43 year-old male anesthesiologist presents to the ED with increasing shortness of breath, tachycardia and lethargy. Patient reports alternating positive and negative COVID tests since early March, job related exposure, as well as travel to Belize and twice to Summit County (considered a hotspot for COVID) since early March. Reported 7 days of fever, cough, illness in the 20’s of March which remitted. The fevers returned-measurably, and he was last measurably febrile on 4/10/2020. Since then he has been intermittently well and unwell, with night sweats, tachycardia. Not much cough at all now, just stuffy nose. Had anosmia, which actually resolved one week prior to this ED visit. He states that he has vague chest pressure, describes it as global non-radiating. He states that he feels his heart is racing when he has any physical activity.
COVID testing reported: Tested pos 3/24, neg test 4/7, pos 4/11 (also had pos RVP at that time for HHV-6), neg 4/22 (presenting to ED again on 4/26).
Constitutional: Positive measured fever. Recent illnesses as noted in HPI.
Eyes: No visual changes.
ENT, Mouth: No sore throat. No changes in hearing.
Respiratory: Positive cough. Positive shortness of breath.
Cardiovascular: Chest pressure as noted in HPI.
Gastrointestinal: No abdominal pain. No nausea. No vomiting. No change in bowel function.
Skin: No rashes.
Musculoskeletal: No back pain. No neck pain.
Neurological: No headache. No syncope. No dizziness. Positive anosmia-resolved.
Pertinent Exam Findings:
General Appearance: Airway intact. Alert, awake & appropriate.
Eyes: Pupils equal.
ENT, Mouth: Mucous membranes are moist.
Respiratory: CTA in all lung fields. No wheezes rales or rhonchi.
Cardiovascular: tachycardia rate and regular rhythm. No murmurs gallops or rubs. No calf tenderness or asymmetry.
Gastrointestinal: Abdomen is soft, non-tender.
Skin: Warm and dry. No Janeway lesions or splinter hemorrhages noted.
Musculoskeletal: Neck is supple. Extremities atraumatic.
Neurological: Awake, alert, oriented. Moving all extremities. CN3-12 intact
CBC 1: Leukocytosis (30K) with lymphocytopenia (0.55 lower normal is 0.85) but also neutrophils (28 and upper limit normal is 6.35) normal platelets and Hg.
CBC 2: Repeated with similar results
Troponin: Negative, repeat troponin negative but up trending.
Lactic Acid: Normal.
EKG: sinus tach, no STEMI, no significant intervals.
Repeat EKG: no changes.
CTA PE: Neg PE, no acute process, no findings of COVID.
CT Abd/Pel: No acute process.
3L IVF and remained tachycardic to the 110s.
Discussed with admitting team, they agreed to hold antibiotics.
Tested for COVID and admitted to medicine service.
ID was consulted and recommended:
- F/U COVID-19 PCR 4/26
- Send RVP given recent positive test – coinfection may explain prolonged waxing/waning course.
- Given travel, will send for malaria thick smear (although very low malaria rate in Belize).
- Given travel hx will send leptospirosis PCR from urine.
- Labs so far not c/w dengue or chikungunya.
- Monitor off of ABX for now, blood cultures obtained.
Patient initially had a white blood cell count of 30 and a heart rate up to 140s in the emergency department. Troponins remained within the normal range. He did not have evidence of endocarditis or myocarditis. By hospital day 3, day of discharge, his white count was 8 and his heart rate was in the 70s. Testing for malaria, mono, leptospirosis, COVID all came back negative. Imaging did not reveal source of infection. It was found in records that on a respiratory viral panel he did test positive for HHV-6 at an urgent care, this could have been contributing to the waxing and waning of the symptoms. He has had several significant life stressors recently and this could have been contributing to his tachycardia, but it is unclear if this would be contributing to his leukocytosis. Patient was to follow with infectious disease 1 week from discharge. He was discharged with the following diagnoses: SIRS versus sepsis stress reaction, recent COVID infection, and anxiety.
We are going to deviate from the typical discussion format on EMM and focus primarily on testing of COVID-19. This is a clinical case that highlights variability within testing for COVID-19 and the limited knowledge present behind testing. We are going to discuss both the diagnostic RT-PCR tests as well as the antibody test. As of 6/2/20, there have been almost 19 million tests conducted and over 2.2 million positive test results reported in the United States (CDC).
Figure 1: Total COVID-19 Tests by Country as of 6/2/20 (Source: Our World in Data)
The diagnostic test used is a reverse transcription-polymerase chain reaction (PCR) test and is commonly conducted using a sample collection from nasopharyngeal or oral pharyngeal swabs (see figure 1). The genome of a disease agent in a clinical sample typically can’t be directly detected because there is an insufficient amount of nucleic acid (DNA or RNA) present. Amplifying nucleic acid is done with polymerase chain reaction. The polymerase enzyme is found inside of cells which make DNA. In the PCR test, the polymerase enzyme is used to synthesize new strands of DNA repeatedly, creating a “chain reaction” to generate a large volume of DNA rapidly. The PCR test not only amplifies DNA to detectable levels, but it can also be used to uniquely identify viruses.
Reverse transcriptase-PCR is identical to PCR except the starting template is RNA instead of DNA. It is necessary to use RT-PCR when detecting nucleic acid from RNA viruses like the novel coronavirus. Reverse transcriptase is a polymerase that can use RNA as a template to make a complementary strand of DNA. After a DNA copy is created, traditional PCR can be used to amplify it. The PCR test only requires intact nucleic acid (DNA or RNA), this is why dead as well as alive organisms can be detected in a clinical sample.
Test characteristics, including sensitivity and specificity, are largely lacking as there is no accepted gold standard. Importantly, clinical sensitivity varies depending on several factors including how when the specimen was obtained, patient viral load and the design of the PCR assay. When the sample is collected, this requires a swab from the posterior nasopharynx which can be very uncomfortable for the patient sometimes limiting the collection process. Viral load can vary depending upon the individual patient, location in the body of specimen collection and where in the course of the illness the patient is during the test being obtained. Lastly, the design of the PCR assay varies by what RNA sequence is targeted.
Data suggests that a single negative PCR test is inadequate to rule out COVID-19, the incidence of infection is much higher than currently reported, and there is a crucial need to increase sensitivity of testing to help with diagnostic and epidemiologic accuracy. This is partially because sensitivity of the PCR test also varies depending on the RNA target sequence.
A study published in the Journal of Clinical Microbiology highlights the variation in sensitivities which were reported to be either 60.8% or 85% depending on the RNA target sequence that was used. RdRp-P2 was 60.8% and RdRp/Hel was 85%. These were specific to nasopharyngeal samples. According to a different study published in JAMA, with over a thousand samples from 200 patients from China, sensitivities were reported to be is following: bronchoalveolar 93%, sputum 72%, nasal swab 63%, pharyngeal swab 32%, feces 29%, blood 1%, urine 0% which highlights variability based on specimen location.
What about recurrent positive tests? There are several plausible explanations for this and while there is not enough evidence at this point to say for sure, it seems less likely to be re-infected by the same strain of virus twice within a few of months. As mentioned above, RT-PCR just detects RNA sequence and is unable to distinguish between alive virus and remnant pieces of viral RNA (this could just be viral junk left over causing positive test). Perhaps, it’s a testing characteristic of being initially falsely negative or even falsely positive on retest. It is also possible to be infected with a different viral strain from mutation, we know of at least two strains so far. Another known characteristic of viruses, such as the herpes virus, is latency. Latency is where the virus essentially hibernates and then at later time wakes. It would be uncharacteristic for a virus to have days to couple weeks for latency, usually it months to years. Lastly, there is also prolonged viral shedding for up to 49 days from symptom onset has been reported by case from Li Tan, et al.
Alternatively, viral load and antibody presence or function could be another factor. Low viral load could occur resulting in mild disease leading to ineffective or insufficient antibody production. In regards to antibodies, in order to be immune to a pathogen one needs a certain titer level of antibodies. If a person is exposed to the same virus in a short period of time, prior to obtaining a critical mass of antibodies that is necessary to prevent illness from the virus then perhaps one could get it again. Ultimately, time and clinical studies will reveal the truth, until then it remains speculation.
Figure 2: PCR testing (Source: JAMA)
Antibody testing is like looking into the past as it is an indirect assessment of prior infection. It is also a measurement of the host response to infection. If people have antibodies it is because they were exposed to the pathogen or a vaccine for the pathogen. The current antibody tests largely target the S1 unit on the spike protein because it is highly immunogenic and its affinity for the ACE2 receptor appears to correlate with infectivity. S1 subunits host the binding domain for the angiotensin converting enzyme2 (ACE2) receptor and this is thought to be the mechanism for SARS-CoV-2 to enter the cell. Currently, there are two types of tests being used, one is a rapid finger stick model with results in as little as 10 minutes and the other requires venipuncture. It is hypothesized that the rapid finger stick tests are likely to be less sensitive based on studies with this method for influenza viruses.
Different types of antibodies exist but we will focus on the pertinent IgM and IgG as these are the ones being tested. In general, most of SARS-CoV-2 specific IgM antibodies were positive after 5 days (3-6 days range) from onset of symptoms and IgG was detectable after 14 days (10-18 days range) from onset of symptoms in a study of 208 patients done in China. In another study by Wolfel, IgG was detected in almost all patients within 28 days from onset of symptoms. According to Gang Li et al, IgM can last up to 12 weeks and IgG lasted at least 3 months in the SARS-Associated Coronavirus (but it is unknown exactly for SARS-CoV-2).
The sensitivity and specificity of these tests has been reported to be above 90% for both IgG and IgM and as high 100% in some studies. Exact test characteristics are still unclear as there is no accepted gold standard. These tests are newer than the diagnostic tests and have little data available to date. Furthermore, most of the data gathered for the serologic tests are largely limited to hospitalized patients. It is thought that patients who are more ill may not have antibody levels that correlate with less ill patients that are not hospitalized. Specifically, lower viral loads may result in lower antibody levels. Adding to the challenge of an accurate test is antibody effectiveness, even in severely ill patients. In a study by Wu et al, roughly 30% of hospitalized patients had antibodies that were not able to neutralize the virus when used on viral growth plaque assays. This implies infected people could get reinfected, possibly due to poor affinity as mentioned above. Hopefully, now it is clear why the Center for Disease Control and regulatory health officials can’t use these results as a passport for return to work. Furthermore, individuals who may have obtained a test and had positive results should interpret these with caution, especially if one was never symptomatic.
- RT-PCR tests are used for diagnosis. They have poor sensitivity but an excellent specificity.
- People can have prolonged positive tests for up to 49 days and for various reason alternate between testing positive and negative.
- Antibody tests can let us know who has been exposed in the past but it remains to be seen if and for how long people have immunity against the virus.
- Antibody test results should be interpreted with caution.
- As of 6/2/20, there have been almost 19 million tests conducted and over 2.2 million positive test results reported in the United States (CDC).
- Bao, Linlin, et al. Lack of reinfection in Rhesus Macaques infected with SARS-CoV-2. doi: https://doi.org/10.1101/2020.03.13.990226.
- Chan JF, YIP CC, To KK, et al. improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific Covid-19-RdRp/Hel real-time reverse transcription polymerase chain reaction assay validated in vitro and with clinical specimens[published online ahead of print, 2020 Mar 4]. Journal of clinical microbiology. 2020.
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Aaron Wolfe, DO, FACEP