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Determination of Etiology and Epidemiology of Viral Central Nervous System Infections by Quantitative Real-Time Polymerase Chain Reaction in Central India Population
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.304080
Keywords: One-step reverse transcriptase polymerase chain reaction, plasmids, real-time polymerase chain reaction, two-step reverse transcriptase polymerase chain reaction, viral encephalitis
Central nervous system (CNS) viral infections impose a substantial disease burden leading to complex neurodegenerative diseases. A wide variety of emerging and re-emerging viruses contribute in severe neurological damage and cause a long-term disability or even death. The spectrum of CNS viral infection is broad, encompassing encephalitis, meningitis, myelitis, etc.[1],[2] In some epidemiological studies, several cases of encephalitis were found to be of unexplained etiology.[3] There are various factors such as immunocompetence of patients, age, weather conditions, geographical location, and seasonal time of the year that contribute to the potential for the development of CNS viral infections.[4] The causative agent has been identified only in 40–70% cases of viral infections.[5] The common infectious agents associated with viral CNS infections are cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), Japanese encephalitis virus (JEV), Dengue virus (DENV), West Nile virus (WNV), and Chandipura virus (CHPV).[6],[7],[8] Among these viruses, about 40–100% of the general population is infected with CMV and its incidence rate is higher in developing countries.[9] VZV is the third most causative agent of viral infections and more than 90% of people are infected, before their adolescence.[10] EBV is also associated with long causing infections of the CNS.[11] Among viral infections, which are seasonal, various outbreaks of Flaviviruses such as JEV, WNV, and DENV are reported worldwide. JEV causes approximately 15,000 death and 50,000 cases annually with a high fatality rate. It is estimated that 50–100 million infections occur every year worldwide because of DENV.[12],[13],[14] Similarly, in India, various outbreaks of Flaviviruses are reported every year in the high epidemic region.[15],[16],[17] Earlier, for definitive diagnosis of viral infections of the CNS, conventional methods such as isolation of virus from cerebrospinal fluid (CSF), detection of viral-specific intrathecal antibody response, and antigen detection were used. The isolation of viruses from CSF or brain tissue is slow and time-consuming. Detection of viral-specific antibody response is useful for diagnosis of viral infections, but there may be cross-reactivity, especially in a case of Flaviviruses. Similarly, antigen detection methods are not useful for viruses that have extensive antigenic heterogeneity and are slow growing. So, all the above assays are challenging, undertaking intensive, and not very specific for routine clinical diagnosis.[18],[19] Since the past decade, nucleic acid amplification has been identified as one of the most valuable tools for diagnosis of CNS viral infections caused by a variety of viruses. The most recent progress in this technique is real-time polymerase chain reaction (PCR), including quantitative real-time PCR.[20] The real-time PCR has numerous advantages as compared to conventional PCR methods, including rapidity, quantitative analysis, minimum contamination rate, high sensitivity, and specificity; therefore, PCR has become the new gold standard for the diagnosis of viral infections of the CNS.[21],[22] Real-time PCR is nowadays considered as the best approach for the molecular analysis of CSF during CNS viral infections.[23],[24] In a case of herpes viruses CNS infections, PCR analysis of CSF has been recommended for diagnosis of herpes simple virus (HSV), CMV, EBV, and VZV infections.[25],[26],[27],[28],[29] In real-time PCR, several chemistries are used for the detection of DNA.[30] Among them, SYBR green-based detection has been found to be the most cost-effective, sensitive, and widely practiced in clinical diagnosis.[31],[32] However, comparative evaluation of SYBR green-based real-time PCR methodologies has not been studied for CNS viral infections. Thus, in the present work, comparative evaluation of SYBR green-based real-time PCR assay was performed for the assessment of viral etiology and epidemiology of CNS viral infections among patients of all age groups admitted to Central India Institute of Medical Sciences, Nagpur, India during a 2-year period. The quantitative PCR (qPCR) assay was also developed and evaluated retrospectively in CSF and blood samples from patients with the aim to study the concentration of virus genome in context to the severity of a disease. The purpose of the work is to develop a SYBR green-based real-time PCR assay by comparing one-step and two-step reverse transcriptase PCR (RT-PCR) methodologies for detection and quantification of the common etiological agent in CSF and blood samples of patients of CNS viral infections in Central India populations. In improver to that, detection limit of real-time PCR was also compared using gDNA, gRNA, and plasmids as standards.
Patient and samples The CSF and blood samples were drawn from patients admitted to Central India Institute of Medical Sciences, Nagpur, India with suspected CNS viral infections. Inclusion criteria involved the presence of fever, headache, altered mental status (low level of consciousness, behavior or personality changes, drowsiness), and other clinical manifestations (e.g., focal neurological deficits, seizures, limb weakness, neck stiffness, skin rash), CSF findings showing the mild increase in protein, glucose often normal, and mild pleocytosis. Neurological diagnostic investigations were performed during the first week of hospitalization; these investigations included staining, bacterial culture, determination of the protein, sugar level and cell counts in CSF, computed tomography scan, and magnetic resonance imaging of the brain. A total CSF of 150 patients and parallel blood samples of 50 patients were tested by real-time PCR assay. In addition to that, 20 samples from patients with other CNS infections such as tuberculous, pyogenic or fungal meningitis, and noninfectious neurological disorders such as hypertension, status epilepticus, stroke, etc., were also analyzed to test the specificity of the PCR assays. Approximately 2 ml of CSF (by a standard lumbar puncture) and blood samples were taken for analysis. All the samples were stored at −20°C until further analysis. Studies involving human subjects were reviewed and approved by the Institutional Ethics Committee of Central India Institute of Medical Sciences, Nagpur. Viral DNA isolation The genomic DNA of all the selected viruses was extracted from 200 μl of CSF samples from patients by using a ZR Viral DNA isolation kit (Zymo Research, CA, USA), according to the manufacturer's protocol. Viral DNA was isolated from blood by using phenol-chloroform extraction method. Viral RNA isolation The genomic RNA of all the selected viruses was extracted from 200 μl of CSF and serum samples from patients by using a ZR Viral RNA isolation kit (Zymo Research, CA, USA), according to the manufacturer's protocol. Two-step RT-PCR cDNA synthesis cDNA was prepared from RNA viruses using random hexamers using the SuperScript® III First-Strand Synthesis System for RT-PCR kit from Invitrogen. Quantitative real-time PCR assay The set of primers for all selected viruses were used as shown in [Table 1]. The amplification reactions were carried out in a total volume of 10 μl, containing 1 μl of template DNA and cDNA, 5 μl of Power SYBR® Green PCR master mix (Applied Biosystems, Foster City, USA), 1 μl each of (0.5 μM) forward and reverse primer, and 2 μl of sterile water. The amplification conditions consisted of preincubation at 95°C for 10 min and two steps (40 cycles) at 95°C for 15 s and 60°C for 1 min for CMV, EBV, VZV virus, 55°C for DENV (1, 2, 3, 4) JEV, WNV, and CHPV. The quantification cycle (CT) was calculated as the cycle number at which the concentration increase became exponential. A negative control was also included in PCR assay.
One-step RT-PCR assay The real-time one-step RT-PCR assay was performed using the RT-PCR master mix (Invitrogen). The amplification assay was done in 25 μl of the reaction volume that contained 12.5 μl of SYBR master mix, 0.5 μl Superscript RT enzyme, 0.5 μl ROX dye, 0.5 μl (0.5 μM) of forward and reverse primers, 5 μl of RNA DNase-RNase free water was used to adjust the volume to 25 μl. The amplification reaction consists of 55°C for 3 min, 95°C for 5 min for cDNA synthesis, 40 cycles of 95°C for 15 s, 60°C for 30 s, and 40°C for 1 min. The target amplification was analyzed by melt-curve analysis of the Applied Biosystems Step One real-time PCR systems. Standard curve Genomic DNA/RNA To quantitate viral DNA and RNA, a standard curve was obtained for each experiment by co-amplification of known amounts of viral DNA and RNA (determined spectrophotometrically by Qubit, Applied Biosystems, USA). Consecutive dilutions (dilution factors 1:10) were prepared for respective viruses. The amounts of viral DNA/RNA samples were obtained by plotting CT values onto the standard curve. Plasmid Standard plasmids of respective viruses were prepared by cloning the PCR products in pMD19 vector cloning kit (Takara, Japan) according to manufacturer's instructions and quantified by spectrophotometric method. Plasmids were serially diluted (1:10) from 106 to 1 copy. The PCR product of all the respective viral plasmids was confirmed by Sanger sequencing on the Illumina platform (Scigenome Labs, Cochin, India). Statistical analysis All the statistical analyses were done by using the Medcalc software (version 10.1.2).
A total CSF of 150 patients and parallel blood samples of 50 such patients of suspected CNS viral infection cases were tested by real-time PCR assay for the detection of viral DNA and RNA. A one-step and two-step real-time PCR for RNA viruses were developed to detect and quantify viral particles in CSF and blood of suspected viral CNS infections patients. A comparative analysis was done to determine which of the moiety, viz., gDNA, gRNA, cDNA, or plasmid is most suitable for an efficient quantitation of DNA or RNA viral particles in clinical samples. [Table 2] shows the detection limits of PCR assays using gDNA, gRNA, cDNA, and plasmid and using SYBR green chemistry. A known amount of gDNA and plasmid DNA were serially diluted from 106 to 101 to generate a standard curve in qPCR assay. The detection limits of gDNA-based standard curves were 100 copies for VZV, CMV, and EBV, respectively. Viral plasmids showed higher detection limits with the detection of 10 copies for VZV.
For RNA virus detection and quantitation in clinical samples, the detection limits of one-step and two-step real-time PCR were developed for JEV, WNV, CHPV, and DENV-1, 2, 3, 4. One-step qPCR assay involved serial dilution of gRNA from 106 to 101 copies to generate a standard curve. The detection limit of one-step qPCR assay showed a higher detection limit with detection of 10 copies for JEV, WNV, CHPV, DENV-1, 2, 3, and 100 copies for DENV-4. In two-step PCR, cDNA and plasmids were separately serially diluted from 106 to 101 copies. With cDNA two-step PCR, the detection limit was 1000 copies for JEV, WNV, CHPV, and DENV-1 and 100 copies for DENV-1, 2, and 3. Two-step PCR using plasmids showed detection limits with the detection of 10 copies for JEV, DENV-1, 2, and 3, and 100 copies for WNV, CHPV, and DENV-4. A viral etiology was confirmed for RNA and DNA viruses in a total of 21 (14%) cases out of 150 CSF and 50 parallel blood samples [Figure 1: Confirmed and Unknown viral etiology in total suspected cases of viral CNS infections studied in Central India population]. Out of 150 suspected cases of viral CNS infections, only 14% samples showed confirmed viral etiology, including 5% for VZV, 3% for EBV, 1% for CMV, 5% for JEV, and unknown viral etiology were found in 86% cases. The CSF samples showed positivity for 2 cases of EBV, 7 cases of VZV, and 8 for JEV. With blood samples, 2 cases were found to be positive for EBV and 1 for VZV. In 2 of the cases, both CSF and serum showed positivity for JEV.
[Table 3] depicts the number of viral DNA and RNA copies detected in clinical samples. The range of EBV, CMV, and VZV viral particles varied from 2.3 Õ 101 to 8 Õ 105 copies/ml in CSF and blood samples. Similarly, the range of JEV RNA varied from 1.5 Õ 102 to 1.2 Õ 103 in CSF and serum samples. The age and gender-wise distribution of patients have also been noted in the mentioned range of viral DNA and RNA.
[Table 4] shows the clinical characteristics of CNS viral infection cases in two groups and confirmed viral CNS infection cases and unknown CNS viral infection cases. On comparing the computed tomography/magnetic resonance imaging (CT/MRI) scan results, it was observed that there was a significant variation between the confirmed and unknown viral CNS infections group (P = 0.001). The assessment of clinical outcome showed that there was a significant difference between the confirmed and unknown viral CNS infections groups (P < 0.0001). It was observed that patients in the confirmed viral CNS infection group of 12 (57%) returned to normal function, whereas 2 patients (10%) expired, 1 (4%) deteriorated, 2 (10%) was static, and 4 (19%) improved with neurodeficit. In the unknown viral CNS infections group, 119 patients (92%) regained normal function, whereas 2 patients (2%) expired, 4 (3%) deteriorated, 4 (4%) static, and none of them improved with neurodeficit. The significant variation was also observed in a reduced level of consciousness, limb weakness, and behavior disturbance between both the groups (P = 0.05, 0.005, 0.05, respectively). Other clinical parameters such as seizures, neck stiffness, fever, vomiting, headache, age, gender, etc., did not show any significant variation between the two groups. CSF in the patients of both the groups was analyzed for total leukocyte count, sugar, and protein [Table 5].
The present work describes the viral etiology in suspected cases of viral CNS infections in a tertiary care hospital in Central India. New viral infections seem to predominate now in the West and developed nations; infection by known viruses is still seen in India. In our study, the viral etiology was confirmed in only 14% of cases, the most common etiological agent was JEV (5%), followed by VZV (5%), EBV (3%), and CMV (1%). Among the suspected viral encephalitis case, 2 cases were positive for JEV in both CSF and serum samples and higher JEV load was detected in CSF of a 10-year-old kid. Likewise, in case of EBV, higher viral load was found in blood samples of the patient, while the parallel CSF sample was found negative, which suggested the active infection of EBV. In a survey conducted in northern India on acute encephalitis, JEV was commonly identified, followed by VZV, DENV, HSV, measles virus, mumps virus.[17] In a rural Central India study, enteroviruses were identified in a maximum number of cases followed by VZV, JEV, and HSV.[33] In a hospital-based study conducted in southern Vietnam, JEV was most commonly identified (12%) followed by VZV (1.7%), CMV (0.3%) enteroviruses, DENV, and HSV.[34] As various subsequent studies reported that JEV the most common causative agent of viral CNS infection in India, JEV positivity has been reportedly high in various parts of the country, especially in a hyper endemic region.[16],[17],[33],[35] This study brings out that the most usual cases of viral CNS infection in hospitalized patients in the Central India population was JEV detected in more number of CSF samples followed by VZV as the most frequent viral agent causing CNS infections. In the present work, despite the wide role of PCR-based testing of selected etiological agent, 86% cases show unknown etiological agent. In some previous studies such as in the California encephalitis project the viral etiology could be identified in only 31 (9%) cases out of 334 enrolled patients. In another study from Finland, the 34% cases remain undiagnosed.[36],[37] The clinical manifestation of confirmed viral CNS infections (PCR-positive) and unknown viral CNS infections (PCR-negative) cases was compared to evaluate and support the findings of PCR results. A more severe disease was found in the PCR-positive group of patients.[38],[39],[40] The numbers of patients with lesions in the brain seen by CT/MRI scan were more in PCR-positive group as compared to the PCR-negative group. CT/MRI increases the likelihood of a positive PCR; however, patients with such findings can be negative by PCR and some PCR-positive patients with less severe forms of viral encephalitis have normal CT scans.[41],[43] After completion of treatment, nearly 3% of the individuals in the unknown viral etiology group had moderate levels of impairment which suggest that there are more probabilities that the patient would develop serious neurological sequelae if the treatment is not streamlined in cases of unknown etiology. Diagnosis of viral infections of the CNS has been highly improved by the use of PCR technique that amplifies viral nucleic acid from CSF. Real-time PCR assay is a useful method for the rapid diagnosis and for monitoring the virus load to measure the efficacy of treatment.[20],[21],[22] In this study, comparison and effectiveness of molecular methods were evaluated for the detection and quantification of viruses causing infections of the CNS. A detection limit of real-time PCR was compared using gDNA, gRNA, and plasmids as standards. In an added approach, one-step and two-step PCR were also compared for RNA viruses and comparative experiments of the methods were performed to evaluate their efficiency, sensitivity, and accuracy and also validated the use of gDNA and plasmid as a standard to facilitate the detection and quantification of any target sequence in viruses. A variety of materials have been used as external standards, including plasmid DNA containing the target sequence. Each has advantages and disadvantages in terms of complexity of the material, cost, accessibility, long-term stability, and gene copy accuracy. Among DNA viruses, plasmids showed higher detection limits for the identification and quantification of viruses causing CNS infections. There are several advantages to using plasmids over gDNA.[42] The quantity of plasmid DNA necessary to achieve a useable copy number is generally in the lower (femtogram) range. Our results show that PCR efficiency is affected by the complexity of the material from which a target is amplified. This includes both the quantity as well as the composition of background DNA. It has been stated that real-time PCR is inhibited at high background gDNA concentrations and/or in the presence of long strands of structurally complex DNA. Quantitative RT-PCR can be carried out as a one- or two-step reaction. However, the choice of method raises controversy from the perspective of the researcher and manufacturer, because of advantages and disadvantages to both systems. Therefore, each laboratory must decide what is more important relative to their outcomes, sensitivity, or speed of the assay protocol. The viral load detection limits were higher and more sensitivity for one-step protocol.[31],[32] Although the two-step RT-PCR reaction provided greater flexibility and better optimization, one-step protocols are powerful, as they minimize handling and therefore, reduce chances of pipetting errors and cross-contamination. Epidemiological studies for the virus are not used in many regions of the nation, due to lack of virology research laboratory. Therefore, the percentage of cases of viral CNS infections are hard to judge. The results presented in this study provide observations regarding the diagnosis of viral CNS infections using the most appropriate molecular technique. Our study recommends an assessment of this technique for use with detecting and quantifying viruses in cases of CNS infections. Collaborative studies should be undertaken to assess the interlaboratory reproducibility of this much-needed DNA/RNA-based assay, if it is to be used extensively for the detection and quantification of viruses causing CNS infections. Our study has certain limitations first, as we only focused on patients referred to our tertiary care center for a limited time period. Second, the follow-ups have not been reported, hence long-term sequelae have not been tested and the CT and MRI images of positive patients and other clinical materials have not been described due to unavailability of data. Third, limited number of common etiological agents were tested. Any commercially available kits were not used to look for other causative agents of CNS viral infections such as, rabies, enteroviruses, fungal, and other parasitic organisms, due to which the viral etiology was confirmed in only 14% cases and most of cases remain undiagnosed. So, further studies are required on stored clinical samples to see the unknown etiology. In the present study, in addition to CSF samples, few parallel blood samples (n = 50) were also gathered to measure the specificity and sensitivity of the developed PCR assay for the quantification of viral load in blood samples of patients. Our results suggested that the blood PCR can be a better alternative as compared to CSF in severe cases of viral CNS infections and also useful to understand the latent or primary infections. Nevertheless, the study demands further validations on a large cohort for better establishment of blood PCR for diagnosis of CNS viral infections. We think that this kind of hospital-based survey can provide the valuable etiological and epidemiological data for state healthcare agencies and public health planners for the evolution of integrated surveillance and vaccination program. The SYBR green-based real-time PCR assay developed in the present study for detection of both the RNA and DNA viruses in a single run is cost-efficient, feasible, and rapid. It is also useful for any diagnostic laboratory as an efficient instrument for clinical diagnosis and quantification of these viral infections. As per our knowledge, this is the first work of this kind in this region where comparison of real-time PCR methodologies has been done. Our findings reveal that VZV and JEV are the most usual cases of CNS viral infection in hospitalized patients in the Central India population, and one-step RT-PCR shows higher viral load detection limits for quantitation of viral genome and more sensitivity in comparison to two-step RT-PCR. Acknowledgments All authors would like to greatly acknowledge National Institute of Virology, Pune, India for providing RNA of JEV, DENV (1, 2, 3, 4), and WNV. Dr. Dhrubajyoti Chattopadhyay, Calcutta University, India for providing CHPV RNA. Dr. H.N. Madhavan, Sankara Nethralaya, Chennai, India for providing CMV and EBV DNA. Source of support None. Conflicts of interest None.
[Figure 1]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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