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.

Genomic epidemiology reveals multiple introductions and spread of SARS-CoV-2 in the Indian state of Karnataka.

Karnataka, a state in south India, reported its first case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection on March 8, 2020, more than a month after the first case was reported in India. We used a combination of contact tracing and genomic epidemiology to trace the spread of SARS-CoV-2 in the state up until May 21, 2020 (1578 cases). We obtained 91 genomes of SARS-CoV-2 which clustered into seven lineages (Pangolin lineages-A, B, B.1, B.1.80, B.1.1, B.4, and B.6). The lineages in Karnataka were known to be circulating in China, Southeast Asia, Iran, Europe and other parts of India and are likely to have been imported into the state both by international and domestic travel. Our sequences grouped into 17 contact clusters and 24 cases with no known contacts. We found 14 of the 17 contact clusters had a single lineage of the virus, consistent with multiple introductions and most (12/17) were contained within a single district, reflecting local spread. In most of the 17 clusters, the index case (12/17) and spreaders (11/17) were symptomatic. Of the 91 sequences, 47 belonged to the B.6 lineage, including eleven of 24 cases with no known contact, indicating ongoing transmission of this lineage in the state. Genomic epidemiology of SARS-CoV-2 in Karnataka suggests multiple introductions of the virus followed by local transmission in parallel with ongoing viral evolution. This is the first study from India combining genomic data with epidemiological information emphasizing the need for an integrated approach to outbreak response.

Continual circulation of ECSA genotype and identification of a novel mutation I317V in the E1 gene of Chikungunya viral strains in southern India during 2015-2016.

Chikungunya, a mosquito-borne disease caused by Chikungunya virus (CHIKV), continues to be a significant public health problem in India. In 2016, 56 000 cases were reported from India, the largest number since the reemergence of CHIKV in this region in 2006. In the present study, using molecular and phylogenetic methods, the circulating strains from southern India during 2015-2016 were characterized in the context of circulating Asian strains. Partial envelope gene (E1) sequencing was performed on 20 serum samples positive for CHIKV by a reverse transcription-polymerase chain reaction. Phylogenetic analysis showed that all the sequences in this study belonged to the East Central South African (ECSA) genotype and clustered together with other strains from India. Bayesian phylogenetic analysis revealed that the sequences from the study grouped into two different subclades. The estimate of divergence times suggests that subclades of the ECSA genotype, share a common ancestor approximately 4 to 12 years ago. Six nonsynonymous mutations-K211E, M269V, D284E, V322A, I317V and V220I were noted in E1. In conclusion, this study revealed the cocirculation of distinct subclades within the ECSA genotype of CHIKV in South India during 2015-2016. The I317V mutation in E1 has only been described in recent CHIKV strains from north-central India and Bangladesh. This study highlights the need for continued molecular surveillance to identify the emergence of novel strains and unique mutations in CHIKV with epidemic potential.

Dengue virus is an under-recognised causative agent of acute encephalitis syndrome (AES): Results from a four year AES surveillance study of Japanese encephalitis in selected states of India.

BACKGROUND: Acute encephalitis syndrome (AES) surveillance in India has indicated that Japanese encephalitis virus (JEV) accounts for 5-35% of AES cases annually; the etiology remains unknown in the remaining cases. We implemented comprehensive AES surveillance to identify other etiological agents of AES, with emphasis on dengue virus. METHODS: Serum and cerebrospinal fluid (CSF) specimens were collected from patients enrolled prospectively in AES surveillance from 2014-2017 at selected sites of three high burden states of India. All samples were initially tested for JEV IgM. Specimens negative for JEV by serology were tested for IgM to scrub typhus, dengue virus (DEN), and West Nile virus; all JEV IgM-negative CSF samples were tested by PCR for S. pneumoniae, N. meningitidis, H. influenzae, herpes simplex virus type 1, enteroviruses and DEN. RESULTS: Of 10,107 AES patients, an etiology could be established in 49.2% of patients including JEV (16%), scrub typhus (16%) and DEN (5.2%) as the top three agents. Amongst the DEN positive cases (359/6892), seven (2%) were positive only for dengue virus RNA: one in serum and six in CSF. CONCLUSION: Amongst the pathogens identified, dengue accounted for 5% of all AES cases and was one of the three common etiological agents. These results underscore the importance of including dengue virus in routine testing of AES cases.

Syndrome Evaluation System for Simultaneous Detection of Pathogens Causing Acute Encephalitic Syndrome in India, Part-2: Validation Using Well Characterized Clinical Samples.

Diagnosis of the aetiological agent in case of acute encephalitic syndrome (AES) continues to pose a challenge in clinical practice as a variety of pathogens are known to cause AES. Here, we report the validation of a Syndrome Evaluation System (SES) developed for simultaneous detection of multiple AES pathogens using a well characterized set of Cerebrospinal fluid (CSF) samples. The validation of the SES was carried out in two phases. In the first phase, the SES was validated using 51 CSF samples obtained from autopsy proven cases and 50 samples obtained from apparently healthy individuals undergoing spinal anesthesia for minor surgeries served as "controls." The SES detected etilogical agent in 48/51 (94.11 %) samples obtained from autopsy proven AES cases while all the 50 CSF samples obtained from "controls" were negative. In the second phase, the SES was validated using well characterized CSF samples obtained from AES patients fulfilling the WHO case definition of AES (Group I; n = 207) and samples that were collected from patients with non-infectious neurological disorder (Group II; n = 90). All the samples were tested using multiple conventional/serological assays and categorized into various groups. Amongst the AES cases fulfilling WHO case definition, the SES detected AES pathogens in 160/207 (77.29%) cases while conventional serological/molecular assays were able to detect AES pathogens only in 77/207 (37.1%) of cases. Further, in 12/83 CSF samples that were positive by SES and negative by conventional serological/molecular tests, the results were additionally confirmed by sequencing the PCR products to rule out non-specific amplification in the SES. In patients with non-infectious neurological disorders the SES detected latent viruses 12/90 CSF samples. These results indicate that the SES, apart being a rapid, sensitive, specific, and cost-effective method provides the major advantage of simultaneous detection of multiple pathogens using as single specimen of CSF.

Molecular Mimicry between Chikungunya Virus and Host Components: A Possible Mechanism for the Arthritic Manifestations.

BACKGROUND: Chikungunya virus (CHIKV), a reemerging pathogen causes a self limited illness characterized by fever, headache, myalgia and arthralgia. However, 10-20% affected individuals develop persistent arthralgia which contributes to considerable morbidity. The exact molecular mechanisms underlying these manifestations are not well understood. The present study investigated the possible occurrence of molecular mimicry between CHIKV E1 glycoprotein and host human components. METHODOLOGY: Bioinformatic tools were used to identify peptides of CHIKV E1 exhibiting similarity to host components. Two peptides (A&B) were identified using several bioinformatic tools, synthesised and used to validate the results obtained in silico. An ELISA was designed to assess the immunoreactivity of serum samples from CHIKV patients to these peptides. Further, experiments were conducted in a C57BL/6J experimental mouse model to investigate if peptide A and peptide B were indeed capable of inducing pathology. FINDINGS: The serum samples showed reactivity of varying degrees, indicating that these peptides are indeed being recognized by the host immune system during CHIKV infection. Further, these peptides when injected into C57BL/6J mice were able to induce significant inflammation in the muscles of C57BL/6J mice, similar to that observed in animals that were injected with CHIKV alone. Additionally, animals that were primed initially with CHIKV followed by a subsequent injection of the CHIKV peptides exhibited enhanced inflammatory pathology in the skeletal muscles as compared to animals that were injected with peptides or virus alone. Collectively these observations validate the hypothesis that molecular mimicry between CHIKV E1 protein and host proteins does contribute to pathology in CHIKV infection.

Correlation of plasma viral loads and presence of Chikungunya IgM antibodies with cytokine/chemokine levels during acute Chikungunya virus infection.

Chikungunya (CHIKV) is an emerging arboviral infection of public health concern in India contributing to widespread morbidity. The precise molecular events occurring early in the infection have not been well understood. Cytokines/chemokines are suspected to play a key role in its pathogenesis. Very few studies have correlated the plasma levels of cytokines/chemokines with diagnostic markers such as viral loads and presence of CHIKV IgM antibodies. Understanding these dynamics in the early phase of CHIKV infection is likely to provide an insight into the evolution of the immune response, identify biomarkers for assessing severity, and for development of newer therapeutic strategies. This study was therefore undertaken to estimate the levels of various cytokines/chemokines in plasma samples of patients infected with CHIKV and correlate to viral load and CHIKV IgM antibodies. Cytokine/chemokine levels and viral loads in plasma were measured using cytometric bead array and TaqMan real time PCR assay, respectively. The findings revealed that acute phase of CHIKV infection is characterized by predominant inflammatory responses mediated by IL-6, IL-8, IP-10, MCP-1, and MIG (P < 0.003). Plasma levels of IL-6 (r = 0.53, P < 0.05) and MCP-1 (r = 0.83, P < 0.05) emerged as reliable biomarkers of high viral loads in Chikungunya patients. Further, presence of elevated levels of MCP-1 and MIG during the chronic phase of the disease suggests that these chemokines may contribute to perpetuation of symptoms. Hence, these chemokines might serve as targets for the development of treatment to ameliorate the symptoms during the acute phase and prevent the development of chronic manifestations.

Utility of real-time Taqman PCR for antemortem and postmortem diagnosis of human rabies.

Rabies, a fatal zoonotic viral encephalitis remains a neglected disease in India despite a high disease burden. Laboratory confirmation is essential, especially in patients with paralytic rabies who pose a diagnostic dilemma. However, conventional tests for diagnosis of rabies have several limitations. In the present study the utility of a real-time TaqMan PCR assay was evaluated for antemortem/postmortem diagnosis of rabies. Human clinical samples received for antemortem rabies diagnosis (CSF, saliva, nuchal skin biopsy, serum), and samples obtained postmortem from laboratory confirmed rabies in humans (brain tissue, CSF, serum) and animals (brain tissue) were included in the study. All CSF and sera were tested for rabies viral neutralizing antibodies (RVNA) by rapid fluorescent focus inhibition test (RFFIT) and all samples (except sera) were processed for detection of rabies viral RNA by real-time TaqMan PCR. All the 29 (100%) brain tissues from confirmed cases of human and animal rabies, and 11/14 (78.5%) CSF samples obtained postmortem from confirmed human rabies cases were positive by real-time TaqMan PCR. Rabies viral RNA was detected in 5/11 (45.4%) CSF samples, 6/10 (60%) nuchal skin biopsies, and 6/7 (85.7%) saliva samples received for antemortem diagnosis. Real-time TaqMan PCR alone could achieve antemortem rabies diagnosis in 11/13 (84.6%) cases; combined with RVNA detection in CSF antemortem rabies diagnosis could be achieved in all 13 (100%) cases. Real-time TaqMan PCR should be made available widely as an adjunctive test for diagnosis of human rabies in high disease burden countries like India.

Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks.