Molecular confirmation & characterization of Rickettsia conorii in north India: A report of three cases.

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 Rickettsia.
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.

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 region123. The mortality rate in spotted fever is variable but can be high if there is a delay in the diagnosis and treatment145. Most studies on the prevalence of spotted fever group rickettsioses (SFGR) in India are based largely on serological tests such as Weil-Felix67, ELISA689 and immunofluorescence assay10. 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 laboratories3. 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 India11. A novel spotted fever Rickettsia was detected in a Japanese traveller returning from India12. Though the tick vector, Rhipicephalus sanguineus sensu lato, has been found in 21 States in India13, 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 al14 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 () to identify the agent. The phylogenetic tree was constructed using MEGA version 715. The evolutionary history was constructed using the neighbour-Joining method, and evolutionary distance matrix was computed using the Maximum Composite Likelihood method15.

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 I. 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 (), 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). 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 India111216. Our sequences clustered in the group completely different from that of Rickettsia sp. reported from the south Indian studies111216. The distance matrix revealed an evolutionary divergence between sequences of north and south Indian isolates of R. conorii (Table II).

Table I

Clinical and laboratory features of three Rickettsia conorii-positive patients

Characteristics	Patient 1	Patient 2	Patient 3
Gender	Male	Male	Male
Age (yr)	14	36	24
Location	Panchkula, Haryana	Solan, Himachal Pradesh	Nawanshahr, Punjab
Fever	+	+	+
Duration of fever (days)	14	1	10
Headache	-	+	-
Cough	-	+	-
Shortness of breath	-	+	-
Rash/eschar/petechia	-	-	-
Jaundice	-	+	-
Myalgia	-	+	-
Bleeding manifestations	-	-	-
Hepatomegaly	+	+	-
Splenomegaly	-	-	-
Chest X-ray	Mild right pleural effusion	Bilateral diffuse infiltrates	-
Abdominal ultrasonography	Hepatomegaly, splenomegaly, mild ascites	Hepatomegaly	Normal
Haemoglobin (g/dl)	8.3	10.1	14
Total leucocytes count (cells per µl)	7600	12900	6600
Platelet count (×103/µl)	50	71	55
Total bilirubin (mg/dl)	4.53	5.6	0.6
Conjugated bilirubin (mg/dl)	4.0	1.2	0.2
Total protein (g/dl)	5.48	5.6	6
Albumin (g/dl)	2.7	3.1	3.4
Urea (mg/dl)	30	51	24
Creatinine (mg/dl)	0.49	1.7	0.6
Other investigations	EBV and CMV IgM - negative	-	-
Organ dysfunction
PaO2/FiO2	410	217	412
ARDS	No	Yes	No
Hypotension	No	No	No
GCS	15	15	15
SOFA score at admission	4	7	2
Therapy	Intravenous ceftriaxone 1 g twice daily (b.i.d) and oral doxycycline 100 mg b.i.d, given for total seven days	Intravenous ceftriaxone 1 g b.i.d and oral doxycycline 100 mg b.i.d, given for total seven days	Intravenous ceftriaxone 1 g b.i.d and oral doxycycline 100 mg b.i.d, given for total seven days
Outcome	Recovered	Recovered	Recovered

PaO2/FiO2, ratio of partial pressure arterial oxygen and fraction of inspired oxygen; ARDS, acute respiratory distress syndrome; GCS, Glasgow coma scale; SOFA, sequential organ failure assessment; EBV, Epstein-Barr virus; CMV, cytomegalovirus

Figure

A phylogenetic tree constructed based on ompA gene using MEGA7 software. The sequences of Coxiella burnetii (AF010129), Orientia tsutsugamushi (735177), Rickettsia akari (L01461) and Rickettsia typhi (DQ317533) used as outgroups. Geometric shapes and colour indicate sequences from the present study and other Indian studies.

Table II

Distance matrix showing the evolutionary divergence between sequences of Indian Rickettsia conorii isolates

Indian R. conorii isolates*	1	2	3	4	5	6	7	8	9	10
Rickettsia_conorii_clone_09_(KR401144)
Candidatus_Rickettsia_kellyi_(DQ080005)	0.1033
Rickettsia_sp._CMCMICRO_1_(HM587248)	0.1033	0.0000
PGI_RC1_KX016792	0.0000	0.1033	0.1033
PGI_RC2_KX016793	0.0000	0.1033	0.1033	0.0000
Rickettsia_sp._CMCMICRO_2_(HM587249)	0.1033	0.0000	0.0000	0.1033	0.1033
Rickettsia_sp._CMCMICRO_3_(HM587250)	0.1033	0.0000	0.0000	0.1033	0.1033	0.0000
Rickettsia_sp._CMCMICRO_4_(HM587251)	0.1033	0.0000	0.0000	0.1033	0.1033	0.0000	0.0000
Rickettsia_sp._CMCMICRO_5_(HM587252)	0.0458	0.1033	0.1033	0.0458	0.0458	0.1033	0.1033	0.1033
Rickettsia_sp._Tenjiku01_(LC089865)	0.1033	0.0000	0.0000	0.1033	0.1033	0.0000	0.0000	0.0000	0.1033

*The distance matrix shows the number of base substitutions per site between sequences after eliminating the gaps and missing data and analysis was conducted using the maximum composite likelihood model

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 test3. 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 cent11171819. 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 patients111820. 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 earlier821. 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 death2223. 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 patients24. However, Colomba et al25 showed 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 India1116, 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 location26. The limitation of our study was that only single gene (ompA) was used instead of three different genes used for identifying the rickettsiae27. 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 rickettsiae14262829. 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 region920. Early initiation of appropriate antibiotic is important for the favourable outcome in spotted fevers caused by a Rickettsia3031. Most of the patients respond well to antibiotics such as doxycycline, and somewhat less effectively to macrolides and chloramphenicol332.

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.