Ocular pathology in NeuroAIDS - An autopsy study.

The central nervous system (CNS) and the eye are involved in Human immunodeficiency virus related disease. Although, optic nerve is considered an extension of the CNS, it has not been systematically evaluated to determine if infections of brain can extend into the eye or vice versa. The brain and posterior compartment of eyeball retrieved at autopsy of patients succumbing to NeuroAIDS, were evaluated with Hematoxylin & Eosin, special stains and immunohistochemistry for infective pathogens. Multiplex PCR was performed in vitreous, CSF and serum for simultaneous detection of bacterial, viral, and protozoal opportunistic infections. Ocular involvement in NeuroAIDS was seen in 93.7% (15/16) with opportunistic infection being the most common 62.5% (10/16); with toxoplasma optic neuropathy in 5 (50%), Cryptococcal optic neuritis in 3 (30%), and Cytomegalovirus chorioretinitis in 2 (20%). Concordance between ocular and CNS pathology was seen in 50% of cases. CSF PCR was more sensitive than PCR in vitreous for detecting ocular infections in posterior compartment of eye.

Syndrome Evaluation System for Simultaneous Detection Pathogens Causing Acute Encephalitic Syndrome in India, Part-1: Development and Standardization of the Assay.

A large number of organisms are known to cause acute encephalitic syndrome (AES). A number of diagnostic tests have to be performed in order to arrive at a probable pathogen causing AES thus making it a very time consuming, laborious and expensive. The problem is further compounded by the lack of availability of sufficient volume of Cerebrospinal fluid (CSF). Thus, there is an urgent need of a diagnostic tool for the simultaneous detection of all probable pathogens responsible for causing AES. Here we report the development of a novel diagnostic method, Syndrome Evaluation System (SES) for the simultaneous detection of 22 pathogens including RNA and DNA Viruses, bacteria, fungi, and parasite all endemic to India and Southeast Asia in a single sample using a novel multiplexing strategy. Syndrome Evaluation System (SES) involves isolation of nucleic acid, multiplex amplification of the DNA, and cDNA followed by identification of the amplified product by sequence specific hybridization on SES platform with the final read out being a visually recordable colored signal. The total time required to carry out this diagnostic procedure is 7 h. The SES was standardized using the commercially available vaccines, panels and cell culture grown quantified viruses/bacteria/fungi. The limit of detection (LOD) of SES ranged between 0.1 and 50 viral particles per ml of CSF and 100 to 200 bacterial cells or 5 parasites per ml of CSF, along with 100% specificity. Precision studies carried out as per the Clinical Laboratory Improvement Amendments (CLIA) guidelines, using two concentrations of each pathogen one the LOD and the other double the LOD, clearly demonstrated, that inter/intra assay variability was within the limits prescribed by the guidelines. SES is a rapid molecular diagnostic tool for simultaneous identification of 22 etiological agents of AES encountered both in sporadic and outbreak settings.

Syndrome Evaluation System for Simultaneous Detection of Pathogens Causing Acute Encephalitic Syndrome in India, Part-2: Validation Using Well Characterized Clinical Samples.

Diagnosis of the aetiological agent in case of acute encephalitic syndrome (AES) continues to pose a challenge in clinical practice as a variety of pathogens are known to cause AES. Here, we report the validation of a Syndrome Evaluation System (SES) developed for simultaneous detection of multiple AES pathogens using a well characterized set of Cerebrospinal fluid (CSF) samples. The validation of the SES was carried out in two phases. In the first phase, the SES was validated using 51 CSF samples obtained from autopsy proven cases and 50 samples obtained from apparently healthy individuals undergoing spinal anesthesia for minor surgeries served as "controls." The SES detected etilogical agent in 48/51 (94.11 %) samples obtained from autopsy proven AES cases while all the 50 CSF samples obtained from "controls" were negative. In the second phase, the SES was validated using well characterized CSF samples obtained from AES patients fulfilling the WHO case definition of AES (Group I; n = 207) and samples that were collected from patients with non-infectious neurological disorder (Group II; n = 90). All the samples were tested using multiple conventional/serological assays and categorized into various groups. Amongst the AES cases fulfilling WHO case definition, the SES detected AES pathogens in 160/207 (77.29%) cases while conventional serological/molecular assays were able to detect AES pathogens only in 77/207 (37.1%) of cases. Further, in 12/83 CSF samples that were positive by SES and negative by conventional serological/molecular tests, the results were additionally confirmed by sequencing the PCR products to rule out non-specific amplification in the SES. In patients with non-infectious neurological disorders the SES detected latent viruses 12/90 CSF samples. These results indicate that the SES, apart being a rapid, sensitive, specific, and cost-effective method provides the major advantage of simultaneous detection of multiple pathogens using as single specimen of CSF.