Investigation of acute encephalitis syndrome with implementation of metagenomic next generation sequencing in Nepal.

BACKGROUND: The causative agents of Acute Encephalitis Syndrome remain unknown in 68-75% of the cases. In Nepal, the cases are tested only for Japanese encephalitis, which constitutes only about 15% of the cases. However, there could be several organisms, including vaccine-preventable etiologies that cause acute encephalitis, when identified could direct public health efforts for prevention, including addressing gaps in vaccine coverage. OBJECTIVES: This study employs metagenomic next-generation-sequencing in the investigation of underlying causative etiologies contributing to acute encephalitis syndrome in Nepal. METHODS: In this study, we investigated 90, Japanese-encephalitis-negative, banked cerebrospinal fluid samples that were collected as part of a national surveillance network in 2016 and 2017. Randomization was done to include three age groups (< 5-years; 5-14-years; >15-years). Only some metadata (age and gender) were available. The investigation was performed in two batches which included total nucleic-acid extraction, followed by individual library preparation (DNA and RNA) and sequencing on Illumina iSeq100. The genomic data were interpreted using Chan Zuckerberg-ID and confirmed with polymerase-chain-reaction. RESULTS: Human-alphaherpes-virus 2 and Enterovirus-B were seen in two samples. These hits were confirmed by qPCR and semi-nested PCR respectively. Most of the other samples were marred by low abundance of pathogen, possible freeze-thaw cycles, lack of process controls and associated clinical metadata. CONCLUSION: From this study, two documented causative agents were revealed through metagenomic next-generation-sequencing. Insufficiency of clinical metadata, process controls, low pathogen abundance and absence of standard procedures to collect and store samples in nucleic-acid protectants could have impeded the study and incorporated ambiguity while correlating the identified hits to infection. Therefore, there is need of standardized procedures for sample collection, inclusion of process controls and clinical metadata. Despite challenging conditions, this study highlights the usefulness of mNGS to investigate diseases with unknown etiologies and guide development of adequate clinical-management-algorithms and outbreak investigations in Nepal.

Aetiological profile of acute encephalitis syndrome in Assam, India, during a 4-year period from 2019 to 2022.

Acute encephalitis syndrome (AES) is a major public health concern in India as the aetiology remains unknown in the majority of cases with the current testing algorithm. We aimed to study the incidence of Japanese encephalitis (JE) and determine the aetiology of non-JE AES cases to develop an evidence-based testing algorithm. Cerebrospinal fluid (CSF) samples were tested for Japanese encephalitis virus by ELISA and polymerase chain reaction (PCR). Multiplex real-time PCR was done for Dengue, Chikungunya, West Nile, Zika, Enterovirus, Epstein Barr Virus, Herpes Simplex Virus, Adenovirus, Cytomegalovirus, Herpesvirus 6, Parechovirus, Parvovirus B19, Varicella Zoster Virus, Scrub typhus, Rickettsia species, Leptospira, Salmonella species, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Plasmodium species and by ELISA for Mumps and Measles virus. Of the 3173 CSF samples, 461 (14.5%) were positive for JE. Of the 334 non-JE AES cases, 66.2% viz. Scrub typhus (25.7%), Mumps (19.5%), Measles (4.2%), Parvovirus B19 (3.9%) Plasmodium (2.7%), HSV 1 and 2 (2.4%), EBV and Streptococcus pneumoniae (2.1% each), Salmonella and HHV 6 (1.2% each) were predominant. Hence, an improved surveillance system and our suggested expanded testing algorithm can improve the diagnosis of potentially treatable infectious agents of AES in India.

Utility of Film Array Meningoencephalitis Panel in Children With Acute Encephalitis Syndrome: A Single Centre Experience from South India.

OBJECTIVE: To describe the utility of film array meningoencephalitis (FAME) panel in the management of children with acute encephalitis syndrome (AES). METHODS: A retrospective audit was conducted between January 2017 to July 2022. We included children aged < 18 years with a diagnosis of AES for whom a CSF analysis study including FAME panel testing performed within 48 hours of admission was available. Electronic medical records were reviewed for details including demographic profile, clinical presentation, investigations and outcome. RESULTS: Out of 157 CSF samples sent for FAME panel testing, 49 were positive (31.4%.) Viral pathogens were identified in 42 (Enterovirus: 31, Human herpes virus 6: 9, Varicella zoster virus: 1, and Cytomegalovirus: 1) Bacterial pathogens were identified in 6 (Streptococcus pneumoniae: 2, Streptococcus agalactiae: 2, Hemophilus influenzae: 1, and Escherischia coli: 1). Fungal etiology (Cryptococcus neoformans) was detected in one child. Antibiotics could be stopped within 72 hours of initiation in 42 children in whom a viral etiology was established. Acyclovir could be stopped in 21 out of 32 children within 72 hours after the FAME panel testing. FAME panel was presumed to be false positive in 4 children. CONCLUSION: Etiology of AES could be established in nearly a third of children with AES using the rapid diagnostic FAME panel testing in CSF and it was found to be effective in reducing empirical antibiotic/antiviral therapy.

Molecular and serological evidence of chikungunya virus infection with high case fatality among pediatric population with acute encephalitis syndrome: first report from Eastern Uttar Pradesh, India.

Acute encephalitis syndrome (AES) outbreaks in children of Eastern Uttar Pradesh (E-UP) region of India have been a longstanding public health issue, with a significant case fatality rate of 20-25%. Since past decade, a rise in chikungunya (CHIK) cases has been occurring, which is a reported etiology of AES. However, the burden of chikungunya virus (CHIKV) among pediatric AES (pAES) is unknown from E-UP. We included 238 hospitalized pAES cases. The presence of IgM antibodies for CHIKV, and Dengue virus (DENV) was tested, and RT-PCR was performed for CHIKV and DENV in serologically confirmed CHIKV and DENV pAES cases. Positive samples were sequenced using Sangers sequencing. Further, to check for co-infection, IgM antibodies for other AES etiologies including Japanese encephalitis virus (JEV), Leptospira and Orientia tsutsugamushi (OT) in serum were also investigated. IgM ELISA demonstrated 5.04% (12) positivity for CHIKV. Among CHIKV IgM positive, 3 (25%, 3/12) pAES patients died. CHIKV genome was detected in 3 pAES specimens. Among which, 2 CHIKV cases were also positive for OT DNA. Partially sequenced CHIKV were genotyped as ECSA. The overall finding indicates evidence of CHIKV infection with high case fatality among pAES patients from E-UP. This study advocates constant serological and molecular surveillance of CHIKV in AES endemic regions of India.

Acute Encephalitis Syndrome in Children and Adolescents: A Five-Year Descriptive Study From South India.

OBJECTIVE: Acute encephalitis syndrome (AES) in children results in significant neurocognitive deficits or mortality. It is pertinent to study the AES patterns periodically to identify the changes in the etiological trends and outcomes. Our objective was to find the etiological agents of AES, mode of diagnosis, treatment given, and outcomes. METHODS: We reviewed the electronic records of children aged 1 month to 15 years who were admitted with AES in our centre from January 2015 to December 2019. We analyzed the the clinical, laboratory, and radiological profile of these children and adolescents in relation to their outcome. Poor outcome was defined as death, discharge against medical advice with neurological deficits, or Glasgow Outcome Score Extended (GOS-E) d≤ 5 at the time of discharge. RESULTS: Among 250 patients admitted with AES during the study period, a definitive etiological diagnosis was established in 56.4% of children (30.4% viral, 22% bacterial). Scrub typhus (11.2%) and dengue (9%) were the two most common underlying illnesses. Serology helped in clinching the diagnosis in 30% of children. A surge in AES cases in the post-monsoon season was observed in our cohort. Third-generation cephalosporin drugs (85.7%) and acyclovir (77.7%) were the most commonly used empiric antimicrobial drugs. About one-third of children (n = 80) had a poor outcome. GCS ≤ 8 at presentation and requirement for invasive ventilation were found to be significant predictors of poor outcome. CONCLUSION: A definitive diagnosis was obtained in about half of the children with AES. Viral (30.4%) and rickettsial infections (22%) were the common etiologies identified. Poor outcome was observed in 32% of patients.

Association of transient mitochondrial functional impairment with acute heat exposure in children from Muzaffarpur region of Bihar, India.

Over the past several years, the Muzaffarpur district of Bihar (India) has witnessed recurrent outbreaks of acute encephalitis illness of unknown etiology, called acute encephalitis syndrome (AES) among young children, especially during the peak-summer season. Pesticide exposure, viral encephalitis, and litchi toxin intake have all been postulated as potential sources of the ailment. However, no conclusive etiology for AES has been identified in the affected children. During recent rounds of the outbreak, metabolic abnormalities have been documented in these children, and a direct correlation was observed between higher environmental temperature during the peak-summer month and AES caseload. The clinical and metabolic profiles of these children suggested the possible involvement of mitochondrial dysfunction during heat stress as one of the several contributory factors leading to multisystem metabolic derangement. The present study observed that mitochondrial function parameters such as cell death, mitochondrial membrane potential, oxidative stress, and mitochondrial pathway-related gene expression in peripheral blood mononuclear cells (PBMCs) isolated from children were affected in peak-summer when compared to post-summer months. Similar observations of mitochondrial function parameters along with impaired bioenergetic parameters were demonstrated in the heat-exposed model of PBMCs isolated from healthy adult individuals. In conclusion, the results suggested that there is an association of transient mitochondrial dysfunction when exposed to sustained heat during the summer months. One may consider mitochondrial dysfunction as one of the important factors leading to an outbreak of AES among the children from affected regions though this needs to be substantiated with further studies.

Emergence of Japanese encephalitis in eastern parts of Madhya Pradesh, India.

BACKGROUND & OBJECTIVES: Emerging zoonotic and vector-borne diseases are posing new challenges to public health authorities. Morbidities and mortalities due to acute encephalitis syndrome (AES) is a serious health problem in paediatric patients. We conducted serological investigations on AES cases from six districts of north eastern Madhya Pradesh (MP), India for Japanese encephalitis (JE). METHODS: The paired serum and CSF samples were collected from paediatric patients having signs and symptoms of encephalitis and admitted at a tertiary care hospital during the study period from August 2020 to October 2021. Demographic and clinical information was collected in predesigned formats. Serum and CSF were subjected to JE IgM specific ELISA. RESULTS: Samples from 110 patients were collected during the study period of which 28 (25.4%) were reactive for JE IgM antibodies. JE IgM positivity was marginally higher in male children (26.6%) as compared to female children (22.8%). Out of 28 positive cases, 11 (39.2%) deaths were attributed to JE. Four districts of north eastern Madhya Pradesh showed JE activity. Maximum cases were observed in post-monsoon season. INTERPRETATION & CONCLUSION: Our results show that JEV is an emerging threat in eastern central India and health authorities need to be vigilant. A systematic molecular and serological survey among humans and animals along with xenomonitoring will help in understanding intricacies of JE epidemiology in the region.

Infectious Causes of Acute Encephalitis Syndrome in India - Decadal Change and the Way Forward.

The diagnosis and management of encephalitis were previously largely based on clinical grounds and minimal laboratory investigations. Japanese encephalitis (JE) gets considered as the probable diagnosis in most encephalitis cases. However, reports of JE in adults and the elderly are increasing after the JE vaccine introduction among children in 2006. The Nipah virus (NiV) emerged in 2002 and continues to afflict humans in new geographic areas. Many other infections cause encephalitis, including Chandipura, chikungunya, dengue, and West Nile. Significant advances in diagnostic testing like multiplex testing panels and metagenomic approaches along with sequencing have helped in the detection of new etiologies. Recent years have witnessed an increase in climate-sensitive zoonotic diseases with encephalitis. This highlights the importance of the One Health approach in studying the impact of climate change-associated infectious diseases on human health. The government of India's efforts to develop health research infrastructure would help future responses to emerging infectious disease epidemics.

Incidence of Japanese Encephalitis and Acute Encephalitis Syndrome Hospitalizations in the Medium-Endemic Region in Central India.

BACKGROUND: We estimated the incidence of Japanese encephalitis (JE) and acute encephalitis syndrome (AES) following routine immunization with the live-attenuated SA 14-14-2 JE vaccine. METHODS: We implemented enhanced surveillance of AES and JE hospitalizations in endemic districts in Maharashtra and Telangana States during 2015-2016 and 2018-2020. We estimated incidence and compared differences in the incidence of JE and AES between two states, and vaccinated and unvaccinated districts during two study periods. We also considered secondary data from public health services to understand long-term trends from 2007 to 2020. RESULTS: The annual AES incidence rate of 2.25 cases per 100,000 children in Maharashtra during 2018-2020 was significantly lower than 3.36 cases per 100,000 children during 2015-2016. The six JE-vaccinated districts in Maharashtra had significantly lower incidence rates during 2018-2020 (2.03, 95% CI 1.73-2.37) than in 2015-16 (3.26, 2.86-3.70). In addition, the incidence of both JE and AES in two unvaccinated districts was higher than in the vaccinated districts in Maharashtra. Telangana had a lower incidence of both JE and AES than Maharashtra. The AES incidence rate of 0.95 (0.77-1.17) during 2018-2020 in Telangana was significantly lower than 1.67 (1.41-1.97) during 2015-2016. CONCLUSIONS: The annual incidence rate of Japanese encephalitis was < 1 case per 100,000 children. It indicated accelerated control of Japanese encephalitis after routine immunization. However, the annual incidence of acute encephalitis syndrome was still > 1 case per 100,000 children. It highlights the need for improving surveillance and evaluating the impacts of vaccination.

Molecular detection of spotted fever group of Rickettsiae in acute encephalitis syndrome cases from eastern Uttar Pradesh region of India.

The Eastern Uttar Pradesh region of India is known for its endemicity of acute encephalitis syndrome (AES). Decades of research have established that Orientia tsutsugamushi, a causative of scrub typhus, is a substantial contributor (>60%) for the AES cases besides other aetiology, but additional factors in the remaining proportion are still unidentified. Rickettsial infections are challenging to diagnose in clinical settings due to overlapping clinical symptoms, the absence of definitive indicators, a low index of suspicion, and the lack of low-cost, rapid diagnostic tools. Hence, the present study was designed to determine the load of rickettsial infections among AES cases. Furthermore, we aim to find out the prevalent rickettsial species in AES cases as well as in the vector population at this location. The study included the whole blood/cerebrospinal fluid of AES patients and arthropod specimens from rodents. The molecular identification was performed using the 23S-5S intergenic spacer region and ompB gene with genomic DNA obtained from studied specimens. We detected 5.34% (62/1160) of rickettsial infection in AES cases. Among these, phylogenetic analysis confirmed the presence of 54.8% Rickettsia conorii (n = 34) and 16.1% of Rickettsia felis (n = 10), while the rest proportion of the isolates was unidentified at the species level. Furthermore, R. felis was identified in one CSF sample from AES patients and three flea samples from Xenopsylla cheopis. Rickettsia spp. was also confirmed in one Ornithonyssus bacoti mite sample. The results of this investigation concluded the presence of spotted fever group Rickettsia spp. among AES identified cases as well as in the mite and flea vectors that infest rodents.

Association of Scrub Typhus in Children with Acute Encephalitis Syndrome and Meningoencephalitis, Southern India.

Scrub typhus is an established cause of acute encephalitis syndrome (AES) in northern states of India. We systematically investigated 376 children with AES in southern India, using a stepwise diagnostic strategy for the causative agent of scrub typhus, Orientia tsutsugamushi, including IgM and PCR testing of blood and cerebrospinal fluid (CSF) to grade its association with AES. We diagnosed scrub typhus in 87 (23%) children; of those, association with AES was confirmed in 16 (18%) cases, probable in 55 (63%), and possible in 16 (18%). IgM detection in CSF had a sensitivity of 93% and specificity of 82% compared with PCR. Our findings suggest scrub typhus as an emerging common treatable cause of AES in children in southern India and highlight the importance of routine testing for scrub typhus in diagnostic algorithms. Our results also suggest the potential promise of IgM screening of CSF for diagnosis of AES resulting from scrub typhus.

Comparison of Clinical Profile and Outcomes of Japanese Encephalitis and Acute Encephalitis Syndrome among Rural Children.

The study compared the clinical profile and outcomes of Japanese encephalitis (JE) and acute encephalitis syndrome (AES) in children. Fifty-six consecutive children with symptoms fulfilling the WHO clinical case definition of AES from June 2018 to June 2020 were included in the study. All patients who tested positive for either serum or cerebrospinal fluid (CSF) anti-JE-IgM antibodies were JE patients (n = 24) and compared with non-JE AES cases (n = 32). Fever, seizures, and altered sensorium were the most common presenting symptoms. Low GCS, status epilepticus, meningeal irritation, raised CSF protein, and INR > 1.5 of JE children showed significant association with mortality (p value < 0.05), whereas only low GCS showed significant association in non-JE AES cases. The JE-specific mortality rate was 29%, which was less than the mortality rate of non-JE AES children at 41%. Both JE and non-JE AES children had a similar clinical profile, but only the JE children's poor clinical and laboratory parameters were associated with adverse outcomes.

Comparison of Full Outline of Unresponsiveness Score and Glasgow Coma Scale for Assessment of Consciousness in Children With Acute Encephalitis Syndrome.

OBJECTIVE: To correlate the Full outline of unresponsiveness (FOUR) score and Glasgow coma scale (GCS) in the assessment of children with acute encephalitis syndrome (AES). METHODS: This observational study was conducted in the department of pediatrics of a public sector tertiary care center from January, 2019 to March, 2020. All consecutive patients of AES admitted during the study period (n=150) were recruited. Subjects were analyzed using the FOUR score and GCS on admission, and then 12-hourly till discharge/death. Treatment-related and demographic variables were collected and analyzed. Correlation between FOUR score and GCS scores was calculated using spearman correlation coefficient. RESULTS: Positive correlation was observed between the GCS score and the FOUR score (n=0.82; P<0.001). CONCLUSION: FOUR score and GCS were comparable to assess the level of consciousness in patients with AES. The possibility of using FOUR score as an alternative to GCS in children with AES needs to be considered.

Infectious causes of acute encephalitis syndrome hospitalizations in Central India, 2018-20.

BACKGROUND: We enhanced surveillance of hospitalizations of all ages for acute encephalitis syndrome (AES) along with infectious aetiologies, including the Japanese encephalitis virus (JEV). METHODS: From October 2018 to September 2020, we screened neurological patients for AES in all age groups in Maharashtra and Telangana States. AES cases were enrolled at study hospitals along with other referrals and sampled with cerebrospinal fluid, acute and convalescent sera. We tested specimens for non-viral aetiologies viz. leptospirosis, typhoid, scrub typhus, malaria and acute bacterial meningitis, along with viruses - JEV, Dengue virus (DENV), Chikungunya virus (CHIKV), Chandipura virus (CHPV) and Herpes simplex virus (HSV). RESULTS: Among 4977 neurological hospitalizations at three study site hospitals over two years period, 857 (17.2%) were AES. However, only 287 (33.5%) AES cases were eligible. Among 278 (96.9%) enrolled AES cases, infectious aetiologies were identified in 115 (41.4%) cases, including non-viral in 17 (6.1%) cases - leptospirosis (8), scrub-typhus (3) and typhoid (6); and viral in 98 (35.3%) cases - JEV (58, 20.9%), HSV (22, 7.9%), DENV (15, 5.4%) and CHPV (3, 1.1%). JEV confirmation was significantly higher in enrolled cases than referred cases (10.2%) (p < 0.05). However, the contribution of JEV in AES cases was similar in both children and adults. JE was reported year-round and from adjacent non-endemic districts. CONCLUSIONS: The Japanese encephalitis virus continues to be the leading cause of acute encephalitis syndrome in central India despite vaccination among children. Surveillance needs to be strengthened along with advanced diagnostic testing for assessing the impact of vaccination.

An algorithmic approach to identifying the aetiology of acute encephalitis syndrome in India: results of a 4-year enhanced surveillance study.

BACKGROUND: Annual outbreaks of acute encephalitis syndrome pose a major health burden in India. Although Japanese encephalitis virus (JEV) accounts for around 15% of reported cases, the aetiology of most cases remains unknown. We aimed to establish an enhanced surveillance network and to use a standardised diagnostic algorithm to conduct a systematic evaluation of acute encephalitis syndrome in India. METHODS: In this large-scale, systematic surveillance study in India, patients presenting with acute encephalitis syndrome (ie, acute onset of fever with altered mental status, seizure, or both) to any of the 18 participating hospitals across Uttar Pradesh, West Bengal, and Assam were evaluated for JEV (serum and cerebrospinal fluid [CSF] IgM ELISA) per standard of care. In enhanced surveillance, JEV IgM-negative specimens were additionally evaluated for scrub typhus, dengue virus, and West Nile virus by serum IgM ELISA, and for Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, dengue virus, herpes simplex virus, and enterovirus by CSF PCR across five referral laboratories. In 2017, chikungunya and Leptospira serum IgM by ELISA and Zika virus serum and CSF by PCR were also tested. FINDINGS: Of 10 107 patients with acute encephalitis syndrome enrolled in enhanced surveillance between Jan 1, 2014, and Dec 31, 2017, 5734 (57·8%) of 9917 participants with available data were male and 6179 (62·7%) of 9856 were children aged 15 years and younger. Among patients who provided a sample of either CSF or serum in enhanced surveillance, an aetiology was identified in 1921 (33·2%) of 5786 patients enrolled between 2014 and 2016 and in 1484 (34·3%) of 4321 patients enrolled in 2017. The most commonly identified aetiologies were JEV (1023 [17·7%] of 5786 patients), scrub typhus (645 [18·5%] of 3489), and dengue virus (161 [5·2%] of 3124). Among participants who provided both CSF and serum specimens, an aetiology was identified in 1446 (38·3%) of 3774 patients enrolled between 2014 and 2016 and in 936 (40·3%) of 2324 enrolled in 2017, representing a 3·1-times increase in the number of patients with acute encephalitis syndrome with an identified aetiology compared with standard care alone (299 [12·9%]; p<0·0001). INTERPRETATION: Implementation of a systematic diagnostic algorithm in an enhanced surveillance platform resulted in a 3·1-times increase in identification of the aetiology of acute encephalitis syndrome, besides JEV alone, and highlighted the importance of scrub typhus and dengue virus as important infectious aetiologies in India. These findings have prompted revision of the national testing guidelines for this syndrome across India. FUNDING: US Centers for Disease Control and Prevention.

Seasonal abundance of Leptotrombidium deliense, the vector of scrub typhus, in areas reporting acute encephalitis syndrome in Gorakhpur district, Uttar Pradesh, India.

The rickettsial pathogen Orientia tsutsugamushi, causing scrub typhus, has been implicated as a major cause of acute encephalitis syndrome (AES) in many places in India including Gorakhpur district of Uttar Pradesh. Seasonal abundance of the principal vector mite of the pathogen, Leptotrombidium deliense, its animal hosts, and prevalence of infection on them are important attributes in the assessment of outbreaks of the disease. Hence, these aspects were investigated, seasonally, in rural villages of Gorakhpur district, where peak incidence of AES cases were reported. A total of 903 animals (rodents/shrews) was collected using 6484 Sherman traps in eight study villages (14% overall trap rate). A sum of 5526 trombiculid mites comprising 12 species was collected from 676 live rodents/shrews screened. Suncus murinus, the Asian house shrew was the predominant species (67%). Among trombiculids, the principal vector mite, L. deliense, was predominant (64.7%) and its infestation index (i.e., average number of chiggers per host animal) was 5.3. The L. deliense infestation index was higher during July to November with a peak in October. Out of 401 animal sera samples screened, 68% were positive for antibodies against O. tsutsugamushi. Of 465 blood samples tested by nested PCR, seven were positive for the 56 kDa gene of O. tsutsugamushi. In conventional PCR, 41 out of 265 samples were positive for the 60 kDa groEL gene of O. tsutsugamushi. Among the 5526 mite samples, tested as 352 pools through nested PCR, four pools were positive for 56 kDa gene. Phylogenetic analysis of 56 and 60 kDa genes confirmed circulation of Karp and TA678 (rodents) and TA678 (mite) serotypes of O. tsutsugamushi in Gorakhpur. Peak incidence of AES in Gorakhpur district occurs during the rainy season (July-October), coinciding with the peak abundance of L. deliense. These results indicate involvement of L. deliense as the vector mite transmitting the scrub typhus pathogen O. tsutsugamushi to humans in the rural areas of Gorakhpur district, India.

Molecular Detection and Genetic Characterization of Orientia tsutsugamushi from Hospitalized Acute Encephalitis Syndrome Cases During Two Consecutive Outbreaks in Eastern Uttar Pradesh, India.

Background: Seasonal outbreaks of acute encephalitis syndrome (AES) have been reported especially in the pediatric population with a high case fatality rate in Eastern Uttar Pradesh, India. Orientia tsutsugamushi (OT) is a causative agent of scrub typhus that has been recently identified as a major cause of AES. However, the specific genotypes of OT responsible for AES cases of this region are not known. Therefore, the present study was undertaken to understand the molecular epidemiology of OT prevailing in the AES endemic Eastern Uttar Pradesh region of India. Methods: The study was conducted on 2529 hospitalized AES cases from August 2016 to December 2017. The presence of antibodies against OT from cerebrospinal fluid (CSF) and serum samples were tested using OT IgM enzyme-linked immunosorbent assay (ELISA), whereas OT DNA was tested from whole blood and CSF specimens targeting the partial gene of 56 kDa using nested PCR. Phylogenetic analysis was conducted with sequences (n = 241) generated in this study. Findings: Among the studied AES cases, 50% were found positive for antibodies against OT, whereas 37% of cases were positive for OT DNA. The genetic analysis study revealed that Gilliam (93.8%) is the prevailing genotype of OT followed by Karp (6.16%) genotype in AES cases. Furthermore, the Gilliam strains of this study showed they were >99% identical to earlier reported Gilliam strains from AES cases. Conclusion: We observed the presence of two main OT genotypes in AES cases, among which the majority of OT genotypes fall under the Gilliam clade. The understanding of predominant genotype will be beneficial for its future implications in vaccine development strategies and the development of rapid diagnostic tests.

Investigations of seasonal outbreaks of acute encephalitis syndrome due to Orientia tsutsugamushi in Gorakhpur region, India: A One Health case study.

Gorakhpur division consisting of Gorakhpur and neighboring districts Deoria, Kushinagar and Maharajganj in Uttar Pradesh, India, have been witnessing seasonal outbreaks of acute encephalitis syndrome (AES) among children for the last three decades. Investigations conducted during 2005 identified Japanese encephalitis (JE) virus as an aetiology of AES. With the introduction of JE vaccination and other control strategies, the incidence of JE in the region declined, however, outbreaks of acute febrile illness with neurological manifestations continued to occur. Subsequent investigations identified Orientia tsutsugamushi, as the major aetiology of AES outbreaks in the region. This review details clinical, epidemiological, animal and entomological investigations conducted for AES due to O. tsutsugamushi during 2015 and 2017 in Gorakhpur region. Surveillance of acute febrile illness among children attending peripheral health facilities identified scrub typhus as an important aetiology of febrile illness during monsoon and post-monsoon months. Population-based serosurveys indicated high endemicity of scrub typhus. Entomological studies demonstrated natural infection of O. tsutsugamushi in small animal hosts and vector mites. Children acquired this infection through recent exposure to outdoor environment, while playing, or visiting fields or defecating in open fields. A few of the children with scrub typhus progress to develop CNS manifestations. Hence, early administration of appropriate antibiotics is crucial in preventing progression of AFI due to scrub typhus to AES. The investigations conducted by the multi-disciplinary team helped understand the transmission dynamics of scrub typhus in Gorakhpur division and recommend strategies for its control.

Association of leptospirosis and scrub typhus in acute encephalitis syndrome in a tertiary care hospital in Odisha, India.

BACKGROUND: Acute encephalitis syndrome (AES) is a major public health concern in India, causing febrile illness principally associated with viral infection. Bacteria-like scrub typhus and leptospirosis also cause acute febrile illness. Therefore, this study was conceived to address the possible etiological agents contributing to sporadic AES in a tertiary care center in Odisha, India. METHOD: This was a prospective hospital-based study that enrolled 92 consecutive patients with clinically diagnosed AES whose blood/cerebrospinal fluid samples were tested for IgM antibodies to dengue, Japanese encephalitis (JE), herpes simplex virus (HSV), Epstein-Barr virus (EBV), leptospirosis and scrub typhus. RESULTS: Viral antibodies to dengue were detected in three (3.26%) cases, HSV1 in four (4.34%) and HSV2 in three (3.26%) cases. Significantly, antibodies to EBV in 22 (23.591%) and to JE in 27 (29.34%) cases were detected. Notably, 30 (32.60%) and 11(12.0%) of patients had IgM antibodies to leptospirosis and scrub typhus, respectively. CONCLUSION: This observation indicates an association of leptospirosis and scrub typhus infection in sporadic cases of AES, besides other viruses.