|Year : 2018 | Volume
| Issue : 2 | Page : 144-150
Serological evidence of spotted fever group rickettsiosis in and around Puducherry, south India—A three years study
Selvaraj Stephen1, Stanley Ambroise2, Dhandapany Gunasekaran1, Mohammed Hanifah1, Balakrishnan Sangeetha1, Jothimani Pradeep1, Kengamuthu Sarangapani2
1 Department of Microbiology, Paediatrics and General Medicine, Mahatma Gandhi Medical College and Research Institute, Sri Balaji Vidyapeeth (Deemed-to-be-University), Puducherry, India
2 Department of General Medicine and Microbiology, Indira Gandhi Govt. General Hospital and Post Graduate Institute, Puducherry, India
|Date of Submission||08-Sep-2017|
|Date of Acceptance||03-May-2018|
|Date of Web Publication||1-Oct-2018|
Professor of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pondy-Cuddalore Main Road, Pillaiyarkuppam, Puducherry-607 403
Source of Support: None, Conflict of Interest: None
Background & objectives: Rickettsial diseases are important re-emerging infections that mostly go unnoticed or are misdiagnosed. Though few case reports of Indian tick typhus have been reported in Indian literature in the past 10 yr, prevalence surveys are few and far between. The objective of this research was to study the seroprevalence of spotted fever (SF) group rickettsiosis and its coinfection with scrub typhus (ST) in Puducherry region of south India, as these two diseases may show similar clinical presentations.
Methods: During 2012–2015, paired sera of 320 febrile patients were examined for Rickettsia conorii IgM/IgG by ELISA and OX19 and OX2 agglutinins by Weil-Felix test. Additionally, patients were screened for ST IgM ELISA. Statistical analysis was performed for clinical and laboratory parameters in children and adults using Fisher’s exact test and chi-square test with Yates correction.
Results: Out of 320 patients, 142 (44.38%) had R. conorii IgM and/or IgG antibodies. Only IgM was present in 72 (22.5%) patients, while 36 patients were positive for IgG only and 34 were positive for both IgG and IgM. A total of 68 patients (21.25%) showed only OX19 and/or OX2 antibodies (titres ≥ 1 : 80). SF and ST coinfection was observed in 47 cases (14.69%).
Interpretation & conclusion: Seroprevalence of SF in Puducherry was found to be quite high (44.38%). ST and SF coinfection was observed in 34.50% of the SG IgG positive patients, however, this require further evaluation by PCR to rule out cross-reaction or false positivity. At present ELISA seems to be an affordable alternative to highly subjective and technically demanding immunofluorescence assay (IFA) for serodiagnosis of SF.
Keywords: ELISA; Indian tick typhus; Rickettsia conorii; scrub typhus; spotted fever group rickettsiosis; Weil-Felix test
|How to cite this article:|
Stephen S, Ambroise S, Gunasekaran D, Hanifah M, Sangeetha B, Pradeep J, Sarangapani K. Serological evidence of spotted fever group rickettsiosis in and around Puducherry, south India—A three years study. J Vector Borne Dis 2018;55:144-50
|How to cite this URL:|
Stephen S, Ambroise S, Gunasekaran D, Hanifah M, Sangeetha B, Pradeep J, Sarangapani K. Serological evidence of spotted fever group rickettsiosis in and around Puducherry, south India—A three years study. J Vector Borne Dis [serial online] 2018 [cited 2018 Oct 21];55:144-50. Available from: http://www.jvbd.org/text.asp?2018/55/2/144/242562
| Introduction|| |
Due to the paucity of seroprevalence studies on Indian tick typhus throughout India, the rickettsiosis caused by Rickettsia conorii, remains under-diagnosed,,. The disease has been reported from the states of Tamil Nadu, Kerala, Karnataka, Andhra, Maharashtra, New Delhi, Uttar Pradesh, Jammu & Kashmir, Himachal Pradesh, Haryana, Assam, Bihar, Rajasthan, West Bengal and Meghalaya and its zoonotic nature has been documented in several studies,,,,,,,,,,,,,,,,,,,,. Clinical features of spotted fever (SF) resemble that of scrub typhus, dengue and other viral diseases and hence may be difficult to differentiate by the clinicians. Few case reports,,,,, and prevalence surveys of SF rickettsioses in both children and adults have appeared in the last few years from south as well as few other areas of India. The serological evidence was based on nonspecific Weil-Felix (WF) tests and/or specific tests with SF antigens,,,,,,,,,,,,,,,,,. Immunofluorescence assay (IFA) is the gold standard serological test for diagnosis of rickettsial infections including spotted fever,,,,,,, but it is highly subjective and technically demanding test. Unlike ELISA, which is a qualitative test, IFA is a quantitative serological test and helpful in interpreting the results based on seroconversion and four-fold changes in titres of paired samples. Molecular diagnosis of spotted fever has been recently reported by Prakash et al. Since, the present status of SF in Puducherry and surrounding Tamil Nadu (south India) is not known, our objective was to study the seroprevalence and epidemiological features of spotted fever group (SFG) rickettsiosis along with its coinfection status with scrub typhus, among the febrile patients as both these rickettsioses present similar clinical presentations.
| Material & Methods|| |
This prospective study was conducted from October 2012 to June 2015 in the Departments of Microbiology, Paediatrics and General Medicine of Mahatma Gandhi Medical College and Research Institute (MGMC & RI), Puducherry; and Departments of Microbiology and General Medicine of Indira Gandhi Government General Hospital and Post Graduate Institute (IGGGH & PGI), Puducherry.
Approval for this ICMR task force ad-hoc research project was obtained from the Institutional Human Ethical Committee (IHEC), MGMC & RI (dated 29 November 2010) and IGGGH & PGI, Puducherry (dated 13 November 2010). This study is in compliance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Patients' informed consent was obtained prior to the collection of blood samples. In case of children, consent was obtained from parents or care takers.
Sample collection, storage and processing
A total of 470 patients were initially registered (including in-patients and out-patients) for this study. However, only 320 patients participated voluntarily to provide paired samples—Acute and convalescent, at intervals of 14–21 days. This included 125 children (≤ 18 yr) and 195 adults (≥ 18 yr). Blood was collected aseptically in a sterile clot activator tube and sera were separated, aliquoted and kept at −20 °C for carrying out SF ELISA IgM/IgG, ST IgM ELISA and WF tests. Blood samples were collected by duty nurses/phlebotomists at IGGGH & PGI and MGMC & RI, Puducherry. Experiments for SF, ST were carried out in the Department of Microbiology, MGMC & RI, Puducherry.
Inclusion and exclusion criteria
These were set as described previously for ST investigation. Briefly, the inclusion criteria were high grade fever with or without chills and rigour; eschar/hepatosplenomegaly/jaundice/lymphadenopathy/thrombocytopenia; malaise, myalgia, nausea, vomiting; and capillary leak syndrome. Exclusion criteria were known cases of immunocompromised patients like AIDS/lymphomas; malignancy secondaries; bleeding disorders and fever of >4 wk duration (pulmonary tuberculosis, etc.).
Initially, haemogram and biochemical tests for liver enzymes were carried out. Then, additional tests were performed whenever there was a suspicion of malaria (peripheral blood smear and malaria antigen detection test—Malaria Pf/Pv Ag, SD Biosensor Healthcare, Haryana), dengue (NS1/IgM/IgG detection—SD Bioline Dengue Duo kit, Seoul, South Korea), leptospirosis (SD Bioline Leptospira IgM/IgG, Seoul, South Korea), typhoid (Widal test—Span Diagnostics Ltd, India), urinary tract infection (Urine culture) etc, according to requirement as described earlier.
Demographic details of patients
The patients came from urban (126), peri-urban (99) as well as rural areas (95) of Puducherry and surrounding Districts of Tamil Nadu, like Cuddalore (Kurinjipadi, Panruti, Chidambaram, Virudhachalam, Kattumannarkovil, Tittakudi and Vadalur taluks), Villupuram, Ariyalur, Nagapattinam and Kanchipuram.
Regarding the occupation of the patients, there were 125 (0–3 yr: 30, 3–5 yr: 19, school going: 76) children and 29 college students. Out of 68 women, 60 were house-wives and eight were labourers. Among the 98 men, 55 were retired personnel, 14 were farmers and nine were labourers. Each of the cleaners, drivers and building construction workers were three in number. There were two mechanics and managers each. Similarly, there was one painter, railway trackman, storekeeper, canteen supplier, foreman, civil supplier and accountant each.
Animal contact: In total 57 patients indicated a history of having domestic animals and pets like cattle, goat, dog, cat, chicken, parrot and pig. Three of them lived near the animal farms.
For the seroprevalence, SF group specific Rickettsia conorii IgM/IgG ELISA (Vircell, Granada, Spain) and Weil-Felix test for OX19 and OX2 antibodies were performed. In order to rule out scrub typhus, ST IgM ELISA (InBios International, Seattle, USA) was done. WF test was carried out with plain OX19 and OX2 antigen purchased from the King Institute of Preventive Medicine and Research, Chennai, Tamil Nadu. Six doubling dilutions ranging from 1 : 20 to 1 : 640 to exclude pro-zone phenomenon were done. Further, titrations were carried out if the titres were ≥1 : 640.
SF IgM and IgG ELISA plates were coated with R. conorii antigen, Moroccan strain (ATCC VR-141). Positive and negative controls were provided by SF ELISA kit manufacturers, Vircell Microbiologica, Spain. Test was performed strictly according to the manufacturer’s instructions. Briefly, for both ELISA tests, the total volume was 105 μl per well. The reagents for R. conorii IgM ELISA were added in the following order: Vircell IgG Sorbent (25 μl) + patients’ samples (5 μl) + serum diluent (75 μl). In R. conorii IgG ELISA about 5 μl patient serum was added to 100 μl serum diluent. For both IgM and IgG ELISA, 5 μl each of positive control, negative control and cutoff control (in duplicates) were included. Plates were incubated at 37 °C for 45 min followed by washing. After the addition of IgM/IgG conjugate (100 μl), plates were incubated at 37 °C for 30 min followed by washing. Substrate (100 μl) was added and incubated for 20 min in the dark. The reaction was stopped by the addition of 50 μl of stop solution. Optical density (OD) readings were taken at 450 nm with 620 nm as reference filter in ELISA reader (Bio-Rad, Japan). The results were calculated using the following formula as outlined in the technical brochure of the kit: Antibody index = (Sample OD)/Cutoff serum mean OD × 10.
The interpretation of the results was made as follows: Antibody index <9 were considered negative, 9–11 were equivocal (needed repeat testing) and >11 were considered as positive for specific antibodies against R. cono-rii/SFG rickettsioses. ST IgM ELISA (Scrub typhus, In Bios International, Seattle, USA) was carried out in accordance with the instructions of the kit manufacturer’s and as described earlier,.
Based on the serological surveys conducted in the early 1970–90, a national prevalence of 25% was considered for sample size (n) calculation using the following formula: n = [t2 × p (1−p)]/m2; where, t = Confidence level at 95% (standard value of 1.96), p = Estimated prevalence of the disease, m = Margin of error at 5% (standard value of 0.05). As per the above formula, the number of patients required were 275, but we added another 45 patients (n = 320).
Patients’ data were analyzed by Fisher’s exact and chi-square test with Yates correction for small number of samples using Graph Pad Quick Calcs (Graph Pad Software Inc, USA) and p-values of ≤ 0.05 were regarded as statistically significant.
The clinical diagnosis of SF was based on the CDC guidelines which defines it as “fever with rash/eschar/headache/myalgia/anaemia/thrombocytopaenia/hepatic transaminase elevation”. As per CDC, If there is fourfold increase in paired sera by IgG IFA, it may be called laboratory confirmed SF, whereas if there is evidence of elevated IgG or IgM by IFA, ELISA, dot-ELISA, or latex agglutination, it may be called laboratory supportive SF.
| Results|| |
The result of serological tests carried out in 320 patients is presented in [Table 1]. Patients’ age ranged from 1–89 yr with a mean ± SD 26.58±19.99 (CI 24.39–28.77). Based on CDC criteria of laboratory supportive SF, a retrospective diagnosis of spotted fever could be made in 142 patients (44.38%). In WF test only, 68 (21.25%) patients were positive with OX19 and/or OX2 with titres ranging from 1 : 80 to 1 : 320 [Table 2]. A total of 37 (11.56%) patients who were positive in SF IgM and/or IgG had OX19 and/or OX2 agglutinins. Among 125 children screened for SF, 51 (40.8%) were seropositive and out of 195 adults, 91(46.6%) were seropositive. Clinical and laboratory parameters of the 142 patients are detailed in [Table 3]. Statistically, significant numbers of adults compared to children, were positive for thrombocytopaenia, increased levels of liver enzymes and serum creatinine. Similarly, more adults exhibited signs and symptoms of headache, myalgia, chills and rigor, nausea and complications (p ≤0.05). However, only 70 patients were positive, according to the criteria of SF IgG positivity (±SF IgM). No statistical difference was observed between children and adults in these 70 patients. This group had only nine children, but 61 adults [Table 4]. SF IgM and/or IgG ELISA positivity in 142 patients with reference to their duration of illness was divided into two groups, viz, those with fever of 1 to 7 days duration and others whose febrile illness was of >7 days [Table 5].
|Table 1: Serological response of patients to SF IgM/IgG/OX19/OX2 (n = 320)|
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|Table 2: Details of patients with significant (≥1 : 80) titres of OX19 and/or OX2|
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|Table 3: Clinical and laboratory profile of SF in children vs adults (SF IgM and/or IgG)|
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|Table 4: Clinical and laboratory profile of SF in children vs adults based on IgG ± IgM positivity|
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|Table 5: Duration of febrile illness and SF IgM and IgG positivity in patients (n = 142)|
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History of contact with domestic animals like cattle, sheep, goat, dog, hen and cat was observed/confirmed in 21 SF seropositive patients. Distribution of SF in different geographical areas was as follows: Urban 50% (63/126), peri-urban 25.25% (25/99) and rural 56.84% (54/95). Statistical analysis of these three groups showed that the difference was highly significant (p = 0.000015).
Based on ST IgM ELISA, 189 (59.06%) among 320 patients screened, were positive for ST. Coinfection/cross reactivity was observed between SF and ST in 49(34.50%) out of 70 SF IgG positive patients. Compared to this, 67(93.06%) out of 72 SF IgM positive patients had coinfection with ST, which is quite high and most likely due to false SF IgM positivity. Mixed infection of SF was observed with leptospirosis, falciparum malaria and vivax malaria, one case each [Table 6]. Patients found positive for malaria were given antimalarial drugs. One DHF positive patients was given supportive treatment and platelets were transfused. ST/SF patients were administered doxycycline (adults)/azithromycin (children).
| Discussion|| |
Clinical case definition of spotted fever is almost similar to scrub typhus, dengue and other viral diseases. CDC criterion of laboratory confirmed SF includes isolation of the rickettsia from the clinical samples/demonstration of SFG DNA by PCR in patient’s blood or eschar/demonstration of SFG antigen in biopsy by immunohistochemistry (IHC)/fourfold change in IgG titres to SFG rickettsia in IFA.
Isolation of SFG rickettsiae requires BSL-3 facilities since these are class ‘B’ pathogens of bioterrorism and hence, are outside the scope of most of the laboratories in India and developing countries,. IFA kits are presently imported and are quite expensive. The test itself is technically demanding and is highly subjective, requiring sufficient expertise and an expensive fluorescence microscope. Practically, SF ELISA test appears an alternative to IFA under the present circumstances, although it is only a qualitative test and titres in serum can not be estimated. R. conorii Vircell IgM/IgG ELISA kit has been satisfactorily validated by Indian as well as foreign workers,,,,. According to the manufacturers of the kit, the sensitivity and specificity of Vircell R. conorii IgM ELISA kits are 94 and 95%; and for IgG ELISA kits, these are 85, 100%, respectively. Do et al, in their study observed that the sensitivity for R. conorii Vircell IgM + IgG ELISA was 90% and specificity was 100%.
IgM positivity alone is not considered as a proof of acute SFG by different Rickettsiologists,,. This is because SFG IgM may be falsely positive in different diseases including Q fever and it may have a prolonged presence in blood,. However, Tripathi et al recently diagnosed cases of acute SF on the basis of R. conorii IgM antibody only, by ELISA and IFA.
In the present study 142 among 320 patients (44.38%) were positive for R. conorii IgM and/or IgG antibodies in acute and/or convalescent blood samples. Analysis of 72 (22.5%) patients with only SF IgM positivity showed that 26 of them were positive in acute, 15 in convalescent (seroconversion) and 31 in both acute and convalescent specimens. The patients showing seroconversion (15) for SF IgM might qualify for consideration as acute SF. If, 72 samples which were only R. conorii IgM positive are excluded as per CDC criteria, then the cases of ‘Laboratory supportive SF’ would be 70 (21.88%) (IgG positivity ± IgM positivity). Among these 70 patients, R. conorii IgG (only) was detectable in 36 patients comprising of eight acute samples, six convalescent sera (seroconversion) and 22 acute + convalescent specimens. R. conorii IgG together with IgM was observed in 34 patients, viz. three acute specimens, one convalescent serum (seroconversion) and 30 paired samples. IgM positivity in single acute specimens cannot be ignored altogether, especially in the early phase of illness. As detailed in [Table 5], 30 patients with only IgM, had fever of 1 to 7 days duration. Perhaps PCR of these samples might have identified SFG DNA. In a similar manner, less percentage of PCR positivity may be expected in patients who had fever of >1 wk duration. Biopsies from eschar samples yield higher PCR positivity than whole blood. Presence of IgG alone in the acute samples may be related to past infection. Antibody response to R. conorii is group specific and not species-specific.
Only few seroprevalence studies based on specific rickettsial tests (ELISA/IFA) have been reported in India during the past 10 yr, 12,. Seroprevalence of SF in children of Tamil Nadu was found to be 7.78% during 2003–2004 by Somashekar et al. Kalal et al, from Karnataka observed that 37.1% hospitalized children were seropositive for SFG rickettsia based on the IgG ELISA result in acute samples and some paired sera. Koraluru et al from Karnataka reported that 2.1% cases were positive for SFG based on IFA. Recently, Tripathi et al from Uttar Pradesh reported seroprevalence of spotted fever in 42.15% in children and 17.10% adults. Their observation was based on IgM ELISA and IgM IFA positivity on acute samples only. While the finding of this study with respect to SF prevalence in children (40.8%) is similar to Tripathi et al, the percentage of adults with SF is quite high (46.6%).
Weil-Felix test is a nonspecific test for rickettsial diseases, which is based on detection of antibodies to various Proteus antigens with cross reacting epitopes to antigens of genus Rickettsiae (except R. akari) The test has a low sensitivity and specificity for diagnosis of these infections.WF test should be used only in those settings where specific tests are not available and results must be interpreted with caution and clinical correlation,,,,,. Therefore, 68 patients who were found positive in WF only were not taken into account. Thirty-seven out of 142 SF IgM and/or IgG positive patients had agglutinins to OX19 and/or OX2.
A recent report by Prakash et al has utilized molecular methodology (PCR) for diagnosis of SFG. It should be noted that though PCR was 100% specific, it showed low sensitivity.
In the present study coinfection of ST with SF was observed in 49 (14.06%) out of 320 patients screened. Similar coinfection rate between ST and SF has been reported by others as well. In a study by Kalal et al from Karnataka, 14 (13.6%) out of 103 children examined showed dual infection with ST and SF. Faruque et al from Bangladesh identified 45 (6%) coinfection cases out of 720 patients examined. These studies require further evaluation to see if this is a cross-reaction or false positivity. Isolation of rickettsiae from patients/animals and arthropod vectors and submitting them to genetic analysis might throw light on this issue.
| Conclusion|| |
The present study has demonstrated a high seroprevalence of SFG rickettsiosis (44.38%) in Puducherry region of south India. A moderately higher rate of ST and SFG coinfection/cross-reactivity (34.50%) was also observed in the SF IgG positive patients. At present ELISA seems to be a viable alternative to IFA against the serodiagnosis of SF. Use of IFA and PCR for ST/SFG rickettsiosis detection might yield a better confirmatory result on their prevalence.
Conflict of interest: None to declare.
| Acknowledgements|| |
This ad-hoc Task-Force research project (Iris ID No. 2008-08180) was funded by the Indian Council of Medical Research, New Delhi, India. The authors are grateful to the Chairman, Vice-Chancellor, Dean (Medical Faculty) and Dean (Research and Allied Health Sciences) of MGMC & RI, Puducherry, India for providing the necessary laboratory facilities.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]