Differential genotypic signatures of Toll-like receptor polymorphisms among dengue-chikungunya mono- and co-infected Eastern Indian patients.

Dengue (DENV) and chikungunya (CHIKV) viral infections trigger high patient morbidity and mortality. Mono-/co-infection of these viruses activates innate immune response, triggering Toll-like receptor (TLR) pathways. The present study investigated the differential role of TLR3, 7 and 8 single-nucleotide polymorphisms (SNPs) between mono- and co-infected Eastern Indian patients. Interaction of TLR polymorphic variants with signal peptidase complex (SPC18) was explored which might affect immune signalling against DENV/CHIKV infections. Out of 550 febrile symptomatic patients, 128 DENV-CHIKV co-infected samples were genotyped for eight SNPs of TLR3 (rs3775290-chr4:186083063), TLR7 (rs179008-chrX:12885540, rs5741880-chrX:12869297, rs179010-chrX:12884766, rs3853839-chrX:12889539) and TLR8 (rs5744080-chrX:12919685, rs3764879-chrX:12906578, rs3764880-chrX:12906707) by PCR-RFLP along with 157 healthy individuals. Statistical analysis established genotypic association of TLR SNPs with DENV-CHIKV co-infection, and difference between mono- and co-infected patients and their role in determining high viral load (HVL) during competitive viral replication among co-infected patients. In silico protein-protein docking evaluated interactive effect of TLR variants with SPC18. The findings revealed patients with CC genotypes of TLR7 and 8 SNPs were significantly susceptible towards co-infection, whereas specific genotypes of TLR7 and 8 imparted protection against co-infection. Differential analysis between mono-/co-infected patients revealed distinct genotypic distribution of TLR3, 7 and 8 SNPs. Co-infected patients with TT-rs179010 exhibited DENV-HVL, whereas CHIKV-HVL was detected among patients with other genotypes. Molecular docking of TLR7-rs179008 Q variant and TLR8-rs3764880 V variant with SPC18 generated better free binding energy. This study underlined the importance of TLR7 and 8 SNPs towards mono-/co-infection of DENV/CHIKV, with certain genotypes associated with co-infection susceptibility. Moreover, it suggested a probable role of specific genotypes of TLR7 and 8 polymorphisms imparting high dengue/chikungunya viral load among co-infected patients.

Diagnostic accuracy of commercially available immunochromatographic rapid tests for diagnosis of dengue in India.

BACKGROUND & OBJECTIVES: There is limited evidence regarding the accuracy of dengue rapid diagnostic kits despite their extensive use in India. We evaluated the performance of four immunochromatographic Rapid Diagnostic Test (RDTs) kits: Multisure dengue Ab/Ag rapid test (MP biomedicals; MP), Dengucheck combo (Zephyr Biomedicals; ZB), SD bioline dengue duo (Alere; SD) and Dengue day 1 test (J Mitra; JM). METHODS: This is a laboratory-based diagnostic evaluation study. Rapid tests results were compared to reference non-structural (NS1) antigen or immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) results of 241 dengue-positive samples and 247 dengue-negative samples. Sensitivity and specificity of NS1 and IgM components of each RDT were calculated separately and in combination (either NS1 or IgM positive) against reference standard ELISA. RESULTS: A total of 238, 226, 208, and 146 reference NS1 ELISA samples were tested with MP, ZB, SD, and JM tests, respectively. In comparison to the NS1 ELISA reference tests, the NS1 component of MP, ZB, SD, and JM RDTs demonstrated a sensitivity of 71.8%, 85.1%, 77.2% and 80.9% respectively and specificity of 90.1%, 92.8%, 96.1 %, and 93.6%, respectively. In comparison to the IgM ELISA reference test, the IgM component of RDTs showed a sensitivity of 40.0%, 50.3%, 47.3% and 20.0% respectively and specificity of 92.4%, 88.6%, 96.5%, and 92.2% respectively. Combining NS1 antigen and IgM antibody results led to sensitivities of 87.5%, 82.9%, 93.8% and 91.7% respectively, and specificities of 75.3%, 73.9%, 76.5%, and 80.0% respectively. INTERPRETATION & CONCLUSION: Though specificities were acceptable, the sensitivities of each test were markedly lower than manufacturers' claims. These results also support the added value of combined antigen-and antibody-based RDTs for the diagnosis of acute dengue.

Increased CRP, anti-CCP antibody, IL-2R, COMP levels in prognosis of post-chikungunya chronic arthritis and protective role of their specific genotypes against arthritic manifestation.

Chikungunya infection leads to acute/chronic polyarthritis/polyarthralgia causing long-term morbidity among patients. Prognosis of post-chikungunya chronic arthritis (PCA) is of utmost necessity for proper disease management. Arthritic and hepatic biomarkers were evaluated among chikungunya patients without arthritis, with acute arthritis and with post-chikungunya chronic arthritis in the study. Serum levels of arthritic [CRP (C-reactive protein), anti cyclic-citrullinated-peptide (anti-CCP) antibody, soluble interleukin-2 receptor (sIL-2R), cartilage oligomeric matrix protein (COMP)] and hepatic [ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), albumin and bilirubin] biomarkers of 167 chikungunya positive patients were determined by sandwich-ELISA/immunoturbidimetry/auto-analyser. 167 chikungunya-patients and 102 healthy controls were genotyped to understand role of CRP-rs3093059/rs3091244, IL-2R-rs743777 and COMP-rs144778694 polymorphisms towards chikungunya virus (CHIKV) infectivity and arthralgic manifestation. CRP, anti-CCP antibody, IL-2R and COMP levels significantly increased among PCA patients. Concentrations of AST, ALT, AST/ALT-ratio, bilirubin and ALP increased among arthritic chikungunya patients. Principal component analysis differentiated PCA groups from acute (AA) and non-arthritic groups. Patients with IL-2R-rs743777-GA, G-allele and COMP-rs144778694-GA genotypes were susceptible to chikungunya infection. Moreover, patients with CRP-rs3093059-CT, rs3091244-TT, IL-2R-rs743777-GA and COMP-rs144778694-AA genotypes were significantly protected from arthralgia, whereas, COMP-rs144778694-GA genotype was susceptible towards it. Patients with certain genotypes of CRP, IL-2R and COMP demonstrated significantly higher biomarker serum-levels among patients suffering from AA with/without PCA. Thus, both serum biomarker levels and polymorphic genotypes of infected patients play decisive role in development of PCA.

Association of C-reactive protein polymorphisms with serum-CRP concentration and viral load among dengue-chikungunya mono/co-infected patients.

India being endemic to Dengue (DENV) and chikungunya (CHIKV) infections faces high patient-mortality and morbidity with overlapping clinical features. C-reactive protein (CRP) acts as early defence system in response to these infections. This study investigated role of CRP single-nucleotide polymorphism (SNP) genotypes and protein levels towards DENV/CHIKV mono and co-infection among eastern Indian patients. 128 DENV-CHIKV co-infected, 206 DENV and 167 CHIKV mono-infected patients were subjected to genotyping of two CRP SNPs by PCR-RFLP along with 102 healthy individuals. CRP levels were determined by immunoturbidimetry. Statistical correlation of CRP genotypes with CRP concentration, DENV-CHIKV mono/co-infection and viral load was performed. Patients with rs3093059-CT and rs3091244-TT were more susceptible to DENV-CHIKV co-infection, whereas, rs3091244-CT might have imparted protection against CHIKV mono-infection. DENV-HVL was more prevalent within rs3093059-TT and rs3091244-CT co-infected patients, whereas, CHIKV-HVL among rs3091244-CC. Acute phase co-infected patients had significantly higher CRP level compared to mono-infections. Both mono and co-infected patients with aches/pain exhibited 2-3-fold higher CRP levels compared to those without. rs3093059-CT and rs3091244-CT co-infected patients had higher CRP concentration compared to rs3093059-TT and rs3091244-CC, respectively. Co-infected patients with WHO-defined warning signs had higher anti-dengue IgG/IgM ratio and serum CRP level compared to those without warning signs. Thus, patient's CRP genotype might play significant role in determining serum-CRP concentration, viral load and DENV-CHIKV mono/co-infection.

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.

Re-emergence of Chikungunya virus infection in Eastern India.

Chikungunya fever is a major public health issue in India. Re-emergence of chikungunya virus (CHIKV) in West Bengal was detected after 32 years in 2006. After 2010, this infection was in apparent decline, but in 2016 a massive outbreak affected the country. Present study was carried out to understand CHIKV infection dynamics during recent outbreaks in West Bengal, Eastern India and its implication on disease manifestations. Blood was collected from 641 symptomatic patients. Patients' sera were serologically diagnosed to detect presence of anti-chikungunya-IgM antibodies. Viral RNA was extracted; presence of CHIKV genome and its respective viral load was determined by real time quantitative reverse transcription-PCR (real-time qRT-PCR). Statistical analysis was performed using EPI INFO software. CHIKV infection was detected in 24.64% of symptomatic patients. Middle-aged patients (31-40 years) were predominantly affected; clinically, both arthralgia and joint-swelling were significantly prevalent among CHIKV-infected patients. Myalgia, joint-swelling, and arthralgic manifestation were found in significantly higher frequency among patients with high chikungunya viral load (> 10,000 copies/ml). Thus, this study clearly indicated the re-emergence of CHIKV in Eastern India. Significant presence of myalgia, joint swelling, and arthralgia among chikungunya patients with high viral load implied association of disease severity with viral load; requiring vigilance for proper management of infected patients as this disease is highly morbid in nature. However, in addition to chikungunya virus, other viral, bacterial, and protozoal infections also occur during post-monsoon season in India, having overlapping symptoms. Hence, continuous monitoring of these infections is required for better clinical management of patients.

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.

Differential Expression and Significance of Circulating microRNAs in Cerebrospinal Fluid of Acute Encephalitis Patients Infected with Japanese Encephalitis Virus.

Changes in circulating microRNAs (miRNAs) in the cerebrospinal fluid (CSF) have been associated with different neurological diseases. Here, we presented results of a pilot study aimed at determining the feasibility of detecting miRNAs in the CSF of Japanese Encephalitis virus (JEV) infected individuals with acute encephalitis syndrome (AES). We demonstrated the circulating miRNA profile in CSF of acute encephalitis patients infected with JEV. Using a quantitative real-time PCR-based miRNA array, we examined the level of 87 miRNAs expressed in human exosomes isolated from CSF. Subsequently, correlation between cytokine level and miRNAs expression in CSF samples was examined. In this study, we identified and validated the upregulated expression of three miRNAs, miR-21-5p, miR-150-5p, and miR-342-3p that were specifically circulated in CSF of acute encephalitis patients infected with JEV. CSF miR-21-5p, miR-150-5p, and miR-342-3p expressions were also elevated in infected mice brain. However, the expression pattern of these miRNAs differed in neuronal cells, microglial cells, and the exosome derived from JEV-infected cell culture supernatant. Interestingly, neuronal cells infected with vaccine strain (SA-14-14) did not lead to any upregulation of these three miRNAs. Further, miR-150-5p expression was found to be negatively correlated(r = -0.5279, p = 0.016) with TNFα level. Pathway analysis of putative target genes of these miRNAs indicated involvement of TGF-ß, NGF, axon guidance, and MAPK signaling pathways in JEV/AES patients. This study for the first time represents the circulating miRNA in CSF of AES patients and identified the upregulated miRNAs in JEV-infected patients and offers the basis for future investigation.

RNA-Seq analysis of peripheral blood mononuclear cells reveals unique transcriptional signatures associated with disease progression in dengue patients.

Patients infected with Dengue virus usually present a mild, self-limiting febrile dengue infection (DI) that occasionally leads to a potentially lethal complication, called the severe dengue (DS). The ability to identify the prognostic markers of DS could allow an improved disease intervention and management. To identify the transcriptional signatures associated with the dengue disease progression, we carried out the high-throughput sequencing of the RNA isolated from the peripheral blood mononuclear cells (PBMCs) of the dengue patients of varying severity and compared with that in the patients with other febrile illnesses (OFIs) or the healthy controls. The transcriptional signatures that discriminated the DS patients from OFI and DI patients were broadly related to the pathways involving glycine, serine, and threonine metabolisms, extracellular matrix organization, ubiquitination, and cytokines and inflammatory response. Several upregulated genes in the inflammatory process (MPO, DEFA4, ELANE, AUZ1, CTSG, OLFM4, SLC16A14, and CRISP3) that were associated with the dengue disease progression are known to facilitate leukocyte-mediated migration, and neutrophil activation and degranulation process. High activity of MPO and ELANE in the plasma samples of the follow-up and recovered dengue patients, as well as and the presence of a larger amount of cell-free dsDNA in the DS patients, suggested an association of neutrophil-mediated immunity with dengue disease progression. Careful monitoring of some of these gene transcripts, and control of the activity of proteins encoded by them, may have a great translational significance for the prognosis and management of the dengue patients.

Incidence of Japanese encephalitis among acute encephalitis syndrome cases in West Bengal, India.

BACKGROUND AND OBJECTIVES: Japanese encephalitis (JE) is the most important cause of acute and epidemic viral encephalitis. Every year sporadic JE cases are reported from the various districts of West Bengal, indicating its endemicity in this state. JE vaccination programme has been undertaken by the State Health Department of West Bengal. This study was aimed at seeing the present scenario of JE among acute encephalitis syndrome (AES) cases in West Bengal. MATERIALS AND METHODS: Blood and/or CSF samples were referred from suspected AES cases to the referral virology laboratory of the Calcutta School of Tropical Medicine from different hospitals of Kolkata. IgM antibody capture ELISA was performed on the CSF and serum samples by JE virus MAC ELISA kit supplied by the National Institute of Virology, Pune. RESULTS: The present study reveals that 22.76% and 5% of the AES cases were positive for JE IgM in 2011 and 2012, respectively. JE is mainly prevalent in children and adolescents below 20 years of age with no gender predilection. Although the percentages of JE positive cases were high in 2011, it sharply decreased thereafter possibly due to better awareness programs, due to mass vaccination, or simply due to natural epidemiological niche periodicity due to herd immunity.