This was a prospective and laboratory based study, which was conducted during the period February 2018 to March 2019 in the Department of Microbiology, Mahatma Gandhi Medical College and Research Institute and Indira Gandhi Government General Hospital and Post Graduate Institute, Puducherry, India. The work started after getting approval from our Institutional Human Ethics Committee (IHEC) (FACULTY/12/2017/28 dated 10/12/2017). A total of 114 paired serum samples were collected from the febrile patients comprising of both acute and convalescent samples taken at intervals of two to three weeks. Forty sera from healthy ante-natal women and 23 samples from voluntary blood donors were included as controls. Inclusion and exclusion criteria were as reported earlier [25].

The clinical features, demographic details and laboratory results were retrieved from the patients’ case records. All sera were subjected to SF Rickettsia conorii IgG IFA test (Fuller Laboratories, Fullerton, California, USA). The procedure was strictly carried out as per the manufacturer’s instructions. Briefly, the serum samples were diluted 1:64 with Phosphate Buffered Saline (PBS). Ten microlitres of 1:64 diluted samples including the positive and negative controls provided in the kit were added to the antigen spots. The slides were coated with killed and acetone fixed Vero cells some of which were infected with Moroccan strain of R. conorii. Slides were kept in a humidity chamber and incubated at 37°C for 30 minutes, followed by washing with gentle stream of PBS for three times and then placed in a beaker containing PBS and magnetic beads and washed gently for five minutes. Slides were removed, dried and 15 μL of conjugate (affinity purified FITC-labeled goat anti-human IgG (heavy chain) with bovine serum albumin and Evans’ blue counterstain) was added to the wells and further incubated in dark in a humidity chamber at 37°C for 30 minutes. After incubation, the slides were again washed as before, dried, a coverslip of 24×50 mm was placed over the smears and mounted using mounting medium. Slides were observed at 400X magnification of the fluorescent microscope (Primo Star iLEDZeiss Microscope, Germany). The positive reactions showed short pleomorphic rods with apple green fluorescence and negative reaction shows red fluorescence [Table/Fig-1]. The smears were scored as 1+ to 4+. According to the technical brochure of the kit, the significant titre being ≥1:64.

[Table/Fig-1]:

Images of SF R. Conorii IgG IFA. a) Positive Control (≥1:128); b) Negative control.

Mean±Standard deviation with 95% confidence interval for the age and duration of illness of the patients who were positive and negative for R. conorii IgG antibodies was calculated using Graph Pad Quick Calcs Software, USA. Chi-square and Fisher’s-exact tests were performed for numerical variables and p-value ≤0.05 was considered as statistically significant.

Out of 114 febrile patients who provided paired sera and screened for SF IgG IFA, 27 were positive in IgG IFA with titres ranging from 1:128 to 1:2048 (23.68%). Among the control group (n=63), only one participant was seropositive for R. conorii IgG IFA (1.59%). Statistical difference in seropositivity between the febrile patients and healthy controls was quite significant (p=0.0001). While all the voluntary blood donors (n=23) were seronegative, a single ante-natal mother among 40 women was seropositive (2.5%). SF seropositive patients were aged between 9 to 60 years and the mean and SD age with 95% confidence interval was 33.96 (SD=13.76) (28.77-39.15) for R. conorii IgG antibodies positive patients. Among 27 seropositive patients, 16 were females and 11 were males. However, there was no statistical difference between the two sexes (p=0.156). Eleven patients had seroconversion upto titres of 1:128/1:256. Three patients showed four-fold (1:512) and another patient sixteen-fold increase (1:2048) in the convalescent sera. Only one ante-natal woman had significant titer of 1:128 [Table/Fig-2]. None of the voluntary blood donors were positive for R. conorii IgG antibodies. [Table/Fig-3] shows the details of clinical and laboratory parameters, which are statistically significant: duration of illness (p=0.0111), chills, rigor (p=0.0388) and thrombocytopenia (p=0.0079). Other clinical and laboratory parameters like presence of eschar, lymphadenopathy, headache, myalgia and other gastrointestinal symptoms were of no statistical significance. Two patients developed complication with acute renal failure and one Myocarditis. Two patients gave a history of contact with domestic animals like dog/goat/cat.

For the diagnosis of SF IgM antibodies in acute samples are not confirmatory, since false positives are too many with the persistence of IgM for a longer time and hence many authors recommend use of SF IgG IFA only [1-3,28,29]. Seropositivity only in the acute sample is not being helpful in the diagnosis of SFG. According to CDC and Council of State and Territorial Epidemiologists (CSTE) case definition has two categories viz., probable or confirmed cases [2].

Febrile patient with one or more of the following symptoms: rash, eschar, headache, myalgia, anaemia, decreased platelets, or elevated hepatic transaminase.

Elevation of IgG or IgM to SFG by IFA, ELISA or Dot-ELISA.

Probable case: Clinical symptoms with laboratory supportive results and

Confirmed case: Clinical symptoms with laboratory confirmed results.

Since ELISA is a qualitative test, the increase in titres between acute and convalescent samples can be inferred by performing the IFA test, which is quantitative. In the present study, 27 febrile patients were seropositive for R. conorii IgG antibodies by the serological ‘gold standard’ IFA. Among them, 13 belonged to the CDC category of confirmed SF (based on clinical evidence and laboratory confirmation). Another 14 patients come under the CDC category of probable cases of SFG (with clinical evidence and laboratory supportive results). [Table/Fig-2] shows the results of IgG IFA titers of 27 febrile patients.

In USA, the prevalence of SFG was relatively high (15.0%) among aged persons from 60-69 years compared to children of <10 years (3.81%) [2]. However, our findings are different with only one patient was in the age of 60 and four children were aged between 1-18 years. Remaining 22 belonged to the age group of 19-59 years. Kantsø B et al., reported that the positivity for SF was 56% in both children and adults [29]. However, in the present study a moderate sero positivity of 23.68% was observed in our patients (both children and adults). About 1% was positive for R. conorii IgG antibodies in blood donors in Netherlands [29], but in our study none of them were positive. Rickettsial diseases are endemic in Sri Lanka and a prevalence of R. conorii infection of 40.9% using IgG IFA was reported by Kularatne SAM et al., [30]. Several researchers who documented the prevalence of SFG from various places of India employed the non-specific Weil-Felix test with a cut-off titer of ≥1:80 [15-20,25]. In a study conducted in Bangalore, 31.0% of children had SF infection with meningoencephalitis, while in the same state, another study reported 26.35% sero positivity among febrile children [15,19]. Similarly, Puducherry recorded SF sero prevalence of 21.25% [25] while Tamil Nadu showed the least positivity of 4.6% [21] all these results were obtained by the application of Weil-Felix. In Uttar Pradesh 37.1% were sero positive for Indian tick typhus in IgM ELISA [22] whereas 40.8% in children from Puducherry were positive in IgM and/or IgG ELISA [25].

Regarding adults, 17.1% were seropositive in Uttar Pradesh for R. conorii IgM by both ELISA and IFA [24] while 46.6% from Puducherry [25] were seropositive for SFG IgM/IgG ELISA.

In the current study we observed a moderate seropositivity of 23.68% among febrile patients for R. conorii IgG antibodies in IFA. The cut-off titer considered by us was ≥1:128, which has been endorsed by several researchers from India and abroad [10-14,22-24,26]. Somashekar HR et al., from Tamil Nadu reported seroprevalence of 32.6% [20] whereas in much contrast, only 2.1% seropositivity was observed in Karnataka [26] both by the application of IgM IFA.

We could not perform molecular tests for the detection of specific gene targets. Construction of a phylogenetic tree, might throw some light regarding circulation of any new species of SFG rickettsiae other than R.conorii in this part of Southern India.

Present study has established a moderate seroprevalence of Indian tick typhus (23.68%) in Puducherry and surrounding Tamil Nadu, by the application of R. conorii IgG, which is considered as a specific serological test, superior to IgM IFA.