Development of a novel lectin-based gold nanoparticle point-of-care immunoassay for rapid diagnosis of patients with severe Dengue infection.

Rapid diagnosis of patients with severe Dengue infection can be useful for the efficient clinical management of cases caused by the Dengue virus. Lateral Flow Immunoassay (LFIA) have been broadly used for rapid Dengue diagnosis, because of their quick readouts with the human eye, simplicity of use, and affordability. Despite the availability of several commercial Dengue point-of-care assays, none has shown to be successful in discriminating between severe and nonsevere forms of Dengue infection. In the current study, for the first time, a novel lectin-based point-of-care assay for the early detection of patients with severe Dengue infection with gold-adorned sheets as detection labels is being reported. In this assay, Dengue severity was diagnosed by detecting the glycosylation profile of vitronectin, a known Dengue severity marker. Two lectins were employed namely DSA (Datura stramonium) and MAA (Maackia amurensis) that can recognize specific glycans like galactose Gal-(1-4) GlcNAc and sialic acid in an (α2-3) linkage, which displayed high sensitivity and high specificity, i.e. 90% and 85% for DSA and 90.91% and 95% for MAA. The new assay has a detection limit of 5 µg µl-1 and enables the quick (30 min) and sensitive detection of severe Dengue cases. The reported point-of-care immunoassay exhibits considerable promise for early identification of patients with Dengue severity.

Recent advances of lateral flow immunoassay components as "point of need".

Lateral flow immunoassay is the leading Point of Care test and is becoming increasingly essential for its versatile properties. The attraction of lateral flow assay (LFA) has reached its prime position during recent SARS-CoV-2 pandemic and Ebola, Zika epidemics in third world countries where primary screening of the disease and financial issues are very important. During the last decade traditional methodology of LFA was limited to visual detection and qualitative assessment only. However, recently researchers are focusing on the development and improvement of this tool to enhance its specificity, assessment power (quantitative) to make it an alternative to traditional lab-based technology. Modifying working principle and instrumentation, combination of different modern molecular techniques such as Reverse transcription loop mediated isothermal amplification (RT-LAMP), Clustered regularly inter-spaced short palindromic repeat (CRISPR-Cas), Recombinase amplification polymerase (RPA), also association of image-based software, involvement of nanotechnology, implementation of LFA ruler have established authenticity and ultra-specific detection level. These leading immunochromatographic techniques offer simultaneous detection of different analytes from a single sample unit into one multiplex strip and provide the necessary information. This review is a foremost attempt to encompass recent advances of lateral flow assays in combination with molecular biology techniques along with improvements of assay components for improved diagnostic sensitivity and specificity. Some infectious disease diagnosis by LFA with its reporter and low detection limit have also been mentioned in this review.

Comprehensive investigation of fever cases enrolled during 2019 dengue outbreaks from three hyperendemic regions of North 24 Parganas district of West Bengal, India.

For the past several decades, dengue fever has been emerging in epidemic proportions in several regions of the world. During August-September 2019, an increasing number of fever cases were being reported from some areas of North 24 Parganas district of West Bengal, India. Accordingly, outbreak investigation of fever cases from these affected areas of Bongoan, Barasat, and Habra was carried out. To characterize clinical and biochemical features of fever cases as well as to investigate the utility of CRP as a Dengue severity marker in resource-limited settings. We systematically enrolled 108 patients from the affected region of North 24 Parganas. Standard diagnostic assays along with routine serological and biochemical parameters were performed. Of the 108 patients, 77 (71%) were confirmed with Dengue infection followed by 22 (20%) DENV seronegative and 9 (8%) coinfected DENV cases. Among the 77 confirmed Dengue patients, 53 (69%) had primary infection while 24 (31%) had secondary infection. Among the DENV clinical symptoms, fever (r = 0.50; p = 0.004), headache (r = 0.40; p = 0.03) and abdominal pain (r = -0.40; p = 0.02) were found to bear significant correlation with DENV viral load. The predominant circulating serotype was found to be DENV2. CRP Dengue severity cut-off level of 10.15 mg/L (AUC: 0.85; 86% sensitivity, 77% specificity) was obtained. CRP had correlation with viral load (r = 0.4, p = 0.05) within febrile phase of infection. The performance of biomarkers can be influenced by local epidemiology, geography, and several patient factors, therefore, CRP Dengue severity cut-off value may be region-specific. This study for the first time attempts to estimate CRP Dengue severity cut-off value based on routine immunoturbidometric evaluation from Dengue Hyperendemic zones of North 24 Parganas, West Bengal, Eastern India.

The relationship between changes in IL-2 / IL-18 and liver enzyme with dengue severity.

A synergistic impact of virulence capacity of dengue viruses and host immune response contributes to the pathogenesis of dengue virus infection (DENV). Elevation of hepatic enzyme and cytokine storm is the major contributory factor producing severity. IL-2 and IL-18 both play a critical role in generating immunogenicity during viral infection. The goal of the study was to evaluate the correlation between alterations in IL-2 and IL-18 levels with alterations in hepatic enzymes in dengue-infected patients. Accordingly, a total of 91 NS1/IgM confirmed DENV infected patients were selected for the IL-2 and IL-18 evaluation using a standard ELISA assay. Biochemical, haematological parameters were recorded at the time of admission. Interestingly, raised levels of IL-2 (p < 0.0001) and IL-18 (p < 0.0001) was obtained in severe dengue as compared to non-severe dengue patients. A significant positive correlation was observed between IL and 2 levels and SGOT (r = 0.3168 with p = 0.002), SGPT (r = 0.569 with p < 0.0001). Similarly IL-18 was also highly associated with SGOT (r = 0.387 with p = 0.0002) and SGPT (r = 0.407 with p < 0.0001) in dengue infected patient.Our data reveal that a rising amount of IL-2 and IL-18 in initial stage of the disease could be predictors for developing severe form of dengue infection.

Increased levels of pentraxins protein and cytokines bear good association in patients with severe dengue infection.

Dengue is an arboviral infection with high rates of morbidity and mortality throughout the tropics and sub-tropics. This work studied the status of pentraxin (CRP/SAP) protein, ferritin, TNF-α and IL-1ß levels in Dengue patients of different pathophysiological manifestations. Accordingly, clinically confirmed Dengue cases (n = 97) were enrolled and subsequently blood parameters were studied by Haematology cell counter and Biochemistry Autoanalyser. CRP, SAP, ferritin, TNF-α and IL-1ß ELISA were done in all the samples by using standard ELISA kits. Statistical Analysis was done in all the experiments. The levels of CRP (p < 0.0001), SAP (p < 0.0001), ferritin (p < 0.0001), TNF-α (p < 0.0001) and IL-1ß (p < 0.0001) were high in patients with Severe Dengue as compared to Dengue without warning signs. High levels of SGOT, SGPT and decreased platelet counts were found in severe patients as compared to Healthy donor. CRP/SAP as well as TNF-α/IL-1ß were independently associated with both dengue severity and overall disease manifestation. Statistically significant increased CRP, SAP, ferritin, TNF-α and IL-1ß titres were correlated in patients with severe clinical manifestations as compared to mild disease forms of dengue. Elevated levels of pentraxin, TNF-α/IL-1ß in blood during dengue infection could act as an early predictor in Severe Dengue infection.

Clinical Proteomics Profiling for Biomarker Identification Among Patients Suffering With Indian Post Kala Azar Dermal Leishmaniasis.

Background: Post Kala Azar Dermal Leishmaniasis (PKDL) is a non-fatal dermal sequel of Visceral Leishmaniasis (VL), affecting individuals worldwide. Available diagnostic tools lack sensitivity and specificity toward identifying macular (MAC) PKDL patients, due to low parasite load in patients' sample. Confirmatory test like punch biopsy are invasive and painful. Considering the rural nature of this disease and the prevailing situation of diagnostic scenario, PKDL patients mostly remains unattended from receiving proper medical care. They in turn act as "mobile parasite reservoir," responsible for VL transmission among healthy individuals (HI). This study aims to identify PKDL disease specific glycated protein biomarkers, utilizing the powerful LC-MS/MS technology, which is the tool of choice to efficiently identify and quantify disease specific protein biomarkers. These identified PKDL disease specific novel glycoproteins could be developed in future as immunochromatographic based assay for efficient case detection. Methodology: Previously our lab had identified importance of glycated (Circulating Immune Complexes) CICs, among PKDL patients. This study aims to further characterize disease specific glycated protein biomarkers, among MAC PKDL patients for both diagnostic and prognostic evaluation of the disease. LC-MS/MS based comparative spectral count analysis of MAC PKDL to polymorphic (POLY) PKDL, HI, and Cured (CR) individuals were performed. Proteins level alterations among all study groups were confirmed by Western blot and enzyme-linked immunosorbant Assay (ELISA). Results: Among MAC PKDL patients 43, 60, 90 proteins were altered compared to POLY PKDL, HI, and CR groups, respectively. Filtering for the most significant proteins, Plasminogen (PLG) and Vitronectin (VTN) were identified which promisingly identified MAC PKDL cases. Active surveillance results from endemic districts of West Bengal revealed drastic rise of MAC PKDL cases, alarming the urgency for field adaptive efficient biomarker. Conclusion: This current study aims to establish PLG and VTN as novel diagnostic and prognostic protein biomarker for MAC and POLY PKDL cases management.

Evaluation of anti-gal enzyme-linked immunosorbent assay for the diagnosis of Indian post-kala-azar dermal leishmaniasis.

BACKGROUND: Elimination of kala azar from India is challenging as there are potential reservoirs of Leishmania donovani in patients with post-kala-azar dermal leishmaniasis (PKDL). The vast repertoire of carbohydrate moieties on L. donovani is known to elicit specific and strong humoral responses in patients with kala azar. AIM: The present study was undertaken to evaluate the diagnostic performances of anti-gal antibodies using enzyme-linked immunosorbent assay for successful serological diagnosis of PKDL in Indian patients and to differentiate cases of past cured visceral leishmaniasis infections. METHODS: We developed Gal enzyme-linked immunosorbent assay to measure specific anti-gal IgG isotype in the sera of 71 Indian patients with PKDL. The diagnostic efficacy of the newly developed assay was evaluated for precision, sensitivity and accuracy. RESULTS: Gal2 enzyme-linked immunosorbent assay revealed three-fold increased anti-gal titers in 71 patients with active PKDL compared to controls. Subclass enzyme-linked immunosorbent assay analysis further revealed enhanced IgG2 and IgG3 anti-gal titers in patients with PKDL compared to control subjects. The rank order for specificity and sensitivity for IgG subclasses was IgG3>IgG2>IgG4>IgG1. The area under the curve values of 0.98 and 0.99 were obtained for IgG and IgG3 Gal2 enzyme-linked immunosorbent assays respectively. Overall sensitivity and specificity were 95.7% (95% CI: 88.1-99.1) and 98.1% (95% confidence interval: 90.1-99.9), and 98.5% (95% CI: 92.4-99.9) and 98.1% (95% CI: 90.1-99.9), respectively. Intra-assay coefficient of variation was 1.5% and inter-assay coefficient of variation was 11.7%. LIMITATIONS: The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. CONCLUSION: Taken together, anti-gal titers detected through Gal2 enzyme-linked immunosorbent assay can serve as an effective diagnostic tool in disease elimination setting and help in better case management in endemic districts.

Study of serum VEGF levels in patients with severe dengue infection admitted in a tertiary care hospital in Kolkata.

Dengue is the most common arboviral infection globally, but its pathogenesis is poorly explored. Vascular endothelial growth factor (VEGF) has an essential role in the host defense against viral infection. However, not much information is available regarding its status in dengue patients from the eastern zone of India. In the present investigation, the level of VEGF was investigated for its possible utility as a dengue severity marker. Accordingly, confirmed dengue cases were enrolled during 2016-2018. Serum from all the study subjects was subjected to the standard enzyme-linked immunosorbent assay test for VEGF analysis. In addition, we assessed the association of VEGF to dengue severity. The study revealed that VEGF titers (P < .0001) were significantly increased in severe dengue (SD) patients in contrast to those with a milder form of dengue. An association was obtained between VEGF and increased SGOT (r = 0.517 with P < .0001) while VEGF had a negative correlation with platelets in SD patients (r = -0.331 with P = .001). Enhanced VEGF titers along with decreased platelets had a good association with SD. The investigation revealed that high VEGF titers are novel indicators of dengue severity. However, our results must be verified in a study evaluating a larger number of dengue patients.

Systemic Redox Imbalance Along with Increased Serum Sialic Acid is Prevalent in Patients with Active Vitiligo: A Study from a Tertiary Care Teaching Hospital of Eastern India.

BACKGROUND: Vitiligo is one of the common depigmenting disorders causing disfigurement and affecting the quality of life. Redox imbalance is known to play a contributory role in melanocyte destruction. Serum sialic acid (SA) is an important marker of the acute-phase response and is associated with oxidative protein damage. AIM: The aim of this study was to analyze the status of oxidative stress markers and serum SA in vitiligo patients and to correlate the same with disease activity. MATERIALS AND METHODS: The different oxidative stress parameters namely superoxide dismutase (SOD), malondialdehyde (MDA), and serum SA were measured spectrophotometrically using standard biochemical methodologies in all the study subjects. RESULTS: Serum SOD and MDA values were higher in patients with active vitiligo (n = 23) as compared to stable vitiligo (n = 20) and healthy controls (n = 20). The MDA/SOD ratio was higher in patients with active vitiligo (P<0.0001). Serum SA was increased in active vitiligo as compared to stable vitiligo and healthy controls (P<0.0001). CONCLUSION: This study indicates that patients with active vitiligo demonstrate enhanced MDA/SOD ratio and increased serum SA. The studied parameters can serve as an important tool to monitor disease activity in vitiligo.

Dermatological Implications of Galectin-3 in Circulation: An Evaluation From the Perspective of Patients With Differential Manifestations of Post-Kala-Azar Dermal Leishmaniasis.

Galectin-3, a ß-galactoside-binding lectin, has been implicated in vast repertoire of inflammatory and immunomodulatory processes including skin diseases. However, galectin-3 has not been comprehensively studied in infectious diseases. This study emphasizes on fascinating aspects of galectin-3 expression in dermal infection by studying post-kala-azar dermal leishmaniasis (PKDL), an intracellular infection caused by Leishmania donovani. Indian PKDL is a well-recognized parasitic dermatosis, with a high risk of anthroponotic transmission of L. donovani in causing leishmaniasis. This study aims to investigate the levels of galectin-3 and galectin-3-binding site expression in circulation of different forms of Indian patients with PKDL. Thirty-seven confirmed untreated PKDL patients, comprising 20 polymorphic and 17 macular PKDL manifestations, were evaluated for the levels of sera galectin-3 with respect to 28 age- and sex-matched healthy controls from endemic areas. Result shows a significant increment (P < 0.001) in circulatory galectin-3 levels in PKDL variants as compared to healthy controls. In addition, there were heightened levels of galectin-3 and galectin-3-binding sites on cellular infiltrates on lesional sites. Furthermore, there was a positive correlation between frequencies of mononuclear cells and galectin-3 during microcirculation in lesions. Data were well corroborated with positive correlation of IL-10 and IFN-γ with sera galectin-3 levels. Moreover, flow cytometry demonstrated the enhanced expression levels of the galectin-3-binding site in circulation in patients with PKDL as compared to healthy controls. Taken together, elevated levels of galectin-3 reflect its involvement in PKDL pathogenesis.

Assessment of chemokine and cytokine signatures in patients with dengue infection: A hospital-based study in Kolkata, India.

Dengue fever is an acute viral infection transmitted by arthropods but may evolve to severe clinical manifestations. Descriptions of the role of circulating immune modulators such as cytokines or chemokines in dengue immunopathogenesis have largely relied on data from South-east Asia and America, while India is poorly represented. This study characterizes dengue cases from West Bengal, eastern India, with respect to clinical profile and pro-inflammatory and inflammatory cytokines. We evaluated the profile of both inflammatory and anti-inflammatory cytokines (IFNγ, IL6, IL10, IL12 and TGFß) and chemokines (IL8, CXCL9, CXCL10 and RANTES) in 100 hospitalized NS1/IgM confirmed Dengue patients during the epidemic in West Bengal during 2017. Additionally, all necessary blood investigations of the study subjects were performed. The patients mostly hailed from Kolkata, followed by Nadia, 24 Parganas (North and South), Murshidabad and Midnapore. The most common presentations apart from fever and bodyache were gastrointestinal symptoms. An elevated levels of cytokines IL6 and IL10 chemokine IL8 and CXCL10 along with decreased RANTES were found in the patients with Severe Dengue as compared to mild forms of dengue (p < 0.0001) during 3-6 days of infections. A significant association was obtained between most of cytokine and increased SGPT, haematocrit, albumin and decreased platelet count, whereas a negative correlation with the level of RANTES to haematocrit (r=-0.220 with p = 0.029) was found in severe dengue cases with altered liver function parameters. This is the first study demonstrating cytokine and chemokine association with dengue severity from the eastern part of India. Taken together, this study demonstrated that the altered expression levels of IL6, IL10, IL8, CXCL10 and RANTES had significant associations with dengue severity parameters.

Intracellular ROS generated in chikungunya patients with persisting polyarthralgia can be reduced by Tinospora cordifolia leaf extract.

Chikungunya (CHIK) is an arboviral infection having huge global burden affecting the life style of the patient badly due to debilitating polyarthralgia. This study aims to evaluate intracellular reactive oxygen species (ROS) in peripheral blood of patients suffering with persisting polyarthralgia post CHIK infection and the potential of Tinospora cordifolia leaf extract in scavenging those free radicals in peripheral blood mononuclear cells (PBMC) of the patient. Peripheral blood was collected from written informed consented patients and intracellular ROS was measured in PBMC of patients suffering with persisting polyarthralgia 3 months post CHIK infection followed by the study of free radical scavenging by T. cordifolia leaf extract in those cells through flow cytometry. Control population comprising healthy donors were also included in the study. As compared to healthy subjects, twofold higher Intracellular ROS (17.89 ± 1.007 vs. 37.96 ± 1.510, P < 0.0001) was found in patient PBMC. Ex-vivo treatment of those PBMC with ethanolic extract of T. cordifolia leaf (1 µg/mL) decreased intracellular ROS significantly by twofold (P < 0.0001). This study reports that CHIK infection produces high level of intracellular ROS in the patients suffering with persisting polyarthralgia, which was significantly scavenged by ex vivo treatment with T. cordifolia leaf extract.

Oxidative damage markers and inflammatory cytokines are altered in patients suffering with post-chikungunya persisting polyarthralgia.

Redox homoeostasis is necessary for the maintenance of living systems. Chikungunya viral infection manifests into joint inflammation and debilitating polyarthralgia affecting the life style of the patient badly. The disease pathophysiology is poorly understood and there is a lack of targeted therapeutics. The pathogenic role of free radicals in arthritis is well established. This study aims for the first time to evaluate the status of several standard oxidative stress markers and their correlation in chikungunya patients suffering with polyarthralgia. Expression of Siglec-9 on monocytes; which can modulate oxidative stress is studied along with intracellular reactive oxygen species (ROS), cellular lipid and protein damage markers in chikungunya patients with/without persisting polyarthralgia along with healthy controls. Furthermore, plasma NO level, antioxidant status was investigated along with some inflammatory cytokines namely IL-6, IFN-γ, CXCL-9, IL-10 and TGFß1. Interestingly, all oxidative damage markers are altered significantly in groups but their alteration levels vary in patients with/without persisting polyarthralgia. Siglec-9 expression level is increased in patients revealing cellular response to manage oxidative stress with respect to controls. Correlation studies reveal that intracellular ROS correlates well with most of the studied parameters but the correlation coefficient (Pearson r) differs with disease manifestation demonstrating strong role of these factors in a pro-oxidant milieu. The presence of free radicals increases the availability of neoantigens continuously, which possibly further cascades oxidative damage and development of persisting polyarthralgia.

Glycoproteins in circulating immune complexes are biomarkers of patients with Indian PKDL: A study from endemic districts of West Bengal, India.

BACKGROUND: Post Kala Azar Dermal Leishmaniasis (PKDL) occurs as dermal consequence of previous Visceral Leishmaniasis (VL) infection and serves as an important reservoir for transmission of VL. Diagnosis of PKDL is often challenging for its symptomatic resemblance to other co-endemic diseases like Leprosy or Vitiligo. Parasitological examination by slit-skin smear and culture are the standard methods but lack high sensitivity. Thus, for efficient control of VL, reliable diagnostic and prognostic assay of PKDL are required. OBJECTIVE: Previously, glycoproteins (9-OAcSA) have been reported as promising biomarkers of Indian VL patients. However, till date, the status of glycans in Indian PKDL patients remains unexplored. Accordingly, in this study, the glyco-profile of PKDL Circulating Immune Complexes (CICs) as compared to other cross diseases like Vitiligo and Leprosyhas been investigated. Further, a novel Glyco CIC assay has been developed for efficient Indian PKDL patient diagnosis. METHODS/PRINCIPAL FINDING: In the present study, 90 PKDL patients were enrolled from 3 VL endemic districts of West Bengal during 2015-16. Glycosylation profile of isolated CICs from sera of PKDL patients were initially analyzed through gradient SDS gel electrophoresis followed by PAS silver double staining, which revealed the presence of several glycan rich PKDL specific proteins of varying molecular weights. To further characterize the glyco-profile of acid dissociated affinity purified immuno-reactive antigens present in the CICs, glycosylation was demonstrated in these purified CIC antigens by DIG glycan differentiation kit with or without glycosidase as well as neuraminidase treatment. Diagnostic evaluation of the newly developed colorimetric Glyco CIC assay through Receiver Operating Characteristic (ROC) curve analysis revealed excellent (0.99) AUC value as compared to other conventional serodiagnostic assays like PEG CIC, Parasite ELISA (IgG and IgM). Additionally, longitudinal monitoring of 18 PKDL patients further revealed its good prognostic utility. CONCLUSION: These results highlight the glycosylation status of CICs among Indian PKDL patients present in all the studied endemic districts of West Bengal. These PKDL biomarkers were completely absent in cross diseases like Vitiligo and Leprosy. Further, the newly developed Glyco CIC assay had an improved sensitivity of 95.6%, specificity of 99.3%, NPV of 97.1% and PPV of 98.9%.

Viral glycoproteins: biological role and application in diagnosis.

The viruses that infect humans cause a huge global disease burden and produce immense challenge towards healthcare system. Glycoproteins are one of the major components of human pathogenic viruses. They have been demonstrated to have important role(s) in infection and immunity. Concomitantly high titres of antibodies against these antigenic viral glycoproteins have paved the way for development of novel diagnostics. Availability of appropriate biomarkers is necessary for advance diagnosis of infectious diseases especially in case of outbreaks. As human mobilization has increased manifold nowadays, dissemination of infectious agents became quicker that paves the need of rapid diagnostic system. In case of viral infection it is an emergency as virus spreads and mutates very fast. This review encircles the vast arena of viral glycoproteins, their importance in health and disease and their diagnostic applications.

Status of circulating immune complexes, IL8 titers and cryoglobulins in patients with dengue infection.

Dengue, a serious viral infection caused by the mosquito vector, Aedes aegyptii, affects about 390 million people annually from more than 125 countries across the globe. However, until now, there is no reliable clinical or laboratory indicator to accurately predict the development of dengue severity. Here, we explored critical pathophysiological determinants like IL8, circulating immune complex (CIC) and cryoglobulin in dengue-infected patients for identification of novel dengue severity biomarker(s). Totally, 100 clinically suspected dengue cases were tested by NS1 ELISA and MAC ELISA for dengue virus aetiology. For control, 49 healthy volunteers were included. Blood profiling (complete hemogram and liver function test) of patient population were done using automated cell counter and standard auto analyzer based biochemical analysis. Serum CIC was quantified by PEG precipitation. Serum cryoglobulins were estimated by Folin assay. Levels of serum IL-8 were assessed by standard sandwich ELISA kits. Patient CIC were further characterized by SDS Gel electrophoresis. Forty per cent of the cases tested positive, of which 11 patients had severe clinical manifestation. The mean ±SEM of cryoglobulin concentration for DHF, DF, and HC were 1.30 ± 0.31, 0.59 ± 0.08 and 0.143 ± 0.009 µg/µl, respectively. Thus, DHF and DF patients have shown 9- and 2.2-fold increase in cryoglobulin levels; and 18- and 5-fold increased CIC, respectively compared to HC patients. The mean ±SEM of CIC-PEG index for DHF, DF and HC were 491 ± 41.22, 146 ± 14.19 and 27.98 ± 2.56, respectively. Raised levels of IL8 titers were also found in all 11 DHF patients. Peak levels of CIC, cryoglobulin and IL8 titers were associated with thrombocytopenia. SDS PAGE analysis of CIC from DHF revealed the presence of at least six protein bands that were not observed in samples from DF and HC. Prediction efficacy of IL8, CIC and cryoglobulin for DHF was determined using the receiver operator characteristic curve (ROC). The area under the curve was 1.00 for IL8, 0.99 for CIC and 0.74 for cryoglobulins. Overall, the results suggest that CIC, IL-8 and cryoglobulins may serve as important laboratory parameters to monitor dengue infection progression.

Identification and glycobiological characterization of circulating immune complexes in patients with visceral leishmaniasis and post kala azar dermal leishmaniasis.

Here, we investigated the quantitative and qualitative differences in antibody classes and subclasses in serum immune complexes (ICs) of Visceral Leishmaniasis (VL), Post Kala-azar Dermal Leishmaniasis (PKDL) and different cross reactive diseases like Malaria, Leprosy, Vitiligo as compared to control subjects. IC levels were measured through a newly developed PEG ELISA, using L. donovani promastigote membrane antigen coated plate. Antibody classes and subclasses were identified using polyspecific sera and monoclonal antibodies, respectively. ICs were purified using polyethylene glycol (PEG) precipitation. Conditional logistic regression showed an association between IgG1-containing ICs and increased risk of PKDL (OR = 75, P < 0.05) and an association of IgG-containing ICs with VL (OR = 621, P = 0.001). PEG ELISA demonstrated almost 13-15 fold higher IgG containing ICs titers in VL as compared to control (P < 0.001). The assay further established a significant (P < 0.05) difference in the IgG containing ICs titers between VL and PKDL. The isolated ICs were further analyzed by subjecting them to one-dimensional PAGE and subsequently stained with combination of periodic acid schiff (PAS) with silver. A differential banding pattern between VL and PKDL was obtained. Four distinct bands with carbohydrate rich glycoconjugates were identified in PKDL ICs, which were absent in VL and control group. It suggests the scope for developing a novel differential diagnostic assay.

Glycosylation of erythrocyte spectrin and its modification in visceral leishmaniasis.

Using a lectin, Achatinin-H, having preferential specificity for glycoproteins with terminal 9-O-acetyl sialic acid derivatives linked in α2-6 linkages to subterminal N-acetylgalactosamine, eight distinct disease-associated 9-O-acetylated sialoglycoproteins was purified from erythrocytes of visceral leishmaniaisis (VL) patients (RBC(VL)). Analyses of tryptic fragments by mass spectrometry led to the identification of two high-molecular weight 9-O-acetylated sialoglycoproteins as human erythrocytic α- and ß-spectrin. Total spectrin purified from erythrocytes of VL patients (spectrin(VL)) was reactive with Achatinin-H. Interestingly, along with two high molecular weight bands corresponding to α- and ß-spectrin another low molecular weight 60 kDa band was observed. Total spectrin was also purified from normal human erythrocytes (spectrin(N)) and insignificant binding with Achatinin-H was demonstrated. Additionally, this 60 kDa fragment was totally absent in spectrin(N). Although the presence of both N- and O-glycosylations was found both in spectrin(N) and spectrin(VL), enhanced sialylation was predominantly induced in spectrin(VL). Sialic acids accounted for approximately 1.25 kDa mass of the 60 kDa polypeptide. The demonstration of a few identified sialylated tryptic fragments of α- and ß-spectrin(VL) confirmed the presence of terminal sialic acids. Molecular modelling studies of spectrin suggest that a sugar moiety can fit into the potential glycosylation sites. Interestingly, highly sialylated spectrin(VL) showed decreased binding with spectrin-depleted inside-out membrane vesicles of normal erythrocytes compared to spectrin(N) suggesting functional abnormality. Taken together this is the first report of glycosylated eythrocytic spectrin in normal erythrocytes and its enhanced sialylation in RBC(VL). The enhanced sialylation of this cytoskeleton protein is possibly related to the fragmentation of spectrin(VL) as evidenced by the presence of an additional 60 kDa fragment, absent in spectrin(N) which possibly affects the biology of RBC(VL) linked to both severe distortion of erythrocyte development and impairment of erythrocyte membrane integrity and may provide an explanation for their sensitivity to hemolysis and anemia in VL patients.

Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis.

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7-8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (10(4)-10(5) M(-1)) and number of binding sites (10(6)-10(7)sites/erythrocyte) for CRP on patients' erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10 microg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two diseases.