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The Viruses
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
Virus isolation remains an important component of procedures used to confirm the identity of viral pathogens. Fluids or tissues likely to contain a viral pathogen are collected and placed in cell cultures capable of supporting the replication of the virus. The procedure can be technically difficult because of the potential for bacterial and fungal contamination of cell cultures used in the procedure. The agent must be detected by procedures outlined earlier in this section, and requires confirmation of the viral agents by specific means (e.g., antigen detection of cultures). The choice of the cell used for isolation is very important. Primary fetal cells are often good hosts, yet they are difficult to maintain. Cell lines are convenient and as a consequence are frequently used for isolation, but it is necessary to establish that they are susceptable to the virus that one is attempting to isolate.
Arthropod-borne virus encephalitis
Published in Avindra Nath, Joseph R. Berger, Clinical Neurovirology, 2020
The presumptive diagnostic test of choice is IgM capture ELISA, with very high sensitivity from one to two weeks after onset in serum and CSF [147]. However, cross-reactions with other serologically closely related viruses require confirmation by diagnostic fourfold rises between acute and convalescent serum samples tested by other assays such as neutralization or complement fixation. Virus isolation can be achieved on some occasions, particularly early in the illness, from CSF and serum. However, the time required to achieve and characterize an isolate renders isolation technologies much less useful in acute disease management than for epidemiologic studies. CSF PCR can also be used to detect Japanese encephalitis virus [148].
Supplemental Tests for HIV-1 Infection
Published in Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts, Retroviral Testing, 2020
Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts
A definitive diagnosis of HIV infection can be made by the use of cell culture to isolate the virus from selected cells, tissues, and body fluids of asymptomatic and symptomatic individuals. A cocultivation system is required in which cultures are prepared from peripheral blood mononuclear cells (PBMC) of HIV seronegative donors. Continuous cell lines derived from neoplastic T cells (HT) also maybe used, but are less susceptible to HIV infection. While PBMCs isolated from an individual suspected of being infected are the preferred specimen for attempting virus isolation, HIV can also be isolated from CSF, serum or plasma, saliva, cervical secretions, semen, tears, brain tissue, organ biopsies, and breast milk. The isolation success rate for HIV from PBMCs may vary from 100% among symptomatic patients to a substantially lower rate for similar specimens obtained for asymptomatic individuals. Variation in the efficiency of donor seronegative PBMC cultures to consistently isolate HIV is likely to reflect existing levels of virus and/or antibody in the sample, as well as the degree of susceptibility of the PBMC culture to infection. These variables, and the fact that culture procedures are laborious, time consuming, dangerous, and costly have limited the use of this technique for diagnosing HIV infection. In addition, virus isolation also depends on sample size, sample freshness, and the laboratory worker’s expertise.
Diagnostic approaches for dengue infection
Published in Expert Review of Molecular Diagnostics, 2023
Gaythri Thergarajan, Shamala Devi Sekaran
The DENV has a single stranded RNA genome coding for three structural proteins and five non-structural proteins (capsid protein, membrane protein, envelope protein, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [10]. The proteins are arranged in the sequence of NH2-C-M-E-NS1-NS2A-NS2B-NS3-NS4-NS4B-NS5-COOH as a single polyprotein precursor consisting nearly 11,000 nucleotides [11,12]. Diagnosis begins with a clinical suspicion which are not unique to dengue and hence limited in its usefulness for early diagnosis. Clinical symptoms have high sensitivities but have poor specificities and hence a confirmatory diagnosis is essential. Currently, markers used include the NS1, IgM and IgG antibodies. Virus isolation is not routinely done except in reference centers and research laboratories. Nucleic acid amplification is not as popular and only utilized in laboratories tracking infections or public health laboratories. Most of them amplify the E regions while some include the pre-M, capsid and/or the NS5 regions. The titers of the IgM and IgG antibodies depend on whether the infection is a primary or a secondary infection. This can only be determined by quantitative or semi-quantitative methods. The main issue with confirmatory assays is the validation of each assay. Hence, early diagnosis remains a challenge, more so the markers to use and the time frame of detection. A combination of clinical markers and laboratory-confirmed assays may serve as predictive markers at different stages of the disease.
Neurological manifestations due to dengue virus infection in children: clinical follow-up
Published in Pathogens and Global Health, 2021
Aline Almeida Bentes, Roberta Maia De Castro Romanelli, Ana Paula Correa Crispim, Paula Eillanny Silva Marinho, Karina Soares Loutfi, Sara Tavares Araujo, Luciana Maria Campos E Silva, Isabela Guedes, Alice Martins Alvarenga, Marcele Almeida Santos, Erna Geessien Kroon
All children included in our study were diagnosed by conventional RT-qPCR in CSF. For RNA extraction, the QIAamp® Viral RNA Kit (QIAGEN®, USA.) was used. The viruses tested by PCR were DENV, Zika virus (ZIKV), Yellow Fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Sant Luis encephalitis virus (SLEV), Mayaro virus (MAYV), Oropouche virus (OROV), Chikungunya virus (CHKV), herpes virus (HHV-1, HHV-2, HHV-3, and HHV-4) and enterovirus (ENV). We achieved viral isolation on mosquitoes from 32% of DENV-positive samples by RT-qPCR. The molecular diagnosis and viral isolation were made at the Virus Laboratory at Universidade Federal de Minas Gerais.
Chikungunya fever: a threat to global public health
Published in Pathogens and Global Health, 2018
Raíza Nara Cunha Moizéis, Thales Allyrio Araújo de Medeiros Fernandes, Paulo Marcos da Matta Guedes, Hannaly Wana Bezerra Pereira, Daniel Carlos Ferreira Lanza, Judson Welber Veríssimo de Azevedo, Josélio Maria de Araújo Galvão, José Veríssimo Fernandes
Clinical diagnosis of Chikungunya fever is very difficult, especially in regions where there is simultaneous circulation of other arboviruses such as Dengue viruses, Zika virus and Mayaro virus, which can cause very similar symptoms to those of the Chikungunya virus. Because of this, laboratory diagnosis becomes extremely necessary. Virus isolation and identification is considered the gold standard technique. However, its complexity, high cost and the delay in obtaining the result make it unsuitable for routine use.