A double-entry table to obtain indicators of diagnostic tests from analyzing a sample showing all possible results. The sensitivity and specificity have distinctive features of diagnostic tests, they are not compromised by the prevalence of the disease, and they are inversely proportional. If a study in which the majority of people are suffering from this disease is carried out, a high sensitivity test is needed in order to identify the highest number of true positive and the lowest number of false negative.
However, it may increase the number of false positive. If you want to obtain a good disease diagnosis, a high specificity test must be used to detect the highest number of true negative. Here, the false positive will also be low. Moreover, positive and negative predictive values of the diagnostic tests are affected by the prevalence of the disease in the study population. The likelihood ratio LR that is independent of prevalence is used when the laboratory tests do not present dichotomous results but cut-off value.
This is another way of assessing the accuracy. Not only one but also many tests can be used to diagnose dengue in the epidemiological studies. It can be done sequentially or in parallel. For example, when performing a test with two sequential tests, all positive people need to be assessed with a second test upfront, and this will cause the reduction of net sensitivity and a net specificity enhancement obtained from both tests. It will be considered as positive if their tests are positive in all tests.
Likewise, the negative ones will have negative results in the confirmatory test. On the other hand, if two simultaneous tests are used, a net sensitivity is gained, while a net specificity is reduced. This is different when tests are done independently. Negative is considered people whose negative results were in all tests and positive the ones whose positive results were in at least one of the tests [ 3 , 4 ].
The type of sample taken in the right moment, storage and transport to the laboratory to be processed, and the appropriate documentation plays a key role to obtain results because if there is a change of sample quality, this can reduce antibody titers, viruses or genetic material resulting in lower titers or concentrations from the real ones in quantitative tests or false negative results in quantitative or qualitative tests.
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The most used samples to diagnose and to search about dengue are whole blood, serum, plasma, and human organs like spleen, liver, and heads of mosquitoes, pools of mosquitoes, brains of mice, serum samples saturated with filter paper, etc. The samples must be taken to the laboratory as soon as possible and preferably dealt with in dry ice or liquid nitrogen [ 5 , 6 ]. Although most infections are asymptomatic or subclinical, a set of symptoms starts after a dengue infection elapses the 4—day incubation period.
When people are infected with the virus on the first time, dengue infections are known as a primary infection in which a viral load and the relevant antibody formation IgM, IgG and IgA are triggered. In a primary infection, the titer of IgM is generally much higher and more specific than in a secondary infection. When people are previously exposed to any serotype or flavivirus, or even after a vaccine i. In secondary infections, the IgG is detected in the highest levels and even on the acute phase.
When a positive IgM occurs in a single serum sample or a positive IgG in a single sample with hemagglutination inhibition titer is the same or higher than , it is considered a suggestive case [ 5 , 6 , 7 , 8 ]. The laboratory tests can be interchangeably used in different researches, both basic and applied ones. We can classify them into direct methods that allow virus detection or part of its structure and indirect methods which identify a reaction produced by the presence of DENV in the organism. The genetic material extraction has a key role for PCR tests so that the quality of a product extracted can vary depending on the type of sample being used, and the extraction method applied will directly affect the test sensitivity.
The dengue RNA can be recovered from serum, blood, urine, plasma samples and other organs. However, the viral load in blood is much higher 7.
Inhibition of West Nile Virus Multiplication in Cell Culture by Anti-Parkinsonian Drugs
It was found that 34 samples out of 47 were positive by using the Chomczynski-Sacchi method, and the remaining 27 samples were positive by using the kit commercial method [ 8 , 9 , 10 ]. Less time to process the results, being able to identify the circulating serotypes of the virus, presenting the highest sensitivity and specificity levels are among its advantages. This type of test has the benefit of obtaining rapid results while identifying circulating serotypes of dengue.
Then, the PCR is carried out when primers are used to amplify prM genes and C virus areas to continue with a specific primer-nested PCR for each virus serotype. There are many different variants in this technique having different sensitivity levels that are used in research laboratories. They are transmitted by the same vector Aedes aegypti y Aedes albopictus facilitating its cocirculation in some areas of the region.
Because of these arboviruses, the affected patients develop similar symptoms but its clinical management and its possible results as the aftermath of the disease, and mortality rates are different. They can even produce coinfection with other microorganisms making a proper identification necessary in an early stage of the disease acute phase. The main advantage of this test is time reduction to obtain results about an hour , discriminating the amount of pathogens, and minimizing cross-contamination problems so that all reactions are carried out among a closed system.
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The cost of the product and a machine analyzing only a sample at a time is within its limiting [ 13 ]. The strengths of this test are the same as the conventional RT-PCR, it also reduces time when releasing the results as well as the cross-contamination risk post PCR, the levels of sensitivity and specificity are higher than the conventional RT-PCR, and overall it allows quantifying the genetic material. There are different commercial kits in the market to diagnose DENV, and its sensitivity and specificity levels vary when they are compared among them [ 14 , 15 ].
Viral isolation in cell cultures or mosquitoes followed by the virus detection using indirect immunofluorescence is considered as gold standard [ 8 , 17 ]. In order to carry out the DENV viral isolation and as a general rule to any virus, it is necessary to consider the following: To know the isolated virus virus characteristics, replication, transmission mechanism, etc. To know which biosafety level a virus can be performed. In the case of DENV, biosafety level 2 is needed [ 18 ]. To determine which cell line to use and to be able to isolate the virus, it is essential to identify the most sensitive cell line from mosquitoes and mammals, and its use for the isolation and DENV propagation, being the most sensitive cell lines of mosquitoes as follows:.
Knowing the viral isolation technique that provides better results to isolation and virus propagation. The standard method is based on the virus propagation in a sensitive cell line for inoculating a previous diluted sample in a cell culture medium. Then, the infected cells are recovered, and the virus presence is determined by an immunofluorescence process, ELISA, molecular techniques, and others.
Bottles, tubes, 6—96 well culture plates, and others are used in order to sustain the binding to cell cultures. This technique can also be used to isolate DENV coinfections. However, it seems not to have good results for VEE isolation [ 19 , 20 , 21 , 22 ]. The sample type to be used. The sample type to be used and its proper preservation until the processing time are extremely important to isolate a virus.
The most used dilutions to a viral isolation vary from to A very concentrated dilution of the sample could generate a toxic effect in the cells. On the other hand, a much diluted sample could cause the inability of isolating the virus because of having a low concentration virus in the inoculum [ 19 , 21 ]. It is the most used test to determine viral vaccine titers so that it quantifies the virus to infect cells.
Inhibition of West Nile Virus Multiplication in Cell Culture by Anti-Parkinsonian Drugs
It is based on the infection of a cell monolayer with different virus dilutions to evaluate. After an incubation period, the viral infection results in lytic plaques. If they are colored, they are displayed as holes in the cell monolayer. Each plaque corresponds with an infectious virus. One of the main disadvantages of this technique is that it can only use viruses being able to produce a cytopathic effect.
Another one is that all native strains of DENV are not always capable of producing well-defined plaques, and the viral titers can vary depending on the cell line used. It is a combination of plaque assay and immunofluorescence. Viruses are inoculated in different dilutions in the cell line, then a cell incubation period is fixed to plaques with any organic solvent, and an immunofluorescence is carried out. Positive cells are observed with fluorescent foci that can be counted.
One of its advantages is to reduce the incubation period in order to obtain the results in comparison with plaque assay. It allows processing a bigger number of samples so that it can be adapted to use well culture plates in comparison with the well plates which are used in the plaque assay. Suckling mice were greatly used because of their easy reproduction and handling to isolate virus as well as the antigen production. About 1—3 neonatal mice and an intracranial inoculation are carried out.
Then, a day daily checking to observe the occurrence of neuromotor symptoms is needed. This technique is starting to cease to use due to a great variety of cell cultures that allow good sensitivity levels in DENV detection. One of the most common practices to carry out euthanasia on suckling mice is using a CO 2 camera. When using this technique, it is necessary to make sure such mice are dead as they are very resilient to lacking of oxygen so it is advisable to continue with other euthanasia techniques like cervical dislocation, decapitation, etc. The mosquitoes like Aedes genus can be used for dengue virus isolation when infection and disease transmission studies are carried out.
The intracerebral and intrathoracic inoculations are used for mosquitoes which are immobilized at low temperatures. The mosquito infection technique is to feed them directly with the dengue-infected patient blood in the acute phase of the disease. The mosquitoes of the Toxorhynchites genus, which are not blood-feeding insects, can be used for the four-serotype dengue isolation, Japanese encephalitis and encephalitis of San Luis are more susceptible than cell culture isolation of dengue virus as well as the Drosophila melanogaster that can be inoculated by micro injection in the abdomen, and it could reach higher titers using less time in comparison with the Aedes aegypti inoculation [ 6 , 25 , 26 ].
Then, it eliminates anything that represents one type of fluorescent [ 27 ]. Some studies have reported a correlation between elevated NS1 protein levels with hemorrhagic dengue cases, and even this technique seems to be effective to detect DENV in the vector. When evaluating three of these commercial tests from different manufacturers with human serum samples, it is found sensitivity between Besides, some particular test characteristics could alter the result of the test as the rheumatoid factor depending on IgM ELISA type causes false positives [ 5 , 7 , 8 , 9 ].
The IgG is detected with low titers when ending the first week of the onset symptoms in humans, and they could even be detected for a lifelong. The tests to detect an IgG using the virus bind to a plate in a smooth antigen way protein cocktail usually present a low specificity so that there is a cross-reaction with other viruses from the same genus due to the proteins found in the antigen, and this test cannot be used to determine the infectious dengue serotype but it can present a higher sensitivity than the hemagglutination inhibition test.
If there are paired samples, a primary infection is considered when the seroconversion in antibody titers from the acute and convalescent phase occurs, and a secondary infection is considered when the antibody titers increase four times or more between the acute and convalescent phase, or in both serums [ 6 ].
It was used in some studies to identify the infected people in an early stage of dengue infection. However, the results were not very favorable for primary infections [ 29 ]. This test is considered the gold standard to detect neutralizing IgG antibodies because they have high sensitivity but can have a cross-reaction among members of flavivirus group. It is based on the binding of antibodies present in the sample which contains a known virus load working dilution. The mixture of both is incubated and inoculated in a cell line until forming lytic plaques that are observed when coloring the cell monolayer.
There are many different variants of a PRNT test that could produce a variation in the results when being compared with using different used reduction rates, different PRNT methods solid or semi-solid , and different cell lines. The use of different genotypes can alter the antibody titer results. Antibody titers were found in higher levels when using the same genotype rather than using the Asian genotype.
PRNT can be used to differentiate dengue infection to yellow fever infection. In a dengue secondary infection, PRNT can have a cross-reaction with other serotypes.
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Dilutions from to 1: were used. Among its advantages, this allows reducing the days to obtain the results and undertaking studies with native strains so that some of them produce tenuous plates that are difficult to count but they make easier to detect fluorescent foci. And, fluorescent foci can be counted with an ultraviolet light microscope or using computerized readers capable of reading fluorescent foci and reducing time to obtain results [ 33 , 34 ].
It is a variable in the PRNT test and uses the same immunological basis with the advantage of being worked on well plates different from the PRNT that generally uses well plates. This allows saving materials and handling a higher number of samples. If there were neutralizing antibodies in the sample, these ones would block the entry from the virus to the cell and would not produce the infection. When this technique was evaluated with serum samples from patients with primary infection, the results were very similar to the ones in PRNT.
However, the result correlation was very poor in comparison with this technique using samples from patients with secondary infections [ 35 ]. Enzyme-linked immunosorbent assay-format microneutralization test for DENV This test can be direct or indirect, and it is the most commonly used to identify the infected cells deriving from cell lines, salivary glands of mosquitoes, etc. It is based on the binding of the actual virus to a sample that can be infected cells with a dengue virus antibody joined to a fluorescent marker named conjugated direct immunofluorescence.
One of the most used fluorochromes for this technique is the fluorescein isothiocyanate FITC. Key paper: Olmstead, A. PLoS Pathogens. Names of authors from my lab are underlined. Other papers of interest from the Jean lab in the field of protein-based therapeutics: Richer M et al. A ; Jean F et al. Targeting viral protease-associated replication complexes for antiviral chemotherapy: From membrane-targeted activity-based probes to novel naturally occurring protease inhibitors. Our study is the first to provide cellular insights into the biological and enzymatic properties of NS3, a prime target for inhibitors of WNV replication [Condotta, S.
Project summary: The long-term goals of this research program targeted at virally encoded proteases are to increase our understanding of the virus host-cell interactions, and to discover how viral enzymatic pathways essential for the virus lifecycle can be interrupted. Over the last 10 years, my research in antiviral drug discovery has been punctuated by important contributions including 2 collective patents and 9 collective applications submitted to the University-Industry Liaison Office UILO at UBC.
My team has discovered novel classes of naturally occurring direct-acting antivirals DAAs targeting viral proteases that are essential for the virus lifecycle of emerging and re-emerging human viruses of great concern around the world e. Andersen; Paper of the Year Hamill P. Jean F. Key paper: Martin, M. Biological Chemistry. The findings of our studies also demonstrate the potential of our experimental approaches based on membrane-targeted activity-based probes for antiviral drug screening directed at complex induced-fit membrane-bound viral proteases in the context of viral infection.
Other papers of interest from the Jean lab in the field of viral proteases as therapeutic targets: Condotta, S. Cover Illustration ; Martin, M. Cover Illustration ; Hamill, P. Biological Chemistry Host-cell microRNA fingerprints reveal new insights into influenza A biology. Journal of Virology ]. Human exosomes — A new treasure chest for viral-disease biomarker discovery?
Delivery of exosome-associated cargos molecules e. Exosomes are microvesicles secreted by both normal and pathological cells and play important roles in intercellular communication. Secretory exosomes provide a rich source for discovering potential blood-based biomarkers through non-invasive blood tests see cover image: Journal of Virology ; June , V. Jean, team leader , my lab has recently gathered experimental evidence of virus-specific circulatory microRNA miRNA expression signatures biomarker fingerprinting on infections with pathogenic human viruses [UBC invention disclosure Dr.
Loveday et al. Jean; Cover Image Vol.