Reeta Subramaniam Mani

BACTERIOLOGICAL PROFILE OF COMMUNITY ACQUIRED ACUTE BACTERIAL MENINGITIS: A TEN-YEAR RETROSPECTIVE STUDY IN A TERTIARY NEUROCARE CENTRE IN SOUTH INDIA

PURPOSE Ten years retrospective study to evaluate the bacteriological spectrum of community acquired acute bacterial meningitis (CAABM). METHODS Cerebrospinal fluid (CSF) samples from 385 clinically suspected cases of pyogenic meningitis were processed for cell counts, cytospin Gram stain, culture, antigen detection by latex agglutination (LAT) and antibiotic susceptibility test. Eighteen of these CSF samples were also subjected to a polymerase chain reaction (PCR) assay for detection of pneumococcal DNA. RESULTS The etiological agent could be identified in 284 (73.8%) of the total 385 cases by culture and/or smear and /or LAT. Streptococcus pneumoniae was the predominant pathogen accounting for 238 (61.8%) cases. Haemophilus influenzae and Neisseria meningitidis accounted for 7 (1.8%) and 4 (1%) cases respectively. Other gram negative bacilli, Streptococcus spp. and Staphylococcus aureus were isolated from 19 (4.9%), 9 (2.3%) and 7 (1.8%) cases respectively. CONCLUSIONS Streptococcus pneumoniae remains the major aetiological agent of CAABM both in adults and children in our set-up. No penicillin resistance was detected among the isolates. Further research should focus on preventable aspects of CAABM, especially pneumococcal vaccines, to help reduce the disease burden.

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. J. Med. Virol. 86:1393–1401, 2014.

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza A H1N1 virus.

The recent emergence of the swine-origin influenza A H1N1 virus (S-OIV) poses a serious global health threat. Rapid detection and differentiation of S-OIV from seasonal influenza is crucial for patient management and control of the epidemics. A one-step, single-tube accelerated and quantitative S-OIV-specific H1 reverse transcription loop-mediated isothermal amplification (RTLAMP) assay for clinical diagnosis of S-OIV by targeting the H1 gene is reported in this article. A comparative evaluation of the H1-specific RTLAMP assay vis-à-vis the World Health Organization-approved real-time polymerase chain reaction (RTPCR), involving 239 acute-phase throat swab samples, demonstrated exceptionally higher sensitivity by picking up all of the 116 H1N1-positive cases and 36 additional positive cases among the negatives that were sequence-confirmed as S-OIV H1N1. None of the real-time RTPCR-positive samples were missed by the RTLAMP system. The comparative analysis revealed that S-OIV RTLAMP was up to tenfold more sensitive than the World Health Organization real-time RTPCR; it had a detection limit of 0.1 tissue culture infectious dosage of 50/ml. One of the most attractive features of this isothermal gene amplification assay is that it seems to have an advantage in monitoring gene amplification by means of SYBR Green I dye-mediated naked-eye visualization within 30 minutes compared to 2 to 3 hours for a real-time reverse transcription polymerase chain reaction. This suggests that the RTLAMP assay is a valuable tool for rapid, real-time detection and quantification of S-OIV in acute-phase throat swab samples without requiring sophisticated equipment.

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.

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. J. Med. Virol. 86: 1804–1812, 2014.

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.

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.

Rapid Diagnosis of Acute Bacterial Meningitis: Role of a Broad Range 16S rRNA Polymerase Chain Reaction

BACKGROUND Acute bacterial meningitis is a significant cause of morbidity and mortality throughout the world. It can be difficult to diagnose, as the symptoms and signs are often non-specific. STUDY OBJECTIVE To evaluate the performance of an in-house semi-nested polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Eubacteria for the rapid diagnosis of acute bacterial meningitis using cerebrospinal fluid (CSF) specimens. METHODS A total of 112 CSF samples from 112 patients were used in the study. Among these, 32 samples were obtained from confirmed cases of Streptococcus pneumoniae, six samples were obtained from confirmed cases of Haemophilus influenzae, one sample from a confirmed case of Neisseria meningitidis, and 10 cases of clinically suspected acute bacterial meningitis. The remaining 63 CSF samples were obtained from patients with non-infectious illnesses (n = 47) of the central nervous system (CNS) and autopsy-confirmed tuberculous meningitis (n = 16). RESULTS The assay had an overall sensitivity of 93% (95% confidence interval [CI] 0.81-0.98, negative predictive value = 95%) and a specificity of 98% (95% CI 0.92-1.0, positive predictive value = 98%). CONCLUSION These preliminary findings suggest that the semi-nested PCR assay targeting the 16S rRNA gene may be used as a rapid test for the diagnosis of acute bacterial meningitis.

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.

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

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.