|Year : 2020 | Volume
| Issue : 1 | Page : 59-64
Molecular confirmation & characterization of Rickettsia conorii in north India: A report of three cases
Manisha Biswal1, Kamran Zaman1, Vikas Suri2, Srikanth Gopi2, Abhay Kumar1, T Gopi1, Shashi Vig1, Navneet Sharma2, Ashish Bhalla2
1 Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research, Chandigarh, India
2 Department of Internal Medicine, Postgraduate Institute of Medical Education & Research, Chandigarh, India
|Date of Submission||12-Jan-2018|
|Date of Web Publication||24-Feb-2020|
Dr Ashish Bhalla
Department of Internal Medicine, Postgraduate Institute of Medical Education & Research, Sector 12, Chandigarh 160 012
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background & objectives: In India, spotted fever group rickettsiae (SFGR) are an underdiagnosed cause of acute febrile illness (AFI). The non-specific Weil-Felix test is the first diagnostic modality for the diagnosis of SFGR in many laboratories due to the lack of advanced diagnostic facilities in developing countries. The aim of this study was to detect SFGR using molecular methods in the patients, presenting with AFI in a tertiary care centre in north India.
Methods: Consecutive patients (>14 yr of age) with AFI were enrolled over a six month period. Standard investigations for common pathogens causing AFI in India (malaria, dengue, scrub typhus, leptospirosis and enteric fever) were carried out. In patients who were negative for all of the above investigations, blood was subjected to polymerase chain reaction (PCR) targeting outer membrane protein A (ompA) gene of
Results: Of the 51 patients with an undiagnosed aetiology, three were positive by ompA PCR. Two of the PCR products produced good sequences and BLAST identification confirmed them as Rickettsia conorii. The sequences of R. conorii reported from south India clustered with two previously reported novel rickettsial genotypes. The study sequences clustered in a group different from that of Rickettsia spp. of the south Indian sequences reported earlier.
Interpretation & conclusions: This study showed the existence of R. conorii in north India. Testing for SFGR may be included in the diagnostic workup of AFI for better disease management.
Keywords: Acute febrile illness - India - Rickettsia conorii - rickettsial infection - spotted fever
|How to cite this article:|
Biswal M, Zaman K, Suri V, Gopi S, Kumar A, Gopi T, Vig S, Sharma N, Bhalla A. Molecular confirmation & characterization of Rickettsia conorii in north India: A report of three cases. Indian J Med Res 2020;151:59-64
|How to cite this URL:|
Biswal M, Zaman K, Suri V, Gopi S, Kumar A, Gopi T, Vig S, Sharma N, Bhalla A. Molecular confirmation & characterization of Rickettsia conorii in north India: A report of three cases. Indian J Med Res [serial online] 2020 [cited 2020 Aug 13];151:59-64. Available from: http://www.ijmr.org.in/text.asp?2020/151/1/59/279143
Present address of Kamran Zaman: ICMR-Regional Medical Research Centre, Gorakhpur, Uttar Pradesh, India
Rickettsia conorii- mediated spotted fever may be an underdiagnosed cause of acute febrile illness (AFI) in India, as it has been sporadically reported from this geographical region,,. The mortality rate in spotted fever is variable but can be high if there is a delay in the diagnosis and treatment,,. Most studies on the prevalence of spotted fever group rickettsioses (SFGR) in India are based largely on serological tests such as Weil-Felix,, ELISA,, and immunofluorescence assay. The serological diagnosis has limitations and accurate disease correlation can be made only by DNA detection or by culture. However, the culture of the organism requires biosafety level 3 (BSL-3) containment facilities and is restricted to reference laboratories. In India, the prevalence of R. conorii in febrile patients has been evaluated with the help of polymerase chain reaction (PCR) and sequencing in a study from south India. A novel spotted fever Rickettsia was detected in a Japanese traveller returning from India. Though the tick vector, Rhipicephalus sanguineus sensu lato, has been found in 21 States in India, there are many gaps in the knowledge about the true burden of this infection in India. The aim of the present study was to detect the presence of SFGR in patients presenting with AFI in a tertiary care hospital in north India using molecular diagnosis.
| Material & Methods|| |
Consecutive patients above the age of 14 yr presenting with AFI to the Emergency Medical Ward of Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India, over a six-month period (June-November, 2014) were enrolled in the study. A detailed clinical history was noted. The patients were thoroughly examined for skin rash, eschar and manifestations of bleeding. The standard workup for fever, which included peripheral blood smear and antigen testing for malarial parasites, blood culture by BACTEC, Widal test for enteric fever, NS1 antigen and/or IgM ELISA for dengue, PCR and/or IgM ELISA for scrub typhus and ELISA and/or microscopic agglutination test (MAT) for leptospirosis was performed in the department of Medical Microbiology. In patients negative for all of the above infections, PCR was carried out for the detection of the outer membrane protein A (ompA) gene of Rickettsia. Primers described by Regnery et al were employed in this study for amplification purpose and the amplification was carried out as described. Nuclease-free water was used as negative control and DNA of R. conorii Malish strain (gifted from Prof. Pierre-Edouard Fournier, URMITE, Marseille, France) was used as the positive control. All measures were taken to avoid cross-contamination during the PCR processing. The amplicons were subjected to gel electrophoresis and band patterns visualized. The purified amplicons were subjected to DNA sequencing using BigDye Terminator Cycle Sequencing (Applied Biosystems, USA). The DNA sequences obtained were subjected to BLAST search (http://blast.ncbi.nlm.nih.gov/blast) to identify the agent. The phylogenetic tree was constructed using MEGA version 7. The evolutionary history was constructed using the neighbour-Joining method, and evolutionary distance matrix was computed using the Maximum Composite Likelihood method.
The protocol was approved by the Institutional Ethics Committee (ECC reference no. NK/1300/
MD/1261), PGIMER, Chandigarh. Written informed consent was obtained from all the patients.
| Results|| |
A total of 135 patients diagnosed with an acute undifferentiated febrile illness, who presented to the emergency medical ward for adults were enrolled in this study. The most common diagnosis was scrub typhus seen in 54 (40%) patients followed by malaria in 13 (9.6%) patients and in 51 patients (37.8%) no definite diagnosis was established. These 51 patients were tested for the presence of R. conorii DNA by ompA PCR; among them three patients turned out to be positive. All three patients were young male, one each hailing from the States of Punjab, Haryana and Himachal Pradesh. All presented with fever with non-specific symptoms. None had a history of travel and no history of a bite by an arthropod (tick). Rash or eschar could not be found in any of these patients diagnosed with R. conorii. The clinical symptoms and signs of the three R. conorii patients are shown in [Table 1]. Of the three ompA-positive amplicons with same band size, only two produced good sequences and BLAST identification confirmed these as R. conorii. On the basis of same amplicon size and in view of that all measures were taken to avoid cross-contamination during the PCR processing, the third amplicon was also considered similar to other two sequences. Both sequences were submitted to the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/), and were assigned the accession numbers PGI_RC1_KX016792 and PGI_RC2_KX016793, respectively. The PGI_RC1 ompA sequence showed 100 per cent similarity to R. conorii clone 09 (KR401144) and PGI_RC2 showed 100 per cent similarity to R. conorii subsp.conorii clone 45(JN182802). A phylogenetic tree constructed by the neighbour-joining algorithm using MEGA7 to compare all the sequences of R. conorii reported in India and other parts of the world. The sequences from these patients, grouped in a cluster consisting of R. conorii and R. conorii subsp.conorii [Figure 1]. The sequences of R. conorii reported from south India, Rickettsia sp. CMC MICRO 1-4 (GenBank accession nos. HM587248-HM587251) clustered with two reported novel Rickettsia genotypes, Candidatus Rickettsia kellyi (DQ080005) and Rickettsia sp. Tenjiku01 (LC089865) from the south India,,. Our sequences clustered in the group completely different from that of Rickettsia sp. reported from the south Indian studies,,. The distance matrix revealed an evolutionary divergence between sequences of north and south Indian isolates of R. conorii [Table 2].
|Figure 1: A phylogenetic tree constructed based on ompA gene using MEGA7 software. The sequences of Coxiella burnetii /i>(AF010129), Orientia tsutsugamushi shi (735177), Rickettsia akari 01461) and Rickettsia typhi Q317533) used as outgroups. Geometric shapes and colour indicate sequences from the present study and other Indian studies.|
Click here to view
|Table 1: Clinical and laboratory features of three Rickettsia conorii-positive patients|
Click here to view
|Table 2: Distance matrix showing the evolutionary divergence between sequences of Indian Rickettsia conorii isolates|
Click here to view
| Discussion|| |
A total of 51 patients with AFI who were negative for the common causes of fever were studied. Rickettsial ompA PCR detected three patients with spotted fever. There is a strong possibility that most of these infections go undiagnosed because of the low index of clinical suspicion due to the non-specific symptoms and lack of a suitable diagnostic test. None of our patients reported rash and eschar. The rash in case of spotted fever appears on 2-5 days after onset of symptoms and may be absent in approximately 9-10 per cent,,,. Two of our patients presented after 10 days of fever. Eschar may not be present in all cases; most often, it is missed and masked by skin complexion in Indian patients,,. The clinical presentation and severity of these infections may differ geographically based on the hypothesis that the pathogenic potential of the infecting strains may differ. The Rickettsia infections are more rampant during post-monsoon season as reported earlier,. In the present study, all three patients presented during the post-monsoon season (July to September), and recovered completely after therapy with oral doxycycline. Doxycycline is the drug of choice for spotted fever and is most effective when initiated within the first five days of illness, as early administration of doxycycline in adults and children can prevent severe illness and death,. Azithromycin, when compared with other macrolides, is more effective in the case of spotted fever and can be used as an alternative. Azithromycin was shown to be ineffective in severe spotted fever patients. However, Colomba et alshowed that azithromycin was the better choice for children with Mediterranean spotted fever.
The sequences obtained from our patients grouped into a cluster composed of R. conorii subsp.conorii and subsp. indica. In the studies from south India,, four Rickettsia sp. sequences clustered with the earlier reported novel Rickettsia genotypes Candidatus Rickettsia kellyi. Our sequences clustered in the group different from that of Rickettsia sp. reported in the different south Indian studies. This showed a diversity in the strains isolated from different parts of our country. Multicentric studies involving a large number of patients are required to elucidate the genetic diversity of all R. conorii strains circulating in different parts of India.
In the present study, the identification was done based on the sequences obtained from the gene encoding surface proteins ompA, which is known for its immunogenicity in humans due to its surface location. The limitation of our study was that only single gene (ompA) was used instead of three different genes used for identifying the rickettsiae. However, several studies have shown ompA to be more specific and capable of demonstrating marked diversity; thereby ompA gene alone can serve as a potential tool for differentiating various SFG rickettsiae,,,. In our study, no serological assay for spotted fever was performed; however, ELISA for scrub typhus was done in all cases as the incidence of scrub typhus was higher in this region,. Early initiation of appropriate antibiotic is important for the favourable outcome in spotted fevers caused by a Rickettsia,. Most of the patients respond well to antibiotics such as doxycycline, and somewhat less effectively to macrolides and chloramphenicol,.
In conclusion, this study showed the existence of R. conorii in north India. Tests for SFGR may be included in the diagnostic workup of AFI in north India also.
Financial support & sponsorship: None.
Conflicts of Interest: None.
| References|| |
Batra HV. Spotted fevers & typhus fever in Tamil Nadu. Indian J Med Res
Mahajan SK, Kashyap R, Sankhyan N, Sharma V, Rolain JM, Prasher BS, et al
. Spotted fever group rickettsioses in Himachal Pradesh. J Assoc Physicians India
Rahi M, Gupte MD, Bhargava A, Varghese GM, Arora R. DHR-ICMR Guidelines for diagnosis management of rickettsial diseases in India. Indian J Med Res
Chapman AS, Bakken JS, Folk SM, Paddock CD, Bloch KC, Krusell A, et al
. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever, ehrlichioses, and anaplasmosis-United States: A practical guide for physicians and other health-care and public health professionals. MMWR Recomm Rep
Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: Emerging diseases challenging old concepts. Clin Microbiol Rev
Farhana A, Bali N, Kanth F, Farooq R, Haq IU, Shah P. Serological evidence of scrub typhus among cases of PUO in the Kashmir valley- A hospital based study. J Clin Diagn Res
Kamarasu K, Malathi M, Rajagopal V, Subramani K, Jagadeeshramasamy D, Mathai E. Serological evidence for wide distribution of spotted fevers & typhus fever in Tamil Nadu. Indian J Med Res
Kalal BS, Puranik P, Nagaraj S, Rego S, Shet A. Scrub typhus and spotted fever among hospitalised children in South India: Clinical profile and serological epidemiology. Indian J Med Microbiol
Sethi S, Prasad A, Biswal M, Hallur VK, Mewara A, Gupta N, et al
. Outbreak of scrub typhus in North India: A re-emerging epidemic. Trop Doct
Khan SA, Bora T, Chattopadhyay S, Jiang J, Richards AL, Dutta P. Seroepidemiology of rickettsial infections in Northeast India. Trans R Soc Trop Med Hyg
Prakash JA, Sohan Lal T, Rosemol V, Verghese VP, Pulimood SA, Reller M, et al
. Molecular detection and analysis of spotted fever group Rickettsia
in patients with fever and rash at a tertiary care centre in Tamil Nadu, India. Pathog Glob Health
Takajo I, Sekizuka T, Fujita H, Kawano A, Kawaguchi T, Matsuda M, et al
. Possible case of novel spotted fever group rickettsiosis in traveler returning to Japan from India. Emerg Infect Dis
Ghosh S, Nagar G. Problem of ticks and tick-borne diseases in India with special emphasis on progress in tick control research: A review. J Vector Borne Dis
Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol
Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol
Rolain JM, Mathai E, Lepidi H, Somashekar HR, Mathew LG, Prakash JA, et al
. “Candidatus rickettsia kellyi
,” India. Emerg Infect Di
s 2006; 12
Mahajan SK. Rickettsial diseases. J Assoc Physicians India
Rathi N, Rathi A. Rickettsial infections: Indian perspective. Indian Pediatr
Sexton DJ, Corey GR. Rocky Mountain “spotless” and “almost spotless” fever: A wolf in sheep's clothing. Clin Infect Dis
Sharma N, Biswal M, Kumar A, Zaman K, Jain S, Bhalla A. Scrub typhus in a tertiary care hospital in North India. Am J Trop Med Hyg
Gopinath KG, Chrispal A, Boorugu H, Chandy S, Prakash JJ, Abraham AM, et al
. Clinico-epidemiological profile of seven adults with spotted fever from a tertiary care hospital in South India. Trop Doct
Todd SR, Dahlgren FS, Traeger MS, Beltrán-Aguilar ED, Marianos DW, Hamilton C, et al
. No visible dental staining in children treated with doxycycline for suspected Rocky Mountain spotted fever. J Pediatr
Zientek J, Dahlgren FS, McQuiston JH, Regan J. Self-reported treatment practices by healthcare providers could lead to death from Rocky Mountain spotted fever. J Pediatr
Raoult D, Drancourt M. Antimicrobial therapy of rickettsial diseases. Antimicrob Agents Chemother
Colomba C, Saporito L, Polara VF, Rubino R, Titone L. Mediterranean spotted fever: Clinical and laboratory characteristics of 415 Sicilian children. BMC Infect Dis
Gilmore RD Jr., Hackstadt T. DNA polymorphism in the conserved 190 kDa antigen gene repeat region among spotted fever group Rickettsiae. Biochim Biophys Acta
Fournier PE, Dumler JS, Greub G, Zhang J, Wu Y, Raoult D. Gene sequence-based criteria for identification of new Rickettsia
isolates and description of Rickettsia heilongjiangensis
sp. nov. J Clin Microbiol
Beati L, Finidori JP, Gilot B, Raoult D. Comparison of serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, and genetic restriction fragment length polymorphism analysis for identification of rickettsiae: Characterization of two new rickettsial strains. J Clin Microbiol
Eremeeva M, Yu X, Raoult D. Differentiation among spotted fever group rickettsiae species by analysis of restriction fragment length polymorphism of PCR-amplified DNA. J Clin Microbiol
Rovery C, Brouqui P, Raoult D. Questions on mediterranean spotted fever a century after its discovery. Emerg Infect Dis
Rovery C, Raoult D. Mediterranean spotted fever. Infect Dis Clin North Am
: 515-30, ix.
Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, et al
. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever and other spotted fever group rickettsioses, ehrlichioses, and anaplasmosis - United States. MMWR Recomm Rep
[Table 1], [Table 2]