Molecular identification and phylogenetic analysis of spotted fever Rickettsiae in peripheral blood from suspected Rickettsiosis cases in Sri Lanka

Abstract

Background

Rickettsioses are an emerging public health concern in Sri Lanka, particularly in the central hills, where the Spotted Fever Rickettsiosis is prevalent. This study aimed to molecularly detect and characterize Rickettsia species in peripheral blood from patients suspected of Spotted Fever Rickettsiosis.

Methods

Peripheral blood samples were collected from 217 patients suspected of Rickettsiosis between January 2020 and June 2021. All samples were screened using the Rickettsia conorii immunoglobulin G (IgG) Indirect Immunofluorescence Antibody Test (IFAT). DNA was extracted from IFAT-positive samples and subjected to nested polymerase chain reaction (PCR) targeting the 17 kDa protein gene of Spotted Fever Rickettsiae. Selected PCR-positive samples were purified, sequenced, and analyzed using nBLAST and phylogenetic analysis.

Results

Of 217 clinically suspected patients, 137 (63.1%) had antibodies reactive with R. conorii antigen. DNA extracted from 113 seropositive blood samples underwent nested PCR targeting the 17 kDa protein gene, yielding 15 (13.2%) positives. Ten amplicons were sequenced, revealing three genotypes. Phylogenetic analysis showed one sequence with 100% identity to R. felis, marking the first molecular detection of this species in human blood in Sri Lanka. Other sequences matched multiple Spotted Fever Rickettsia species, including R. rickettsii, R. conorii, and R. parkeri, though species-level resolution was limited due to conserved gene regions. The majority of seropositive patients were middle-aged females from rural areas.

Conclusion

Findings underscore the presence of diverse, potentially virulent Rickettsia species and highlight the need for enhanced molecular surveillance. This study provides critical insights into the epidemiology of rickettsioses in Sri Lanka and supports the use of molecular tools for accurate pathogen identification, crucial for diagnosis, treatment, and vector control efforts.

Clinical trial

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-026-13147-3.

Background

Rickettsiae are a diverse group of gram-negative, non-motile, obligate intracellular bacteria known for their ability to infect a wide range of host species, including humans and animals [1–10]. Rickettsia species are broadly classified based on genomic and phylogenetic analysis: the Spotted Fever Group (SFG) and the Typhus Group (TG) [11, 12]. The former group includes ~ 24 human pathogenic species such as R. rickettsii, R. africae, R. parkeri, R. japonica and R. conorii etc., whereas the latter group includes species such as R. prowazekii, and R. typhi [13]. Additional lineages that do not fit neatly into either group have been identified as the Transitional Group (TRG), which includes species such as R. felis, R. akari, R. australis, R. honei, and R. heilongjiangensis. There is another group of Rickettsiae known as the Scrub Typhus Group (STG), with Orientia tsutsugamushi being the main species involved [12].

Rickettsiae are primarily transmitted through the bite of infected ticks, although other arthropods, such as fleas, lice and mites, can also serve as vectors [14]. Across the world, a variety of wild and domestic animals, such as rodents, small mammals, and birds, have been recognized as important reservoirs of Rickettsia infection [7]. Rickettsial infections are emerging as significant public health threat in many parts of the world, particularly in Asia [15–19], Africa [20, 21] and the America [22, 23], where tick vectors are prevalent.

The incidence is approximately 1 case per 100,000 people per year [24]. Fatality rates of certain untreated Rickettsioses, such as Rocky Mountain Spotted Fever, are reported to be as high as 30% [25]. Thus, Rickettsiosis is particularly problematic in tropical and subtropical areas where it is endemic, the vectors thrive, and the disease often presents as an underdiagnosed or misdiagnosed illness [26, 27].

Sri Lanka is endemic for Rickettsioses, and numerous investigations have recognized Rickettsiosis as an emerging and re-emerging public health concern [9, 27–31]. Ample serological evidence exists in Sri Lanka showing that people are being infected with SFG Rickettsia, TG Rickettsia and STG of Rickettsia [27–32]. These rickettsial infections demonstrate spatial clustering in Sri Lanka, with Spotted Fever Rickettsiosis being prevalent in central hills of the country [27, 29, 31, 32], while STG is predominately found in most lowland areas [30, 33, 34]. TG has been reported in both geographical locations, but with very low frequencies [27, 29].

While serology provides broad evidence of Rickettsia infections in humans, molecular characterization of Rickettsia species infecting humans using human specimens has rarely been done in Sri Lanka. To date, successful molecular detection of Rickettsia species in infected human blood has not been reported in Sri Lanka. Identifying the predominant Rickettsia species infecting humans in Sri Lanka would help better define disease epidemiology, understand the specific clinical signs associated with each Rickettsia species, and identify the most relevant vectors and reservoirs. Understanding the Rickettsia species infecting humans is crucial for comprehending disease dynamics and formulating effective control strategies. Therefore, this study aimed at molecular detection of etiology, characterization of the identified, and phylogenetic analysis Rickettsia species present in peripheral blood samples from suspected Rickettsiosis cases in Sri Lanka.

Methods

Study population and samples

All patients included in this study were referred to the Rickettsia Diagnostic Unit, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, by the clinicians based on clinical evidence suggesting Spotted Fever Rickettsioses from the Teaching Hospital, Peradeniya and private clinics. Clinical features, including fever, headaches, discrete maculopapular erythematous rash, swelling of legs, along with ankle joint arthritis and atypical manifestations that responded well to anti-rickettsial antibiotics, were taken in multiple combinations to suspect the diagnosis. The inclusion criteria also required that patients suspected of having Spotted Fever Rickettsia submit blood samples during the period from January 2020 to June 2021. Written informed consent was obtained from all participants.

Ethics approval and consent to participate

Ethical approval for the current study was obtained from the Ethical Committee, Faculty of Medicine, University of Peradeniya (Ethical Approval No: 2019EC63). Furthermore, all patients included in the study voluntarily provided a blood sample to the Rickettsia Diagnostic Unit of the Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka.

Identification of Rickettsia IgG sero positives

Indirect immunofluorescence assay (IFA) was performed on the blood samples using the Vircell^® Rickettsia Conorii IFA IgG immunofluorescence assay (Vircell S.L., Granada, Spain), following the manufacturers’ instructions. Only whole blood samples in EDTA tubes were included for the molecular study and after IFA EDTA blood samples were pelleted by centrifuging 1500 rpm for 10 min and the pellets were stored at -20 °C until further processing.

Retrospective analysis of socio-demographic factors associated with spotted fever Rickettsiosis seropositivity

A retrospective descriptive study was conducted to assess the association between Rickettsia seropositivity and selected socio-demographic factors, including age (≤ 20, 21–40, 41–60, and ≥ 61 years), gender (male or female), and residential location (urban, peri-urban, or rural). Data from patients clinically suspected of having Spotted Fever Rickettsiosis were analyzed using descriptive and proportional statistics. Associations between categorical variables were evaluated using the Chi-square test. All statistical analyses were performed using SPSS software, version 14.0 (SPSS Inc., 2005), with statistical significance defined as p ≤ 0.05. To visualize the spatial distribution of suspected Rickettsiosis cases and identify potential clusters, residential locations were mapped using Quantum GIS (QGIS) version 3.3.5 (Open Source Geospatial Foundation, Boston, USA).

Molecular diagnosis of SFG Rickettsia in blood samples

Pelleted blood samples from patients with antibodies reactive to the R. conorii antigen were used for DNA extraction using the DNeasy^® Blood & Tissue Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Extracted DNA was subjected to Nested PCR to detect the 17 kDa protein gene of SFG Rickettsia. Previously published primers, R17-122 [5’-CAGAGTGCTATGAACAAACAAGG-3’] and R17-500 [5’-CTTGCCATTGCCCATCAGGTTG-3’], were used as primers in the first round, and TZ15 [5’-TTCTCAATTCGGTAAGGGC-3’] and TZ16 [5’-ATATTGACCAGTGCTATTTC-3’] were used as primer for the second round [35, 36].

Each PCR reaction consisted of a 25µL reaction, which included 12.5µL of GoTaq^® Green Master Mix, 0.4µM of each forward and reverse primer, 5µL of template DNA, and 5.5 µL of nuclease-free water. The first round of the nested PCR used the extracted DNA as the template, while the second round of the nested PCR used a 1:10 dilution of the first-round PCR product as the template. This dilution served to inactivate the first-round primers prior to the secondary PCR. To minimize the risk of contamination, barrier tips were used, dilutions and template additions were performed in a biosafety cabinet, samples were handled individually, and negative controls were included throughout. While dilution may increase the risk of false negatives, it reduces non-specific amplification and facilitates PCR product purification, yielding higher-quality amplicons than gel-based methods.

First round of nested PCR involved an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The final extension was 5 min at 72 °C, and the PCR products were visualized using electrophoresis in a 2% agarose gel. Samples that did not yield the expected ~ 378 bp band were subjected to a second round of nested PCR.

The second round of PCR consisted an initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The final extension was at 72 °C for 5 min, and the products were visualized using gel electrophoresis to confirm a ~ 247 bp amplicon. In each PCR run, DNA extracted from the R. conorii Moroccan strain (ATCC VR-141) served as the positive control. This DNA was obtained from scrapings of intact Vircell^® Rickettsia conorii IFA IgG immunofluorescence assay slides. Nuclease-free water was included as the negative control to verify the specificity and reliability of the amplification results.

Sequencing of partial 17 kDa protein gene and sequence analysis

From the PCR positive samples, 10 amplicons were selected and purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Bi-directional Sanger sequencing of the purified amplicons was performed at Macrogen Inc. (Seoul, South Korea). The resulting sequences were quality-trimmed, assembled, and aligned using Geneious Prime version 2025.1 (Biomatters Ltd., Auckland, New Zealand). During sequence quality control, low-quality terminal regions, including primer sequences, were trimmed from all raw reads prior to downstream processing. BLAST (Basic Local Alignment Tool) analysis was performed using the National Center for Biotechnology Information (NCBI) database to identify the closest matching 17 kDa protein gene sequence. Consequently, sequence homology estimates were not influenced by primer-derived nucleotides.

Multiple sequence alignment and phylogenetic analysis

Multiple sequence alignment was performed using Geneious Alignment to compare the obtained sequences with established reference sequences and to identify potential nucleotide polymorphisms. As no Rickettsia 17 kDa protein gene sequences from Sri Lanka were available in GenBank, well-characterized sequences from type or reference isolates were selected for comparative analysis. Specifically, the Rickettsia conorii isolate MUWRP-0040 17 kDa antigen gene (GenBank accession no. OP974440.1), the Rickettsia felis 17 kDa antigen gene (GenBank accession no. AF195118.1) and the Rickettsia typhi isolate AB3169 17 kDa antigen gene (GenBank accession no. OR567470.1) were used as reference sequences based on prior studies that identified them as representative for their respective species. Phylogenetic tree was constructed using the MEGA 11 [37], employing the maximum likelihood method with 100 bootstrap replicates. A total of 33 reference sequences were retrieved from the NCBI database and incorporated into the analysis. Rickettsia typhi isolate AB3169 17 kDa antigen gene (GenBank accession no. OR567470.1) was used as the outgroup for the phylogenetic reconstruction.

Results

Demographic characteristics of patients with suspected spotted fever Rickettsioses

A significantly higher proportion of the suspected patients were female (44.7%, p = 0.02). The majority of cases (36.4%) were in the 41–60 years age group, followed by those aged over 61 years (29.0%). There was no significant difference in the number of suspected cases reported from urban (30.9%) and rural (38.2%) areas (Table 1).

Table 1.

Demographic distribution of participants

Variables	Frequency	Percentage
Gender
Female	97	44.7%
Male	74	34.1%
Missing Data	46	21.2%
Age groups (years)
≤ 20	24	11.1%
21–40	40	18.4%
41–60	79	36.4%
≥ 60	63	29.0%
Missing Data	11	5.1%
Living area
Rural	83	38.2%
Periurban	22	10.1%
Urban	67	30.9%
Missing Data	45	20.7%

Clinical manifestations of the suspected patients

Clinical records were available for 171 patients with suspected Rickettsiosis. Fever was the predominant symptom (n = 138, 81%), lasting 1–5 days before admission, with rapid defervescence within three days of doxycycline or chloramphenicol therapy. Other common symptoms included loss of appetite (n = 130, 76%), myalgia (n = 118, 69%), acute arthritis (n = 108, 63%), maculopapular rash (n = 109, 64%), and malaise (n = 67, 40%). Less frequent symptoms were abdominal pain (n = 18, 11%) and hearing impairment (n = 14, 8%).

A typical fever-rash presentation occurred in 102 patients (60%), while atypical features such as apathy, confusion, and extrapyramidal signs were noted in 28 (16%). Fibromyalgia or subacute arthritis occurred in 32 (19%), and tick-bite eschars in 16 (9%). Six patients (3%) showed vasculitic rashes on the toes and feet with ulcerations. All recovered following treatment, with convalescence lasting 3–14 days.

Prevalence of spotted fever Rickettsiosis in suspected patients

Of the 217 patients with suspected Rickettsiosis, 137 (63.1%) had antibodies reactive with R. conorii antigen. Seropositivity was not significantly associated with gender, age group or residential area (Table 2).

Table 2.

Demographic characteristics of Rickettsia conorii IgG-seropositive patients as determined by indirect immunofluorescence assay performed at a 1:40 serum dilution

Variable	IFA results Positive	IFA results Negative	p-value (p) associated with Chi square analysis# (X2)
Gender
Male Female	50 57	24 40	X2=1.39 p = 0.238
Age Groups (years)
≤ 20 21–40 41–60 ≥ 60	13 26 50 42	11 14 29 21	X2= 1.22 p = 0.749
Living Area
Urban Periurban Rural	48 11 52	19 11 19	X2= 3.64 p = 0.162

^#Underpowered chi-square values may be attributable to missing patient data

Molecular characterization of the Rickettsia strains

Of the patients with antibodies reactive to the R. conorii antigen, 113 provided EDTA blood samples, which were used for DNA extraction and PCR analysis. Among these, 15 (13.2%) tested positive for Rickettsia DNA. Of the 15 who tested positive for Rickettsia DNA, 14 had fever, acute arthritis and maculopapular rash; however, one patient had only recurrent fibromyalgia without fever. Ten of the positive amplicons were bidirectionally sequenced. Nucleotide sequence alignment revealed three distinct genotypes among the analyzed samples. Genotype 1, identified in a single sample, was identical to the R. felis reference sequence (AF195118.1) and exhibited 14 single nucleotide polymorphisms (SNPs) relative to the other local sequences. In addition, the Genotype 1 sequence identified in this study demonstrated 100% sequence identity with multiple R. felis strains available in the NCBI database upon BLAST analysis. Identical matches included isolates derived from diverse hosts and geographical regions, such as KR709306.1 (blood of Didelphis sp., Mexico), LC764839.1 (human blood, Japan), ON053295.1 (Ixodes ricinus nymph, Slovakia), MK242047.1 (Ctenocephalides felis, Hungary), and MT012721.1 (Ctenocephalides felis, Brazil). Genotype 2, detected in two samples, differed from the R. conorii reference sequence by a single SNP, while Genotype 3, observed in seven samples, was identical to the R. conorii reference sequence (Fig. 1).

Fig. 1.

Nucleotide alignment of 10 bidirectional sequences showing SNPs relative to Rickettsia conorii (OP974440.1), R. felis (AF195118.1), and R. typhi (OR567470.1) using Geneious Prime v2025.1

BLAST analysis of the Genotype 1 sequence revealed 100% identity with multiple R. felis isolates available in GenBank. Genotype 3 sequences exhibited 100% homology with the partial 17 kDa protein gene of the majority of SFG Rickettsia species, reflecting the highly conserved nature of this gene within the group. The SNP observed in Genotype 2 sequences showed 100% similarity to certain Rickettsia species reported from both the SFG and TRG, including R. jingxinensis, R. japonica, R. honei, and R. heilongjiangensis. Given the limited discriminatory power of the 17 kDa gene among core SFG Rickettsia, these findings were interpreted at the group level rather than at the individual species level.

Phylogenetic analysis of the local Rickettsia strains

Phylogenetic analysis demonstrated that the local Rickettsia sequence representing Genotype 1 (Sample 2 F; Fig. 2) formed a sister clade with the reference sequence LC764841.1, identified as R. felis previously detected in human blood samples from Japan, and KM006831.1, a R. felis strain isolated from fleas collected from cats and dogs in Sicily, southern Italy. The local sequence corresponding to Genotype 2 (Sample 8 F; Fig. 2) clustered closely with Candidatus Rickettsia vulgarisii (PP117200.1), which was originally identified from ticks collected in Ningxia, northwestern China. In contrast, the local Rickettsia sequence representing Genotype 3 (Sample 4 F; Fig. 2) grouped with ON987324.1, identified as R. conorii isolated from the blood of a patient with Spotted Fever Rickettsiosis in Jammu and Kashmir, India, and PP442019.1, corresponding to Rickettsia sibirica subsp. sibirica isolated from a human patient in urban Beijing, China.

Fig. 2.

Phylogenetic tree constructed using MEGA 11 software illustrating the evolutionary relationships between the three local Rickettsia genotypes detected in the blood of Sri Lankan patients and 33 references Rickettsia sequences retrieved from the NCBI database

Geographical distribution of the suspected patients, seropositives and PCR positives

Figure 3 shows the geographical distribution of patients suspected of having Rickettsioses, categorized as seropositive, seronegative and PCR positive. The figure clearly indicates a higher concentration of cases in the central hills, with sporadic Cases in the lowlands. These latter cases were likely referrals to central hospitals or consultants.

Fig. 3.

Geographical distribution of suspected Spotted Fever Rickettsiosis cases, Rickettsia IgG seropositives, and PCR-positive patients mapped using QGIS v3.3.5

Discussion

This study provides novel molecular and serological insights into the epidemiology of SFG Rickettsia infections in Sri Lanka, where Rickettsioses are increasingly recognised as an emerging public health concern. The observed IgG seroprevalence of 63.1% among clinically suspected patients likely overestimates true population exposure, as participants were pre-selected for suspected infection, introducing selection bias. Nevertheless, this finding is consistent with previous reports from the central hill region, where seroprevalence increased from 26% in 2000–2001 [27] to 40.9% in 2002–2007 [29] and exceeded 60% in later surveys [9, 31]. The burden of Spotted Fever Rickettsiosis appears to be higher in the Central Province of Sri Lanka than in many other regions globally. Comparable studies in other countries targeting IgG antibodies against SFG Rickettsia in human patients have reported seroprevalence rates of 10% in Kenya [38], 31.2% in Kazakhstan [39], and 37.1% in Karnataka, India [40]. Findings of this study indicate that Spotted Fever Rickettsioses remain highly endemic in Sri Lanka, likely driven by ecological and socio-behavioural factors that promote sustained human–vector contact.

Although the IFA remains the serological reference standard, reliance on IgG alone limits the diagnostic interpretation of acute infection, as antibodies may persist long after exposure, making it difficult to distinguish between past and current disease. IgG antibodies may also persist in patients with chronic rickettsial infections. Because IgM testing is less specific, clinicians in Sri Lanka often rely on paired IgG assays. However, patients seldom return for convalescent-phase sampling unless symptoms persist after treatment, largely due to financial constraints and limited diagnostic facilities. Therefore, these serological findings should be interpreted in conjunction with molecular evidence to provide a more accurate understanding of active infection dynamics [41]. PCR positivity for rickettsial DNA provides stronger evidence of active infection when interpreted alongside a compatible clinical presentation.

Molecular screening using nested PCR targeting the 17 kDa protein gene detected Rickettsia DNA in 13.2% of seropositive blood samples. Although this detection rate is lower than those reported from South Korea, Brazil, and Japan [42–44], it represents the first molecular evidence of Rickettsia in human blood from Sri Lanka. One of the ten sequenced amplicons showed 100% identity with R. felis, confirming the first documented detection of this pathogen in humans in Sri Lanka. Identical matches with isolates from diverse hosts and geographical regions further support the classification of this genotype as R. felis and strengthen the species-level identification based on the 17 kDa gene sequence. Previous serological work by Angelakis et al. [28] detected R. felis-specific antibodies in 1.6% of patients, but molecular confirmation was lacking. R. felis has been identified in human blood in several other countries, including Kenya (3.7%) [45], Vietnam (2.53%) [46], China (2.14%) [47], Bangkok (15.48%) [48], and Bangladesh (42%) [49]. However, these studies varied in the biological samples used for molecular detection, including whole blood [49], serum [45], buffy coat [46, 48], plasma, and bronchoalveolar lavage fluid [48]. R. felis is recognised as an emerging pathogen of global concern, with an increasing number of human infections reported worldwide [50, 51].

The remaining nine sequences showed identity to SFG Rickettsia species such as R. conorii, R. jingxinensis, R. japonica, and R. honei, or to TRG Rickettsia such as R. heilongjiangensis. However, as the 17 kDa antigen gene is highly conserved among SFG and TRG Rickettsia, these findings should be considered provisional and confirmed by multilocus sequence typing (MLST) using more variable loci such as ompA, ompB, gltA, or sca4 [52]. Previous molecular work by Cordier et al. [53] identified Rickettsia sibirica mongolitimonae from an eschar skin biopsy of a traveller returning from Sri Lanka. Additionally, several pathogenic Rickettsia species have been detected in local tick populations, including R. africae and R. massiliae, both recognized human pathogens [2, 6]. Local genotypes 2 and 3 in the present study exhibited unique SNPs distinguishing them from R. africae (OP974426.1), R. massiliae (MF098402.1), and R. sibirica (NZ_AHZB01000018.1), suggesting the co-circulation of multiple zoonotic Rickettsia species in Sri Lanka. Together with the molecular detection of R. felis, these results highlight the central hills as a potential hotspot of Rickettsia diversity [9, 31, 32].

Phylogenetic analysis clustered the local representative sequences with isolates obtained from patients with Spotted Fever Rickettsiosis in countries such as India, China, Italy, and Japan, indicating that Sri Lanka harbours rickettsial strains closely related to globally distributed human-pathogenic Rickettsia species. However, because the 17 kDa antigen gene provides limited phylogenetic resolution, particularly among SFG these relationships should be considered tentative and further validated through multilocus sequence typing using more variable loci.

No statistically significant associations were found between seropositivity and demographic variables however, most seropositive individuals were middle-aged rural females, consistent with previous studies linking infection risk to occupational and environmental exposure [29, 54]. Ecological factors in these regions such as dense vegetation, the presence of reservoir hosts, and frequent human–vector interactions likely contribute to the sustained transmission of Rickettsia in central hills.

Several methodological limitations warrant consideration. The use of a single conserved gene (17 kDa) restricted species-level resolution; sample collection may not have coincided with peak bacteraemia; only IgG IFA was performed; demographic data were incomplete; and the absence of concurrent vector or reservoir sampling precluded assessment of transmission pathways. Future integrative surveillance combining multilocus sequencing, serology and vector–host investigations will be crucial to elucidate transmission routes and the broader epidemiological landscape.

Despite these constraints, this study provides the first molecular evidence of R. felis in human blood in Sri Lanka, together with genetic indications of other SFG Rickettsia species. These findings underscore a potentially under-recognised public-health threat and emphasise the need for continued molecular and ecological surveillance to guide targeted control measures for rickettsial diseases in endemic regions of the country.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

⁰C

Degree Celsius

µL

Microliter

µM

Micromolar

NCBI

National Center for Biotechnology Information

BLAST

Basic Local Alignment Search Tool

bp

Base pair

DNA

Deoxyribonucleic Acid

EDTA

Ethylenediaminetetraacetic Acid

GIS

Geographic Information System

IFAT

Indirect Fluorescent Antibody Test

IgM

Immunoglobulin M

IgG

Immunoglobulin G

IFA

Indirect immunofluorescence assay

kDa

Kilodalton

MEGA11

Molecular Evolutionary Genetics Analysis version 11

PCR

Polymerase Chain Reaction

rpm

Revolutions per minute

STG

Scrub Typhus Group

SFG

Spotted Fever Group

SNPs

Single Nucleotide Polymorphisms

SL

Sri Lanka

SPSS

Statistical Package for the Social Sciences

TG

Typhus Group

TRG

Transitional Group

Author contributions

Resources & Conceptualization- DS. Investigation & data analysis- SS, DS. Supervision- RPVJ, SAM, DS. Writing original draft- SS, Review & editing- RPVJ, SAM, DS.

Funding

University Research Grant (URG/2022/67/V), University of Peradeniya, Sri Lanka.

Data availability

All data generated or analyzed during this study are included in this published article. Additional datasets are available from the corresponding author upon reasonable request.

Declarations

Ethical approval and consent to participate

Ethical approval obtained from the Ethical Committee, Faculty of Medicine, University of Peradeniya (No: 2019EC63). Written informed consent obtained from all participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.