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Siraj A Khan, Trishna Bora, Jeromie Wesley Vivian Thangaraj, Manoj V Murhekar, Spotted Fever Group Rickettsiae among Children with Acute Febrile Illness, in Gorakhpur, India, Journal of Tropical Pediatrics, Volume 67, Issue 3, June 2021, fmaa031, https://doi.org/10.1093/tropej/fmaa031
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Abstract
Seasonal outbreaks of acute encephalitis syndrome have been occurring in Gorakhpur division in the Indian state of Uttar Pradesh during monsoon and post-monsoon months. Orientia tsutsugamushi was identified as the major aetiology of these outbreaks. Orientia tsutsugamushi was also identified as one of the important aetiology of febrile illness among children attending peripheral health facilities. The present study was undertaken to detect antibodies against spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) among children with acute febrile illness presenting at peripheral health facilities in Gorakhpur district. Of the 224 blood samples tested, SFGR infection was detected in 13 (6%) patients. None of the samples tested positive for TGR.
INTRODUCTION
Among the emerging diseases, rickettsial diseases have attracted a worldwide attention in recent times. Rickettsial diseases are caused by intracellular Gram-negative bacteria belonging to the genus Orientia and Rickettsia and are transmitted by an infected mite, tick, flea and lice [1]. Scrub typhus (ST), caused by Orientia tsutsugamushi, is one of the most reported rickettsial diseases in India [2]. Gorakhpur and neighbouring districts in the eastern Uttar Pradesh, India have recorded seasonal outbreaks of acute encephalitis syndrome (AES) with high mortality. A few reports have identified ST as one of the predominant aetiology of AES and a major cause of acute febrile illness (AFI) in Uttar Pradesh and other states of India [3, 4]. Beside ST, other major rickettsial diseases spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) have been identified as causes of AFI in limited studies [5, 6]. These diseases if left untreated can lead to severe consequences. The present study was undertaken to detect antibodies against SFGR and TGR among children with AFI presenting at peripheral health facilities in Gorakhpur district.
METHODS
A surveillance for AFI (defined as acute onset of fever for ≥3 days among children aged 1–15 years) was established in three primary/community health centres in Gorakhpur during August–October 2016 [7]. Three millilitre blood samples were collected from children attending these facilities after obtaining written informed consent. Our previous study demonstrated 40 (17.9%, 95% confidence interval 13.3–23.3) sera to be positive for IgM antibodies against O. tsutsugamushi [7]. This study was approved by the Institutional Ethical Committee, Indian Council of Medical Research-National Institute of Epidemiology, Chennai.
In the present study, serum samples were screened for SFGR and TGR by using an indirect ELISA assay employing purified whole-cell antigens of Rickettsia conorii (for SFGR) and Rickettsia typhi (for TGR) for detection of Immunoglobulin G (IgG) antibodies as described elsewhere [8–14]. Briefly, plates were coated with individual antigens and incubated at 4°C for 48 h. After incubation, plates were washed with freshly prepared wash buffer (1× Phosphate Buffer Saline containing 0.1% Tween-20). Following this, the coated plates were blocked with 5% skimmed milk and incubated for an hour at room temperature. Serum samples are initially subjected for the screen ELISA at 1:100 serum dilution followed by a stringent titre ELISA at 4-fold dilutions (1:100 to 1:6400). After plate incubation, plates were washed thrice with wash buffer and 100 µl of prepared serum samples were added to the respective wells of the plate with and without the antigen. The plates were again incubated for an hour at room temperature. After incubation, 100 μl of horseradish peroxidase-conjugated goat anti-human IgG (Promega, Madison, WI, USA) as a secondary antibody was added to each well. The plates were then incubated for an hour. The plates were again washed thrice with wash buffer followed by addition of 100 µl of 3, 3′, 5, 5′-tetramethylbenzedine substrate. After a 10 min incubation, 1N H2SO4 (stop solution) was added. Concentration was determined by measuring the optical density (OD) at 450 nm. For each assay, one positive and three negative controls were used in every test.
The screen ELISA positive samples whose OD >0.5 were further subjected for the titre ELISA. Here, serum samples were tested at 4-fold dilutions (1:100, 1:400, 1:1600 and 1:6400). Samples with net absorbance (sum of the OD of all the four dilutions) of ≥1.000 were taken as confirmed positive. The inverse of the highest dilution where OD ≥0.200 was obtained was taken as the ELISA titre. All samples were also screened for Immunoglobulin M (IgM) against dengue and Leptospira.
RESULTS
A total of 224 blood samples were collected of which 118 were male and 106 were female. Antibodies against SFGR and TGR were tested for 217 patients’ sera. IgG antibodies against SFGR were detected in 13 (6%) patients, of whom 8 were boys. Titre ELISA demonstrated SFGR antibodies at dilution 400 in 8 samples; 1600 in 4 samples and 6400 in 1 sample. Fever was recorded in 100% [duration ranging from 4 to 15 days (median 7 days)], followed by headache (n = 6), vomiting (n = 3), skin rash (n = 2), cough (n = 4), conjunctival congestion (n = 3), hepatomegaly (n = 2), lymphadenopathy (n = 1), splenomegaly (n = 1) and facial swelling (n = 1) (Table 1). None of the cases tested positive for TGR. IgM antibodies against dengue and leptospirosis were detected in 7.6% (17/224) and 3.1% (7/224), respectively. Mixed infections were also recorded. Three cases had antibodies against dengue and SFGR, while one case was positive for dengue, leptospirosis and SFGR. The presence of antibodies against these diseases either suggests that the patient had been infected with multiple infections at a time or possibly due to the presence of background sero-prevalence against SFGR.
| . | Patient 1 . | Patient 2 . | Patient 3 . | Patient 4 . | Patient 5 . | Patient 6 . | Patient 7 . | Patient 8 . | Patient 9 . | Patient 10 . | Patient 11 . | Patient 12 . | Patient 13 . |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Gender (male/female) | M | M | F | M | M | M | M | M | F | M | F | F | F |
| Age | 15 | 12 | 7 | 12 | 15 | 4 | 13 | 10 | 5 | 2 | 8 | 8 | 5 |
| Signs and symptoms | |||||||||||||
| Fever | + | + | + | + | + | + | + | + | + | + | + | + | + |
| Headache | + | + | − | − | + | − | − | + | + | − | + | − | − |
| Vomiting | − | + | + | − | − | + | − | − | − | − | − | − | − |
| Skin rash | − | − | − | − | − | − | − | − | − | − | + | − | + |
| Cough | + | − | − | + | − | − | − | − | − | + | + | − | − |
| Conjunctival congestion | − | − | + | − | − | + | − | − | − | − | + | − | − |
| Hepatomegaly | − | − | + | − | − | + | − | − | − | − | − | − | − |
| Lymphadenopathy | − | − | − | − | − | − | − | − | − | − | + | − | − |
| Splenomegaly | − | − | − | − | − | + | − | − | − | − | − | − | − |
| Facial swelling | − | − | + | − | − | − | − | − | − | − | − | − | − |
| Peri-orbital oedema | − | − | + | − | − | − | − | − | − | − | − | − | − |
| Abdominal pain | − | − | − | − | − | − | − | − | − | − | + | − | − |
| . | Patient 1 . | Patient 2 . | Patient 3 . | Patient 4 . | Patient 5 . | Patient 6 . | Patient 7 . | Patient 8 . | Patient 9 . | Patient 10 . | Patient 11 . | Patient 12 . | Patient 13 . |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Gender (male/female) | M | M | F | M | M | M | M | M | F | M | F | F | F |
| Age | 15 | 12 | 7 | 12 | 15 | 4 | 13 | 10 | 5 | 2 | 8 | 8 | 5 |
| Signs and symptoms | |||||||||||||
| Fever | + | + | + | + | + | + | + | + | + | + | + | + | + |
| Headache | + | + | − | − | + | − | − | + | + | − | + | − | − |
| Vomiting | − | + | + | − | − | + | − | − | − | − | − | − | − |
| Skin rash | − | − | − | − | − | − | − | − | − | − | + | − | + |
| Cough | + | − | − | + | − | − | − | − | − | + | + | − | − |
| Conjunctival congestion | − | − | + | − | − | + | − | − | − | − | + | − | − |
| Hepatomegaly | − | − | + | − | − | + | − | − | − | − | − | − | − |
| Lymphadenopathy | − | − | − | − | − | − | − | − | − | − | + | − | − |
| Splenomegaly | − | − | − | − | − | + | − | − | − | − | − | − | − |
| Facial swelling | − | − | + | − | − | − | − | − | − | − | − | − | − |
| Peri-orbital oedema | − | − | + | − | − | − | − | − | − | − | − | − | − |
| Abdominal pain | − | − | − | − | − | − | − | − | − | − | + | − | − |
| . | Patient 1 . | Patient 2 . | Patient 3 . | Patient 4 . | Patient 5 . | Patient 6 . | Patient 7 . | Patient 8 . | Patient 9 . | Patient 10 . | Patient 11 . | Patient 12 . | Patient 13 . |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Gender (male/female) | M | M | F | M | M | M | M | M | F | M | F | F | F |
| Age | 15 | 12 | 7 | 12 | 15 | 4 | 13 | 10 | 5 | 2 | 8 | 8 | 5 |
| Signs and symptoms | |||||||||||||
| Fever | + | + | + | + | + | + | + | + | + | + | + | + | + |
| Headache | + | + | − | − | + | − | − | + | + | − | + | − | − |
| Vomiting | − | + | + | − | − | + | − | − | − | − | − | − | − |
| Skin rash | − | − | − | − | − | − | − | − | − | − | + | − | + |
| Cough | + | − | − | + | − | − | − | − | − | + | + | − | − |
| Conjunctival congestion | − | − | + | − | − | + | − | − | − | − | + | − | − |
| Hepatomegaly | − | − | + | − | − | + | − | − | − | − | − | − | − |
| Lymphadenopathy | − | − | − | − | − | − | − | − | − | − | + | − | − |
| Splenomegaly | − | − | − | − | − | + | − | − | − | − | − | − | − |
| Facial swelling | − | − | + | − | − | − | − | − | − | − | − | − | − |
| Peri-orbital oedema | − | − | + | − | − | − | − | − | − | − | − | − | − |
| Abdominal pain | − | − | − | − | − | − | − | − | − | − | + | − | − |
| . | Patient 1 . | Patient 2 . | Patient 3 . | Patient 4 . | Patient 5 . | Patient 6 . | Patient 7 . | Patient 8 . | Patient 9 . | Patient 10 . | Patient 11 . | Patient 12 . | Patient 13 . |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Gender (male/female) | M | M | F | M | M | M | M | M | F | M | F | F | F |
| Age | 15 | 12 | 7 | 12 | 15 | 4 | 13 | 10 | 5 | 2 | 8 | 8 | 5 |
| Signs and symptoms | |||||||||||||
| Fever | + | + | + | + | + | + | + | + | + | + | + | + | + |
| Headache | + | + | − | − | + | − | − | + | + | − | + | − | − |
| Vomiting | − | + | + | − | − | + | − | − | − | − | − | − | − |
| Skin rash | − | − | − | − | − | − | − | − | − | − | + | − | + |
| Cough | + | − | − | + | − | − | − | − | − | + | + | − | − |
| Conjunctival congestion | − | − | + | − | − | + | − | − | − | − | + | − | − |
| Hepatomegaly | − | − | + | − | − | + | − | − | − | − | − | − | − |
| Lymphadenopathy | − | − | − | − | − | − | − | − | − | − | + | − | − |
| Splenomegaly | − | − | − | − | − | + | − | − | − | − | − | − | − |
| Facial swelling | − | − | + | − | − | − | − | − | − | − | − | − | − |
| Peri-orbital oedema | − | − | + | − | − | − | − | − | − | − | − | − | − |
| Abdominal pain | − | − | − | − | − | − | − | − | − | − | + | − | − |
Basic haematological and biochemical investigations were not routinely done in the study health facilities and also were not part of the AFI surveillance. All the 13 cases were treated symptomatically with acetaminophen. Additionally, 4 of the 13 patients were treated with cephalosporin. None of them received doxycycline or azithromycin. Only 8 of the 13 patients could be followed up telephonically and all the 8 patients recovered from the illness.
DISCUSSION
Gorakhpur division has been experiencing periodic outbreaks of AES since several years [3, 15, 16]. ST has been increasingly reported as a major cause of AES and AFI [3, 16]. There, however, is scanty information on occurrence of other rickettsial diseases in this region. A community-based sero-survey conducted among healthy individuals in Gorakhpur in 2016 documented the first evidence of SFGR and TGR sero-positivity in both paediatric and adult population during October and November months, when the AES cases in the region peaked [5]. Our study provides additional evidence of the presence of SFGR as a potential cause of AFI in paediatric population. We found sero-positivity of SFGR in 6% of the AFI patients.
Across the Southeast Asia region, 17 species of rickettsiae causing SFGR have been recognized [17]. However, in India, the disease is underestimated due to lack of facilities for laboratory diagnosis, as well as overlapping clinical presentation with ST. Due to lack of specific clinically distinguishing features of the two diseases and sero-diagnostic cross-reactivity, it is quite possible that ST may have been over-represented while SFGR remains underreported. Thus, the actual case burden of SFGR remains unclear. More information needs to be generated in terms of clinical and laboratory spectrum of rickettsial diseases for differential diagnosis in India.
The present study had few limitations. The study was cross-sectional in nature and blood samples were collection one-time point, hence definitive diagnosis of current infection could not be established. Second, we could not conduct molecular tests to confirm SFGR infection, due to sample constraints. Nevertheless, this study as well as the study conducted by Mane et al. [5] provides credible evidence about the presence of SFGR in this region.
In conclusion, the present study highlights the endemicity of SFGR in Gorakhpur. There is an urgent need to understand the seasonality and disease epidemiology, together with the development of affordable diagnostic tools. Comprehensive knowledge about the diseases, role of vectors and dissemination of preventive measure among the clinicians as well as the local populace needs to be prioritized.
Acknowledgements
The authors thank Jahnabi Saikia, Kumud Sonar and Deep Panika for technical assistance in the laboratory and they also thank Dr Christina M. Farris, Naval Medical Research Center (NMRC), Maryland, USA, for supplying antigen for the tests.
Funding
This work was supported by the Indian Council of Medical Research.
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