Dear colleagues,
We work at a laboratory of diagnosis of COVID 19 with qPCR.
As intended by the protocol, we recieve samples of positive patients until the second negative PCR (second negative controle).
The problem is that some patients still positive for long time, very often, with CTs very close to the upper limit (I mean Cts of 36 or 37, when the threshold of the PCR kit indicate 38 as the upper limit of positivity).
As, positive patients are confined at hospitals in our country, this causes a serious problem.
Thank you in advance
Yuan-Yeu Yau, Ill answer each point here as best I can
"In Ireland, if a person confirmed COVID-19 positive, will s/he take to the hospital for isolation and treatment (enforced; mandatory) or is s/he advised to stay at home for self-isolation until symptoms become worse (then go to hospital for treatment)? In the US, it takes the later approach."
So currently if they are sick but not requiring hospitalisation then they self isolate for 2 weeks after symptoms subside in their own homes. If the person needs to be hospitalised then that is done. These people are tested for the virus. All of Irelands quarantine points are government guidelines, there is very little in the way of enforcement as that has always been our countries way of policing where we can. It has been very effective to date. Though to the negative tests then positive after infection a lot of them are case study/anecdotal I believe so I would be interested to get truer figures for this
"Will different real-time PCR machines (from different companies) and different enzymes (DNA polymerases) and components (from different companies) will affect the ct number? In other words, the same sample will have very close Ct values in machines made from different companies and with PCR components from different companies? "
100% but to what degree I am not sure, some companies have different cut offs as to what defines a positive like a cut of ct of 35 or 40 or 45 and this will all change the sensitivity and specificity of the test depending on what is required of it. But yes, there will always be variance and that is completely fine and known for medical scientists. Every test we ever use has variance and its just something we have to live with. There is no perfect test.
"Have you heard that they picked up other coronaviruses, not the novel coronavirus SARS-CoV-2? For example, coronaviruses responsible for 4 human flu or common cold. Antibody derived from SARS-CoV-2 from some popularly used kits (i.e. 'Antibody Fast Testing Kits') can pick up these 4 coronaviruses. It will show 'positive' if a person is previously infected with these 4 coronaviruses. Fine print of warning is usually printed on such boxes. I wander if current SARS-CoV-2 RT-PCR tests can pick up these 4 coronaviruses if they present in a patient? "
So all the accredited tests have done analytical specificity testing to see if there are any cross reactive species that may give us false positives and I believe they are all fine. However in most tests there are two genes being looked for, a specific and non specific one. A lot of tests detect the E-gene which is positive in SARS-CoV-2 but also in other coronavirus's we may see during the cold season (accounts for 15/30% of the common cold cases). However we also have the specific gene to look for like N2 which is specific to just SARS-CoV-2. So when we get a result back with N2 with/without a positive E gene we know its SARS-CoV-2. If its just the E gene on its own we would term that Indeterminate or a Presumptive Positive for SARS-CoV-2 but would require more testing. The non-specific target there on purpose to catch most coronavirus and not miss a SARS-CoV-2 due to a mutation etc in the novel/specific regions
Ab kits have had some issues and here our government isnt using them for a few reasons not withstanding that it may not indicate immunity and we may ver rely on that. Plus previous coronavirus's have only had immunity post infection for a year or two so what this one will do we dont know.
Hope that answered the questions :)
More Questions than definite answers about COVID-19 recurrence
At the outset I would say "It is very easy to ask questions, but very difficult to answer them correctly".
This problem of recurrence issue or positive results based on RNA RT-PCR tests for SARS-Cov-2 in the management of COVID-19 is still common, and has many possible explanations, some proven, other hypothetical. The possible
These issues and possible explanations (my view points, as all of the answers are not evidence-based) include:
1. A discharged patient on the basis of improvement of symptoms, and two consecutive RT-PCR based tests, becoming positive again. In a study (1) such patients were negative due to a substandard kit used, and were positive with a standard test kit. However, such patients had evidence of anybody formation, had no worsening of symptoms or lab tests, and did not infect any contacts. Other similar cases, where patients are asymptomatic or mildly symptomatic with no progression of the disease, all reflect a recovered patient. This is because the RNA based test will be positive irrespective of whether virus is active (active disease), or inactive (virus litter or garbage) in recently recovered patients.
2. Positive RT-PCR tests on fecal samples in children or adults reported for many weeks even after improvement (2). However, these patients developed SARS-CoV-2 specific antibodies (when it was done) and did not develop any worsening of symptoms or progression of disease by lab and radiology. There was no report of infection in the contacts. Whether such patients are carriers, is a debatable issue, needing further follow up of such patients, immune status by antibody testing, and any contagiousness.
3. Reinfection after initial recovery from COVID-19 due to poor immunity or by another SAR-CoV-2 strain: There are reports of cases who recovered and developed symptoms again with positive RT-PCR tests for SARS-Cov-2. This could have been because of poor immune status of such patients, use of immunosuppressants (steroids or other drugs)(3) or a different mutant strain of SARS-Cov-2. There are reports of many mutant strains causing COVID-19 in Europe (4). Again more information about the history of use of immunosuppre-ssant drugs in such patients, course of the illness by follow up and investigations for poor immunity will help.
Because there are not enough study or follow of such patients, my explanations may be proven wrong later on, so keep following the literature for answers in this evolving COVID-19 pandemic.
1. Preprint Clinical characteristics of the recovered COVID-19 patients ...
2. Preprint Prolonged presence of SARS-CoV-2 in feces of pediatric patie...
3. Preprint Breadth of concomitant immune responses underpinning viral c...
4. https://www.biorxiv.org/content/10.1101/2020.04.29.069054v1.full.pdf
Yea it can be a challenge and Muhammad Yousuf made some very good points. If we break it into two groups 1) requiring hospitalisation and 2) not requiring hospitalisation then we can potentially say Group 1, even at a high ct should be continuing treatment and precautions.
Group 2 is trickier, they are not too sick and may even be healthy now but have elevated ct for a long period. The main issue here is they may be infectious to others during this, even with positive antibody response or they may disprove later. Either way a risk assessment for these people is needed. Is the situation such that they can manage to remain quarantined until their ct values move to a negative? What is the risk of allowing them back to work/public if we are not sure if they can spark a new cluster of infections? For these people is the viral load very low and how would a mask deal with these people (important for asymptomatic but potentially positive individuals)
As it stands with lack of treatments/vaccines etc we have to take the safer course and keep this group quarantined and support them in that. Also hope that they are the exception to the rule which they seem to be from my experience.
@ Stephen Reynolds
In fact, the discharge criteria for SARS-CoV-2 confirmed COVID-2 patients (1-2) admitted to the hospital includes:
1. Resolution of the symptoms (particularly fever) for at least 3 days
2. Improvement in the laboratory tests and chest findings radiologically
3. Two consecutive negative RT-PCR tests on representative samples (nasopharyngeal, oropharyngeal or anal swabs).
After discharge the patients are recommended to have two weeks of quarantine at home with distancing and hand hygiene measures.
Ideally, discharged patients should again be tested for RT-PCR test as often as appropriate (every 3 days, or weekly) and if the patient becomes symptomatic again, or another test returns positive, two week quarantine period should be extended. However, all the countries are not following a uniform policy for this due to logistic issues.
Patients who are old, immunocompromised, or are on immunosuppressive medications need extended testing and protection.
Most importantly, a SARS-CoV-2 specific validated sensitive antibody test need to be done to check for the development of protective antibodies (not being done or not available in many countries due to logistic, or quality issues)
1. https://www.ecdc.europa.eu/sites/default/files/documents/COVID-19-Discharge-criteria.pdf
2. https://www.cdc.gov/coronavirus/2019-ncov/hcp/disposition-hospitalized-patients.html
In our country, till date, we used to wait for 2 negative PCR reports. But, now with rising cases and to maintain the availability of beds in the hospital, new guidelines recommend keeping only sick in hospital and rest in home isolation
So we have had about 3-4 patients who are persistently positive and treat them in isolation. This is not good for their rehabilitation and their mental well being. They do not have symptoms of Covid-19 anymore, but have other problems such as fraitly, cardiovascular issues etc. There is nothing you can do, except continue to isolate them, and manage them as they come, with clear decesions about escalation procedures etc. Happy to help further if you want to message me directly
I think we should look at the antioxidant supplementation. A high viral load is still suggestive of oxidative stress. We must arrive at a balance. If you cant increase the vitamins why don't you go for regular administration of antioxidant beverages
No evidence for any of those. It is important NOT to give such treatments that are not proven
What exactly is not proven? Viraemia causes oxidative stress? Oxidative stress weakens the immune system? Antioxidant beverages (made of fruits and vegetables) can relieve oxidative stress? Why do we administer Vitamin C? If something is safe (no acute toxicity at 5,000mg/kg) then why not? We seem to look at viral inhibition, immunomodulation and other supportive strategies for the management of COVID-19. Good! but I suggest we open up to other safe approaches. The virus suppresses Interferon type 1 production, does Vitamin C have any effect on this? The NK cells unusually take the back stage in the fight against SARS-CoV-2 , what is the effect of bioactive compounds from fruits and vegetables on the stimulation of these cells ? I guess we might be missing out a lot. Even WHO is calling for investigation of the “COVID-19 Organics” of Madagascar. Why? Undeniable results! But while we await the outcome lets keep it at the level of just fruits and veggies
Muhammad Yousuf, again some good points though I am also speaking in relation to asymptomatic individuals with long positive periods with no symptoms and low viral loads. Can be hard to maintain them in quarantine if they feel they dont have to stay there.
On the point of Ab tests "Most importantly, a SARS-CoV-2 specific validated sensitive antibody test need to be done to check for the development of protective antibodies", an issue with this currently is we are not 100% sure if these denote long lasting immunity or a shorter acting immunity. Hopefully the latter
Dear Professors,
Thank you for your answers, I quote from them the expressions I think explain exactly what I wanted to say:
"asymptomatic individuals with long positive periods with no symptoms and low viral loads. Can be hard to maintain them in quarantine if they feel they dont have to stay there".( Stephen Reynolds )
" This problem of recurrence issue or positive results based on RNA RT-PCR tests for SARS-Cov-2 in the management of COVID-19 is still common, and has many possible explanations, some proven, other hypothetical" (Muhammad Yousuf )
More research is needed on this subject especially in relation to risk assessement. Meanwhile, as mentioned Stephen Reynolds, we need to take the safer course at the moment.
Please to share any useful information about this issue.
Also Hamza RAHAB PCR cannot give us information on infectivity. It may be the people with high ct values are not infections but we cannot rule this out.
It is the same issue with a lot of the studies on how long the virus can stay on a surface. Its positive on PCR for say 3 days but is it still infectious at this point?
So much work to do
I agree with Stephen Reynolds . PCR is not a definitive way to assess infection levels. Viral broken sequences might still be roaming in the bloodstream andwhen you sample that from blood you might just get false positives.
Oxidative stress might not be a solution for the same reason described above, however IFN1 levels might be indicative and suggest a good counter-protocol as Adekunle B Rowaiye suggested (reducing oxidative stress, increase vitamin intake etc...).
A possible marker to analyse could be related to the impaired ACE-2 activity or to angiotensin or aldosterone levels...which still, however, might be affected by the quantity of sclerotic tissue damaged in the lungs inflicted by the infection.
An overall increased level of angiotensin (as compensatory homeostatic mechanism), paired with lung NMR scan, might give some hints of an ongoing viral infection with a recent objective examination of the sympthoms.
It's not an easy situation, and the best solution should be tailored very specifically for each case.
Wish you all good luck.
Hamza RAHAB
This problem did not happen in your country only. There are some reports existing for this problem already. In some regions, they need more strict requirement-- at least 3 negatives-- to declare a patient virus-free. For example, in Taiwan. Rare events were also observed and reported-- some patients can have positive results from real time RT-qPCR for a long time. I watched an interview on TV, a doctor said that "even the virus is dead, their genetic materials can still be amplified for a while." For my own PCR experience (not on virus), the statement and concern is legitimate.
yes, 3 negatives is a true negative.....it depends on whether you think your test has a high false negative rate. here, rate is very low, so 1 negative is enought
Keeping them in quarantine while continuing to give treatment in severe cases, but in cases that are not severe, it is sufficient to quarantine the house
A mixed algorithm is useful for decision, if you answer these questions:
1- Positive cases, with or without treatment?
2- Treated cases, in-patients or out-patients?
3- Serologic tests, IgM and IgG?
4- Current symptoms and signs?
5- Recent chest CT or CXR?
6- Recent exposure to positive cases?
7- Comorbidities?
Please estimate
- Mortality risk
- Morbidity risk
- Virus spreading risk
in each situation (not only for each clinical case). Each situation contains each posotive patient or healthy person, home, workplace, homemate, family, exposure,....
Asymptomatic people with positive PCR donot need hospitals . Only isolation not necessary in hospitals.
Thank you for your question Hamza RAHAB !
I agree with all the statements. Being novel, we really cannot have the right answers yet. But we need to keep on asking. I had a patient with 3 consecutive negative RT-PCR tests, then had a positive IgG Antibody. At the end of the day, we had to go clinical. Hopefully we can harmonize RT-PCR for COVID-19 soon and/or discover a better solution.
Avinash Aujayeb, 3 negatives do not make a true negative. There are too many unknowns currently and we do not know the true false negative, ppv, npv, snes and spec of these tests. Three negatives would denote a very low risk but we cannot say 'true negative' yet. We have found some tests to be much less sensitive than others
Ludovico Pipito Thanks for that, on the other potential markers I would have the concern that one, they would be very non specific especially as flu and cold season swings around and two, that post infection they could remain elevated for a period as repair gets underway (though maybe not as long as some PCR's). Potentially a combination testing approach but I cant see the efficacy atm
Yuan-Yeu Yau currently in Ireland if we have a high ct value and they patient has had and cleared the infection clinically they are told to wait 7 more days before returning out of quarantine. There is a lot of issues with very sensitive platforms like the GeneXpert and how we interpret them clinically for best management and risk mitigation. Bloody tricky problem to work through
Hamza RAHAB I would also count values below a ct of 40 as positive and over 40 as a high ct value that needs interpretation based on their clinical picture (new presentation or old etc)
Traci L Testerman The PCR tests on the market currently are quite specific, they dont seem to be picking up non-coronavirus's as false positives. There is of course the question of the other coronavirus's but PCR has the non specific E-gene (or other) and specific e.g. N2, ORF etc genes to help in the differential. We get a lot of these prolonged high ct's from staff in the hospital so it is very unlikely this is a defect in immunity, more likely perhaps it is non-infectious remnant virus or its last hurrah before removal from the system (we just have to hope they are not infectious at this low viral load). Unfortunately serology has been a mix bag so its used are limited at the moment, at a national level we are not using serology.
I do fully agree we dont want super spreaders from false negatives but unfortunately it is going to happen when we dont know the limitations of the tests we use for very valid reasons currently
Stephen Reynolds Yes, I have seen reported that patients become 'positive' after 3 negatives test.
In Ireland, if a person confirmed COVID-19 positive, will s/he take to the hospital for isolation and treatment (enforced; mandatory) or is s/he advised to stay at home for self-isolation until symptoms become worse (then go to hospital for treatment)? In the US, it takes the later approach.
Stephen Reynolds < I would also count values below a ct of 40 as positive and over 40 as a negative....>
Will different real-time PCR machines (from different companies) and different enzymes (DNA polymerases) and components (from different companies) will affect the ct number? In other words, the same sample will have very close Ct values in machines made from different companies and with PCR components from different companies?
Stephen Reynolds < The PCR tests on the market currently are quite specific, they dont seem to be picking up non-coronavirus's as false positives. >
Have you heard that they picked up other coronaviruses, not the novel coronavirus SARS-CoV-2? For example, coronaviruses responsible for 4 human flu or common cold. Antibody derived from SARS-CoV-2 from some popularly used kits (i.e. 'Antibody Fast Testing Kits') can pick up these 4 coronaviruses. It will show 'positive' if a person is previously infected with these 4 coronaviruses. Fine print of warning is usually printed on such boxes. I wander if current SARS-CoV-2 RT-PCR tests can pick up these 4 coronaviruses if they present in a patient?
Traci L Testerman, I totally agree, even high ct values which indicate a low load cannot rule out you are still infectious. But a continuing high ct value over the course of the week and the nasty clinical decision has to be made for health care workers and in most cases they are too valuable to not have in. Its a tough call but we have had to make it.
Yuan-Yeu Yau, Ill answer each point here as best I can
"In Ireland, if a person confirmed COVID-19 positive, will s/he take to the hospital for isolation and treatment (enforced; mandatory) or is s/he advised to stay at home for self-isolation until symptoms become worse (then go to hospital for treatment)? In the US, it takes the later approach."
So currently if they are sick but not requiring hospitalisation then they self isolate for 2 weeks after symptoms subside in their own homes. If the person needs to be hospitalised then that is done. These people are tested for the virus. All of Irelands quarantine points are government guidelines, there is very little in the way of enforcement as that has always been our countries way of policing where we can. It has been very effective to date. Though to the negative tests then positive after infection a lot of them are case study/anecdotal I believe so I would be interested to get truer figures for this
"Will different real-time PCR machines (from different companies) and different enzymes (DNA polymerases) and components (from different companies) will affect the ct number? In other words, the same sample will have very close Ct values in machines made from different companies and with PCR components from different companies? "
100% but to what degree I am not sure, some companies have different cut offs as to what defines a positive like a cut of ct of 35 or 40 or 45 and this will all change the sensitivity and specificity of the test depending on what is required of it. But yes, there will always be variance and that is completely fine and known for medical scientists. Every test we ever use has variance and its just something we have to live with. There is no perfect test.
"Have you heard that they picked up other coronaviruses, not the novel coronavirus SARS-CoV-2? For example, coronaviruses responsible for 4 human flu or common cold. Antibody derived from SARS-CoV-2 from some popularly used kits (i.e. 'Antibody Fast Testing Kits') can pick up these 4 coronaviruses. It will show 'positive' if a person is previously infected with these 4 coronaviruses. Fine print of warning is usually printed on such boxes. I wander if current SARS-CoV-2 RT-PCR tests can pick up these 4 coronaviruses if they present in a patient? "
So all the accredited tests have done analytical specificity testing to see if there are any cross reactive species that may give us false positives and I believe they are all fine. However in most tests there are two genes being looked for, a specific and non specific one. A lot of tests detect the E-gene which is positive in SARS-CoV-2 but also in other coronavirus's we may see during the cold season (accounts for 15/30% of the common cold cases). However we also have the specific gene to look for like N2 which is specific to just SARS-CoV-2. So when we get a result back with N2 with/without a positive E gene we know its SARS-CoV-2. If its just the E gene on its own we would term that Indeterminate or a Presumptive Positive for SARS-CoV-2 but would require more testing. The non-specific target there on purpose to catch most coronavirus and not miss a SARS-CoV-2 due to a mutation etc in the novel/specific regions
Ab kits have had some issues and here our government isnt using them for a few reasons not withstanding that it may not indicate immunity and we may ver rely on that. Plus previous coronavirus's have only had immunity post infection for a year or two so what this one will do we dont know.
Hope that answered the questions :)
We had patients admitted in March 2020 who had persistent PCR positive over weeks. We had to keep them admitted in the hospital isolation ward under the national policy. The new national policy allows us to discharge such patients now if they are asymptomatic. But, the challenge is to strictly advise them home isolation to mitigate the spread further to household members. The presymptomatic transmission and post-recovery transmission are very difficult to control at the community level. The social distancing, hand hygiene, and PPE are the best barriers for such forms of spread
Until the protocol is updated, you are most likely picking up dead virus particles. This is a problem of PCR based testing.
Antigen testing is needed.
Traci L Testerman true, but antigens are not exponentially increased by the assay protocol thereby skewing results. Then there are the false positives (or stronger positives) from PCR based assays because sooner or later there is amplified DNA everywhere.
It has been a while since I worked at an HIV research lab in Maryland, but it was a state of the art P3 certified lab and we still had problems with amplified product showing up everywhere, even in the pre-PCR room. We had to regularly stop lab work to deal with "escaped templates".
Today, I regularly run qPCRs on mRNA and cDNA, and a CT of 38 is bordering on negative for my protocol. In fact, for one of my cell lines I use a control whose CT is 36-39 as my low limit positive control, but for another cell line that is already in negative result territory and I have to use a different expression control.
Leaving aside the peculiarities of the course of Covid infection in humans, and without knowledge of the lab and work flow, I can only point at lab problems often encountered when dealing with PCR and its peculiarities. With those, I have tons of experience. However, I had time to deal with them and the freedom to adjust, and they were not a matter of life and death unlike now. And it doesn't help that politics has invaded science in many places around the world concerning Covid-19.
RNA Shedding, Re-occurrence and or New Infection of COVID-19.
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