key: cord-0019033-b46e71oj authors: Tavakolian, Shaian; Goudarzi, Hossein; Eslami, Gita; Darazam, Ilad Alavi; Dehghan, Golnaz; Faghihloo, Ebrahim title: Detection of Enterovirus, Herpes Simplex, Varicella Zoster, Epstein‐Barr and Cytomegalovirus in cerebrospinal fluid in meningitis patients in Iran date: 2021-06-21 journal: J Clin Lab Anal DOI: 10.1002/jcla.23836 sha: 6481e0fa73ab5b9ec78754701889a88243f5dfb5 doc_id: 19033 cord_uid: b46e71oj BACKGROUND: Despite medical advances, central nervous system (CNS) diseases put a pressure on the health care system. A number of risk factors, especially infectious agents can accelerate the progression of meningitis. As viruses probably account for most cases of meningitis, the diagnosis of them can reduce antibiotic prescriptions. Among various types of infectious diseases, the relationship between two important virus families, including Picornaviridae and Herpesviridae, and meningitis has attracted attraction. METHODS: In this study, one hundred and two samples were collected from patients who experienced symptoms, such as the loss of consciousness, seizures, muscle weakness, fever, headache, rash, and severe dementia, between November 2018 and September 2019. After RNA and DNA extraction, the prevalence of Enterovirus (EV), Cytomegalovirus (CMV), Epstein–Barr virus (EBV), Herpes simplex virus type 1 (HSV‐1), Herpes simplex virus type 2 (HSV‐2), and Varicella zoster virus (VZV) was evaluated using PCR, multiplex PCR, and nested PCR. RESULTS: Results indicated that there were two VZV DNA‐positive specimens, while six and five samples were infected with HSV‐1 and EBV, respectively. CONCLUSION: We reported that the prevalence of EBV, HSV‐1, and VZV in patients, suffering from meningitis cannot be ignored; however, further investigation is needed. for blood-brain barrier (BBB) destruction and brain tissue necrosis. At least five members of Herpesviridae, including CMV, EBV, HSV-1, HSV-2, and VZV, which spread around the world, can cause tumorigenic transformation, and gene overexpression. 8 The most common clinical symptoms of patients, suffering from these viral health problems, are blisters, fever, meningitis, changes in an individual's behavior, cognitive disability, aphasia, seizures, and neuropsychological deficits. 9, 10 Immunocompromised people, such as AIDS patients and organtransplant recipients, usually have these symptoms as these viruses are opportunistic pathogens 11, 12 Moreover, Enterovirus (EV) is another virus that can be considered one of the leading causes of aseptic meningitis in children. [13] [14] [15] This virus, accounting for 60%-80% of all aseptic meningitis cases, belongs to Picornaviridae. 16 EV infections cause a wide variety of symptoms, such as pleocytosis (an increase in the number of lymphocytes in cerebrospinal fluid). 17 As viruses probably account for most cases of meningitis, the diagnosis of them can reduce antibiotic prescriptions. 18 In the light of what is mentioned above, we attempted to investigate the prevalence of these viral diseases in CSF of patients, suffering from meningitis. The present study is financially supported by "Infectious Diseases The RNA and DNA of filtered CSF were extracted by High Pure Viral Nucleic Acid Kit (Roche Diagnostics), in which 200 μl of filtered CSF sample was eluded by 50 μl of elution, according to manufacturer's protocol. In this study, we used a Bio fact cDNA kit. After combining 25 μl of eluded purified viral nucleic acids with 24 μl reverse transcriptase and 1 μl Random hexamer, the incubation was carried out for 40 min at 50℃ and 10 min at 95℃ in Bio Intellectica PCR. The cDNA was then diluted twice in sterile water. In this study, we evaluated EV prevalence with nested PCR. In the first step, 12.5 µl master mix PCR, 1 µl forward primer, 1 µl reverse primer (Table 2) , 5 µl DNA, and 5.5 µl sterile water were mixed. The first PCR schedule was 3 min initial denaturation at 94°C, 40 cycles of denaturation at 94℃ for 30 s, annealing at 44°C for 30 s, extension at 72°C for 30 s and final extension at 72°C for 5 min. Then, in the second step, 12.5 µl master mix, 1 µl forward primer, 1 µl reverse primer, 8.5 µl sterile water, and 2 µl DNA of the first PCR products were mixed. The PCR schedule was as follow: 3 min initial denaturation at 94°C, 40 cycles of denaturation at 94 0 C for 30 s, annealing at 51 0 C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 5 min. The second PCR product was about 360 μl, and run into 2% agarose gel electrophoresis. Multiplex PCR was employed to investigate the prevalence of VZV, HSV-1, and HSV-2 in CSF samples. 25 In this step, the DNA sequences were amplified and the glycoprotein gB, glycoprotein gG, and glycoprotein gB were encoded to detect CMV, HSV-1,2, VZV respectively. Indeed, after subcloning these genes in Escherichia coli (strain BL21), we used them as positive controls. The results, obtained from the present study, were analyzed to find the correlation between the prevalence of VZV, HSV-1, EBV and patient's sex and age by IBM SPSS statistics software version 22, and Chi-square test. Moreover, ANOVA test was employed to confirm the relation between age and VZV, HSV-1, EBV infections. Experimental data were expressed by mean ±standard deviation of three independent assays. p-value less than (p < 0.05) was used for the differences. Our results indicated that 6, 5, and 2 samples (7 male and 6 female) were infected with HSV-1, EBV, and VZV, respectively. The mean age in these patients was 11 months and fever was seen in all of them. In patients, suffering from VZV, headache and nausea or vomiting were reported, and one of them had a rash. (Table 1 ). Central nervous system (CNS) diseases can be considered one of the leading causes of death all around the world. Statistics have shown that meningitis is responsible for 0.6% of all deaths, and survivors usually suffer from various types of disabilities. 19 There are a number of tight relationships between different risk factors and central nervous system disorders, but the role of viral infections has drawn much attention. 13, [20] [21] [22] As symptoms of this disease are nonspecific, molecular analysis is essential to identify causative agents. 23, 24 In this study, the prevalence of Herpesviridae and Picornaviridae was investigated in CSF of patients, suffering from meningitis. In this study, we detected 6, 5, and 2 samples infected with HSV-1, EBV, and VZV, respectively, but we did not find CMV [36] [37] [38] In the light of what was mentioned above, the HSV-1, EBV, and VZV prevalence is high in Iran and can play a pivotal role in the progression of meningitis, requiring immediate detection and treatment. The limitation of this study was the low number of samples. The present study is financially supported by "Infectious Diseases Not applicable. Not applicable. The authors declare no conflicts of interest. All data generated or analyzed during this study are included in this published article. Ebrahim Faghihloo https://orcid.org/0000-0002-8669-305X Case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium MicroRNAs in the interaction between host-bacterial pathogens: a new perspective In silico analysis of a chimeric fusion protein as a new vaccine candidate against Clostridium perfringens type A and Clostridium septicum alpha toxins Proliferative effect of FadA recombinant protein from Fusobacterium nucleatum on SW480 colorectal cancer cell line. 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