Abstract
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.
Highlights
Acute Encephalitic Syndrome (AES) is a major public health problem in several parts of Asia, especially India
ATCC strains of S. pneumoniae, N. meningitidis, H. influenzae, C.neoformans, and a standard strain of M.tuberculosis (H37Rv) available in the laboratory were used as standards
The primers were initially evaluated for their ability to amplify the nucleic acid obtained from the standards for which they were designed in an uniplex format using agarose gel electrophoresis as the end read out
Summary
Acute Encephalitic Syndrome (AES) is a major public health problem in several parts of Asia, especially India. A variety of pathogens are known to cause AES. They include DNA and RNA viruses, bacteria, fungi, and parasites [1]. Despite the availability of several modalities, the etiological diagnosis of AES remains a challenge. Amongst the methods available at present for diagnosis of AES, routine Cerebrospinal fluid (CSF) cell count, protein, and glucose estimation as well as smear examinations, virus culture, and immunological tests are neither sensitive nor specific enough to provide a precise etiological diagnosis [2, 3]. CSF is the specimen of choice, it is most often not available in all cases of AES, and when available the volume quite often is insufficient for carrying out sequential diagnosis of a variety of pathogens that can cause CNS infections. The diagnostic tools available at present are expensive, time consuming [4] and are not designed for simultaneous detection of all pathogens in a single assay
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