|Year : 2013 | Volume
| Issue : 4 | Page : 343-348
First case series of emerging Rickettsial neonatal sepsis identified by polymerase chain reaction-based deoxyribonucleic acid sequencing
P Aarthi1, R Bagyalakshmi1, KR Mohan2, M Krishna2, M Nitin2, HN Madhavan1, S Kalyani2
1 Larsen and Toubro Microbiology Research Centre, Kamal Nayan Bajaj Research Centre, Vision Research Foundation, Nungambakkam, Chennai, Tamil Nadu, India
2 Department of Neonatology, Kanchi Kamakoti Childs Trust Hospital, Nungambakkam, Chennai, Tamil Nadu, India
|Date of Submission||02-May-2013|
|Date of Acceptance||16-Aug-2013|
|Date of Web Publication||25-Sep-2013|
H N Madhavan
Larsen and Toubro Microbiology Research Centre, Kamal Nayan Bajaj Research Centre, Vision Research Foundation, Nungambakkam, Chennai, Tamil Nadu
Source of Support: Vision Research Foundation, Conflict of Interest: None
Purpose: To detect and identify the aetiological agent in the peripheral blood from the cases of neonatal sepsis. Materials and Methods: Four neonates from geographically different regions of South India presented with signs of neonatal sepsis and all the routine clinical and laboratory investigations were performed. Blood culture by Bac T Alert 3D was negative. To establish the aetiology, polymerase chain reaction (PCR) for eubacterial genome and subsequent amplification with Gram positive and Gram negative primers were performed followed by deoxyribonucleic acid (DNA) sequencing. Results: PCR for the detection of eubacterial genome was positive in all the four neonates and further amplification with designed Gram positive and Gram negative primers revealed the presence of Gram negative bacteria. The amplicons were identified as Orientia tsutsugamushi in three neonates and Coxiella burnetti in the other neonate. Multalin analysis was done to further characterise the strain variation among the three strains. Conclusion: PCR-based DNA sequencing is a rapid and reliable diagnostic tool to identify the aetiological agents of neonatal sepsis. This is the first case series of emerging Rickettsial neonatal sepsis in India .
Keywords: 16S ribosomal ribonucleic acid region, Coxiella burnetti, neonatal sepsis, Orientia tsutsugamushi, doxycycline, PCR-based DNA sequencing
|How to cite this article:|
Aarthi P, Bagyalakshmi R, Mohan K R, Krishna M, Nitin M, Madhavan H N, Kalyani S. First case series of emerging Rickettsial neonatal sepsis identified by polymerase chain reaction-based deoxyribonucleic acid sequencing. Indian J Med Microbiol 2013;31:343-8
|How to cite this URL:|
Aarthi P, Bagyalakshmi R, Mohan K R, Krishna M, Nitin M, Madhavan H N, Kalyani S. First case series of emerging Rickettsial neonatal sepsis identified by polymerase chain reaction-based deoxyribonucleic acid sequencing. Indian J Med Microbiol [serial online] 2013 [cited 2017 Dec 13];31:343-8. Available from: http://www.ijmm.org/text.asp?2013/31/4/343/118874
| ~ Introduction|| |
Rickettsial infections are re-emerging and are prevalent throughout the world. In India, they are reported from Maharashtra, Tamil Nadu, Karnataka, Kerala, Jammu and Kashmir, Uttaranchal, Himachal Pradesh, Rajasthan, Assam and West Bengal. In view of low index of suspicion, nonspecific signs and symptoms and absence of widely available sensitive and specific diagnostic test, these infections are notoriously difficult to diagnose. Failure of timely diagnosis leads to significant morbidity and mortality. With timely diagnosis, treatment is easy, affordable and often successful with dramatic response to antimicrobials.  Scrub typhus caused by Orientia tsutsugamushi (formerly known as Rickettsia tsutsugamushi until 1995) is the most prevalent human rickettsial infection, with over one billion people living in endemic areas and as many as one million infections annually.  It remains an important public health problem as a cause of febrile illness that is difficult to diagnose clinically as well as by conventional microbiological methods. Virulent strains of O. tsutsugamushi can cause a fulminant illness with pneumonia, myocarditis and disseminated intravascular coagulation often leading to death. McGready et al.,  have reported a case of O. tsutsugamushi TA716-like strain causing abortion in a pregnant woman (first trimester).
Coxiella burnetii is an obligate intracellular bacterial pathogen and is the causative agent of Q fever.  The most important reservoirs of this infection are sheep, cattle and goats. Infection of humans usually occurs by inhalation of infected particles and by consumption of contaminated milk. Two forms of the disease, acute and chronic, are recognised. The most common clinical presentations of acute Q fever are fever, pneumonia, granulomatous hepatitis, and meningoencephalitis, although acute Q fever may be asymptomatic.  So far, there are no reports on O. tsutsugamushi and C. burnetii neonatal sepsis from India. This report describes the first case series of Rickettsial neonatal sepsis in India highlighting the importance of nucleic acid based assays, which proved extremely useful in identifying the aetiological agent of neonatal sepsis and its successful treatment.
| ~ Materials and Methods|| |
Four neonates were clinically diagnosed with neonatal sepsis and hospitalized in Neonatal Intensive Care Unit (NICU) in a tertiary paediatric hospital. The clinical history of the four neonates consisting of place of birth, birth defects and clinical findings at the time of admission are provided in [Table 1] . The various haematological, biochemical and microbiological and serological investigations performed are provided in [Table 2]. Peripheral blood specimens collected from the four neonates was subjected to bacterial culture BAC T Alert 3D (Biomerieux, France). BAC T Alert culture did not reveal any bacterial growth.
|Table 1: Clinical history of the four neonates clinically diagnosed with neonatal sepsis|
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|Table 2: The various laboratory investigations performed on the neonates|
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Since the blood culture revealed no bacterial aetiology, polymerase chain reaction (PCR) targeting the 16S ribosomal ribonucleic acid (rRNA) gene of eubacterial genome  was performed on the peripheral blood specimen. Eubacterial genome was detected in the peripheral blood and further amplification was carried out to identify the aetiology using indigenously designed Gram positive and Gram negative primers. 
| ~ Results|| |
PCR for the detection of eubacterial genome was positive in all the four neonates and further amplification was carried out with designed Gram positive and Gram negative primers. There was no amplification obtained with Gram positive primers and the results were suggestive of the presence of Gram negative bacteria (presence of 262 bp specific amplified product) [Figure 1]. The amplicons were further subjected to PCR-based DNA sequencing and the bacteria were identified as O. tsutsugamushi in three neonates and C. burnetti in one neonate. The results of PCR-based DNA sequencing (electrophoretogram) performed on the 16S rRNA amplicon is represented in [Figure 2].
|Figure 1: Agarose gel electrophoretogram representing the results of Gram negative PCR performed on peripheral blood specimen Lane 1 Molecular Weight Marker 100 bp ladder Lane 2 Negative control Round‑2 Lane 3 Negative control Round‑1 Lane 4 Peripheral blood revealing the presence of Gram negative genome (262 bp) Lane 5 Positive control – Escherichia More Details coli|
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|Figure 2: Electrophoretogram of PCR‑based DNA sequencing targeting 16SrRNA region The 16SrRNA sequence of VRF 388 revealed 99% BLAST score matching with Orientia tsutsugamushi (Genbank Accession Number AF478127)|
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To further characterise and detect the strain variation existing among O. tsutsugamushi, a multalin analysis (multalin.toulouse.inra.fr/multalin/) was carried out and the results of the same are shown in [Figure 3]. The multalin analysis revealed nucleotide polymorphisms in the second strain while the first and third strains were genetically identical. A dendrogram was constructed using the clustalW software (http://www.clustal.org) to identify the genetic homology among the three strains of O. tsutsugamushi. The dendrogram [Figure 4] revealed that O. tsutsugamushi strain 1 and strain 3 were genetically identical with that of O. tsutsugamushi, (Accession No AF478127.1) while strain 2 was genetically close with that of O. tsutsugamushi AF479302.1.
|Figure 3: Multalin analysis performed on the three strains of Orientia tsutsugamushi identified by PCR‑based DNA sequencing Multalign analysis representing the nucleotide polymorphisms exhibited by Orientia tsutsugamushi (Nellore strain VRF 1584/12‑GenBank Accession No: JX852621). The other two strains from Thirunelveli (VRF 388‑GenBank Accession No: JX852620) and Thanjavur (VRF 1773‑GenBank Accession No: JX852622) were genetically identical|
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|Figure 4: Dendogram representing the genetic homology among the three strains of Orientia tsutsugamushi Dendrogram representing the genetic similarity of Orientia tsutsugamushi strains identified from Thirunelveli (388‑GenBank Accession No: JX852620) and Thanjavur (1773‑GenBank Accession No: JX852622), Tamil Nadu which clustered together while the Nellore (1584‑GenBank Accession No: JX852621) strain, Andhra Pradesh, India was unique and clustered away from the Tamil Nadu strains|
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The clinical outcome and the prognosis of the four neonates are provided in [Table 3].
| ~ Discussion|| |
According to World Health Organisation (WHO) estimates, there are about five million neonatal deaths a year, 98% occurring in the developing countries. In the developing countries, neonatal mortality (deaths in the first 28 days of life per 1,000 live births) from all causes is about 34; most of these deaths occur in the first week of life, most on the first day. Neonatal mortality in Asia is about 34, in Africa about 42, and in Latin America and the Caribbean about 17, although there are wide variations between different countries in these regions as well as within the countries themselves. 
Rickettsial infections are re-emerging and are prevalent through out the world. O. tsutsugamushi causes scrub typhus, which is a rural zoonosis endemic in the Asia Pacific region. In India, they are reported from Maharashtra, Tamil Nadu, Karnataka, Kerala, Jammu and Kashmir, Himachal Pradesh, Rajasthan, Assam and West Bengal. 
PCR-based DNA sequencing proved to be extremely useful in identification of O. tsutsugamushi causing neonatal sepsis. Dutta et al.,  have emphasised the role of PCR-based DNA sequencing targeting the 16S rRNA region in detection of aetiological agents causing neonatal sepsis. Once the aetiology was identified, a course of doxycycline (5 mg/kg of body weight per day) was administered. The neonates one and two recovered well due to the timely institution of doxycycline and supportive treatment, while the third neonate had a more severe course and failed to respond to doxycycline, azithromycin and aggressive supportive management and died on 10 th day of hospitalisation. In literature, two cases (China and Japan) of neonatal scrub typhus have been reported and its transmission route from mother to the foetus was still uncertain. Two possible routes of transmission were suggested, one being trans-placental and the other perinatal blood borne transmission. 
In the present study, multalin analysis of O. tsutsugamushi revealed sequence variation in strain 2 (1584-GenBank Accession No: JX852621) while the remaining two strains (strain 1 and strain 3) were identical. The nucleotide polymorphisms detected in the second strain were in the nucleotide positions 555 (A to C), 556,560,600,602 (A to T), 557 (G to T) and nucleotide position 687 (C to T). Due to the nucleotide variation, the second strain clustered away from the first and the third strain, which were genetically close with O. tsutsugamushi (Accession No AF478127.1).
Phylogenetic analysis on O. tsutsugamushi by Nakayama et al.,  revealed the presence of O. tsutsugamushi lineage with higher potential for virulence, which may prove useful in establishing epidemiological differences among O. tsutsugamushi strains. As far as the geographical distribution of the strains are concerned, the first and the third strain originated from Tamil Nadu (Tirunelveli and Thanjavur), while the second strain's origin was from Andhra Pradesh (Nellore district). The geographical difference prevailing among O. tsutsugamushi is reflected in the genetic analysis since the second strain exhibited multiple nucleotide polymorphisms and clustered away from the other two strains.
The usual source of human Coxiella infection is farm animals. C. burnetii is highly infectious and is currently considered a potential warfare agent, classified as a category B biological agent by the Centre for Diseases Control and Prevention. In humans, infection most often results from inhalation of contaminated aerosols from amniotic fluid, placenta  or contaminated wool.  The diagnosis of Q fever relies mainly on serological examination, the most commonly used method being the indirect immunofluorescence assay. Fenollar et al.,  have developed a molecular assay for rapid detection of C. burnetii causing endocarditis and vascular infection. In the present study, C. burnetii was identified as the aetiological agent of neonatal sepsis and doxycycline was administered and improved, but unfortunately died 3 months after due to unknown cause.
The present study shows that universal primer PCR is a sensitive and specific method for diagnosing neonatal bacterial infections in culture-negative specimens. In clinical situations, the use of universal primer PCR has been shown to significantly reduce the time for initiation of suitable antibiotic therapy and the hospital stay. The only limitation is that information about antibiotic drug resistance is not available. Further, molecular assays have to be designed to determine the sensitivity and resistant pattern of the infecting agent to different antibiotics.
To the best of our knowledge, this is the first case series of Rickettsial neonatal sepsis being reported in literature from India diagnosed by PCR-based DNA sequencing.
| ~ Acknowledgement|| |
We extend our sincere gratitude to Vision Research Foundation for supporting the study and the physicians of Child Trust Hospital for referring the clinical specimen to carry out the study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]