• Users Online: 376
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 
Table of Contents
RESEARCH ARTICLE
Year : 2020  |  Volume : 57  |  Issue : 2  |  Page : 147-152

Scrub typhus in Puducherry, India: Application of nested PCR targeting three different genes – 56 kDa, 47 kDa and groEL of Orientia tsutsugamushi and comparison with ST IgM ELISA


1 Department of Microbiology, Mahatma Gandhi Medical College & Research Institute, Sri Balaji Vidyapeeth, Puducherry, India
2 Department of Genomics and Proteomics, Central Interdisciplinary Research Facility, Mahatma Gandhi Medical College and Research Institute, Puducherry – 607 403, India

Date of Submission31-May-2018
Date of Acceptance08-Jan-2019
Date of Web Publication14-Jul-2021

Correspondence Address:
Dr Selvaraj Stephen
Professor of Microbiology, Mahatma Gandhi Medical College & Research Institute, Sri Balaji Vidyapeeth, Pondy-Cuddalore Main road, Pillaiyarkuppam, Puducherry
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.310866

Rights and Permissions
  Abstract 

Background & objectives: Scrub typhus (ST), an important zoonosis caused by Orientia tsutsugamushi, is now prevalent throughout India. While demonstration of IgM antibody by Indirect Immunofluorescence Assay (IFA) is the gold standard serological test, IgM ELISA is an alternative. Demonstration of O. tsutsugamushi DNA in the blood or eschar confirms infection in the early febrile period.
Methods: Scrub typhus nested PCR (n-PCR) for 56 kDa, 47 kDa and groEL genes and ST IgM ELISA were performed for 210 clinically suspected ST patients. As healthy controls, 70 voluntary blood donors were included. Statistical analysis was performed for laboratory parameters using Fisher exact test/chi-square test. Ninety-five PCR products of n-PCR positive samples were purified and submitted for gene sequencing.
Results: PCR was positive for one or more gene targets in 75.71% of IgM ELISA positive patients and 10% of antibody negative patients. All voluntary blood donors were negative for both antibodies and DNA. Gene sequences of 95 n-PCR positive products confirmed the presence of Orientia tsutsugamushi DNA in the samples and NCBI database accession numbers MG601875 to MG601969 were obtained.
Interpretation & conclusion: Compared to IgM ELISA, sensitivity of three PCRs was 30, 51.43 and 61.43% for 56 kDa, 47 kDa and groEL targets, respectively. Since IgM ELISA positivity can persist up to one year, PCR confirms ST diagnosis in the acute phase of the illness, in the presence of IgM and even before IgM appears. Inclusion of all three genes – 56 kDa, 47 kDa and groEL, instead of a single 56 kDa target, identifies and confirms maximum number of ST patients.

Keywords: Nested PCR; Puducherry; scrub typhus; ST ELISA IgM


How to cite this article:
Anitharaj V, Stephen S, Pratheesh P. Scrub typhus in Puducherry, India: Application of nested PCR targeting three different genes – 56 kDa, 47 kDa and groEL of Orientia tsutsugamushi and comparison with ST IgM ELISA. J Vector Borne Dis 2020;57:147-52

How to cite this URL:
Anitharaj V, Stephen S, Pratheesh P. Scrub typhus in Puducherry, India: Application of nested PCR targeting three different genes – 56 kDa, 47 kDa and groEL of Orientia tsutsugamushi and comparison with ST IgM ELISA. J Vector Borne Dis [serial online] 2020 [cited 2021 Aug 5];57:147-52. Available from: https://www.jvbd.org/text.asp?2020/57/2/147/310866


  Introduction Top


Scrub typhus (ST) caused by Orientia tsutsugamushi is a zoonotic infection, transmitted by the larval stage of a mite known as chiggers. According to Watt and Parola[1], every year one million cases of ST are reported throughout the world. Isaac et al[2] observed that in India, scrub typhus accounts for 50% of undifferentiated febrile cases. The Orientia genus itself is highly variable which is the basis for strain differentiation. For serological diagnosis, follow-up visit is needed to collect convalescent blood sample. Serological tests include Passive Hemagglutination Assay (PHA), Immunofluroescence Assay (IFA), Immunoperoxidase Assay (IPA) or Enzyme-Linked ImmunoSorbent Assay (ELISA)[3]. The antibodies may not have appeared in the early days of acute illness, thus necessitating the paired sera testing. Several weeks are required to identify O. tsutsugamushi in cultured cells, which also requires Biosafety level 3 containment facilities and can be performed only in Rickettsial research laboratories. So, a simple, specific and rapid diagnostic test is needed. PCR assay has proven to be very useful in the early diagnosis of ST. Saisongkorh et al[4] observed that nested PCR (n-PCR) is more sensitive than the serological tests. One or more among the four common gene targets are employed by different rickettsiologists-16SrRNA, 56 kDa, 47k Da and groEL genes[3],[5],[6],[7]. In this study, we have used nested PCR, targeting three different genes, 56 kDa, 47 kDa and groEL as recommended by Paris et al[8], in their Scrub Typhus Inclusion Criteria (STIC).


  Material & Methods Top


Ethical statement

This study was approved by our Institute Human Ethics Committee (IHEC). Archived serum samples from suspected cases of ST patients who presented themselves at Mahatma Gandhi Medical College and Research Institute, Puducherry, India, a tertiary care teaching hospital during November 2012 to March 2017 and for whom ST IgM ELISA was done were included. The work was carried out at the Department of Microbiology and Department of Genomics and Proteomics, Central Interdisciplinary Research Facility, Mahatma Gandhi Medical College and Research Institute, Puducherry, India. The patients were enrolled based on inclusion and exclusion criteria as described by us earlier[9]. Only anonymised samples were included in this research, by way of excluding the identity and personal details of the patients.

Statistical analysis

Based on the 85% sensitivity of the antibody tests, sample size (n) was calculated using the formula:



P = Sensitivity of the antibody tests

Q = 1-p

d2=Margin of error at 6%.

The sample size was therefore 136 and it was rounded up to 140. The samples included: 140 antibody positive samples and 70 antibody negative samples from febrile patients with clinical suspicion of ST. Additionally, 70 healthy voluntary blood donors were included as controls. Thus, a total of 280 serum samples were included in the study.

After obtaining written informed consent, 7mL of venous blood was collected from each of the 280 volunteers (2mL in plain tube + 5mL in EDTA tube). Buffy coat was prepared from EDTA treated samples and it was collected between the upper plasma layer and lower erythrocyte layer after centrifugation of 3000 rpm for 15 min. Serum/ buffy coat was separated and stored at -20°C for further use.

DNA extraction

DNA was extracted from 200μL of buffy coat sample using a QIA amp DNA Blood mini kit (Qiagen, Hilden, Germany) and the final eluted volume was stored at -80°C. Primers were purchased from Sigma-Aldrich, Chennai, India.

ST IgM ELISA

Clinically suspected scrub Typhus positive cases were confirmed by IgM ELISA test (Scrub Detect TM IgM ELISA, In Bios International, Seattle, USA). O. tsutsugamushi antigen is incorporated in the ELISA wells. The test was carried out strictly in compliance with the procedure outlined in the technical brochure and as reported earlier[9]. OD values above 0.5 were taken as positive[10].

ST nested PCR

Molecular diagnosis was made by performing three nested PCRs targeting 56 kDa, 47 kDa, and groEL gene respectively with 140 ST IgM antibody positive, 70 ST IgM antibody negative febrile patients with clinical suspicion of ST and 70 healthy blood donors.

56 kDa nested PCR

The primers used were as reported by Furuya et al[6]: Outer set of primers p34 (5’-TCAAGCTTATTGCTAGT-GCAATGTCTGC-3’), p 55 (5’-AGGGATCCCTGCT-GCTGTGCTT GCTGCG-3’) and inner set of primers were p10 (5’- GATCAAGCTlTCCTCAGCCTACTATA-ATGCC-3’), p11 (5’-CTAGGGATCCCGACAGATG-CACT ATTAGGC-3’). The first round PCR amplification mixture contained:

Taq amplicon PCR master mix - 12.5μL, primers (p34 and p55)-2μL, milli q water - 8.5μL, template DNA-2μL. (final volume of 25μL).

For the second round PCR, it contained all the same ingredients as in the first PCR except the primers p10 and p11 were substituted by primers p34 and p55 and the template DNA was 2μL from the first PCR amplified product. The reaction was carried out in a Thermal Cycler ABI veriti PCR (Applied Biosystems, USA). Cycling conditions were same for both PCRs and the amplification of 35 cycles consisted of; Denaturation: 95°C for 30 sec, Annealing: 60°C for 40 sec, Extension: 72°C for 30 sec and the final extension step at 72°C for 5 min. Finally, the PCR products were electrophoresed in 1.5% agarose gel and the band at 483bp was visualised in UV transilluminator.

47 kDa Nested PCR

ST Nested PCR for 47 kDa was performed as per Kim et al[3], with slight modification: Outer primers otsu 555 (5’-TCCTTTCGGTTTAAGAGGAACA-3’), otsu 771 (5’-GCATTCAACTGCTTCAAGTACA-3’) and inner set of primers: otsu 630 (5’-AACTGATTTTATTCAAC-TAATGCTGCT-3’), otsu 747 (5’- TATGCCTGAG-TAAGATACRTGAATRGAATT-3’) were the inner set of primers used in the second round PCR to amplify the 364bp sequence from the DNA of scrub typhus infected patients.

The first PCR amplification mixture contains a final volume of 20μL, Amplicon master mix: 10μL, Primers: 2μL, Template DNA: 2μL, milli q water: 6μL. Except primers, all the other ingredients were same as in first PCR and the DNA template from the first product was used for the sec. Cycling conditions for 1st round PCR (30 cycles) Initial denaturation– 94°C for 10 mts, Denaturation – 94°C for 1 mt, Annealing – 56°C for 1 mt, Extension – 72°C for 1 mt and the final extension 72°C for 7 mts. Cycling conditions for IInd round PCR (25 cycles): Initial denaturation – 94°C for 10 mts, Denaturation – 94°C for 30 sec, Annealing – 60°C for 30 sec, Extension – 72°C for 1 mt and the final extension 72°C for 7 mts. Finally, the band at 118bp was visualised by transilluminator.

groEL gene Nested PCR

ST Nested PCR targeting groEL gene was performed as per the method of Li et al[7]. Gro-1, 5’-AAGAAGGA/ CGTGATAAC-3’ and Gro-2, 5’-ACTTCA/CGTAG-CACC-3’ were the outer set of primers used in the first PCR and TF1, 5′-ATATATCACAGTACTTTGCAAC-3′ and TR2, 5′-TTCCTAACTTAGATGTATCAT-3′ were the inner set of primers used in the second round PCR to amplify the 364bp sequence from the DNA of scrub typhus infected patients.

The first round of PCR reactions was carried out under the following conditions; Initial denaturation at 94°C for 40 sec followed by 35 cycles, denaturation at 94°C for 40 sec, annealing at 38°C for 40 sec, extension 72 °C for 40 sec and the final extension at 72°C for 4 min. And for the second round PCR, it contained all the ingredients as in first PCR except that the primers and the annealing temperature (52°C) were changed to amplify the 364bp sequence from the DNA of scrub typhus infected patients. N-PCR positive samples were purified using QIAquick PCR Purification kit (Qiagen, Germany). Gene sequencing of PCR products were done by Eurofins Genomics India Pvt. Ltd, Bangalore, India.

Statistical analysis

Mean and Standard deviation with 95% confidence interval for days of febrile illness and age of patients was calculated using online calculator. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) between n-PCR and IgM ELISA was calculated using GraphPad QuickCalcs (GraphPad Software Inc, USA).


  Results Top


Among 140 ST IgM ELISA positive patients, 106 were positive (75.71%) and among 70 antibody negative patients, 7 were positive (10%) in PCR targeting 56 kDa/47 kDa/groEl gene targets. Nested PCR positivity of these 113 patients in various combinations is analysed in [Table 1]. All 70 voluntary blood donors were negative for O. tsutsugamushi targeting all three genes. Gene sequences of 95 n-PCR positive products were submitted to NCBI database and accession numbers MG601875 to MG601969 were allotted respectively, thus confirming the presence of O. tsutsugamushi DNA in the samples.
Table 1: n-PCR positivity of three gene targets in various combinations

Click here to view


56 kDa

Out of 140 ST IgM ELISA antibody positive cases 42 (30%) were positive for 56 kDa gene (band at 483bp). Among those 42, 26 were adults16 were children. Out of 70 ST IgM ELISA antibody negative febrile patients only three (4.28%) were positive (one child and two adults). In total 45 (21.42%) cases were detected among 210 patients [Figure 1].
Figure 1: Nested PCR targeting 56 kDa gene.

Click here to view


47kDa

Among 140 antibody positive cases 72 (51.42%) (24 children, 48 adults) were positive for 47 kDa gene (band 364bp). Four out of 70 antibody negative febrile patients, (5.71%) (0 children, 4 adults) Thus among 210 clinically suspected ST patients, 76 (36.19%) were positive for 118bp [Figure 2].
Figure 2: Nested PCR targeting 47 kDa gene.

Click here to view


groEL gene

Among 140 ST IgM ELISA antibody positive cases, 86 (61.42%) (26 children, 60 adults) were detected targeting the groEL gene. Among 70 ST IgM ELISA negative patients, three (4.28%) (1 child, 2 adults) were positive for groEL gene. Out of 210 buffy coat specimens, 89 (42.38%) extracts were able to show the band at 364bp [Figure 3].
Figure 3: Nested PCR targeting groEL gene.

Click here to view


There is no statistical significance among children and adults as well as male and female with reference to their positivity in PCR (p = ≥0.05). The mean age of our patients was 30.56 ± 19.48 yr (95% confidence interval = 27.92 to 33.19) and the mean duration of illness at the time of blood collection was 8.86 ± 2.78 days (95% confidence interval = 8.48 to 9.24) in IgM ELISA positive and IgM ELISA negative febrile patients.


  Discussion Top


The non-specific Weil Felix test with poor sensitivity is still being used by resource poor laboratories, due to the non-availability of IFA test. IgM ELISA is considered to be an alternative serological diagnostic assay to the gold standard IgM IFA, with sensitivity 85 to 95% and specificity of 90 to 100%[3],[11],[12],[13],[14]. Seven IgM ELISA negative patients were positive in nested PCR. The duration of fever among these seven patients ranged from 7 to 10 days and probably IgM antibody has not appeared in these patients to detectable level. Eschar was not present in these seven patients. On the other hand, 34 IgM ELISA positive patients were negative in all three n-PCRs. However, ST eschar could be observed in 9 out of these 34 patients. The duration of their febrile illness ranged from three to 14 days (9.13 ±2.7). The failure to demonstrate O. tsutsugamushi DNA in these patients may be due to initiation of antibiotic therapy before acute blood sample collection or due to the presence of only few copies of DNA which may be below the detection limits or could be because of prior infection in the past.

Furuya et al[6] noted that ST diagnosis is possible in the early stage by PCR, i.e, before the antibody appears. Saisongkorh et al[4] observed that without antibiotic treatment, O. tsutsugamushi DNA persists in blood of ST patients up to 22 days of fever. According to Kim et al[11], nested PCR of O. tsutsugamushi using buffy coat of blood has high degree of sensitivity and specificity.

Silpapojakul et al[15] observed that 16SrRNA is conserved among different strains, but with a wider detection range of 1000–29,000 copies/L of blood. Ohashi et al[16] reported that 56 kDa is a type specific antigen (TSA) gene that occupies for 10 to 15% of total protein of O.tsutsugamushi. According to Furuya et al[6], 56 kDa nested PCR utilizes oligonucleotide primers based on the nucleotide sequences of a gene encoding 56 kDa antigen of a Gilliam strain, and it is a major protein antigen of this rickettsia. Stover et al[17] observed that Karp strain was the first reported 56 kDa TSA nucleotide sequence of O. tsutsugamushi. Karp, Kato, Gilliam, Boryong and TH187 strains are incorporated in the internal primers used in detecting the 47kDa gene O. Tsutsugamushi[3]. Continuous persistence of groEL gene throughout the course of ST infection was recorded by Paris et al[8]. Kelly et al[18] reported that groEL gene is a member of the chaperonin family, required for protein folding after translation.

[Table 2] compares the sensitivity and specificity reports of ST nested PCR targeting 56 kDa/47 kDa/groEL genes against IgM ELISA/IgM IFA as reported by different authors. Kim et al[3] observed that the sensitivity of nested PCR targeting 56 kDa and 47 kDa were similar viz., 87.8 and 85.4% and both scored 100% specificity. But in our study, 56 kDa showed least sensitivity of 30%, whereas 47 kDa had 51.43% and groEL gene showed the highest sensitivity of 61.43% against IgM ELISA as the reference. Recently, Patricia et al[19] reported similar low sensitivity of 27.6% for 56 kDa and 68.2% for groEl gene from Puducherry. The experience of rickettsiologists from India and abroad with reference to sensitivity and specificity of the three gene targets is summarised in [Table 2]. The specificity was excellent ranging from 88 to 100%. Most of the reports[3],[8],[11],[12],[13],[19],[20],[21],[22] were based on 56 kDa with sensitivity ranging from 27.6 to 90.47%. The highest percentage of 100% was reported by Usha et al[23], but the number was only 40. Only two reports record the application of nested PCR targeting 47 k Da gene by Kim et al[3] and Lim et al[22]. groEl gene target was utilized by two researchers Patricia et al[19] and Lim et al[22]. Nested PCR targeting all three genes was carried out only by Lim et al[22], and by us now for the first time in India.
Table 2: Reports of Sensitivity and Specificity of ST nested PCR targeting 56kDa/47kDa/groEL genes against IgM ELISA/IgM IFA

Click here to view


According to scrub typhus inclusion criteria (STIC) of Paris et al[8], any one of the following three criteria are to be fulfilled to confirm a case of ST: Culture positivity/ IgM IFA titre of ≥1:12,800 in acute or convalescent samples/PCR positivity in any two of the three targets – 56 kDa, 47 kDa and groEL. IgM ELISA is not taken into consideration. Since isolation of O. tsutsugamushi mandates the use of Biosafety level 3 containment facilities, this criterion cannot be met by most of the laboratories. IFA being an expensive, highly subjective and technically demanding technique requiring paired serum samples, PCR is the third option. Going by this strict guideline, out of 140 ELISA positive samples, 23 were positive in all three gene targets and 48 were positive for two genes, thus reaching a percentage positivity of 50.71 (71/140). Recently Varghese et al[24] recorded the persistence of IgM in ST patients for up to one year. Hence, laboratory confirmation of ST cases on the sole evidence of ST IgM positivity needs to be revised, since this could be due to present/past infection. Clinical correlation is a must for treating ST patients.

Limitation of the study

We could not perform the detection limit for O. tsutsugamushi positive cases.


  Conclusion Top


Looking for all three targets namely 56 kDa, 47 kDa and groEL genes will result in identifying more number of ST patients, although application of PCR targeting only 56 kDa has been recommended by many authors in the past. However, due to its relative poor sensitivity, a re-appraisal of 56 kDa as a single target is needed. Development of a Multiplex PCR with the incorporation of primers for all three gene targets would perhaps confirm maximum number of ST patients. Seven out of 70 (10.00%) antibody negative patients were positive in one or two PCR, which would have gone as ST negative cases if PCR was not performed. Hence, carrying out both PCR and IgM ELISA would be the ideal option to get maximum positive cases of ST.

Conflict of interest: None


  Acknowledgements Top


The authors acknowledge with thanks the Chairman, Vice-Chancellor, Dean (Medical Faculty) and Dean (Research), Mahatma Gandhi Medical College & Research Institute, Sri Balaji Vidyapeeth (Deemed-to be-University) Puducherry, India for funding this Faculty Research Project.

 
  References Top

1.
Watt G, Parola P. Scrub typhus and tropical rickettsiosis. Curr Opin Infect Dis 2003; 16(5): 429–36.  Back to cited text no. 1
    
2.
Issac R, Varghese GM, Mathai E, Manjula J, Joseph I. Scrub typhus: Prevalence and diagnostic issues in rural southern India. Clin Infect Dis 2004; 39(9): 1395–6.  Back to cited text no. 2
    
3.
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(2): 607–12.  Back to cited text no. 3
    
4.
Saisongkorh W, Chenchittikul M, Silpapojakul K. Evaluation of nested PCR for the diagnosis of scrub typhus among patients with acute pyrexia of unknown origin. Trans R Soc Trop Med Hyg 2004; 98(6): 360–6.  Back to cited text no. 4
    
5.
Kim C-M, Cho MK, Kim D-M, Yun N-R, Kim SW, Jang SJ, et al. Accuracy of conventional PCR targeting the 16S rRNA gene with the Ot-16sRF1 and Ot-16sRR1 primers for diagnosis of scrub typhus: a case-control study. J Clin Microbiol 2016; 54(1): 178–9.  Back to cited text no. 5
    
6.
Furuya Y, Yoshida Y, Katayama T, Yamamoto S, Kawamura AJ. Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase chain reaction. J Clin Microbiol 1993; 31(6): 1637–40.  Back to cited text no. 6
    
7.
Li W, Dou X, Zhang L, Lyu Y, Du Z, Tian L, et al. Laboratory diagnosis and genotype identification of scrub typhus from Pinggu district, Beijing, 2008 and 2010. Am J Trop Med Hyg 2013; 89(1): 123–9.  Back to cited text no. 7
    
8.
Paris DH, Blacksell SD, Nawtaisong P, Jenjaroen K, Teeraratkul A, Chierakul W, et al. Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute scrub typhus infection. PLoS Negl Trop Dis 2011; 5(9): e1307.  Back to cited text no. 8
    
9.
Stephen S, Sangeetha B, Ambroise S, Sarangapani K, Gunasekaran D, Hanifah M, et al. Outbreak of scrub typhus in Puducherry & Tamil Nadu during cooler months. Indian J Med Res 2015; 142(5): 591–7.  Back to cited text no. 9
    
10.
Rahi M, Gupte MD, Bhargava A, Varghese GM, Arora R. DHR-ICMR guidelines for diagnosis and management of Rickettsial diseases in India. Indian J Med Res 2015; 141(4): 417–22.  Back to cited text no. 10
    
11.
Kim DM, Yun NR, Yang TY, Lee JH, Yang JT, Shim SK, et al. Usefulness of nested PCR for the diagnosis of scrub typhus in clinical practice: A prospective study. Am J Trop Med Hyg 2006; 75(3): 542–5.  Back to cited text no. 11
    
12.
Manosroi J, Chutipongvivate S, Auwanit W, Manosroi A. Early diagnosis of scrub typhus in Thailand from clinical specimens by nested polymerase chain reaction. Southeast Asian J Trop Med Public Health 2003; 34(4): 831–8.  Back to cited text no. 12
    
13.
Gupta N, Chaudhry R, Kabra SK, Lodha R, Mirdha BR, Das BK, et al. Comparative evaluation of serological and molecular methods for the diagnosis of scrub typhus in Indian settings. Jpn J Infect Dis 2017; 70(2): 221–2.  Back to cited text no. 13
    
14.
Blacksell SD, Tanganuchitcharnchai A, Nawtaisong P, Kantipong P, Laongnualpanich A, Day NPJ, 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(2): 148–54.  Back to cited text no. 14
    
15.
Silpapojakul K. Scrub typhus in the Western Pacific region. Ann Acad Med Singap 1997; 26(6): 794–800.  Back to cited text no. 15
    
16.
Ohashi N, Tamura A, Ohta M, Hayashi K. Purification and partial characterization of a type-specific antigen of Rickettsia tsutsugamushi. Infect Immun 1989; 57(5): 1427–31.  Back to cited text no. 16
    
17.
Stover CK, Marana DP, Carter JM, Roe BA, Mardis E, Oaks EV. The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: Molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope. Infect Immun 1990; 58(7): 2076–84.  Back to cited text no. 17
    
18.
Kelly DJ, Fuerst PA, Ching WM, Richards AL. Scrub typhus: The geographic distribution of phenotypic and genotypic variants of Orientia tsutsugamushi. Clin Infect Dis 2009; 48(suppl 3): 203–28.  Back to cited text no. 18
    
19.
Patricia KA, Hoti SL, Kanungo R, Jambulingam P, Shashikala N, Naik A. Improving the Diagnosis of Scrub typhus by combining groEL based polymerase chain reaction and IgM ELISA. J Clin Diagn Res 2017; 11(8): 27–31.  Back to cited text no. 19
    
20.
Prakash JAJ, 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(1): 47–50.  Back to cited text no. 20
    
21.
Usha K, Kumar E, Kalawat U, Kumar BS, Chaudhury A, Gopal DVRS. Molecular characterization of Orientia tsutsugamushi serotypes causing scrub typhus outbreak in s outhern region of Andhra Pradesh, India. Indian J Med Res 2016; 144(4): 597–603.  Back to cited text no. 21
    
22.
Lim C, Paris DH, Blacksell SD, Laongnualpanich A, Kantipong P, Chierakul W, et al. How to determine the accuracy of an alternative diagnostic test when it is actually better than the reference tests: A re-evaluation of diagnostic tests for scrub typhus using Bayesian LCMs. PLoS One 2015; 10(5): e0114930.  Back to cited text no. 22
    
23.
Usha K, Kumar E, Kalawat U, Kumar BS, Chaudhury A, Gopal DVRS. Molecular detection of scrub typhus in Tirupati, Andhra Pradesh, India. J Vector Borne Dis 2015; 52(2): 171–4.  Back to cited text no. 23
    
24.
Varghese GM, Rajagopal VM, Trowbridge P, Purushothaman D, Martin SJ. Kinetics of IgM and IgG antibodies after scrub typhus infection and the clinical implications. Int J Infect Dis 2018; 71: 53–5  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Material & M...
Results
Discussion
Conclusion
Acknowledgements
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed664    
    Printed1    
    Emailed0    
    PDF Downloaded61    
    Comments [Add]    

Recommend this journal