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

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.

Keywords: acute encephalitis syndrome; development; molecular diagnostics; simultaneous detection; syndrome evaluation system.

Figure 1

(A) Template depicting the position of various probes used for detection of DNA pathogens on the Syndrome Evaluation System platform. Row 1:HgD = Glycoprotein D of HSV 1 and 2; HUL = UL 44 of HSV 1; HDP = DNA polymerase of HSV 1 and 2; 1st Comp = complementary strand of HgD gene of HSV 1 and 2; Row 2: CGO = Glycoprotein O of CMV; CUL = UL 63 of CMV; CMT = Morphological transformation Region of CMV; 5th Comp = complementary strand of CMT gene of CMV; Row 3: VO = ORF 29 gene of VZV; VDP = DNA polymerase gene of VZV; MBT = M. tuberculosis; TG = T.gondii; Row4: HI = H. influenzae; Nm = N. meningitidis; SP = S. Pneumoniae and JC = JC virus; Row 5: CN = C. neoformans; Row 6: HHV-6 = Human Herpes Vvirus-6; IC = Internal control. (B) Template depicting the position of various probes used for detection of RNA pathogens on the Syndrome Evaluation System platform. Row 1: FLA1 = Flavivirus; MES = Measles; FLA2 = Complimentary strand of FLA1 gene of Flavivirus; MUM = Mumps; Row 2: CHIK = Chikungunya; JEV = Japanese encephalitis virus; NIP = Nipah; CHA = Chandipura; Row 3: RAB = Rabies; ENT1 = Enteroviruses; RUB = Rubella; ENT 2 = Complimentary strand of ENT1 of Enteroviruses; Row 4: Den1 = Dengue type1and 3 virus; Den2 = Dengue Type 2 and 4 virus and IC = Internal control.

Figure 2

Ethidium bromide stained agarose gel image depicting 156 base pair uniplex PCR amplified product obtained from JE viral cDNA prepared by two methods. (A) Depicts PCR amplified products obtained using cDNA prepared with random primers, while (B) depicts PCR amplified products obtained using cDNA prepared with JEV specific primers. Serially log dilutions of cell culture grown JE virus ranging from10⁶ to 0.1 particle/ml was used to extract RNA and preparation cDNA. Lanes 1 and 11, depict results obtained with Negative control, Lanes 10 and 20, represents 100 bp molecular weight markers (DNA ladder), Lanes 2 to 9 and 12 to 19 represent the PCR amplified product of log dilutions of JEV 10⁶ particles/ ml to 0.1 JEV particles/ml respectively. The PCR sensitivity obtained with cDNA prepared using specific primers (lane 19, 0.1 particles/ml) is higher than the cDNA prepared using random primers (lane 6, 10² particles/ml).

Figure 3

Specificity of Chikungunya primer and probes set in SES. cDNAs were prepared for various RNA viruses as described in Materials and Methods and subjected to amplification and hybridization using Chikungunya primer and probe sets. Each panel depicts results obtained thereof. Panel 1 Depicts results obtained with negative control while Panel 16 depicts results obtained with Positive Control i.e., Chikungunya cDNA. Panels 2–15 depict results obtained with Dengue 2(Panel 2), JEV (Panel 3), West-Nile(Panel 4), Polio(Panel 5), Coxsackie B1(Panel 6), Coxsackie B2(Panel 7), Coxsackie B3(Panel 8), Coxsackie B4(Panel 9), Measles(Panel 10), Mumps(Panel 11), Rubella(Panel 12), Rabies(Panel 13), Nipah(Panel 14), and Chandipura (Panel 15) viruses respectively. Lack of signals in Panels 2 to 15 indicates that no cross amplification and/or hybridization was noted with cDNA's/ plasmid DNA of all RNA viruses, thereby indicating specificity in the RNA SES for Chikungunya primer and probe set.

Figure 4

Specificity of Herpes Simplex Virus and Cytomegalovirus primer and probes set used in the SES. 1. Herpes Simplex Virus DNA was amplified in multiplex format and hybridized on a SES platform. 2. Cytomegalovirus DNA was amplified in multiplex format and hybridized on a SES platform. Only signals specific to the organism identified is observed indicating no cross reactivity with genomically identical virus.

Figure 5

Determination of Limit of detection (LOD) for DNA pathogen on the SES platform. DNA extracted from defined concentrations of viruses/bacteria /fungi/parasites were subjected to multiplex PCR and hybridization on the SES platform (Vide Materials and Methods for details). As indicated in the template provided on the right, multiple genes were amplified and hybridized with respective probes for HSV (row 1 of template), CMV (row 2 of template),VZV (row 3 of the template), and single genes were amplified and hybridized in case of M. tuberculosis and T. gondii (row 3 of the template), H influenza, N. meningitidis, S. pneumoniae and JCV (row 4 of template), C. neoformans (row 5 of the template), and HHV-6 and Internal control (row6 of the template). Each panel depicts NC = negative control. PC = Positive Control and the numbers written below each SES indicate the number of particles/CFU/organisms/ml that has been used for DNA extraction, amplification and hybridization in a multiplex format. (refer to Table 4).

Figure 6

Determination of Limit of detection (LOD) for RNA viruses on the SES platform. RNA extracted from defined concentrations of viruses was subjected to, cDNA conversion, multiplex amplification and hybridization on the SES platform (Vide Materials and Methods for details). As indicated in the template provided on the right side, the Limit of Detection obtained in the SES for various RNA viruses in the various panels above. NC = Negative control and the numbers written below each SES indicate the number of viral particles/ml that has been used for RNA extraction, amplification and hybridization in a multiplex format. The limit of detection for Measles, Mumps, Rubella, Rabies, Entero and Chikungunya viruses was 1 particle /ml. The limit of detection for JEV using panflavivirus primers was 100 particles/ml whilst it was 10 particle/ml when JEV specific primers were used. The limit of detection for Nipah and Chandipura plasmids was 2.7 fg and 1.3 fg respectively.

Figure 7

Scatter plot depicting the results of the Standard Precision assay for Measles virus. X axis indicates the number of days, Y axis indicates the average spot intensity scores obtained for each of the two concentrations of template used; blue = concentration of template at LOD and red = concentration of template double that of LOD. A representative hybridization image depicting a score of 5 on the spot intensity scale obtained with twice the LOD of Measles Virus is depicted adjacent to the graph. As evident from the graph there is no significant inter or intra assay variability obtained in the SES for Measles Virus over a ten day period.

Figure 8

Scatter plot depicting the results of the Standard Precision assay for JC virus. X axis indicates the number of days, Y axis indicates the average spot intensity scores obtained for each of the two concentrations of template used; blue = concentration of template at LOD and red = concentration of template double that of LOD. A representative hybridization image depicting a score of 4 on the spot intensity scale obtained with the LOD of JC Virus is depicted adjacent to the graph. As evident from the graph there is no significant inter or intra assay variability obtained in the SES for JCVirus over a tenday period.