Dengue Virus and its Microbiological diagnosis.
By Aditya Tripathi and Abhay Raj Chauhan
(2nd Year Medical Students)
Introduction
In this article we are going to study about dengue Virus Dengue which is an increasingly common mosquito-borne, flaviviral disease of significant public health concern, The disease is endemic throughout tropical and subtropical regions, placing almost half of the world’s population at risk, and each year approaching 100 million people in around 130 countries are infected as well the methods of it's diagnosis.
Background
There is acknowledged to be four antigenically distinct serotypes of the virus, and arguably a fifth, each of which elicits a full spectrum of clinical disease. This ranges from asymptomatic self-limiting infection to life-threatening severe manifestations characterized by plasma leakage, bleeding, and/or organ failure. Recovery from primary infection by one serotype provides life-long immunity against reinfection by that particular serotype, whereas with subsequent infections by other serotypes, the risk of developing severe dengue is increased. Rising mortality and morbidity rates caused by infection in recent years are attributable partly to a lack of availability of effective antiviral therapies and vaccines. In this context, early detection of infection with sensitive and specific laboratory tools and the prompt clinical management of this disease is a health care priority. Although a variety of techniques are currently used for laboratory diagnosis of dengue, no single methodology satisfies the ideal requirement for both sensitivity and specificity, while also being rapid and inexpensive now further we will discuss about different microbiological diagnosis of dengue virus.
Methods of Microbiological diagnosis
Virus Isolation
Virus isolation is considered to be the gold standard technique in dengue diagnosis, Isolation is highly successful when specimens are collected in the viraemic period, which starts 2–3 days before the onset of fever and lasts for a further 2–3 days . Routinely, serum is the primary choice of sample. In addition, other types of specimen, including plasma, peripheral blood, cerebrospinal fluid, pleural fluid, and tissues of reticuloendothelial origin such as liver, spleen, lymph nodes, lung, and thymus, are of substantial importance. Diagnostic sensitivity is dependent upon the timing of specimen collection, proper storage, and transportation. Since the DENV virus particle is heat-labile, appropriate handling and prompt delivery to the laboratory is required. When storage is warranted, a standard refrigerator temperature (~4°C) is quite appropriate in the short term, while for a longer duration, freezer temperatures of −20 or −70°C are recommended. In the latter case, they should be maintained in order to prevent thawing .There are different methods available for the isolation of DENV including
Inoculation of specimens into mosquitoes (adults or larvae).
Mosquito or mammalian cell cultures.
Injection intra-cerebrally in mice.
Viral nucleic acid detection
DENV is a single-stranded positive-sense RNA virus of approximately 50 nm in length. The 11 kb genome of each virion encodes three structural proteins (capsid, C; precursor membrane, prM; and envelope, E) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). These non-structural proteins play roles in viral replication and assembly. Structurally, a virion consists of a nucleocapsid, enveloped by an outer glycoprotein shell and an inner lipid bilayer. Surface projections in the lipid membrane consist of E and membrane (M) glycoproteins.
Nucleic acid amplification tests and identification of virus antigen or antibody serve as the predominant means of detection of DENV, based on the molecular or immunological response to specified viral structural components. Commonly used methods are
1. Transcription polymerase chain reaction (RT-PCR)
2. Nucleic acid sequence-based amplification (NASBA)
3. Transcription-mediated amplification (TMA).
Serological methods
Antigen detection –
During the febrile phase of dengue illness, DENV antigens may be detected in peripheral blood leukocytes, liver, and lung at autopsy, and less often in the thymus, lymph nodes, skin, spleen, bone marrow, and serosa. Immunohistochemistry and enzyme immunoassay (EIA) are useful techniques to detect dengue antigens in tissue sections (frozen, paraffin-embedded) using labeled monoclonal antibodies that are visualized with markers such as fluorescent dyes (fluorescent antibody), enzymes (immunoperoxidase and avidin-biotin enzyme) or colloidal gold. Given the inconvenience of collecting samples on which to apply these techniques, their utility has been limited for routine laboratory tests. Instead, assays have been developed against intact virus particle and recombinant viral proteins, with NS1 and E protein detection available commercially.
Non-structural proteins (NS1 and NS5)-based assay –
Non-structural gene, NS1, is a highly conserved glycoprotein produced by all flaviviruses in both membrane-associated and secreted forms, and which is essential for viral replication and viability. Localized to cellular organelles, this antigen is secreted abundantly in sera of patients during virus replication and the early stage of infection. A unique feature of this protein is its secretion by mammalian cells as hexamers (dimer subunits only), while it is not secreted by insect cells, including those of mosquitoes. Glycosylation is believed to be an important step for protein secretion. It can be identified in peripheral blood prior to formation of antibodies, and the detection rate is higher in acute primary infection from the day of onset of fever to day 9. NS1 is also a complement-fixing antigen that elicits a very strong humoral response. Several recent studies have addressed the use of NS1 antigen and anti-NS1 antibodies as a tool for dengue diagnosis, with higher specificity for determination of homologous serotypes by serotype- and group-specific NS1 capture ELISA, and a detection sensitivity of 1–4 ng/ml NS1 in blood. This tool can be utilized to differentiate between primary and secondary dengue virus infections, as proven by the highly correlated results of NS1 serotype-specific IgG (determined by ELISA) and PRNT. The NS1 serotype-specific IgG ELISA worked reliably for serotyping dengue virus in convalescent phase sera from patients with primary infection and also in acute phase sera from patients with secondary infection (in which the serotype that caused the first infection would be detected), but not so with convalescent phase sera from patients with secondary infections. Different commercial assays to detect NS1 antigen are available, including Panbio dengue virus Pan-E NS1 early ELISA (Alere); dengue virus NS1 antigen ELISA (Standard Diagnostics); Platelia NS1 antigen ELISA (Bio-Rad). However, none of these have excellent sensitivity, ranging from 45 to 57%. NS1 ELISA has a noted lower sensitivity in sera from patients infected with DENV-4. This may be due to possible quantitative differences in the secretion of NS1 antigen by distinct serotype. Dengue non-structural protein NS5, which plays a vital role in dengue replication, is the largest and most well conserved of the flavivirus proteins. One study demonstrated reliable results in diagnosing and differentiating DENV from West Nile virus and St. Louis encephalitis (SLE) virus using an immunoassay designed to targeting NS5 antigen.
4. Antibody detection
DENV-specific IgM antibodies start to appear after 4–5 days of primary infection and are measurable for up to 3 months. IgG antibodies appear about a week after onset of fever, persist at high titers for several weeks and then decline; however, IgG may be detected for decades. IgG1 and IgG3 are the subclasses that are mainly induced. Following secondary infection, memory B cells are stimulated in response to secrete DENV-specific IgG that is measurable even on the first day of symptoms and the titre remains much higher compared to during primary infection. In secondary dengue, the IgM response is variable and, in some cases, even undetectable. The most commonly used antibody detection techniques for dengue are
Hemagglutination inhibition.
E/M-specific capture IgM and IgG ELISA.
5. Rapid diagnostic test
Immunochromatographic tests are developed to meet the need for rapid methods of dengue diagnosis. The value of the Immunochromatographic format is its rapid attainment of a result by virtue of a color change that is visible to the naked eye within 10–15 min. Tools are based on the detection of anti-dengue IgM and IgG antibodies and of dengue antigens. A number of studies have evaluated dengue rapid diagnostic tests (RDTs). Commonly used RDTs are Duo IgM and IgG Rapid Test Strip (Panbio); Bioline Dengue IgG/IgM (Standard Diagnostics); VScan (Minerava); Smartcheck (GlobaleMed); Denguecheck-WB (Tulip); and Dengue IgG/IgM (Core). With acute phase samples, the diagnostic accuracy of the tests has not been established reliably, but manufacturers’ claimed performance ranges from 76 to 100% sensitivity and exceeding 99% specificity. In contrast, markedly different results were claimed independently, suggesting most RDTs are unsuitable for dengue diagnosis as they have poor sensitivity and specificity. A preliminary trial of dengue from Nepal has reported a similar finding when rapid immunochromatographic tests are comparing with ELISA; the former tool did not prove sufficiently reliable with regard to either sensitivity or specificity. In order to overcome the limitations posed by RDTs, techniques have been modified whereby dengue NS1 antigen is detected in combination with anti-glycoprotein E IgM and IgG antibodies.
6. Dengue virus serotyping and genotyping
Detection and serotyping of dengue viruses are performed by molecular methods such as PCR (nested, semi-nested, and multiplex RT-PCR) using serotype-specific primers. Following RT-PCR, the amplified product is subjected to nucleotide sequencing. The most commonly used commercial sequencing technique for amplicon purification and sequencing is QIAquick PCR Purification Kit (Qiagen). Both strands of the PCR product are sequenced in order to avoid discrepancies. Gene sequences obtained for DENV strains are submitted to the GenBank database. A basic local alignment search tool (BLAST) algorithm performed against information stored in the database finds regions of local similarity between nucleotide sequences. This may yield functional and evolutionary clues about the structure and function of any novel sequence.
Conclusion
A lack of sensitive, specific, rapid, and cost-effective assays remains the major hurdle to the development of diagnostic tools for dengue virus detection. Although virus isolation is more specific, it is limited by being time-consuming and expensive, needing expertise, an inability to differentiate primary and secondary infections, and requiring acute samples (0–5 days post onset of fever). Nucleic acid (RNA) detection, although sufficiently sensitive and specific to identify both serotype and genotype in a short time is also restricted by the need for an acute sample, skilled handling, and by its incapacity to distinguish primary from secondary infections. Furthermore, it is an expensive tool that is not affordable by all laboratories. While these limitations are being overcome by the detection of NS1 antigen, its sensitivity compared to genome detection methods is poor and cannot be guaranteed. Tests that measure anti-dengue IgM antibodies are useful only for the first 4–6 days of infection. Also, IgM levels in secondary infection are quite low and could be below the threshold for detection. Moreover, IgG specificity is reduced due to cross-reactivity among flaviviruses, so there should be more effort made for finding cheap, efficient and less time consuming diagnostic method.
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