Assessment of larval and pupal indices of dengue mosquito vectors in a North-Eastern state of Tripura, India.

BACKGROUND OBJECTIVES: Dengue is a major vector-borne disease having public health importance. It is caused by Dengue Virus (DENV) and is transmitted by mosquitoes of Aedes species. With the unavailability of a vaccine, vector control remains the only preventive measure for dengue. Studies have already been conducted to establish the presence of dengue vectors in the north-eastern states of India. However, limited studies have been conducted in Tripura state. In the present study we aimed to identify the preferred breeding habitats of dengue vectors in the state. METHODS: Clinical case data of dengue since the last five years was studied and the areas with the highest case numbers were identified. Entomological investigation was carried out in areas reporting the highest number of cases. Larvae were collected from the breeding habitats using standard protocol followed by morphological and molecular identification. Further, House index (HI), Container index (CI) and Pupal index (PI) were determined. The positive pools were then processed for incrimination for the presence of dengue virus. Calculation of entomological indices was done. RESULTS: Of the total 815 containers searched, 36.80% containers were positive for mosquito larvae. Among the immature mosquito collection, 836 adults emerged and were identified as Aedes albopictus using standard taxonomic keys followed by molecular methods. HI, CI and PI, varied from 15.38% to 100%, 21% to 31.04 %, and 2.93% to 110.53% respectively. However, none of the pools was positive for dengue virus. INTERPRETATION CONCLUSION: The present study identified Ae. albopictus as a potential vector of dengue in Tripura. The study gave important insights on the preferred larval habitats and provides information on the indication of displacement of Ae. albopictus from rural to urban and semi-urban areas. However, longitudinal studies for longer time frame are necessary for any conclusive remarks.

Persistence of West Nile Virus circulation in North-East region of India: A prospective facility-based study spanning over a decade.

BACKGROUND OBJECTIVES: To understand the distribution and presence of West Nile (WN) among the acute encephalitis syndrome (AES) patients, a prospective facility-based study was conducted over 13 years (2007-2019). METHODS: During the period, 8957 patients hospitalized with AES in northeastern India were tested for the evidence of WN virus infection by IgM ELISA followed by Plaque reduction neutralization test (PRNT). RESULTS: Of the samples tested 324 (3.62%) were found positive for WN IgM antibodies and 73 paired sera showed a fourfold rise in neutralizing antibody titre by PRNT. The study establishes WN as a noteworthy cause of encephalitis in an erstwhile endemic area for Japanese encephalitis (JE). INTERPRETATION CONCLUSION: Therefore, it is important that WN is recognized as an important acute encephalitis syndrome (AES) causing aetiology in this region and the National centre for vector borne disease control (NCVBDC) guidelines for detection of AES may be modified accordingly. Thus, inclusion of WN in routine diagnosis, along with establishment of an integrative surveillance network with one-health approach will be important.

Clinical Utility of Pro-inflammatory Oligomeric Glycoprotein Tenascin-C in the Diagnosis of Seropositive and Seronegative Rheumatoid Arthritis.

Owing to limited usefulness of Rheumatoid Factor and anti-CCP in rheumatoid arthritis, there is a need to identify a more sensitive and specific biomarker to detect rheumatoid arthritis (RA), particularly seronegative RA cases. Tenascin-C is an extracellular matrix glycoprotein, which has been implicated in the pathophysiology of RA. The objective of our study was to evaluate the diagnostic utility of serum Tenascin-C in seropositive and seronegative rheumatoid arthritis patients. We conducted a cross-sectional case control study. Sixty patients who fulfilled the ACR 2010 criteria for rheumatoid arthritis were included in the study. Thirty patients were found to be positive for RF and/or anti-CCP and 30 were negative for both RF and anti-CCP. Thirty age and gender-matched healthy subjects were taken as controls. Serum Tenascin-C was measured by quantitative sandwich enzyme immunoassay technique. The mean serum concentration of Tenascin-C in controls, seronegative and seropositive cases was 0.66 ng/ml, 20.54 ng/ml and 23.42 ng/ml, respectively. Tenascin-C levels were significantly higher in RA cases compared to controls (p < 0.0001). There was no significant difference in Tenascin-C between seropositive and seronegative cases (p = 0.603). ROC curve analysis showed a sensitivity of 96.6% and specificity of 100% with AUC of 0.98 at 2.21 ng/ml as cut-off value for diagnosing RA. Tenascin-C is elevated in both seronegative and seropositive RA, which indicates that it can be used as a sensitive marker for RA. The addition of Tenascin-C to the existing RF and anti-CCP may help in identifying a large number of patients with RA, particularly seronegative rheumatoid arthritis cases.

Global emergence of West Nile virus: Threat & preparedness in special perspective to India.

West Nile virus (WNV) is a mosquito-borne single-stranded RNA neurotropic virus within the family Flaviviridae. The virus was first reported in the West Nile province of Uganda in 1937. Since then, sporadic cases have been reported until the last two decades when it has emerged as a threat to public health. The emergence of WNV with more severity in recent times is intriguing. Considering this phenomenon, the WNV-affected areas of the world were distinguished as old versus new in a depicted world map. The present review showcases the historical and epidemiological perspectives of the virus, genetic diversity of prevailing lineages and clinical spectrum associated with its infection. Emergence of the virus has been discussed in special context to India because of co-circulation of different WNV lineages/strains along with other flaviviruses. Recent laboratory diagnostics, vaccine development and clinical management associated with WNV infection have also been discussed. Further, the research gaps, especially in context to India have been highlighted that may have a pivotal role in combating the spread of WNV.

Spotted Fever Group Rickettsiae among Children with Acute Febrile Illness, in Gorakhpur, India.

Seasonal outbreaks of acute encephalitis syndrome have been occurring in Gorakhpur division in the Indian state of Uttar Pradesh during monsoon and post-monsoon months. Orientia tsutsugamushi was identified as the major aetiology of these outbreaks. Orientia tsutsugamushi was also identified as one of the important aetiology of febrile illness among children attending peripheral health facilities. The present study was undertaken to detect antibodies against spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) among children with acute febrile illness presenting at peripheral health facilities in Gorakhpur district. Of the 224 blood samples tested, SFGR infection was detected in 13 (6%) patients. None of the samples tested positive for TGR.

Experimental study of dengue virus infection in Aedes aegypti and Aedes albopictus: A comparative analysis on susceptibility, virus transmission and reproductive success.

Epidemiology of dengue fever has substantially changed over the years with respect to prevalent strains, affected geographical locations and severity of disease. Mosquito vectors show variable response in terms of susceptibility to four different serotypes of dengue virus. Although studies have postulated that, the vectors Ae. aegypti and Ae. albopictus are crucial for transmission of dengue virus, comparative efficacy of these species for viral transmission and tolerance is still enigmatic. In this study, these two vectors were infected orally with four serotypes of the dengue virus viz. DENV-1 to DENV-4 and their co-infection. It was observed that Ae. aegypti harbors multiple serotype infections more efficiently than Ae. albopictus. We suggest that transovarial transmission is of low importance in the epidemiology of the virus due to low infection rates in the filial generation, and also that reduced fecundity and fertility in both vectors after dengue virus infection affect the ecology of the pathogen.

Adaptive Neuro-Fuzzy Inference System-Based Exploration of the Interrelationships of 25-Hydroxyvitamin D, Calcium, Phosphorus with Parathyroid Hormone Production.

The rationale of the current study was to assess the prevalence of 25-hydroxyvitamin D (25-OHD) deficiency and hyperparathyroidism in South Indian population and to explore interrelationships of 25-OHD, Ca, P towards parathyroid hormone (PTH) production using adaptive neuro-fuzzy inference system (ANFIS). A total of 407 subjects (228 men 179 women) with the mean age 56.8 ± 14.1 were tested for these parameters. In view of the skewed distribution of biochemical variables, data segregation was performed in tertiles and this data was trained to generate fuzzy interference system based on subclusters. The optimized model had 358 nodes and followed 44 fuzzy rules for prediction. This ANFIS model demonstrates that the deficiency of 25-OHD and Calcium triggers PTH production. PTH elevation is significant when Phosphorus is in the highest tertile. The associations observed by this model were consistent with the Kendall-Tau correlation matrix, which revealed inverse associations of Ca with P; and Ca with PTH and positive associations of P with PTH, and Ca with 25-OHD. Furthermore, the association statistics of the machine learning algorithm were also consistent, which suggested that depletion of Ca below 8.245 mg/dl was shown to elevate PTH levels greater than 167 pg/ml when P > 4.66. Subnormal depletion in 25-OHD (9.3-16.2 ng/ml) is associated with subnormal elevation in PTH (47-73.6 pg/ml). To conclude, ANFIS and machine learning algorithm are in agreement with each other in stating that 25-OHD deficiency triggers lower calcium levels, lower calcium and higher phosphorus trigger PTH production.

Dengue virus serotypes and genotypic characterization from northeast India.

Dengue is a rapidly spreading acute arboviral infection transmitted through a human and Aedes mosquito cycle. Though northeast region of India has been experiencing dengue outbreaks regularly for over a decade, reports on genetic characterization of the virus from this region are limited. The present study was undertaken to detect the genotype and genetic composition of circulating dengue virus (DENV) in this region. Blood samples were collected from 918 suspected dengue patients of five northeast Indian states. Serological investigations, viz, nonstructural 1 (NS1) enzyme-linked immunosorbent assay (ELISA), immunoglobulin M (IgM) ELISA, and immunoglobulin G (IgG) ELISA were performed followed by molecular detection. Sequence analysis and phylogenetic tree construction based on capsid-premembrane (C-prM) gene junction was done by BioEdit and MEGA6 software, respectively. Serological detection showed 35.34% NS1 and 18.12% IgM positivity. Secondary infection was observed in 24.53%. All four serotypes were detected. Phylogenetic analysis demonstrated circulation of genotype III of DENV-1, genotype IV of DENV-2, and genotype III of DENV-3. Sequences from this region form distinct clades in the phylogenetic tree. Characterization of the C-prM gene junction reveals divergence among the DENV strains. As genetic variation within the DENV is known to be associated with diverse clinical outcomes, information regarding the genetic composition of circulating virus could be beneficial in designing an effective intervention strategy.

Hepcidin and Ferritin: Important Mediators in Inflammation Associated Anemia in Systemic Lupus Erythematosus Patients.

Systemic Lupus Erythematosus is an autoimmune disease with female preponderance. Anemia is found in 50% of Systemic Lupus Erythematosus patients. This is a cross sectional case control study with 30 female Systemic Lupus Erythematosus patients having inflammation associated anemia (Hemoglobin < 10.0 gm/dl) and 30 age matched controls with the aim to measure serum hepcidin and ferritin levels, correlate and study their role as homeostatic regulators of iron metabolism and utility as markers. Serum transferrin, ferritin, iron, total iron binding capacity, hsCRP, liver enzymes and renal parameters were analyzed by using automated analyser. Hepcidin levels were estimated by Sandwich-ELISA method. There was significant decrease in Iron (p < 0.0001), Iron Binding capacity (p < 0.0001), Transferrin (p < 0.0001) in patients, and a significant increase in inflammatory markers: hs-CRP (p < 0.0001), ESR (p < 0.0001) compared to controls. Significant increase in both Hepcidin (p < 0.0001) and Ferritin (p < 0.0001) was observed in patients with significant positive correlation (r = 0.711) with each other. Additionally, ferritin and hepcidin significantly positively correlated with hs-CRP and ESR (r = 0.526, 0.735); (r = 0.427, 0.742) respectively. Negative correlation with hemoglobin, iron, total iron binding capacity and transferrin with hepcidin (r = - 0.80, - 0.307, - 0.553, - 0.584) and ferritin (r = -0.722, - 0.22, - 0.654, - 0.728) was observed respectively. On ROC analysis both hepcidin and ferritin has sensitivity of 96.7%, specificity of 100% at cut-off values of 110 and 49 respectively. AUC of hepcidin was 0.993 and ferritin was 0.978. We have established a positive linear correlation between Hepcidin and Ferritin levels in disease activity and the changes correlated with the inflammatory state and anemia in patients, making them important mediators and potential markers of inflammation associated anemia.

Scrub Typhus Leading to Acute Encephalitis Syndrome, Assam, India.

To determine the contribution of Orientia tsutsugamushi, the agent of scrub typhus, as a cause of acute encephalitis syndrome (AES) in Assam, India, we conducted a retrospective study of hospital patients with symptoms of AES during 2013-2015. Our findings suggest that O. tsutsugamushi infection leads to AES and the resulting illness and death.

Enhanced cytopathic effect of Japanese encephalitis virus strain SA14-14-2: Probable association of mutation in amino acid of its envelope protein.

Japanese encephalitis (JE) is a mosquito-borne viral disease. It is a global public health concern since it causes an acute encephalitis syndrome (AES). A large number of JE/AES cases are reported to occur in areas with established or developing JE vaccination program. Partial vaccine coverage and emergence of new variants of Japanese encephalitis virus (JEV) might be playing an important role. The envelope protein (E) of JEV is a major antigenic determinant and responsible for immunogenic responses as well as membrane fusion and virion assembly. In the present study, we have characterized the JEV live attenuated vaccine strain SA14-14-2 in baby hamster kidney cells (BHK-21). The vaccine strain showed enhanced replication following its passage in BHK-21 cells. Nucleotide sequence analysis of the E protein gene of the cell-culture adapted vaccine strain showed an important point mutation. The mutation in the E protein gene was identical to its wild-type parent strain SA14. This study suggests the possibility of reversion mutation and exaltation of vaccine strains following adaptation in the host cells.

Using in silico techniques: Isolation and characterization of an insect cuticle-degrading-protease gene from Beauveria bassiana.

Cuticle-degrading-proteases (CDPs) secreted by Beauveria spp. are pivotal biocontrol substances, possessing commercial potential for developing bio-pesticides. Therefore, a thoughtful and contemplative understanding and assessment of the structural and functional features of these proteases would markedly assist the development of biogenic pesticides. Computational molecular biology is a new facile alternative approach to the tedious experimental molecular biology; therefore, by using bioinformatics tools, we isolated and characterized an insect CDP gene from Beauveria bassiana 70 s.l. genomic DNA. The CDP gene (1240 bp with GeneBank accession no. KT804651.1) consisted of three introns and four CDS exons, and shared 74-100% sequence identity to the reference CDP genes. Its phylogenetic tree results showed a unique evolution pattern, and the predicted amino acid peptide (PAAP) consisted of 344 amino acid residues with pI, molecular weight, instability index, grand average hydropathicity value and aliphatic index of 7.2, 35.4 kDa, 24.45, -0.149, and 76.63, respectively. The gene possessed 74-89% amino acid sequence similarity to the 12 reference strains. Three motifs (Peptidase_S8 subtilase family) were detected in the PAAP, and the computed 3D structure possessed 79.09% structural identity to alkaline serine proteases. The PAAP had four (three serine proteases and one Pyridoxal-dependent decarboxylase) conserved domains, a disulfide bridge, two calcium binding sites, MY domain, and three predicted active sites in the serine family domains. These results will set the groundwork for further exploitation of proteases and understanding the mechanism of disease caused by cuticle-degrading-serine-proteases from entomopathogenic fungi.

Immunogenicity & safety of a single dose of live-attenuated Japanese encephalitis vaccine SA 14-14-2 in adults.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) caused by mosquito-borne Flavivirus is one of the leading causes of viral encephalitis in Asia. Control strategies include vector control and human vaccination. Due to lack of immunization programmes in endemic regions, there are still high mortality and morbidity. A live-attenuated SA 14-14-2 JE vaccine (LAJEV) has been licensed and used in Asian countries, including India. We report the assessment of immunogenicity and safety of the vaccine in adults during the first mass adult vaccination campaign carried out in Assam, India. METHODS: One thousand and seventy five adults (aged ≥15 yr) who received LAJEV were monitored for adverse events following immunization for one year. The safety assessment of vaccinated population was evaluated till 28 days and at 6 and 12 months. Blood samples collected from the enrolled participants were tested by plaque reduction neutralization test (PRNT 50 ) to assess the neutralizing antibody titres (NATs) before vaccination and 28 days, six and 12 months post-vaccination (PV). RESULTS: Among the 1075 vaccinated individuals, four reported minor adverse effects from 30 min to 28 days PV. Based on the pre-vaccination NAT, the study participants were categorized as seronegative, moderately seropositive and strongly seropositive. Nearly 85.5 per cent of JE seronegative participants seroconverted by 28 days PV. The geometric mean titre (GMT) in all the three groups increased by 28 days and decreased by six and 12 months PV. Nearly 60 per cent of the moderately positive individuals exhibited four-fold rise in GMT, 28 days PV. Almost 95.5 per cent of the participants in the study population remained seroprotected at the end of 12 months PV. INTERPRETATION & CONCLUSIONS: This study on immunogenicity and safety of LAJEV in adults showed that a single dose of the live-attenuated vaccine was safe and induced protective immunity to both JE seronegative and naturally seropositive adults. Further study is required to find out long term protective efficacy of this vaccine.

Chikungunya outbreak in Garo Hills, Meghalaya: An epidemiological perspective.

BACKGROUND & OBJECTIVES: Chikungunya (CHIK) fever is a mosquito-borne disease caused by chikungunya virus (CHIKV). Chikungunya infection was first reported from India in 1963 from Kolkata. We report the serological and molecular evidence of an outbreak of chikungunya in northeast India that occurred in Tura, a hilly and forested terrain in Garo Hills district of Meghalaya. METHODS: Blood samples (3 ml) collected from hospitalized patients during the outbreak were tested for IgM antibodies against CHIKV and followed up four months later. A repeat survey was carried out in the same area after four months from where cases had been reported. Blood samples were also collected from people with history of fever and body ache in the last four months. Persons showing IgM positivity against CHIKV in the repeat survey were followed up one and a half years later. All samples were also processed by RT-PCR assay for CHIK Envelope (E) 1 gene. Immature mosquitoes were collected, link reared and identified with standard keys. Virus incrimination studies were done on Aedes aegypti and Ae. albopictus mosquitoes collected during the survey. RESULTS: Fever, headache and joint pain were the primary clinical presentations. Twenty three (35.93 %) of 64 samples reported during the outbreak were IgM positive for CHIK. Three samples showed PCR amplification. All these were IgM positive. The sequenced E1 gene revealed that the strains belonged to East Central South African (ECSA) genotype. INTERPRETATION & CONCLUSIONS: Field survey done after four months revealed that some individuals still had joint pain associated with episodes of headache and fever. It could be inferred that these persons might have contracted infection during the CHIK outbreak four months ago or during the intervening period which caused persistence of sequelae. ECSA genotype was found to be involved in the outbreak. Aedes albopictus was the predominant mosquito species collected during the outbreak.

First evidence of dengue virus infection in wild caught mosquitoes during an outbreak in Assam, Northeast India.

BACKGROUND & OBJECTIVES: Dengue is one of the major public health problems worldwide, transmitted mainly by Aedes aegypti and Ae. albopictus mosquitoes. Rapid urbanisation and industrialisation have led to an increase in vector population in Northeastern states of India. In 2013, Guwahati, the capital city of Assam, India experienced an outbreak of dengue. This study was undertaken with an objective to determine infection rates of dengue viruses (DENV) in both the established vectors present in this region. METHODS: During the outbreak (2013), adults and larvae of both the vector species were collected from different container habitats found in case reporting areas and container index was also recorded. The mosquitoes were first pooled, homogenised and processed for NS1-ELISA. This was followed by RT-PCR of the mosquito pools. RESULTS: Both Ae. aegypti and Ae. albopictus were found breeding in containers with container index in the range of 29.41 to 80%. Six pools of Ae. aegypti were found to be positive for NS1 antigen. RT-PCR assay revealed positivity in only the NS1-ELISA positive pools, exhibiting circulation of serotype DENV-2. Minimum infection rate of female and male Ae. aegypti was recorded as 10.87 and 11.03 respectively. INTERPRETATION & CONCLUSION: This is the maiden report of detection of DENV in wild caught Ae. aegypti mosquitoes from Northeastern Region of India. The study also demonstrates the presence of transovarial transmission of dengue virus in this part of country. This information is useful in respect of both entomological as well as epidemiological point of view for taking appropriate vector control measures.

Dengue outbreak in a hilly state of Arunachal Pradesh in Northeast India.

Dengue has been reported from plains as well as hilly regions of India including some parts of Northeast India. In July-August 2012, outbreak of fever with unknown origin (FUO) indicative of Dengue was reported in Pasighat, East Siang district of Arunachal Pradesh (AP) state. Serum samples (n = 164) collected from patients from Health Training and Research Centre General Hospital, Pasighat, were tested for NS1 antigen and IgM antibodies. NS1-positive samples were analyzed by RT-PCR assay and entomological surveys were carried out. The majority of suspected cases reported NS1 antigen positivity. Females and young adults were mostly affected. The majority of the amplified NS1-positive samples showed Dengue serotype 3 infection. Aedes (Stegomyia) albopictus, known as semiurban breeding mosquitoes, was the only potential vector species identified from the affected areas of Pasighat which single handedly contributed to the outbreak. Thus, the present work identifies Dengue as an emerging arboviral infection in hilly state of AP along with a looming risk of its spread to neighbouring areas.

Forecasting Japanese encephalitis incidence from historical morbidity patterns: Statistical analysis with 27 years of observation in Assam, India.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is one of the dreaded mosquito-borne viral diseases mostly prevalent in south Asian countries including India. Early warning of the disease in terms of disease intensity is crucial for taking adequate and appropriate intervention measures. The present study was carried out in Dibrugarh district in the state of Assam located in the northeastern region of India to assess the accuracy of selected forecasting methods based on historical morbidity patterns of JE incidence during the past 22 years (1985-2006). METHODS: Four selected forecasting methods, viz. seasonal average (SA), seasonal adjustment with last three observations (SAT), modified method adjusting long-term and cyclic trend (MSAT), and autoregressive integrated moving average (ARIMA) have been employed to assess the accuracy of each of the forecasting methods. The forecasting methods were validated for five consecutive years from 2007-2012 and accuracy of each method has been assessed. RESULTS: The forecasting method utilising seasonal adjustment with long-term and cyclic trend emerged as best forecasting method among the four selected forecasting methods and outperformed the even statistically more advanced ARIMA method. Peak of the disease incidence could effectively be predicted with all the methods, but there are significant variations in magnitude of forecast errors among the selected methods. As expected, variation in forecasts at primary health centre (PHC) level is wide as compared to that of district level forecasts. INTERPRETATION & CONCLUSION: The study showed that adopted forecasting techniques could reasonably forecast the intensity of JE cases at PHC level without considering the external variables. The results indicate that the understanding of long-term and cyclic trend of the disease intensity will improve the accuracy of the forecasts, but there is a need for making the forecast models more robust to explain sudden variation in the disease intensity with detail analysis of parasite and host population dynamics.

Efficient leukocytes detection and classification in microscopic blood images using convolutional neural network coupled with a dual attention network.

Leukocytes, also called White Blood Cells (WBCs) or leucocytes, are the cells that play a pivotal role in human health and are vital indicators of diseases such as malaria, leukemia, AIDS, and other viral infections. WBCs detection and classification in blood smears offers insights to pathologists, aiding diagnosis across medical conditions. Traditional techniques, including manual counting, detection, classification, and visual inspection of microscopic images by medical professionals, pose challenges due to their labor-intensive nature. However, traditional methods are time consuming and sometimes susceptible to errors. Here, we propose a high-performance convolutional neural network (CNN) coupled with a dual-attention network that efficiently detects and classifies WBCs in microscopic thick smear images. The main aim of this study was to enhance clinical hematology systems and expedite medical diagnostic processes. In the proposed technique, we utilized a deep convolutional generative adversarial network (DCGAN) to overcome the limitations imposed by limited training data and employed a dual attention mechanism to improve accuracy, efficiency, and generalization. The proposed technique achieved overall accuracy rates of 99.83%, 99.35%, and 99.60% for the peripheral blood cell (PBC), leukocyte images for segmentation and classification (LISC), and Raabin-WBC benchmark datasets, respectively. Our proposed approach outperforms state-of-the-art methods in terms of accuracy, highlighting the effectiveness of the strategies employed and their potential to enhance diagnostic capabilities and advance real-world healthcare practices and diagnostic systems.