- Why does rt pcr take time
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Why does rt pcr take time
- Why does rt pcr take time
- Why does rt pcr take time
Therefore, the Ministry of Health and Family Welfare has come up with a new policy regarding the testing. Patient can be discharged after full clinical recovery and after they have tested negative once by RT-PCR after there are no symptoms.
Now, in most of the mild and asymptomatic COVID cases, the virus dies after the 7th or the 8th day of getting infected. At that time, it cannot get transmitted to other people. But the dead virus, or the particles of the dead virus, can be picked up in an RT-PCR test for COVID and there is a possibility of a positive report, even if the person has become free from Covid infection.
This can increase your isolation time period and can cause non required stress and anxiety. Many other countries also have changed the discharge and isolation policies. Know Its Importance. The definition of recovery is different for a treating doctor, Cirologist, and the general population. For severe cases, it might take a longer time. However, this information tells us that even after the virus is dead and ineffective in the body, some people can still have after-effects.
But that does not mean that these people can spread the virus to others. Wearing masks, maintaining social distancing and handwashing are the necessary precautions for months to come and just don't let your guards down. Read more articles on Miscellaneous. All possible measures have been taken to ensure accuracy, reliability, timeliness and authenticity of the information; however Onlymyhealth. False-negative results in a screening test can have serious implications during a pandemic, such as COVID because a proportion of true infected cases are categorized as disease-free and can unintentionally transmit the disease.
Unfortunately, there is no single molecular test that can guarantee the infection free status for a suspected case; therefore, the clinical history and social contacts of the individual should be always taken into account in the assessment of the infection probability. Repetition of the molecular tests over time also helps to increase the selectivity. Reports have described RT-PCR on various specimens obtained from the respiratory tract; however, there are accumulating reports indicating the lack of adequate sensitivity for the test.
For instance, Yang et al. Similarly, Zhao et al. One of the main reasons for such a high false-negative rate in RT-PCR results, is the time of sampling after the onset of symptoms. The time of sampling is important because it was shown that the false-negative rate of the test varies over time [ 14 ]. The false-negative rate of RT-PCR testing on nasopharyngeal NP and oropharyngeal samples was described as "shockingly high" in a study of confirmed cases.
In their investigation, the authors pooled the data on the confirmed COVID cases from seven previously published studies. They analyzed these data using a Bayesian hierarchical model to estimate the false-negative rate from 5 days before the onset of symptoms up to days post-emergence of symptoms. Consequently, the false-negative rate of the test changes over time depending on when the samples were obtained from the onset of symptoms, and even at best, the RT-PCR fails to detect a considerable fraction one out of five of the infected cases [ 14 ].
This could vary among different specimens and patients. The highest viral loads are found in the lower respiratory tracts of COVID patients compared to the upper respiratory tract [ 15 ]. However, sampling from the lower respiratory tract is difficult in patients with severe respiratory symptoms who are receiving oxygenation intervention [ 16 ].
In the upper tract, nasopharyngeal and oropharyngeal swaps or aspirates are recommended for early diagnosis of the infection. NP samples exhibited much higher viral loads compared to OP samples, giving a better chance detecting SARS-CoV-2 infection and lowering the risk of missing the infection [ 17 ].
Moreover, false-negative results occurred in some patients with gastrointestinal symptoms. Therefore, some false-negative results are inevitable depending on the specimen chosen and the patient clinical symptoms. Given the imperfect selectivity of the RT-PCR test, other diagnostic information should be taken into account to achieve the desirable sensitivity for true-positives or true-negatives for COVID These factors include the clinical symptoms, immunodiagnostic test results, and prevalence of the disease within the community.
These factors can help clinicians to better estimate how likely any particular case is to have disease. For instance, whether or not a case demonstrates the typical clinical symptoms of COVID can give a primary estimate of the probability of the case being infected, and successive addition of the molecular test results e.
RT-PCR and serologic tests will increase the confidence to distinguish between disease-free or infected. Furthermore, RT-PCR in combination with an immunodiagnostic test will improve the overall selectivity [ 18 ]. For example, in a retrospective study of patients, the combined selectivity of RT-PCR and antibody testing was significantly higher compared to each test alone.
Lastly, the prevalence of the disease should be taken into account in deciding whether or not a particular result is enough to send a person home as disease-free.
However, when the prevalence of disease increases throughout a community, that level of sensitivity is less valuable to ensure a suspected patient is disease-free. In technical terms, the negative predictive value NPV of the test decreases with an increase in the prevalence of the disease [ 19 ].
To sum up, the false-negative rate of RT-PCR is significant and varies across different specimens and time periods. However, the false-negative rate can be minimized when immunodiagnostic tests and clinical symptoms are considered along with the RT-PCR test result. Moreover, it is importannt to stick to social distancing and recommended hygiene protocols to keep the prevalence of the disease as low as possible, in order to maintain the NPV of the tests at a high level.
Otherwise, the negative results of PCR tests will no longer give us enough confidence that the suspected case is disease-free. Firstly, the viral load can be low or absent within the samples [ 19 ]. The viral load governs the amount of RNA in the samples. The higher the viral load in the sample, the more RNA with a better chance for a test to get a truly positive result. Secondly, the viral RNA might be subjected to denaturation or degradation in the samples due to improper manipulation or storage, which lowers the final amount of intact RNA for the test [ 20 ].
Thirdly, a sufficient viral load is limited to specific time periods when the virus rapidly replicates itself and is shed from the cells.
Fourthly, the viral load has also shown to vary in terms of the anatomical site from which the specimen is obtained Lastly, the virus is present at low numbers or is absent in some specimens from some patients, while other specimens might have a higher viral load in the same patients [ 21 ]. Therefore, the variability of the false-negative rate depends on the viral load, which in turn, fluctuates over the course of the disease, and between specimens from patients with different clinical characteristics.
Given the mentioned viral load variability over time, specimen, and patients, an improved RT-PCR test should be more simple, rapid, and cost-effective to allow frequent repettition [ 22 ].
This will increase the chance of detecting the infection if the test is repeated over time and on different samples. If the test can be made rapid and less labor-intensive, the sampling-to-PCR gap time will be shortened, which will reduce the loss of viral RNA due to denaturation during this period. Moreover, a simplified test will require less sophisticated laboratory equipment. These simplified tests could enable rapid point-of-care sample manipulation and analysis, with a higher throughput.
Pooling different samples from either the same patient or the patient's family members can reduce the number of tests and lower the costs positive rate of the test. Because in some patients the viral infection is limited to the lower respiratory tract, combining sputum, nasal and pharyngeal swabs coulsd be useful. In other patients with gastrointestinal involvement, the virus was only found in fecal material, while RT-PCR of the NP swabs and sputum were negative.
Therefore conducting the test on pooled samples from different specimens can improve the probability of getting a sample with sufficient viral load to increase the accuracy of RT-PCR. The other benefit of pooling samples is to allow better at-home quarantine decisions amongst communities. For instance, pooled samples from the whole family of a suspected case can provide guidance on strict quarantine for the entire family, to reduce disease transmission in the community [ 23 ].
Therefore, the repetition of the RT-PCR test in pooled samples might offset the high false-negative rate of the test. Also, the conduction of the test in pooled samples appears to increase the utility of the test for screening purposes. To this end, recent cutting-edge technology has attempted to provide simple point-of-care or at home RT-PCR kits. By overcoming these obstacles, the laboratory RT-PCR test can be turned into a convenient, rapid, and budget-friendly kit that can be used more widely in clinics.
Technically, the RT-PCR procedure for SARS-CoVinfected samples consists of several steps, and needs laboratory equipment that makes the process tedious and difficult to be conducted outside the laboratory setting. First of all, the RNA material must be extracted from the cells and the virions viral particles and preserved from destruction by RNase enzymes. This step needs laboratory equipment such as a centrifuge and a laminar flow cabinet, and might lose some of the RNA materials due to denaturation.
Secondly, the process of PCR requires thermal-cycling equipment for creating a cyclic temperature change during the process of RNA amplification. The third difficulty is the readout method used, which in most cases required expensive sophisticated spectrofluorometric equipment [ 24 ].
During this process, certain laboratory chemicals and equipment are used for specific purposes. Firstly, the infected cells and the virions are disintegrated by the addition of lysis buffer typically containing detergents Tween 20 or Triton X The lysis of the cells and virions causes all the biomolecules, including viral RNAs to be released into the medium and be readily available for the test.
The lysis buffer also contains salts such as sodium iodide NaI or guanidinium thiocyanate GuSCN that facilitate the separation of the viral RNA from other biomolecules e. Centrifugation of samples containing these salts assists in the separation of these proteins from the viral RNA fraction.
Besides, cellular RNase enzymes are inactivated by the addition of detergents and thermal treatment. This cycle is repeated several times e. The thermocycler apparatus that provides this accurate cycle of temperature changes is expensive equipment that is often confined to a laboratory [ 25 ]. Finally, the increasing number of C-DNA replicates is monitored using a real-time spectrofluorimetric technique that is also expensive and not always available.
This technique offers a readout of the C-DNA amplification on a computer screen based on the fluorescent signal that changes increases in line with C-DNA numbers. This fluorescent probe de-quenches upon the separation of the C-DNA strands from each other. In both techniques, a spectrofluorimetric apparatus coupled to a computer is required for the final readout of the RNA amount in the samples.
These pieces of equipment are expensive and may not be available everywhere in large numbers [ 25 ]. Given the aforementioned difficulties of the RT-PCR test, enormous efforts have been made to produce an easier, faster, and more convenient test capable of being used outside the laboratory environment.
A simple and rapid test can reduce the sampling-to-result time SRT and encourage its wider application. The test procedure should require fewer steps and laboratory tools.
A shorter SRT and easier manipulation of the sample will have some other benefits, including an increase in the test sensitivity. One important simplification in the nucleic acid amplification procedure was the invention of an isothermal PCR method that eliminated the need for a thermal cycling apparatus.
This allowed the amplification of RNA or DNA using a widely available kitchen oven maintained at a specific constant temperature. Instead, the DNA polymerase itself displaces one of the strands of the DNA as it acts on the other strand and synthesizes a new copy. Therefore, the technique is called the loop-mediated isothermal amplification LAMP technique, described in reference [ 26 ].
The provision of a constant temperature is technically much easier than a temperature cycling program that is required for conventional PCR [ 19 ]. This reduction in the number of steps of the test offers some advantages.
Firstly, a single step preparation of RNA reduces the SRT and increases the potential of the test for wider application. A shorter SRT decreases the probability of disease transfer by individuals whose test results have yet to be determined. Secondly, a one-step preparation of the RNA samples is much easier for potential users to learn how to use the test correctly.
Thirdly, during the extraction of RNA from the sample, there is a risk of viral transmission from the samples to the laboratory staff, and cross-sample contamination due to unintentionally errors in sample manipulation. A shorter and easier process of RNA preparation can minimize the mentioned risks.
Lastly, the combination of the steps has been shown to eliminate the need for apparatus that limits the test to a lab environment [ 19 ]. In the case of COVID infection, it is only necessary to know whether or not viral RNA is present in the samples; therefore, there is no need for expensive quantification methods like spectrofluorimetry. Instead of quantitation, qualitative readouts such as a color change are much easier to achieve, and are more appropriate for diagnosis of SARS-CoV-2 infection.
By using these kinds of readout, one can simply observe the results with the naked eye [ 19 ]. For instance, Yu et al. In this test, the positive samples with Genefinder dye turned bright white, while the negative samples remained blue under blue light. In this technique, the sample color changes from white to blue if the samples contained the amplified RNA material. The method contains a kit with a lateral flow visual readout using a strip of paper.
In this test, one just needs to dip the correct end of the designed strip in the vial of the final RT-LAMP product and wait to observe either a positive or negative result. These results appear in the form of a band at specific distances from the starting point. The FAM-biotin trans-reporter is already placed and affixed to the strip. As the sample flows laterally across the strip, the remaining target sequence interacts with the FAM-biotin trans-reporter molecules on the strip producing the band.
Reprinted from ref. Taken together, the RT-LAMP methodology has provided a new alternative for rapid, simple, and home-use molecular diagnostic tests. Being rapid and simple has enabled wider and more frequent use of these tests for COVID detection bymembers of the public, therefore, overcoming the high negative rate of RNA-based tests.
On the other hand, the false-positive rate of these tests poses some issues regarding the management of the COVID pandemic that will be discussed in the following section. Another question that needs to be addressed is to be certain that a positive PCR test result for COVID truly reflects the infected status of the patient.
To this end, a positive PCR test result can be confirmed when the sample is examined by the gold standard viral culture test. Although data on viral culture results are sparse, there is some evidence that can help us to evaluate the predictive value of the PCR test as a screening method under different conditions.
To what extent a positive PCR result predicts the chance of someone being infectious may be governed by different factors. These factors include the time after symptom onset, symptom severity, and the specimen used when the PCR test is carried out [ 30 ]. First of all, we should consider the time of symptom onset when interpreting the probability of being infectious according to RT-PCR results.
It has been reported that the viral load is maximum by the 3rd day from the onset of symptoms in samples from the upper respiratory tract, and that live virus can still be detected at 8 days after the onset of the disease symptoms by the viral culture test.
However, beyond this period the virus might no longer be infectious, although RT-PCR results continue to detect the presence of viral RNA material [ 31 ]. In one study [ 32 ] conducted on hospitalized patients with COVID, RT-PCR testing showed that the duration of virus shedding was longer, and ranged from 0 to 20 days post-onset of symptoms.
However, there is some evidence from serum samples suggesting that the RT-PCR could give positive results by detecting viral RNA remnants long after infectious virus had disappeared. Therefore, it is possible that the RT-PCR result was positive even after the infectious virus had been neutralized by the immune system.
The source of the specimen can also reflect the disease progression. Viral shedding can be detected only during a specific period that varies according to the sampling site. For example, within 5—6 days from the onset of symptoms, high viral loads were reportedly found in the upper and lower respiratory tracts in COVID patients.
As a result, nasopharyngeal NP and oropharyngeal OP swabs are recommended for early diagnosis of the infection. However, upper tract respiratory samples might fail to give sufficient viral load for detection purposes in a given time point of the infection [ 16 ]. For instance, one case report showed that the virus was only detected within the first 18 days from the onset of respiratory specimens [ 33 ], while the presence of the virus in fecal samples was detected for a longer period after respiratory samples became negative [ 34 ].
Some patients with COVID pneumonia exhibited a longer-lasting shedding of the virus in the respiratory tract, whereas there had been high loads of SARS-CoV-2 in their fecal samples from the beginning of the symptoms [ [35] , [36] , [37] , [38] ].
The fecal shedding of the viral RNA continued between days 1—33, while at least 3-days post-onset of symptom was identified as the optimum timepoint for a high positive rate of RT-PCR test in upper respiratory tract samples [ 34 ]. Consequently, RT-PCR positive results in fecal and upper respiratory tract samples will continue for a specific period of time probably longer for fecal samples , but the infectious status of the patient might be limited to the period when active virus can be detected in serum samples.
Lastly, the initial viral RNA load in the specimen can influence the likelihood of getting a positive PCR result and can result in the test being oversensitive. This detection limit can be improved lowered by making modifications in the test, such as improving the viral RNA extraction method and the fluorescent probes. However, reducing the detection limit of the test might also increase the false positive rate of the test in the later stages of the infection, because lower amounts of remnant RNA from the inactivated virus would be sufficient to give a positive result.
Therefore, other molecular and clinical evidence in combination with RT-PCR results should be used to confirm the status of the infection [ 39 , 40 ]. Taken together, the PCR results for COVID should be carefully considered to confirm the infection, and special attention should be paid to the stage of disease development and the type of specimens collected for the test.
The false-positive rate of the diagnostic tests might at first glimpse, seem not to be as important as the false-negative rate, given the current global prevalence of the disease. However, erroneous positive results are indeed important, and can have serious implications for public health services [ 3 ].
Currently, the global health policy is to maintain COVID transmission as low as possible within communities. When the PCR test remains positive over time, the positive results will be taken seriously, and the suspect patient is recommended for stay-at-home quarantine as long as the repetition of the test gives positive results.
For instance, as of September 19, , the false positive rate of the swab tests was estimated to be between 0. Despite the low positive predictive value for the test, patients are still recommended to follow a strict quarantine which will not cause a serious social problem.
However, the low positive predictive value of RT-PCR tests causes problems for health and social services. The prevalence of the disease is likely to be much higher in the health-care environment, and the high false-positive rate of PCR tests will lead to the quarantine of significant numbers of social health-care workers and health-care personnel, that might have been avoided.
This could cause a serious shortage of health-care workers especially at the peak of waves of disease transmission [ 3 ]. Therefore, the high false-positive rate of the RT-PCR test is indeed a problem among health-care personnel and the results of the test should be confirmed based on other clinical evidence.
Moreover, the RT-PCR and serologic tests display opposite trends in sensitivity during the infection, in which one test can cover the failure of the other as the disease progresses [ 18 ]. The combination of these techniques has already been shown to improve the sensitivity in the early stages. Guo et al. While the RT-PCR was highly sensitive during the first week after symptoms emerge, the serological tests had higher sensitivity in the second week, underlining the advantage of the combination [ 17 ].
During the course of the infection, Zhao et al. Besides, these tests follow opposite trends in the sensitivity during the infection period; therefore, the use of both tests can improve the. The results of serologic tests depends on the amount of antibodies produced, which may vary according to the severity of the disease.
Some studies proposed the monitoring of antibody titers as a prognostic indicator for early aggressive treatment of the disease. Other studies observed higher antibody titers in elderly patients compared to other age groups regardless of the disease severity. Moreover, it is traditionally believed that seroconversion from IgM to IgG takes place during the development of the humoral immune response; however, some reports found that the expression of IgG and IgM could occur simultaneously, and the median time of appearance from onset of symptoms varied in different patients [ 18 ].
The median time of seroconversion of antibody isoforms may vary according to the type of immunodiagnostic test and the choice of the target antigen. In a large multicenter study conducted by Long et al. Zhao et al.
The median seroconversion time varied more widely in other studies.
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