotypic characterisation of Orientia tsutsugamushi causing scrub typhus


1 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Medical Gastroenterology, Trivandrum Medical College and Hospital, Trivandrum, Kerala, India
4 Department of Paediatric Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Web Publication14-Nov-2018

Correspondence Address:
Dr. Manisha Biswal
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_8

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 ~ Abstract 

Scrub typhus caused by Orientia tsutsugamushi presents as an acute febrile illness with a varied presentation from mild illness to fatal disease in the absence of appropriate antibiotic treatment. Performing polymerase chain reaction (PCR) on eschar sample acts a rapid diagnostic tool in the early stage of scrub typhus when blood is negative. A total of eight patients from whom both whole blood and eschar samples were collected and tested by nested PCR targeting 56 kDa trichostatin A (TSA) gene to detect O. tsutsugamushi DNA. All (100%) eschar samples and three whole blood samples tested positive. Genetic analysis of the 56 kDa TSA gene sequences showed that the majority were related to Karp reference strains, while one clustered with Kawasaki strain. When present, eschar should be favoured as a diagnostic sample over whole blood in the early phase of infection.


Keywords: Diagnosis, eschar, febrile illness, genetic diversity, India, Orientia tsutsugamushi, polymerase chain reaction, scrub typhus


How to cite this article:
Biswal M, Zaman K, Suri V, Rao H, Kumar A, Kapur G, Sharma N, Bhalla A, Jayashree M. Use of eschar for the molecular diagnosis and genotypic characterisation of Orientia tsutsugamushi causing scrub typhus. Indian J Med Microbiol 2018;36:422-5

How to cite this URL:
Biswal M, Zaman K, Suri V, Rao H, Kumar A, Kapur G, Sharma N, Bhalla A, Jayashree M. Use of eschar for the molecular diagnosis and genotypic characterisation of Orientia tsutsugamushi causing scrub typhus. Indian J Med Microbiol [serial online] 2018 [cited 2019 Nov 11];36:422-5. Available from: http://www.ijmm.org/text.asp?2018/36/3/422/245399



 ~ Introduction Top


Scrub typhus, caused by the bacterium Orientia tsutsugamushi, has re-emerged in India with multiple regions in India reporting outbreaks in the past 2 years.[1],[2],[3] The clinical features can vary from mild acute febrile illness to severe, life-threatening illness.[2],[3],[4] In India, the diagnosis of scrub typhus is made serologically by the non-specific Weil Felix (OXK, Ox 2 and Ox 19 antigens, Plasmatec, CA, USA) test or by a commercially available ELISA (Scrub Typhus Detect Immunoglobulin M (IgM) ELISA System, InB ios International, Inc., Seattle, WA, USA), or in few centres by immunofluorescent assay and polymerase chain reaction (PCR).[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14] While the most common sample is blood (whole or buffy coat) for PCR analyses, eschar which is the pathognomonic sign of scrub typhus, is a much less common sample.[8],[15],[16],[17] Here, in this study, we describe eight patients of scrub typhus where the eschar sample was positive for O. tsutsugamushi DNA and the genetic characterisation enhancing the knowledge of the existing diversity among the circulating strains of O. tsutsugamushi in India.


 ~ Materials and Methods Top


Whole blood and eschar were collected from the eight patients for PCR and serum for IgM ELISA. ELISA for IgM antibody was carried out using Scrub Typhus Detect IgM ELISA (InBios International, Inc., Seattle, WA, USA) following the manufacturer's instructions, an optical density of (OD) >0.5 was considered positive. The total genomic DNA was extracted from the whole blood (1 ml) and homogenised eschar sample by the phenol–chloroform method.[8] Nested PCR assay was performed according to the method that Furuya et al. used.[18] The nucleotide primers were based on the nucleotide sequence encoding the 56-kDa major wall protein antigen of the O. tsutsugamushi Gilliam species.[17],[18] The primers 34 (5'-TCAAGCTTATTGCTAGTGCAATGTCTGC-3') and 55 (5'-AGGGATCCCTGCTGC TGTG CTTGCTGCG-3') were used for the first PCR, and the nested PCR primers 10 (5'-GATCAAGCTTCCTCAGCCTACTATAATGCC-3') and 11 (5'-CTAGGGATCCCGA CAGATGCACTATTAGGC-3') were used for the second PCR.[18] The amplification reaction was carried out in a 25-μl volume containing PCR buffer (10 mM Tris, 50 mM NaCl, 10 mM MgCl2, 200 μg of bovine serum albumin per ml), 0.5 mM of deoxynucleotide triphosphate, 100 pM forward and reverse primers, and 0.3 U Taq polymerase (Thermo Scientific, Waltham, MA, USA). To monitor for contamination, controls were used in each set of reaction. Amplification consisted of initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 1 min and extension at 70°C for 1 min was used. For the second PCR, the amplicons (5 μl) obtained from the first PCR was used as the template with the second set of primers and the same amplification protocol. The amplified products of both amplification rounds were visualised under ultraviolet transilluminator following electrophoretic separation (2% agarose gel). Sequencing PCR was performed using the BigDye Terminator Cycle sequencing ready reaction kit, version 3.1 (Applied Biosystems, Foster City, CA).

The sequences obtained were identified by comparison with sequences available in GenBank by using BLAST (http://blast.ncbi.nlm.nih.gov). These sequences were submitted to GenBank (accession nos. MF457888-MF457895) and molecular evolutionary analyses were conducted using MEGA version 7.[19] The study sequences and reference sequences downloaded from GenBank were aligned by using Clustalx 2 (http://www.clustal.org) and trimmed to the appropriate size. A phylogenetic tree with 1,000 bootstrap replications was constructed using the Neighbor-Joining method.[20],[21] The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site.

Ethical statement: The study was carried out on patient samples after obtaining written informed consent, and the study was approved by Institute Ethical Committee.


 ~ Results Top


The DNA extracted from eschar varied from 44.7 to 323 ng/μl and that from blood from 50.5 to 695.4 ng/μl. The mean value for purity at 280/260 ratio absorbance was 1.81 for eschar and 1.72 for blood. The PCR from eschar sample was positive in all eight (100%) patients, while blood samples in only three (37.5%) patients were positive. In seven patients, anti-scrub typhus therapy had already been initiated at the time of sampling. Six patients had already been started on doxycycline and one patient had received azithromycin. In one patient, the sample was collected before doxycycline treatment was initiated. The IgM ELISA for antibodies against O. tsutsugamushi was positive in five patients, negative in two and was not performed in one patient. The clinical features, investigations performed and the final outcome is listed in [Table 1].
Table 1: Clinical features, investigations performed and final outcome of patients in the present studys

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The PCR amplicons from eschar samples were sequenced and analysed to correlate the phylogenetic association. Six of the sequences gave good sequences and were found to have high similarity to O. tsutsugamushi Karp-like strains (MF45789-MF457891, MF457894/95) and one clustered close to a Kawasaki-like strain (MF457892). The eschar sequences from a study from the south of India show similar circulating strains from an eschar sample [Figure 1].[10]
Figure 1: Phylogenetic tree inferred using the Neighbor-Joining method with 1000 replicate bootstrap test and the evolutionary distances were computed using the maximum composite likelihood method

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 ~ Discussion Top


The eschar is the pathognomonic lesion associated with scrub typhus. It is the initial lesion which develops at the site of chigger (Leptotrombidium mite) bite. Eschar has been shown to be a better sample compared to blood for PCR assay because eschar is the area where O. tsutsugamushi is proliferating, and a large number of organisms are present there. Furthermore, eschar can be readily collected. Eschar can remain positive for up to 1 week even after appropriate therapy is initiated.[16],[22] The low load of the organism in blood, amount of blood used for extraction, the presence of PCR inhibitors such as heme and bacterial clearance by antibiotic therapy and/or immune response against Orientia can possibly explain the reduced sensitivity of PCR assay from blood samples.[8],[23] Similar findings, we noted in our study, were the PCR from blood was negative in five samples while the eschar samples were positive for O. tsutsugamushi DNA.

The nested PCR targeting 56kDA type-specific antigen has better sensitivity then the conventional PCR, thereby nested PCR was used in the present study.[6] The sensitivity of nested PCR from eschar samples has been reported to be 86.5% (83/96) with a specificity of 100% which is as high as that reported from buffy coat samples (82.2%).[16],[22] In a recent study from Vietnam, swab collected from the base of eschar has also been used to detect O. tsutsugamushi DNA. Among the 20 patients with clinically suspected scrub typhus in whom both eschar and whole blood were available, positivity from eschar was much higher compared to blood (17 vs. 5).[24] This method has been shown to be far less invasive than skin biopsy and does not require much expertise making it a very convenient sample to collect.

In the present study, Karp like strains was reported the maximum (5 eschar samples) followed by Kawasaki strain (1 eschar sample). Karp like strains is the most prevalent genotype in Southeast Asian countries. In a study from India to study the genotypic diversity of O. tsutsugamushi in India in three different geographical regions (Shimla, Shillong and Vellore) based on the eschar sequences, Karp-like strains was reported to be in higher proportion compared to Kato-like strains from Shimla in comparison to Vellore and Shillong.[9] Similar findings were noted in our study. Studies from Sri Lanka and Vietnam also report a predominance of Karp-like strains.[24],[25] Varghese et al. reported that Kato-like strains predominate in (77.5%) in South India.[9] We did not find a single Kato-like strain in our eschar samples. The Kawasaki strain in our study showed similarity with the Kawasaki strain from Vietnam and the Kawasaki reference strain (GQ332755).[24]

One of the main limitations of using eschar as a diagnostic specimen for scrub typhus is that this pathognomonic sign does not present in all patients. Eschar has been reported to be present in 46% and 60% of South Vietnamese and Taiwanese patients, respectively.[17],[18] In a study conducted in north Indian patients in our hospital, it was found that the eschar was found in 14% patients and was mainly confined to the trunk, inguinal, genital and axially areas of the patients.[4] Second, an eschar is typically painless and non-pruritic, and hence its presence is not reported by patients. The eschar can also be missed in a cursory physical examination, especially in dark-skinned patients or mistakenly reported to be due to trauma, etc.[18],[19] Therefore, a thorough physical examination is warranted. In above all cases, buffy coat is probably a more practical and suitable alternative for improving detection by nested PCR.[26]

It is important to study regional antigenic diversity in O. tsutsugamushi as this information has implications for the success of diagnostic immunoassays and vaccines against scrub typhus. Genetic analysis of the 56 kDa type-specific antigen showed that the majority clustered close to Karp reference strains, while one was similar to the Kawasaki reference strain. Our paper adds to the evidence of the genotypic diversity of this organism in India and further emphasise the importance of eschar as a diagnostic sample.


 ~ Conclusion Top


Eschar should be favoured as a diagnostic sample over whole blood in the early phase of infection. The knowledge about the existing antigenic diversity in a geographical region helps in establishing an effective diagnostic assays and vaccines against scrub typhus.

Acknowledgements

We would like to acknowledge Mrs. Shashi Vig and Mr. Shamanth Adekhandi for their assistance in the molecular work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Sethi S, Prasad A, Biswal M, Hallur VK, Mewara A, Gupta N, et al. Outbreak of scrub typhus in North India: A re-emerging epidemic. Trop Doct 2014;44:156-9.  Back to cited text no. 1
    
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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. 2
    
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Varghese GM, Trowbridge P, Janardhanan J, Thomas K, Peter JV, Mathews P, et al. Clinical profile and improving mortality trend of scrub typhus in South India. Int J Infect Dis 2014;23:39-43.  Back to cited text no. 3
    
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Stephen S, Sangeetha B, Ambroise S, Sarangapani K, Gunasekaran D, Hanifah M, et al. Outbreak of scrub typhus in Puducherry and Tamil Nadu during cooler months. Indian J Med Res 2015;142:591-7.  Back to cited text no. 5
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Janardhanan J, Prakash JA, Abraham OC, Varghese GM. Comparison of a conventional and nested PCR for diagnostic confirmation and genotyping of Orientia Tsutsugamushi. Diagn Microbiol Infect Dis 2014;79:7-9.  Back to cited text no. 6
    
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Prakash JA, Kavitha ML, Mathai E. Nested polymerase chain reaction on blood clots for gene encoding 56 kDa antigen and serology for the diagnosis of scrub typhus. Indian J Med Microbiol 2011;29:47-50.  Back to cited text no. 8
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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. 9
    
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Mahajan SK, Raina R, Singh B, Singh DV, Kanga A, Sharma A, et al. Pattern of clinical presentation, laboratory findings and mortality risk among patients of scrub typhus in Western Himalayas. J Assoc Physicians India 2016;64:26-30.  Back to cited text no. 12
    
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Blacksell SD, Tanganuchitcharnchai A, Nawtaisong P, Kantipong P, Laongnualpanich A, Day NP, et al. Diagnostic accuracy of the inBios scrub typhus detect enzyme-linked immunoassay for the detection of IgM antibodies in Northern Thailand. Clin Vaccine Immunol 2016;23:148-54.  Back to cited text no. 13
    
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Lim C, Blacksell SD, Laongnualpanich A, Kantipong P, Day NP, Paris DH, et al. Optimal cutoff titers for indirect immunofluorescence assay for diagnosis of scrub typhus. J Clin Microbiol 2015;53:3663-6.  Back to cited text no. 14
    
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Kim DM, Park G, Kim HS, Lee JY, Neupane GP, Graves S, et al. Comparison of conventional, nested and real-time quantitative PCR for diagnosis of scrub typhus. J Clin Microbiol 2011;49:607-12.  Back to cited text no. 15
    
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Kim DM, Kim HL, Park CY, Yang TY, Lee JH, Yang JT, et al. Clinical usefulness of eschar polymerase chain reaction for the diagnosis of scrub typhus: A prospective study. Clin Infect Dis 2006;43:1296-300.  Back to cited text no. 16
    
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