Proinflammatory chemokines are major mediators of exuberant immune response associated with Influenza A (H1N1) pdm09 virus infection.

In India, the case fatality ratio of the pandemic A (H1N1) pdm09 influenza was relatively higher when compared to seasonal Influenza A infection. Hypercytokinemia or "cytokine storm" has been previously implicated in the pathogenesis of other influenza viruses. The present study was undertaken to compare the cytokine profiles of A (H1N1) pdm09 influenza and seasonal H3 infection in Indian population and to correlate the findings with disease severity. Plasma levels of 18 cytokines/chemokines were measured by flow-cytometry using a bead based assay in patients infected with A (H1N1) pdm09 virus (n = 96) and Influenza A seasonal H3 virus (n = 30) categorised into mild, moderate, and severe groups along with healthy controls (n = 36). There was an overall trend indicating an exuberant cytokine/chemokine response in A (H1N1) pdm09 as compared to seasonal H3 influenza, which was more evident in severe cases, suggesting a role for these cytokines/chemokines in the pathogenesis of A(H1N1) pdm09. Increased levels of CXCL-8/IL-8, IL-10, IL-6, and IL-17A were seen in both A(H1N1) pdm09 influenza and seasonal H3 cases when compared to healthy controls. However, dysregulated production of proinflammatory chemokines was seen more pronounced in A (H1N1) pdm09 influenza cases as compared to seasonal H3 cases. This study has brought forth the potential role of chemokines as prognostic indicators of disease severity and outcome. Further research on modulating the host immune response to limit severity of the disease could help in the treatment and management of influenza.

Human rabies in India: an audit from a rabies diagnostic laboratory.

OBJECTIVES: Rabies, an acute progressive encephalomyelitis, continues to be a serious public health problem in India and many other countries in Asia and Africa. The low level of commitment to rabies control is partly attributable to challenges in laboratory diagnosis and lack of adequate surveillance to indicate the disease burden. A laboratory audit of human rabies cases was undertaken to disseminate information on the clinical, demographic, prophylactic and most importantly the laboratory diagnostic aspects of rabies. METHODS: A retrospective analysis of all clinically suspected human rabies cases, whose samples were received at a rabies diagnostic laboratory in South India in the last 3 years, was performed. Clinical and demographic details of patients were obtained. The clinical samples included cerebrospinal fluid (CSF), serum, saliva and nuchal skin biopsy collected antemortem, and brain tissue obtained post-mortem. Various laboratory tests were performed for diagnosis. RESULTS: Clinical samples from 128 patients with suspected rabies, from 11 states in India, were received for diagnostic confirmation. About 94% of the victims reported dog-bites, more than a third of them were children and most of the victims did not receive adequate post-exposure prophylaxis. Antemortem confirmation of rabies by a combination of laboratory diagnostic assays (detection of viral RNA in CSF, skin and saliva, and neutralising antibodies in CSF) could be achieved in 40.6% cases. CONCLUSIONS: Increasing awareness about adequate post-exposure prophylaxis, additional rabies diagnostic facilities, and enhanced human and animal rabies surveillance to indicate the true disease burden are essential to control this fatal disease.

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.

Laboratory diagnosis of human rabies: recent advances.

Rabies, an acute progressive, fatal encephalomyelitis, transmitted most commonly through the bite of a rabid animal, is responsible for an estimated 61,000 human deaths worldwide. The true disease burden and public health impact due to rabies remain underestimated due to lack of sensitive laboratory diagnostic methods. Rapid diagnosis of rabies can help initiate prompt infection control and public health measures, obviate the need for unnecessary treatment/medical tests, and assist in timely administration of pre- or postexposure prophylactic vaccination to family members and medical staff. Antemortem diagnosis of human rabies provides an impetus for clinicians to attempt experimental therapeutic approaches in some patients, especially after the reported survival of a few cases of human rabies. Traditional methods for antemortem and postmortem rabies diagnosis have several limitations. Recent advances in technology have led to the improvement or development of several diagnostic assays which include methods for rabies viral antigen and antibody detection and assays for viral nucleic acid detection and identification of specific biomarkers. These assays which complement traditional methods have the potential to revolutionize rabies diagnosis in future.

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.

Case Report: Failure of Therapeutic Coma in Rabies Encephalitis.

Rabies encephalitis is a fulminant, almost universally fatal infection involving the central nervous system. A unique treatment protocol, including anti-exicitotoxic therapy and induced coma was credited with the survival of a vaccinated teenager with bat rabies encephalitis in 2005. However, multiple efforts to replicate this expensive and intense protocol have not been successful. In this article, we report the failure of the protocol in Indian patients with canine-acquired rabies and elucidate the potential explanations for the failure of the protocol in our patients.

A randomized non-inferiority clinical study to assess post-exposure prophylaxis by a new purified vero cell rabies vaccine (Rabivax-S) administered by intramuscular and intradermal routes.

BACKGROUND: Rabies is a 100% fatal disease but preventable with vaccines and immunoglobulins. We have developed a new purified vero cell rabies vaccine (Rabivax-S) and evaluated its safety and immunogenicity in post-exposure prophylaxis by intramuscular (IM) and intradermal (ID) routes. METHODS: This was a randomized active-controlled non-inferiority study in 180 individuals (age 5years and above) with suspected rabies exposure (90 each with WHO Category II and Category III exposures). The participants received either Rabivax-S (1mL IM; five doses), Rabivax-S (0.1mL ID; eight doses) or purified chick embryo cell vaccine (PCEC, Rabipur®) (1mL IM; five doses). The IM doses were given on Day 0, 3, 7, 14 and 28 while the ID doses were given on days 0, 3, 7 and 28. Category III patients also received a human rabies immunoglobulin (HRIG) on Day 0. Adverse events (AEs) were recorded with diary cards till day 42. Rabies neutralizing antibody levels were measured on day 0, 7, 14, 28 and 42. RESULTS: In both the category II and III patients, the geometric mean concentration (GMC) ratios of Rabivax-S IM and Rabivax-S ID groups to PCEC IM were more than 1, thus proving the non-inferiority. GMCs were similar or higher in Rabivax-S groups at all the time points. Seroresponse against rabies (RFFIT titre⩾0.5IU/mL) was achieved in all participants. Mostly mild local and systemic adverse events were reported across the three groups and all resolved without sequelae. CONCLUSIONS: Rabivax-S was well tolerated and showed immunogenicity comparable to a licensed rabies vaccine by both IM and ID routes in post-exposure prophylaxis. Registry No.: CTRI/2012/11/003135.

Natural Rabies Infection in a Domestic Fowl (Gallus domesticus): A Report from India.

BACKGROUND: Rabies is a fatal encephalitis caused by viruses belonging to the genus Lyssavirus of the family Rhabdoviridae. It is a viral disease primarily affecting mammals, though all warm blooded animals are susceptible. Experimental rabies virus infection in birds has been reported, but naturally occurring infection of birds has been documented very rarely. PRINCIPAL FINDINGS: The carcass of a domestic fowl (Gallus domesticus), which had been bitten by a stray dog one month back, was brought to the rabies diagnostic laboratory. A necropsy was performed and the brain tissue obtained was subjected to laboratory tests for rabies. The brain tissue was positive for rabies viral antigens by fluorescent antibody test (FAT) confirming a diagnosis of rabies. Phylogenetic analysis based on nucleoprotein gene sequencing revealed that the rabies virus strain from the domestic fowl belonged to a distinct and relatively rare Indian subcontinent lineage. SIGNIFICANCE: This case of naturally acquired rabies infection in a bird species, Gallus domesticus, being reported for the first time in India, was identified from an area which has a significant stray dog population and is highly endemic for canine rabies. It indicates that spill over of infection even to an unusual host is possible in highly endemic areas. Lack of any clinical signs, and fewer opportunities for diagnostic laboratory testing of suspected rabies in birds, may be the reason for disease in these species being undiagnosed and probably under-reported. Butchering and handling of rabies virus- infected poultry may pose a potential exposure risk.

Atypical rabies encephalitis in a six-year-old boy: clinical, radiological, and laboratory findings.

A 6-year-old boy from India developed an atypical form of rabies following a stray dog bite and as a consequence of not receiving the standard World Health Organization recommended post-exposure prophylaxis for category III wounds. Serial rising rabies virus neutralizing antibody titres in serum and cerebrospinal fluid by rapid fluorescent focus inhibition test helped confirm the diagnosis of rabies. The child has survived for 4 months since the onset of illness, albeit with neurological sequelae.

Cellular immune response following pre-exposure and postexposure rabies vaccination by intradermal and intramuscular routes.

PURPOSE: Immunization against rabies in humans induces protective neutralizing antibodies; however, the induction of type 1 or type 2 cytokine mediated cellular immune responses following rabies vaccination is not understood. Hence, the present study investigated cellular cytokine responses in vaccinated individuals. MATERIALS AND METHODS: The study groups included healthy rabies antigen naive controls (n=10), individuals who received intradermal primary (n=10) or booster pre-exposure vaccination (n=20) and subjects who received postexposure rabies vaccination either by intradermal (n=18) or intramuscular (n=20) routes. The antigen specific cellular responses were analyzed by stimulating peripheral blood mononuclear cells with a rabies vaccine antigen in the interferon-γ (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunospot (ELISpot) assay. These responses were compared to the rabies virus neutralizing antibody (RVNA) titers that were measured by rapid fluorescent focus inhibition test. RESULTS: We observed that cellular and humoral immune responses to primary intradermal rabies vaccination could be greatly enhanced by a booster vaccine; and both type 1 and type 2 cytokine responses were significantly elevated. The magnitude of type 1 and type 2 cytokine responses did not differ significantly among the intramuscular and intradermal routes of postexposure vaccination. The number of cells producing IFN-γ and IL-4 correlated significantly with the levels of RVNA. CONCLUSION: Both type 1 and type 2 cellular cytokine responses are strongly induced after rabies vaccination and directly correlate with levels of RVNA titers. The neutralizing antibody as well as the type 1 and type 2 cytokine responses may be important for vaccine induced protective responses against rabies.

Intradermal vaccination for rabies prophylaxis: conceptualization, evolution, present status and future.

Rabies is a fatal viral encephalitis which can be effectively prevented by prophylactic measures. The currently available cell culture vaccines used for rabies prophylaxis are expensive for use by the standard intramuscular route of administration. In the last 3 decades, intradermal (ID) routes of vaccination using lesser amounts of vaccine as compared to that used for standard intramuscular vaccination have been used extensively in some Asian countries which has reduced the economic burden of rabies prophylaxis and also contributed in achieving a decline in the incidence of human rabies. ID vaccination is based on sound immunological principles and has been found to be safe and immunogenic. New short duration regimens to further economize the cost and enhance patient compliance, and novel non-invasive devices for ID vaccine delivery are being evaluated. Considering the success of ID rabies vaccination in Asian countries, its implementation in rabies endemic African countries should be encouraged.

Cerebral phaeohyphomycosis caused by Scytalidium dimidiatum: a case report from India.

We report a case of cerebral phaeohyphomycosis caused by Scytalidium dimidiatum (synanamorph Nattrassia mangiferae) in a young, apparently immunocompetent Indian male. Etiological diagnosis was made by recovery of the fungus in culture and histopathological examination. The infection proved fatal despite aggressive antifungal therapy.