Application of Nested PCR and Loop-Mediated Isothermal Amplification (LAMP) to Target 56kDa gene in Scrub Typhus Patients and Phylogenetic Analysis to Identify Orientia tsutsugamushi Strains Circulating In and Around Puducherry

Scrub typhus (ST) is a re-emerging zoonotic disease caused by Orientia tsutsugamushi and is transmitted by chiggers. Serological tests targeting IgM and/or IgG antibodies play a major role in the detection of ST cases. Orientia 56kDa genome is common target for the molecular diagosis of ST to identify the prevalence of specific serotypes of O. tsutsugamushi in and around Puducherry by targeting 56kDa gene with the application of phylogenetic analysis. This prospective laboratory-based study was conducted in a tertiary care teaching hospital, from November 2105 to March 2018. Blood samples were collected from out-patients/ in-patients, and those tested positive for ST IgM ELISA (n=140) and an equal number of negative samples were archived and anonymized. Genomic DNA was extracted and analyzed by using Nested PCR and LAMP assay. The positive products were purified and sequenced. Phylogenetic tree was constructed based on the sequences. Among 280 samples, 45 (16.1%) N-PCR and 102 LAMP (36.43%) positivity was observed for 56kDa gene. Forty-one N-PCR positive products were sequenced and accession numbers were obtained (MG601875 to MG601917) for the isolates. Phylogenetic analysis was done by Maximum Likelihood methods and this study has showed that 32.3% are similar to the Karp isolates. Molecular diagnosis of Scrub typhus has become essential in case of doubtful serology and early acute phase of illness. Gene sequencing result indicates that most of them were different from the existing ones, which may belong to the newer strains. The identification of newer strains will be helpful in future for development of scrub typhus vaccine.


INTRODUCTION
2][3][4] The infection is mostly caused by the chiggers' larvae breed on human's blood in contrast to the adults on plants.Children may get affected accidentally by walking with bare foot and other activities like sitting or lying on the grass vegetation where the larval mite breeds.Patient's symptoms start from 6-21 days after mite bite. 1 Other non-specific findings like fever, headache, rash, lymphadenopathy etc. can be observed in different febrile conditions such as Dengue, Malaria, Leptospirosis, and other hemorrhagic fevers.][5][6][7][8][9][10][11][12][13][14][15][16][17][18] LAMP is one of the additional molecular methods for the rickettsial diagnosis and easy to perform, with four to six primers in isothermal temperature and it does not require any costly instruments.4][5] Hence, ST N-PCR and LAMP were employed in this study by targeting 56kDa, to identify the prevalent genotypes circulating in and around Puducherry by using Phylogenetic analysis.

Study Participants
The Out-patients/In-patients attending Mahatma Gandhi Medical College and Research Institute(MGMCRI), Puducherry during the time period from November 2015 to March 2018 were included in the study.The study was conducted with the approval from Institutional Human Ethical Committee (IHEC), (IHEC/24/2020) MGMCRI, Puducherry.After getting consent from the patients, the blood specimens were collected from suspected febrile patients from General Medicine and Pediatrics were analyzed for ST IgM ELISA at Department of Microbiology and the molecular work at Centre for Interdisciplinary Research Facility (CIDRF), MGMCRI Campus, Puducherry.

Molecular diagnosis of Scrub Typhus Buffy coat preparation
About 2 ml of whole blood was collected in a tube containing Ethylenediaminetetraacetic acid (K2EDTA) anti-coagulant.This was transferred into 15ml falcon tubes and 4ml of phosphate buffered saline (PBS) and inverted 2 to 3 times.Blood sample was diluted by adding 1ml of HiSep LSM to the new falcon tube and centrifuged at 1500 rpm for 30 minutes at 15-25°C.Transfer the upper layer of plasma into a separate Eppendorf tube.Collected the white layer (Buffy coat) in a separate falcon tube and added 3 more volumes of PBS.Centrifuged at 260g for 10 mins and without any brake.Remove the supernatant and finally add 500µl of PBS immediately transferred to 1.5ml Micro centrifuge tubes and stored at -80°C for the extraction of genomic DNA. 19About 200µl buffy coat samples were used for the genomic DNA extractions as per the procedure of DNA Blood Mini Kit (Qiagen, Germany).The purity of the extracted DNA was determined by absorbance (A) using Eppendorf Spectrophotometer.A260/A280 ratios were in the range of 1.7-1.8 for the 280 samples.The samples were aliquoted and stored in 0.2ml PCR tubes at 80°C till the completion of the molecular tests.

Nested PCR
N-PCR outer and inner primer sets targeting 56kDa gene and cycling conditions were followed as described by Furuya et al., 20 in25.0µlreactionvolume.PCR was carried out in Applied Biosystem, VeritiDx 96-wells Thermal cycler (Thermo Fisher Scientific, Singapore) and post-amplification, the amplicon with specific product size of 483-bp was visualized by 1.5 % agarose gel electrophoresis and documented by Gel Documentation system (Bio-Rad, USA).

Loop-Mediated Isothermal Amplification (LAMP)
The LAMP primers (forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3) and backward outer primer (B3)) targeting 56kDa gene were designed from a highly conserved region of O. tsutsugamushi Hualien -5 strain (Accession no.AY243357.1)using the Primer Explorer version 4.0 http:// loopamp.eiken.co.jp/e/:,is using for screening specimens.The location and the sequences of the primers were shown in Figure 1 and Table represents the custom synthesized LAMP primers by Sigma Aldrich, Bangalore.LAMP Reaction was conducted in 25µl of reaction volume comprising of 7.5µlNuclease free water, 3.5µl 1.4mM DNTPs 1.0µl of each 40µM of Forward and Reverse Innerprimersand1.0µl of each 5µM of Forward and Reverse Outer Primers,3.5µl of 5M Betaine, 2.5µl10X Isothermal Amplification Buffer II, 1.5µl of 8mM MgSo4, 1.0µlBst 3.0 DNA Polymerase (8000 Units/ml), 0.5µl 3mM HydroxyNaphthol blue and 1.0µl genomic DNA.After amplification, primarily visual observation of the reaction was done and interpretation was made based on the color change from Sky blue to purple for positive and negative reactions, respectively (Figure 2).Further confirmation was made by loading the LAMP amplicon in 1.5% agarose gel electrophoresis stained with Ethidium bromide solution for the presence of ladder like pattern in positive samples (Figure 3).

Phylogenetic analysis
Both N-PCR and LAMP positive products were purified by Qiagen DNA Purification kit and the products were sequenced by Applied Biosystems ABI3730XL (Eurofins Genomics India Private  Limited, Whitefield, Bangalore) for specificity of the amplicon.The sequences obtained from the study was submitted and accession numbers were procured from the GenBank database (National Centre for Biotechnology Information -NCBI).The sequences were aligned using Clustal W and the Evolutionary analysis was constructed using of MEGA version 11.0. 8The 56-kDa TSA gene of O. tsutsugamushi isolates were aligned with reference sequences with a total of 68 nucleotide sequences.One thousand bootstrap value replicates were analyzed to estimate the reliability of the node.The evolutionary tree was inferred by using the Maximum Likelihood method and Kimura 2-parameter model.Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.Bootstrap values more than 75% were considered to be significant.

Statistical analysis
Percentages were calculated for categorical variables.Mean and standard deviations (SD) were calculated with 95% confidence interval for numerical variables.Sensitivity, specificity, positive and negative predictive value was calculated using Med Calc's online software.Chisquare test was applied for numerical variables and Fisher's exact test for small number samples.Statistical tests were analyzed by Quick Calcs, GraphPad Prism.P values ≤ 0.05 were considered statistically significant.Phylogenetic tree was constructed by Maximum Likelihood method based on 56kDa sequences of O.tsutsugamushi and presented in the Figure 4.In this study, only 12 sequences had significant bootstrap value more than 90% and remaining 29 sequences are less than 75% which are nonsignificant.Geographical locations of participants reporting to hospital at Puducherry and flow of study procedures were shown in Figure 5.

DISCUSSION
Molecular diagnosis of Scrub typhus has become essential in case of doubtful serology and early acute phase of illness.Presence of O. tsutsugamushi DNA could be demonstrated either in the Buffy coat or eschar biopsy samples.Conventional PCR, Nested PCR and Real Time PCR are being regularly used for this accurate detection. 3Recently, LAMP assay has been introduced as an additional molecular test. 4The LAMP primers were newly designed in this study and further used for screening the specimens.
According to Kala et al., 21 56 kDatsa gene is mostly used for the characterization of the O. tsutsugamushi strains.
The study sequences have 98-100% of maximum identity with the other reference strains available in the NCBI database.In the present study, most of our 56kDa isolates are closely related to Karp and Gilliam, followed by Boryong and some strains are closely related to Kawasaki strains.In a total of 42 patients, 55% of sequences were related to the Karp serotype followed by 17%, were Gilliam type in Japan variant respectively.One of the studies from Pondicherry also proved that most of the sequences were matched with the Karp group strains. 11A study was conducted by Patricia et al., 11 in Puducherry comprised that 15 of their sequences were correlated with the Karp group and another 6 from Vellore strains, respectively.They also indicated that 12 of their sequences form a separate group and did not match with the other genotypes that were included as a reference strain.Phylogenetic analysis of 16 sequences from Sri Lanka was related with Karp, Kato, JGV and JGA (Kawasaki group), Gilliam, Kuroki-Boryong genotypes.Karp is one of the major dominant genotypes of O. tsutsugamushi varying from 40-64.4% and it was reported in many countries.Regarding the genotypes of O. tsutsugamushi circulating in India, Patricia et al., 11 from Puducherry reported positivity of 42.9% for Karp and 17.1% for Kato.Usha et al., 10 from Andhra Pradesh also had a higher prevalence of Karp strains (70.4%) followed by Kawasaki (29.6%).Varghese et al., 13 from Tamil Nadu and other parts of India observed that they have Kato as the majority strain with 61.5% followed by Karp (27.1%),Neimang (8.7%) and Ikeda (2.3%).Recently, Thakur et al., was recorded that majority of the circulating strains of O. tsutsugamushi in New Delhi were close to Gilliam and Karp. 20According to Biswal et al., 14 from Chandigarh, Boryong occupied the highest prevalence with 63.4% followed by Karp (23.6%),Gilliam (11.8%) and Kawasaki (1.2%).A similar observation of highest positivity of Boryong genotype was recently reported by Ramaiah et al., 16 from Manipal, Karnataka.The present study had recorded 32.29% moderate positivity for Karp like strains and 17.7% are grouped with a Kawasaki strains.Most of our isolates (50.01%) have highest homology with that of the strains reported from India, Korea, Japan and China.Extension of the research work to cover a wider geographical area in regions surrounding Puducherry might help in identifying additional genotypes of O. tsutsugamushi.Development of a Multiplex PCR targeting all three genes would help in the rapid confirmation for the diagnosis of ST.The present study concluded that the study sequences have 98-100% maximum identity with the other reference strains available in the GenBank database.Gene sequencing result indicates that most of the study sequences were related to those who submitted from India as well as overseas.Few of our study sequences were different from the existing ones,

Figure 2 .Figure 3 .
Figure 2. Image for Visual Detection of LAMP products: Upper row: 1 & 2 -DNA Samples positive for 56kDa gene target for O. tsutsugamushi, 3 and 4 -Sample negative for O. tsutsugamushi DNA; Lower row: NC -Negative control, PC -Positive control

Figure 4 .
Figure 4. Geographical locations of participants reporting to MGMCRI, Puducherry and flow of study procedures.The image depicts the geographical locations of patients reporting to MGMCRI, Puducherry.The map includes number of cases reported from each location

Table .
Details of LAMP primers used for 56kDa gene targets Note: F3 & B3 -Forward and Backward primers; FIP & BIP -Forward and Backward Inner Primers