|Year : 2020 | Volume
| 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
Velmurugan Anitharaj1, Selvaraj Stephen1, Pooja Pratheesh2
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 Submission||31-May-2018|
|Date of Acceptance||08-Jan-2019|
|Date of Web Publication||14-Jul-2021|
Dr Selvaraj Stephen
Professor of Microbiology, Mahatma Gandhi Medical College & Research Institute, Sri Balaji Vidyapeeth, Pondy-Cuddalore Main road, Pillaiyarkuppam, Puducherry
Source of Support: None, Conflict of Interest: None
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 2022 May 21];57:147-52. Available from: https://www.jvbd.org/text.asp?2020/57/2/147/310866
| Introduction|| |
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, every year one million cases of ST are reported throughout the world. Isaac et al 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). 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 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,,,. In this study, we have used nested PCR, targeting three different genes, 56 kDa, 47 kDa and groEL as recommended by Paris et al, in their Scrub Typhus Inclusion Criteria (STIC).
| Material & Methods|| |
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. Only anonymised samples were included in this research, by way of excluding the identity and personal details of the patients.
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 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. OD values above 0.5 were taken as positive.
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: 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, 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. 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.
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|| |
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.
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].
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].
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].
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|| |
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%,,,,. 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 noted that ST diagnosis is possible in the early stage by PCR, i.e, before the antibody appears. Saisongkorh et al 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, nested PCR of O. tsutsugamushi using buffy coat of blood has high degree of sensitivity and specificity.
Silpapojakul et al 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 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, 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 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. Continuous persistence of groEL gene throughout the course of ST infection was recorded by Paris et al. Kelly et al 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 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 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,,,,,,,, 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, but the number was only 40. Only two reports record the application of nested PCR targeting 47 k Da gene by Kim et al and Lim et al. groEl gene target was utilized by two researchers Patricia et al and Lim et al. Nested PCR targeting all three genes was carried out only by Lim et al, 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|
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According to scrub typhus inclusion criteria (STIC) of Paris et al, 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 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|| |
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|| |
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.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]